]> 199904 eng REGULATION OF ETHANOL INDUCIBLE CYTOCHROME P450 2E1 (CYP2E1) Research RPROJ We have demonstrated multiple regulatory mechanisms for CYP2E1: induction via transcription, mRNA stabilization, activation of mRNA translation and protein stabilization, suppression via transcription, mRNA degradations and protein degradation. We have recently reported transcriptional suppression of CYP2E1 gene by an exogenous compound, YH439. The potential beneficial effect of YH439 were studied in an in vivo model of acute hepatitis by treatment with carbon tetrachloride. In vivo hepatobiliary imaging analyses revealed that YH439 efficiently protects liver injury from carbon tetrachloride. These results were confirmed by the corresponding changes in the levels of serum transaminases and by histological evaluations. These data were recently published in Nucl. Med. Biol. Because of numerous recent reports about the role of enzymes involved in early signal transduction in cell death, activities of these enzymes in carbon tetrachloride-treated rat livers were measured. The level of c-jun kinase activity was transiently (1-2 hr) and selectively activated while p-38 protein kinase or mitogen activated protein kinase remained unchanged. Consistent with the in vivo data, we also observed that c-jun kinase in PC12 and COS-7 cells was selectively activated by 4-hydroxynonenal (HNE), another cytotoxic metabolite from the CYP2E1-mediated metabolism of long chain fatty acids. The selective c-jun activation was dependent upon HNE concentration and transient (within 15-30 min) upon HNE treatment, long before the apoptotic cell death. To further elucidate the relationship between the c-jun kinase activation and cell death in an in vitro model, we have recently transfected human CYP2E1 cDNA into HepG2 and Neuro2 cells via stable transfection. Using these cells with high level of CYP2E1 protein and activity as an in vitro model of cell death, we are testing the effects of various CYP2E1 substrates including ethanol, arachidonic acid and acetaminophen (Tylenol). In addition, changes in the level of DNA-adducts in the CYP2E1-transfected cells, upon treatment of CYP2E1 substrates, are being determined by HPLC. SONG BJ NIAAA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol acetaldehyde laboratory rat adduct drug withdrawal enzyme inhibitor animal food genetic regulatory element genetic transcription gene induction /repression carbon tetrachloride poisoning cytochrome P450 immunocytochemistry alcoholic hepatitis disease model nutrition related tag protein degradation ubiquitin DNA damage oxidative stress aromatic hydrocarbon receptor CRISP RPROJ A CRISP/99/AA00036-12 CRISP/99/AA00036-12 199904 eng DRUG EFFECTS ON MEMORY AND RELATED COGNITIVE FUNCTIONS Research RPROJ The studies that make up this project have several related objectives that include: 1) an understanding of the specific acute and chronic cognitive effects of alcohol; 2) the use of drug probes and models to provide a complementary view of the determinants of learning and remembering using brain imaging techniques; 3) the development of pharmacological models of impaired cognition as expressed in different neuropsychiatric disorders; 4) uncovering cognitive deficits in alcoholics that ordinarily might not be readily apparent but would be expressed under drug challenge conditions. To meet these objectives normal volunteers, alcoholics and other neuropsychiatric disorder patients were studied in repeat measure crossover designs using several contrasting classes of drugs including benzodiazepines, serotoninergic drugs, drugs that affect the cholinergic nervous system and drugs that interact with the NMDA receptor. Previously validated cognitive neuroscience methods were used to assess and contrast the cognitive effects of these agents in different populations of patients. Detoxified alcoholics, treated with benzodiazepines (which mimics many of the effects of alcohol in the alcoholic), demonstrate a dramatic impairment in reflective cognitive functioning and inhibitory functions. This form of impairment is not nearly as apparent under placebo test conditions; furthermore, this deficit is not secondary to changes in many other cognitive domains, such as those involved in learning and remembering. In addition, access to what is in knowledge memory is qualitatively different under benzodiazepine test conditions (compared to placebo) and this is not the case in normal volunteers. Similarly, polydrug abusing patients demonstrate a pattern of cognitive changes in response to both benzodiazepine and stimulant (amphetamine) drug challenges. These findings provide some new perspective, in cognitive terms, of ways that alcohol and other psychoactive drugs alter mental functions in alcoholics and polydrug abusers that are distinguishable from the effects of alcohol in normals. Parallel studies have explored the role of awareness in memory using benzodiazepines as a tool for altering cognitive functioning. Other studies have been designed to examine whether the effects of alcohol on attention are selective and parallel the changes seen in other cognitive domains (such as those involved in learning and remembering). This research has the dual goal of utilizing drug probes for exploring the differentiated nature of attention (as well as learning and memory) while at the same time providing new, and clinically relevant, information about the cognitive effects of alcohol. These attentional-cognitive studies were designed to contrast the effects of alcohol on goal-directed (or top down) control processes with stimulus-driven (or bottom-up) cognitive functions. Goal-directed attention requires the inhibition of the stimulus-driven information. Because WEINGARTNER H NIAAA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol drug adverse effect human subject cognition memory cognition disorder memory disorder psychopharmacologic agent psychopharmacology clinical research CRISP RPROJ G CRISP/99/A00060-07 CRISP/99/A00060-07 199904 eng INVOLVEMENT OF BRAIN PROTEINS IN ACUTE ETHANOL ACTIONS Research RPROJ This proposal is based on 1) findings in humans that initial insensitivity to ethanol is a predictor of later problems with ethanol in Caucasians 2) our knowledge of the genetic mechanisms underlying sensitivity and insensitivity to ethanol in animals. This proposal will expand to Native Americans and to determine whether or not they have similar reactions to ingested ethanol as do Caucasians. If Native Americans don't have the same or similar risk factors for the development of alcoholism as found in Caucasians then, prevention and treatment of alcoholism for them should be altered. The second portion is to locate genes in selectively bred rodent populations that are responsible for the initial sensitivity or insensitivity to ethanol. Quantitative Trait Loci (QTL) in our selectively bred rat colony (High and Low Alcohol Sensitive as well as in Long and Short sleep mice by others) will be located and candidate genes contained within these QTL will be identified. Finally we will search human data bases, for those QTL or genes, found in rodents, that are responsible for ethanol sensitivity and insensitivity and determine their role in risk factors for alcoholism. During the candidate's career he has been involved in the pharmacogenetic aspects of ethanol's action in animal models and has served as Scientific Director and then Principal Investigator of the University of Colorado Alcohol Research Center. This Center's major focus is the Behavioral Pharmacogenetics of Ethanol Action. Thus the current proposal is a culmination of much of the work of the candidate's work in this Center and in separate grants for rat studies. More recently the candidate has become involved in testing of humans for their reactions to ethanol with an emphasis on Native Americans. The environment is optimal for the studies proposed. DEITRICH RA UCHSC AT FITZSIMMONS, BUILDING 500, MS F428 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse alcoholism /alcohol abuse therapy laboratory rat brain disease /disorder proneness /risk gene expression pharmacogenetics genetic polymorphism statistics /biometry racial /ethnic difference human genetic material tag animal genetic material tag neurotoxicology behavioral /social science research tag quantitative trait loci CRISP RPROJ COLORADO G CRISP/99/AA00093-12 CRISP/99/AA00093-12 199904 eng ETHANOLS ACTIONS IN GAMMA-PKC NULL MUTANTS Research RPROJ The goal of this RCA is to further the investigation and development of techniques for the investigation of the genetic basis of alcohol dependence. Short-term studies are designed to address the role of protein phosphorylation in regulating sensitivity and neuroadaptive processes. Protein phosphorylation is a basic regulator of cellular processes. One gene family the calcium/lipid activated protein kinase C (PKC) encodes at least 11 isotypes. Among these isotypes, the y-PKC isotype, is the only PKC solely expressed in neurons of the brain and spinal cord. Until the creation of null mutant mice ("knock-outs") that carry a disrupted y-PKC gene, it has been difficult to analyze the role of specific isotypes in behavioral, biochemical, and physiological processes. Y-PKC null mutants differ in their responses to ethanol but these responses are mediated by polygenic systems and it is unknown whether the impact of the null mutation in y-PKC is influenced by genetic background and varying gene-gene interactions. The goal of this proposal is to investigate the question of epistatic interactions by introgressing the null y-PKC or wild-type on to a variety of inbred strains which are known to differ in their responses to ethanol to create congenic lines. These backgrounds include: C57/BL6J, DBA/2J, and 129/Svev. Initial sensitivity to ethanol, the development of ethanol tolerance, and drinking of ethanol will be examined in these congenic lines as well as ligand-gated ion channel function. Long-term studies are designed to establish the molecular technique of RNA differential display to be used to address the functional nature of neuroadaptive processes that underlie tolerance development to ethanol and the lack of such tolerance in y-PKC null mutants. WEHNER JM UNIVERSITY OF COLORADO, CAMPUS BOX 447, BOULDER, CO 80309-0447 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol transgenic animal genetic strain drug interaction drug hypersensitivity drug tolerance gene expression gene interaction genetic regulation genotype gene mutation neurochemistry phosphorylation protein kinase C membrane channel psychobiology behavior test receptor sensitivity GABA receptor phenotype enzyme activity protein isoform neurogenetics quantitative trait loci CRISP RPROJ COLORADO G CRISP/99/AA00141-07 CRISP/99/AA00141-07 199904 eng ETHANOL EFFECTS ON MUSCLE DEVELOPMENT Research RPROJ The long-term objectives of the project are to determine the mechanisms responsible for ethanol-induced cardiomyopathies during fetal development and to devise treatments which will reverse or prevent the occurrence of alcohol-related myopathies. The specific aims are designed to investigate possible mechanisms involved in the production of mitochondrial abnormalities in ethanol-exposed muscle. Transcriptional regulation will be investigated by determining the relative levels of nuclear and mitochondrial mRNAs encoding different subunits of cytochrome c oxidase (COX). Polyclonal antibodies will be raised against nuclear and mitochondrial COX subunits and used to investigate the translational regulation of COX by measuring the steady state levels and synthesis rats of COX subunits. Techniques to identify the subcellular distribution of COX mRNAs and subunits will be developed in order to investigate their localization in abnormal mitochondria. A model employing the chronic stimulation of skeletal muscle will be developed to determine if ethanol exposure disrupts the normal events of mitochondrial biogenesis in trained muscle. KENNEDY JM UNIV OF ILLINOIS AT CHICAGO, 835 S WOLCOTT AVE 901, CHICAGO, IL 60612-7205/RADIOTHERAPY 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol chick embryo laboratory rat mitochondria genetic transcription genetic translation myocardium disorder striated muscle complementary DNA messenger RNA nucleic acid sequence cytochrome oxidase embryo /fetus embryo /fetus toxicology enzyme activity membrane biogenesis CRISP RPROJ ILLINOIS G CRISP/99/AA00179-05 CRISP/99/AA00179-05 199904 eng ETHANOL EFFECT ON ENDOCYTOSIS IN LIVER Research RPROJ APPLICANT'S ABSTRACT: This proposal represents a request for an ADAMHA Research Scientist Development Award (RSDA). The central hypothesis of the proposed investigation is that faulty acidification of endosomes and other acidic organelles after ethanol administration is a major mechanism by which ethanol impairs RME and other protein trafficking pathways. Acidification of endocytic vesicles is critical to efficient sorting and delivery of ligands and/or receptors to various compartments in the cell. We have used asialoglycoproteins as model ligands for studying RME and have identified several steps of the multi-step pathway that are affected by ethanol treatment, including impaired receptor recycling, altered dissociation of intracellular receptor-ligand complexes, and impaired internalization of the complexes into coated pits. Mechanisms which have been proposed to explain defective RME include formation of acetaldehyde adducts to tubulin resulting in impaired microtubule function, improper acidification of endosomes and defective receptor clustering in coated pits. The initial objective of our study is to demonstrate that ethanol administration impairs ATP-dependent acidification of prelysosomal endosomes after 1-5 weeks of ethanol feeding. In addition we plan to determine the effect of ethanol administration on ATP-dependent acidification in other subcellular organelles which are known to contain acidic interiors, namely Golgi apparatus, endoplasmic reticulum and lysosomes. A third objective is to examine potential impairment of acidification which may be induced by acetaldehyde. In these studies, we hope to establish whether the major metabolite of ethanol induces alterations in acidification similar to previously reported alterations in protein trafficking. Investigation of whether faulty acidification could lead to inactivation of the asialoglycoprotein receptor and subsequently alter synthesis and turnover of the receptor will be the goal of the fourth specific aim. A final objective is to clarify the effects of ethanol administration on the clathrin-coated vesicle population, since initial internalization via coated pits is impaired by ethanol treatment. Clathrin-coated vesicles are acidic organelles with an ATP dependent proton pump and we will measure acidification in these vesicles as well as rates of vesicle uncoating, a process which is necessary before vesicle fusion and subsequent transport of material across the cell can occur. The proposed studies should give valuable information concerning the basic molecular mechanism(s) of alcohol-induced hepatotoxicity. In addition to the research plans outlined above, the principal investigator will also maintain an active interest in teaching and training students in alcohol-related research. These additional areas of participation include lecturing in graduate level course as well as actively participating in other educational activities at the University of Nebraska Medical Center including journal clubs and problem solving days. CASEY CA VA MEDICAL CTR, 4101 WOOLWORTH AVE, OHAMHA, NE 68105 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 68105 Crisp Data Base National Institutes Of Health acidity /alkalinity ethanol acetaldehyde laboratory rat endoplasmic reticulum Golgi apparatus lysosome organelle hydrogen transport immunochemistry liver toxic disorder ligand clathrin glycoprotein adenosine triphosphate receptor coupling endocytosis CRISP RPROJ NEBRASKA G CRISP/99/AA00185-05 CRISP/99/AA00185-05 199904 eng INTERLEUKIN 8 AND ALCOHOLIC LIVER INJURY Research RPROJ Alcoholic liver disease (ALD) is an important public health problem. Patients with alcoholic hepatitis (AH) frequently have endotoxemia and elevated plasma tumor necrosis factor (TNF) levels which correlate with clinical indicators of liver dysfunction and mortality. Hepatic neutrophil (PMN) infiltration is an early manifestation of AH. Interleukin-8 (IL-8), a cytokine also known as neutrophil chemotactic factor, is an 8 kDa polypeptide produced by monocytes and hepatocytes in response to endotoxin (LPS) and TNF. We recently reported elevated IL-8 levels in acute AH that decreased as patients improved clinically. The grade of immunohistochemical staining for IL-8 in liver tissue correlates with the degree of hepatic PMN infiltration in AH. A positive effect of treatment with corticosteroids in AH can be predicted by hepatic PMN infiltration, and corticosteroids downregulate IL-8 production. Ethanol metabolism produces reactive oxygen intermediates and chronic alcohol abuse is associated with decreased levels of nutritionally dependent free radical scavengers such as glutathione. NF(kappa)B is a transcription factor for several cytokines including IL-8. NF(kappa)B is activated by reactive oxygen intermediates and its activation can be blocked by antioxidants. It is our working hypothesis that IL-8 plays an etiologic role in the neutrophilia, hepatic PMN infiltration and PMN-induced liver damage in AH. The overall research goals of our laboratory are to further define mechanisms and modulatory pathways whereby cytokines such as IL-8 induce metabolic disturbances and liver injury in ALD, with the ultimate goal being to develop a specific and anti-cytokine therapy for ALD. In this proposal, we will evaluate the role of oxidative stress and antioxidants in regulating IL-8 gene expression. We will determine whether plasma IL-8 levels increase in normal volunteers given alcohol or LPS, and if so, can this response be decreased by giving antioxidants such as vitamin E. We will also determine whether the moderately increased plasma levels of IL-8 present in alcohol-dependent patients without clinical liver disease normalized with abstinence. In vitro, we will determine whether or not Hep G2 liver cells cultured in alcohol, have increased NF(kappa)B activity, mRNA for IL-8 and secreted IL-8 protein in response to recombinant human TNF or monocyte supernatants (containing TNF) from patients with AH. We will also study Hep G2 cells transfected to overexpress cytochrome P450 2E1 to determine if they have increased production of reactive oxygen intermediates, NF(kappa)B activity and IL-8 gene expression when cultured with alcohol. These studies should provide new insights into the mechanism(s) of the increased IL-8 activity seen in ALD, both in vivo and in vitro on the transcription factor level. The knowledge gained should be useful in the development of "anticytokine" therapy for ALD. HILL DB UNIVERSITY OF KENTUCKY, 800 ROSE ST, RM MN654, LEXINGTON, KY 40536-0084 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse mass screening gastrointestinal absorption /transport transfection gene expression cytochrome P450 human subject cytokine tumor necrosis factor alpha endotoxin alcoholic liver cirrhosis liver metabolism antioxidant free radical oxygen tissue /cell culture tocopherol interleukin 8 oxidative stress CRISP RPROJ KENTUCKY G CRISP/99/AA00190-05 CRISP/99/AA00190-05 199904 eng ORGANOTYPIC CULTURE OF ETHANOL SENSITIVE REGIONS OF THE CNS--NEUROTOXICITY MODEL Research RPROJ A series of whole animal studies have clearly demonstrated selective brain neurodegeneration in a binge-type alcohol rodent model. Pyramidal cells of layer III of the entorhinal cortex (ENT) and hippocampal (HIPP) dentate gyrus granule cells are particularly vulnerable to the cytotoxic consequences of binge-type alcohol exposure. The main purpose of this project was to establish an experimental model to investigate alcohol-induced neurodegeneration in vitro that would complement our in vivo studies. Organotypic rat brain slices provide a culture system that preserves the main morphological and neurochemical features of brain tissue.Furthermore, organotypic slice cultures retain their native circuitry, cell types and synaptic density with conservation of main afferents.Ongoing studies have revealed that the ENT-HIPP explants are optimal for study at 2-3 weeks post harvest when slices are obtained from 8-day old neonates. The ENT-HIPP explants were treated with ethanol (0.2-0.6%) over 6 days using several different alcohol exposure paradigms. Alcohol-induced neuronal injury and/or death were assessed by the extent of uptake of propidium iodide(cytotoxic marker) and the amount of lactate dehydrogenase(LDH), another cytotoxic marker, in the explant medium. Treatment of ENT-HIPP slices with 0.4% alcohol for 6 days in the absence of antioxidants (glutathione, vitamin E,catalase and superoxide dismutase) caused a consistent, significant increase in medium LDH which paralleled propidium iodide uptake. Inclusion of antioxidants was cytoprotective.These results suggest that oxidative stress is an important mediator of alcohol-induced brain damage. Additional studies are needed to determine whether or not alcohol treatment of organotypic brain cultures will provide a useful model for our whole animal studies. ESKAY R NIAAA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcohol ethanol laboratory rat biomarker hippocampus cell death cell type model design /development neural degeneration afferent nerve synapse antioxidant lactate dehydrogenase superoxide dismutase glutathione catalase organ culture cytotoxicity tocopherol entorhinal cortex oxidative stress nerve injury neurotoxicology CRISP RPROJ G CRISP/99/A00212-01 CRISP/99/A00212-01 199904 eng AFFECTIVE AND CONATIVE CHANGES IN ALCOHOLISM Research RPROJ This is an application for an ADAMHA Senior Scientist Award (SSA). The SSA would permit the PI (a) to devote all of her research efforts to alcoholism; (b)to expand her research and mentoring activities concerned with gender issues in alcoholism; and (c) to gain valuable experience with structural and functional neuroimaging techniques. In conjunction with 2RO1 AA 07112-09, investigations are planned to examine changes in affect (emotion) and conation (intention) in abstinent alcoholics. Secondary aims of the research are to expand studies of age-related changes and gender differences in emotional and intentional functions. The importance of the research is fourfold: (1) Putative sites of alcohol-related brain damage involve separate frontal systems which are tied to different perceptual/cognitive aspects of emotional and intentional behaviors; (2) gender differences in alcohol- related neurobehavioral functions are ripe for experimental exploration; (3) the literature on whether emotional changes have reciprocal effects on perception and cognition in alcoholism is equivocal and controversial; and (4) even though affective and conative abnormalities have been clinically apparent in alcoholic groups, neuropsychological studies have focused primarily on cognitive changes unrelated to emotion and intention. In the proposed experiments we will enlist the participation of right- handed male and female research subjects ranging in age from 20 to 75 years. The experimental groups will include abstinent alcoholics with and without Korsakoff's syndrome. Patterns and levels of performances by the alcoholics will be compared to those of age-matched nonalcoholic subjects, in order to evaluate the ways in which behavioral consequences of aging and alcoholism are parallel, divergent, or interactive. Additionally, patients with right-frontal or bilateral frontal lobe damage from cerebrovascular accidents will provide the necessary control comparisons for neurobehavioral changes linked directly to focal brain damage. These groups were chosen specifically to clarify frontal system contributions to deficits of Korsakoff and non-Korsakoff alcoholics. We also will be able to evaluate hypotheses about greater right- than left-hemisphere functional decline in the alcoholic and aging groups, and in women compared to men. It is expected that results of the proposed studies will show clear evidence of frontal-mediated affective and conative changes in alcoholics (most notably in the Korsakoff patients), but that these changes will not be conspicuous in aging populations uncomplicated by alcoholism. By contrast, certain aspects of perceptual functioning will be compromised by aging - whether or not a history of alcohol abuse exists. Finally, women will display different performance patterns than men. BERMAN MO BOSTON UNIV SCH OF MEDICINE, 715 ALBANY ST, M 902, BOSTON, MA 02118 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 02118 Crisp Data Base National Institutes Of Health adult human (19+) alcoholism /alcohol abuse computer processing of clinical data cerebral dominance human subject Wernicke Korsakoff syndrome attention behavior test neuropsychology neuropsychological test cognition disorder mood disorder handedness organic brain syndrome perception disorder sex difference age difference neurotoxicology clinical research CRISP RPROJ MASSACHUSETTS G CRISP/99/AA00219-02 CRISP/99/AA00219-02 199904 eng ALCOHOLISM RISK--STUDIES IN HUMAN AND ANIMAL MODELS Research RPROJ The candidates research goals have focused on the use of electrophysiological and neuroendocrinological techniques for the evaluation of brain function. In the research program described in this grant, physiological and behavioral methods are used to study risk factors for ethanol abuse as well as the CNS consequences of ethanol exposure. Studies are proposed in clinical populations and in appropriate animal models. The use of parallel studies in humans and animals allows for the identification of clinically relevant measures in humans and provides a basis to explore the neural systems underlying such measures in animals models. This program involves four sets of studies that will be conducted over the next five years: l) identification of genetic risk factors at baseline and in response to ethanol exposure in Native Americans. 2) Electrophysiological studies of P and NP rats aimed at understanding brain mechanisms underlying alcohol preference. 3) Evaluation of the long term physiological effects of alcohol exposure during the perinatal period. 4) Development of new theoretical approaches to alcoholism based on principles from nonlinear dynamics. The Scripps Research Institute has an active program with six major laboratories dedicating a major portion of their research effort to alcohol research. In addition TSRI has an Alcohol Research Center that draws not only from the Scripps faculty but also from faculty in the neighboring institutions of SDSU, UCSD, and the Salk institute. Therefore Dr. Ehlers has wide access to research collaboration, as well ample possibilities to train and mentor young investigators in alcohol research. EHLERS CL SCRIPPS RESEARCH INSTITUTE, 10550 N TORREY PINES RD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 92037 Crisp Data Base National Institutes Of Health young adult human (19-34) alcoholism /alcohol abuse laboratory rat brain adolescence (12-18) middle childhood (6-11) disease /disorder proneness /risk electrophysiology evoked potential genotype human subject statistics /biometry longitudinal human study fetal alcohol syndrome behavioral genetics behavior test intelligence test neuropsychological test Native American caucasian American racial /ethnic difference human genetic material tag family genetics neurotoxicology clinical research CRISP RPROJ CALIFORNIA G CRISP/99/AA00223-02 CRISP/99/AA00223-02 199904 eng MODULATION OF CALPASTATIN INTERACTIONS BY ETHANOL Research RPROJ Exposure of PC12 cells to ethanol results in large changes in calcium-stimulated protease activities. Since these proteases are critical modulators of both neurotransmitter release as well as cellular toxicity and death, we are exploring the hypothesis that ethanol-mediated neural toxicity may result from calcium- activated protease dysregulation. Calpastatin, an inhibitor of calpain, is an acidic, hydrophobic protein which interacts with the hydrophobic active site(s) of mu- and m-calpains. A series of post-translational modifications of calpastatin have been described which alter the binding affinity to calpains, among them, PKC-mediated phosphorylations. Using a PC12 model, we are examining the effects of ethanol exposure and withdrawal on protein-protein interactions, and will determine what, if any, post-translational modifications occur to alter these interactions. A variety of techniques are used for these studies, including gel filtration, fluorescence-based protease assays, identification of protein-protein complexes using a variety of immunochemical techniques and mass-mapping of isolated protein complexes. Because of the hydrophobic nature of calpastatin-calpain interactions, the possibility that ethanol might modify protease-inhibitor complex stability is being entertained. DEPETRILLO PB NIAAA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol drug withdrawal enzyme inhibitor immunochemistry intermolecular interaction phosphorylation protein kinase C calpain endopeptidase posttranslational modification protein sequence cytotoxicity PC12 cell neurotoxicology protein binding CRISP RPROJ G CRISP/99/A00226-01 CRISP/99/A00226-01 199904 eng STRESS AXIS, ACTIVATION, SITE SPECIFIC CNS NEURODEGENERATION AND ETHANOL Research RPROJ Consumption of moderate to large amounts of ethanol (Et) activates the hypothalamic-pituitary-adrenal axis (HPAA). Activation of the HPAA or hypercortisolism accompanies both short- and long-term consumption of Et and the Et withdrawal syndrome. Alcoholics often present with a pseudo-Cushing's syndrome in which some 17-40 percent of alcoholics do not respond to the dexamethasone suppression test during the first week of abstinence, suggesting an ongoing hypercortisolemic state in this group of patients. Since a relative state of elevated glucocorticoids (chronic continuous or chronic intermittent) can lead to neural changes and even cell death, particularly in the hippocampus, the progressive loss of cognitive capacity in many alcoholics may indeed be due in part to hypercortisolemia and subsequent irreversible neural damage in the hippocampus and other areas of the central nervous system. Using an intragastric cannulated rodent model and short-term (4 days) intermittent or binge-type Et administration, we have demonstrated site-specific CNS neurodegeneration in the dentate gyrus of the hippocampus, the entorhinal cortex and the piriform cortex.The observed Et-induced neurodegeneration was functionally validated as noted by the decline in learning and memory capacity in the Et-treated animals in the hippocampal-dependent Morris water maze test. Ongoing efforts to define the mechanism of Et's cytotoxicity continue by us and others. Surprisingly, the co-administration of glutamate receptor subtype antagonists or calcium uptake blocking drugs with Et are not neuroprotective, which argues against an excitotoxic basis for the neurodegeneration; however, elevated glucocorticoids exacerbate the Et-induced neurodegeneration presumably through excitotoxic mechanisms. To date the most potent cytoprotective agent in the binge-type rodent model has been shown to be furosemide, an anion transport inhibitor; however, our finding that LY-644,711, an equally potent anion transport inhibitor, is not neuroprotective would argue against a primary edema-based mechanism of neurotoxicity. Finally, with the knowledge that certain cannabinoids are neuroprotective we co-administered cannabidiol with Et and found an attenuation of neurodegeneration. Since in vitro studies have demonstrated that cannabidiol blocked glutamate-NMDA, -AMPA or -kainate receptor- mediated toxicity, it would appear that the cannabidiol site of action is downstream of receptor activation and perhaps has a generalized metabolic mechanism of neuroprotection. The ability of cannabidiol to protect against the toxicity of reactive oxygen species may underlie its cytoprotection in our binge-type Et rodent model. ESKAY R NIAAA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health glucocorticoid cortisol Cushing's syndrome alcoholic beverage consumption alcoholism /alcohol abuse ethanol Rodentia lateral olfactory area hippocampus dentate gyrus calcium metabolism drug withdrawal hormone regulation /control mechanism disease model neural degeneration neuropharmacology neurotoxin cognition disorder dexamethasone suppression test stress entorhinal cortex hypercortisolism hypothalamic pituitary adrenal axis CRISP RPROJ G CRISP/99/A00287-08 CRISP/99/A00287-08 199904 eng NERVE CELL EXCITABILITY AND ALCOHOL ACTIONS Research RPROJ Alcohol is classified pharmacologically as a central nervous system depressant. The cellular mechanisms that underlie this alcohol-induced depression of nervous system excitability, however, are poorly understood. This project investigated voltage-dependent membrane ion channels in neuronal precursor cells. Precursor cells from the anterior subventricular zone (SVZa) of neonatal rat forebrain express neuron-specific markers and divide while migrating along the path to the olfactory bulb, where they differentiate into granule and periglomerular interneurons. SVZa cells also express neuron-specific tublin and divide in vitro. Voltage-dependent potassium currents were studied using whole-cell patch-clamp recording in SVZa cells isolated from newborn rats and cultured for 1 day. A-type potassium current (I-K[A]) was recorded in the presence of 300 nM tetrodotoxin and 20 mM tetraethylammonium (TEA); it was identified by its properties of steady-state half-inactivation (-90 mV) and rapid recovery from inactivation (20 ms at -130 mV). Inactivation of I-K[A] was voltage-independent and had a time-constant of 15 ms. The second potassium current identified resembled a delayed rectifier (I-K[DR]) by inactivating slowly over several seconds and being blocked reversibly by external TEA (IC50 4.1 mM). I-K[DR] exhibited steady-state half-inactivation of -50 mV and sigmoidal activation kinetics, with time-constants ranging from 11 ms at -40 mV to 1.5 ms at 100 mV. Although SVZa cells undergo division in culture, their properties resemble those of postmitotic cerebellar granule neurons. Future experiments are planned to study the effect of alcohol on these currents. WEIGHT FF NIAAA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat patch clamp electrophysiology ganglion axon neuropharmacology neural inhibition neural conduction potassium channel sodium channel voltage gated channel tetrodotoxin CRISP RPROJ A CRISP/99/AA00480-15 CRISP/99/AA00480-15 199904 eng ALCOHOL INTAKE DURING PREGNANCY--OFFSPRING DEVELOPMENT Research RPROJ This proposal is for years 20 through 23 of the Seattle Longitudinal Prospective Study on Alcohol and Pregnancy. The overall objective of the research is to determine the long-term consequences of maternal alcohol use during pregnancy on the health and development of the offspring. The present proposal would permit the first examination of young adults whose prenatal alcohol exposure had been determined by maternal self-report in mid-pregnancy 21 years ago. The basic hypothesis for this long-term study of the teratogenic effects of alcohol in humans is that prenatal alcohol exposure exerts an enduring dose-dependent effect on adult neurobehavioral function. Five Specific Aims will be addressed: 1. Four domains of cognitive function will be examined in 21-year-old offspring, in relation to prenatal alcohol exposure as mediated by earlier manifestations of cognitive dysfunction and modified by appropriate covariates. The four domains include: Attention/Concentration, Executive Function, Memory, and Information Processing. 2. Adaptive and emotional functioning in 21-year old offspring will be examined in relation to prenatal alcohol exposure as mediated by earlier measures of behavioral dysfunction and modified by appropriate covariates. 3. Contextual/environmental factors assessed throughout the lifespan from mid-pregnancy through 21 years will be examined in relation to offspring function at 21 years. 4. Morphologic and physical size dimensions in 21-year old offspring will be examined in relation to prenatal alcohol exposure, as modified by appropriate covariates. 5. The full spectrum of fetal alcohol effects, measured in this study through the first 21 years of life, will be examined to identify those earlier patterns of alcohol-related deficits that could serve as clinical markers for individual children at risk for adverse outcome. These proposed studies have far-reaching public health implications. Alcohol remains the teratogenic drug most frequently ingested during pregnancy. This would be the first opportunity to examine the adult consequences of a wide variety of patterns and levels of alcohol use and abuse using a cohort that has manifested subtle neurobehavioral effects of prenatal alcohol exposure at many earlier ages. Findings from this study have direct implications for public policy, efforts to prevent fetal alcohol effects, and methods for the detection and appropriate remediation of children and adults at risk for adverse effects of prenatal alcohol exposure. STREISSGUTH AP UNIVERSITY OF WASHINGTON, 180 NICKERSON ST, SUITE 309, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 98195 Crisp Data Base National Institutes Of Health adult human (19+) biomarker child physical development child psychology child mental disorder child behavior disorder child (0-11) adolescence (12-18) teratogen dosage postnatal growth disorder human subject longitudinal human study embryo /fetus toxicology prenatal stress fetal alcohol syndrome behavior test intelligence test cognition neuropsychology neuropsychological test intelligence behavior disorder cognition disorder psychometrics behavioral /social science research tag CRISP RPROJ WASHINGTON G CRISP/99/AA01455-23 CRISP/99/AA01455-23 199904 eng ALCOHOL-INDUCED BEHAVIORAL TERATOGENESIS Research RPROJ Although a considerable amount of data has been published regarding the morphological and behavioral effects associated with prenatal alcohol exposure, very little information is known regarding the underlying mechanisms for these effects. Recently, alcohol's interaction with the prostaglandins has been implicated as one possible mechanism for some of the consequences associated with alcohol exposure in utero. Support for this hypothesis stems from studies showing that pretreatment with the prostaglandin synthetase inhibitors, aspirin and indomethacin, prior to alcohol exposure reduces fetal hypoplasia and the incidence of fetal death and malformations in mice. In this proposal, we plan to look at the role of prostaglandins in some of the behavioral and physiological effects that are found following prenatal alcohol exposure. In the first set of experiments, we will examine the effects of aspirin pretreatment will ameliorate some of the effects associated with prenatal alcohol exposure. In the first set of experiments, we will examine the effects of aspirin pretreatment will ameliorate some of the effects associated with prenatal alcohol exposure. In the first set of experiments, we will examine the effects of aspirin pretreatment and acute and chronic alcohol exposure on fetal movement, since alcohol exposure has been shown to result in a suppression of fetal movement. Also, reduced fetal movement has been suggested as one possible cause for some of alcohol's morphological effects. In the second series of experiments, we will examine the effects of aspirin pretreatment on sexually dimorphic behaviors and neuroanatomical structures that have been shown to be sensitive to prenatal alcohol exposure. The arachidonic acid cascade, which results in the production of prostaglandins, has been implicated in some of the effects of testosterone during fetal development and thus, may play a role in some of the alcohol-related changes in sexually dimorphic behaviors that have been reported. In the third series of experiments, the effects of aspirin pretreatment on a number of behaviors that are known to be sensitive to prenatal alcohol exposure will be examined to determine if this pretreatment can ameliorate some of the behavioral dysfunctions that are typically associated with prenatal alcohol exposure. RILEY EP SAN DIEGO STATE UNIVERSITY, 6363 ALVARADO CT SUITE 209, SAN DIEGO, CA 92182 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 92182 Crisp Data Base National Institutes Of Health infant animal alcoholic beverage consumption alcoholism /alcohol abuse laboratory rat congenital nervous system disorder dosage inhibitor /antagonist drug interaction enzyme inhibitor enzyme mechanism maternal behavior prostaglandin neuroanatomy neuroendocrine system prostaglandin endoperoxide synthase aspirin longitudinal animal study embryo /fetus toxicology prenatal stress fetal alcohol syndrome behavior test behavior exploratory behavior play ethology avoidance behavior behavior disorder learning disorder memory disorder psychomotor disorder psychomotor reaction time psychopharmacology behavioral /social science research tag CRISP RPROJ CALIFORNIA G CRISP/99/AA03249-22 CRISP/99/AA03249-22 199904 eng ETHANOL AND ACETALDEHYDE EFFECTS ON LIVER FUNCTION Research RPROJ The overall goal is to evaluate the role of reactive oxygen intermediates (ROl) in the molecular mechanisms by which ethanol is toxic. Major focus will be on the role of P4502E1, interaction with iron, and reactivity of NADH and NADPH as cofactors. Specific Aim 1 - Our laboratory developed a HepG2 cell line which stably expresses human P4502E1. Ethanol was toxic to these cells, but not to control cells. These cells appear to represent a novel model to assess the role of P4502E1 or ROl generated during the metabolism of ethanol by P4502E1 in the hepatotoxic actions of ethanol. Studies will be carried out to characterize ethanol toxicity, effect of P4502E1 inhibitors, antioxidants, iron chelators, enrichment of cell membranes with PUFA and the ability of ethanol, acting via P4502E1,to produce a state of oxidative stress: Lipid, protein, and DNA targets for ROl produced by ethanol will be studied. Acetaldehyde and lipid adducts will be immunochemically detected. A final study will be to assess whether ethanol toxicity involves an apoptotic mechanism; if so, protection by BCl-2 will be studied. Specific Aim 2 - The role of NADH-cyt b5 reductase, cyt b5, and cyt P450 in the NADH-dependent production of ROl by microsomes will be studied employing reconstituted systems containing the purified enzymes, and inhibitors and antibodies against these enzymes. Specific Aim 3 - The interaction of NADPH-P450 reductase and P4502E1 with various ferric complexes to produce ROl will be investigated to test the hypothesis that the ability of the reductase versus P450 to interact with iron is not only dependent upon the chelated form of the iron, but also the concentration of iron. Specific Aim 4 - The production of ROl by microsomes and mitochondria isolated from periportal and pericentral hepatocytes of control and ethanol-treated rats will be determined since P4502E1 is present at highest levels in the PC zone and ethanol injury originates here. Specific Aim 5 - Studies will be carried out to compare the production of ROl by human P4502E1 with other human P450 isozymes. Ethanol toxicity and the role of 2E1 and ROl will be studied in immortalized normal human liver cells. These studies will help define the role of P4502E1 and ROl in the mechanism by which ethanol is hepatotoxic, help to design therapeutic interventions to prevent or ameliorate this toxicity, and provide basic mechanistic information of value to the oxygen radical and P450 areas of research. CEDERBAUM AI MOUNT SINAI SCHOOL OF MEDICINE, ONE GUSTAVE L LEVY PL BOX 102, NEW YORK, NY 10029 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 10029 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol acetaldehyde laboratory rat microsome mitochondria adduct isozyme unsaturated fatty acid cytochrome P450 iron hepatotoxin liver function liver metabolism metal complex chelating agent antioxidant xanthine oxidase free radical oxygen nicotinamide adenine dinucleotide NAD(H) phosphate tissue /cell culture oxidative stress CRISP RPROJ NEW YORK G CRISP/99/AA03312-19 CRISP/99/AA03312-19 199904 eng MATERNAL ALCOHOLISM AND CNS DEVELOPMENT OF OFFSPRING Research RPROJ Many studies of fetal alcohol syndrome (FAS) emphasize the relationship between the severity of abnormalities and the timing of ethanol administration relative to vulnerable periods in brain development. Chronic in utero ethanol exposure markedly impairs the early development of the serotonin (5-HT) system by encompassing a critical vulnerable period. During this period, 5-HT neurons are generated, differentiate, and extend projections to target areas; also during this period 5-HT normally exerts neurotrophic effects by stimulating astrocyte 5HT/1A receptors to increase production of S100beta, a neurotrophic factor (NTF) that is essential for the normal development of 5-HT neurons. Despite the frequency of FAS and the tremendous public health costs associated with FAS, there is no therapeutic treatment that prevents the CNS damage. This grant will investigate a mechanism by which ethanol may impair the development of the 5-HT system and a therapeutic intervention that may prevent this damage. A potential mechanism underlying the aberrant development of the 5-HT system is an ethanol-induced reduction of S100beta. This grant will investigate the use of a 5-HT/1A agonist because research suggests that this agonist prevents ethanol-associated damage to the developing 5-HT system in rats, and because this agonist increases production of S100beta. This grant proposal will investigate the following questions: Does ethanol impair the astrocyte-mediated neurotrophic stimulation of the development of serotonergic neurons? Can the damaging effects of ethanol be prevented by stimulation with a 5-HT/1A agonist? These questions will be examined using both an in vivo and in vitro experiments. In vivo experiments use a rat model to assess the effects of in utero ethanol exposure on the development of 5-HT neurons and astrocyte production of S100beta; Separate in vitro studies will use astrocytes, neurons, and astrocyte-neuron co-cultures to identify the cellular level at which these effects are mediated. Analyses will include in situ hybridization, northern and western blots, immunohistochemistry, [3H]5-HT reuptake, HPLC, and cell culture. DRUSE-MANTEUFFEL MJ LOYOLA UNIVERSITY, 2160 SOUTH FIRST AVE, MAYWOOD, IL 60153 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 60153 Crisp Data Base National Institutes Of Health ethanol laboratory rat serotonin drug screening /evaluation neurotrophic factor immunocytochemistry developmental neurobiology neuropharmacology in situ hybridization norepinephrine high performance liquid chromatography embryo /fetus toxicology fetal alcohol syndrome nonhuman therapy evaluation tissue /cell culture embryo /fetus cell culture chemoprevention neuroprotectant neurotransmitter transport neurotransmitter agonist CRISP RPROJ ILLINOIS G CRISP/99/AA03490-18A1 CRISP/99/AA03490-18A1 199904 eng CONTROL OF DRUG AND ETHANOL METABOLISM Research RPROJ Three new and exciting findings from this laboratory from the foundation for this proposal. 1) We demonstrated that Kupffer cells participate in the mechanism of elevated oxygen metabolism in hepatic parenchymal cells caused by acute ethanol treatment, 2) that inactivation of Kupffer cells prevents early injury due to ethanol in the Tsukamoto-French model, and 3) that hypoxia and free radicals are formed in vivo in response to ethanol in this model. Collectively, this new work has led us to hypothesize that early alcohol-induced liver damage is due to an oxygen- dependent reperfusion injury involving both hypoxia due to hypermetabolism and/or impaired microcirculation with subsequent O2- dependent free radical formation. We are eager to test this hypothesis using the clinical relevant Tsukamoto-French model of alcohol treatment exclusively employing a combination of specialized techniques (e.g. miniature O2 electrodes) unique to this laboratory. This will allow us to fill critical gaps in our knowledge which will lead to our important long-term goal -- the prevention of early alcohol-induced liver injury in the alcoholic. The first major goal will be to determine if hepatic nonparenchymal cells are involved in regulation of O2 uptake and if they can explain alcohol-induced hypoxia due to chronic ethanol treatment. Kupffer and endothelial cells will be selectively inactivated and the effect of chronic alcohol treatment and oxygen concentration on oxygen uptake will be assessed in the perfused liver at two week intervals for up to 4 months during development of early liver disease in Tsukamoto- French rats. We recently demonstrated that cultured Kupffer cells produce mediators which stimulate oxygen uptake in parenchymal cells, so we will determine which eicosanoids and/or selected cytokines produced by Kupffer cells are involved in an oxygen sensor mechanism which is stimulated by O2 and Tsukamoto-French ethanol treatment. The possible role of Kupffer cell Ca2+ channels in the O2 sensing mechanism will be assessed from depolarization-induced Ca2+ influx determined fluorometrically in cultured Kupffer cells. Our second major goal will be to determine if an oxygen-dependent reperfusion injury is a critical event in early alcohol-induced liver injury. The role of Kupffer cells in an oxygen-dependent reperfusion injury in a low-flow, reflow model of liver perfusion will be assessed. Subsequently, we will exploit temporal fluctuations in blood ethanol in the Tsukamoto-French model in vivo to dissect components of a reperfusion injury. When blood ethanol is high, we expect to detect hypoxia with miniature surface O2 electrodes as well as purine accumulation due to inefficient energetics predominantly in pericentral regions of the liver lobule. We predict that free radicals will be formed as blood ethanol declines and oxygen reenters the previously hypoxic tissue. Collectively, the new approach embodied in these experiments wil THURMAN RG UNIV OF NC AT CHAPEL HILL, CB# 7365,ROOM 1124, CHAPEL HILL, NC 27599-7365 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol Peromyscus laboratory rat reperfusion microcirculation isolation perfusion drug metabolism microelectrode free radical hormone regulation /control mechanism eicosanoid arachidonate fatty acid metabolism cytokine Kupffer's cell liver circulation liver toxic disorder liver metabolism scanning electron microscopy oxidation /reduction catalase CRISP RPROJ NORTH CAROLINA G CRISP/99/AA03624-20 CRISP/99/AA03624-20 199904 eng ALCOHOLIC LIVER INJURY--COVALENT BINDING OF ACETALDEHYDE Research RPROJ Recent studies have shown that acetaldehyde and the lipid peroxidation-derived aldehyde, malondialdehyde, can react together with proteins in a synergistic manner to form distinct hybrid adducts which have been designated as MAA adducts. MAA adducts are immunogenic, generating antibodies which recognize MAA epitopes on proteins. MAA adducts were shown to be formed in the lives of ethanol-fed rats. Because both the covalent binding of acetaldehyde to proteins and increased lipid peroxidation have been proposed as possible mechanisms of ethanol-induced liver injury. MAA protein-adduct formation represents an event dependent on both of these mechanisms, indicating a common or unifying process (i.e. MAA adduct formation) that may account for the hepatotoxic effects of alcohol. Therefore, the following hypothesis has been proposed. MAA adducts, resulting from the synergistic reaction of acetaldehyde an malondialdehyde with proteins, represent a major species of aldehyde adducts formed in the liver during ethanol oxidation. MAA adduct formation with specific target proteins results in selective functional impairment of thee proteins and ultimately leads to liver injury. The objectives of the proposed studies are to further characterize the chemistry of MAA adduct formation and develop sensitive and reliable immunochemical based assays for the detection of MAA adducts in biological samples. Further critical objectives include describing the functional consequences of MAA adduct formation in the liver and evaluating the role of MAA adduct in ethanol-induced liver injury. Therefore, to attain these objectives the following specific aims are proposed: 1) To further characterize the chemistry of MAA adduct formation and provide structural assignments for the major defined MAA adducts; 2) To generate and characterize specific antibodies against defined MAA adducts in order to develop immunological methods for MMA adduct detection and quantification; 3) To determine MAA protein adduct formation and identify target proteins in the liver and other organs during chronic ethanol administration; 4) To ascertain the role of MAA adducts in the ethanol-induced impairment of hepatic protein trafficking pathways; 5) To evaluate the relationship between MAA adducts and hepatotoxicity in combined models of chronic ethanol ingestion and administration of agents that induce lipid peroxidation. Overall, these proposed studies should provide valuable information concerning the basic molecular mechanisms of alcohol-induced hepatotoxicity. TUMA DJ VA MEDICAL CENTER, 4101 WOOLWORTH AVENUE, OMAHA, NE 68105 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 68105 Crisp Data Base National Institutes Of Health ethanol acetaldehyde laboratory rabbit laboratory rat adduct molecular pathology drug metabolism immunochemistry immunologic assay /test lipid peroxide hepatotoxin liver metabolism intermolecular interaction peroxidation protein structure protein transport method development CRISP RPROJ NEBRASKA G CRISP/99/AA04961-18 CRISP/99/AA04961-18 199904 eng IN UTERO ALCOHOL EXPOSURE AND FETAL B CELL DEVELOPMENT Research RPROJ Infants born with fetal alcohol syndrome often suffer from an increased incidence of illness due to immunodeficiency at both the humoral and cell- mediated levels. Recent work from this lab suggests that exposure to ethanol in utero causes a delay in B cell development in the fetal liver, then in the spleen and bone marrow of neonatal animals. Our working hypothesis is that a delay in the development of B lineage cells in the fetal liver would account for the diminished number of B cells in the spleen and bone marrow of neonatal mice exposed to ethanol in utero. The goals of this proposal are to determine the effects of in utero ethanol exposure has on: 1) The appearance of lymphoid precursors, in the fetal liver, which can commit to the B cell lineage. 2) The number of B cell developmental intermediates in the fetal liver at various time points during gestation. 3) The functional ability and expansion of B cell developmental intermediates in the fetal liver. BIBER KL LSU MEDICAL CTR, 1501 KINGS HIGHWAY P O BOX 339, SHREVEPORT, LA 71130 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 71130 Crisp Data Base National Institutes Of Health ethanol laboratory mouse hematopoiesis B lymphocyte cell differentiation flow cytometry polymerase chain reaction gene rearrangement histogenesis immunoglobulin gene enzyme linked immunosorbent assay immunodeficiency liver function surface property embryo /fetus toxicology tissue /cell culture phenotype developmental immunology CRISP RPROJ LOUISIANA G CRISP/99/AA05441-03 CRISP/99/AA05441-03 199904 eng TESTOSTERONE AND ETHANOL WITHDRAWAL NEUROTOXICITY Research RPROJ Alcohol is known to cause selective neuronal degeneration. In the hippocampus, pyramidal neurons are selectively vulnerable to alcohol, especially during withdrawal. In addition, in males, alcohol acutely decreases circulating testosterone(T) levels. Recent evidence suggests that T can be neuroprotective. Therefore, alcohol-related reduction in circulating T may contribute to the pyramidal cell's vulnerability during alcohol withdrawal. The goal of this project is to assess the contribution of lowered testosterone levels, which accompany alcohol consumption, to alcohol withdrawal-related neurotoxicity. Specific aim 1 will address the ability of DHT (a highly active form of T) to attenuate toxicity to cultured hippocampal pyramidal cells following alcohol withdrawal. Specific aim 2 will address the same question in vivo. Specific aim 3 will address the specific molecular mechanism by which DHT is attenuating neurotoxicity following alcohol withdrawal. The information gained from this research will be-valuable in understanding the mechanisms by which alcohol is causing specific neuronal degeneration. PRICE RH COLORADO STATE UNIVERSITY, FORT COLLINS, CO 80523 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 80523 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse testosterone laboratory rat pyramidal cell brain disorder polymerase chain reaction drug withdrawal neural degeneration phase contrast microscopy toxicant interaction steroid hormone receptor male RNase protection assay neuroprotectant neurotoxicology CRISP RPROJ COLORADO G CRISP/99/AA05461-03 CRISP/99/AA05461-03 199904 eng MINORITY PREDOCTORAL FELLOWSHIP PROGRAM Research RPROJ NO ABSTRACT AVAILABLE BARNES SL UNIVERSITY ILLINOIS, 905 SOUTH GOODWIN AVENUE, URBANA, IL 61801 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 61801 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol acetaldehyde adduct benzanthracene benzopyrene drug metabolism training health science research human tissue chemical related neoplasm /cancer nutrition related neoplasm /cancer neoplasm /cancer genetics chemical carcinogenesis carcinogen DNA repair nutrition related tag career animal tissue DNA damage MCF7 cell CRISP RPROJ ILLINOIS G CRISP/99/AA05479-03 CRISP/99/AA05479-03 199904 eng ALCOHOL EFFECTS ON SIGNAL TRANSDUCTION IN ASTROCYTES Research RPROJ NO ABSTRACT AVAILABLE YAGLE KJ Kevin J. Yagle, 16676 AGATE PASS RD, BAINBRIDGE ISLAND, WA 98110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 98110 Crisp Data Base National Institutes Of Health ethanol laboratory rat biological signal transduction pharmacokinetics developmental genetics regulatory gene disease model astrocyte fetal alcohol syndrome mitogen activated protein kinase neurotoxicology cell proliferation neurogenetics CRISP RPROJ WASHINGTON G CRISP/99/AA05488-02 CRISP/99/AA05488-02 199904 eng PRENATAL ETHANOL EXPOSURE AND POSTNATAL HANDLING Research RPROJ The proposed research is designed to investigate the effects of early postnatal handling as an environmental manipulation which may attenuate the adverse behavioral and physiological consequences of prenatal ethanol exposure. This research will examine the possible mechanisms mediating the hormonal hyperresponsiveness to stressors observed in animals prenatally exposed to ethanol (E) as well as the possible influence of postnatal handling on these mechanisms. In addition, experiments will investigate the correspondence between prenatal ethanol-induced alterations in behavior and HPA activity. Toward achievement of these aims, experiments will involve behavioral and hormonal assessments. Tasks involving behavioral inhibition (conditioned taste aversion) and spatial learning (Morris water maze) will explore further the learning and/or performance deficits previously demonstrated in E animals as well as investigate the correspondence between behavior and HPA activity. Functional manipulations of the hypothalamic-pituitary-adrenal (HPA) axis will examine possible mechanisms of the altered hormonal responsiveness to stress observed in E animals as well as explore the effects of handling on this HPA hyperresponsiveness. Lastly, experiments will investigate the effects of prenatal ethanol exposure and postnatal handling on activity of the HPA axis under basal conditions to determine if altered steady state activity contributed to HPA differences following stress. GABRIEL KI UNIV OF BRITISH COLUMBIA, 2177 WESBROOK MALL, VANCOUVER BC, CANADA V6T 1Z3 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health newborn animal ethanol laboratory rat hormone regulation /control mechanism environmental adaptation embryo /fetus toxicology fetal alcohol syndrome psychological test ethology learning behavioral habituation /sensitization performance touch stimulus /response stress behavioral /social science research tag hypothalamic pituitary adrenal axis CRISP RPROJ CANADA G CRISP/99/AA05499-01 CRISP/99/AA05499-01 199904 eng ETHANOL EFFECTS ON GLIAL GROWTH FACTORS Research RPROJ FAS children exhibit behavioral deficits that are indicative of impaired development of neurotransmitter systems. In rodents, ethanol exposure produces a remarkable (45-65 percent) decrease of serotonin content in the raphe neurons of the fetal brainstem. This reduction is important because 5-HT is an essential trophic factor for serotonergic neurons; reductions in 5-HT impair the development of raphe neurons and the density of their projects. Maternal administration of buspirone, a 5- HT1A agonist, prevents many of the 5-HT deficits that arise from in utero ethanol exposure. This proposal will test the hypothesis that buspirone prevents the damaging effects of ethanol on developing 5-HT neurons through its stimulation of astroglial 5-TH1A receptors and subsequent release of astroglial trophic factors, notably the glial growth factor S-100beta. S-100beta plays an essential role in the development of the serotonergic system. In vivo studies have shown that disruption of S-100beta production impairs the development of the 5-HT system. The three aims of this proposal will: (1) determine whether astroglial production of S-100beta in the Midline Raphe Glial Structure is impaired by in utero ethanol exposure, and whether buspirone prevents this effect of ethanol, (2) determine whether ethanol inhibits the paracrine effects of astroglia factors that normally result in increased production of S-100beta, and whether buspirone prevents these effects of ethanol, and (3) determine whether buspirone treatment of astrocytes prevents the reduced production/secretion of astrocytic neurotrophic factors. Results from these studies will contribute to our understanding of the mechanism by which ethanol exerts its damaging effects on the 5-HT system. ERIKSEN JL LOYOLA UNIVERSITY, 2160 S FIRST AVENUE, MAYWOOD, IL 60153 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 60153 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat serotonin gene induction /repression growth factor immunocytochemistry neurogenesis astrocyte neuron developmental neurobiology embryo /fetus toxicology fetal alcohol syndrome receptor expression serotonin receptor tissue /cell culture buspirone chemoprevention animal genetic material tag neurotoxicology behavioral /social science research tag neurogenetics CRISP RPROJ ILLINOIS G CRISP/99/AA05501-01A1 CRISP/99/AA05501-01A1 199904 eng NMDA RECEPTOR EXPRESSION AFTER FETAL ALCOHOL EXPOSURE Research RPROJ ADHD, a widely diagnosed disorder in the school-aged population, is correlated with a variety of causes, but its etiology remains a mystery. Alcohol exposure during pregnancy has been correlated with the occurrence of ADHD in childhood. As ADHD may have several subtypes, one of which is the result of hippocampal dysfunction, and alcohol acts primarily on the hippocampus, alcohol-induced damage to the hippocampus may describe a subtype of ADHD. The project is also a further extension of the investigation of the effect of alcohol on the NMDA receptor, a primary receptor in the hippocampus. The project determines the composition of NMDA receptor protein subunits in rats exposed to alcohol during the third trimester equivalent and second and third trimesters combined. The secondary subunits of the NMDA modulate its function by altering Mg2+ affinity to the attached calcium channel, which in specific configurations may promote either excitotoxicity or apoptosis. Differential NMDA receptor function is observed after exposure to EtOH, as well as cell death, but the means by which cells die has not been described. This project seeks to answer whether altered NMDA receptor subunit composition is responsible for the change in NMDA receptor function, whether it causes apoptosis or excitotoxicity, and its subsequent effects on behavior. NIXON K UNIVERSITY OF TEXAS IN AUSTIN, MEZES 330, AUSTIN, TX 78712 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 78712 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat hippocampus embryo /fetus toxicology fetal alcohol syndrome protein structure /function frustration learning short term memory developmental psychology psychopharmacology receptor expression NMDA receptor programmed cell death behavioral /social science research tag CRISP RPROJ TEXAS G CRISP/99/AA05509-01 CRISP/99/AA05509-01 199904 eng PERINATAL ALCOHOL EXPOSURE AND RESPONSE INHIBITION Research RPROJ This proposal contains a series of experiments designed to evaluate preservation with respect to the response inhibition hypothesis. The response inhibition framework has received considerable attention both in animal and human research, with little attention paid to alternative possibilities that might be able to provide a more functional explanation tied to animal learning theory. Identifying whether or not the behavioral deficits identified as perseverative is important not only because it has implications for the treatment of FAS but because this distinction may be able to provide a tool to differentiate FAS from other learning disorders. BELL MC 7693 PALMILLA DR, #2217, SAN DIEGO, CA 92122 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 92122 Crisp Data Base National Institutes Of Health ethanol laboratory rat embryo /fetus toxicology ethology animal developmental psychology behavioral /social science research tag CRISP RPROJ CALIFORNIA G CRISP/99/AA05514-01 CRISP/99/AA05514-01 199904 eng KUPFFER CELLS AND ALCOHOLIC LIVER DISEASE Research RPROJ Liver, which plays a major role in regulating bodily metabolic functions, is often the target of systemic insults arising from endotoxins, xenobiotics etc., resulting in hepatic injury. Recent studies have shown that hepatic injury is closely linked to the way Kupffer cells react to such insults and that obliteration of Kupffer cells can prevent liver injury. The Kupffer cell-mediated liver injury has been attributed to the excessive release of cytokines like tumor necrosis factor-alpha, interleuken-1, IL-6 and prostanoids, during endotoxemia. Endotoxins have been implicated in alcoholic liver diseases, since, high levels of endotoxins have been reported in the blood of alcoholic patients with liver disease. The objective of this proposal therefore, is to develop a method by which to control the production of cytokines and prostanoids in Kupffer cells. To achieve this goal, a method will be developed in which antisense and/antigene molecules, which are specifically targetted to the inflammatory cytokine/prostanoid-gene products will be encapsulated in anionic liposomes and delivered in vivo to Kupffer cells. The efficacy of delivery will be assessed by monitoring a) the tissue distribution of the delivered molecule, b) its biological half-life and c) cellular levels of targetted mRNA. Based on other reports, during the final year of the fellowship period, the molecular species of the liposomes will be modified to target other cell types such as hepatocytes and splenic macrophages. Positive data from such exploratory experiments will set the stage for future investigations with wider scope. DEY I THOMAS JEFFERSON MEDICAL COLLE, 1020 LOCUST STREET, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse liposome eicosanoid gene therapy cytokine liver cell Kupffer's cell liver disorder hepatotoxin antisense nucleic acid tissue /cell culture method development transfection vector CRISP RPROJ PENNSYLVANIA G CRISP/99/AA05529-01 CRISP/99/AA05529-01 199904 eng DENDRITIC PARAMETERS--AGE AND ETHANOL EFFECTS Research RPROJ DESCRIPTION: The purpose of this study is to characterize and quantitate effects of ethanol treatment on neurons in the aging central nervous system. Previous data showed (1) that ethanol induced dendritic degeneration in cerebellar Purkinje cells (PC) that resulted in longer surviving dendritic segments, (2) that ethanol depressed the number of synapses on PC dendrites reversibly such that significantly more synapses were formed on surviving dendrites during an extended recovery period, and (3) that ethanol did not alter the number of granule cells (GC), the source of most afferents to PC. The latter result contrasted with data from studies of younger ethanol-treated rats. Preliminary data also suggested that ethanol induced hypertrophy of the smooth endoplasmic reticulum (SER), an organelle involved in regulation of intracellular calcium levels in PC. Experiments are proposed to confirm and to build on the above results. First, immunocytochemical labeling of reactive astroglia and microglia, accepted markers of neuronal damage, will be used to confirm the extent and distribution of the dendritic degeneration. Second, immunocytochemical labeling of GABA+ synapses will be used to determine whether the increased number of synapses following recovery signaled an increase in inhibitory input to PC. Third, systematic morphometric measurements of SER profiles in shafts and spines of terminal dendrites will be used to determine whether the SER in dendritic shafts, especially at branch points, was preferentially altered by exposure to ethanol. Finally, the discrepancy stemming from the absence of cell loss in our old F344 rat model will be studied further to determine whether GC in young and old F344 rats differ in vulnerability to ethanol or whether GC in F344 rats are less vulnerable than GC in other rat strains at all ages. Results from this study are expected to contribute to our understanding of relationships between aging processes, alcohol abuse, and brain damage. This information is expected to promote the development of pharmacological interventions that will contribute to the health of elderly individuals in need of pharmacological assistance during recovery from long term alcohol dependency. PENTNEY RJ STATE UNIVERSITY OF NEW YORK, 317 FARBER HALL, BUFFALO, NY 14214-3000 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol gamma aminobutyrate laboratory rat genetic strain cerebellar Purkinje cell cell age intercellular connection endoplasmic reticulum immunocytochemistry neural degeneration synapse glia astrocyte microglia dendrite neural inhibition age difference granule cell neurotoxicology CRISP RPROJ NEW YORK G CRISP/99/AA05592-12 CRISP/99/AA05592-12 199904 eng MECHANISM AND ROLE OF MICROSOMAL ETHANOL OXIDATION Research RPROJ We plan to pursue our studies of factors that affect activity and inducibility of the microsomal ethanol oxidizing systems (MEOS) and associated functions, and hence their pathologic role in alcohol-induced liver injury. We will take advantage of the availability of liver specimens reflecting a large spectrum of human disease, and subject them to Western blot and PCR analysis for P4502E1 (2E1) protein and mRNA, respectively. We will first determine how the induction of microsomal 2E1 is regulated in man, with focus on possible roles of dose and type of alcoholic beverage, age and associated medications and/or deficiencies. We will also asses the role of dietary lipids, especially the species of phosphatidylcholine, in the activity of MEOS in vitro and its inducibility in vivo after chronic ethanol consumption. Specifically, we will evaluate in vitro how the fatty acid composition of the phospholipids may affect 2E1 catalytic activity. In vivo, we will determine how changes in dietary lipids alter the activity of enzymes (including 2E1) in liver microsomes and how the latter correlates with changes in phospholipid composition. The studies will be conducted in our experimental models of alcoholic liver injury in rodents and nonhuman primates and corroborated in human liver tissue, which we will obtain from a parallel study evaluating the effects of supplementation of dietary phospholipids on the outcome of alcoholic liver disease. Associated changes in microsomal phosphatidylethanolamine methyltransferase activity and membrane fluidity will be determined. At present, measurement of 2E1 activity requires liver tissue. However, since 2E1 metabolizes not only ethanol but also chlorozoxazone (CZX), a widely used drug, the suitability of CZX clearance in vivo as a marker of 2E1 induction will be evaluated. These studies will be extended to P4501A2(1A2), found to be able to sustain ethanol metabolism, inducible by factors commonly present in the heavy drinker (smoking, omeprazole treatment) and capable of activating carcinogens. A potentially important factor that may affect 2E1 and 1A2 activity is the extent of adduct formation between the proteins and acetaldehyde. The potential impact of this adduction on the catalytic conversion of ethanol to acetaldehyde, and other 2E1 and 1A2 mediated activities, will be investigated. The planned studies, by revealing some of the determining factors of enzyme activity, may provide the information needed to minimize ethanol's toxicity, or that resulting from the activation of other hepatotoxins. Finally, we will also evaluate the interaction of ethanol with microsomal P450s (especially P4502C8, 1A2 and also 3A3/4) in retinoid metabolism and hepatic retinol depletion, which may contribute to the increased incidence of various cancers in the alcoholic. The broad aim of our proposed studies is to define biochemical differences between heavy drinkers and more moderate consumers. We will focus LIEBER CS VA MEDICAL CENTER 151-2, 130 WEST KINGSBRIDGE ROAD, BRONX, NY 10468 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 10468 Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol acetaldehyde laboratory rabbit laboratory rat membrane permeability microsome adduct polymerase chain reaction pathology drug metabolism enzyme mechanism cytochrome P450 human tissue western blotting alcoholic liver cirrhosis hepatotoxin liver metabolism dietary lipid vitamin A deficiency dietary supplement oxidation phosphatidylcholine vitamin metabolism enzyme activity smoking omeprazole CRISP RPROJ NEW YORK G CRISP/99/AA05934-13 CRISP/99/AA05934-13 199904 eng ETHANOL EFFECTS ON NEUROTRANSMISSION Research RPROJ The broad, long-term, objectives of this proposal are to sensitivity to excitotoxicity. Stimulation of excitatory amino acid receptors, particularly those of the N-methyl-D-aspartic acid (NMDA) and kainate subtype of glutamate receptor, can trigger delayed neuronal death that occurs over a period of hours to days. This process, commonly referred to as excitotoxicity, is thought to involve excessive and prolonged increases of intracellular calcium that ultimately lead to calcium-induced neuronal death. Recent studies have shown that ethanol has potent inhibitory actions on NMDA and kainate receptor mediated calcium influx. Preliminary data contained in this proposal shows that in primary neuronal cultures, acute in vitro ethanol can potently inhibit NMDA receptor mediated excitotoxicity. The first portion of this proposal (specific aims 1 and 2) consist of in vitro studies using primary rat neuronal cultures that will fully characterize the acute and chronic effects of ethanol on excitotoxicity. Acute studies will examine the effects of ethanol on calcium influx, the role of voltage-dependent calcium channels in excitotoxicity and their interaction with ethanol in this process, and the differential sensitivity of certain neurons to excitotoxicity and to excitoprotection by ethanol. The effects of chronic in vitro ethanol treatment on neuronal cultures, including the ability of chronic ethanol to sensitize neurons to excitotoxicity via upregulation of both excitatory amino acid-operated and voltage-operated calcium channels, will also be examined. The in vitro studies will provide a foundation for the in vivo studies (specific aims 3 and 4) contained in the second portion of this proposal. A rat model of ishemic brain damage is proposed that will allow investigations of the interactions of both acute and chronic ethanol consumption with ischemia induced delayed neuronal death. Excitatory amino acid receptors, ion channels and neuronal cell death will be quantitated by histopathological and autoradiographic analysis of brain sections. The interaction of ethanol with ischemic brain damage could yield important and clinically significant information on the neurotoxic effects of ethanol and the neuropathology of chronic alcohol abuse. CREWS FT UNIV OF NC AT CHAPEL HILL, CHAPEL HILL, NC 27599-7178 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat hippocampus calcium flux calcium indicator artery occlusion cell death cell type cerebral ischemia /hypoxia molecular site kainate glutamate dosage arachidonate histopathology disease model neural degeneration neuropharmacology neural inhibition neural transmission neurotoxin protein kinase C calcium channel voltage gated channel autoradiography receptor binding receptor sensitivity NMDA receptor tissue /cell culture excitatory aminoacid CRISP RPROJ NORTH CAROLINA G CRISP/99/AA06069-15 CRISP/99/AA06069-15 199904 eng ALCOHOL METABOLISM--ROLE OF P450 OXYGENASES Research RPROJ APPLICANT'S ABSTRACT: The alcohol-inducible forms of cytochrome P450 contribute to alcohol oxidation and are of biomedical importance because they are involved in the chemical toxicities, mutagenesis, and carcinogenesis associated with alcohol abuse. In addition, they catalyze the reductive cleavage of lipid hydroperoxides and may thereby cause a loss in the integrity of biological membranes. Our goals are as follows: 1. To determine the role of alcohol-inducible P450 oxygenases (rabbit liver microsomal isozymes 2E1 and 2E2) in the oxidative cleavage of esters and amides, with the eventual goal of finding physiologically important substrates for the reaction. Compounds of metabolic importance to be considered include retinyl esters, thiol esters, phosphate esters of carbohydrates, triglycerides, phospholipids, nucleotides, and various peptides. Xenobiotic esters and amides that form aldehydes or other potentially toxic products will also be examined as possible substrates. 2. To compare the activities of P450 2E1 and 2E2 in the reductive beta-scission of various lipid hydroperoxides and determine the metabolic fate and possible function of the products. The long-range aim of the work is to determine whether chronic exposure of animals to ethanol, by causing induction of P450s, leads to enhanced degradation of membrane lipids and eventual loss of membrane integrity. In addition to determining the metabolic fate of the products of reductive cleavage, we will look for the presence of hydroxylated fatty acids, which are alternative products of hydroperoxide reduction. 3. To study the inactivation of ethanol-inducible P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal (HNE) and related products of membrane lipid peroxidation. We will also study the biosynthesis of HNE in mammalian tissues and examine the possible inhibitory effects on P450s 2E1 and 2E2 of a series of other alpha, beta-unsaturated carbonyl compounds produced by ionizing radiation and drug metabolism as well as by lipid peroxidation. 4. To determine the mechanism of induction of P450 2E1 in diabetes, which may involve release of down-regulation of gene expression by insulin. We will first establish a cell line with stably expressed P450 2E1 under the control of its own promoter, in Hep G2, which does not express detectable P450 2E1 endogeneously, and then investigate the effect of insulin in the transfected Hep G2 cells. The mechanism of regulation by insulin will also be examined by nuclear run-on assays and mRNA half-life determination. In addition, we will determine whether induction in animals by the diabetic state and by alcohol administration give additive effects on the P450 2E level in liver microsomes. If so, the oxidative damage and chemical pathology (including nitrosamine activation and acetaminophen toxicity) associated with these cytochromes may be comparably elevated. COON MJ UNIVERSITY OF MICHIGAN, 1301 CATHERINE RD, ANN ARBOR, MI 48109-0606 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol nitrosamine laboratory rabbit microsome diabetes mellitus enzyme induction /repression enzyme inhibitor enzyme reconstitution enzyme substrate isozyme gene expression genetic regulation transcription factor cytochrome P450 liver metabolism membrane activity oxidation /reduction insulin peroxidation acetaminophen protein purification detoxification cell line enzyme activity oxidative stress CRISP RPROJ MICHIGAN G CRISP/99/AA06221-15 CRISP/99/AA06221-15 199904 eng EFFECTS OF ALCOHOL ON CENTRAL NEUROENDOCRINE SYSTEMS Research RIVIER C SCRIPPS RESEARCH INSTITUTE, 10550 N TORREY PINES ROAD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 92037 Crisp Data Base National Institutes Of Health corticosterone ethanol laboratory mouse laboratory rat experimental brain lesion endocrine pharmacology hormone regulation /control mechanism gene expression regulatory gene pharmacogenetics immunocytochemistry neuroendocrine system neuropharmacology adrenocorticotropic hormone corticotropin releasing factor pituitary gonadal axis embryo /fetus toxicology reproductive development sex difference stress behavioral /social science research tag hypothalamic pituitary adrenal axis CRISP RPROJ CALIFORNIA G CRISP/99/AA06420-150006 CRISP/99/AA06420-150006 199904 eng ALCOHOL AND PYRAZOLE REACTION AND METABOLISM BY CYP2E1 Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) There is much current interest in the role of CYP2E1 in alcohol-induced liver injury. We propose to characterize CYP2E1- and ethanol-mediated cytotoxicity in HepG2 cell models either transduced or transfected to express human CYP2E1. We have recently developed a new HepG2 cell line which over-expresses CYP2E1 at levels 5 to 10-fold greater than previously established cell lines. The CYP2E1 over-expressing E47 cells grow at a slower rate than control cells but remain viable. When GSH is depleted, marked toxicity is observed with the E47 cells but not control cells. Sp. Aim I is designed to characterize and evaluate mechanisms involved in this growth inhibition effect caused by over-expression of CYP2E1 and in the cytotoxic effect observed when GSH is depleted. The ability of antioxidants and iron chelators to prevent the growth inhibition and cytotoxicity, and the ability of iron and polyunsaturated fatty acids to exacerbate these effects will be determined. Impairment of mitochondrial function and development of oxidative stress will be studied. Whether injury is apoptotic in nature and the ability of bcl-2 to "rescue" the cells from CYP2E1-catalyzed toxicity will be evaluated. Calcium plays a critical role in cell injury produced by oxidative stress and hepatotoxins. Aim II will evaluate the ability of metabolites derived from CYP2E1 oxidation of ethanol and other substrates and CYP2E1-catalyzed formation of reactive oxygen species to activate Ca2+ channels in microsomes and promote release of Ca2+. The role of Ca2+ in toxicity exhibited by ethanol, PUFA or over-expressed CYP2E1 in HepG2 cells will be evaluated. Aim III will evaluate whether oxidative stress mediated by CYP2E1 itself or CYP2E1 interactions with ethanol, PUFA, acetaminophen activates the transcription factor NF-kB, and if so, whether the HepG2 cells can initially be protected against injury by upregulation of NF-kB-activated genes. Special emphasis will be on cellular GSH levels. TNF-CYP2E1 interactions and induction of IL-8, a powerful chemoattractant will be evaluated. Selected experiments will be carried out with cultures containing hepatocytes isolated from chronic ethanol-fed rats and pair-fed controls to allow extrapolation of results with the HepG2 cells to normal hepatocytes. It is hoped that these studies will help to define the role of CYP2E1 and CYP2E1-derived reactive oxygen species in the hepatotoxic actions of ethanol. CEDERBAUM AI MOUNT SINAI SCHOOL OF MEDICINE, ONE GUSTAVE L LEVY PL BOX 102, NEW YORK, NY 10029 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 10029 Crisp Data Base National Institutes Of Health ethanol laboratory rat mitochondria cell growth regulation enzyme mechanism unsaturated fatty acid growth inhibitor cytochrome P450 tumor necrosis factor alpha iron hepatotoxin liver metabolism chelating agent antioxidant glutathione acetaminophen toxicant interaction calcium channel cytotoxicity toxin metabolism cell line interleukin 8 oxidative stress nuclear factor kappa beta BCL2 protein CRISP RPROJ NEW YORK G CRISP/99/AA06610-13A1 CRISP/99/AA06610-13A1 199904 eng ALCOHOL USE DURING PREGNANCY--A LONGITUDINAL STUDY Research RPROJ In the Maternal Health Practices and Child Development (MHPCD) Project, we have found that prenatal alcohol exposure negatively affects the growth, morphology, behavioral, and cognitive development of children through the age of six. It is important to evaluate the impact these deficits will have on the continued development of the children and to determine whether additional effects will be detected at older ages. This information is necessary for counseling parents and educating pregnant women, as well as for identifying and treating the problems of school-aged children who have been exposed to alcohol prenatally. The MHPCD project is a prospective epidemiologic study of the pregnancy outcomes of 650 women. It is one of few studies that have followed a cohort from early in pregnancy through the child's sixth year. The women who were selected for the study represented the entire spectrum of alcohol use, although the majority were moderate drinkers and moderate users of other substances. Thus, we have a unique opportunity to study alcohol effects across the entire exposure curve while concentrating on assessment of the effects of moderate levels of drinking during pregnancy. The next phase of this longitudinal study will assess growth, morphology, cognitive development, behavior, motor skills, and neuropsychological performance of the children at age ten. We will determine whether the observed effects of prenatal alcohol exposure persist as the children mature and examine the way in which environmental factors moderate the effects of prenatal alcohol exposure. New measures have been added to the child assessment to evaluate these relationships in greater depth and to detect new effects that may become evident as the children mature. We anticipate that additional effects of prenatal alcohol exposure will be detected at older ages as more complex demands are made on the children for mastery of increasingly difficult levels of cognitive tasks and motor skills. DAY NL WESTERN PSYCHIATRIC INST, 3811 O'HARA STREET, PITTSBURGH, PA 15213 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 15213 Crisp Data Base National Institutes Of Health alcoholic beverage consumption child physical development child psychology middle childhood (6-11) mother child interaction postnatal growth disorder human subject developmental neurobiology longitudinal human study embryo /fetus toxicology fetal alcohol syndrome behavior test cognition early experience neuropsychology psychomotor function female behavioral /social science research tag substance abuse epidemiology CRISP RPROJ PENNSYLVANIA G CRISP/99/AA06666-12S1 CRISP/99/AA06666-12S1 199904 eng ETHANOL INGESTION ACTIVATION OF INDUSTRIAL POLLUTANTS Research RPROJ A major goal is to study the toxicity of environmental pollutants, and the interactions of these pollutants with other toxins. One of the most abused toxins is ethanol. Because there is increasing evidence to link alcohol consumption with carcinogenesis, the overall objective of this proposal is to study the metabolism (fate and effect) of a variety of environmental toxins from industrial sources as a function of chronic ethanol ingestion. Studies will be carried out to correlate various metabolic alterations that result from chronic alcohol consumption, i.e., changes in cytochrome P-450 isoenzyme population, changes in the lipid environment upon which critical membrane-protein interactions of the microsomes are dependent, increased production of oxyradicals that contribute to the alterations of lipid membranes, with the metabolic fate and effects of industrial pollutants that lead to carcinogenesis. To this end, enzyme fractions (S9, microsomal and purified) from chronic ethanol-fed rats will be compared with pair-fed controls in the Ames test for their relative abilities to activate several EPA priority pollutants known to be of significant exposure to the industrial work force where alcohol consumption is high. The studies will attempt to determine which of the various alcohol mediated alterations are most important in the carcinogenic action of industrial pollutants by assessing the individual roles of mixed-function oxidase activity, bioreductants, i.e., oxidoreductases and oxyradicals. The differential effects of specific P-450 inhibitors, competing substrates, specific chelating agents that augment or inhibit the iron-catalyzed Haber-Weiss reaction, competing hydroxyl radical scavengers, enzymes such as catalase and superoxide dismutase as well as antioxidants will be compared and contrasted in alcohol-fed vs. pair-fed rats. Using difference spectroscopy, the relative capacity of alcohol-induced microsomes to activate promutagens and to deactivate direct-acting mutagens will be compared with that of microsomes induced by other agents. New information concerning the role of alcohol-consumption in mediating the carcinogenic action of industrial pollutants will be gained and thus, may prove of value in understanding the ramifications of alcohol ingestion in conjunction with exposure to such pollutants. WINSTON GW NORTH CAROLINA STATE UNIVERSIT, COLL OF AGRICULTURE & LIFE SCI, RALEIGH, NC 27695 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 27695 Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol laboratory rat industrial waste enzyme inhibitor cytochrome P450 membrane lipid antioxidant oxidoreductase free radical oxygen toxicant interaction environmental toxicology toxin metabolism free radical scavenger CRISP RPROJ NORTH CAROLINA G CRISP/99/AA06758-10 CRISP/99/AA06758-10 199904 eng EXPERIMENTAL FETAL ALCOHOL SYNDROME Research RPROJ Studies of human with fetal alcohol syndrome (FAS) and rats with experimental FAS show that the structure and the function of the brain are profoundly affected by prenatal exposure to ethanol. Ethanol-induced defects include microencephaly, a thinner cerebral cortex, and reductions in the number of cortical neurons. Each of these findings may result from a single cause-toxic effects of ethanol that cause neuronal death. The number of neurons in the central nervous system (CNS) is determined by the addition of neurons via cell proliferation and migration and by the loss of neurons due to their death. We will test thy hypothesis that prenatal exposure to ethanol increases the amount of neuronal death in the developing nervous system. Three groups of rats will be used: rats will be fed a liquid ethanol-containing diet, pair-fed an isocaloric liquid control diet, or fed chow and water. In the first experiment, the effect of prenatal exposure to ethanol on neuronal death in cerebral cortex will be examined. We will (a) perform a light microscopic analysis of the morphology of dying neurones identified with a monoclonal antibody, ALZ-50 or a silver staining technique and (b) use an immuno-electron microscopic method and a gold- toning technique to analyze the ultrastructure and synaptology of dying neurons. (c) We will determine the spatiotemporal sequence of neuronal death by documenting the changes in the total number of cortical neurons in a defined segment of cortex. In addition, we will calculate the effects of ethanol on the numbers of ALZ-50-positive, addition, silver- stained, and pyknotic neurons in the same cortical segment. (d) We will perform a biochemical examination of the temporal expression of ALZ-50- immunoreactivity. In the second experiment, we will determine if other CNS structures are affected as is cortex. We will repeat most of the above studies using the principal sensory nucleus of the trigeminal nerve (PSN). The advantage of studying the PSN is that it is a simpler structure containing a more homogeneous population of neurons than cortex and it is composed of a countable number of neurons. Moreover, the PSN represents the focus of CNS and craniofacial malformations characteristic of FAS. In the third experiment, we will test the hypothesis that ethanol-induced neuronal death result from alterations occurring early in development. We will examine the effects of ethanol upon the time of origin of dying neurons and upon the relationship of neuronal migration and neuronal death. We will examine the effects of ethanol on the survival of ectopic neurons and appropriately migrated neurons and the extracellular matrix which impacts on neuronal migration. The proposed experiments are a focussed study into a mechanism of FAS and a test of the hypothesis that the CNS defects associated with FAS result from ethanol-induced increases in neuronal death. MILLER MW UNIV OF IOWA COLL OF MED, 500 NEWTON RD MEB, IOWA CITY, IA 52242 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 52242 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse ethanol laboratory rat cerebral cortex cell death cell cycle cell growth regulation cell migration teratogen growth /development synapse neuron developmental neurobiology neurotransmitter microscopy electron microscopy cell population study fetal alcohol syndrome autoradiography craniofacial CRISP RPROJ IOWA G CRISP/99/AA06916-13 CRISP/99/AA06916-13 199904 eng STRUCTURAL CHARACTERIZATION OF ALDEHYDE DEHYDROGENASE Research RPROJ APPLICANT'S ABSTRACT: Our objective is to understand the molecular basis of aldehyde dehydrogenase (ALDH) function. Liver ALDHs act in human alcohol metabolism, to clear ethanol-derived acetaldehyde. The soluble class 3 enzyme, present in nonhepatic tissues, also clears toxic products of lipid peroxidation which, in turn, are elevated by chronic ethanol intake.Toward this goal, we have now determined the first three-dimensional structure of an ALDH the class 3 rat enzyme, with a current resolution of 2.6A. The a-carbon chain has been traced in the electron density map and sidechains have been fitted. Each monomer has two distinct domains. NAD is identified bound in an open a/b structure characteristic of NAD-binding domains, but which differs in certain details from the "Rossmann folds" of other dehydrogenases. Residue conservations indicate that this is characteristic of all ALDHS. A deep channel (for aldehyde binding) contains the catalytic cysteine and other conserved residues. In the funding period, work will focus on refinement of the binary complex (E-NAD) structure and analysis of the cyclopropanone ternary complex crystals, to better understand the active site geometry. We will also model the active site cavities of the present structure, using residues from class I and 2 ALDHS, according to our multiple alignment. We are also poised to continue site-directed mutagenesis from a fresh perspective. With the structure and alignment, we now have the abillty to select residues which will be far more likely to alter, but not abolish activity. Implications of this work for human health are the obvious - that our data should help solve the structure and understand the function of ALDHs more directly involved in ethanol metabolism. Also, with this structure we have solved a structure that is relevant to tumor biology by virtue of class 3 ALDH activity on cyclophospharnide metabolites. We have also solved a structure of immediate relevance to the now-demonstrated enzymatic defect responsibleible for Sjogren-Larsson Syndrome. HEMPEL JD UNIVERSITY OF PITTSBURGH SCH M, 301 CLAPP HALL, PITTSBURGH, PA 15260 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 15260 Crisp Data Base National Institutes Of Health X ray crystallography enzyme mechanism enzyme structure isozyme site directed mutagenesis liver metabolism aldehyde dehydrogenase protein sequence protein structure /function detoxification CRISP RPROJ PENNSYLVANIA G CRISP/99/AA06985-10 CRISP/99/AA06985-10 199904 eng MOLECULAR BASIS OF ETHANOL ACTION ON CALCIUM CHANNELS Research CAVARRUBIAS M THOMAS JEFFERSON UNIVERSITY, 1020 LOCUST STREET, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol computer processing of laboratory data biophysics calcium channel blocker antiarrhythmic agent polymerase chain reaction microinjection drug interaction patch clamp electrophysiology hormone regulation /control mechanism site directed mutagenesis cardiotoxin myocardium disorder human embryo /fetus tissue membrane lipid molecular biology nucleic acid sequence phosphorylation protein kinase C toxicant interaction calcium channel voltage gated channel PC12 cell Xenopus oocyte disease /disorder etiology CRISP RPROJ PENNSYLVANIA G CRISP/99/AA07186-130022 CRISP/99/AA07186-130022 199904 eng EFFECTS OF ETHANOL ON APOPTOSIS Research RUBIN R THOMAS JEFFERSON UNIVERSITY, 1020 LOCUST STREET, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse ethanol chick embryo laboratory mouse laboratory rat flow cytometry polymerase chain reaction enzyme inhibitor molecular cloning gene expression insulinlike growth factor western blotting immunoprecipitation developmental neurobiology protein tyrosine kinase toxicant interaction fetal alcohol syndrome receptor expression receptor sensitivity tissue /cell culture cytotoxicity programmed cell death 3T3 cell PC12 cell CRISP RPROJ PENNSYLVANIA G CRISP/99/AA07186-130023 CRISP/99/AA07186-130023 199904 eng ACETALDEHYDE DEHYDROGENASE GENE EXPRESSION Research ISWRAEL Y THOMAS JEFFERSON UNIVERSITY, 1020 LOCUST STREET, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse therapy laboratory rabbit laboratory mouse laboratory rat nucleic acid chemical synthesis nuclear runoff assay polymerase chain reaction enzyme inhibitor gene expression genetic transcription genetic transduction liver cell antisense nucleic acid messenger RNA nucleic acid inhibitor nucleic acid sequence oligonucleotide aldehyde dehydrogenase high performance liquid chromatography DNA binding protein thiophosphate tissue /cell culture toxin metabolism enzyme activity gel mobility shift assay CRISP RPROJ PENNSYLVANIA G CRISP/99/AA07186-130024 CRISP/99/AA07186-130024 199904 eng ETHANOL AND PLACENTAL TOXICITY--SIGNAL CROSS TALK Research RPROJ The placenta is a multifunctional organ of fetal origin, which is critical to normal fetal growth and development. In addition to direct fetal toxicity, ethanol may be toxic to the placenta as well. Ethanol-induced placental toxicity could contribute to the pathophysiology of alcohol related fetal injury. During the course of the current grant, this laboratory has characterized human placental trophoblasts in culture and demonstrated several alterations in cellular physiology. The human trophoblast is the principal functional cell of the human placenta, but it also expresses physiologic properties which are found in other human cell types. The ethanol-treated, cultured trophoblast model system, developed in this laboratory, enables evaluation of molecular biochemistry in readily available, nontransformed human cells. Using this system, we propose to test the fundamental hypothesis: Ethanol alters intracellular signal transduction. In particular, we will concentrate on the effect of ethanol on signal "cross-talk" mediated by protein kinase C (PKC), an important regulatory factor in normal intracellular signal transduction. This laboratory has shown that chronic exposure to ethanol, within a dose range found in man, results in enhancement of ligand-stimulated cAMP production by cultured trophoblasts. Preliminary data indicate that this alteration is not due to quantitative changes in G protein expression. Rather, there is an effective increase in adenylyl cyclase (AC) activity. We propose that this may be due, at least in part, to ethanol-induced activation of PKC and subsequent interaction of PKC with components of the adenylyl cyclase system. This proposal will investigate the biochemical basis for ethanol-induced enhancement of ligand-stimulated cAMP production in the context of PKC mediated signal "cross talk." Using the general model system of the cultured human placental trophoblast, exposed to ethanol in vitro, we propose to: Investigate ethanol-induced changes in components of the AC signalling system which are modulated by PKC "cross- talk;" Use inhibition of PKC to evaluate the role of PKC in ethanol- induced changes in the AC system; Evaluate the effect of ethanol on PKC activity, as it relates to the several phospholipases and diacylglycerol production; Determine the role of phosphatidylethanol as an activator of PKC and its role in signal "cross-talk." The studies should provide new information on the cellular mechanisms by which ethanol alters placental function and, hence, advance our understanding of the pathophysiology of the Fetal Alcohol Syndrome. Additionally, these findings should contribute to the general understanding of the mechanisms by which ethanol affects the biology of many human tissues, including those of the fetus. FISHER SE SUNY HEALTH SCIS CTR AT BROOKL, BOX 49, BROOKLYN, NY 11203 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 11203 Crisp Data Base National Institutes Of Health ethanol biological signal transduction human tissue diacylglycerol ligand adenylate cyclase phosphorylation protein kinase C embryo /fetus toxicology placental transfer fetal alcohol syndrome trophoblast G protein cyclic AMP receptor binding receptor expression tissue /cell culture enzyme activity protein isoform CRISP RPROJ NEW YORK G CRISP/99/AA07284-12 CRISP/99/AA07284-12 199904 eng MECHANISMS OF ALCOHOL RELATED INHIBITION OF NK ACTIVITY Research RPROJ It is estimated that alcohol dependency affects over 10 million Americans and that the cost of alcohol related problems totals over 100 billion dollars. Alcohol consumption impairs both humoral and cell-mediated immunity. Natural killer (NK) cells play an important role in immunosurveillance against certain infectious diseases and cancer and are especially active in preventing tumor metastasis. NK cells also control the rate of progression of aids. We showed previously that alcohol consumption specifically results in impaired NK and lymphokine activated killer (LAK) cytolytic activity when mice consume between 25-40% of their calories from ethanol administered in the drinking water. The objective of this proposal is to determine the mechanism(s) underlying this impaired cytolytic activity and to specifically test the hypothesis that alcohol consumption impairs cytolytic activity through disruption of protein kinase C cell signaling, which in turn modulates granule and cytotoxic mediator function. We will examine the effect that alcohol consumption has on NK granule cytotoxicity and the specific function of the cytolytic mediators, perforin, granzyme A, and granzyme B. We will also determine the effect of alcohol consumption on the activity and amount of these proteins in the two primary subpopulations of NK and LAK cells that contribute to inherent NK cell cytolytic activity (NK1.1+LGL1-cells) and to LAK cytolytic activity (NK1.1+LGL-cells). In vitro colorimetric enzymatic assays will characterize granzyme A and granzyme B proteolytic activity. Isolation of enriched NK1.1+ cells and the LGL1 subpopulations will utilize magnetic bead separation technology. Western blot analysis will be used to determine protein expression in granzymes, and reverse transcriptase polymerase chain reaction (RT-PCR) will be used to determine mRNA. Protein kinase C activity will be determined using standard assays. Cytolytic granules will be isolated from NK/LAK cells with nitrogen cavitation. MEADOWS GG WASHINGTON STATE UNIVERSITY, PULLMAN, WA 99164-6510 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health corticosterone alcoholic beverage consumption alcoholism /alcohol abuse athymic mouse laboratory mouse biological signal transduction natural killer cell calcium flow cytometry growth inhibitor cellular immunity cytokine interleukin 6 interleukin 2 humoral immunity enzyme linked immunosorbent assay melanoma neoplastic cell neoplastic growth metastasis toxicology cytotoxicity lymphocyte proliferation CRISP RPROJ WASHINGTON G CRISP/99/AA07293-11A1 CRISP/99/AA07293-11A1 199904 eng PRENATAL ALCOHOL EXPOSURE--IMMUNE INTERACTION Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) Adverse effects of maternal alcohol consumption during pregnancy include the immune-deficiencies reported in children with fetal alcohol syndrome. Animal models have also shown that fetal alcohol exposure (FAE) produces T-cell-specific immunosuppression that likely arises from altered thymic development. In the investigator model, this T-cell dysfunction is prevalent in the male FAE offspring. The prevalence of relative immune-deficiency in males in general makes this model appropriate and clinically significant. The investigator's overall hypothesis is that the male-specific T cell dysfunction likely originates in altered thymic development due to elevated maternal corticosterone during a critical period followed by the prenatal surge of testosterone on the fetal male thymus. Their original observation that maternal adrenalectomy (ADX) reverses to T-cell dysfunction in the male FAE offspring, but induces a T-cell dysfunction in the FAE female, suggests that maternal adrenal factors, in addition to alcohol, are involved in these effects. Maternal ADX eliminates maternal plasma CORT, the newly identified surge of maternal plasma dehydroepiandrosterone (DHEA), and attenuates the prenatal surge of plasma testosterone (T) in the male fetus. Thus, some of these steroids may disrupt developmental processes in the male, but protect the female fetal thymus in the presence of alcohol. Therefore, this proposal seeks to determine whether: 1.) the T-cell dysfunction observed in the male FAE offspring is solely due to elevated maternal CORT levels, and what is the critical period of exposure; 2.) inhibiting the maternal CORT response to alcohol in the intact pregnant rat will attenuate the immunosuppressive effects of FAE; 3.) the surge of maternal DHEA contributes to the protection of the FAE female fetal thymus; 4.) the thymolytic characteristics of T-cells contribute to the increased susceptibility to FAE in the male fetus; and 5.) these steroids affect directly the production of specific cytokines by fetal thymic epithelial cells in culture. The results of these studies could contribute to our understanding of the mechanism of FAE-induced immune-deficiency, and in a broader context, the male predominance in the incidence of infectious diseases in children. REDEI EE NORTHWESTERN UNIVERSITY MED SC, 303 E CHICAGO AVE 9-142, CHICAGO, IL 60611-3008 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health adrenalectomy alcoholism /alcohol abuse ethanol dehydroepiandrosterone testosterone laboratory mouse laboratory rat T lymphocyte endocrine pharmacology gene induction /repression pharmacogenetics cytokine immunogenetics immunopharmacology gestational age embryo /fetus toxicology fetal alcohol syndrome pregnancy immunology sex difference tissue /cell culture chemoprevention animal genetic material tag developmental immunology CRISP RPROJ ILLINOIS G CRISP/99/AA07389-07A2 CRISP/99/AA07389-07A2 199904 eng ETHANOL EFFECT ON CELL PROLIFERATION Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) This is a resubmitted revised application for renewal of an ongoing project relating to the consequences of exposure to ethanol during nervous system development. The proposed studies are designed to test two major hypotheses: That a primary target of ethanol during nervous system development is proliferating cells, and that ethanol-induced changes in proliferation of neuronal and glial cells or precursors result from disruption of actions of growth factors. The proposed studies will use primarily in vitro analyses to examine the interactions of ethanol and several growth factors which have been shown to affect cellular proliferation. These factors include the mitogens basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and the anti-mitogen transforming growth factor beta-1(TGF-beta1). The cellular populations to be used to assay the interactions of ethanol with these substances will be C6 astrocytoma cells, primary cortical astrocyte cultures, and B104 neuroblastoma cells. A wide variety of procedures will be applied to these studies, ranging from quantitative molecular techniques to neuroanatomical analyses. Specific experiments will determine the influence of ethanol exposure on expression and secretion of growth factors; effects of ethanol on the expression of specific growth factor receptors; the influences on these growth factors on regulation of cell proliferation and cell cycle kinetics; the effects of ethanol on binding characteristics of growth factor receptors; ethanol influences on growth factor and growth factor receptor MAP kinase activity and expression; and a developmental analysis of growth factor and growth factor receptor expression in vivo. These studies are designed to examine many levels at which ethanol may act, including ligands, receptors, receptor-mediated signal transduction, secretion, transcription and translation. MILLER MW UNIV OF IOWA COLL OF MED, 500 NEWTON RD MEB, IOWA CITY, IA 52242-1000 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat cell growth regulation gene expression genetic transcription growth factor fibroblast growth factor platelet derived growth factor transforming growth factor astrocyte developmental neurobiology receptor binding receptor expression growth factor receptor tissue /cell culture mitogen activated protein kinase neurotoxicology cell proliferation CRISP RPROJ IOWA G CRISP/99/AA07568-09 CRISP/99/AA07568-09 199904 eng FETAL ALCOHOL EXPOSURE--FROM MECHANISM TO PREVENTION Research RPROJ Prenatal alcohol exposure remains a leading known cause of neurobehavioral disorders -- the most severe and least amenable to treatment of the known alcohol-related birth defects (ARBD). Much has been learned about ARBDs since they were first described 19 years ago. Opportunities now exist for new and exciting studies in this area ranging from alcohol's effects on embryonic gene transcription to laboratory and clinical tests for attenuating and preventing ARBDs. These research opportunities are the cynosure of the projects and pilot studies contained in this renewal application for a Fetal Alcohol Research Center (FARC) at Wayne State University (WSU). This current proposal involves investigators at WSU and collaborations with investigators at other universities, and provides an optimal environment for an integrated, broad-based, multidisciplinary, multifaceted and focused attack on ARBDs. Three areas of shared resources: Administrative Core (including Data Management, Statistics, Education and Training Components), Clinical Research Core and Laboratory Animal Research Core, make this FARC highly cost-effective and efficient. The breadth and focus of this FARC are reflected in 5 major research components and 4 pilot projects. The 5 components range from molecular mechanistic studies to clinical prevention and include: 1) Studies of GENETIC POLYMORPHISMS in ADH genotype and alcohol intake as determinants of alcohol-related birth defects; 2) Molecular biological studies of alcohol's actions on PRE-IMPLANTATION EMBRYOS and subsequent peri- implantation; 3) Molecular biological, neurophysiological and pharmacological studies of midbrain DOPAMINERGIC NEURAL SYSTEMS affected by in utero alcohol exposure; 4) ATTENUATION of neurobehavioral deficits through postnatal environmental enrichment; and 5) A PREVENTION STRATEGY to reduce alcohol consumption and ARBDs in the clinic. Our pilot projects range from molecular studies to early identification studies and include: a) Studies of alcohol's effects on components of the MYC-GENE family in the developing hippocampus; b) NEURAL CYTOSKELETAL alterations resulting from in utero alcohol exposure; c) The role of SLEEP DISTURBANCES in learning/memory dysfunction associated with in utero alcohol exposure; and d) SCREENING for in utero alcohol exposure via brain stem auditory potentials. The cadre of scientists participating in this FARC includes many experienced in alcohol research and several newly attracted to the field. There are clear plans for and commitment to extensive education and training activities, including sponsorship of regular research conferences. Finally, long term commitment to this research area is demonstrated by plans for studies following up on findings from those initially proposed, as well as entirely new investigations. SOKOL RJ WAYNE STATE UNIVERSITY, 540 EAST CANFIELD AVE, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health ethanol embryo /fetus toxicology fetal alcohol syndrome CRISP RPROJ MICHIGAN G CRISP/99/AA07606-10S1 CRISP/99/AA07606-10S1 199904 eng ADH GENOTYPE AND ALCOHOL INTAKE AS DETERMINANTS OF ALCOHOL-RELATED BIRTH DEFECTS Research MAY G WAYNE STATE UNIVERSITY, 540 EAST CANFIELD AVE, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol acetaldehyde blood test chemical kinetics child psychology infant human (0-1 year) teratogen disease /disorder proneness /risk drug metabolism isozyme maternal behavior allele genotype genetic polymorphism human subject nucleic acid hybridization alcohol dehydrogenase gas chromatography pregnancy embryo /fetus toxicology postpartum fetal alcohol syndrome neuropsychological test female African American human genetic material tag phenotype clearance rate substance abuse epidemiology genetic susceptibility CRISP RPROJ MICHIGAN G CRISP/99/AA07606-10S10009 CRISP/99/AA07606-10S10009 199904 eng IN-VITRO ETHANOL EXPOSURE OF MOUSE PRE-IMPLANTATION EMBRYOS--MOLECULAR CHANGES Research ARMANT DR WAYNE STATE UNIVERSITY, 540 EAST CANFIELD AVE, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health ethanol laboratory mouse biological signal transduction calcium flux cell differentiation mitogen cell growth regulation flow cytometry early embryonic stage mammalian embryology developmental genetics gene expression protooncogene genetic library transcription factor pharmacogenetics immunocytochemistry messenger RNA fluorescence microscopy phospholipase C protein kinase C protein tyrosine kinase embryo /fetus culture embryo /fetus toxicology embryo implantation growth factor receptor embryogenesis subtraction hybridization transmission electron microscopy CRISP RPROJ MICHIGAN G CRISP/99/AA07606-10S10010 CRISP/99/AA07606-10S10010 199904 eng DOPAMINE FUNCTION IN MIDBRAIN Research CHIODO LA WAYNE STATE UNIVERSITY, 540 EAST CANFIELD AVE, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health newborn animal laboratory rat biological signal transduction mesencephalon flow cytometry teratogen electrophysiology action potential gene expression immunocytochemistry developmental neurobiology enkephalin substance P neuropharmacology neurotransmitter biosynthesis in situ hybridization tyrosine 3 monooxygenase dopamine high performance liquid chromatography pregnancy embryo /fetus toxicology G protein membrane transport protein receptor binding receptor sensitivity dopamine receptor embryo /fetus cell culture northern blotting RNase protection assay neurotransmitter transport dopamine transporter CRISP RPROJ MICHIGAN G CRISP/99/AA07606-10S10011 CRISP/99/AA07606-10S10011 199904 eng AMELIORATION OF FETAL ALCOHOL EFFECTS Research HANNIGAN J WAYNE STATE UNIVERSITY, 540 EAST CANFIELD AVE, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health newborn animal weanling animal ethanol laboratory rat blood test hippocampus teratogen dosage electrophysiology childrearing neuroanatomy developmental neurobiology ataxia pregnancy embryo /fetus toxicology prenatal stress fetal alcohol syndrome behavior test open field behavior avoidance behavior learning memory isolation /deprivation chordate locomotion long term potentiation gait behavioral /social science research tag CRISP RPROJ MICHIGAN G CRISP/99/AA07606-10S10012 CRISP/99/AA07606-10S10012 199904 eng PILOT PROJECTS Research SOKOL RJ WAYNE STATE UNIVERSITY, 540 EAST CANFIELD AVE, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health newborn animal ethanol laboratory mouse laboratory rat biomarker hippocampus brain electrical activity electroencephalography infant human (0-1 year) teratogen dosage sensorineural hearing loss otitis media evoked potential gene expression protooncogene pharmacogenetics human subject immunocytochemistry western blotting developmental neurobiology sleep disorder embryo /fetus toxicology fetal alcohol syndrome posttranslational modification cytoskeletal protein learning disorder cocaine CRISP RPROJ MICHIGAN G CRISP/99/AA07606-10S10014 CRISP/99/AA07606-10S10014 199904 eng CORE--CLINICAL COMPONENT Research SOKOL RJ WAYNE STATE UNIVERSITY, 540 EAST CANFIELD AVE, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health alcoholic beverage consumption biomedical facility child psychology infant human (0-1 year) teratogen disease /disorder proneness /risk maternal behavior patient /disease registry human subject interview mother /embryo /fetus nutrition nutrition related tag pregnancy embryo /fetus monitoring prenatal stress fetal alcohol syndrome behavior test female African American prenatal care CRISP RPROJ MICHIGAN G CRISP/99/AA07606-10S19001 CRISP/99/AA07606-10S19001 199904 eng HUMAN GENETICS RESEARCH Research CHRISTIAN JC INDIANA UNIVERSITY, 545 BARNHILL DRIVE, INDIANAPOLIS, IN 46202-5124 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health young adult human (19-34) alcoholic beverage consumption alcoholism /alcohol abuse biomarker blood test electroencephalography breath test drug hypersensitivity drug resistance sibling twin /multiplet genetic marker genetic polymorphism human subject behavioral genetics psychophysiology personality test cognition neuropsychological test personality phenotype family genetics behavioral /social science research tag genetic susceptibility CRISP RPROJ INDIANA G CRISP/99/AA07611-110005 CRISP/99/AA07611-110005 199904 eng ALCOHOL AND STRESS--INTERACTIVE EFFECTS Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) The ability to respond to stress is an important basic adaptive mechanism; hypothalamic-pituitary-adrenal (HPA) activation is known to be a central feature of this response. Hyperresponsiveness and/or deficits in recovery of HPA activity following stress could have adverse behavioral and physiological consequences for the organism and thus hyperresponsiveness, as well as altered behavioral and immune responsiveness, particularly following exposure to stressors. The present proposal will investigate mechanisms mediating the effects of prenatal ethanol exposure on HPA regulation as well as the role of the HPA hormones in mediating the alterations in immune function seen in E offspring. The Specific Aims are 1) to explore further the mechanisms underlying the increased HPA activity observed in E animals. Experiments will test two possible and not incompatible hypotheses: a) that HPA hyper-responsiveness in E animals result, at least in part, from deficits in feedback regulation of the HPA axis. Experiments will examine effects of repeated exposure to restraint (increased HPA activity and feedback) and of adrenalectomy (adx, removal of the feedback signal) and corticosterone (CORT) replacement (restoration of the feedback signal) on plasma hormone levels, hypothalamic and pituitary gene expression, and hippocampal glucocorticoid receptor gene expression ND activation; b) that HPA hyper-responsiveness in E animals results, at least in part, from enhanced stimulatory input to the HPA axis. Experiments will examine effects of acute and repeated restraint stress and/or of adx and CORT replacement on hypothalamic and pituitary gene expression, corticotropin releasing fracture (CRF) and CRF1 receptor expression, and pituitary CRF binding protein expression. 2) to examine effects of prenatal ethanol exposure on immune function of offspring. Experiments will test the hypothesis that the HPA hyper-responsiveness induced by prenatal ethanol may mediate at least in part the adverse changes in immune competence of E animals. Experiments will examine the role of HPA hormones in mediating altered antibody responses, and the role of the transcription factor NF-kB in mediating possible CORT-induced immunosuppression. The proposed work will have relevance to our understanding of the adverse effects of prenatal ethanol on adaptive functioning in adulthood. WEINBERG JK UNIVERSITY OF BRITISH COLUMBIA, 2177 WESBROOK MALL, VANCOUVER, BC CANADA V6T 1Z3 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health corticosterone adrenalectomy ethanol laboratory rat hormone regulation /control mechanism immunoregulation immunosuppression embryo /fetus toxicology prenatal stress fetal alcohol syndrome behavior test stress restraint stressor nuclear factor kappa beta hypothalamic pituitary adrenal axis CRISP RPROJ CANADA G CRISP/99/AA07789-10A1 CRISP/99/AA07789-10A1 199904 eng CHEMOKINES AS MEDIATORS OF ALCOHOLIC LIVER INJURY Research RPROJ DESCRIPTION: (Adapted from Investigator's Abstract) Alcoholic hepatitis is a serious and potentially fatal complication of chronic alcohol abuse. The condition is marked by recruitment of inflammatory cells, in particular neutrophils, to the liver parenchyma. Once neutrophils invade the liver they can actively attack and destroy hepatocytes. The overall objectives of this research project are to determine how chronic alcohol consumption signal neutrophils to invade the liver, and how neutrophils and their chemoattractants cause liver damage. The main focus of the research will be on C-X-C chemokines. C-X-C chemokines are potent neutrophil chemoattractants and activators; when produced in the liver in vivo, they cause a severe neutrophilic hepatitis. In the setting of chronic ethanol abuse, C-X-C chemokines are induced in the liver and are believed to promote alcoholic hepatitis. However, the mechanism by which chronic ethanol consumption stimulates chemokine production is uncertain. Based on work to date, the investigators propose that ethanol alone is insufficient to induce hepatic chemokine production in vivo. The P.I. will explore the possibility that a cofactor, namely endotoxin, acts in conjunction with ethanol to stimulate C-X-C chemokine production by liver cells in intact rats. The investigators will address this question by administering low-dose endotoxin to ethanol-fed rats over a 4-wk interval, and by determining whether both insults induce C-X-C chemokines even if each alone does not. In separate studies they will investigate how C-X-C chemokines actually cause liver injury. Based on preliminary data, they propose that they induce liver damage by two independent mechanisms: one involving neutrophil recruitment and activation, and the other involving direct cytotoxicity toward hepatocytes. The investigators intend to explore this further by examining chemokine effects on liver cells in culture and in vivo. Cell culture studies will permit them to determine whether C-X-C chemokines cause death by apoptosis or necrosis, and whether they exert their effects intracellularly or by binding to cell-surface receptors. Studies in vivo will allow them to determine the extent to which neutrophils and direct cytotoxicity each contribute to chemokine-induced liver damage. Finally, they will investigate whether the adverse effects of chemokines are enhanced by chronic ethanol consumption. MAHER JJ SAN FRANCISCO GENERAL HOSPITAL, 1001 POTRERO AVENUE, SAN FRANCISCO, CA 94110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94110 Crisp Data Base National Institutes Of Health ethanol laboratory rat neutrophil cellular pathology inflammation transfection gene expression leukocyte activation /transformation chemoattractant immunocytochemistry endotoxin liver cell alcoholic hepatitis hepatotoxin in situ hybridization tissue /cell culture cytotoxicity RNase protection assay chemokine CRISP RPROJ CALIFORNIA G CRISP/99/AA07810-10 CRISP/99/AA07810-10 199904 eng CELLULAR MECHANISM OF ACTION OF ALCOHOL Research RPROJ The long-term objectives of the proposed study are to elucidate the mechanisms by which alcohols exert their toxic actions on the nervous system. Specific attention is focused on the mechanisms underlying the interactions of ethanol and longer-chain alcohols with ion channels by using patch clamp techniques as applied to mammalian neurons. Our previous studies as well as those by other investigators have laid out the groundwork along this line, establishing the phenomenological aspects of alcohol interactions with certain types of ion channels including those activated by GABA, excitatory amino acid (EAA), acetylcholine (ACh) and 5-hydroxytryptamine (5-HT), and voltage-activated sodium, potassium and calcium channels. However, conflicting data have been obtained for certain types of ion channels, and in many cases no detailed mechanisms of alcohol action have been elucidated. The proposed study is aimed at solving some of these problems. Thus the proposed projects will focus on elucidation of the mechanisms of ethanol and longer-chain alcohols on the GABA/A receptor-channel complex. Both whole-cell and single-channel patch clamp techniques will be applied to rat dorsal root gang lion, hippocampal and cortical neurons, and cerebellar Purkinje and granular layer neurons. The specific aims will be concerned with the modulation of open and/or closed channels, dependence of alcohol action on neuron type, animal age and experimental temperature, the role of intracellular components in alcohol action, and the comparison of subtypes and subunits of receptor-channel complex. For the GABA receptor subunit study, human embryonic kidney (HEK-293) cells, in which various combinations of GABA subunits have been transfected, will be used to determine the role of each subunit in alcohol action. Attention will be focused not only on changes in tee amplitude of GABA-induced currents, but also on changes in the rate of desensitization of the currents. The results of the proposed study will provide the basis for the mechanisms of alcoholism and for approaches to prevention and cure of the disorder. NARAHASHI T NORTHWESTERN UNIV MED SCH, 303 E CHICAGO AVENUE, CHICAGO, IL 60611-3008 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat serotonin hippocampus acetylcholine patch clamp electrophysiology transfection human embryo /fetus tissue kidney cell spinal ganglion neuron dorsal root neurotoxin phosphorylation protein kinase membrane channel chloride channel cyclic AMP GABA receptor temperature tissue /cell culture active site excitatory aminoacid age difference CRISP RPROJ ILLINOIS G CRISP/99/AA07836-09 CRISP/99/AA07836-09 199904 eng NEGATIVE IMPACT OF ALCOHOL ON ANTIHYPERTENSIVE THERAPY Research RPROJ Alcohol intake has been implicated as a cause for refractory hypertension which constitutes a major health problem. The nature and the mechanism(s) of the hemodynamic interaction between alcohol and different antihypertensive medications, which may vary both in magnitude and direction, have received little attention. The general aim of the proposed studies is to focus on the mechanism(s) of the antagonistic hemodynamic interaction between alcohol and centrally acting antihypertensive drugs. Renewed interest in utilizing this class of drugs and particularly the second generation drugs, e.g. rilmenidine, as a first line monotherapy for the treatment of hypertension makes the proposed studies noteworthy. The newer drugs lack the sedative side effect of clonidine but lower blood pressure by the same mechanism. The proposed studies will therefore focus on the action of ethanol on the primary processes that lead to the hypotensive responses elicited by this class of drugs which are triggered by activation of the imidazoline receptor complex at their major site of action, the rostral ventrolateral medulla. Given the widespread use of alcohol, studies that deal with its action on the central pathways involved in the hemodynamic responses elicited by centrally acting drugs are warranted. The first aim is intended. to gain support to the hypothesis that the adverse effect of alcohol is targeted against the subclass of centrally acting drugs that lower blood pressure by activating the imidazoline receptor complex. The second aim is to investigate the mechanism(s) of this adverse hemodynamic interaction. The hypothesis is tested that the adverse hemodynamic effects of ethanol involve reversal of the electrochemical responses triggered by clonidine or rilmenidine at their major site of the hypotensive action, the rostral ventrolateral medulla (RVLM) via a selective interaction with the nonadrenergic imidazoline receptor complex. Further studies will determine the site and receptor selectivity of the action of ethanol. The third aim is to test the hypothesis that attenuation of the hypotensive responses elicited by clonidine administered concurrently with alcohol or acutely to alcohol-fed rats involves alcohol evoked modification of the binding characteristics of the imidazoline receptor complex in the rostral ventrolateral medulla, the major site of the hypotensive action of clonidine and the second generation drugs. Whether a pharmacokinetic interaction is involved in the chronic infraction between alcohol and clonidine will be investigated. The experiments proposed herein are intended to test these hypotheses in unrestrained conscious spontaneously hypertensive and normotensive rats chronically instrumented for hemodynamic and electrochemical measurements. These studies are expected to provide significant new insights about the effect of ethanol intake on the hypotensive responses elicited by activating central imidazoline receptors and the mechanism(s) of this health related hemodynamic interaction. In addition to gaining basic knowledge about the neurobiological effects of alcohol on the cardiovascular system, these studies are expected to yield clinically relevant information that could have significant outcome on the treatment of hypertension in alcohol using individuals. ABDEL-RAHMAN AA EAST CAROLINA UNIVERSITY, SCHOOL OF MEDICINE, GREENVILLE, NC 27858 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 27858 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat locus coeruleus antihypertensive agent hypertension cardiovascular disorder chemotherapy hemodynamics electrochemistry inhibitor /antagonist pharmacokinetics drug adverse effect drug interaction clonidine ligand centrally acting drug densitometry autoradiography drug receptor alpha adrenergic receptor CRISP RPROJ NORTH CAROLINA G CRISP/99/AA07839-09 CRISP/99/AA07839-09 199904 eng ETHANOL EFFECTS ON ENDOCYTOSIS IN THE LIVER Research RPROJ Ethanol abuse with its resulting liver injury is a major health problem in this country and worldwide. Our previous studies have shown that the process of receptor-mediated endocytosis (RME) mediated by the hepatic asialoglycoprotein receptor (ASGP-R) is especially susceptible to the deleterious effects of ethanol. The defects in endocytosis are accompanied by defects in receptor function, in that ligand binding is impaired without a decrease in total receptor protein content. These proposed studies will continue to explore this phenomenon, and in this regard, the following hypothesis has been formulated: "Ethanol-induced inactivation of the ASGP-R leads to altered receptor function and impaired RME. Impaired function of the this receptor could lead to alteration of membrane internalization events after ethanol administration and contribute to aberrations such as increased apoptosis in the liver". To examine this hypothesis we have proposed 5 specific aims. In the first two aims, we will continue our examination of altered function of the ASGP-R at a biochemical level, by studying post- translational modifications of the receptor (acylation and phosphorylation) as well as examine in detail the effect of ethanol on the two subpopulations which exist for this receptor (Specific Aim 1). The two pathways for these subpopulations are differentially regulated and have different intracellular "itineraries" post-internalization. In Specific Aim 2 we will determine whether the ethanol-induced post- translational modifications of the ASGP-R result in impaired intracellular receptor-ligand segregation and routing in hepatocytes. In Specific Aim 3 we will examine the effect of ethanol on another cell type in the liver, the Kupffer cell. Kupffer cells possess a receptor (the galactosyl receptor) which has similar binding properties to the ASGP-R, except that this receptor on Kupffer cells is responsible for binding and internalizing particles larger than those internalized by the hepatic ASGP-R. We will study the galactosyl receptor on Kuppfer cells to determine if ethanol administration impairs the function of this receptor similarly to the ASGP-R. In the 4th and 5th specific aims, we will examine two potentially important functional consequences of impaired ASGP-R and galactosyl receptor function. We will determine if the ASGP-R and/or the galactosyl receptors are involved in clearing apoptotic bodies in the liver in studies proposed for Specific Aim 4. Our final specific aim is to investigate whether impaired ASGP-R function is associated with increased levels of soluble asialo- glycoconjugates in the plasma. All together, these proposed studies should provide valuable information concerning the basic molecular mechanisms of alcohol-induced hepatotoxicity. CASEY CA VA MEDICAL CTR, 4101 WOOLWORTH AVE, OHAMHA, NE 68105 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 68105 Crisp Data Base National Institutes Of Health acylation ethanol laboratory rat receptor mediated endocytosis molecular pathology immunoprecipitation liver cell Kupffer's cell liver toxic disorder membrane activity ligand electron microscopy phosphorylation posttranslational modification protein transport glycoprotein receptor programmed cell death CRISP RPROJ NEBRASKA G CRISP/99/AA07846-11 CRISP/99/AA07846-11 199904 eng ETHANOL, CALCIUM CHANNELS, AND PROTEIN KINASE C Research RPROJ A major challenge in alcohol research is to determine the molecular events that underlie alcohol abuse. Much evidence implicates ethanol- induced increases n voltage-dependent calcium channels in alcohol dependence and manifestations of alcohol withdrawal in animals. Studies in our laboratory, using the neural cell line PC12, demonstrated that exposure to 25-200 mM ethanol for 2-6 days increases depolarization- stimulated calcium uptake, which remains elevated for several hours following removal of ethanol. This is associated with an increase in the number of binding sites for dihydropyridine calcium channel antagonists, indicating that ethanol increases the number of functional L-type calcium channels. The process by which this occurs requires protein kinase C (PKC) and chronic ethanol exposure appears to activate PKC by increasing levels of two isozymes, deltaPKC and epsilon PKC. The first goal of this project is to determine which of these isozymes mediates up-regulation of L-channels by ethanol. Since L-channels are composed of multiple subunits, a second goal is to identify which subunits are regulated by ethanol. The final goal is to determine whether similar PKC isozymes and L-channel subunits are regulated by ethanol in experimental animals. Studies are planned to use antisense DNA and RNA to inhibit PKC isozyme expression and block ethanol's effects on L-channels, and to make stable transfectants of PC12 cells that over-express PKC isozymes to mimic or enhance ethanol's effects. Antibodies will be made against L-channel subunits which will be analyzed for ethanol-induced changes in abundance by Northern analysis, Western analysis, and immunoassay. Finally, strains of mice bred for susceptibility or resistance to alcohol withdrawal seizures will be provide specific information about which PKC isozymes and L-channel subunits are involved in up-regulation of calcium channels by ethanol. In addition, they may uncover differences in regulations of L-channels by PCK that underlie genetic differences in the development of alcohol dependence and susceptibility to withdrawal seizures. This will advance the search for genes involved in the development of dependence on alcohol. MESSING RO ERNEST GALLO CLINIC C, 1001 POTRERO AVENUE, BLDG 1, SAN FRANCISCO, CA 94110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94110 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory mouse genetic strain drug withdrawal isozyme neuron antisense nucleic acid messenger RNA nucleic acid sequence phosphorylation protein kinase C membrane protein calcium channel toxicology northern blotting dihydropyridine PC12 cell CRISP RPROJ CALIFORNIA G CRISP/99/AA08117-09 CRISP/99/AA08117-09 199904 eng MECHANISMS OF ACTIONS OF ALCOHOL AND OMEGA3 FATTY ACIDS Research RPROJ APPLICANT'S ABSTRACT: Plasma apolipoprotein E (apoE) is a glycoprotein which plays important roles in "Reverse Cholesterol Transport" (RCT) functions of HDL, viz., (i) cholesterol removal from peripheral tissues and/or (ii) its delivery to the liver. Significantly, ethanol decreased plasma HDL apoE, whereas omega3-fatty acids prevented this decrease. Our preliminary work shows that plasma HDL apoE is also reduced in human alcoholics. We have further shown that chronic ethanol inhibits hepatic sialation of apoE. Sialic acid deficiency in apoE may affect its association with HDL. In turn, the loss of apoE from HDL may impair its RCT functions. Therefore, it is important to confirm whether apoE concentration in HDL and sialic acid content of apoE are reduced in alcoholics and then show how these changes affect HDL metabolism and functions. 90% of the study will utilize humans and only the remainder will use rats:Human Study: ApoE and HDL will be from sera of human alcoholics and non-alcoholics and non-alcoholics. The specific questions are: Does the degree of sialic acid deficiency in apoE affect (I) the association of apoE with HDL and (II) dissociation of apoE from HDL & association of HDL- apoE with cholesterol. III. does the loss of apoE from HDL affect its ability to remove cholesterol from peripheral tissues? Do human alcoholics compared to non-alcoholics have decreased: IV. concentration of apoE in HDL? V. sialic acid content of HDL-apoE? VI. HDL ability to remove cholesterol from the peripheral tissues using human macrophages?VII. HDL ability to deliver cholesterol to liver using the human HepG2 liver cells?Rat Study: If apoE sialation is crucial in the above important aspects of HDL it is equally important to determine the post-translational modifications of apoE in the liver. This can only be done in experimental animals like rats.Similarly, if omega3- fatty acids do correct the above defects caused by ethanol it is simpler and less time consuming to test their effects on HDL-apoE functions in rats than in humans. Thus, the questions asked are what are the influences of ethanol and omega3-fatty acids on: VIII. the posttranslational modifications of apoE at the subcellular level? IX. the mechanism of inhibition of hepatic sialyltransferase activity? Specifically, does ethanol inhibit the synthesis of sialyltransferase and by down regulation of its hepatic mRNA levels? LAKSHMAN R VA MEDICAL CENTER, 50 IRVING ST NW RM 1F154, WASHINGTON, DC 20422 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 20422 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse laboratory rat omega 3 fatty acid fatty acid biosynthesis human subject triglyceride hyperlipidemia lipid metabolism apolipoprotein chylomicron high density lipoprotein very low density lipoprotein blood lipoprotein metabolism fatty liver messenger RNA dietary lipid nutrition related tag ion exchange chromatography gel electrophoresis posttranslational modification toxicology sialyltransferase enzyme activity apolipoprotein E CRISP RPROJ DIST OF COL G CRISP/99/AA08149-09 CRISP/99/AA08149-09 199904 eng ETHANOL AND PHOSPHOINOSITIDES DURING BRAIN DEVELOPMENT Research RPROJ DESCRIPTION: The profound and deleterious effects that ethanol exerts on the developing brain are well established, yet the mechanism(s) involved in its neurotoxic effects remain elusive. Receptors for neurotransmitters and growth factors and their cell signaling systems are increasingly recognized as playing relevant roles in various aspects o rain development. In the last few years we have pursued the hypothesis that the metabolism of phosphoinositides coupled to activation of acetylcholine muscarinic receptors may represent a relevant target for the developmental neurotoxicity of ethanol. Our studies have shown that ethanol exerts a dose-, age-, brain region- and neurotransmitter - specific inhibition of this biochemical response, and have demonstrated a strong correlation between disruption of muscarinic receptor - stimulated phosphoinositide metabolism during the brain growth spurt and long-lasting microencephaly. As proliferation of glial cells is a major event occurring during the in growth spurt, and following observations that acetylcholine can act as a mitogen in astrocytes, we have formulated the hypothesis that disruption of acetylcholine - driven glial cell proliferation may play a primary role in the developmental neurotoxicity of ethanol, and may provide a direct causal link between the observe inhibition of phosphoinositide metabolism and the ensuing microencephaly. The focus of the propose -experiments has shifted from investigations of the effects of ethanol in vivo or in vitro in brain slices or mixed cultures, to the study of its interactions with a selected glial cell population (astrocytes) in vitro. We believe that this shirt represents a logical progression toward a more comprehensive understanding of the sequence of event :hat may link our initial observations of the effects of ethanol on phosphoinositide metabolism to the observe neurotoxic endpoints, such as microencephaly. The specific aims of this proposal are: 1. To investigate selected signal transduction pathways involved in the mitogenic effect of acetylcholine in cultured cortical astrocytes. These will include the hydrolysis of phosphoinositides and phosphatidylcholine, mobilization of intracellular calcium, activation of protein kinase C and induction of immediate - early - genes. 2. To investigate the molecular pathways involved in the observed inhibition by ethanol of the mitogenic action of acetylcholine in astrocytes. COSTA LG UNIVERSITY OF WASHINGTON, BOX 354695, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 98195 Crisp Data Base National Institutes Of Health ethanol laboratory rat calcium flux mitogen cell growth regulation acetylcholine micrencephaly cellular pathology developmental genetics regulatory gene gene induction /repression astrocyte developmental neurobiology phospholipase C phosphatidylinositol protein kinase C embryo /fetus toxicology fetal alcohol syndrome receptor binding muscarinic receptor tissue /cell culture mixed tissue /cell culture neurotoxicology cell proliferation CRISP RPROJ WASHINGTON G CRISP/99/AA08154-06 CRISP/99/AA08154-06 199904 eng NEUROPHARMACOLOGY OF ETHANOL REINFORCEMENT Research RPROJ Ethanol has reinforcing properties in man that are thought to contribute to alcohol abuse and alcoholism. In rats ethanol is readily self-administered orally when taste factors are minimized by preexposing the rats to sweetened solutions. Non-dependent and non- fluid or non-food deprived rats will choose ethanol over water in limited access situations and will lever press to ingest sufficient quantities to produce meaningful blood ethanol levels. Work in the previous funding period was directed at examining the role of a limbic-nucleus accumbens-extrapyramidal circuit in ethanol reinforcement. Results show that while dopamine in the nucleus accumbens may modulate ethanol reinforcement, other systems such as GABA and glutamate may have important if not crucial roles. The purpose of the present proposal will be to extend these earlier results in non-dependent rats to other brain sites such as the amygdala and to other neurotransmitter systems. A further purpose will be to examine the neuropharmacological basis of ethanol reinforcement in dependent rats. Finally, possible sex differences in ethanol reinforcement and the neuropharmacological modulation of ethanol reinforcement will be explored. Non-food or non-fluid deprived male and female rats will be trained to self-administer ethanol in daily 30 minute sessions using a saccharin fade out procedure. The rats then will be implanted with bilateral cannulas aimed at specific brain sites implicated by previous work on ethanol reinforcement. Following establishment of stable ethanol self-administration, the rats will be microinjected with neurotransmitter antagonists and agonists prior to ethanol self-administration sessions. Dose effect functions will be generated using Latin square within subjects designs for dopamine, serotonin, opioid peptide, GABA and glutamate antagonists, and in some cases agonists. Experiments yielding significant results will be repeated in control experiments where rats are trained to self-administer saccharin (without ethanol). Following establishment of critical sites and neuropharmacological effects in non-dependent rats, similar experiments will be repeated in dependent animals and in female rats. The hypothesis under test is that chronic ethanol may result in adaptive changes in those brain mechanisms critical for ethanol reinforcement in the non-dependent animal and that this adaptation may be a crucial motivational part of the dependence process. Knowledge of the neurobiological basis of the motivational aspects of ethanol dependence will provide key information for the development of diagnosis, treatment, and prevention strategies for alcoholism. KOOB GF SCRIPPS RESEARCH INSTITUTE, 10550 N TORREY PINES ROAD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 92037 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol gamma aminobutyrate laboratory rat serotonin nucleus accumbens glutamate disease model neuroanatomy neurobiology neuropharmacology neurotransmitter self stimulation preference operant conditioning psychological reinforcement dopamine receptor sex difference toxicology opioid behavioral /social science research tag CRISP RPROJ CALIFORNIA G CRISP/99/AA08459-09 CRISP/99/AA08459-09 199904 eng ALCOHOL DIRECT AND INDIRECT EFFECTS ON DRUG METABOLISM Research RPROJ Alcohol induced liver disease (ALD) is the fourth leading cause of death among adult men 24-65 years of age residing in urban areas, the eleventh leading cause of deaths overall in the United States, and accounts for billions of dollars annually in medical expenditures. The investigators developed a rat model in which an ethanol-containing diet is infused intragastrically as part of a total enteral nutrition (TEN) system to study the nutrition/ethanol relationships to ALD and ethanol metabolism. They have made several important observations using the TEN model. First, they have identified a diet that prevents ethanol-induced liver injury, even during high ethanol and high unsaturated fat intake for periods reported by others to produce significant hepatic injury. Second, the investigators have identified another diet that produces ethanol-induced liver injury at the same ethanol intake as the first diet. They feel that these two observations together are exciting because study of the differences between these diets could reveal important mechanism underlying ethanol-induced liver injury leading to ALD in humans. Third, the investigators have studied what they feel is an interesting consequence of constant intragastric infusion of ethanol- containing diets, the pulsatile BECs that appear to be due to "cyclic" ethanol metabolism. They do not know the biological significance of this phenomenon in the development of human ALD. However, it is of scientific importance for two reasons: a) ethanol metabolism is thought to be a contributing factor in adverse effects of ethanol; and b) the intragastric rat models are the only practical models that produce ethanol-induced liver injury within a reasonable time frame and expense. Thus, understanding how ethanol is metabolized in this model may be important. Fourth, beer has different metabolic effects than pure ethanol. This is an important observation, because 57 percent of U.S. alcohol drinkers consume their ethanol as beer, not the pure ethanol used in most alcohol research. Their data suggest that there are possible consequences upon the clearance, efficacy and toxicity of medications that may be taken concomitantly with alcoholic beverages. The principal aims of this renewal are to study in detail: 1) the interactions of ethanol and diet on the biochemical and cellular basis of ethanol induced liver injury by proposing novel nutritional and cellular mechanisms involving carbohydrate-regulated and oxygen radical- regulated gene expression, respectively; 2) a unique pulsatile aspect of ethanol metabolism revealed by chronic intragastric infusion of ethanol-containing diets; and 3) the comparative effects of laboratory ethanol and alcoholic beverages (especially beer). BADGER TM ARKANSAS CHILDREN'S HOSP RES I, 1120 MARSHALL STREET, LITTLE ROCK, ARK 72202 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 72202 Crisp Data Base National Institutes Of Health alcoholic beverage alcoholic beverage consumption ethanol laboratory rat pharmacokinetics drug metabolism cytochrome P450 liver disorder hepatotoxin liver metabolism disease model pathogenic diet parenteral feeding nutrition related tag nutrient drug interaction toxin metabolism CRISP RPROJ ARKANSAS G CRISP/99/AA08645-09 CRISP/99/AA08645-09 199904 eng ROLE OF OPIATES IN ALCOHOL-INDUCED NEUROTOXICITY Research RPROJ Central opioid peptides are known to be involved in the neurological and behavioral complications of alcoholism. The cellular mechanism involved in ethanol-regulated endorphin activity is elusive at present because of a lack of reliable experimental model systems. However, we have recently evaluated the interaction between ethanol and the hypothalamic opioid peptide beta-endorphin, by using primary cultures of isolated rat fetal hypothalamic neurons. We have found, for the first time, that beta- endorphin neurons in primary cultures show secretory responses to acute alcohol, adaptive responses to chronic alcohol, secretory responses following withdrawal from chronic ethanol and hypersecretory responses to intermittent ethanol treatment. These ethanol-induced responses of cultured opioid neurons are similar to those observed in opioid neurons and receptors in the brain. Thus, this neuron culture system appears to be a unique and useful model to study cellular actions of ethanol on PEP neurons. The goal of this proposal is to determine whether this in vitro hypothalamic neuron culture system can be used to identify some of the transmembrane signaling processes involved in ethanol regulated beta- endorphin secretion. Our preliminary study suggests that some of the ethanol actions on beta-endorphin secretion parallel that of ethanol- action on steady-state proopiomelanocortin (POMC) mRNA levels. Therefore, the role of various cell signaling pathways in alcohol modulated POMC gene expression will also be characterized. The specific aims are: 1) to determine whether ethanol affects the level of POMC mRNA by altering the half-life, the stability or transcription of the POMC mRNA; 2) to study the role of calcium channels and intracellular calcium in alcohol regulated beta-endorphin secretion and POMC mRNA expression; 3) to elucidate the role of cAMP in alcohol modulated beta-endorphin secretion and POMC mRNA expression; and 4) to evaluate whether the protein kinase C system plays any role in the ethanol-regulated B-endorphin activity. Two- prong approaches will be applied to study the role of these signal transduction systems; one to pharmacologically manipulate the signal transduction system, another to measure the intracellular level of these signal transducers. Several methodological approaches will also be utilized. These will include: a) biochemical assays for measurement of beta-endorphin, cAMP, adenylyl cyclase, translocation of PKC and alcohol levels; b) RNA protection assays for estimation of mature, processing intermediates and primary transcripts of POMC mRNA levels; c) fura-2 dye incorporation techniques for determination of intracellular calcium and d) double-labeled immunocytochemistry techniques to identify cAMP-activated early gene, c-fos, in the beta-endorphin cells. These studies should increase our understanding of the cellular and molecular mechanisms of ethanol actions on opioid peptides and may indicate effective therapi SARKAR DK WASHINGTON STATE UNIVERSITY, 205 WEGNER HALL, PULLMAN, WA 99164-6520 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat biological signal transduction hypothalamus nuclear runoff assay gene expression genetic transcription immunocytochemistry western blotting radioimmunoassay biological model neuron endorphin neurotoxin messenger RNA adenylate cyclase protein kinase C proopiomelanocortin calcium channel cyclic AMP tissue /cell culture opioid RNase protection assay CRISP RPROJ WASHINGTON G CRISP/99/AA08757-07 CRISP/99/AA08757-07 199904 eng ETHANOL AND ACETALDEHYDE ALTERED CILIARY MOTILITY Research RPROJ Alcoholics have an increased incidence of pulmonary diseases that are in part due to altered lung host defense functions related to alcohol toxicity. There is also a strong tendency for alcoholics to smoke heavily. A major airway defense function that is impaired during both alcohol ingestion and cigarette smoking is the mucociliary clearance system which is dependent on the coordinated beating of cilia that line the airways. One possible mechanism of mucociliary impairment common to both alcohol ingestion and smoking is acetaldehyde exposure since acetaldehyde is produced during the metabolism of ethanol, both by the liver and locally in the airways, and is also found in significant amounts in the vapor phase of cigarette smoke. Recent studies from our lab indicate that ethanol stimulates ciliary motility through a nitric-oxide dependent mechanism. Additional findings indicate that this NO-dependent mechanism is especially sensitive acetaldehyde injury. In this context we hypothesize that: ETHANOL AND ACETALDEHDYE ALTER NO-DEPENDENT CILIARY MOTILITY IN AIRWAY EPITHELIA. To test this hypothesis we will perform experiments focused around four specific aims: 1) Characterize the effects of ethanol on airway epithelial NO synthases (NOS) and correlate NOS activation with ethanol-induced ciliary motility changes; 2) Determine how ethanol stimulates the ciliary motility signal transduction pathway by evaluating sequential steps in the cilia activation pathway; 3) Confirm and characterize the effects of acetaldehyde on NO-dependent changes in ciliary motility; 4) Determine the mechanisms(s) by which acetaldehyde impairs NO-dependent stimulation of ciliary motility. The impact of alcohol and smoking-related respiratory illnesses on society is immense. The studies we have outlined in this proposal will explore a novel NO-dependent mechanism by which ethanol and acetaldehyde enhance or impair ciliary function, respectively. Establishing both the mechanisms by which ethanol appears to stimulate and acetaldehyde impairs ciliary function in airways cells will provide meaningful insight into the roles alcohol ingestion and smoking play in the pathogenesis of bronchitis, pneumonia and lung cancer. SISSON JH UNIV OF NEBRASKA MED CENTER, 600 SOUTH 42ND STREET, OMAHA, NE 68198-5300 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol acetaldehyde laboratory rat biological signal transduction cilium /flagellum motility adduct metabolism vapor dynein ATPase respiratory disorder respiratory function respiratory airway clearance respiratory epithelium toxicology nitric oxide synthase smoking CRISP RPROJ NEBRASKA G CRISP/99/AA08769-08 CRISP/99/AA08769-08 199904 eng LIVER AND THE IMMUNODEFICIENCY OF ALCOHOLICS Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) This proposal is an extension (as an R01) of the currently "FIRST award"-funded project, entitled "The role of the liver in the immunodeficiency of alcoholics (R29 AA08846). Studies supported by this grant demonstrated that the effect of alcohol on the liver is a paradox. Based on these observations, the overall objective of this proposal is to examine the mechanisms by which acute or chronic alcohol intoxication regulates some aspects of the hepatic immune system that contribute to the development of hepatic injury and immunosuppression. Hypothesis 1: Chronic alcohol intoxication induces endotoxin influx in the circulation which in turn stimulates hepatic macrophages to produce macrophage inflammatory protein-2(MIP2), a potent stimulus for PMN chemotaxis, superoxide release and hepatotoxicity. Consequently, there is enhanced expression of adhesion molecules on leukocytes and hepatic cells, which can lead to increased migration of inflammatory PMNs (neutrophils) in the liver, resulting in hepatic injury. Specific aim 1: To determine the effect of chronic alcohol intoxication on serum endotoxin, MIP2 production in vivo and in vitro, adhesion molecule expression on peripheral PMNs and hepatic cells, and hepatotoxicity and to determine LPS clearance. Hypothesis 2: Alcohol withdrawal, following an acute alcohol binge exacerbates the toxic effects of endotoxic shock. Specific aim 2: To determine the effect of endotoxemia during alcohol withdrawal on TNF and MIP2 production, hepatic migration of PMNs, and spontaneous superoxide anion production by hepatic non-parenchymal cells. The effect of alcohol withdrawal on LPS-induced mortality and hepatic injury will be studied. Hypothesis 3: Immunosuppression is more frequently encountered among chronic alcoholics, which may be partially due to the down regulation of antigen or ligand-induced respiratory burst. Specific aim 3: To study the effect of prolonged consumption of alcohol on opsonized-Escherichia coli-induced production of extracellular superoxide anion and intracellular H2O2 and Fc receptor expression on Kupffer cells. The microbicidal activity of these cells or, lack thereof, will be tested by using viable E. Coli as target cells. This proposal is unique and novel because it will elucidate the role of a newly discovered chemokine, macrophage inflammatory protein-2 (MIP2) on the induction of alcohol-associated liver injury. The proposed research also includes studies on the role of alcohol-mediated regulation of Fc receptor expression and respiratory burst in Kupffer cells. Results that will be generated from this competitive renewal are expected to provide new and exciting information that may have an important application for the immunotherapy of alcoholics whose livers are further compromised by trauma or infection. BAUTISTA AP LOUISIANA STATE UNIV MED CTR, 1901 PERDIDO STREET, NEW ORLEANS, LA 70112-1393 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat drug withdrawal protease inhibitor leukotriene cytokine chemoattractant endotoxin immunodeficiency liver disorder liver function liver metabolism superoxide superoxide dismutase ibuprofen elastase endopeptidase mixed tissue /cell culture enzyme activity cathepsin G CRISP RPROJ LOUISIANA G CRISP/99/AA08846-06A2 CRISP/99/AA08846-06A2 199904 eng ALCOHOL INTERACTIONS ON THE HPA AXIS Research RPROJ As part of the functional relationship which exists between the immune and the neuroendocrine systems, proteins released by activated immune cells, called interleukins (ILs) or cytokines, convey the occurrence of immune stimulation to the hypothalamic-pituitary-adrenal (HPA) axis. The resulting increased activity of the HPA axis induced by ILs is essential to allow the organism to mount proper immune, endocrine and metabolic responses to an antigenic challenge. Consequently, pathological secretory rates of corticotropin-releasing factor (CRF), ACTH and/or corticosteroids during antigenic challenges can result in increased susceptibility immunologic disorders. Alcoholics and children of alcoholic mothers suffer from an increased occurrence of both infectious and inflammatory diseases, conditions that are associated, respectively, with abnormally elevated or blunted activity of the HPA axis. The distribution of these diseases in predisposed individuals indicates a distribution that is skewed by gender. Based on our animal studies, we hypothesize that alcohol-induced changes in the normal response of the HPA axis to immune signals participate in these pathologies, and that the sex steroid milieu influences both the stimulatory effect of cytokines on neuroendocrine functions, and the ability of alcohol to alter these responses. Our studies are therefore aimed at gaining a better understanding of the mechanisms responsible for the ability of alcohol alters the normal functional link between the immune system and the HPA axis. We have shown that exposure to alcohol during embryonic development or postnatally alters the stimulatory effect of ILs on ACTH and corticosterone release. Studies described in the present proposal will extend these results and explore the mechanisms responsible for the influence of alcohol. We will use IL-1beta, endotoxins or a small volume of turpentine to investigate the effect of a single cytokine, of a cascade of cytokines, or of a true inflammatory response, to probe the mechanisms through which alcohol disrupts the response of the HPA axis to immune signals. Under Specific Aim 1, we will study the influence of gender and sex steroids, the role of CRF, vasopressin and their receptors, and of nitric oxide, in mediating the influence of prenatal alcohol exposure on the response of the HPA axis to immune challenges. Under Specific Aim 2, a similar approach will be used to investigate the ability of post-natal alcohol administration to alter the response of the HPA axis of peripubertal and mature rats to cytokines. In both sets of experiments, sophisticated surgical approaches will be combined with techniques of molecular biology to provide a comprehensive investigation of the hypotheses we propose to test. We believe that the concept we present is original, and that the information gained from our studies will form the basis for a novel approach to the treatment of alcohol-related immune dysfunction. RIVIER CL SALK INST FOR BIOLOGICAL STUDI, PO BOX 85800, SAN DIEGO, CA 92186-5800 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health glucocorticoid alcoholic beverage consumption alcoholism /alcohol abuse laboratory rat hormone regulation /control mechanism postnatal growth disorder immunity interleukin 1 radioimmunoassay secretory immune system messenger RNA corticotropin releasing factor vasopressin lipopolysaccharide embryo /fetus toxicology prenatal stress hormone receptor sex difference nitric oxide northern blotting hypothalamic pituitary adrenal axis CRISP RPROJ CALIFORNIA G CRISP/99/AA08924-08 CRISP/99/AA08924-08 199904 eng ADENYLYL CYCLASE IN ALCOHOL Research RPROJ Over the past 26 months our laboratory has investigated G-protein expression and AC activity in alcoholics, nonalcoholics and the nonalcoholic family members of both groups. Our 'work in progress' demonstrates differential expression of Gsalpha levels in nonalcoholic individuals with a family history of alcoholism [family history positive (FHP) compared to nonalcoholic individuals without a family history of alcoholism (family history negative (FHN)]. These data suggest an intrinsic abnormality in the AC system of alcoholics and those biologically vulnerable to this disorder. In our renewal, we shall confirm our important observation that FHP nonalcoholic subjects have enhanced expression of Gsalpha in erythrocyte and lymphocyte membranes by increasing the sample size and including female subjects. We will examine cell lines from control, alcoholic, and unaffected children of alcoholic subjects. From the promising elements, we shall examine the particularly informative stage IV families for appropriate linkage analysis. We shall investigate the potential significance of differential expression of Gsalpha in FHP and FHN nonalcoholic subjects by correlating membrane G-protein expression/function (conducted at JHU) with sophisticated studies of tolerance/activation. The significance of membrane Gsalpha levels in the development of tolerance will be pursued utilizing two manipulatable models of G-protein expression. In the first model, we will employ several stable cell lines created from S49 cells which were designed to have varying degrees of Gsalpha expression. These cell lines recapitulate the spectrum of G-protein expression in humans. We shall test the hypothesis that the degree of ethanol-induced activation/tolerance in the AC system is proportional to amount of Gsalpha expression. We will also utilize a transgenic mouse model of enhanced Gsalpha expression to determine the effects of varying amounts of cellular Gsalpha on the acquisition of tolerance on the whole animal level. Lastly, we shall continue our studies determining if genetic linkage exists between the inheritance of the alcoholism phenotype and specific Gsalpha gene alleles. To date, our analysis has allowed us to detect several polymorphisms in the Gsalpha. We shall obtain DNA from cell lines of control, alcoholic, and unaffected children of alcoholic subjects. From the promising elements, we shall examine the particularly informative stage IV families for appropriate linkage analysis. WAND GS JOHNS HOPKINS UNIV SCH OF MED, 720 RUTLAND AVE ROSS 1032, BALTIMORE, MD 21205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 21205 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse laboratory mouse transgenic animal biological signal transduction biomarker polymerase chain reaction disease /disorder proneness /risk drug tolerance genetic marker linkage mapping gene expression ADP ribosylation human subject bacterial toxin erythrocyte membrane messenger RNA adenylate cyclase G protein membrane protein denaturing gradient gel electrophoresis enzyme activity family genetics CRISP RPROJ MARYLAND G CRISP/99/AA09000-08 CRISP/99/AA09000-08 199904 eng ETHANOL AND NMDA RECEPTOR FUNCTION Research RPROJ DESCRIPTION: (Adapted from Investigator's Abstract) Prenatal ethanol exposure can cause the Fetal Alcohol Syndrome, which is characterized by, among other symptoms, microencephaly and mental retardation. Ethanol exposure can reduce neuronal numbers in several brain areas during development, with the cerebellum being one of the most sensitive regions, in which Purkinje and granule neurons are lost. The neuronal loss results, at least in part, from direct effects of ethanol on neuronal survival, but the mechanism of ethanol's action is not known. During development, neurons are subject to apoptosis, a form of programmed cell death that has been attributed to a loss of trophic support. Cells undergoing apoptotic death display characteristic morphological and biochemical changes which distinguish them from cells undergoing necrotic death. Necrotic death can often be equated with excitotxicity, which results from overstimulation of the cells by the excitatory amino acid neurotransmitter, glutamate, acting at the N-methyl-D-aspartate (NMDA) receptor. Ethanol has been shown to modulate apoptosis in certain neuronal populations, and ethanol can also affect necrotic cell death by altering the properties of NMDA receptors. Cerebellar granule neurons, which are lost as a result of in vivo ethanol exposure during development, provide an excellent model in which to study the mechanism by which ethanol affects apoptosis and necrosis during development. In primary culture, these cells undergo apoptotic death in the absence of depolarization, and their survival is promoted by exposure to depolarizing concentrations of K+ or to NMDA, which are thought to mimic the innervation of granule cells by glutamatergic mossy fibers in vivo. Initial results suggest that ethanol reduces the trophic effect of NMDA in these cells, i.e., increases apoptosis. We hypothesize that the ability of ethanol to inhibit NMDA receptor function in the cerebellar granule cells leads to this enhanced apoptotic death. We will characterize the acute and chronic effects of ethanol on NMDA receptor characteristics and function (measured as increases in intracellular CA2+) and on apoptosis (measured with cell morphology, DNA fragmentation, fluorescein flororescence and the ApopTag method), as well as necrosis, in cerebellar granule cells during their development in vitro. We will also evaluate the role of the cyclic AMP system, which has been reported to inhibit apoptosis in cerebellar granule cells, in the trophic effect of NMDA, by measuring NMDA-stimulated cyclic AMP production in the presence and absence of ethanol, and attempting to influence the trophic effect of NMDA with protein kinase A modulators. Understanding the mechanism of ethanol's effects on neuronal death during development may lead to therapies to ameliorate this damage. HOFFMAN PL UNIV OF COLORADO HLTH SCI CTR, 4200 EAST 9TH STREET, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 80262 Crisp Data Base National Institutes Of Health ethanol laboratory rat cerebellar cortex calcium flux glutamate fluorescent dye /probe membrane potential histology developmental neurobiology neurotoxin cyclic AMP receptor binding NMDA receptor tissue /cell culture excitatory aminoacid programmed cell death neurotoxicology CRISP RPROJ COLORADO G CRISP/99/AA09005-07 CRISP/99/AA09005-07 199904 eng GENETIC ASPECTS OF ALCOHOLS BEHAVIORAL ACTIVATION Research RPROJ One of the hallmarks of alcohol's many actions is it's frequent biphasic effect. Low doses often produce activation of behavioral and physiological characters. The behavioral activation is variously described as a stimulant effect, euphoria, and disinhibition. Although clear resolution of its dimensions is not yet available, such activating effects are now receiving focus in some genetically based theories of alcoholism. In Cloninger's scheme of alcoholic subtypes, Type II is argued to use alcohol for these euphoriant effects, implying a genetically based sensitivity to this domain of ethanol action. A general working hypothesis states that these activational effects are somehow related to the abuse potential of ethanol, and therefore represent an important addiction liability. A small literature on neurobiological aspects of the activation effect implicate brain dopamine systems, one neurotransmitter known to be important in brain reward systems. Laboratory studies of behavioral activation by ethanol have shown clear genetic influences on this type of initial sensitivity. Mouse locomotor activity has been the most highly investigated phenotype for this question of ethanol activation. The studies outlined here have three general goals. The first is the use of detailed breeding studies to continue our genetic characterization of the activational effect. Our work has suggested that a simple genetic system with as few as three genes might influence this character, and several breeding designs will further test this hypothesis. The second goal is the production of congenic strains of mice where the genetic alleles producing activation in three different stocks, are backcrossed onto the background of the C57BL/6 strain which shows no ethanol-induced activation. Congenic strains are an extremely powerful technique, not heretofore used in alcohol research. They will, in the future, permit tests of specific hypotheses concerning the behavioral, and neurobiological foundations of ethanol's activating actions, and provide a strong tool for mapping and molecular biological study. A third goal involves characterizing the psychopharmacological phenomenology of the paradoxical activational effect of ethanol. We are attempting to define the range of behaviors, such as aggressive behavior, which are commonly genetically influenced in the domain of these activational effects of ethanol. DUDEK BC UNIVERSITY AT ALBANY-SUNY, 1400 WASHINGTON AVE, ALBANY, NY 12222 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 12222 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory mouse genetic strain chemical stimulation allele inbreeding biological model model design /development genetic model molecular biology neurobiology behavioral genetics aggression sedative /hypnotic tranquilizer psychopharmacology chordate locomotion toxicology phenotype behavioral /social science research tag CRISP RPROJ NEW YORK G CRISP/99/AA09038-08 CRISP/99/AA09038-08 199904 eng ETHANOL INFLUENCES ON TROPHIC FACTORS IN CNS DEVELOPMENT Research RPROJ DESCRIPTION: The proposed research will investigate the hypothesis that an important mechanism leading to certain of the CNS anomalies seen in the fetal alcohol syndrome (FAS) is a disruption of normal neurotrophic interactions during neurogenesis. We propose that chronic prenatal or early postnatal ethanol treatment results in alterations in the synthesis, availability, delivery, and/or biological activity of normally occurring neutrophic factors, or may alter the capacity of neurons to respond appropriately to these factors. In addition, we hypothesize that cell death effector or repressor genes may modulate ethanol neurotoxicity during development, and that early ethanol exposure may affect this gene expression. These relationships will be studied in the fetal and neonatal rat and mouse septohippocampal system. This system was chosen because its importance to normal cognitive functioning makes it likely that disruptions in its development may be associated with the severe intellectual impairment seen in FAS. Experiments will examine the following: The influence of early postnatal ethanol treatment (PNET) on cholinergic, GABAergic, and neurotrophic factor receptor-positive basal forebrain neurons (of the medial septal/vertical diagonal band of Broca nuclei); the influence of PNET on neurotrophic factor (NTF) biological activity in the hippocampus (HC); the effect of chronic prenatal ethanol treatment (CPET) and PNET on NTF and NTF receptor gene expression in HC and septum, using quantitative Northern blot analyses of NTF and NTF receptor mRNAs and ELISA analyses of protein content; the influence of CPET on neuronal survival in NTF gene-deleted animals; the potential of overexpression of the bcl-2 anti-apoptosis gene to ameliorate ethanol neurotoxicity in vivo; the relative vulnerability of septohippocampal neurons from bcl-2 gene-deleted animals to ethanol neurotoxicity; and the influence of CPET and PNET on calcium homeostasis in septal and HC neurons, and on NTF stabilization of calcium homeostasis. In addition we will extend our tissue culture model system for ethanol neurotoxicity to include additional concomitants of in vivo ethanol exposure (e.g., hypoxia, hypoglycemia). These studies have the potential to elucidate important cellular mechanisms underlying the devastating CNS damage seen in FAS, and could eventually lead to therapeutic intervention in FAS mental retardation via neurotrophic factor replacement procedures. HEATON MB UNIVERSITY OF FLORIDA, BOX 100244 JHMHSC, GAINESVILLE, FL 32610-0244 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health newborn animal ethanol laboratory mouse laboratory rat transgenic animal brain mapping calcium flux hypoglycemia cellular pathology gene expression neurotrophic factor disease model neurogenesis developmental neurobiology embryo /fetus embryo /fetus toxicology fetal alcohol syndrome growth factor receptor neurotransmitter receptor hypoxia tissue /cell culture northern blotting programmed cell death CRISP RPROJ FLORIDA G CRISP/99/AA09128-07 CRISP/99/AA09128-07 199904 eng NEURAL SUBSTRATES OF THE ACUTE EFFECTS OF ETHANOL Research RPROJ DESCRIPTION: (from applicant's abstract) Although alcohol abuse and alcoholism are significant health problems in our society, our understanding of the neurobiological actions of alcohol in the central nervous system remains incomplete. The overall goal of our research continues to be the expansion of knowledge of the neuroanatomical basis of the effects of alcohol using neuroimaging methods. During the past funding period we have demonstrated that the neuroanatomical circuits activated by the administration of alcohol are dependent on the dose of alcohol, as well as the length of time since ingestion. In addition, we have identified a circuit comprised mainly portions of the mesocorticolimbic system that mediates the effects of alcohol self-administration. The purpose of the studies proposed here is to build on this work, thereby extending our understanding specifically of the neural substrates of alcohol self-administration. Using the quantitative auto-radiographic 2- [14C]deoxyglucose method, our studies will, first, identify the role of the dose of alcohol in determining the characteristics of the circuitry underlying self-administration. Second, changes in the pattern of functional activation that result from a longer post-ingestion intervals after self-administration will be determined. A third goal of these studies is to identify the neuroanatomical substrates of the anticipatory response to alcohol. Metabolic mapping will be applied to rats in the presence of cues that predict the availability of alcohol. The final goal of these studies to determine the circuits through which dopamine is involved in the voluntary ingestion of alcohol. The studies proposed in the present application focus on the definition of the neural circuitry that mediates various aspects of the alcohol self-administration by combining neuroimaging methods with well-controlled behavioral paradigms in order to further our understanding of the effects of alcohol in behaviorally relevant contexts. PORRINO LJ WAKE FOREST UNIVERSITY SCHOOL, MEDICAL CENTER BOULEVARD, WINSTON SALEM, NC 27012-1083 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol laboratory rat brain /spinal pathway /tract brain mapping brain metabolism glucose metabolism dosage self medication deoxyglucose neuropharmacology oral behavior cue autoradiography dopamine antagonist neurotoxicology behavioral /social science research tag CRISP RPROJ NORTH CAROLINA G CRISP/99/AA09291-06 CRISP/99/AA09291-06 199904 eng ALCOHOL HYDROLYSIS/CELL GROWTH Research RPROJ The long-term goal of this project is to determine the role of protein kinase C (PKC) and the phospholipid-hydrolyzing enzymes in the mediation of growth regulatory effects of ethanol with a special focus on fetal alcohol syndrome (FAS) and the "co-carcinogenic" effects of ethanol. The proposed work will initially determine the mechanisms by which ethanol stimulates both transient and prolonged hydrolysis of phosphatidylethanolamine (PtdEtn) by phospholipase D (PLD) and phospholipase C (PLC), respectively. Both effects require concomitant activation of PKC-epsilon (and perhaps PKC-delta by PKC activators, including phorbol 12-myristate-13-acetate (PMA), but PKC-epsilon regulates the PLD and PLC activities differently. With respect to the regulation of PLD (Aim 1), the proposed work will examine the possibility that potentiation of the stimulatory PMA effect by ethanol is due to the inhibition of p21/ras, a negative regulator of PLD. As an important tool to verify this mechanism, the effect of ethanol on the activity state of p21/ras will be determined by measuring binding of GTP to immunoprecipitated p21/ras. With respect to the regulation of PLC (Aim 2), a major task will be to determine whether PKC-epsilon potentiates the effect of ethanol per se, or PKC-epsilon stimulates the synthesis of fatty acid ethyl esters (FAEE), the possible ultimate regulator of PLC. This latter mechanism is possible because 1-chloro-2,4-dinitrobenzene (CDNB), an inhibitor of FAEE-synthase III, was found to inhibit the effect of ethanol on PLC-mediated PtdEtn hydrolysis. Various molecular species of FAEE will be synthesized to determine their possible effects on PLC activity. The work in Aim 3 will examine the role of PKC-epsilon, PKC-delta, and PLC in the mediation of inhibitory ethanol effect on nerve growth factor-induced neurite formation, a measure of differentiation, in PC12 neural cells. PKC-epsilon and PKC-delta will be overexpressed in PC12 neural cells to examine if they potentiate the inhibitory effect of ethanol on neurite formation. To probe the mediatory role of PLC, the effect of ethanol on neurite formation in control and PKC-overexpressing cells will be determined in the presence of CDNB. In the C3H/10T1/2 fibroblast system, a cellular model for carcinogenesis, ethanol potentiated the formation of transforming foci induced by a suboptimal concentration (0.25 microg/ml) of 7,12-dimethylbenz[a]anthracene. Work in Aim 4 will examine the role of PLC in the mediation of this apparent co-carcinogenic effect of ethanol (or FAEE), by using the inhibitor CDNB. The projected work will likely reveal that the effects of ethanol (or FAEE) on neural cell differentiation and carcinogenesis involve specific PKC isozymes as well as increased PtdEtn hydrolysis. This outcome could identify PtdEtn-specific PLC as a possible site of intervention to alleviate these effects of ethanol. KISS Z UNIVERSITY OF MINNESOTA, 801 16TH AVE NE, AUSTIN, MN 55912 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 55912 Crisp Data Base National Institutes Of Health ethanol cell growth regulation hydrolysis isozyme western blotting chemical carcinogenesis cocarcinogen phospholipase C phospholipid phosphatidylethanolamine fetal alcohol syndrome enzyme activity 3T3 cell oncoprotein p21 CRISP RPROJ MINNESOTA G CRISP/99/AA09292-05 CRISP/99/AA09292-05 199904 eng ROLE OF LIPID ALDEHYDES IN ETHANOL INDUCED LIVER DAMAGE Research RPROJ The aim of this project is to investigate the ability of chronic ethanol ingestion to stimulate hepatic production of chemically reactive aldehydic products having the potential to function as intermediates in alcohol- induced liver damage. The proposed studies are designed to identify correlations of alcohol-induced changes in lobular histology and immunocytochemistry with biochemical and molecular alterations at the level of zonal-specific hepatocytes and isolated mitochondria. Specifically, experiments are described to measure concentrations of "free" malondialdehyde, 4-hydroxynonenal and hexanal in livers of rats following chronic ethanol feeding. Lipid- and protein-complexed carbonyls will also be measured to quantitate aldehydic functions hound to hepatocellular constituents. The mechanisms of production of these aldehydic products will be examined in isolated periportal and perivenous hepatocytes and isolated mitochondria obtained from rats chronically ingesting ethanol. The ability of ethanol and acetaldehyde metabolism to stimulate lipid peroxidation will be evaluated in zonal specific hepatocytes and isolated mitochondria. Steady-state concentrations of malondialdehyde, 4-hydroxynonenal and hexanal will be quantitated as indices of lipid peroxidation in hepatocytes and mitochondrial for determination of correlations between the magnitude of lipid peroxidation, alterations in glutathione concentrations and disruption of ethanol and acetaldehyde oxidation. The ability of aldehydic lipids to interfere with mitochondrial acetaldehyde oxidation will be evaluated in order to test the hypothesis that these biogenic aldehydes inhibit aldehyde dehydrogenase mediated oxidation of acetaldehyde. A final series of experiments are proposed to evaluate the hypothesis that specific proteins in perivenous or periportal hepatocytes are targets for adduct formation with reactive aldehydic products of lipid peroxidation. This will be determined by using antibodies against aldehydelysine and cysteine adducts for immunocytochemical identification of lobular, cellular and subcellular proteins predisposed for formation of aldehyde adducts. Collectively, the design and statistical analyses employed in the proposed experiments will allow identification of significant changes in lobular histochemistry and immunocytochemical parameters causally related to biochemical and molecular changes at the hepatocellular level. The long-term goal of this proposed research is to establish the role of aldehydic products of lipid peroxidation and their adducts in alcohol-induced liver injury and determine the utility of their measurement as an early indicator of hepatocellular liver damage. PETERSEN DR UNIV OF COLORADO HLTH SCIS CTR, 4200 EAST 9TH AVE, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 80262 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol aldehyde laboratory rat adduct molecular pathology free radical molecular cloning genetic regulation histology immunocytochemistry immunotoxicity autoantibody lipid peroxide liver toxic disorder liver metabolism glutathione peroxidation protein sequence toxicology CRISP RPROJ COLORADO G CRISP/99/AA09300-05 CRISP/99/AA09300-05 199904 eng ETHANOL EFFECTS ON PROTEOLYTIC SYSTEMS IN THE LIVER Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) The hypotheses of this proposal are: 1) chronic ethanol consumption impairs lysosome biogenesis by preventing processing and trafficking of lysosomal hydrolases, causing their placement at other intracellular or extracellular sites; 2) ethanol administration alters the ubiquitin-proteasome pathway by inactivating the proteasome which could lead to accumulation of modified proteins. Ethanol may differentially influence the proteasome in Kupffer cells. In Specific Aim 1, lysosome biogenesis will be measured by examining the processing, and compartmentalization of the protease, cathepsin L in hepatocytes isolated from control and ethanol-fed rats. This enzyme follows a specific pathway through vesicular compartments en route to the lysosome and the investigator's Aim 1 is to determine the step(s) at which this process is impaired by ethanol, because misrouting of cathepsin L and other hydrolases could potentially cause cell damage due to their potent hydrolytic capacities. In Specific Aim 1b they will examine whether ethanol influences the distribution of the mannose-6-phosphate receptor by subcellular fractionation immunocytochemistry and functional assay studies. This receptor mediates lysosome assembly by targeting cathepsin L and other hydrolases to the lysosome. The investigators postulate that ethanol may change its intracellular distribution as well as its ligand affinity for procathepsin L. In Specific Aim 2, the components of the ubiquitin-proteasome pathway will be examined in whole livers as well as parenchymal and Kupffer cells of control and ethanol-fed rats subjected to both ad lib and intra gastric feeding regimens. This nonlysosomal proteolytic pathway has a crucial role in: 1) the degradation of altered proteins; and 2) the activation of the transcription factor NFkappaB which initiates transcription of genes involved in the inflammatory and immune responses. The investigators postulate that while ethanol may down-regulate the proteasome in liver parenchymal cells, its activity may be regulated differentially in Kupffer cells, since they play a paracrine role in the pathogenesis of alcoholic liver disease. DONOHUE TM VETERANS AFFAIRS MEDICAL CENTE, 4101 WOOLWORTH AVE, OMAHA, NE 68105 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 68105 Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol laboratory rat intracellular transport lysosome gene expression immunocytochemistry liver cell liver metabolism endopeptidase protein metabolism ubiquitin protein transport proteolysis carbohydrate receptor detoxification membrane biogenesis mannose 6 phosphate nuclear factor kappa beta proteasome CRISP RPROJ NEBRASKA G CRISP/99/AA09384-06 CRISP/99/AA09384-06 199904 eng ALCOHOL RADICAL INTERACTIONS AS DETECTED BY ESR Research RPROJ APPLICANT'S ABSTRACT: There is much current interest in the role of oxidative stress and ethanol generation of reactive radical species in the mechanism(s) by which ethanol is toxic. It has been difficult to establish direct linkage between CYP2El, oxidative stress, and ethanol toxicity. Our laboratory has established a HepG2 cell line which constitutively expresses the human CYP2El. Ethanol or a polyunsaturated fatty acid (PUFA) was toxic to the E9 cells which express CYP2El, but not to control cells. Toxicity by ethanol and PUFA was prevented by antioxidants. These cells appear to be a valuable model to assess the role of CYP2El-dependent formation of reactive species such as the 1-hydroxyethyl radical (HER) in the hepatotoxic actions of ethanol. SPECIFIC Aim I is designed to study the role of HER, lipid, and other free radicals in the toxic actions of ethanol and PUFA to cells expressing CYP2El. The major analytical technique to be used is ESR spectroscopy. Studies are planned to study the mechanism of HER formation by CYP2El, and the role of HER and lipid radicals in the toxicity exerted by ethanol and PUFA; the interaction of HER with cellular constituents including adduct formation with CYP2El will be evaluated; activation of the transcription factor, NF-kB, by ethanol or CYP2El derived HER and other radical species will be determined. These studies should identify production of HER and lipid radicals in cells expressing CYP2El and assess the role of these radicals in the toxicity by ethanol and PUFA. Aim II will characterize NADPH- and NADH-dependent formation of HER, 02-, OH, and other reactive intermediates by microsomes from cell lines which express only one human P450 isoform. Toxicity by ethanol or PUFA to these cells will be evaluated, to indicate if human CYP2El is uniquely reactive in activating ethanol to HER, in formation of free radicals, and in promoting ethanol toxicity via a free radical, oxidative stress type of mechanism. The effect of NO on CYP2El catalytic activity and generation of HER and other free radicals will be evaluated in Aim Ill. If NO inhibits CYP2El, NO may prove to be useful as a protectant against the toxicity of ethanol and other toxins which are activated by CYP2El; this will be directly determined with the E9 cells. These studies will provide new information on the ability of NO to modulate CYP2El catalytic activity, generation of reactive intermediates, and ethanol and PUFA toxicity. CEDERBAUM AI MOUNT SINAI SCHOOL OF MEDICINE, ONE GUSTAVE L LEVY PL BOX 102, NEW YORK, NY 10029 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 10029 Crisp Data Base National Institutes Of Health ethanol laboratory rat microsome electron spin resonance spectroscopy unsaturated fatty acid cytochrome P450 hepatotoxin free radical oxygen nicotinamide adenine dinucleotide NAD(H) phosphate cytotoxicity toxin metabolism nitric oxide cell line oxidative stress hydroxyl radical CRISP RPROJ NEW YORK G CRISP/99/AA09460-06 CRISP/99/AA09460-06 199904 eng COTERATOGENICITY OF ALCOHOL AND OTHER DRUGS OF ABUSE Research RPROJ Alcohol abuse during pregnancy is recognized as a significant risk factor to normal fetal growth and development, and alcohol is considered to be a potent teratogenic agent in both humans and animals. Given that alcohol frequently is used concomitantly with other drugs of abuse by pregnant women, it is important to know how the combined use of alcohol and other drugs of abuse affect alcohol's teratogenic effects. The "other" drug of abuse this project will focus on is cocaine, since its use by pregnant women has increased dramatically in the last decade. In Series I, an established mouse model of alcohol-induced birth defects will be employed. Studies are designed to evaluate the effect of combined alcohol and cocaine administration (either chronically or acutely) on pregnancy outcome in C57BL/6J mice. The primary dependent variables will include the effect on fetal weight, Utter size, and type and number of birth defects. The studies in Series II and III take this research one step further. In Series II, the C57BL/6J mouse model system will be used to measure the effects of concomitant alcohol and cocaine administration on alterations in the vasoactive prostaglandins (thromboxane and prostacyclin) which regulate placental blood flow. Since both alcohol and cocaine have been shown to decrease intrauterine and placental blood flow, alterations in the vasoactive prostaglandins or their "balance" might be a common site where concomitant alcohol and cocaine exposure may have an interactive effect. Studies manipulating endogenous thromboxane and prostacyclin levels in vivo are proposed, as well. A unique aspect of this proposal is that in Series III, we propose to use the human placenta and human umbilical artery to address the effect of concomitant alcohol and cocaine exposure on thromboxane and prostacyclin production or levels. Thus, we will utilize both animal and human tissue to address a potential mechanism for the co-teratogenicity of alcohol and cocaine. In summary, the proposed research will begin to fill a void in the literature regarding the effect of concomitant alcohol and cocaine administration on pregnancy outcome. Moreover, it win begin to address issues related to a mechanism of action which might be able to explain at least some of the teratogenic effects of the drug combination. Since there is virtually no information in the literature on this topic, the results generated will be of major significance to the prenatal field. RANDALL CL MEDICAL UNIV OF SOUTH CAROLINA, 171 ASHLEY AVE, CHARLESTON, SC 29425 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 29425 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse laboratory mouse vasoactive agent teratogen prostacyclin thromboxane human tissue disease model pregnancy embryo /fetus toxicology substance abuse cocaine behavioral /social science research tag CRISP RPROJ SOUTH CAROLINA G CRISP/99/AA09487-06 CRISP/99/AA09487-06 199904 eng FETAL ALCOHOL EXPOSURE AND SCHOOL AGE COGNITIVE FUNCTION Research RPROJ Children exposed prenatally to alcohol are at risk for learning deficits and behavioral problems in school even when they do not exhibit full Fetal Alcohol Syndrome. Although decrements in Full Scale IQ are modest, deficits have been noted particularly in arithmetic, spatial reasoning, and nonverbal short-term memory. Poorer sustained attention has been documented by objective test procedures, but other aspects of attention have not been evaluated systematically. We have recently completed a study of 480 black inner city infants, who were assessed on both the Bayley Scales and a new battery of domain-specific tests with better predictive validity for school-age performance. Prenatal alcohol exposure was associated with poorer Bayley Scale performance and slower, less efficient processing of information in two domains: visual recognition memory and cross-modal transfer. The proposed study will re-evaluate these children at 7 years (a) to determine the degree to which the processing efficiency deficit seen during infancy continues to be evident at school age; (b) to assess effects of this exposure on four distinct dimensions of attention using standard tests adapted for young children; (c) to determine the degree to which the alcohol-related deficits in IQ test performance seen in the moderately-exposed, white middle class Seattle sample also hold for our cohort of black inner city children exposed at similar levels; and (d) to determine the degree to which alcohol-related cognitive deficits at 7 years are attributable to processing efficiency and/or attentional impairment. If processing efficiency is implicated, we will test the hypothesis that slower processing in Infancy may provide a marker for early identification of affected children in need of remedial Intervention. Assessment of fetal alcohol exposure will be based on a series of 2-week, day-by-day drinking histories obtained contemporaneously during pregnancy, which proved highly sensitive In the infant study. A broad range of control variables will be assessed, including prenatal exposure to Illicit drugs, quality of parental stimulation, and current maternal drinking and drug use. Alcohol effects will be inferred only after statistical adjustment for confounders. Dose dependence and exposure thresholds will be evaluated, as well as the degree to which observed statistically significant effects are also clinically meaningful. JACOBSON JL WAYNE STATE UNIVERSITY, 71 W WARREN, DETROIT, MI 48202 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48202 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse child psychology attention deficit disorder middle childhood (6-11) dosage childrearing parent offspring interaction human subject mental health epidemiology longitudinal human study embryo /fetus toxicology fetal alcohol syndrome attention neuropsychology cognition disorder African American low income clinical research behavioral /social science research tag CRISP RPROJ MICHIGAN G CRISP/99/AA09524-06 CRISP/99/AA09524-06 199904 eng GENETICS OF INTERACTIONS BETWEEN ETHANOL AND NICOTINE Research RPROJ Co-dependence on ethanol and nicotine is the most common of polydrug addictions. Previous studies have suggested that sensitivity of these two drugs on certain measures are genetically correlated; those animals which are more sensitive to one agent are also more sensitive to the other. This correlation may have arisen because ethanol acts at specific nicotinic receptor subtypes to modulate their function. Preliminary evidence supports this. The overall hypothesis of this research is that ethanol acts at nicotinic cholinergic receptors and that this interaction is the basis for the observed genetic correlation in sensitivities between ethanol and nicotine and the common co-use of these agents. The long term goal of this research is to determine the specific mechanism(s) by which ethanol and nicotine interact to produce co-dependence. We will work toward this goal by confirming that nicotine and ethanol sensitivities are genetically correlated. Potential mechanisms for interactions between ethanol and nicotine will be examined and characterized by focusing on the effects of ethanol and interactions with nicotine on nicotine acetylcholine receptors (nAChRs). Previous studies suggest highly that ethanol and nicotine sensitivities are genetically correlated. Studies are proposed to replicate, confirm and expand on our preliminary studies with the HAS/CAS/LAS selectively bred rat lines. Because studies in inbred strains of mouse have demonstrated that numbers of nicotinic binding sites are correlated with nicotine sensitivity, the numbers of binding sites for [3H]nicotine and alpha-[125]bungarotoxin will be measured in these animals. Pharmacokinetic analyses with nicotine will also be conducted. Evidence suggests that ethanol may act at nAChRs. We will use a Xenopus oocyte expression/recording system to screen specific nAChR subtypes for responsiveness to ethanol. By conducting dose-response and time course analyses we will be able to determine at which receptor subtypes ethanol acts (efficacy studies) and to assess the degree to which these subtypes are sensitive (potency studies). Additionally, we will be able to assess the nature of ethanol's actions at a particular subtype. We hypotheize that ethanol will modulate the responsiveness of nAChR subtypes to agonists in a subtype- and concentration-dependent fashion. Specifically, we hypothesize that the actions of ethanol will include both attenuation and enhancement of nicotinic responses. DE FIEBRE CM UNIV OF NORTH TEXAS HLTH SCIS, 3500 CAMP BOWIE BLVD, FORTH WORTH, TX 76107-2699 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory mouse laboratory rat genetic strain pharmacokinetics drug addiction drug interaction electrophysiology genotype centrally acting drug nicotine receptor binding receptor expression nicotinic receptor bungarotoxin Xenopus oocyte CRISP RPROJ TEXAS G CRISP/99/AA09585-05 CRISP/99/AA09585-05 199904 eng NEONATAL ETHANOL EXPOSURE AND DRUG INTERACTIONS Research RPROJ Prenatal alcohol exposure can have a variety of behavioral and physical consequences for the developing offspring. While the incidence of alcohol consumption during pregnancy may be decreasing, many clinical studies suggest that women that continue to consume alcohol during pregnancy are also more likely to consume other drugs as well. One of the more frequent drug combinations consumed is alcohol and cocaine. The purpose of this application is to use a rodent model to assess the effects of "third trimester" exposure to this drug combination. The third trimester is a period of rapid CNS growth and proliferation commonly known as the "brain growth spurt" and evidence suggests that the CNS is vulnerable to injury during this period. In rats, the analogous period of development actually occurs during the first two weeks after birth. Therefore, in order to study"third trimester" effects, the drugs must be given to the neonatal rat. An artificial-rearing procedure will be employed in which pups are implanted with an intragastric cannula an fed a milk formula with the drug added to the milk. A major advantage of this artificial rearing procedure is that it allows the amount of food consumed (and drug consumed) to be controlled by the experimenter. This allows complete control over drug exposure without any concomitant undernutrition or change in maternal behavior. Alcohol and cocaine will be administered both separately and in various combinations to allow us to study the interactive effects of these drugs on development. Many of the studies will focus on clinically- relevant behaviors that are believed to be sensitive to prenatal/neonatal exposure to one of these drugs. This strategy will allow us to focus on the possible interactions when exposure occurs to known quantities of both of these drugs. In addition, tasks that have not yet been examined following neonatal ethanol or cocaine exposure have been proposed. The results from these studies should provide important information regarding the interactive effects of neonatal exposure to ethanol and cocaine as well as providing new information regarding the effects of neonatal exposure to each of these drugs independently. These findings should also provide a basis for further direction regarding the mechanisms for these drugs effects during early development. BARRON S UNIVERSITY OF KENTUCKY, KASTLE HALL, RM 208, LEXINGTON, KY 40506-0044 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health juvenile animal ethanol laboratory rat drug interaction growth /development neurochemistry longitudinal animal study embryo /fetus toxicology oral behavior cognition problem solving learning social behavior dopamine receptor cocaine gait CRISP RPROJ KENTUCKY G CRISP/99/AA09723-03 CRISP/99/AA09723-03 199904 eng GENETIC DISSECTION OF ALCOHOL METABOLIC PATHWAYS Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) Vitamin A (retinol) must be metabolized to an active retinoid ligand in order to fulfill all of its roles in vertebrate development. During retinoid signaling retinol is first converted to retinal followed by conversion of retinal to the active ligand retinoic acid which modulates nuclear retinoic acid receptors. The alcohol dehydrogenase (ADH) enzyme family may function in the metabolism of retinol, the alcohol form of vitamin A, as well as ethanol metabolism. Some members of the ADH family prefer retinol as a substrate over ethanol, and the ability to oxidize retinol is competitively inhibited by intoxicating levels of ethanol. Likewise, there exists an aldehyde dehydrogenase (ALDH) family containing members preferring retinal, the aldehyde form of vitamin A, as a substrate over acetaldehyde. The spatiotemporal expression patterns of mouse ADHs and ALDHs overlap, suggesting that these enzymes may cooperate to upregulate retinoic acid synthesis during development. Retinoic acid synthesis may be decreased by excess ethanol consumption due to the ability of ethanol to act as a competitive inhibitor of ADH-catalyzed retinol oxidation. This suggests a mechanism whereby ethanol damage may occur during alcohol abuse. Treatment of mouse embryos at the neurulation stage with an intoxicating amount of ethanol leads to a reduction in retinoic acid levels, thus suggesting ADH participates in the retinoic acid synthetic pathway. This may be a contributing factor in fetal alcohol syndrome, characterized by malformations of neural and craniofacial tissues known to require retinoic acid for proper development. The in vitro properties and gene expression profiles of the ADH and ALDH enzyme families suggest a role in both alcohol and retinol metabolism, but there is a need for genetic loss-of-function studies in mice to address their true physiological roles. The mouse ADH gene family consists of three classes (ADH-I, ADH-III, and ADH-IV), with only ADH-I and ADH-IV known to oxidize retinol in vitro. The extent of the mouse ALDH gene family is unknown, but ALDH-I has been shown to oxidize retinal in vitro and has an expression pattern overlapping that of ADH-I and ADH-IV. Mutational analysis of all three mouse ADHs and ALDH-I is proposed here. Goals: (1) Complete the genetic analysis of ADH now in progress by preparing mice carrying knockout mutations of ADH-I, ADH-III, and ADH-IV, as well as mice carrying mutations of multiple ADHs since redundancy of function is suspected. (2) Analyze the phenotype of mice carrying mutations in single or multiple ADH genes for morphological defects during development and adulthood, for the ability to metabolize ethanol and retinol, and for the ability to survive and reproduce during vitamin A deficiency. (3) Prepare and ALDH-I knockout mouse plus mice mutated for one or more ADHs and ALDH-I, then analyze their phenotype as above. DUESTER GL THE BURNHAM INSTITUTE, 10901 NORTH TORREY PINES ROAD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 92037 Crisp Data Base National Institutes Of Health ethanol laboratory mouse enzyme mechanism enzyme substrate isozyme developmental genetics gene expression disease model nutrition related tag vitamin A deficiency alcohol dehydrogenase aldehyde dehydrogenase embryo /fetus toxicology fetal alcohol syndrome toxin metabolism retinoid retinoate vitamin metabolism gene targeting vitamin receptor CRISP RPROJ CALIFORNIA G CRISP/99/AA09731-04 CRISP/99/AA09731-04 199904 eng ALCOHOL CONSUMPTION AND LUNG CANCER Research FREUDENHEIM J RESEARCH INSTITUTE ON ADDICTIO, 1021 MAIN STREET, BUFFALO, NY 14203 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 14203 Crisp Data Base National Institutes Of Health alcoholic beverage consumption blood chemistry cholesterol passive smoking case history human subject interview lung neoplasm neoplasm /cancer epidemiology cancer risk diet dietary lipid nutrition related tag occupational hazard antioxidant life style sex difference racial /ethnic difference socioeconomics carotenoid smoking behavioral /social science research tag substance abuse epidemiology CRISP RPROJ NEW YORK G CRISP/99/AA09802-050003 CRISP/99/AA09802-050003 199904 eng ETHANOL AND HOST DEFENSE RELATED HEPATIC FUNCTION Research SPITZER JJ LSU MEDICAL CENTER, 1901 PERDIDO STREET, NEW ORLEANS, LA 70112 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 70112 Crisp Data Base National Institutes Of Health ethanol laboratory rat blood toxicology glucose metabolism glucose transport phagocytosis enzyme induction /repression gene expression bactericidal immunity cytokine immunosuppressive western blotting endotoxin liver cell liver metabolism superoxide glucose 6 phosphate dehydrogenase insulin glutathione hexokinase lipopolysaccharide insulin receptor tissue /cell culture nitric oxide oxidative stress CRISP RPROJ LOUISIANA G CRISP/99/AA09803-050001 CRISP/99/AA09803-050001 199904 eng PILOT--EFFECT OF ALCOHOL AND TRANSLOCATION ON PULMONARY DEFENSES Research MASON CM LSU MEDICAL CENTER, 1901 PERDIDO STREET, NEW ORLEANS, LA 70112 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 70112 Crisp Data Base National Institutes Of Health ethanol laboratory rat antibiotic Escherichia coli gram negative bacteria Staphylococcus aureus Pseudomonas aeruginosa lymph node neutrophil blood toxicology mesentery phagocytosis gastrointestinal absorption /transport gastrointestinal epithelium histopathology bactericidal immunity tumor necrosis factor alpha lung lavage bacterial pneumonia alveolar macrophage CRISP RPROJ LOUISIANA G CRISP/99/AA09803-050004 CRISP/99/AA09803-050004 199904 eng PILOT--EFFECT OF ALCOHOL AND SEPSIS ON CARDIAC RATE Research MC DONOUGH KH LSU MEDICAL CENTER, 1901 PERDIDO STREET, NEW ORLEANS, LA 70112 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 70112 Crisp Data Base National Institutes Of Health ethanol blood toxicology blood glucose blood pressure reperfusion arrhythmia myocardial ischemia /hypoxia electrocardiography heart rate heart metabolism endotoxin blood lipid peroxidation isoproterenol high performance liquid chromatography physiologic stressor CRISP RPROJ LOUISIANA G CRISP/99/AA09803-050005 CRISP/99/AA09803-050005 199904 eng PILOT--EFFECT OF ETHANOL ON THE INTESTINAL IMMUNE SYSTEM Research JERRELLS TR LSU MEDICAL CENTER, 1901 PERDIDO STREET, NEW ORLEANS, LA 70112 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 70112 Crisp Data Base National Institutes Of Health ethanol Salmonella typhimurium macrophage helper T lymphocyte blood toxicology mesentery phagocytosis flow cytometry polymerase chain reaction gastrointestinal infection intestinal mucosa bactericidal immunity cytokine tumor necrosis factor alpha immunosuppressive lipopolysaccharide cell population study T cell receptor gut associated lymphoid tissue lymphocyte proliferation nitric oxide northern blotting CRISP RPROJ LOUISIANA G CRISP/99/AA09803-050007 CRISP/99/AA09803-050007 199904 eng THERAPEUTIC MOTOR TRAINING AND FETAL ALCOHOL EFFECTS Research RPROJ Human and animal studies have shown that exposure to moderate peak blood levels of ethanol during development results in permanent morphological changes in the cerebellum and cerebrum and in motor and other behavioral deficits. In the rat "motor learning," as opposed to simple motor activity, results in morphologically-detectable increases in synapse numbers, vasculature and glial cell processes in the cerebellar cortex and improved performance in motor tasks. This project is assessing the therapeutic effects of exposure to a program of "rehabilitative motor training upon 1) the behavioral symptoms of brain dysfunction in tests of motor skill and 2) synaptic, glial, and vascular morphology in adult rats following alcohol exposure on days 4-9 postnatal. This period of exposure corresponds to third trimester fetal alcohol exposure in humans. We have found that the rehabilitation procedure, when administered in adulthood, 1) improves motor performance and 2) increases the number of synapses per Purkinje cell in the cerebellar paramedian lobule (PML) of the alcohol exposed rats. A motor control procedure, walking in a closed alleyway, run only for the behavioral study, had only small effects on motor performance. Proposed goals for the coming period are to 1) continue study of material from these animals exposed to rehabilitation in adulthood, to understand the potentially distributed nature of the rehabilitation process and 2) investigate whether intervention at an earlier age, immediately after weaning, will have greater impact, an issue of great relevance with regard to clinical applications. Data collection from the adult rehabilitation rats will focus upon the lateral cerebellar nucleus (the primary recipient of PML output, which is damaged by neonatal alcohol treatment and does not respond to this experience in normal animals), motor cerebral cortex (a separate region that may well be involved in compensation for cerebellar damage) and dorsal hippocampus (a non-motor structure), as well as upon Golgi impregnation studies of PML neuronal dendritic fields (initially cerebellar Purkinje and stellate neurons). Initial morphological studies of the postweaning rehabilitation rats will focus upon cerebellar PML and vermis lobule 1. Behavioral evaluation will involve locomotion on a rotating rod, climbing of ropes and a parallel bar walking test, motor skill tests upon which performance has been shown to be sensitive to alcohol exposure during brain development, as well as spontaneous alternation and reinforced conditional spatial delayed alternation as tests of hippocampal functioning. Morphological assessment will involve the use of state-of- the-art stereological methods, including the optical disector, to determine cell loss following alcohol exposure, the double disector to assess synapse number, and appropriate method for vasculature and glia. The overall goals of the study are 1) to obtain the most accurate possible view of the effects of postnatal alcohol exposure upon brain organization and behavioral performance and 2) to obtain a similarly accurate view of the effects of the program of motor skill intervention training upon these same measures. The long-range goal is to assess the potential therapeutic value of intervention programs in human offspring suffering from alcohol-related neurodevelopmental disorder. GREENOUGH WT UNIVERSITY OF ILLINOIS, 405 N MATHEWS AVENUE, URBANA, IL 61801 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 61801 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat cerebral cortex training physical therapy neural degeneration embryo /fetus toxicology fetal alcohol syndrome behavior test performance psychomotor function psychopharmacology human therapy evaluation functional ability behavioral /social science research tag CRISP RPROJ ILLINOIS G CRISP/99/AA09838-05 CRISP/99/AA09838-05 199904 eng FETAL ALCOHOL EFFECTS ON IMMUNE DEVELOPMENT Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) The teratogenic potential of alcohol is well known and the offspring of mothers who abuse alcohol have been shown to have a broad spectrum of anomalies that have been termed alcohol related birth defects (ARBD). The manifestation of ARB span from reduced birth weight to the unique constellation of features termed fetal alcohol syndrome (FAS). FAS children are the offspring of chronic alcoholic women. However, clinical studies have shown that even moderate drinking during pregnancy can affect fetal development suggesting that alcohol abuse may be one of the leading causes of birth defects. Most studies of the teratogenic effects of alcohol have focused on the morphological and neurological features of the affected infants. However, recent studies have shown ARBD children to be at high risk of having some degree of immune deficiency and consequent increased incidence and severity of infection. Since the immune system is not fully developed at birth the infant's ability to cope with infection is fragile. Therefore, it is important to identify environmental factors that might delay normal immune development and put infants at risk. Recent studies from this laboratory using a murine model of ARBD have shown that in utero exposure to alcohol caused a retarded development of B lymphocytes in fetal liver and neonatal bone marrow and spleen. Phenotypic analysis of developmental intermediates in the B lineage showed several to be affected by in utero alcohol exposure. In particular, the investigator's observed that a previously unreported B cell precursor was decreased in neonatal marrow and spleens of animals exposed in utero to alcohol. In this proposal the applicants will use a model consisting of fetal alcohol exposed and pair-fed and chow-fed control animals to assess the effects of alcohol on B lymphopoiesis during fetal and neonatal life. They will use multiparameter flow cytometry to ascertain the absolute number of B cell intermediates and the phenotype of these cells within the fetal liver and neonatal bone marrow and spleen. The developmental potential of the B cell intermediates will be determined by sorting B-lineage cells and other hematopoietic precursors and stem cells and using clonal analysis to determine if alcohol exposure alters the frequency of cells that are capable of differentiation. They will also follow fetal alcohol exposed animals in to adulthood to determine if the exposure affected the function of the humoral immune system and the longevity of the effect. WOLCOTT RM LSU MED CTR SCH OF MED, PO BOX 33932, SHREVEPORT, LA 71130-3932 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory mouse B lymphocyte cell differentiation flow cytometry teratogen mammalian embryology humoral immunity immunosuppression disease model fluorescence microscopy cell population study embryo /fetus toxicology fetal alcohol syndrome immune system phenotype embryonic stem cell developmental immunology lymphopoiesis CRISP RPROJ LOUISIANA G CRISP/99/AA09876-04A1 CRISP/99/AA09876-04A1 199904 eng LIFE LONG EFFECTS OF PRENATAL ALCOHOL EXPOSURE Research RPROJ DESCRIPTION: Most of the research involving the Fetal Alcohol Syndrome (FAS) and alcohol-related birth defects (ARBD) has been conducted on young children and young animals. Research on long-term morbidities associated with prenatal alcohol exposure in humans will not be forthcoming for many years. We may be able to anticipate such long-term morbidities through animal studies, but as yet there are few relevant studies from which one can make such inferences. Those which have been conducted suggest that animals prenatally exposed to alcohol show a changing pattern of impairment across their lifespan, particularly with respect to auditory and neurobehavioral functioning. We propose an in-depth evaluation of select morbidities across the lifespan in an animal model of prenatal alcohol exposure. To this end we will study the effects of prenatal alcohol exposure on the brainstem auditory evoked potential, histologies of select auditory structures, and longevity. The subjects will be the offspring of female rats who have received liquid diets containing either 0 %, 17.5 % or 35 % ethanol-derived calories (EDC) from gestation days 7 to parturition. Animals receiving the 0% and 17.5% EDC will be pair-fed to those receiving the 35% EDC dose. An untreated ad-lib fed control group will also be included. All litters will be removed at birth and placed with non-treated surrogate dams to eliminate possible confounding between gestational alcohol administration and postnatal residual effects on maternal behavior or lactational performance. The offspring will be evaluated cross-sectionally at different stages of their lifespan. This study will advance our understanding about the long-term effects of in utero alcohol exposure on sensory and neural functioning and help clinicians to anticipate the special needs of aging individuals with FAS. CHURCH MW WAYNE STATE UNIVERSITY, 275 E HANCOCK, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health longevity ethanol laboratory rat auditory pathway brain electrical activity hearing auditory feedback hearing test electrophysiology evoked potential histology sound frequency longitudinal animal study embryo /fetus toxicology fetal alcohol syndrome neuropsychology CRISP RPROJ MICHIGAN G CRISP/99/AA09991-02 CRISP/99/AA09991-02 199904 eng GENES THAT REGULATE ETHANOL RESPONSES IN DROSOPHILA Research RPROJ A growing body of evidence is emerging from studies in animal and cellular model systems that indicates that the effects of ethanol on a variety of cellular functions are mediated by changes in specific proteins. In these systems, however, it is difficult to establish whether these proteins directly or indirectly mediate ethanol-induced changes in nervous system function. It is therefore important to establish a simple model system for alcoholism that is easily accessible to genetic and molecular analyses. We have recently initiated studies using the fruit fly Drosophila as a potential model system for.alcoholism. Preliminary studies have shown that flies display many of the behaviors observed in humans after both acute and chronic exposure to ethanol. Flies display signs of hyperactivity and incoordination, followed by sedation and anesthesia. In addition, flies develop tolerance after single or multiple exposures to ethanol. We propose to generate and isolate Drosophila mutants that have altered responses to ethanol. For this purpose an "inebriometer" has been constructed, which allows the separation of flies with different sensitivity to ethanol. A genetic screen for mutants with increased or decreased sensitivity to an acute ethanol exposure will be carried out. In addition, mutant flies that fail to become tolerant or become excessively tolerant to ethanol will be isolated. Several secondary behavioral assays will be carried out to determine whether the phenotype is ethanol-specific and whether the focus of the mutation is in the central nervous system. Mutations will be mapped to specific chromosomal locations. Some of the genes affected will be isolated and sequenced. These genes will serve as tools to study molecular and biochemical mechanisms underlying ethanol-induced responses, and may in the future serve as genetic markers for alcoholism or targets for potential therapeutic intervention. HEBERLEIN UA ERNEST GALLO CLINIC RESEARCH C, BLDG 1 RM 101/1001 POTRERO AVE, SAN FRANCISCO, CA 94110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94110 Crisp Data Base National Institutes Of Health ethanol Drosophilidae drug tolerance chromosome walking molecular cloning genetic regulation genotype mutagen gene mutation mutant alternatives to animals in research central nervous system nucleic acid sequence tissue /cell culture phenotype CRISP RPROJ CALIFORNIA G CRISP/99/AA10035-04 CRISP/99/AA10035-04 199904 eng ETHANOL, NEURAL DIFFERENTIATION, AND PROTEIN KINASE C Research RPROJ Chronic ethanol exposure can damage the nervous system, in part, by altering the growth of neural processes (neurites). In some regions of the brain, ethanol increases neurite growth. This could harm the nervous system by slowing conduction down dendrites to the cell body, and by disturbing the balanced development and organization of synapses. PC12 cells, which form neurites in response to nerve growth factor (NGF), have been used to study mechanisms involved in neurite outgrowth. Ethanol enhances NGF-induced neurite outgrowth by a process that is dependent on protein kinase C (PKC). Chronic ethanol exposure increases levels of two PKC isozymes, delta and epsilon, and increases PKC activity in PC12 cells. Studies outlined in this proposal will examine whether these PKC isozymes mediate enhancement of neurite outgrowth by ethanol in PC12 cells and primary cultures of cerebellar Purkinje cells which are known to respond to NGF and ethanol with increased neurite growth. In addition to increasing NGF-induced neurite outgrowth, ethanol also enhances NGF- induced stimulation of mitogen-activated protein kinases, suggesting that ethanol regulates NGF signal transduction. Studies are planned to investigate whether ethanol enhances NGF signaling by promoting the activity of the GTP-binding protein Ras or the kinase Raf-1, which are critical for NGF-induced neural differentiation. Methods to be used include PKC assay in cell homogenates and permeabilized cells, MAP kinase assays, assays for Ras-GTP formation, Ras GTPase activity, and Raf-1 activity, Western analyses, and stable transfection of PC12 cells to over- express PKC isozymes and to express antisense RNA and dominant-negative mutants. In addition, oligodeoxynucleotides and new PKC antagonists will be studied for isozyme-specific activity in PC12 cells. These will then be used to inhibit specific PKC isozymes in Purkinje cell cultures. These studies will provide new information about a biochemical mechanism that may be important in the pathogenesis of brain damage in alcoholics and of neurologic abnormalities in children born to alcoholic mothers. In addition, these studies will identify and test PKC isozyme-selective agents that may eventually prove useful as drugs in the treatment of ethanol neurotoxicity. MESSING RO ERNEST GALLO CLINIC C, 1001 POTRERO AVENUE, BLDG 1, SAN FRANCISCO, CA 94110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94110 Crisp Data Base National Institutes Of Health ethanol biological signal transduction cerebellar Purkinje cell cell type isozyme transfection western blotting neurogenesis neurotoxin antisense nucleic acid protein kinase C guanine nucleotide binding protein tissue /cell culture enzyme activity mitogen activated protein kinase CRISP RPROJ CALIFORNIA G CRISP/99/AA10036-04 CRISP/99/AA10036-04 199904 eng PRIMATE MODEL OF PRENATAL LOW-LEVEL ALCOHOL EXPOSURE Research RPROJ This research will investigate whether low-level ethanol and/or psychological stress exposure constitutes a danger to the developing fetus and what long-term effects emerge in and/or extend to adolescence. This question is difficult, if not impossible, to resolve in human studies. Our previous research, using a monkey model, has documented that prenatal stress and/or low-level ethanol exposure alter neonatal and infant development, as reflected by impaired neuromotor coordination, diminished attention span, and cognitive impairments. Evidence for enhanced stress reactivity in the juvenile stage suggests that problems also persist later in life. The proposed study will replicate and extend our nonhuman primate model of prenatal exposure to low-level ethanol and/or psychological stress during pregnancy. Specific Aim 1 will be accomplished by examining the behavior and physiology of juvenile rhesus monkeys exposed in utero to ethanol, psychological stress, or ethanol and stress. We will monitor growth patterns, social and cognitive development and sires reactivity in these monkeys. Cognitive tests that identify damage to the hippocampal formation, a brain region particularly susceptible to prenatal ethanol and/or stress exposure will be used. Specific Aim 2 will evaluate patterns of ethanol consumption in control and prenatally-exposed adolescent rhesus monkey offspring by examining physiologic responses to fixed quantities of ethanol as well as amount consumed using a voluntary 2-bottle choice paradigm. Specific Aim 3 will involve the generation of an additional cohort of low-level ethanol exposed infants to determine the effects of gestational timing of exposure to ethanol. Because of the ability to ascertain timing and level of ethanol exposure during pregnancy, to separate the effects of ethanol from other life-style factors, and to explore possible mediating factors, these results will provide unique information on the long-term impact of early ethanol and/or stress exposure on offspring outcome.| SCHNEIDER ML UNIVERSITY OF WISCONSIN, 1300 UNIVERSITY AVENUE, MADISON, WI 53706-1532 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol Macaca mulatta hippocampus adolescence (12-18) dosage growth /development model design /development disease model neurogenesis neuroanatomy neurophysiology longitudinal human study embryo /fetus toxicology prenatal stress fetal alcohol syndrome cognition social behavior psychological stressor behavioral /social science research tag CRISP RPROJ WISCONSIN G CRISP/99/AA10079-04 CRISP/99/AA10079-04 199904 eng ALCOHOL AND BRAIN DEVELOPMENT Research RPROJ The overall purpose of this proposal is to determine whether there are critical periods during which the developing brain is expressly vulnerable to alcohol exposure. The first hypothesis to be tested is that the developing brain is vulnerable to alcohol-induced damage as a consequence of the timing of the alcohol exposure; this is the hypothesis of temporal vulnerability. A second major hypothesis to be addressed is that specific regions of the fetal brain are differentially vulnerable to alcohol exposure; this is the hypothesis of regional vulnerability. The proposal is divided into two series of experiments. The objective of the first series will be document dose-dependent fetal alcohol-induced alterations in regional volume and neuronal loss in selected brain regions across major periods of prenatal development in the rat: the first trimester equivalent (embryonic days E1-E10), the second trimester equivalent (E11-E21), and the combined first and second trimesters equivalent (E1-E21). once we have identified the temporal risks to the fetal brain from alcohol exposure on a trimester basis, we will narrow the focus of our temporal vulnerability hypothesis. The second series of experiments will address a third hypothesis that is closely related to the first one; that alcohol exposure interferes with neurogenesis (i.e., the specific period when the neurons are generated) preventing the acquisition of a normal complement of neurons at this particular stage of their development. All of the hypotheses in this proposal are intimately linked to an important postulate: the severity of a deficit is a function of the peak blood alcohol concentration (BAC) to which the fetus is exposed. Therefore, to optimize control over the peak BACs, a gastric intubation technique will be used to administer daily doses of alcohol that will produce low, medium, or high BACs. In order to accomplish the goals stated above, it is necessary to use dependent variables that are appropriate for comparisons across all brain regions. Thus, state-of-the-art three-dimensional stereological methods will be used to determine any alterations in regional volumes and in neuronal numbers in seven important brain regions including the cerebellum, hippocampus, locus coeruleus, substantia nigra, ventrolateral thalamus, entorhinal cortex, and the septum. Taken together, the results from these experiments will facilitate the formulation of tentative conclusions regarding temporal vulnerability of developing neurons to alcohol exposure. From an experimental point of view, information concerning temporal windows and regional differences in vulnerability of the developing brain to alcohol exposure will contribute significantly to the formulation of research strategies directed toward discovering the mechanism(s) underlying fetal alcohol-induced brain damage. Equally important from a clinical perspective, knowledge of critical periods during development, when the brain is especiall WEST JR TEXAS A&M UNIVERSITY, COLLEGE STATION, TX 77843-111 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat dosage morphology neurogenesis neuron neurochemistry cell population study gestational age embryo /fetus toxicology fetal alcohol syndrome intubation CRISP RPROJ TEXAS G CRISP/99/AA10090-05 CRISP/99/AA10090-05 199904 eng PRENATAL ALCOHOL EXPOSURE--CONSEQUENCES IN ADOLESCENCE Research RPROJ The proposed study will follow into early adolescence, a cohort of children affected by prenatal alcohol exposure due to maternal drinking. While the negative consequences of alcohol exposure for the infant and the young child are fairly well established, there is little systematic data about long-term effects. Clinical studies of adolescents and adults with alcohol syndrome (FAS) or alcohol effects (FAE) suggest very negative consequences for these groups, including deficits in neurocognitive functioning, academic performance, social and emotional functioning and a greatly increased probability of alcohol and drug abuse. While such consequences do exist, it is not clear what are the relative contributions of direct neurological damage, familial factors and secondary effects (e.g., mental retardation). This study will examine four kinds of outcomes (neurocognitive functioning, social functioning, alcohol and drug use and physical status) in 14 year old, low income, predominantly African American children (N=270) and social and physical functioning and drug and alcohol use in their caregivers. Four groups will be employed. Three groups will be drawn from a longitudinal cohort first identified in the prenatal period. These are: 1) Exposed/affected (FAS/FAE); 2) Exposed- nondysmorphic; and 3) Nonexposed, social economic status (SES) controls. In addition, a fourth group ("Special Education Contrast") will be recruited through the Fulton County Board of Education. This group will be matched for race SES and cognitive and educational status with the children in the FAS/FAE group to control for the effects of intellectual limitations on the outcome variables. Outcome variables will include standard cognitive measures, specific neuropsychological tests, measures of social competence and emotional functioning, and measures of alcohol and drug use. In addition, assessment will be made of environmental factors including maternal attitudes and functioning and environmental stress including exposure to familial and community violence. It is anticipated that adolescents with significant alcohol pathology will show deficits relative to contrast groups in cognitive, emotional and social functioning and that negative environmental factors will exacerbate these deficits. It is also hypothesized that adolescent with alcohol effects will show a specific pattern of functioning which is different from that seen in the more heterogeneous Special Education contrast group. COLES CD EMORY UNIVERSITY, 1256 BRIARCLIFF RD NE, ATLANTA, GA 30306 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 30306 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse child physical development child psychology adolescence (12-18) disease /disorder proneness /risk family structure /dynamics human subject longitudinal human study embryo /fetus toxicology prenatal stress fetal alcohol syndrome cognition neuropsychology social behavior African American behavioral /social science research tag CRISP RPROJ GEORGIA G CRISP/99/AA10108-04 CRISP/99/AA10108-04 199904 eng TNF ALPHA AND RECOVERY FROM ALCOHOLIC LIVER INJURY Research RPROJ DESCRIPTION: (Adapted from Investigator's Abstract) Ethanol produces liver disease by injuring the liver and by impairing the regenerative response. The investigator's goal is to identify mechanisms responsible for ethanol's anti-regenerative actions so that treatments can be designed to restore regeneration. Evidence obtained during the last grant indicates that tumor necrosis factor alpha (TNF) (an endotoxin-inducible cytokine involved in alcoholic liver injury) is a key mitogenic factor during liver regeneration. Ethanol-treated animals require TNF for liver regeneration after partial hepatectomy (PH), exhibiting almost no induction of hepatocyte DNA synthesis when treated with agents that neutralize TNF. Preliminary data suggest that specific TNF-dependent proliferative events are inhibited by chronic ethanol consumption although ethanol increases hepatic expression of TNF mRNA. This discrepancy between TNF expression and biological activity suggests that chronic ethanol exposure decreases biologically active TNF protein (despite increasing TNF mRNA) and/or changes hepatocyte sensitivity to TNF. Since both TNF expression and target cell sensitivity to TNF are regulated by other cytokines, this suggests that chronic ethanol exposure disturbs a network of interactive, endotoxin-inducible cytokines that regulate the regenerative response to liver injury. This project will test the hypothesis that PH permits endotoxemia which triggers a cascade of cytokines that cooperate to regulate hepatocyte proliferation. Chronic exposure to ethanol disturbs this cytokine network, resulting in a change in the hepatocyte phenotype that prevents proliferation despite enhanced local accumulation of TNF. Three specific aims are proposed: 1) To identify the TNF-related cytokines that are induced in the liver after PH, characterize the temporal pattern of their expression, and clarify the role of gut-derived endotoxin (LPS) in PH-induction of these genes; 2) To determine if acute or chronic consumption of ethanol alters expression of any of these cytokines before or after PH, and if so, whether this can be explained by increased portal or systemic endotoxemia; 3) To identify which TNF-regulated events in proliferative signaling are inhibited by ethanol, determine if inhibited proliferation predisposes hepatocytes to TNF toxicity, and assess whether proliferative signaling can be restored by "normalizing" the cytokine network. DIEHL AM JOHNS HOPKINS UNIVERSITY, 720 RUTLAND AVE, BALTIMORE, MD 21205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 21205 Crisp Data Base National Institutes Of Health ethanol laboratory rat chemical stimulation cytokine tumor necrosis factor alpha liver cell liver toxic disorder hepatotoxin liver pharmacology liver regeneration hepatectomy lipopolysaccharide tissue /cell culture toxin metabolism cell proliferation CRISP RPROJ MARYLAND G CRISP/99/AA10154-05 CRISP/99/AA10154-05 199904 eng STRUCTURE-BASED LIGAND DESIGN FOR ALCOHOL DEHYDROGENASE Research RPROJ The overall goal of this project is to identify specific inhibitors and substrates for the human beta, gamma, and sigma alcohol dehydrogenase (ADH) isoenzymes. Dietary and biogenic alcohols and aldehydes are primarily metabolized by the NAD(H) dependent alcohol dehydrogenase isoenzymes within the cell. There has been considerable debate concerning the involvement of alcohol dehydrogenase isoenzymes in the metabolism of ethanol. The primary reason for this controversy is the lack of specific inhibitors or substrates with which to evaluate their activities in vivo or in vitro. We propose to utilize a structure-based approach toward the identification of specific inhibitors and substrates for the human beta, gamma, and sigma ADH isoenzymes. We have recently solved the three-dimensional structures of all three of these ADH isoenzymes. We have finished the structure refinement of the human beta forms and are continuing the refinement of the gamma and sigma ADH structures. We propose to use these structures to identify inhibitors based on their active site characteristics with the recently developed computer programs DOCK and AUTODOCK. The three- dimensional structures of these isoenzymes will be used in docking experiments to identify specific substrates and inhibitors for these important ADH isoenzymes. Our initial studies will characterize the determinants of specific binding to the respective isoenzymes using a variety of in vitro methods, such as steady-state and stopped-flow kinetic measurements, as well as X-ray crystallography of enzyme-inhibitor complexes. A long term goal of this proposal is to develop stably transformed cell lines expressing one or more of these isoenzymes for evaluation of in vivo inhibitors and toxicological properties of potential inhibitors and substrates. These studies should contribute to the basic understanding of the involvement of these human ADH isoenzymes in the overall rate of ethanol metabolism and may identify important intracellular substrates for these ADH isoenzymes. HURLEY TD INDIANA UNIV SCH OF MED, 635 BARNHILL DR, INDIANAPOLIS, IN 46202-5122 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol aldehyde computer processing of laboratory data chemical kinetics X ray crystallography computer program /software oxidoreductase inhibitor enzyme mechanism enzyme structure enzyme substrate enzyme substrate complex isozyme transfection HeLa cell alcohol dehydrogenase crystallization stop flow technique nicotinamide adenine dinucleotide toxin metabolism active site human genetic material tag CRISP RPROJ INDIANA G CRISP/99/AA10399-03 CRISP/99/AA10399-03 199904 eng ETHANOL AND EXCITATORY AMINO ACID FUNCTION IN BRAIN Research RPROJ Prenatal ethanol exposure in the developing human CNS can result in severe mental retardation linked to fetal alcohol syndrome or associated attention disorders and hyperactivity. There is an enhanced sensitivity to the teratogenic effects of ethanol during the third trimester which correlates with the onset of the brain growth spurt period in humans. Animal studies have confirmed that ethanol exposure during the brain growth spurt induces severe neuronal loss. The cerebellar region of the developing rat CNS is highly sensitive to ethanol, with decreases in the number of granule and Purkinje cells and cerebellar size, changes in neuronal excitability, and delayed neuronal differentiation and synapse formation all consequences of ethanol treatment. Although the mechanisms underlying ethanol action in the developing CNS remain to be established, several transmembrane signaling systems in neuronal membranes are known to be affected by ethanol exposure. Recent evidence has indicated that excitatory amino acid (EAA) receptors, particularly the NMDA receptor, may be especially sensitive neurochemical targets for the pharmacological properties of ethanol in brain. In addition, many EAA receptor second messenger responses display a transient developmental expression that peaks during the brain spurt, and coincides with a period of heightened sensitivity to the teratogenic effects of ethanol. This has generated great interest in the potential of EAA receptors as potential targets for some of the neurotoxic properties of ethanol in the developing CNS. In this project we will examine the mechanism(s) by which ethanol interferes with excitatory amino acid (EAA) neurotransmission. An increase in cytosolic free calcium ([Ca2+]/i) is central to many of the physiological and pathophysiological properties of EAAs in the developing central nervous system (CNS), and can be initiated by activation of both ionotropic and metabotropic EAA receptors. Ionotropic receptors can stimulate Ca2+ influx directly via receptor-operated ion channels or indirectly through voltage-sensitive Ca2+ channels (VSCC), whereas metabotropic receptors are coupled to a G-protein-regulated phospholipase C (PLC). This enzyme generates the second messengers inositol 1,4,5- trisphosphate (InsP/3) and diacylglycerol (DAG), resulting mobilization of [Ca2+]/i and activation of protein kinase C (PKC), respectively. We will use the cerebellar granule cell as an in vitro model system to determine the effects of acute ethanol treatment on the expression of these EAA transmembrane signaling systems and their physiological functions in developing neurons of the cerebellum. ROONEY TA THOMAS JEFFERSON UNIVERSITY, 1020 LOCUST ST, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health ethanol laboratory rat biological signal transduction second messenger membrane permeability calcium flux single cell analysis divalent cation neurogenesis neuron neurophysiology neural transmission neurotoxin protein kinase C calcium channel receptor coupling glutamate receptor inositol phosphate tissue /cell culture excitatory aminoacid phosphoprotein phosphatase granule cell CRISP RPROJ PENNSYLVANIA G CRISP/99/AA10413-04 CRISP/99/AA10413-04 199904 eng MOLECULAR MECHANISMS UNDERLYING FETAL ALCOHOL SYNDROME Research RPROJ APPLICANT'S ABSTRACT: The long-term goal of the proposed research is to determine the molecular mechanisms by which ethanol produces teratogenic effects on the central nervous system. Specific features of ethanol-induced CNS damage are remarkably consistent among clinical and animal studies, suggesting a common underlying mechanism(s) for these defects. A common characteristic of the damage observed in ethanol-exposed brains is neuronal agenesis and defects in cell migration. Several kinds of studies indicate that at least a subset of ethanol-induced CNS damage may be due to ethanol effects on thyroid hormone function. Independently, studies have indicated that ethanol effects on CNS development may be mediated by blocking the de novo synthesis of retinoic acid. Considering that thyroid hormone receptors are ligand-dependent transcription factors that are known to form functional heterodimers with receptors for retinoic acid, the working hypothesis is that ethanol interferes with the interaction of these two signalling systems at precise times during CNS development. This hypothesis forms the basis for the prediction that brain areas which express both thyroid hormone receptors and receptors for retinoic acid are those that are the most sensitive to the deleterious effects of prenatal ethanol exposure. The first specific aim of the proposed work is to determine whether these receptors are co-expressed by neurons of the cortex and hippocampus during the developmental periods they are known to be sensitive to the deleterious effects of ethanol. To accomplish this, we will use a sensitive dual-label in situ hybridization procedure. Next, we will determine whether alcohol exposure can affect the expression of these receptors in cortex and hippocampus. We will use a model of acute ethanol administration to pregnant females so that we can determine the temporal relationship between the effect of ethanol on CNS damage and alterations in the expression of TRs and RAR/RXRs within known windows of vulnerability to the deleterious effects of ethanol. We will evaluate three doses of ethanol and will control for possible nutritional effects using a "pair-feeding" paradigm and for possible stress effects by including an untreated group. In addition, we will confirm our findings using a chronic ethanol treatment model for which substantial information exists concerning cortical development. We will also confirm that ethanol effects on expression of TR and RAR/RXR mRNAS affect thyroid hormone and retinoic binding. Finally, we will also determine whether T3 and/or RA can ameliorate the effects of ethanol on CNS damage and on TR and RAR/RXR expression. The proposed experiments will potentially identify a mechanism of FAS. The results could explain why clinical and experimental FAS brains exhibit neuronal agenesis and defective neuronal migration. and why there are windows of vulnerability to the deleterious effects of ethanol. These studies will also leave clear implications to problems of FAS, and these issues can be effectively explored based on the initial, proposed studies. ZOELLER RT UNIVERSITY OF MASSACHUSETTS, MORRILL SCIENCE CENTER, AMHERST, MA 01003-5810 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health laboratory rat polymerase chain reaction teratogen radioimmunoassay statistics /biometry central nervous system disorder developmental neurobiology in situ hybridization fetal alcohol syndrome retinoid binding protein autoradiography receptor expression hormone receptor thyroid hormone retinoid RNase protection assay CRISP RPROJ MASSACHUSETTS G CRISP/99/AA10418-03 CRISP/99/AA10418-03 199904 eng PROTEIN METABOLISM AND NUTRITION IN ALCOHOLIC CIRRHOSIS Research RPROJ In alcoholic cirrhosis, malnutrition is a ubiquitous finding which results from, as yet, incompletely defined abnormalities in protein and energy metabolism. Because this malnutrition is an independent and unfavorable risk factor for both long term survival and successful liver transplantation in patients with this disease, empiric nutritional therapies have been instituted without sound scientific rationale and with only marginal benefits obtained. Therefore, the major objectives of this proposal are twofold. The first is to study the normal response of protein metabolism during feeding and how the presence of cirrhosis effects that response. The second is to further delineate the causes of altered protein metabolism in alcoholic cirrhosis; while testing specific nutritional and pharmacologic therapies in vivo to correct these abnormalities. Using a combination of stable and radioactive isotope tracers along with kinetic modeling, the effects of feeding, beta-adrenergic blockade, exercise, insulin resistance and endotoxemia on phenylalanine, leucine, leucine's ketoacid (keto-isocaproic acid), and glycine kinetics will be examined. Based on the kinetics of these amino and keto acids, estimates of whole body protein degradation and hepatic synthesis will be made. Furthermore by measuring blood flow and arterial-venous gradients of amino acids across the human forearm, protein turnover in the skeletal muscle compartment (based on the human forearm model) will be calculated and compared to whole body protein turnover. The need to use protein for energy as an alternative to glucose in these patients with 'accelerated starvation' will also be assessed by measuring leucine oxidation and gluconeogenesis using a novel new technique based on the incorporation of hydrogen molecules of water into different carbons of glucose. In addition, the efficacy of specific nutritional therapies designed to improve protein metabolism by decreasing endotoxemia (glutamine supplements), by increasing protein while decreasing urea synthesis (BCKA supplements), and by improving insulin sensitivity and nitrogen balance (complex carbohydrate, low fat diet) will be studied. In addition, the importance of the route (intestinal vs intravenous) of nutrient administration in determining postprandial protein metabolism will be investigated. Finally, absolute rates of protein turnover will be normalized for body cell mass (based on intracellular water determinations); thereby allowing for the first time a valid comparison of these processes between normal controls and patients with cirrhosis; a disease associated with abnormalities in extracellular compartmentation. It is anticipated that by measuring outcomes from specific nutritional interventions designed to correct abnormalities in protein metabolism, the nutritional management of alcoholic cirrhosis will be optimized and its prognosis improved. MC CULLOUGH AJ METROHEALTH MEDICAL CENTER, 2500 METROHEALTH DRIVE, CLEVELAND, OH 44109-1998 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health aminoacid ketoacid analytical method insulin sensitivity /resistance breath test glutamine prognosis exercise gastrointestinal nutrient absorption gluconeogenesis human subject endotoxin alcoholic liver cirrhosis antiadrenergic agent nitrogen balance tube feeding diet therapy dietary carbohydrate dietary fiber protein metabolism human therapy evaluation CRISP RPROJ OHIO G CRISP/99/AA10445-04 CRISP/99/AA10445-04 199904 eng MECHANISMS OF HEPATIC INJURY IN CHRONIC ALCOHOLICS Research RPROJ The overall objective of this proposal is to elucidate the role of inflammatory neutrophils and activated Kupffer and endothelial cells on the induction of hepatotoxicity in chronic alcoholics, with a view to developing immunotherapeutic and biotechnological methods for the treatment of tissue injury after prolonged alcohol consumption. This proposal is based on the hypothesis that chronic alcohol intoxication regulates the expression of adhesion molecules and leukocyte infiltration into the liver. Such event may also be mediated by the release of chemotractant factors released by hepatocytes, Kupffer and endothelial cells after exposure to ethanol. As a consequence, a wide spectrum of bioactive substances are released that may contribute to the initiation of hepatic injury in susceptible individuals. It is also hypothesized that such events may be exacerbated by endotoxin. Specific aim I will investigate the expression of adhesion molecules, i.e., Beta2-integrins and selectins on neutrophils and their counterreceptors on Kupffer, endothelial cells and hepatocytes. These molecules are important for the sequestration of inflammatory neutrophils into the liver after an alcohol insult. Specific aim 2 will examine the formation of oxygen-derived radicals, cytolytic proteases, proinflammatory cytokines and chemokines in the liver, which are considered to contribute to the induction of tissue injury in chronic alcoholics. Based on the results of specific aims 1 & 2, specific aim 3 examines the hypothesis that by neutralizing the deleterious effects of these metabolites and their sources (e. g. neutrophils and macrophages), hepatic injury will be attenuated or inhibited. This will be achieved by using free radical scavengers, protease inhibitors, monoclonal antibodies against adhesion molecules and liposome encapsulated dichloromethylene diphosphonate (which specifically targets Kupffer cells). This proposal is unique and novel, because it will use modern immunological and biotechnological approaches for the treatment of liver disease in chronic alcoholics. The use of free radical scavengers, liposomes and monoclonal antibodies in the immunotherapy of endotoxemia, cancer, ischemia-reperfusion and immunosuppression is gaining acceptance, and may also be applied to the treatment of tissue injury in the liver and other organs during chronic alcohol intoxication. BAUTISTA AP LOUISIANA STATE UNIV MED CTR, 1901 PERDIDO STREET, NEW ORLEANS, LA 70112-1393 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse transaminase laboratory rat bioassay liposome neutrophil flow cytometry chronic disease /disorder inflammation gene expression histology cytokine chemoattractant immunotherapy monoclonal antibody radioimmunoassay endotoxin liver toxic disorder liver function free radical oxygen diphosphonate cell adhesion molecule enzyme activity chemokine CRISP RPROJ LOUISIANA G CRISP/99/AA10466-03 CRISP/99/AA10466-03 199904 eng CALCIUM, TROPHIC FACTORS, AND ETHANOL NEUROTOXICITY Research RPROJ APPLICANT'S ABSTRACT: Although it is generally conceded that ethanol can be toxic to neurons, it is not known by what mechanisms this toxicity is mediated. The sensitive, reciprocal interactions between calcium and neurotrophic factors, and the importance of each of these with respect to neuronal survival, led the applicants to investigate whether ethanol might produce toxicity through effects on these molecules. Based on a wealth of biochemical studies of ethanol effects on calcium, on preliminary results that ethanol can alter both resting and potassium depolarization-stimulated intracellular calcium, and on more established effects on brain neurotrophic activity, a more detailed investigation is proposed of ethanol effects on intracellular calcium regulation. It is hypothesized that: (1) ethanol may act to disrupt normal calcium homeostasis (resting and/or stimulated); (2) this disruption may (like other treatments that alter [Ca+2]i) affect the expression of neurotrophic factor or receptor genes; (3) ethanol may interfere with the ability of neurotrophic factors to modulate Ca+2i regulation; and (4) neurotrophic factors may be able to offset the effects of ethanol on Ca+2i regulation. These ideas will be tested by measuring Ca+2i in living alcohol- treated neurons, and in situ hybridized neurotrophic factor and receptor mRNAs. Assessment will follow both acute and chronic ethanol treatments, before and after depolarization challenge, in the presence or absence of neurotrophic factors, with a range of ethanol doses, in 3 distinct in vitro systems. The use of 3 culture models offers comparison of different neuronal types which demonstrate different neurotrophic response characteristics, as well as a sequential approximation of in vivo organization. If ethanol is found to disrupt normal Ca+2i metabolism in a manner that is ameliorated by neurotrophic factors, the investigators will test the ability of prolonged neurotrophic factor treatment to reduce the neuropathological consequences of chronic ethanol treatment. WALKER DW UNIVERSITY OF FLORIDA, GAINESVILLE, FL 32610-0244 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat brain septal area hippocampus calcium flux calcium metabolism gene expression pharmacogenetics neurotrophic factor glucose divalent cation neuropharmacology neurotoxin in situ hybridization receptor expression growth factor receptor hypoxia tissue /cell culture organ culture neuroprotectant CRISP RPROJ FLORIDA G CRISP/99/AA10480-04 CRISP/99/AA10480-04 199904 eng ALCOHOL LIVER DISEASE AND S-ADENOSYLMETHIONINE Research RPROJ Chronic ethanol abuse is a serious public health problem which has a significant role in the development of liver injury as well as in enhanced susceptibility to HIV and development of AIDS. However, biochemical mechanisms to explain the role of ethanol in the pathophysiology of hepatic injury or AIDS are not completely understood. Increasing evidence suggests that a systemic deficiency of S-adenosylmethionine (Adomet) has a pivotal role in the pathogenesis of both diseases. This proposal will examine this role of Adomet deficiency and will evaluate exogenous Adomet as a therapy in these diseases. Systemic Adomet deficiency was first identified by us as an acquired metabolic disorder in alcoholic subjects. Similar observations were made in animals administered ethanol rich diets. Adomet deficiency results in depletion of the intracellular antioxidant tripeptide glutathione and patients with alcoholic liver disease and AIDS have subnormal plasma glutathione. We postulate that deficiencies of Adomet and glutathione increase intracellular oxidative stress and activate the redox-sensitive transcription factor NFkappaB which increases production of the inflammatory cytokine tumor necrosis factor alpha(TNF). TNF has been postulated to play a role in the development of alcoholic liver disease, the progression of AIDS, and the clinical complications of both processes. Adomet deficiency also alters membrane fluidity and integrity. Further, we propose that exogenous Adomet attenuates glutathione depletion, oxidative stress, NFkappaB, TNF production, and membrane integrity and fluidity. These objectives will be tested by the following specific objectives: (1) Determine changes in hepatic Adomet concentrations and Adomet synthetase activity in animals administered ethanol rich Tsukamoto-French diet and examine whether exogenous Adomet attenuates this liver injury. (2) Determine whether Adomet deficiency produced in three animal models developed in our laboratory - viz., choline deficient, malnourished, or hypoxic - enhances lipopolysaccharide hepatotoxicity and determine whether Adomet supplementation attenuates this injury. (3) Determine whether Adomet administration improves hepatic functions and attenuates cytokine production and immunocompetence in patients with Adomet deficiency. (4) Examine the mechanisms by which Adomet deficiency may increase TNF production and sensitize cells to TNF cytotoxicity. Our long-term goal is to evaluate the effectiveness of Adomet therapy especially since Adomet may not only act as a glutathione precursor but also improve its mitochondrial transport by normalizing membrane functions. CHAWLA RK UNIVERSITY OF KENTUCKY MED CTR, 800 ROSE STREET, LEXINGTON, K Y 40536-0084 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health laboratory rat clinical trial intravenous administration human subject tumor necrosis factor alpha AIDS therapy HIV infection alcoholic hepatitis hepatotoxin liver disorder chemotherapy S adenosylmethionine diet therapy nutrition related tag choline deficiency malnutrition protein deficiency dietary supplement hypoxia human therapy evaluation clinical research CRISP RPROJ KENTUCKY G CRISP/99/AA10496-03 CRISP/99/AA10496-03 199904 eng NALMEFENE MAINTENANCE TREATMENT OF ALCOHOLISM Research RPROJ This project aims to integrate advances in the field of psychiatry (i.e. continuation/maintenance pharmacotherapy for chronic, recurrent disorders) with alcoholism treatment. Although alcoholism is for many a chronic, recurrent syndrome, most clinical trials of pharmacologic treatments are designed for determining acute efficacy only. The acute efficacy of opiate antagonist treatment of alcoholism has been supported in studies with two simIlar compounds, naltrexone and nalmefene, in double-blind, placebo- controlled, 12-week studies. Subjects who remain in, and benefit from, acute nalmefene treatment are logical candidates for long-term nalmefene treatment, given the extremely high risk of relapse in chronic alcoholism. We propose to admit 184 alcohol dependent patients to 12 weeks of open treatment with fixed daily doses of 80 mg oral nalmefene and weekly coping skills therapy. Projections based on data from our current study indicate 114 patients will meet study criteria for response. Responders to short term open treatment will be randomized to a one year double-blind, placebo-controlled maintenance study to test the safety and efficacy of long-term nalmefene treatment of chronic alcoholism. All study participants will receive individual coping skills cognitive-behavioral therapy in keeping with good clinical practice and in recognition of the psychosocial aspects of chronic alcoholism. Drinking data will be collected from multiple sources, and standardized ratings of functioning, medical and emotional health status, and health-care utilization, will be obtained in a repeated measures design, with a six month post-treatment follow-up. Non responders will be given appropriate treatment referrals and will be paid for completing follow-up interviews. This study will provide vital information about (l) the efficacy of long-term nalmefene treatment in patients who respond to acute treatment; (2) evidence for tolerance to therapeutic effects during long-term treatment; (3) quantification of the costs and benefits of long-term treatment, in terms of side effects, health-related quality of life issues, and health services utilization; (4) whether long-term treatment with opiate antagonists alters the course of chronic alcoholism post treatment; and (5) predictors of acute and long-term outcome for potential use by clinicians to improve the matching of patients with treatments and thereby improve alcoholism treatment outcome. MASON BJ UNIVERSITY OF MIAMI SCH OF MED, 1400 NW 10TH AVENUE, MIAMI, FL 33136 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 33136 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse chemotherapy clinical chemistry clinical trial breath test relapse /recurrence chronic disease /disorder drug administration rate /duration drug adverse effect drug screening /evaluation health care service utilization human subject longitudinal human study coping human therapy evaluation cognitive behavior therapy narcotic antagonist oral administration quality of life combination therapy CRISP RPROJ FLORIDA G CRISP/99/AA10518-04 CRISP/99/AA10518-04 199904 eng MAGNETIC RESONANCE SPECTROSCOPY (MRS) STUDIES OF ALCOHOL INDUCED BRAIN DAMAGE Research RPROJ DESCRIPTION: Chronic alcohol depended patients have a significant reduction in brain volume and concomitant neurobehavioral deficits which may recover during abstinence. However, the pathophysiology of these well-documented findings remains to be elucidated. Using proton magnetic resonance spectroscopy [MRS], which allows noninvasive quantitation in vivo of brain metabolites within anatomically localized volumes of interest [VOI], we have found an increase with duration of abstinence in the concentration of MR visible choline [Cho]- containing compounds relative to the neuronal marker, n-acetylaspartate [NAA], within the midline cerebellum of alcoholic patients. Our preliminary data suggest that the ratio Cho/NAA may be related to clinical variables, particularly to the severity of brain dysfunction. We propose to characterize the longitudinal course of these metabolic changes in regions of the brain recognized to be sensitive to alcohol-induced damage (cerebellar vermis, frontal cortex, and frontal white matter). Alcohol dependent patients will be studied using proton MRS when signs of acute alcohol withdrawal have abated (within 3 to 5 days of their last consumption of alcohol), after 3 weeks of inpatient monitored abstinence, and 9 weeks after discharge from hospital. High resolution MR images will be acquired concurrently to determine the contributions to the intracranial volume of gray matter, white matter, and cerebrospinal fluid, the volume of the cerebellum, and the tissue composition of each VOI. Also, cognitive (attention, psychomotor speed, and memory) and cerebellar (postural sway) functions will be evaluated. Healthy controls will also be studied at the same time intervals as the alcoholics to account for potential MRS/MRI repositioning error and learning effects of neurobehavioral testing. After 3 weeks of monitored abstinence, a comprehensive neuropsychological test battery will be administered to determine whether patients have enduring, clinically meaningful cognitive dysfunction. The longitudinal progression and interrelationships among metabolic characteristics of brain VOIs, volumetric changes, cognitive/cerebellar functions, and relevant clinical variables will be analyzed. If our hypotheses are confirmed, it would implicate white matter injury in the pathophysiology of alcohol-induced brain damage, and an increase in Cho-containing compounds in the recovery of CNS functions observed with abstinence. In the future, it may be possible to chemically identify the exact compounds involved by in vitro studies using autopsied human brain tissue or using available preclinical animal models, with significant implica-tions for prevention, differential diagnosis, and treatment of organic mental disorders associated with alcoholism. MARTIN PR VANDERBILT UNIVERSITY, 21ST AVE SO & GARLAND AVE, NASHVILLE, TN 37232-2647 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol biomarker cerebellum brain injury brain mapping choline noninvasive diagnosis aspartate drug withdrawal histopathology human subject magnetic resonance imaging neuropsychology neuropsychological test cognition disorder excitatory aminoacid neurotoxicology clinical research CRISP RPROJ TENNESSEE G CRISP/99/AA10583-03 CRISP/99/AA10583-03 199904 eng ALCOHOL ACTION ON A CLONED POTASSIUM CHANNEL Research RPROJ APPLICANT'S ABSTRACT: The biological basis of ethanol intoxication and general anesthesia are believed to involve, in part, effects on ion channels that regulate nerve excitability. However, it is not known how ethanol and general anesthetics alter ion channel function at the molecular level and to what extent they share a common mechanism. The long-term goal of this proposal is to understand the molecular mechanism underlying inhibition of a cloned potassium channel by ethanol and other aliphatic alcohols (n-alcohols). Aliphatic alcohols behave as general anesthetics. The channel under study is encoded by Shaw2, a gene cloned from Drosophila. Previous studies in this laboratory have shown that Shaw2 potassium channels are selectively inhibited by clinically-relevant concentrations of ethanol in a manner consistent with a direct drug-channel interaction. Recombinant DNA technology, expression in frog oocytes or insect cells and patch-clamp recording will be used to address the following aspects: 1) The action of n-alcohols on the electrophysiological properties of Shaw2 potassium channels. 2)Identification of Shaw2 protein domains and amino acid residues that may interact with n-alcohols. 3)Overexpression, purification and reconstitution of recombinant Shaw2 potassium channels. The first two aspects will help us to understand the biophysical and molecular basis of channel inhibition. The third aspect will allow purification of sufficient quantities of the channel protein to permit the use of biochemical and biophysical methods to study more directly the interaction between n-alcohols and the channel. A comprehensive study of the molecular mechanism underlying inhibition of a potassium channel by n-alcohols is an important step towards understanding how ethanol can affect ion channel function in general, causing general anesthesia and other acute alterations affecting the brain and other organs. COVARRUBIAS ML THOMAS JEFFERSON UNIV COLL, 1020 LOCUST ST, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health alcohol ethanol molecular cloning neurophysiology neural inhibition neural transmission protein purification potassium channel general anesthesia toad neurotoxicology CRISP RPROJ PENNSYLVANIA G CRISP/99/AA10615-02 CRISP/99/AA10615-02 199904 eng FETAL ALCOHOL INDUCED CHANGES IN THE INSULIN RESPONSE Research RPROJ Insulin is an important source of regulatory signals during fetal growth and development but alcohol exposure alters the metabolic and mitogenic responses of the fetus to insulin. The growth inhibition associated with fetal alcohol exposure is correlated with a lowered tissue glucose content and with decreases in basal glucose transport by the placenta and the fetus. However, alcohol-exposed embryos have a marked increase in glucose uptake in the presence of exogenous insulin when compared with vehicle-treated embryos. Insulin binding by the plasma membrane is increased in the alcohol-treated embryos but the binding of insulin-like growth factors is unchanged. The increase in insulin binding and in insulin-dependent glucose uptake notwithstanding, exposure to exogenous insulin further inhibits growth in alcohol-treated embryos but not in vehicle-treated embryos. Furthermore, fetal alcohol exposure leads to an abnormal insulin response in the adult offspring. these effects of alcohol cannot be completely overcome by glucose or caloric supplementation, suggesting that factors in addition to glucose uptake and metabolism are involved. Using nonplacental (chick) and placental (rat) embryos, cultured embryonic cells and adult animal exposed to alcohol in utero, the goals of the proposed research are to determine the molecular mechanism(s) by which alcohol alters the fetal and adult responses to insulin and to examine the physiological consequences of these molecular changes. The proposed studies will provide a basic understanding of the biochemical and physiological changes in the insulin response and in glucose homeostasis that result from fetal alcohol exposure. Because of their connection to human health, the studies are a logical prelude to the treatment or prevention of the effects of alcohol exposure during fetal development. PENNINGTON SN EAST CAROLINA UNIVERSITY, MOYE BOULEVARD, GREENVILLE, NC 27858-4354 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol chick embryo laboratory rat glucose metabolism molecular pathology hormone regulation /control mechanism disease model insulin embryo /fetus embryo /fetus toxicology fetal alcohol syndrome prenatal growth disorder receptor binding insulin receptor CRISP RPROJ NORTH CAROLINA G CRISP/99/AA10681-02 CRISP/99/AA10681-02 199904 eng NALTREXONE DOSE TITRATION STUDY Research RPROJ NO ABSTRACT AVAILABLE FARREN CK YALE UNIVERSITY SCH OF MEDICIN, 1 LONG WHARF, NEW HAVEN, CT 06519 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 06519 Crisp Data Base National Institutes Of Health cortisol alcoholism /alcohol abuse alcoholism /alcohol abuse chemotherapy naltrexone blood pressure blood chemistry urinalysis dosage drug adverse effect drug interaction drug withdrawal human subject interview questionnaire statistics /biometry hypothalamic pituitary axis adrenocorticotropic hormone prolactin opioid receptor human therapy evaluation clinical research behavioral /social science research tag outcomes research CRISP RPROJ CONNECTICUT G CRISP/99/AA10727-02 CRISP/99/AA10727-02 199904 eng RESPIRATORY BURST, CYTOKINES AND HIV IN ALCOHOLICS Research RPROJ APPLICANT'S ABSTRACT: Increased susceptibility to human immunodeficiency virus-I (HIV-1) infection, and complications associated with AIDS among alcoholics is widely recognized. The mechanism for increased propensity to infection is thought to be due to ethanol-mediated dysregulation of lymphocyte and monocyte functions. However, studies that deal alcohol-associated changes in vivo in various functions of hepatic macrophages (Kupffer cells) and other phagocytes during HIV-1 infection are few or lacking. Kupffer cells are primarily involved in clearance of particulate and soluble materials (HIV-1 glycoproteins, endotoxin). Thus, any alteration in phagocyte function during viral entry into the host is a major limiting factor in the survival of HIV. Based on these considerations, the overall objective of this proposal is to examine the mechanisms by which alcohol modulates HIV-1 gp120-induced non-specific immune defense mechanisms, i.e., respiratory burst and cytokine release, by Kupffer cells, endothelial cells and neutrophils in the presence or absence of endotoxemia. This proposal is based on the hypothesis that alcohol is a predisposing factor in HIV-1 infection, endotoxemia and liver disease that could hasten its progression to immunodeficiency/AIDS. Specifically, ethanol-induced spontaneous formation of reactive oxygen intermediates (ROI) leads to enhanced TNF and IL-1 production in hepatic macrophages, that could contribute to the immunopathogenesis of HIV-1 infection and AIDS. Proteins derived from HIV-1, e.g., HIV 1 gp12O, may exacerbate cytokine production in the alcoholic liver. Alcohol induces immunodeficiency through attenuation of antigen (HIV 1 gp12O, zymosan)-induced free radical formation in hepatic phagocytes. Thus, specific aim 1 will examine the effect of chronic and acute alcohol intoxication in the rat model (male Sprague-Dawley rats) on HIV-GP-120-induced oxygen-derived free radical formation and cytokine release (IL-1, TNF) by isolated Kupffer cells, endothelial cells, hepatic and blood neutrophils. Since alcohol withdrawal may also have an impact on these cells, studies on superoxide and cytokine release at appropriate interval following withdrawal or recovery from chronic or acute alcohol intoxication will be performed. The relationship between alcohol-mediated alteration of free radical release and cytokine production induced by HIV gpl20 will be studied, by the use of inhibitors of reactive oxygen species, i.e., superoxide dismutase and catalase. Specific aim 2 will elucidate the effect of alcohol with or without endotoxemia on the activities of protein kinase c and NADPH oxidase and glucose use during HIV-GP-120-(plus PMA or zymosan)-mediated respiratory burst, by analyzing the translocation and activities of these enzymes in cytosolic and membrane fractions of phagocytic cells. HIV GP 120 is currently being tested in one of the major international clinical trials as a potential vaccine against BAUTISTA AP LOUISIANA STATE UNIV MED CTR, 1901 PERDIDO STREET, NEW ORLEANS, LA 70112-1393 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse laboratory rat neutrophil glucose metabolism vascular endothelium cellular immunity leukocyte oxidative burst cytokine interleukin 1 immunoregulation monoclonal antibody radioimmunoassay endotoxin immunopathology AIDS HIV infection Kupffer's cell alcoholic liver cirrhosis statistics /biometry free radical oxygen protein kinase C virus infection mechanism human immunodeficiency virus 1 HIV envelope protein gp120 NAD(P)H oxidoreductase enzyme activity CRISP RPROJ LOUISIANA G CRISP/99/AA10746-03 CRISP/99/AA10746-03 199904 eng CARBAMAZEPINE AND LORAZEPAM IN OUTPATIENT DETOXIFICATION Research MALCOLM RJ MEDICAL UNIV OF SOUTH CAROLINA, 171 ASHLEY AVE, CHARLESTON, SC 29425-0742 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse alcoholism /alcohol abuse chemotherapy lorazepam carbamazepine urinalysis breath test drug adverse effect drug withdrawal outpatient care electrocardiography human subject tremor psychological test cognition anxiety depression neuropsychological test psychometrics psychomotor function psychomotor reaction time human therapy evaluation detoxification clinical trial phase II /IV clinical research CRISP RPROJ SOUTH CAROLINA G CRISP/99/AA10761-030002 CRISP/99/AA10761-030002 199904 eng NEUROANATOMICAL SUBSTRATES OF ALCOHOL INDUCED AMNESIA Research RPROJ The mechanisms by which alcohol exerts deleterious effects on human memory are still poorly understood. Recent data indicate that alcohol blocks experience-dependent gene activation in the hippocampus, a brain structure which plays a critical role in the acquisition of memory. The goal of the present proposal is to test whether alcohol suppresses memory because of its selective inhibition of hippocampal activity and to investigate the neurochemical and pharmacological mechanisms of this inhibition. The first specific aim of the proposed research is to investigate the effects of acute alcohol intoxication on hippocampus-dependent and -independent learning and experience-induced neural activity. A series of behavioral studies using the contextual conditioning paradigm in rats will determine the selectivity of suppressive effects of ethanol on hippocampal functions and dissect the sensory specificity of these effects of alcohol. Experiments using expression of immediate early genes (IEGs) as an activity-dependent neuronal maker will address whether alcohol&#176;s effects on hippocampal activity, determined by behavioral means, are accompanied by parallel changes in hippocampal gene expression. The second specific aim of this study is to investigate the neurochemical mechanisms leading to alcohol's suppression of hippocampus-dependent learning and neural activity. Experiments with pharmacological agents will determine whether GABAA receptor agonist and antagonists can modulate alcohol effects on hippocampus-dependent forms of fear conditioning and experience- induced hippocampal activity. Double-labeling studies will establish the neurotransmitter nature of hippocampal neurons in which alcohol changes experience-induced IEG expression. Western blotting and immunohistochemical analysis will be performed to assess what second messenger system mediates alcohol&#176;s effects on hippocampal IEG expression and memory. Finally, the DNA binding activity of transcription factor complexes from hippocampus after learning and alcohol intoxication will be investigated in a series of band-ship experiments. Taken together, these studies will provide valuable insight at several levels of analysis of alcohol&#176;s action on cognitive functions and eventually allow to start understanding of the mechanisms involved in alcohol-induced amnesia. RYABININ AE OREGON HEALTH SCIENCES UNIV, 3181 S W SAM JACKSON PARK RD, PORTLAND, OR 97201-3098 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol gamma aminobutyrate laboratory rat hippocampus gene expression regulatory gene transcription factor neurochemistry neurophysiology neural inhibition behavioral genetics behavior test conditioning fear learning memory disorder GABA receptor neurotoxicology neurotransmitter agonist neurotransmitter antagonist behavioral /social science research tag CRISP RPROJ OREGON G CRISP/99/AA10810-02 CRISP/99/AA10810-02 199904 eng IMPROVING AND ESTABLISHING THE BRAC CLAMP Research RPROJ DESCRIPTION: The proposed work will improve a method for maintaining a steady-state breath alcohol concentration (BAC) while a variety of dependent measures of brain function are assessed. Recovery towards baseline values during the steady state assesses acute tolerance to alcohol, and this study will establish the feasibility of future studies. In addition, the rate of alcohol administration in the clamped and equilibrated steady-state yields a direct measure of the alcohol elimination rate (AER). Health is directly affected by both acute tolerance and the AER because both may be genetically determined and differences among individuals may reinforce or discourage future drinking. In pre studies, adjustments to the rate of an intravenous (iv) infusion of 6% v/v ethanol maintained BrAC within 5 mg% of a 50 mg% target BrAC for prescribed intervals, beginning 42 minutes after an oral loading dose of alcohol in ten male subjects. Event-related potential (ERP) amplitudes provided significant and reliable indices of acute tolerance to alcohol. Three items from the Subjective High Assessment Scale showed significant acute tolerance in subjective perceptions. Retesting AER with the clamp in the ten subjects yielded high reliability, reflected in a coefficient of variation of less than 7%. Accelerating the induction phase of the clamping method should provide increased sensitivity to early phases of the brain's adaptation to alcohol. Thus, the first specific aim is to develop rapid intravenous infusion as a method for achieving a 25 minute start time for the BrAC clamp. The second specific aim is to conduct a pilot study of the sensitivity of acute tolerance indices in subjective, behavioral, and neuropsychological measures, and in a single ERP at two target BrAC. Third, the data will define the sample size of future studies assessing acute tolerance to alcohol in subjects at high risk for alcoholism. Two small studies are proposed. In Study #1, one sample (N=10) is employed once to optimize the new iv induction method; another sample (N=10) is tested twice at 60 mg% to compare the iv induction method to the existing method using oral loading. In Study #2, separate sessions are conducted at target BrACs of 40 and 80 mg% for each of 20 additional subjects, with a third session using an infusion containing no alcohol as a control. Once before and twice during every clamping session of both studies, the battery of dependent measures of brain function are obtained with results reduced to scalar indices, and examined for sensitivity to the effect of alcohol and acute tolerance to alcohol, using the control session to account for learning and fatigue. The indices and the AER will be tested for a preliminary estimate of their dose dependency. Results of this study should provide a firm foundation for application of the clamping technique and dependent measures to important questions in the etiology, genetics and brain function associated with acute tolerance to alcohol. O'CONNOR SJ INDIANA UNIVERSITY, DEPT OF PSYCHIATRY, INDIANAPOLIS, IN 46202-4887 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol brain metabolism breath test dosage drug adverse effect drug tolerance evoked potential fatigue human subject behavior learning neuropsychology neuropsychological test perception method development clinical research behavioral /social science research tag CRISP RPROJ INDIANA G CRISP/99/AA10831-02 CRISP/99/AA10831-02 199904 eng NEUROANATOMIC ANALYSES OF FAS/FAE DEFICITS Research RPROJ APPLICANT'S ABSTRACT: This research proposes to quantify the neuroanatomic abnormalities underlying neuropsychological deficits among people with brain damage caused by prenatal alcohol exposure. Although Fetal Alcohol Syndrome (FAS) is a distinct diagnostic entity, it conceals substantial variability of deficit. Furthermore, many others with prenatal alcohol exposure exhibit dysfunctional behaviors that appear to be CNS-based, but do not have facial manifestations of FAS. These are referred to as possible Fetal Alcohol Effects (FAE). We hypothesize that: (1) New image analysis methods will reveal significant mean differences in brain form between FAS and Controls that are not due solely to microcephaly and are not detectable from clinically-read magnetic resonance image (MRI); (2) Neuropsychological testing will demonstrate significant differences in the neuropsychological profiles of FAS vs. Controls; (3) The average FAE brain and behavior will more closely resemble the average FAS brain and behavior than that of Controls; (4) There are significant correlations between brain dysmorphology and neuropsychological deficits; and (5) These associations will be strong enough to classify the extent of damage and predict prognosis in the individual case. Over 5 years we plan to: 1. Gather MRI data (including landmark locations) and neuropsychological data on 180 research subjects in three groups (60 with FAS, 60 with FAE, and 60 Controls group-matched for age, sex and race.) 2. Characterize 2D and 3D differences in shapes of landmark configurations and other patterns of differences in MRI's among these groups, with due attention to age, sex and ethnicity. 3. Characterize differences in neuropsychologic profiles among the three groups, controlling of IQ as well as to age, sex and ethnicity. 4. Determine those aspects of brain shape variation and focal anatomic abnormalities most highly associated with profiles of specific neuropsychological deficit within and among groups. 5. Combine the findings from Aims 2-4 into clinically useful protocols for the detection of individuals with brain damage from prenatal alcohol exposure. This will be the first systematic study to utilize new morphometric methods to localize neuroanatomic abnormalities associated with neuropsychological deficits in patients with FAS/FAE. Detecting neuroanatomic anomalies will permit proper identification and appropriate service delivery to these patients. STREISSGUTH AP UNIVERSITY OF WASHINGTON, 180 NICKERSON ST, SUITE 309, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 98195 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse brain disorder brain disorder diagnosis human subject data collection methodology /evaluation neuroanatomy image processing magnetic resonance imaging embryo /fetus toxicology fetal alcohol syndrome neuropsychological test sex difference Native American African American Hispanic American caucasian American racial /ethnic difference age difference clinical research CRISP RPROJ WASHINGTON G CRISP/99/AA10836-03 CRISP/99/AA10836-03 199904 eng ENHANCED LINKAGE OF ALCOHOL ABUSERS TO PRIMARY CARE Research RPROJ Alcohol abusers suffer significant health consequences as a result of their substance use, including increased risk for HIV infection, yet they are particularly difficult to engage in primary medical care. Because of this, they often miss opportunities to receive preventive healthcare, including HIV risk reduction interventions, addictions behavioral counseling, prevention of the complications of HIV, and ongoing treatment for illnesses. Substance abuse treatment presents an opportunity to link alcohol abusers to primary medical care. Engaging alcohol abusers with or at-risk of HIV infection in health issues during detoxification may promote their subsequent linkage to primary medical care. This linkage, the addition of primary medical care to their addiction care, may result in more appropriate utilization of HIV and other health services, reduced alcoholism severity, decreased HIV risk behaviors, and improved health status. To test these hypotheses, a cohort of 240 individuals undergoing detoxification from alcohol, including 40 who are HIV-infected (the remaining individuals categorized as at high or low risk for HIV), will be identified and followed for two years. The cohort will be randomized into two groups. One group will receive standard care, including information about available primary care. The other group will attend the Health Evaluation and Linkage to Primary care (HELP) Clinic based at the detoxification unit, which will include a comprehensive medical, substance abuse, and social service assessment, referral to a primary care physician at a site where patients are seen regardless of ability to pay; and nurse contacts to remind them of primary care appointments. To study secondary prevention issues, the study will also enroll and follow a longitudinal cohort of 100 HIV-infected alcohol abusers presenting for initiation of ongoing medical care to the HIV Diagnostic Evaluation Unit at Boston City Hospital. All patients will be assessed at baseline regarding alcohol abuse severity using the alcohol factor score from the Addiction Severity index and the Alcohol Dependence Scale; health status using the EuroQol and the SF-36 Health Survey; and history of high-risk HIV behavior. The subjects will participate in interviews at 6, 12, 18, and 24 months after enrollment. The linkage outcome measure will be connecting to primary care follow-up after enrollment. It is expected that subjects randomized to the HELP Clinic will be more likely to attend visits with a primary care physician. Barriers to primary care and measures of primary care continuity, comprehensiveness and coordination will be documented. We hypothesize that the resultant linkage with primary care will result in improved addiction, health status, and HIV risk behavior outcomes, and more appropriate utilization of medical and addiction treatment services over two years. By establishing the HELP Clinic and rigorously assessing patient outcomes, we pl SAMET JH BOSTON MEDICAL CENTER, 91 EAST CONCORD STREET, BOSTON, MA 02118 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 02118 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse biomedical registry /referral center health care facility information system disease /disorder proneness /risk AIDS education /prevention comprehensive care health care service evaluation health care service utilization primary care physician human subject HIV infection psychosocial service nursing care longitudinal human study therapy compliance detoxification clinical research behavioral /social science research tag outcomes research CRISP RPROJ MASSACHUSETTS G CRISP/99/AA10870-03 CRISP/99/AA10870-03 199904 eng OVINE MODEL SYSTEM FOR ALCOHOL RELATED BIRTH DEFECTS Research RPROJ DESCRIPTION (Adapted from the APPLICANT'S ABSTRACT): The overall purpose of this proposal is to use an ovine (sheep) model system to address FAS questions in conjunction with another of NIAAA's areas of special interest, moderate drinking. Specific Aim 1 will test the hypothesis that maternal alcohol exposure levels during the third trimester that are too low to produce fetal hypoxia will produce substantial brain damage. Second, we will determine if high doses of alcohol that mediate hypoxia result in disproportionate increases in brain injury. We will focus on microencephaly, area measurements of the corpus callosum and on stereo logical cell counts in the cerebellum, neocortex, hippocampal formation, locus coeruleus and the principal sensory nucleus of the trigeminal nerve, structures that include populations of neurons known to exhibit differential sensitivity to both fetal alcohol exposure and hypoxia. Data also will be gathered related to a second popular hypothesis, that heavy maternal alcohol consumption produces altered prostaglandin levels that mediate alcohol related birth defects. Additional dependent variables to be assessed include circulating levels of prostaglandins and hormones, hemodynamic measures, blood gases, gross somatic anomalies and prenatal growth measures. Specific Aim 2 will determine the effects of long-term prenatal alcohol exposure (all three trimesters) on brain development. The design for this experiment is driven by drinking patters reported in moderate and heavy drinkers during pregnancy. Specific Aim 3 will test the hypothesis that brain damage will be qualitatively as well as quantitatively different when comparing alcohol exposure throughout gestation with exposure restricted to third trimester. The questions raised in this proposal have not yet been answered due in part to the lack of a suitable animal model system. The sheep model system has several distinct advantages that will be exploited in the proposed studies. These include: 1) the stages of brain development comparable to that which occurs throughout gestation in humans also occur entirely prenatally in the sheep; 2) a large maternal/fetal unit tolerant of chronic indwelling cannulae; and 3) a long gestation making it much easier to evaluate critical periods, threshold doses, and patterns of alcohol exposure. The most compelling reason for developing the ovine model system is that these advantages are particularly valuable for evaluating mechanisms of damage. Together, these studies will provide new, important data related to fetal alcohol exposure and brain damage that have not been addressed adequately with other animal model systems. CUDD TA TEXAS A&M UNIVERSITY, COLLEGE STATION, TX 77843 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 77843 Crisp Data Base National Institutes Of Health ethanol sheep cephalometry chronic brain damage congenital disorder micrencephaly dosage prostaglandin eicosanoid metabolism disease model developmental neurobiology gestational age embryo /fetus toxicology fetal alcohol syndrome hypoxia CRISP RPROJ TEXAS G CRISP/99/AA10940-02 CRISP/99/AA10940-02 199904 eng BAEP SCREENING OF NEONATES EXPOSED TO ALCOHOL Research RPROJ DESCRIPTION: (Adapted from the Applicant's Abstract) Prenatal drug exposure is known to cause a variety of neurodevelopmental disorders, yet many drug-exposed neonates go undetected in the newborn nursery. Consequently, valuable intervention for the affected child is often delayed or neglected. Early identification, tracking and intervention are important in achieving a favorable outcome. The brainstem auditory evoked potential (BAEP), which is both a measure of brain maturity and integrity and a measure of sensory (auditory) function, has already gained wide acceptance as a simple, objective and non-invasive screening test for neonates at risk for auditory and neurodevelopmental disorders resulting from certain perinatal complications (e.g., low birth weight, perinatal hypoxia, low Apgar scores, craniofacial anomalies). We will apply this technology, in combination with antenatal maternal interviews, for the screening of neonates prenatally exposed to drugs. Auditory, language and neurodevelopment follow-up evaluations will be conducted at 12-months of age. The relationship between the neonatal BAEP and subsequent outcome will determine if the BAEP is a useful screening tool, leading to the early identification and intervention of drug-exposed infants with neurodevelopmental disorders. The subject population will be primarily the hard-to-reach, low-income, inner-city minority (African American) women and their infants. This project will focus on the effects of maternal alcohol, cocaine, and cigarette consumption because they are the most commonly abused drugs and because of their negative influence on the child's subsequent hearing, language and intelligence. While these drugs are frequently used in combination, little has been done in the human literature to evaluate the independent, interactive and dose-dependent effects of these drugs. This proposal will address these issues as well. Potentially confounding variables will be gathered and their influence assessed and statistically controlled. Appropriate clinical interventions for mother and infant will be made. CHURCH MW WAYNE STATE UNIVERSITY, 275 E HANCOCK, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48201 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse child physical development child psychology newborn human (0-6 weeks) mass screening drug abuse tobacco abuse auditory stimulus evoked potential human subject developmental neurobiology epidemiology embryo /fetus toxicology neuropsychology neuropsychological test substance abuse cocaine clinical research behavioral /social science research tag substance abuse epidemiology CRISP RPROJ MICHIGAN G CRISP/99/AA10941-02 CRISP/99/AA10941-02 199904 eng ALCOHOL AND MESOLIMBIC DOPAMINE PATHWAY Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) The long-term goal of this project is to elucidate further the neurochemical basis of the reinforcing effects of ethanol and to develop new strategy aimed at decreasing alcoholism in man. The proposed research will be carried out on freely moving rats by using intracerebral microdialysis technique to monitor in vivo dopamine (DA) release in the nucleus accumbens (NACC), a terminal area of the mesolimbic DA pathway. Pharmacologically relevant doses of ethanol will be administered systemically. First, DA and 5-HT in 5-min dialysate samples from the NACC will be measured simultaneously following acute ethanol to detect any changes in extracellular DA and 5-HT which occur during the ascending phase of the brain ethanol concentration curve. In addition, Ethanol-induced DA release in the NACC will be compared with and without the++ treatments with: 1) perfusion with tetrodotoxin (TTX) or Ca -free medium into the NACC, both of which are known to interrupt membrane depolarization; 2) perfusion with quinpirole, a D2 receptor agonist, into the ventral tegmental area (VTA) to suppress the mesolimbic DA neuronal activity; 3) nomifensine, a DA uptake blocker. This will enable us to determine whether and to what extent a carrier-dependent mechanism is involved in ethanol-induced DA release in the NACC. Furthermore, the effects of suppression (Lesioning with 5,7-DHT) or enhancement (pretreatment with 5-hydroxytryptophan and carbidopa) of the serotonergic transmission on ethanol-induced DA release will be evaluated. This will allow us to asses the possible involvement of the 5-HT system in the reinforcing effects of ethanol, the effects of agonists and antagonists at these subtypes of 5-HT2 receptors in the reinforcing effects of ethanol, the effects of agonists and antagonists at these subtypes of 5-HT receptor on ethanol-induced DA release in the NACC will be investigated. This will elucidate the relative contribution of 5-HT2 receptors to the regulation of ethanol-induced DA release and help to explain the effects of 5-HT2 antagonists on alcohol consumption reported in the literature. YAN Q UNIVERSITY OF ILLINOIS, BOX 1649, PEORIA, IL 61656-1649 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat serotonin limbic system nucleus accumbens brain /spinal pathway /tract experimental brain lesion neuropharmacology neural transmission dopamine reinforcer tetrodotoxin microdialysis dopamine antagonist CRISP RPROJ ILLINOIS G CRISP/99/AA10946-01A2 CRISP/99/AA10946-01A2 199904 eng OLIGONUCLEOTIDE TARGETING OF THE KUPFFER CELL Research RPROJ Recent studies indicate that the Kupffer cell plays a major role in the production of liver injury induced by a number of hepatotoxins and pathological conditions. Obliteration of Kupffer cells virtually abolishes cell necrosis and inflammation induced by xenobiotics such as ethanol, carbon tetrachloride and acetaminophen. Similarly, liver damage induced by endotoxemia or liver storage prior to transplantation is markedly reduced by experimental conditions that reduce Kupffer cell function. Data suggest that tumor necrosis factor-alpha released by Kupffer cells plays a major role in cell damage. The synthesis of tumor necrosis factor- alpha is initiated by gene-transcription factors, which are activated by oxygen radicals and endotoxin. Tumor necrosis factor-alpha triggers a cytokine cascade involving cytotoxic, inflammatory and fibrogenic mediators. The studies proposed (a) test the hypothesis that triple-helix targeting of the tumor necrosis factor-alpha gene in Kupffer cells results in marked reduction of liver damage induced by a number of hepatotoxins and pathogenic conditions; (b) develop methodologies for the design and delivery of triple-helix forming oligonucleotide drugs into Kupffer cells, for possible therapeutic use. Kupffer cells are specialized in the phagocytic removal of particulate matter from the circulation. Studies in this application utilize this phagocytic action to deliver liposome-entrapped triple-helix antigene oligonucleotides into Kupffer cells. The proposed research is designed to (i) suppress the synthesis of tumor necrosis factor-a and the cytokine transcription factor NF-kappaB in Kupffer cells, (ii) reduce the expression of genes involved in the generation of oxygen-radicals in Kupffer cells, and (iii) test the protection afforded by triple-helix drugs against liver necrosis and inflammation induced by the chronic administration of ethanol and other hepatotoxins. Overall, the proposed research investigates at the gene level the existence of central mechanisms of liver injury by new approaches with therapeutic potential for alcoholic liver disease in humans. ISRAEL Y THOMAS JEFFERSON UNIV COLL, 1020 LOCUST STREET, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health ethanol laboratory rat liposome nuclear runoff assay polymerase chain reaction drug screening /evaluation drug design /synthesis /production gene expression carbon tetrachloride tumor necrosis factor alpha Kupffer's cell liver toxic disorder hepatotoxin nucleic acid inhibitor oligonucleotide synthetic nucleotide NAD(P)H dehydrogenase acetaminophen lipopolysaccharide thiophosphate northern blotting chemoprevention gel mobility shift assay nuclear factor kappa beta triple helix CRISP RPROJ PENNSYLVANIA G CRISP/99/AA10967-03 CRISP/99/AA10967-03 199904 eng ALCOHOL EFFECTS ON SUBCELLULAR ORGANELLES OF THE LIVER Research RPROJ APPLICANT'S ABSTRACT: This project is part of an IRPG application that represents a continuation of previous studies carried out under a program project grant of the same title that in turn continued studies on a longstanding RO1 grant (also of the same title) originally funded in 1966. This program project included several projects focused on the interactions of ethanol with G protein-coupled phospholipase C (PLC) signalling system in the liver. We identified multiple sites of interaction of ethanol with the control of this signalling process in liver cells resulting in the suppression by ethanol of the response to hormones. This ethanol inhibition originated in at least two mechanisms, one resulting from the direct interaction of ethanol with the receptor-G protein-PLC complex, the other resulting from the potentiation by ethanol of the feedback inhibition mediated by protein kinase C (PKC). In this project, we will pursue the identification of the molecular mechanisms underlying these effects of ethanol and characterize possible physiological consequences for liver function. First, we will further characterize the role of PKC in the inhibitory effects of ethanol. We will interfere in multiple ways with PKC activity in intact cells, by the use of multiple inhibitors, enzyme downregulation and microinjection of specific peptides that interfere with its normal function. In addition, we will use longer-term hepatocyte cultures to suppress specific PKC isoforms using antisense strategies. The phosphorylation of potential protein kinase C target proteins of receptor-Gq/G11 protein-PLCbeta complex in intact hepatocytes will also be studied. The second aim is to obtain molecular information on the direct interactions of ethanol with this signaling complex. For this purpose, we will obtain highly purified vasopressin V1A receptor, Gq protein and phospholipase Cbeta1 protein and reconstitute these proteins in artificial phospholipid vesicle systems, both as individual or paired functional units and as a functional complex. This reconstitution will enable us to focus on individual protein-protein interactions in the complex and investigate the effects of ethanol on these interactions and the effect of treatment with purified PKC isoforms. The third aim is concerned with the functional implications of the effects of ethanol on Ca2+ -mediated signalling in the liver. We will utilize newly developed fluorescence imaging techniques that allow dynamic measurements of intracellular Ca2+ concentrations in hepatocytes in the intact perfused liver. The effects of ethanol and hormones on spatial and temporal organization of intracellular Ca2+ changes in individual cells across the functional hepatic lobule will be examined. We will also investigate the ethanol-induced disruption of hepatic Ca2+ signalling on bile flow and secretion and to study the metabolic interactions between ethanol and hormonal stimulation of mitochondrial Ca2+ met REVIEW A RUBIN E THOMAS JEFFERSON UNIVERSITY, 1020 LOCUST ST, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health ethanol laboratory rat bile liposome calcium flux fluorescence spectrometry hormone regulation /control mechanism isozyme liver cell hepatotoxin liver function liver metabolism membrane reconstitution /synthesis antisense nucleic acid phospholipase C phosphorylation protein kinase C vasopressin G protein receptor coupling receptor sensitivity hormone receptor tissue /cell culture toxin metabolism enzyme activity CRISP RPROJ PENNSYLVANIA G CRISP/99/AA10968-03 CRISP/99/AA10968-03 199904 eng CHRONIC ALCOHOL EXPOSURE AND CALCIUM SIGNALING Research RPROJ APPLICANT'S ABSTRACT: Ethanol exposure is known to perturb many signal transduction pathways, including those that involve adenylate cyclase and phosphoinositide turnover. It has been hypothesized that ethanol-mediated alterations in signal transduction pathways may reflect one mechanism by which cells adapt and develop tolerance to the acute effects of ethanol. Previous studies have established that isolated hepatocytes from alcohol-fed animals show an enhanced responsiveness to Ca2+ -mobilizing hormones and that this enhancement is associated with an increased sensitivity of intracellular Ca2+ stores for mobilization, by D-myo-inositol 1,4,5 trisphosphate ( IP3 ). A family of receptor proteins ( IP3R ) serve to recognize IP3 and act as ligand-gated Ca2+ channels that discharge Ca2+ from internal stores. Our principal hypothesis is that an alteration in the regulatory properties and/or expression of IP3 receptors underlies the observed effects of alcohol on Ca2+ signalling. In order to analyze the mechanism of these effects in more detail, we propose to utilize a cultured rat liver epithelial cell line ( WB cells) as an experimental model. These cells mimic the effects of alcohol - exposure on Ca2+ signalling seen in the animal model and also contain high levels of IP3R protein. Preliminary studies in these cells show that IP3R protein can be down-regulated by chronic stimulation with Angiotensin-II or TGF-B. We suggest that these agonists and ethanol may interact with common mechanisms that regulate IP3R expression. The specific aims of this project are: [1] To further characterize the effects of chronic ethanol exposure on Ca2+ signalling in WB cells in response to hormones and growth factors; [2]. To test the hypothesis that chronic ethanol exposure modifies the regulatory properties of IP3 receptors; [3]. To determine if chronic ethanol, Angiotensin-II and TGF-B change IP3R expression by translational mechanisms; [4]. To determine if chronic ethanol and agonist treatment alter the transcription of IP3R genes. Delineating the molecular mechanism by which chronic ethanol exposure alters hormonal Ca2+ signalling will aid in understanding the effects of alcohol on liver cell growth and injury. JOSEPH SK THOMAS JEFFERSON UNIV COLL, 1020 LOCUST STREET, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 19107 Crisp Data Base National Institutes Of Health ethanol laboratory rat biological signal transduction calcium flux hormone regulation /control mechanism genetic transcription transforming growth factor liver cell liver toxic disorder liver metabolism angiotensin II calcium channel behavioral habituation /sensitization receptor expression inositol phosphate tissue /cell culture CRISP RPROJ PENNSYLVANIA G CRISP/99/AA10971-02 CRISP/99/AA10971-02 199904 eng ETHANOL AND NITRIC OXIDE FORMATION IN BRAIN Research RPROJ DESCRIPTION: Nitric oxide (NO) is an important messenger molecule in the vascular system, immune system, and central nervous system. In brain, NO can act as a neurotransmitter/neuromodulator and increasing evidence suggests that NO, or closely related molecules, may also play an important pathophysiological role. The Broad, Long-Term objective of this project is to investigate the effects of ethanol on NO formation in brain. Glial cells and a subpopulation of neurons possess two distinctly different forms of NO synthase (NOS) that produces NO from the precursor amino acid L-arginine. Neuronal NOS is constitutively expressed and is activated by calcium. In contrast, glial NOS is not normally present in the cell but is synthesized de novo in response to stimulating agents such as cytokines and bacterial endotoxins. Once synthesized, the activity of this "inducible" form of the enzyme is independent of calcium and is regulated at the level of gene transcription. Our preliminary studies in cell culture show that chronic ethanol exposure can increase NO formation in neurons but reduce NO formation in astroglia. The potentiating effect of chronic ethanol on NO formation in neurons is most likely due to an increase in NMDA receptor function while its inhibitory effect on NO formation in astroglia appears to be due to reduced expression of the NOS protein. Given the apparent importance of NO during both normal and pathological conditions, it is important to clearly understand how both acute and chronic ethanol exposure affects NO formation in brain. The Specific Aims of this project are to: (1) identify NMDA receptor subunits that colocalize with and couple to activation of NOS in neurons; (2) determine the effects of chronic ethanol on expression of NMDA receptors that couple to NOS activation and the role of NO in the neuropathology of alcohol abuse; (3) determine the mechanism(s) of how chronic ethanol exposure can inhibit NOS expression in cultured astroglia; and (4) determine the effects of ethanol on NOS expression in reactive astrocytes during response to injury. The project involves studies to be performed in primary cultures of astroglia and neurons as well as studies with brain slice preparations. We hypothesize that ethanol induced alterations in brain function and pathology relate, in part, to altered NMDA receptor function and NO formation. These studies are likely to yield important and clinically significant information on the role of NO in the actions of ethanol in the brain, and may lead to more effective pharmacological treatments for acute alcohol intoxication, withdrawal and neurodegeneration associated with chronic alcohol abuse. CHANDLER LJ LSU MEDICAL CENTER, 1501 KINGS HIGHWAY, SHREVEPORT, LA 71130-3932 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat brain metabolism NMDA receptor nitric oxide nitric oxide synthase neurotoxicology neuropathology CRISP RPROJ LOUISIANA G CRISP/99/AA10983-02 CRISP/99/AA10983-02 199904 eng CRANIOFACIAL MORPHOGENESIS IN PRENATAL ALCOHOL EXPOSURE Research RPROJ NO ABSTRACT AVAILABLE SMITH SM UNIVERSITY OF WISCONSIN-MADISO, 1415 LINDEN DRIVE, MADISON, WI 53706 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 53706 Crisp Data Base National Institutes Of Health ethanol chick embryo laboratory mouse transgenic animal cell differentiation congenital oral /facial /cranial defect teratogen molecular pathology dosage early embryonic stage neural crest genetic marker gene expression fibroblast growth factor disease model developmental neurobiology embryo /fetus toxicology fetal alcohol syndrome bone development programmed cell death CRISP RPROJ WISCONSIN G CRISP/99/AA11085-03 CRISP/99/AA11085-03 199904 eng BRAIN GLUCOSE TRANSPORT AND UTILIZATION IN FAS Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) Mechanisms of ethanol-induced damage of developing brain remain to be elucidated. In cultures of rat astrocytes or intact embryos, ethanol inhibits both glucose transport and glucose oxidation via the pentose phosphate pathway (PPP). Developmental changes in the expression of brain glucose transporters, GLUT 1 and GLUT 3, suggest that these genes are regulated to meet the changing metabolic requirements of immature brain. Glucose is not only the primary energy substrate of brain, but its oxidation via the PPP is essential for production of ribose-5-phosphate which is necessary for synthesis of nucleosides and polysaccharides. The PPP is also involved in the production of NADPH for removal of free radicals and synthesis of lipids and neurotransmitters. Brief exposure to ethanol during the brain growth spurt reduces glucose transport, as well as GLUT 1 and GLUT3 proteins of neurons and astrocytes. It is proposed that these reductions result from specific regulation of the GLUT1 and GLUT3 genes by ethanol. This regulation may differ for the two transporter isoforms, and is likely to involve post-transcriptional mechanisms. In vitro studies with astrocytes and rat embryos also show that ethanol specifically inhibits activity of the PPP. It is proposed that PPP is particularly vulnerable to reduced glucose transport and that reduced activity of this pathway has significant toxicological impact on cells with critical requirements for its products. Goals of the proposed research will be to identify and characterize the mechanism by which ethanol alters glucose transporter expression in neurons and glia of developing brain. Proposed studies will use immunocytochemistry and in situ hybridization histochemistry for detecting regional changes in transporter proteins and mRNAs and for quantitation of mRNAs by northern analysis and nuclear transcription assays in specific brain regions. Transporter proteins and mRNA stabilities and post-transcriptional mechanisms will be studied in cultured neurons, astrocytes and oligodendroglia. The second major aim will be to characterize alterations in glucose utilization, indicators of oxidative stress and parameters of cell function and viability under the same conditions and to define the toxicological significance of these effects, including those on intercellular relations which might affect growth or survival. SNYDER AK FINCH UNIV OF HLTH SCI, 3333 GREEN BAY ROAD, NORTH CHICAGO, IL 60064 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 60064 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat brain metabolism glucose metabolism glucose transport gene induction /repression pharmacogenetics immunocytochemistry neurogenesis glia neuron developmental neurobiology neuropharmacology embryo /fetus toxicology fetal alcohol syndrome glucose transporter CRISP RPROJ ILLINOIS G CRISP/99/AA11103-01A2 CRISP/99/AA11103-01A2 199904 eng EFFECTS OF ALCOHOL ON HEART FUNCTION--GENDER DIFFERENCES Research RPROJ DESCRIPTION (Adapted from the Investigator's Abstract): The long-term objective of this proposal is to understand the changes in cardiac structure and pathophysiological mechanisms that are associated with alcoholic heart disease. The specific aims of this investigation are: (1) to determine in male and female rats the evolution of alcohol-induced changes in cardiac structure (hypertrophy and dilation/cardiomyopathy) and (2) to determine if the development of alcohol-induced cardiomyopathy is associated with corresponding changes in myocardial contraction and activation of specific hemodynamic and neuroendocrine mechanisms in male and female rats. It is well established that chronic alcohol consumption produces functional changes in the myocardium which lead to the development of cardiomyopathy. However, little is known about the progression of alcohol-induced cardiomyopathy, specifically how changes in myocardial structure correlate to changes in contractile function and activation of specific neurohumoral or hemodynamic mechanisms. In addition, there have been many studies documenting the adverse effects of alcohol in male hearts, while the effects alcohol consumption in the female heart are relatively unknown. Male and female Sprague-Dawley rats will receive either the Lieber- DeCarli alcohol or control diet, while a third group will receive rat chow. Animals will be maintained on this protocol for 3 and 6 months. Changes in left ventricular size and chamber dilation will be assessed by echocardiography at 4 week intervals. The indirect tail-cuff method will be used to measure blood pressure at 2 week intervals. Animals will be sacrificed at 3 and 6 months in order to evaluate alcohol- induced changes in myocardial contractility and morphology. To evaluate the latter light and electron microscopy will be performed and changes in contractility will be measured in an isolated papillary muscle preparation. Also at the time of sacrifice, plasma renin and plasma and tissue angiotensin-converting enzyme (ACE) activity will be measured using a radioimmunoassay technique and fluorimetric assay, respectively. Finally, all of the above variables will be re-examined in ethanol-fed and control animals after they have received long-term therapy with either an ACE inhibitor or B-adrenergic blocker. The results of this study will lead to a better understanding of the pathophysiology of alcoholic cardiomyopathy and rational development of therapeutic innervations to prevent and treat alcohol-induced cardiomyopathy. PIANO MR UNIV OF ILLINOIS, 845 S DAMEN, CHICAGO, IL 60612-7350 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol laboratory rat ACE inhibitor heart enlargement myocardium disorder heart function echocardiography histopathology radioimmunoassay muscle contraction beta antiadrenergic agent neurotransmitter renin peptidyl dipeptidase A sex difference toxicology enzyme activity CRISP RPROJ ILLINOIS G CRISP/99/AA11112-03 CRISP/99/AA11112-03 199904 eng CELL AND MOLECULAR EFFECTS OF ETHANOL IN LS AND SS MICE Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) Fetal alcohol syndrome is one of the leading causes of mental retardation in the western world. Ethanol exposure has a wide range of effects on the developing nervous system with the severity of the effects mediated by any of a number of factors; one important factor is the genetic composition of the organism. To asses the role of genetics in the severity of ethanol s effects, the following 2 experiments will be conducted on the recombinant inbred long-sleep and short-sleep strains of mice. Both of these experiments will examine embryos that have been exposed to a single bolus of ethanol on embryonic day 9. The first experiment will examine whether embryos from recombinant inbred long-sleep mice exhibit different levels of cell death compared to embryos from short-sleep mice following in utero ethanol exposure to the same dose of ethanol. Further, these experiments will examine whether the genotype of the embryo affects the regional variability in the levels of cell death and whether the means of cell death is through apoptosis. The level of cell death will be quantified in sections that have been stained with a Nissl stain and a recently developed method for nick-end labeling dying cells, termed TUNEL. The second experiment will examine the changes in messenger RNA (mRNA) expression using the technique of differential display. Changes in mRNA expression will be examined at 1, 4, and 12 hours after ethanol exposure. It is anticipated that this work will provide candidate mRNAs that have possible neuroteratogenic or neuroprotective effects. To assist in defining potential genes that have these effects, the sequence and localization of these mRNAs will be examined as well as their presence or absence in strains of mice that are sensitive or insensitive to the effects of in utero ethanol exposure. HAMRE KM UNIV OF TENNESSEE, 855 MONROE AVE, MEMPHIS, TN 38163 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 38163 Crisp Data Base National Institutes Of Health ethanol laboratory mouse genetic strain cell death disease /disorder proneness /risk cellular pathology mammalian embryology developmental genetics histology disease model developmental neurobiology messenger RNA embryo /fetus toxicology fetal alcohol syndrome programmed cell death neurogenetics CRISP RPROJ TENNESSEE G CRISP/99/AA11113-02 CRISP/99/AA11113-02 199904 eng ETHANOL, NICOTINE AND BRAIN NICOTINIC RECEPTORS Research RPROJ APPLICANT'S ABSTRACT: Alcoholics are almost invariably heavy smokers and smoking enhances alcohol use, but explanations for these interactions are lacking. Observations made in our laboratory indicate that chronic nicotine treatment results in tolerance to ethanol, reverses enhanced excitability seen in ethanol-withdrawing mice, and nicotine increases ethanol self-selection in a two-bottle choice experiment have been seen. The experiments described in this proposal will build on these observations by assessing whether three commonly-used inbred mouse strains differ in these interactions. Since ethanol affects the activity of "peripheral-type" nicotinic receptors (nAChRs) found in the electric organ of Torpedo californica, particularly activation and desensitization, the effects of ethanol on brain (nAChR) function will be assessed using several neurochemical assays that we have developed which can monitor the activation and desensitization of several different (nAChRs). Preliminary data suggest that some of the brain (nAChR) subtypes are resistant to ethanol whereas others, particularly alpha-7-containing, are inhibited by low concentrations of ethanol. Effects of in vitro and chronic in vivo ethanol on activation, desensitization, and recovery from desensitization of several types of brain (nAChRs) will be measured. Some of the chronic treatment experiments will involve short-term/high-dose treatments (1-2 weeks) while others will use long-term (6-8 months) treatment. The chronic studies will build on our observation that chronic nicotine treatment results in a long-term desensitization of brain (nAChRs) in some brain regions, and not in others. Hopefully, these studies will provide a rigorous test of the hypothesis that some of the basis of the co-abuse of ethanol and nicotine arises because of interactions between these two drugs at brain (nAChRs). COLLINS AC UNIVERSITY OF COLORADO, CAMPUS BOX 447, BOULDER, CO 80309-0447 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory mouse genetic strain brain metabolism drug interaction drug tolerance drug withdrawal longitudinal animal study behavioral genetics behavior test choice nicotine receptor expression receptor sensitivity nicotinic receptor oral administration neurotoxicology CRISP RPROJ COLORADO G CRISP/99/AA11156-03 CRISP/99/AA11156-03 199904 eng ETHANOL-CAROTENOID INTERACTIONS--BENEFITS AND RISKS Research RPROJ Substantial amounts of beta-carotene are being administered to thousands of volunteers in ongoing preventive studies and are being consumed by the general public in an uncontrolled undocumented fashion. Beta-carotene is being taken for its anti-cancer effects or as a precursor of (and substitute for) vitamin A, which has known intrinsic hepatotoxicity, exacerbated by ethanol. However, recent experimental studies and epidemiological observations in man revealed toxic interactions between ethanol and beta-carotene. Since moderate alcohol consumption is being advocated as possible health benefits, we determine such intake can adversely interact with beta-carotene supplementation. Established animal models in rats and nonhuman primates, as well as judicious clinical studies, will be used to determine the minimally effective dose of beta- carotene needed to restore hepatic vitamin A levels, whether this is affected by associated moderate alcohol consumption (1 or 2 drinks per day), whether beta-carotene, given in amounts commonly used at present, is influenced by moderate alcohol consumption in terms of absorption, disposition, and possible toxic manifestations, and whether the latter are exacerbated by beta-carotene beadlets. We will further ascertain the activity of the enzyme responsible for intestinal cleavage of beta-carotene is altered by chronic supplementation of beta-carotene and/or associated ethanol usage. Toxic manifestations will be assessed by measurements of circulating liver enzymes (AST, ALT) and of glutamic dehydrogenase (GDH), a marker of mitochondrial injury, as well as by ultrastructural changes in hepatic tissue. Additional experiments will be conducted to determine whether beta-carotene can attenuate the oxidant stress associated with alcohol (with or without iron), while avoiding toxic ethanol/beta-carotene interactions. Parameters of lipid peroxidation, such as F2-isoprostanes and 4-hydroxynonenal, determined by GC/MS will be assessed, Beta Carotene and other carotenoids will be measured by a state-of-the art HPLC method. Associated vitamin E changes will also be evaluated since beta-carotene may act as an antioxidant either directly (by trapping singlet-oxygen or other free radicals) or indirectly, by enhancing tissue and plasma, vitamin E. Possible synergism between beta-carotene and vitamin E supplementation will be evaluated. Ubiquinones 9, 10 (reduced and oxidized forms) shown to be affected earlier than the tocopherols in the plasma of humans exposed to oxidant stress, will also be measured by HPLC and electrochemical detection. The ultimate goal is to define the interactions between ethanol (especially moderate intake) with beta-carotene in terms of bioavailability and possible toxicity, to assess the advantages and possible disadvantages of beta-carotene substitution for vitamin A, and to formulate recommendations for optimal supplementation for that vast segment of our population drinking alc LEO MA VA MEDICAL CENTER-(151-2), 130 W KINGSBRIDGE RD, BRONX, NY 10468 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 10468 Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol laboratory rat gas chromatography mass spectrometry dosage human subject lipid peroxide hepatotoxin liver metabolism nutrient bioavailability nutrition related tag nutrient requirement dietary supplement glutamate dehydrogenase nutrient drug interaction high performance liquid chromatography carotene retinoid tocopherol vitamin metabolism enzyme activity oxidative stress clinical research CRISP RPROJ NEW YORK G CRISP/99/AA11160-03 CRISP/99/AA11160-03 199904 eng FETAL ALCOHOL AND NICOTINE INDUCED GROWTH RETARDATION Research RPROJ APPLICANT'S ABSTRACT: Fetal alcohol syndrome is a constellation of birth defects caused by maternal alcohol use during pregnancy, and is characterized by intrauterine and postnatal growth deficits, and CNS dysfunctions in the offspring. Tobacco use during pregnancy is also an established cause of fetal growth deficiency, although the toxicological effects of prenatal nicotine exposure on the CNS are not clear. Since tobacco use is highly correlated in women that abuse alcohol during pregnancy, exposure to the combination of these substances may exacerbate the deficiencies associated with alcohol or tobacco use alone. While intrauterine and postnatal growth deficiencies are the most common symptoms of fetal alcohol or tobacco exposure, the cause of these deficiencies unknown. Studies have shown a consistent long-term reduction of insulin-like growth factor-1 (IGF-1), a major mediator of developmental growth, in prenatally ethanol-exposed offspring. The goal of this application is to investigate the actions of in utero ethanol, nicotine, and ethanol/nicotine co-exposure, on the regulation of the IGF and somatotropin gene families, and assess the relationship of changes in tissue and brain IGF and GH regulation, to that of the growth and CNS deficits observed in these offspring. The hypothesis is that fetal exposure to ethanol and nicotine inhibits fetal and neonatal IGF-1 gene expression, thereby reducing tissue availability to IGF-1, and causing or exacerbating the observed growth deficits observed in these offspring. The proposed studies to test this hypothesis include: 1) Examining the effect of fetal ethanol, nicotine and alcohol/nicotine co-exposure, on plasma and somatic tissue specific IGF and GH peptide and gene regulation; 2) Assessing the effect of fetal ethanol and nicotine exposure and co-exposure on changes on CNS neurotrophic expression, with particular emphasis on the IGF and neurotrophic gene families; 3) Examining the specific actions of ethanol and nicotine exposure on growth factor induced cellular function and second messenger systems, in organ culture systems of affected tissues; and 4) Assessing changes in gene expression by differential display PCR, to identify additional candidate genes in these disorders. These studies will provide valuable data which correlate with the endocrine and neuropathological changes seen in fetal alcohol syndrome and smoking in human populations. BREESE CR AUBURN UNIVERSITY, SCHOOL OF PHARMACY, ROOM 401, AUBURN UNIVERSITY, AL 36849-5 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat second messenger polymerase chain reaction drug interaction hormone regulation /control mechanism genetic regulation postnatal growth disorder insulinlike growth factor neurotrophic factor somatotropin growth hormone releasing hormone embryo /fetus toxicology fetal alcohol syndrome prenatal growth disorder nicotine organ culture CRISP RPROJ ALABAMA G CRISP/99/AA11164-03 CRISP/99/AA11164-03 199904 eng SERTRALINE AND NALTREXONE FOR ALCOHOL DEPENDENTS Research RPROJ Naltrexone has been shown to be of significant benefit in increasing abstinence and decreasing relapse in conjuction with various psychotherapies in alcohol dependent subjects over a 12 week period. There is however a significant number of alcohol dependent subjects that do not respond to natrexone. The serotonin system has been implicated in the pathogenesis of alcoholism and various serotonergic compounds have has tried with modest success in its treatment. The aim of this double blind placebo controlled outpatient trial is to improve the abstinence and relapse rates in alcohol dependent subjects on naltrexone through the addition of sertraline, a serotonin reuptake inhibitor. We propose to recruit 124 non-depressed alcohol dependent subjects from the Substance Abuse Treatment Unit (SATU) at Yale ask them to take naltrexone for one week and then randomize them into two groups. The first group will take naltrexone 50 mg daily plus placebo for 12 weeks, and the other will take naltrexone 50 mg plus sertraline 50 mg daily, increasing to sertraline 100 mg daily after one week, and ontinue at this does, if tolerated, for a further 11 weeks. All subjects will receive weekly relapse prevention group psychotherapy at SATU. Subjects will be monitored weekly for breath alcohol and drug screens, and also for compliance psychopathology and side effects. At the end of the 13 week trial subjects will be sent for appropriate follow up treatment if required. Subjects will be followed up at 6 and 12 months. The investigators hypothesize an increase in abstinence and a decrease in relapse in the alcohol dependent subjects through a synergistic effect of the opiate antagonist and the serotonin reuptake inhibitor. FARREN CK SATU, 1 LONG WHARF, NEW HAVEN, CT 06511 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 06511 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse prevention alcoholism /alcohol abuse chemotherapy naltrexone glutamyltransferase clinical trial relapse /recurrence combination chemotherapy drug abuse drug adverse effect outpatient care human subject longitudinal human study psychotherapy placebo therapy compliance human therapy evaluation drug testing sertraline clinical research behavioral /social science research tag outcomes research CRISP RPROJ CONNECTICUT G CRISP/99/AA11222-02 CRISP/99/AA11222-02 199904 eng RAT MODEL OF ALCOHOLIC PANCREATITIS Research RPROJ Alcohol abuse is the most important cause of chronic pancreatitis, an inflammatory process characterized by destruction of the exocrine pancreas, fibrosis and ductular stenosis. While acute pancreatitis has been studied extensively, mechanisms responsible for chronic alcoholic pancreatitis are largely unknown in large part due to the lack of an appropriate animal model. Previous work in cultured cells and in liver has provided strong evidence for the role of free radicals in increased expression of extracellular matrix genes and subsequent increased production of type I and fibrillar collagen, the same types observed clinically in alcoholic pancreatitis. In exciting new pilot work, we have developed a unique method to collect pancreatic juice and analyze it for free radicals. After only one month of exposure of rats to enteral ethanol using the Tsukamoto-French model, clear evidence for production of a-hydroxy ethyl radical from ethanol was obtained. This occurred without evidence of tissue injury or changes in rates of collagen mRNA formation. Thus, free radical formation in the pancreas is an early event following chronic exposure to ethanol. Although the Tsukamoto-French model has been used to study alcoholic: pancreatitis; the frequency of pathology was too low to make it a useful model for mechanistic studies. Therefore, the primary goal of this research is to develop a useful model of alcoholic pancreatitis. This goal will be achieved by evaluation of three separate modifications of the Tsukamoto-French enteral ethanol delivery protocol: I) Addition of carbonyl iron to the diet; 2)chronic injection of the CCK analog, caerulein; 3) administration of an adenovirus vector expressing TGFbeta1 via the pancreatic duct. At one, two and five months after the above treatments, free radicals will be evaluated using the shin trapping technique and EPR, while pancreatic pathology will be assessed from histology, pancreatic enzymes, cellular atrophy, collagen protein and mRNA levels. Any procedure causing significant chronic elevation in enzymes 3-fold above basal with a pancreatic histology score of +2 or greater will be judged as successful. We expect these important studies to lead to the development of a new clinically relevant animal model of chronic alcoholic pancreatitis which is required for studies on mechanism and development of new therapeutic strategies. BRENNER DA UNIV OF NC @ CHAPEL HILL, CB #7080, CHAPEL HILL, NC 27599-7080 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat carbonyl compound electron spin resonance spectroscopy cholecystokinin transforming growth factor model design /development disease model dietary iron nutrition related tag pancreatitis toxicology Adenoviridae iron compound transfection vector CRISP RPROJ NORTH CAROLINA G CRISP/99/AA11226-02 CRISP/99/AA11226-02 199904 eng ABUSE LIABILITY OF ETHANOL AS A HYNOTIC BY INSOMNIACS Research RPROJ Chronic insomnia is reported by 10 percent of the population and a large percentage report self-medication using alcohol. Our preliminary data indicate that insomniacs are more likely to self-administer ethanol before bedtime than are non-insomniacs despite similar levels of moderate to light daytime social drinking. However, there is no information regarding the effects of ethanol on the sleep of insomniacs and the data regarding ethanol effects in normals and in alcoholics have used high ethanol doses and may not generalize to insomniacs. Critically, there is no information about the reinforcing properties of ethanol when self administered at bedtime by insomniacs. Do insomniacs use ethanol before sleep to improve their sleep, mood, or both and does that in turn increase daytime ethanol intake? Three experiments are proposed. They will employ standard sleep laboratory methods to diagnose insomnia and document the effects of ethanol on sleep and will use behavioral self-administration and subjective mood and drug effect assessments to determine the reinforcing and subjective effects of ethanol in insomniacs. During the first year, the study will focus on the question of whether, at what doses, and for how long ethanol has effects on sleep and mood. In year two, the study will assess how hypnotic ethanol self administration in insomniacs relates to ethanol dose, duration of nightly effects, and to the presence of an insomnia complaint versus an objective sleep disturbance. In the last two years, the grant focus will be on the extent to which hypnotic ethanol self-administration generalizes to daytime ethanol use. ROEHRS TA CWRU HENRY FORD HLTH SCI CENTE, 1 FORD PLACE, 1D, DETROIT, MI 48202 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48202 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse ethanol drug administration rate /duration self medication pharmacokinetics drug adverse effect human subject sleep disorder sleep REM sleep behavior modification emotion sedative /hypnotic placebo clinical research substance abuse epidemiology CRISP RPROJ MICHIGAN G CRISP/99/AA11264-01A1 CRISP/99/AA11264-01A1 199904 eng ETHANOL EFFECTS ON GLUTAMATE RECEPTORS Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) The excitatory amino acid pathways in the brain are widespread and are involved in synaptic plastic events such as long-term potentiation in the hippocampus. Long-term potentiation is thought to be part of the process of learning and memory. Excitatory amino acids are thought to also play a role in excitotoxicity as related to seizures and may be involved in ethanol withdrawal induced seizures. At least two glutamatergic receptor systems are thought to regulate long-term potentiation in the hippocampus. These receptor systems are the metabotropic receptors and the NMDA receptor complex. The metabotropic receptors are G-protein linked receptors that are coupled to several intracellular enzymes such as phospholipase C. The NMDA receptor is a complex of several protein subunits that form a glutamate gated ion channel that has high permeability for calcium. Each subunit imparts a different character to the NMDA receptor function. Acute and chronic treatment with ethanol alters the function of these receptors. Prenatal exposure to ethanol can affect the offspring even when they are quite old. Alteration of these receptors in the hippocampus by ethanol can influence the process of learning and memory. We now know that chronic ethanol treatment increases the function of NMDA receptor in the hippocampus and that an increase in the protein for NR1 NMDA subunit is observed as well as an increase in binding sites for NMDA. Prenatal exposure to ethanol causes a decrease in both NMDA receptor function and metabotropic receptor function. The specific aims of these investigations are to examine what metabotropic receptor subtypes and/or what NMDA receptor subunits change after chronic ethanol exposure or prenatal ethanol exposure and how these changes in protein expression correllate with functional chages. We will be using receptor subtype and subunit specific antibodies to identify and quantify metabotropic receptor subtypes and NMDA receptor NR1 splice variants and NR2 subunits that may change after such an insult. My long-term goal is to understand how differences in the expression of metabotropic receptors and NMDA receptor subunits occur during chronic and prenatal ethanol treatments. These studies will help us to understand some of the underlying mechanism in fetal alcohol syndrome as well as the effects of chronic alcoholism. YASUDA RP GEORGETOWN UNIVERSITY MED CTR, 3900 RESERVOIR ROAD NW, WASHINGTON, DC 20007 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 20007 Crisp Data Base National Institutes Of Health ethanol laboratory rat hippocampus brain mapping neural plasticity neurotoxin embryo /fetus toxicology prenatal stress protein structure /function receptor binding glutamate receptor NMDA receptor long term potentiation CRISP RPROJ DIST OF COL G CRISP/99/AA11284-02 CRISP/99/AA11284-02 199904 eng ETHANOL OXIDATION EFFECTS ON EGF SIGNAL TRANSDUCTION Research RPROJ DESCRIPTION: (Adapted from Investigator's Abstract) One of the more pronounced hepatic dysfunctions observed in animals chronically fed ethanol is a impairment in receptor-mediated endocytosis. Receptor-mediated endocytosis is responsible for the uptake and degradation of many biologically important molecules including hormones, growth factors and cytokines. Receptor-mediated endocytosis is also involved in the regulation of the expression of surface receptors and signal transduction. Thus, alterations in hepatic receptor-mediated endocytosis could be detrimental to the liver. Although, impairment in receptor-mediated endocytosis is well established in hepatocytes isolated from ethanol fed animals, the mechanism(s) of this impairment have yet to be elucidated. Furthermore, the direct involvement of ethanol metabolism in this defect has yet to be demonstrated. Many mechanisms for the hepatic cell injury associated with chronic ethanol abuse have been proposed. One attractive, though unproven mechanism, proposes that acetaldehyde forms acetaldehyde-protein adducts and through their accumulation eventually causes hepatic dysfunction. One of the main reasons that these underlying mechanisms of chronic ethanol oxidation have not been determined has been the lack of a stable in vitro hepatic model system for the study of chronic ethanol oxidation. The PI recently developed a cell line of hepatic origin that metabolized ethanol to acetaldehyde and produces acetaldehyde-protein adducts and proposes to use this cell line (HAD cells) to investigate impairments in epidermal growth factor signal transduction. The hypothesis is that alcohol dehydrogenase mediated oxidation of ethanol impairs the biological activities of the epidermal growth factor receptor. Inactivation of this receptor dramatically alters the ability of hepatocytes to respond appropriately to extracellular signals. Epidermal growth factor is an important hepatic mitogen. The binding of epidermal growth factor to its receptor activates an intrinsic tyrosine kinase, which initiates a signal transduction cascade resulting in cell proliferation. The Specific Aims of this proposal are: 1) Determine what processes of epidermal growth factor receptor-mediated endocytosis are impaired by ethanol oxidation and exposure to acetaldehyde; 2) Investigate the effect of ethanol oxidation on the signal transduction pathway of the epidermal growth factor receptor in the recombinant HAD cells; 3) Investigate the possibility that acetaldehyde-protein adducts are responsible for the observed ethanol induced impairment of the epidermal growth factor receptor; and 4) Investigate the effects of ethanol oxidation on the mitogenic activities of epidermal growth factor in the recombinant HAD cells. By completing these studies, it is hoped that the investigators can determine the effects of chronic ethanol oxidation and exposure to acetaldehyde on the signal transduction of epidermal growt CLEMENS DL VA MEDICAL CENTER, 4101 WOOLWORTH AVE, OMAHA, NE 68105 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 68105 Crisp Data Base National Institutes Of Health ethanol acetaldehyde biological signal transduction cell growth regulation receptor mediated endocytosis cellular pathology epidermal growth factor liver cell hepatotoxin oxidation /reduction alcohol dehydrogenase toxin metabolism cell line CRISP RPROJ NEBRASKA G CRISP/99/AA11291-02 CRISP/99/AA11291-02 199904 eng DORSAL RAPHE NUCLEUS SEROTONIN NEURONS IN ALCOHOLISM Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) Serotonin deficiency in alcoholics is hypothesized based principally on observations of reduced serotonin metabolites in the cerebrospinal fluid, the efficacy of serotonin agonists and uptake inhibitors in reducing alcohol consumption in humans and animal models and biochemical abnormalities in alcohol preferring rat strains. Preliminary postmortem evidence is presented of a loss of serotonin-synthesizing neurons in the dorsal raphe nucleus (DRN) and a decrease in the number of serotonin nerve terminals in the prefrontal cortex of alcoholics. A comprehensive postmortem study of the serotonin system is proposed to determine the changes in the serotonin system in alcoholics. To further evaluate the significance of any findings we will determine whether these changes are correlated with the duration or severity of alcohol dependence. The proposal has been extensively modified to meet the criticisms of the previous review. The brains of 20 subjects meeting DSM-IV criteria for alcohol abuse or dependence and 20 nonpsychiatric controls will be examined. Alcohol abuse/dependence will be diagnosed by psychological autopsy. Computer-assisted stereology and morphometry of DRN serotonin neurons labeled with antiphenylalanine hydroxylase antibodies in alcoholics and non-psychiatric controls will determine: the number, density, distribution and morphology of DRN serotonergic neurons as measures of neurodegeneration. In the ventrolateral prefrontal cortex, primary motor cortex and visual cortex, measurement of serotonin transporter sites and 5-HT1D sites, 5-HT1A and 5-HT2A receptors and the density of neurons and astrocytes will characterize target neuron integrity. Biological changes related to the duration or severity of alcoholism are consistent with alcohol toxicity; whereas changes independent of duration or severity of alcoholism but related to the age of onset might suggest a biological predisposition to alcoholism. The data gathered will suggest mechanisms involved in the pathogenesis of alcoholism. The demonstration of serotonergic neuropathology in the brain of alcoholics may suggest possible pharmacologic therapeutics and new diagnostic approaches using functional brain imaging. UNDERWOOD MD RES FDN FOR MENTAL HYGIENE, IN, 722 WEST 168TH STREET, NEW YORK, NY 10032 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 10032 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse serotonin serotonin inhibitor dorsal raphe nucleus brain mapping postmortem histopathology human subject human tissue interview neurotransmitter metabolism neurotransmitter transport neurotoxicology serotonin transporter clinical research neuropathology CRISP RPROJ NEW YORK G CRISP/99/AA11293-02 CRISP/99/AA11293-02 199904 eng EFFECTS OF CHRONIC ALCOHOL ABUSE ON THE AGING BRAIN Research RPROJ The primary goal of this proposal is to test two opposing models of how age modulates the effects of chronic alcohol abuse on CNS function and structure in humans: the age-related vulnerability model vs. the cumulative effects model. Both attempt to explain the fact that the morbid CNS effects of alcohol abuse are greatest in the elderly alcoholic, independent of duration and total amount of alcohol consumption. If one acknowledges that the younger brain can compensate/mask damage done by alcohol until the more than linear losses of normal aging make that impossible, it follows that the functional consequences of the cumulative effects of age and alcohol abuse would be much more apparent in the older individual, even if the person quit abusing alcohol before the onset of old age. There might be a threshold for CNS change, where persistent (or permanent) functional or structural change is only apparent if this threshold is surpassed. Indeed, the literature suggests that permanent CNS damage is only rarely evident in (the most severely affected) young adult or middle aged alcoholics. We will test these two opposing models using state-of-the-art structural brain imaging, (supplemented with magnetization transfer imaging to specifically quantitate changes in the white matter), electrophysiological and neuropsychological assessment procedures. The study will use a cross- sectional design with five study samples of 30 males and 30 females each: (1) 50-55 year old chronic (25-40 years of abuse) alcoholics abstinent 15 months - 4 years, (2) 65-70 year old chronic alcoholics abstinent at least since age 55, (3) 65-70 year old chronic alcoholics abstinent 15 months - 4 years, (4) 50-55 year old lifetime light/non-drinking controls, and (5) 65-70 year old lifetime light/non drinking controls. A secondary, aim of this project is to determine whether there are gender differences in the effects of chronic alcohol abuse on CNS function and in the manner in which age modulates these effects. A potential bias in this study exists if an individual's ability to remain abstinent is associated with the extent of CNS morbidity. The requirement for one year of abstinence in the alcohol abuser samples could result in samples biased toward individuals with less morbid CNS effects of chronic alcohol abuse. To the extent that CNS morbidity of chronic alcohol abuse is associated with age, this bias would be greatest in individuals who became abstinent in their 60s. To assess this bias and (as a secondary aim) to determine recovery of function and brain structure during a year of abstinence in elderly individuals, we will study a sixth sample of 40 male and 40 female 65-70 year old chronic alcoholics in very early abstinence (I-e., at the end of a 28 day treatment program) and will follow them for 15 months with regard to their maintenance of abstinence, with abstinent individuals restudied at the end of the 15 month interval. FEIN G SAN FRANCISCO VA MEDICAL CENTE, 4150 CLEMENT STREET, SAN FRANCISCO, CA 94121 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94121 Crisp Data Base National Institutes Of Health human middle age (35-64) human old age (65+) alcoholic beverage consumption alcoholism /alcohol abuse chronic brain damage electroencephalography brain visualization disease /disorder proneness /risk chronic disease /disorder electrophysiology human subject interview neuropsychological test sex difference age difference neurotoxicology clinical research CRISP RPROJ CALIFORNIA G CRISP/99/AA11311-02 CRISP/99/AA11311-02 199904 eng INHIBITION OF CELL PROLIFERATION BY ETHANOL Research RPROJ DESCRIPTION (Adapted from the Investigator's Abstract): The habitual consumption of even moderate quantities of alcoholic beverages (one to two drinks per day) can result in a host of abnormal clinical, biochemical, and physiologic findings that stem from the toxic effects of alcohol on the liver, marrow, brain and skeleton among others. A pathological effect of ethanol on cellular proliferation has been described in all of these target tissues. Yet, the investigation of alcohol-induced growth suppression at the cellular level has, thus far, failed to uncover the mechanism of this adverse effect. The PI's laboratory has confirmed the inhibitory effect of clinically relevant concentrations of ethanol on cell proliferation in vitro in a well-defined osteoblast model system. Additional study of this effect revealed that the growth suppression induced by ethanol was paralleled by suppression of mitogen-activated protein (MAP) kinase activity. The MAP kinase protein phosphorylation cascade plays an essential role in triggering cell proliferation and activation of MAP kinase occurs through successive stimulation of specific proteins in the Ras activation pathway (Ras, Raf-1, and MAP kinase). However, ethanol exerts no inhibitory effect on MAP kinase activity in cells overexpressing a constitutively active Ras gene. These observations indicate that ethanol interferes with an important site in the proliferation pathway that is proximal to the action of Ras. Ras activation is necessary for mitogenesis induced by the receptor tyrosine kinases and, recently, the involvement of adapter molecules and GTP exchange proteins for Ras has been experimentally described. Based on these intriguing observations, it is hypothesized that ethanol inhibits cellular proliferation by interfering with a specific site in the tyrosine kinase phosphorylation pathway that leads to the activation of Ras. The specific objectives of this proposal are : (1) to characterize the intracellular signaling pathway necessary for growth factor-dependent activation of Ras in the osteoblast(s) that is perturbed by ethanol; (2) to identify the site(s) of in the growth factor receptor tyrosine kinase pathway affected by ethanol; (3) to determine the molecular mechanism(s) by which ethanol interferes with growth factor tyrosine kinase signaling pathway in the osteoblast. Ethanol inhibits growth in a wide variety of tissues and the IGF axis is ubiquitous, regulating growth and development of all cell types. Our observations in this model bone cell system are likely to provide fresh insights into the anti-proliferative actions of ethanol in general. KLEIN RF PORTLAND VA MEDICAL CENTER, 3710 SW US VETERANS HOSPITAL R, PORTLAND, OR 97201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 97201 Crisp Data Base National Institutes Of Health ethanol laboratory rat biological signal transduction drug adverse effect gene expression insulinlike growth factor growth inhibitor human tissue molecular biology protein tyrosine kinase guanine nucleotide binding protein growth factor receptor osteoblast tissue /cell culture animal tissue cell line enzyme activity mitogen activated protein kinase cell proliferation CRISP RPROJ OREGON G CRISP/99/AA11317-02 CRISP/99/AA11317-02 199904 eng FETAL ALCOHOL EXPOSURE AND BRAIN DEVELOPMENT Research RPROJ DESCRIPTION: The corpus callosum (CC) can be absent (agenesis) in children with fetal alcohol syndrome (FAS). Preliminary data show that the CC is reduced, and surviving connections are abnormal, in rats with fetal alcohol exposure. Preliminary data also show that CC development is significantly abnormal following moderate or heavy level, during first or second human trimester equivalent in utero alcohol exposure. Studies in humans have shown that CC abnormalities are correlated with affective and/or developmental disorders. Previous studies have shown that normal development of transitory CC connections in cat is critical for normal functional development of the visual system. Rat data show that transitory CC connections are also present for auditory and somatosensory cortex. Cat and rat results show that CC development includes a transitory extension of many axons and dendrites into all neocortical layers. These cortical CC connections are restructured during a finite period of development that is characteristic for each species. Thus, many of the neurological and psychological consequences of fetal alcohol exposure in humans may be linked to negative consequences of altered CC development. The primary goals of this proposal are to determine the extent to which the rat CC is a model for the affects of fetal alcohol exposure in humans, and to determine if there is a critical threshold and time period for alcohol exposure to produce CC abnormalities. Alcohol doses that produce heavy, moderate and light blood alcohol concentrations (BACs) in pregnant mothers will be tested. Alcohol exposure during human trimester equivalents 1, 2, and 1-2 will be tested in rat. Preliminary data show that each BAC/timing combination leads to differential consequences for CC development. The proposed studies will determine the nature and consistency of the CC abnormality related to timing and BAC level, and quantify the effects on CC development. CC axonal and dendritic morphology, as well as CC cellular polarity and orientation, will be quantified; preliminary data show that all these parameters of CC development are altered by prenatal alcohol exposure. Specific parameters of abnormal CC development will be correlated with different doses and timing of alcohol exposure. The effect of alcohol on initial generation of CC cells will also be determined to provide an index of the consistency of prenatal alcohol effects in humans and rats, as well as determine whether alcohol produces CC abnormalities by indirect, as well as direct, effects on CC cells. These investigations will evaluate the effectiveness of the rat CC model for human FAS and fetal alcohol effects (FAE), also called alcohol related birth defects (ARBD). The results will also provide extensive information that will be useful for studying possible mechanisms by which fetal alcohol exposure affects the developing brain. The results of the proposed studies will be clinically relevant in providing detailed data by which to evaluate the timing and extent of alcohol exposure on fetal development, and may be used by health care professionals in counseling pregnant women on the potential effects of alcohol consumption. ELBERGER AJ UNIVERSITY OF TENNESSEE, 855 MONROE AVE, MEMPHIS, TN 38163 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 38163 Crisp Data Base National Institutes Of Health ethanol laboratory rat corpus callosum dosage histology model design /development disease model developmental neurobiology embryo /fetus toxicology fetal alcohol syndrome prenatal growth disorder confocal scanning microscopy CRISP RPROJ TENNESSEE G CRISP/99/AA11325-02 CRISP/99/AA11325-02 199904 eng ARND--IMPACT ON SYNAPTIC PLASTICITY MECHANISMS Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) Learning disabilities are among the most subtle yet most pervasive deficits related to prenatal alcohol exposure in children. These learning deficits, which may not become apparent until a child is school-aged, can occur in the absence of other physical evidence of alcohol-related birth defects. Offspring of rats exposed to moderate levels of alcohol during gestation also show a significant impairment in learning when tested as young adults, validating the use of this animal model in studies of the effect of fetal alcohol exposure (FAE). Initial studies by our integrative research program suggested that these deficits are related to specific alterations in synaptic plasticity mechanisms which correlated with a failure in the establishment of long-term potentiation (LTP). The long-term objectives of this Integrated Research Program Grant (IRPG) are two-fold: 1)to delineate more clearly the molecular and neurochemical alterations of synaptic plasticity mechanisms caused by prenatal alcohol exposure and 2) to explore new treatment strategies to overcome these deficits. The overall hypothesis for the IRPG is that prenatal exposure to moderate levels of ethanol produces multiple defects in the mechanisms underlying glutamate levels of ethanol produces multiple defects in the mechanisms underlying glutamate receptor-dependent LTP in the hippocampus and medial frontal cortex. The specific goal of Project 3 in this IRPG is to define the impact of FAE on the levels and function of proteins involved in synaptic plasticity mechanisms. Based upon preliminary studies, our hypothesis is that protein kinase C (PKC) activity and the levels and phosphorylation of GAP-43 and other important plasticity-associated proteins is altered in specific brain regions of FAE rats. To test this idea, we propose the following Specific Aims: 1)to study PKC activity and the phosphorylation of GAP-43 and other PKC substrate proteins in the hippocampus and medial frontal cortex of FAE rats, both under basal conditions and after electrical stimulation 2)to examine the impact of FAE on activity-dependent changes in GAP-43 phosphorylation and gene expression during behavioral conditioning, 3) to characterize the causes for the deficit in PKC activity in FAE rats and to evaluate its significance in synaptic plasticity mechanisms and 4) to investigate the effects of different pharmacological treatments on PKC activity and GAP-43 phosphorylation in control and FAE rats and 5) to relate these to the behavioral and electrophysicological properties of the animals. The identification of the neurochemical basis for the alterations in synaptic plasticity in the hippocampus and medial frontal cortices of FAE rats will improve our understanding of the effects of prenatal alcohol exposure in the function of these important brain structures. Ultimately, this information will help design better therapeutic strategies PERRONE-BIZZOZERO NI UNIV OF NEW MEXICO HLTH SCI CT, 915 CAMINO DE SALUD, NE, ALBUQUERQUE, NM 87131-5221 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat frontal lobe /cortex hippocampus developmental genetics gene induction /repression synapse synaptogenesis neuropharmacology neural plasticity phosphorylation protein kinase C embryo /fetus toxicology fetal alcohol syndrome ethology conditioning animal developmental psychology psychopharmacology neural growth associated protein enzyme activity animal genetic material tag behavioral /social science research tag CRISP RPROJ NEW MEXICO G CRISP/99/AA11336-01A1 CRISP/99/AA11336-01A1 199904 eng ALCOHOL AND BRAIN INTERLEUKIN 1 Research RPROJ APPLICANT S ABSTRACT: Alcohol (ethanol-ETOH) abuse produces pathological effects including various brain dysfunction and immune impairment associated with increased susceptibility to various infections. One central abnormality is EtOH activation of the hypothalamic-pituitary-adrenal (HPA) axis. Although stimulation of hypothalamic corticotropin-releasing hormone (CRH) producing neurons is critical for EtOH-induced HPA activation, a precise understanding of how this occurs remains to be established. Of potential relevance, interleukin (IL)-l potently stimulates the HPA axis, is produced in hypothalamus, and stimulates common cellular signals as EtOH. Preliminary results in our laboratory show that EtOH increases IL-1beta mRNA in primary hypothalamic neuronal and glial cultures. Thus, EtOH could activate the HPA axis by increasing hypothalamic IL-1 signaling. The proposed hypothesis is that EtOH induces hypothalamic IL-1, in vitro and in vivo studies will demonstrate: 1) effects of acute and chronic EtOH on expression of IL-1beta mRNA(RNase protection assay) and IL-1beta (immunocytochemistry-ICC) and on immune/cytokine stimulation in fetal rat hypothalamic neuronal, astrocytic and microglial cultures, and 2) effects of an EtOH diet on adult rat hypothalamic IL-1beta mRNA and IL-1beta and HPA axis activation. HPA axis activation occurs in alcoholics and can cause peripheral immunosuppression via glucocorticoid-dependent and -independent mechanisms. Recognizing that central effects of IL-1 can also cause peripheral immunosuppression via both these pathways, EtOH induction of brain IL-1 could lead to immunosuppression via these central mechanisms. The well recognized impaired immunity in alcoholics could be further attenuated by mechanisms involving central cytokine dysfunction which could contribute to the high prevalence of HIV infection in alcoholics and progression to AIDS. Long term goals are to learn about mechanisms mediating EtOH effects on the brain IL-1 system and to determine the role and significance of these alterations relative to EtOH-induced HPA axis activation and peripheral immunosuppression. SELMANOFF MP UNIV OF MD @ BALTIMORE, 655 WEST BALTIMORE STREET, BALTIMORE, MD 21201-1559 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol laboratory rat hypothalamus gene induction /repression interleukin 1 immunocytochemistry astrocyte microglia neuron nutrition related tag hypothalamic pituitary axis embryo /fetus toxicology embryo /fetus cell culture artificial immunosuppression neuroimmunomodulation RNase protection assay CRISP RPROJ MARYLAND G CRISP/99/AA11415-02 CRISP/99/AA11415-02 199904 eng ETHANOL AND NEURONAL DEVELOPMENT Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) The long term objectives of this research are to identify and characterize the cellular mechanisms underlying ethanol induced changes in neuronal development, and to assess the role of these changes in the etiology of CNS abnormalities associated with Fetal Alcohol Syndrome (FAS). Key neuropathologic features of FAS include altered neuronal morphogenesis and synapse formation in the hippocampus. Recent studies have elucidated some of the molecules and processes responsible for the distinct growth characteristics of axons and dendrites, thus providing the basis for novel experiments to determine the mechanisms underlying ethanol's disruption of neuronal development. The use of primary cultures of embryonic rat hippocampal pyramidal neurons is integral to the objectives of the project. Neurons in these cultures develop axons, dendrites, and synapses in a sequence of events that mimics their development in vivo. Experiments in this proposal are designed to use immunofluorescent cytochemistry coupled with quantitative morphometric analysis, time lapse videomicroscopy, and Fura-2 intracellular free calcium measurements in the following specific aims: (1) Compare the sensitivity of cultured hippocampal neurons exposed to ethanol at different times relative to development of axons, dendrites and synapses, (2) Distinguish whether ethanol induced changes in neuronal development result from direct effects of ethanol on neurons, or indirect effects on neurons mediated by astrocytes, (3) Determine whether ethanol's effects on process outgrowth involve altered regulation of intracellular calcium levels. The results of these studies will establish whether a disruption of process outgrowth and molecular compartmentalization is a key aspect of ethanol's neurodevelopmental toxicity and will provide important insight into the mechanism(s) underlying these actions. This fundamental knowledge can be expected to provide a basis for improving the identification and treatment of affected individuals. FLETCHER TL ALBANY MEDICAL COLLEGE, 47 NEW SCOTLAND AVE, ALBANY, NY 12208 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 12208 Crisp Data Base National Institutes Of Health ethanol laboratory rat hippocampus cell cell interaction neurogenesis astrocyte neuron developmental neurobiology embryo /fetus toxicology fetal alcohol syndrome tissue /cell culture embryo /fetus cell culture video microscopy neurotoxicology CRISP RPROJ NEW YORK G CRISP/99/AA11416-01A1 CRISP/99/AA11416-01A1 199904 eng ANTIBODIES TO SIGNALING PROTEINS ALTERED BY ETHANOL Research RPROJ DESCRIPTION (APPLICANT'S ABSTRACT): Both acute and chronic ingestion of ethanol result in multiple effects in the central nervous system (CNS) as well as in a variety of other organs such as liver and heart. In the CNS, there are both short and long term effects of chronic alcohol consumption such as intoxication, memory loss, tolerance to the acute intoxicating effects of ethanol, addiction, and dependence. The pleiotropic effects of ethanol exposure can be also observed at a molecular level; amounts and activities of components of several signal transduction systems are altered as a result of exposure to ethanol in a time- and dose-dependent manner. The molecular targets for ethanol and the mechanism by which ethanol-induced effects occur are largely unknown. However, studies from several laboratories indicate that the effects of ethanol are specific.This proposal is focused on protein kinase C (PKC) signaling pathway, a key pathway in the CNS. Short- and long-term exposures to ethanol specifically alter the level and activity of components of this key signal transduction system. There are multiple PKC isozymes that regulate important cellular functions and preliminary studies using a model of neuronal cells in culture suggest that ethanol exposure also results in changes in the subcellular localization of a specific PKC isozyme and its anchoring protein, RACK. The study of ethanol-induced changes in the CNS and their functional consequences is a particular challenge because individual brain regions and various cell types in each region express different PKC isozymes and have different sensitivities to ethanol. Here the investigator describes her plan to devise tools to identify the effects of ethanol in individual cells in situ. They will raise novel monoclonal antibodies (mcAbs) that will distinguish between states of activity of individual PKC isozymes as well as mcAbs to RACK. These mcAbs will be used in immunocytochemical studies of model neuronal cell culture systems as well as in brain of control and ethanol-treated mice. Such mcAbs will allow, for the first time, the determination of how ethanol affects the localization and activity of the components of this key signal transduction system in specific regions and cells in the brain. These studies will help elucidate the molecular basis of ethanol effects and alcoholism in man. MOCHLY-ROSEN DD STANFORD UNIV SCHOOL OF MED, STANFORD, CA 94305-5332 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol laboratory mouse laboratory rat biological signal transduction hybridoma isozyme immunocytochemistry monoclonal antibody enzyme linked immunosorbent assay western blotting immunofluorescence technique immunoprecipitation neuron protein kinase C physical /chemical interaction cytotoxicity enzyme activity CRISP RPROJ CALIFORNIA G CRISP/99/AA11417-03 CRISP/99/AA11417-03 199904 eng ETHANOL EFFECTS IN CNS TISSUE IN LAS AND HAS RATS Research RPROJ An understanding of neuronal mechanisms which mediate the behavioral effects of ethanol is critical to the understanding of alcohol intoxication, alcohol abuse, and alcoholism. The influences of the noradrenergic innervation on neuronal function may well regulate alterations in neuronal responsiveness to ethanol with e.g. stress or arousal, and the phenotype of the individual in terms of neuronal sensitivity to ethanol and regulation of neuronal ethanol effects by central sympathetic neurons will likely influence an individual's response to, and abuse of, alcohol in those behavioral states. The proposed studies will focus on LAS and HAS rats, which are genetic variants that differentially manifest an alcohol-related behavior, as a model system for examining acute ethanol interactions with neurotransmitter actions norepinephrine and GABA in the cerebellum, and rapid acute neuronal tolerance (RANT) to those effects. The effects of ethanol exposure on mammalian central nervous system function will be pursued using extracellular recordings of action potential activity of single neurons, whole-cell patch clamp recordings GABAA/C1 channel function, and in vivo electrochemistry to monitor presynaptic release and uptake of norepinephrine and other monoamines. Recordings will be carried out in rat brain in vivo and in brain slices in vitro, and drugs and transmitters will be applied by pathway activation, superfusion (in vitro), systemic administration (in vivo) and locally to the microenvironment of the cell from multibarrel micropipettes. The long- term objectives of this research program are three fold. First, to identify neuronal ethanol actions that are relevant to the behavioral ethanol sensitivities bred into LAS and HAS rats. Second, to characterize the mechanisms of these ethanol actions. Third, to characterize changes in neuronal responses to ethanol, as well as alterations in the function of neurotransmitter systems, which occur with the induction of RANT, explore the neuronal mechanisms of those changes and to determine the role of this phenomenon in the mechanisms of acute ethanol sensitivity. The specific aims for this project period are as follows: 1) To investigate the hypothesis that neuronal EtOH sensitivity and RANT in the cerebellum are behaviorally relevant EtOH phenotypes. 2) To determine if RANT to EtOH in the cerebellum is mediated by a desensitization of b-adrenergic mechanisms. 3) To study the presynaptic role of cerebellar catecholamine synapses in RANT. PALMER MR UNIV OF COLORADO HLTH SCI CTR, 4200 EAST NINTH AVE, C236, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 80262 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol gamma aminobutyrate laboratory rat genetic strain cerebellum brain metabolism drug tolerance patch clamp synapse neurophysiology neurotoxin norepinephrine psychic activity level behavioral genetics behavioral habituation /sensitization stress behavioral /social science research tag CRISP RPROJ COLORADO G CRISP/99/AA11465-01A2 CRISP/99/AA11465-01A2 199904 eng MAGNETIC RESONANCE STRUCTURAL AND PERFUSION IMAGERY Research DEICKEN R SAN FRANCISCO VA MEDICAL CENTE, 4150 CLEMENT STREET, SAN FRANCISCO, CA 94121 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94121 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse membrane permeability brain metabolism pathologic process dosage pharmacokinetics human subject neuroanatomy magnetic resonance imaging longitudinal human study human morbidity neurotoxicology clinical research AIDS neuropathy CRISP RPROJ CALIFORNIA G CRISP/99/AA11493-01A10001 CRISP/99/AA11493-01A10001 199904 eng MAGNETIC RESONANCE SPECTROSCOPIC IMAGING Research MYERHOFF D SAN FRANCISCO VA MEDICAL CENTE, 4150 CLEMENT STREET, SAN FRANCISCO, CA 94121 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94121 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse brain metabolism pathologic process dosage human subject magnetic resonance imaging longitudinal human study human morbidity neurotoxicology clinical research AIDS neuropathy CRISP RPROJ CALIFORNIA G CRISP/99/AA11493-01A10002 CRISP/99/AA11493-01A10002 199904 eng ELECTROPHYSIOLOGY Research FEIN G SAN FRANCISCO VA MEDICAL CENTE, 4150 CLEMENT STREET, SAN FRANCISCO, CA 94121 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94121 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse brain electrical activity electroencephalography intercellular connection pathologic process dosage evoked potential human subject HIV infection neural information processing longitudinal human study human morbidity stimulus /response neurotoxicology clinical research AIDS neuropathy CRISP RPROJ CALIFORNIA G CRISP/99/AA11493-01A10003 CRISP/99/AA11493-01A10003 199904 eng PERIPHERAL NEUROPATHY Research OLNEY RK SAN FRANCISCO VA MEDICAL CENTE, 4150 CLEMENT STREET, SAN FRANCISCO, CA 94121 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94121 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse postural hypotension blood pressure pathologic process dosage heart rate human subject polyneuritis neurophysiology neural conduction longitudinal human study human morbidity neuropsychological test sensory discrimination vibration perception temperature neurotoxicology clinical research AIDS neuropathy CRISP RPROJ CALIFORNIA G CRISP/99/AA11493-01A10004 CRISP/99/AA11493-01A10004 199904 eng PHARMACOKINETICS AND PHARMACODYNAMICS Research GAMBERTOGLIO J SAN FRANCISCO VA MEDICAL CENTE, 4150 CLEMENT STREET, SAN FRANCISCO, CA 94121 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94121 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse ethanol antiviral agent pharmacokinetics drug metabolism drug interaction protease inhibitor human subject HIV infection antiAIDS agent cerebrospinal fluid neurotoxicology clinical research AIDS neuropathy CRISP RPROJ CALIFORNIA G CRISP/99/AA11493-01A10005 CRISP/99/AA11493-01A10005 199904 eng EXCITOTOXIC MECHANISMS OF ETHANOL--NMDA RECEPTOR FUNCTION Research RPROJ DESCRIPTION: Evidence from several independent laboratories using different experimental approaches indicates that acute ethanol selectively inhibits N-methyl-D-aspartate receptor (NR) function at pharmacologically relevant concentrations, whereas chronic ethanol produces NR supersensitivity due, at least in part, to receptor upregulation. As a consequence, excessive activation of NRs during alcohol withdrawal is thought to contribute to the associated seizures, autonomic instability and neurotoxicity. Pharmacologic treatment with available NR antagonists is undesirable due to generalized inhibition of these ubiquitous receptors. However, NRs are functionally diverse due to differences in subunit composition and associated signal transduction systems. This diversity may account for the selective effects of ethanol on NRs in different brain regions, and in different neuronal populations within a particular region. By exploring the mechanistic basis for this regional selectivity, it should be possible to design and utilize in the alcoholic patient more selective antagonists that are targeted to vulnerable neuronal populations. To further define the role of NRs in chronic ethanol-induced neurotoxicity in hippocampus, the following hypotheses will be tested in adult rats: (I) increased levels of specific NR subunits, and possibly mRNAs, will be observed in hippocampal subfields (CA1, CA3, DG) of ethanol-treated rats, relative to pair-fed control rats, in a manner that corresponds to development of ethanol dependence. Moreover, increased levels of NR subunits will be positively correlated with increased expression or activity of a type II Ca2+/calmodulin-dependent protein kinase (CaM KII) that is co-localized with NRs at excitatory synapses and has been implicated in other forms of excitotoxicity; (ii) chronic ethanol ingestion will differentially sensitize CA1, CA3 and DG neurons to NR-mediated excitotoxicity, relative to pair-fed controls, in a reversible manner. This effect will be mediated by CaM KII. To test these hypotheses, ethanol will be administered to rats for 1-12 weeks and, in some cases, will include a withdrawal period. In ai-m 1, hippocampal subfields from ethanol-treated and control rats will be compared using NRI, NR2 and CaM KII immunoblotting and RT-PCR assays. In aim 2, hippocampal slices will be compared for regional differences in vulnerability to glutamate- or NMDA-mediated excitotoxic damage using a "live-dead" assay in conjunction with confocal imaging analysis. VALLANO ML SUNY HEALTH SCIENCE CENTER, 750 E ADAMS STREET, SYRACUSE, NY 13210 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 13210 Crisp Data Base National Institutes Of Health mature animal ethanol laboratory rat biological signal transduction hippocampus dentate gyrus brain mapping polymerase chain reaction glutamate drug withdrawal western blotting neurotoxin calmodulin dependent protein kinase NMDA receptor tissue /cell culture enzyme activity CRISP RPROJ NEW YORK G CRISP/99/AA11517-01A1 CRISP/99/AA11517-01A1 199904 eng DIURETIC PROTECTION FROM ALCOHOL INDUCED BRAIN DAMAGE Research RPROJ Adult rats episodically intoxicated with alcohol (ethanol)-e.g., 8-12 g/kg/d in fractional doses for 4 days, or simply one daily dose (4-6 g/kg) for 6-10 days-incur brain damage, as revealed by a specific neurodegeneration stain, in selected limbic cortical regions and the olfactory bulbs. Also, organotypic rat brain slice cultures treated episodically with alcohol (100-200 mM) for 6 days show evidence of induced cytotoxic damage. The underlying mechanism(s) is apparently not excitotoxic or seizure-related, because (a) the brain damage in vivo was not significantly diminished by glutamate receptor antagonists, a Ca++ channel blocker, or by nitric oxide synthase inhibitors; and (b) hippocampal CA regions, which are highly vulnerable to seizure damage, were largely unaffected. However, the brains of rats treated once daily with alcohol for a week were edemic and showed Na+ and K+ accumulation. Suspecting a role for edema, we examined the effect of furosemide, a potent diuretic and inhibitor of the C1- transporter that nonsynaptically blocks brain cell swelling in animal epilepsy models. Indeed, furosemide co-treatment significantly reduced alcohol-induced cortical neurodegeneration by 75-85 percent as it prevented the brain edema and electrolyte accumulation. Furthermore, furosemide completely blocked cytotoxicity in organotypic rat brain slice cultures exposed episodically to alcohol. This appears to be the first demonstration of significant neuroprotection during binge alcohol exposure. Hypothesizing brain edema as an key early step in the neurodegeneration, we propose in this R21 application to explore the generality of neuroprotection by studying three other diuretics, acetazolamide, chlorthalidone and torsemide along with a nondiuretic furosemide analog (L-644, 711) in our in vivo/in vitro approach. In Aim 1, neuroprotection from brain edema and degeneration for four agents will be examined in male rats intoxicated once daily with alcohol for an 8 day period. Concurrently, plasma vasopressin, and plasma and urine osmolality will be assessed to facilitate interpretation of results. Also, brain cell swelling and number will be measured stereologically in the alcohol-treated rats. In Aim II, the four agents will be examined with mature organotypic rat cortical/hippocampal slice cultures, in which both cytotoxicity (LDH release) and brain cell swelling by biochemical (taurine release) and morphometric (stereology) measurements will be determined. We anticipate that reductions in brain edema and neurodegeneration in vivo will tend to be reproduced in vitro, accompanied by prevention of cell swelling. The results can potentially provide new therapeutic approaches to neuroprotection in alcoholism, and can lay the groundwork for future detailed studies on possibly novel mechanisms underlying brain damage due to episodic alcohol exposure. COLLINS MA LOYOLA UNIVERSITY CHICAGO, 2160 S FIRST AVENUE, MAYWOOD, IL 60153 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 60153 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse laboratory rat electrolyte balance chronic brain damage brain edema brain injury brain metabolism urinalysis diuretic neural degeneration neuropharmacologic agent neuropharmacology osmotic pressure furosemide acetazolamide benzenesulfonamide tissue /cell preparation tissue /cell culture cytotoxicity chemoprevention neuroprotectant neurotoxicology CRISP RPROJ ILLINOIS G CRISP/99/AA11543-01A1 CRISP/99/AA11543-01A1 199904 eng ALCOHOL EXPOSURE, SOCIAL BEHAVIOR AND DEVELOPMENT Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) People with Fetal Alcohol Syndrome (FAS) exhibit striking deficits in social behavior; these alterations in social behavior are independent of changes in intelligence and may be very damaging with respect to day-to-day functioning. The goal of this proposal is to use a model of FAS in which the rat is exposed to alcohol during development to investigate the mechanism by which alcohol alters social behaviors in the rat. The model that is used is novel and exposes rats to alcohol during all three trimester equivalents or, in other words, during both the prenatal and postnatal periods. The first set of experiments characterizes the model by comparing the effects of alcohol exposure during both the prenatal and postnatal period to exposure during the first half of the prenatal period alone, the second half of the prenatal period alone, and the postnatal period alone on activity, passive avoidance learning and spatial learning. After characterizing the model in the first year, the next two years examine the effect of alcohol exposure during development on social motivation. The second set of experiments focuses on the social motivation in an affiliative context. The experiments use distress vocalizations under different conditions of isolation, running speed to gain access to a conspecific after varying degrees of social deprivation, and social preference for conspecifics with different levels of aggressiveness. The third set of experiments examines social motivation in an aggressive context using the resident/intruder paradigm for both males and females; the motivation is varied by changing the context in which the aggression occurs. All of the experiments on social motivation use the additive factors technique to tease apart effects on motivation from effects on perception or the response mechanism; generally this technique examines the behavior under varying degrees of motivation and predicts that if groups differ in social motivation, then they will differ in how their behavior is altered by alterations in the level of social motivation. Female rats exposed to alcohol during development are predicted to have enhanced social motivation whereas male rats exposed to alcohol during development are predicted to have deceased social motivation. KELLY SJ UNIV OF SOUTH CAROLINA, COLUMBIA, SC 29208 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 29208 Crisp Data Base National Institutes Of Health ethanol laboratory rat disease model embryo /fetus toxicology fetal alcohol syndrome aggression avoidance behavior motivation animal developmental psychology social behavior isolation /deprivation sex difference behavioral /social science research tag CRISP RPROJ SOUTH CAROLINA G CRISP/99/AA11566-01A1 CRISP/99/AA11566-01A1 199904 eng ALCOHOL AND MONOCYTE SIGNALING Research RPROJ DESCRIPTION: (Adapted from the investigator's abstract). Increased susceptibility to infections after acute alcohol use is associated with decreased monocyte production of tumor necrosis factor, (interleukin-1 and interleukin-6), cytokines regulated by transcription factor NF-kB. In contrast with enhanced nuclear translocation of the activating p65/p50 NF-kB heterodimer following LPS stimulation, the investigator's preliminary data show that acute alcohol treatment uniquely enhances nuclear binding of the p50/p50 inhibitory homodimer of the NF-kB/Rel family. Thus, they hypothesize that clinically relevant concentrations of alcohol disrupt NF-kB signaling in monocytes by inducing nuclear translocation of the inhibitory p50/p50 homodimer. Unique activation of p50/p50 homodimers by acute alcohol ingestion would result in inhibition rather than activation of NF-kB regulated inflammatory cytokine genes. Moreover, preferential induction of the p50/p50 homodimers by ethanol would desensitize monocytes to additional stimuli of the NF-kB pathway similar to that described for LPS tolerance, which utilizes p50/p50 mediated inhibition of p65/p50-induced genes. The specific aims of this application are to: (1). investigate the effects of in vivo acute ethanol on monocyte NF-kB/Rel activation, and the mechanisms by which ethanol regulates nuclear translocation and DNA-binding of NF-kB/Rel complexes by evaluating: (a) the kinetics and reversibility of NF-kB/Rel (p50, p65, RelB) and precursor (p105, p100) induction by in vivo acute ethanol in human monocytes; (b) synthesis, nuclear translocation and binding of different NF-kB/Rel dimers in northern (RNA), western blots (protein), gelshift and supershift experiments; (c) role of oxygen radicals. (2) Evaluate the significance of p65- and p50-specific inhibitory kB (IkB) proteins in ethanol-induced changes in NF-kB/Rel activation: Increased stabilization of the IkB molecules that bind p65 will be assessed by investigating the effect of ethanol on IkB tyrosine phosphorylation, degradation, and re-synthesis. The role of alcohol-induced decreases in nuclear levels of Bcl-3 or cytoplasmic levels of IKB (p50-specific IkB molecules) in increased nuclear binding of p50/p50 homodimer will be tested. (3) Evaluate the role of increased DNA-binding of the inhibitory p50/p50 homodimers in ethanol-induced desensitization of monocytes to subsequent stimulation: (a) test whether in vivo acute ethanol exposure desensitizes monocytes to subsequent induction through the NF-kB pathway, (b) study amelioration of ethanol induced monocyte desensitization after IFN priming. These studies should delineate the effect of in vivo acute alcohol on NF-kB signaling in monocytes relevant to inflammation, cirrhosis, atherosclerosis, and HIV infection. SZABO G UNIV OF MASSACHUSETTS MED CENT, 55 LAKE AVENUE NORTH, WORCESTER, MA 01655 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 01655 Crisp Data Base National Institutes Of Health ethanol biological signal transduction monocyte molecular pathology gene induction /repression human subject leukocyte activation /transformation interferon immunotoxicity free radical oxygen tissue /cell culture environmental toxicology nuclear factor kappa beta clinical research CRISP RPROJ MASSACHUSETTS G CRISP/99/AA11576-01A1 CRISP/99/AA11576-01A1 199904 eng ETHANOL INDUCED CELL DEATH--ROLES OF NITRIC OXIDE, CYCLIC GMP AND CALCIUM ION Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) One of the most detrimental effects of ethanol exposure during nervous system development is depletion of neuronal cells. Ethanol may deplete neurons by several means, one of them is ethanol-induced cell death, which is the focus of this proposal. Ethanol exposure of primary neuronal cultures of cerebellar granule cells (CGC) produces cell death and this cell loss is similar to that observed in animal studies. A valuable property of this model is that both ethanol-sensitive and ethanol-resistant cultures can be obtained in two ways. First, as CGC mature in culture they become ethanol-resistant, called time-dependent ethanol resistance. Second, treating ethanol-sensitive CGC with either NMDA or growth factors makes cells ethanol-resistant, called neuroprotection-dependent ethanol resistance. Of considerable value, ethanol-sensitive and ethanol-resistant cells can be compared to identify molecular differences which may be linked to ethanol-induced cell death. Specific Aim 1 will determine whether ethanol causes both necrotic and apoptotic cell death in CGC cultures. Our previous studies determined that the nitric oxide-cGMP (NO-cGMP) pathway is essential for NMDA-mediated neuroprotection against ethanol-induced cell death in CGC cultures. Specific Aim 2 will study whether the pathway plays an essential role in both time-dependent and neuroprotection-dependent ethanol resistance. CGC cultures will be established from mutant mice which lack the NO-cGMP pathway, in order to evaluate the function of the pathway in ethanol neurotoxicity and resistance. This specific aim may establish that the NO-cGMP pathway is activated by diverse signals (NMDA, growth factors, and time) and it plays an essential role in ethanol resistance. Since Ca2+ has been linked to cell death, Specific Aim 3 will examine the role of Ca2+ in ethanol-induced cell death in CGC cultures. Both ethanol-sensitive and ethanol-resistant cultures will be compared to determine whether there are differences in the maintenance of intracellular Ca2+, which may be linked to ethanol-induced cell death. In summary, this proposal will use ethanol-sensitive and ethanol-resistant cultures to obtain information about the nature of ethanol-induced cell death (necrotic versus apoptotic), the functional role of the NO-cGMP pathway in protecting neurons against this cell death, and the role of Ca2+ in this cell death. These closely-linked specific aims will greatly enhance our knowledge about molecular mechanisms involved in one of ethanol's most detrimental effects, neuronal cell death. PANTAZIS NJ UNIVERSITY OF IOWA, IOWA CITY, IA 52242-1109 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory mouse laboratory rat cerebellum calcium flux neurotoxin cyclic GMP tissue /cell culture cytotoxicity nitric oxide programmed cell death granule cell neuroprotectant calcium ion CRISP RPROJ IOWA G CRISP/99/AA11577-01A1 CRISP/99/AA11577-01A1 199904 eng MATERNAL ALCOHOL AND FETAL THYMIC GENE EXPRESSION Research RPROJ DESCRIPTION (Adapted from the Investigator's Abstract): Maternal alcohol consumption during pregnancy can adversely affect T-cell function in the offspring. This has been demonstrated in both man and animals. T-cell differentiation occurs in the thymus and involves interactions with regulatory factors produced by thymic stromal cells. It is proposed that prenatal exposure to alcohol and/or alcohol induced changes in maternal hormones induce a permanent change in expression of one or more of the stromal factors required for normal T-cell maturation, resulting in an immunosuppressive "imprinting" of the fetus. Thus, the overall goal of the proposal is to identify the genes encoding these factors, and determine their role in prenatal alcohol-induced immunosuppression. The strategy for identifying these genes is based upon previously described observations that maternal adrenalectomy reverses the T-cell dysfunction observed in fetal alcohol-exposed male rats. Therefore, this proposal will examine the developing fetal thymus for changes in gene expression brought about by FAE and maternal adrenalectomy. The specific aims are: 1) to analyze expression of known candidate genes in the fetal thymic stromal and lymphoid compartments that may contribute to FAE-induced T-cell dysfunction. 2) Identify new genes with altered expression of fetal thymic stromal cells due to FAE. 3) Establish a fetal thymic cell culture model of FAE. 4) Manipulate expression of specific genes in the fetal thymic cell culture model. Expression of genes identified in experiments 1 and 2 will be manipulated using antisense oligonucleotides to inhibit their expression, or expression vectors to overexpress these genes. Thus, a causal relationship may be established between altered expression of these genes and the observed changes in thymocyte differentiation in response to FAE. AIRD F NORTHWESTERN UNIV MED SCHOOL, 303 E CHICAGO AVE, CHICAGO, IL 60611 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 60611 Crisp Data Base National Institutes Of Health ethanol laboratory rat T lymphocyte cell differentiation transfection developmental genetics gene expression immunogenetics disease model antisense nucleic acid embryo /fetus toxicology prenatal stress fetal alcohol syndrome embryo /fetus cell culture developmental immunology CRISP RPROJ ILLINOIS G CRISP/99/AA11580-02 CRISP/99/AA11580-02 199904 eng DEVELOPING HEART AND FETAL ALCOHOL EXPOSURE Research RPROJ DESCRIPTION: The cardiovascular (CV) system is a common site of spontaneous as well as pharmacologically-induced birth defects that are associated with high morbidity and mortality. Nevertheless, a rat, fetal alcohol exposure (FAE) model that recapitulates some of the FAS CV-defects found in man has not been firmly established. This is our immediate and primary goal. Our preliminary data suggest that several biochemical and molecular indices of heart development are altered in ventricles from FAE male and female offspring. Based on these data, AND information available from the literature, we hypothesize that in utero FAE will modify heart development by mechanisms that remain to be fully elucidated. Because cardiomyocyte proliferation occurs almost exclusively during embryonic and fetal heart development, we postulate that FAE will reduce the total myocyte cell number in the ventricle. For this postulate to be critically tested, we must establish the rat FAE model. To do this, we will focus on ethanol's effects upon in utero cardiomyocyte development and ventricular formation with a potential impact upon structure-function relationships. FAE exposure during near full-term rat gestation (Days 7-to-Term) will be the dietary paradigm used to establish that FAE negatively influences: 1) the cardiomyocyte lineage's highly regulated proliferative expansion OR 2) effects cardiomyocyte population expansion and concomitant differentiation. We anticipate FAE-induced deficits in final ven-tricular myocyte cell number of 5-20%. Such deficits would blunt myocyte-influenced postnatal capillary angiogenesis and ventricular remodeling with structure-function, pathophysiologic consequences. To establish the rat FAE exposure conditions that induces developmental heart defects, pregnant dams will ingest liquid diets containing ethanol (6.5% vol/ vol; approximately 100 mg/dl blood alcohol) to provide 35% of their total caloric intake for the indicated period. At gestational days 17 and 20, AND postnatal days 1, 4, 7, and 14, ventricles from control, Pair Fed and FAE animals will be examined according to this aim: Specific aim #1 will Establish the Rat Model wherein near full-term FAE reproducibly blunts anatomical, biochemical, molecular and/or flow cytometric indices of cardiomyocyte proliferation and/or differentiation AND, overall heart growth/morphogenesis in the late fetal-to-neonatal animal, independent of any underlying gender- or nutritional-based influences. The proposed studies are required to establish the rat as an obliging animal model of human FAE-induced alterations in the CV system, with an emphasis upon the heart. The experimental design will test AND resulting data clarify the validity of our postulate that FAE-induced heart developmental defects that are manifest by reduced myocyte proliferation and/or differentiation, with potential underlying gender influences. Mechanistic insights into FAE-induced alterations may be ascertained from the results of our proposal AND they will be used to direct future cause-and-effect studies. ENGELMANN GL LOYOLA UNIV MEDICAL CTR, 2160 S FIRST AVE, MAYWOOD, IL 60153 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 60153 Crisp Data Base National Institutes Of Health ethanol laboratory rat angiogenesis cell differentiation flow cytometry congenital heart disorder heart myocardium heart ventricle heart dimension /size western blotting myogenesis toxicant interaction cell population study gestational age embryo /fetus toxicology northern blotting SDS polyacrylamide gel electrophoresis protein isoform cell proliferation CRISP RPROJ ILLINOIS G CRISP/99/AA11584-02 CRISP/99/AA11584-02 199904 eng ALCOHOL AND HYPERPROLACTINEMIA Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) In humans, excess alcohol intake often causes reproductive abnormalities including infertility and loss of libido. Some alcoholic men also shown evidence of gynecomastia. These pathological conditions in many human alcoholics are secondary to elevated levels of plasma prolactin (PRL), a condition known as hyperprolactinemia. Owe chronic alcohol intake induces hyperprolactinemia is not yet established. The present proposal will address this issue by studying the actions of ethanol on PRL secreting lactotropic cells in the pituitary gland of rats. Three specific aims are proposed for addressing this interest. Specific Aim 1 is to evaluate the action of ethanol on lactotropic cell proliferation and PRL secretion. Preliminary data indicated that ethanol administration using liquid diet for two weeks elevates blood ethanol levels to 0.1 mg/dl and promotes estrogen-induced lactotropic cell proliferation in Fischer 344 rats. The aim of this study is to confirm these data and to further characterize the time-course of ethanol action on lactotropic cell's growth and secretion. Specific Aim 2 is to determine the site of action of ethanol. Our working hypothesis is that ethanol effects lactotropic cell proliferation by acting at the level of pituitary. This possibility will be tested by determining the action of ethanol on PRL synthesis and secretion and cell proliferation in the primary cultures of enriched lactotropes. Specific Aim 3 is to elucidate the mechanisms by which ethanol enhances lactotropic cell proliferation. The working hypothesis is that ethanol increases lactotropic cell proliferation and secretion by preventing expression of the growth-inhibitory peptide transforming growth factor B1 (TBF-B1) in the pituitary gland. This hypothesis will be tested by I) characterizing ethanol-induced changes of TGF-B1 and TGF-B type II receptor in the pituitary; ii) determining the effect of ethanol on TGF-B1 secretion from the lactotropic cells in primary cultures; iii) evaluating whether ethanol-induced lactotropic cell growth and secretion is altered following overexpression or repression of TGF-B1 and it's TGF-B type II receptor in these cells. The proposed research will yield an increased understanding of ethanol effects on prolactinomas and hyperprolactinemia. Such knowledge should help to better manage these neuroendocrine diseases in alcoholic patients. SARKAR DK WASHINGTON STATE UNIVERSITY, 205 WEGNER HALL, PULLMAN, WA 99164-6520 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat hormone regulation /control mechanism estrogen transforming growth factor prolactin hyperprolactinemia pituitary gonadal axis female tissue /cell culture toxin metabolism cell proliferation CRISP RPROJ WASHINGTON G CRISP/99/AA11591-01A1 CRISP/99/AA11591-01A1 199904 eng NICOTINE AND ETHANOL INDUCED NEUROTOXICITY Research RPROJ NO ABSTRACT AVAILABLE DE FIEBRE CM UNIV OF NORTH TEX HLTH SCI CTR, 3500 CAMP BOWIE BLVD, FORT WORTH, TX 76107-2699 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse laboratory rat tobacco abuse drug interaction drug design /synthesis /production statistics /biometry behavior test learning memory nicotine scintillation counter receptor binding receptor expression nicotinic receptor tissue /cell culture organ culture PC12 cell neuroprotectant neurotoxicology smoking CRISP RPROJ TEXAS G CRISP/99/AA11597-02 CRISP/99/AA11597-02 199904 eng MECHANISMS OF ETHANOL INDUCED TERATOGENICITY Research SULIK KK UNIV OF NC @ CHAPEL HILL, CB 7178 THURSTON-BOWLES BLDG, CHAPEL HILL, NC 27599-7178 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory mouse calcium flux reperfusion mitochondrial membrane teratogen cytology cellular pathology fluorescent dye /probe free radical neural crest mammalian embryology cytochrome P450 embryo /fetus embryo /fetus toxicology embryo /fetus cell culture confocal scanning microscopy programmed cell death oxidative stress CRISP RPROJ NORTH CAROLINA G CRISP/99/AA11605-010004 CRISP/99/AA11605-010004 199904 eng ASTROCYTE-MEDIATED ETHANOL NEUROTOXICITY Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) Neurophysiological and pathological effects of ethanol (EtOH) exposure are mediated in part via the glutamatergic system. Withdrawal from chronic EtOH dependence is associated with increased extracellular fluid concentration of glutamate, contributing to hyperactivity of excitatory neurotransmission. Neither the cellular pool of glutamate, nor the mechanism underlying its increased extracellular concentration during EtOH withdrawal are presently known. The extracellular fluid composition of the CNS is primarily regulated by the astrocytes which comprise approximately 25 percent of the total brain volume. This proposal is based on the hypothesis that astrocytes chronically exposed to EtOH will accumulate compensatory organic solutes to maintain cell volume in lieu of a hyperosmotic extracellular fluid (EtOH-induced hypernatremia), as well as a direct stimulatory effect of EtOH on organic osmolyte uptake. During EtOH withdrawal, the plasma and extracellular fluid hypertonicity will be corrected. In response, the intracellular levels of electrolytes and EtOH will rapidly decrease, with a much slower adaptation to the release of accumulated compensatory organic solutes. Therefore, the cells will swell, behaving in a fashion similar to that of "naive" astrocytes exposed to hypotonic solution, because effectively, their cytoplasm is hypertonic to the normotonic extracellular fluid. Astrocytic swelling will lead to the release of endogenous excitatory amino acids (EAA), and regulatory osmolytes such as taurine, myoinositol, and K+. The cumulative effects of glutamate release over a life-time could result in permanent neuronal damage. The initial approach to testing this hypothesis will be to determine the abundance of compensatory organic solute transporter genes and/or their products (taurine, myoinositol, and glycerophoshorylcholine) in response to chronic EtOH exposure ( hypernatremia) in a well-characterized in vitro model of neonatal rat primary astrocyte cultures. Correlative in vitro studies will determine if withdrawal from chronic EtOH exposure is associated with astrocytic swelling. The time course of astrocytic swelling and the associated release of EAA (glutamate) and compensatory osmolytes (taurine, myoinositol, and K+) will be defined. Finally, it will be determined in in vitro studies and in in vivo microdialysis studies if astrocytic swelling and EAA release during EtOH withdrawal can be attenuated by anion transport blockers (SITS, DIDS, furosemide, and L-644,711). The long term objectives of the proposal are to characterize astrocytic adaptation processes in response to chronic EtOH exposure, with particular emphasis on swelling-associated glutamate release during EtOH withdrawal and to examine mechanistically-based modalities for attenuating glutamate release during EtOH withdrawal. ASCHNER M BOWMAN GRAY SCHOOL OF MEDICINE, MEDICAL CENTER BLVD, WINSTON-SALEM, NC 27157-1083 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol taurine laboratory rat electrolyte balance hypernatremia cell volume glutamate drug withdrawal astrocyte neuropharmacology neurotoxin glycerophosphorylcholine membrane transport protein myoinositol tissue /cell culture excitatory aminoacid microdialysis neurotransmitter transport neurotoxicology CRISP RPROJ NORTH CAROLINA G CRISP/99/AA11617-01A1 CRISP/99/AA11617-01A1 199904 eng MOLECULAR AND CELLULAR MECHANISMS OF ALCOHOL INDUCED LIVER DISEASE (ALD) Research RPROJ DESCRIPTION: Alcoholism is a major health problem in the United States since more than 15 million people are dependent on or abuse alcohol. A significant proportion of alcohol-related morbidity and mortality is a consequence of alcohol-induced liver disease (ALD). Studies have suggested that heavy alcohol consumption results in disruption of the mucosal intestinal epithelium which, in turn, leads to increased Gram negative bacteria] translocation and release of endotoxin (LPS) derived from these bacteria into the liver via the portal vein. Although the exact mechanisms of liver injury in ALD are currently unclear, reports have suggested that production and release of inflammatory cytokines by hepatic macrophages (Kupffer cells) contribute to and help to sustain ALD. However, it is also possible that lamina propria macrophages are a primary target of alcohol effects, given their proximity to alcohol-induce epithelial cell damage. Apart from studies showing altered barrier function and increased bacterial translocation, little is known about the effects of alcohol on the capacity of intestinal epithelial cells or lamina propria macrophages to be stimulated by either translocating bacteria or their LPS to secrete inflammatory products. Circulation of these intestinal-derived cytokines to the liver could contribute to hepatic damage either directly or indirectly via their effects on Kupffer cells, hepatocytes, or endothelial cells within the liver. Thus, intestinal macrophages could play a much broader role in the initiation of ALD than heretofore considered. To address these issues, the following Specific Aims are proposed: 1) To evaluate the effects of alcohol and normal intestinal bacteria or LPS in co-cultures of intestinal epithelial cells and macrophages. In vitro cocultures of epithelial cells and macrophages will be analyzed in the absence or presence of ethanol for cytokine secretion, nitrite production, and expression of specific LPS-inducible genes upon stimulation with E. coli or E coli LPS; and, 2) Mice fed alcohol-enriched or isocalorically-matched control diets will be euthanized at various times to validate augmented translocation and to analyze intestinal and liver sections for induction of inflammatory (LPS-inducible) gene products by semi-quantitative RT-PCR. It is anticipated that these studies will identify specific cytokines and/or genes whose expression is (are) dysregulated by alcohol. Such results would suggest that tissue damage associated with ALD could be mitigated by targeting specific LPS-induced cytokines or gene products. METCALF ES UNIFORMED SERV UNIV SCIS, 4301 JONES BRIDGE ROAD, BETHESDA, MD 20814-4799 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse laboratory mouse enteric bacteria macrophage cell migration polymerase chain reaction pathologic process gastrointestinal absorption /transport gastrointestinal infection gastrointestinal epithelium cytokine endotoxin liver cell Kupffer's cell liver disorder hepatotoxin lipopolysaccharide mixed tissue /cell culture CRISP RPROJ MARYLAND G CRISP/99/AA11622-01A1 CRISP/99/AA11622-01A1 199904 eng BEHAVIORAL EFFECTS OF EARLY ETHANOL AND MK801 EXPOSURE Research RPROJ DESCRIPTION: Prenatal alcohol exposure can produce central nervous system dysfunction, resulting in a wide range of behavioral alterations. The mechanisms by which ethanol disrupts brain and behavioral development, however are currently unknown. Preliminary data demonstrate that administration of MK-801, a noncompetitive NMDA receptor antagonist, during ethanol withdrawal in neonatal rats can attenuate ethanol-induced deficits on a spatial discrimination reversal task. These results suggest that withdrawal-related NMDA receptor-mediated excitotoxicity may be one mechanism producing alcohol's adverse effects on behavioral development. This project proposes to investigate the factors that determine the effectiveness of MK-801 in mitigating ethanol induced behavioral alterations. Rat pups are exposed to ethanol during the neonatal brain growth spurt, a period of development equivalent to a portion of the human's third trimester in utero, via an artificial rearing procedure. MK-801 is administered during withdrawal and behavioral performance is examined with two behavioral tasks: open field activity and serial spatial discrimination reversal learning. These two tasks are sensitive to early alcohol exposure and are believed to rely on the functional integrity of the hippocampus, an area sensitive to both neonatal ethanol exposure and NMDA receptor-related excitotoxicity. Preliminary evidence suggests that MK-801 can either exacerbate or protect against ethanol-related teratogenic effects, depending on the dose and timing of administration. Thus, the dose-response and time course of the effects of MK-801 are evaluated to determine the parameters that control the interaction of ethanol and MK-801. Secondly, given that chronic ethanol treatment has been shown to produce a greater upregulation of NMDA receptors, the comparative effects of MK-801 following acute vs. chronic ethanol exposure is examined. Finally, a preliminary exploration into the effects of postnatal ethanol and MK-801 administration on hippocampal neuropathology is conducted. Characterization of the behavioral consequences of blocking NMDA receptors during developmental ethanol withdrawal lays an important foundation for future studies on the role of withdrawal-related neurotoxicity in fetal alcohol effects. THOMAS JD SAN DIEGO STATE UNIVERSITY FND, 6363 ALVARDO COURT STE 209, SAN DIEGO, CA 92120 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 92120 Crisp Data Base National Institutes Of Health newborn animal alcoholism antagonist alcoholism /alcohol abuse chemotherapy ethanol laboratory rat pharmacokinetics drug withdrawal disease model neuroanatomy developmental neurobiology embryo /fetus toxicology fetal alcohol syndrome psychological test reversal learning developmental psychology NMDA receptor sensory discrimination space perception dizocilpine neuroprotectant behavioral /social science research tag CRISP RPROJ CALIFORNIA G CRISP/99/AA11634-01 CRISP/99/AA11634-01 199904 eng CYP2E1 POLYMORPHISM--MECHANISM AND IMPACT ON DISEASE Research RPROJ Intrauterine ethanol (ETOH) exposure is a common cause of congenital mental retardation. The mechanism of varying susceptibility of exposed offspring may include genetic-or environmentally-induced variation in ETOH metabolizing enzymes. The PI has shown that the alcohol dehydrogenase 2*3 allele (ADH2*3), unique to African Americans and associated with increased ETOH metabolism, is protective against adverse offspring outcome after intrauterine ETOH exposure. A role for CYP2E1 in alcohol related birth defects (ARBD) is supported by the enzyme's activity at high ETOH concentrations, its presence in areas of the brain affected in intrauterine exposure and its inducibility by ETOH intake. The PI recently found a restriction fragment length polymorphism (RFLP) in the regulatory region of human CYP2E1 that correlates with increased in vivo CYP2E1 activity in the presence of obesity or ETOH intake. The RFLP occurs in about 30 percent of African Americans and 10 percent of Caucasians. We propose to assess the mutation as a risk factor for ARBD, as well as the mechanism whereby the mutation alters CYP2E1 expression. We will test the hypothesis that, controlling for ETOH intake and ADH2 genotype, the mutation correlates with better offspring outcome. This study is both time- and cost-effective because previously collected data and blood samples will be used. Available data includes ETOH intake during pregnancy, maternal and offspring ADH genotype and offspring outcome at birth and 1 year of age. These results will further define those at greatest risk for ARBD; however, utilizing this information for intervention will require an understanding of the molecular mechanism. We will test the hypotheses that the RFLP is an insertion mutation that affects CYP2E1 transcription by either disruption of a negative regulatory element or by insertion of a positive regulatory element. The precise sequence and location of the mutation will be defined. The ability of the mutation to alter gene expression will be tested by comparing the ability of wild type and mutated sequences to direct the transcription of a reporter gene in the presence and absence of potential mediators. Electrophoretic mobility shift assays will test the effect of the mutation on the binding of trans-acting regulatory elements and may allow determination of the involved transcription factors. Understanding the mechanisms and underlying molecular basis for ARBD is the first step in planning future intervention, as well as targeted prevention protocols. The results of these studies also will have application to multiple diseases in which CYP2E1-mediated xenobiotic activation occurs and for which ETOH intake and CYP2E1 induction may alter risk. MAY DG WAYNE STATE UNIVERSITY, 656 W KIRBY, DETROIT, MI 48202 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 48202 Crisp Data Base National Institutes Of Health alcoholic beverage consumption ethanol congenital disorder gene expression transcription factor gene frequency genetic polymorphism restriction fragment length polymorphism cytochrome P450 human pregnant subject alcohol dehydrogenase embryo /fetus toxicology fetal alcohol syndrome female African American racial /ethnic difference toxin metabolism human genetic material tag enzyme activity gel mobility shift assay protein isoform gene /environment interaction clinical research genetic susceptibility CRISP RPROJ MICHIGAN G CRISP/99/AA11636-01 CRISP/99/AA11636-01 199904 eng ETHANOL EFFECTS ON THE DEVELOPMENT OF OLIGODENDROGLIA Research RPROJ DESCRIPTION: Among the severe health consequences that have been correlated with alcohol abuse are defects in myelin formation during fetal development, and demyelination during adulthood. The long term goal of the research being initiated in this proposal is to understand the cellular and molecular mechanisms underlying myelin defects that accompany alcohol abuse. Myelin is a multi-lamellar membrane which serves as an electrical insulator to facilitate neurotransmission. Defects in myelin as a result of insult or injury can dramatically affect neurotransmission. In the central nervous system (CNS), myelin is elaborated by oligodendroglial cells. Specific Aim 1 of this proposal is to determine when in their development oligodendroglia are sensitive to ethanol, and which aspects of their development (proliferation, cell death, and/or differentiation are affected by ethanol. Oligodendroglial progenitors, pre-oligodendroglia, and mature oligodendroglia (immunoselected using antibodies to stage- specific antigens)will be cultured under control conditions or with clinically relevant levels of ethanol. The effects of ethanol exposure will be determined by assessment of cell proliferation, cell death, and differentiation. Alcohol, which has been shown to interfere with cellular responses to growth factors, may disrupt oligodendroglial responses to growth factors which regulate their proliferation and maturation, and thus, prevent myelin formation and/or maintenance. Specific Aim 2 of this proposal is to determine if oligodendroglial responses to PDGF and IGF-1 are disrupted by ethanol exposure in vitro. Immunopanned populations of oligodendroglial progenitors, pre-oligodendroglia, and mature oligodendroglia will be cultured with PDGF or IGF-1 in the presence or absence of ethanol. Cellular responses will be evaluated as for Specific Aim 1. Experiments in Aim 2 are designed to test one possible mechanism that could underlie myelin defects that accompany exposure to alcohol, and contribute to design of therapeutic strategies to ameliorate these defects. INGRAHAM CA ALBANY MEDICAL COLLEGE, 47 NEW SCOTLAND AVE, ALBANY, NY 12208 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 12208 Crisp Data Base National Institutes Of Health ethanol laboratory rat cell death cellular pathology insulinlike growth factor platelet derived growth factor immunocytochemistry immunologic assay /test myelinopathy myelination oligodendroglia developmental neurobiology neurotoxin cell population study tissue /cell culture programmed cell death CRISP RPROJ NEW YORK G CRISP/99/AA11641-01A1 CRISP/99/AA11641-01A1 199904 eng ACUTE LUNG INJURY--ALCOHOLISM AND GLUTATHIONE DEPLETION Research RPROJ The Acute Respiratory Distress Syndrome (ARDS) is a common and severe form of lung injury with a mortality of approximately 50 percent. A prospective study of 351 critically ill patients recently identified that a history of chronic alcohol abuse increased the incidence and severity of ARDS regardless of the at-risk diagnosis. This observation distinguishes chronic alcohol abuse as the first reported co-morbid variable that significantly increases a patient's risk of developing ARDS and raises questions about the pathophysiology and specific treatment of acute lung injury. This project will test the hypothesis that chronic alcohol abuse decreases alveolar type II cell levels of glutathione, an important antioxidant, thereby impairing surfactant secretion and function and rendering the lung susceptible to injury. In ARDS the alveolar type II cells are severely damaged, and their ability to secrete glutathione and surfactant into the alveolar lining fluid are critical to patient survival. Because sepsis is the most common risk factor for ARDS, this project will focus on the sepsis syndrome in both patients and in an animal model. Preliminary studies presented in this proposal show that chronic alcohol ingestion in rats decreases type II cell glutathione levels and, in parallel, decreases type II cell surfactant secretion both in vitro and in vivo, and predisposes to endotoxin-mediated acute lung injury. In addition, we determined that otherwise healthy alcoholics have markedly decreased levels of glutathione in their lung lavage fluid compared to control subjects. The fundamental mechanisms by which chronic alcohol use decreases lung glutathione levels and how this affects type II cell function will be examined in a rat model of sepsis in vivo and in isolated type II cells in vitro. Parallel clinical studies in both healthy subjects and in critically ill patients with sepsis will examine the effects of chronic alcohol abuse on lung glutathione homeostasis and surfactant production both in isolated type II cells and in lung lavage fluid. We will thereby test the clinical relevance of the fundamental mechanisms elucidated in the animal model. Importantly, our preliminary studies indicate that glutathione replacement can decrease ethanol- mediated lung injury in our animal model, and this project will ultimately focus on developing a glutathione replacement regimen that reduces the harmful effects of chronic alcohol ingestion on the lungs of patients. GUIDOT DM ATLANTA VAMC, 1670 CLAIRMONT RD, DECATUR, GA 30033 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 30033 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse laboratory rat blood toxicology human subject glutathione adult respiratory distress syndrome respiratory epithelium pulmonary surfactant toxin metabolism oxidative stress clinical research CRISP RPROJ GEORGIA G CRISP/99/AA11660-01A1 CRISP/99/AA11660-01A1 199904 eng PREDICTORS OF SCREENING AND BRIEF INTERVENTION Research RPROJ APPLICANT'S ABSTRACT: Drinking related problems are highly prevalent among patients using primary health care. Although many proven techniques and instruments exists, research evidence indicates that little identification and intervention is provided for these populations, and important opportunities to intervene are lost. The primary aim of this study is to examine factors that may predict practice of screening and brief intervention in 1) a random sample of physicians and nurses in Contra Costa County, California, collected as part of this application; and 2) practitioners and support staff from eight managed care organizations, involving secondary analysis of data collected as part of an ongoing study of screening and brief intervention at the University of Connecticut. Both data sets address each of the research questions in this application. Several explanations of why health professionals do not often screen for alcohol problems exist; many of these reflect their beliefs about alcohol problems, screening and brief intervention. Hypotheses examine beliefs about the effectiveness of intervention; time involved, resources available, self-efficacy and organizational constraints; normative beliefs regarding support from reference groups; and individual knowledge about substance abuse as they relate to the actual practice of screening and brief intervention. Data collection involves self-administered questionnaires. Analysis includes a path model taking into account behavioral, control and normative beliefs; intention; and the role of objective knowledge as it relates to screening and brief intervention. This research has important implications for improving the application of screening and brief intervention strategies in health care organizations. GASSMAN RA ALCOHOL RESEARCH GROUP, BERKELEY, CA 94709 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 94709 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse alcoholism /alcohol abuse prevention diagnosis service alcoholism /alcohol abuse education health care professional practice health care service planning human subject interview questionnaire data collection methodology /evaluation model design /development behavior prediction toxicant screening clinical research behavioral /social science research tag health services research tag outcomes research CRISP RPROJ CALIFORNIA G CRISP/99/AA11713-03 CRISP/99/AA11713-03 199904 eng ALCOHOL AND HIV1 GP120 INDUCED BRAIN DAMAGE Research RPROJ APPLICANT'S ABSTRACT: Progressive brain damage leading to dementia is a major concern in AIDS. Furthermore, a high percentage of HIV-infected individuals consume alcohol (ethanol), often in abusive amounts. However, alcohol's effects on neurodegeneration associated with HIV appear largely unstudied. The HIV-1 coat protein, gp120, is a possible neurotoxin underlying AIDS-related brain damage. GP120 promotes glutamate (NMDA) receptor-mediated excitotoxicity, presumably via stimulating glia to release release factors (esp. Arachidonic acid) that increase extracellular glutamate. Likewise, NMDA receptors and glia are known alcohol targets. Our goal is to better understand how alcohol affects the extent, nature and mechanisms of neurotoxicity due to gp120. This will be approached in AIMS I and II with recombinant gp120 and rat brain organotypic slice cultures, and in AIM III with gp120 transgenic mice. Our overall hypothesis is that alcohol modifies brain neurodegeneration associated with gp120 in diametrically opposite directions, depending on alcohol concentrations and mode of exposure. Limited exposure to subneurotoxic alcohol concentrations may actually reduce gp120-dependent neurotoxicity; indeed, this is indicated by our preliminary in vitro experiments. Whether this effect of alcohol is due to reduced glial number, impairment of gp120's glial actions, or inhibition of MNDA receptor function will be tested. In contrast, chronic alcohol exposure may worsen CNS deficits in AIDS, and experimentally it exacerbates excitotoxicity due to MNDA or glutamate. Thus we anticipate that long-term or severe episodic alcohol exposure will increase neuronal (apoptotic) damage associated with gp120. If so, we will examine if augmentations in NMDA receptors and function, and/or brain edema (which we find to be a key factor in cytotoxicity caused by episodic alcohol alone) underlie the phenomenon. Specific Aim I is: To determine with rat brain organotypic slice cultures the outcome of alcohol exposure on the extent and type (apoptotic, necrotic) of gp120-mediated neurodegeneration; Aim II: To unravel the mechanisms through which alcohol affects the gp120 neurotoxicity in the brain slice cultures; and AIM III: To ascertain if the effects and actions of alcohol on gp120-induced neurodegeneration in vitro are reproduced in vivo in transgenic mice that express brain gp120 and show neuronal damage. The studies have the potential of providing information on alcohol/gp120 neurotoxicity that could be useful in understanding dementia in alcohol-consuming AIDS patients. COLLINS MA LOYOLA UNIVERSITY CHICAGO, 2160 S FIRST AVE, MAYWOOD, IL 60153 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 60153 Crisp Data Base National Institutes Of Health ethanol laboratory mouse laboratory rat transgenic animal brain edema neural degeneration in situ hybridization NMDA receptor furosemide HIV envelope protein gp120 programmed cell death recombinant protein neurotoxicology neurotransmitter antagonist CRISP RPROJ ILLINOIS G CRISP/99/AA11723-02 CRISP/99/AA11723-02 199904 eng NALTREXONE PLUS CBT/MET LIGHT FOR ALCOHOLISM Research RPROJ DESCRIPTION: Naltrexone and acamprosate have shown efficacy as adjuncts to psychosocial/behavioral alcoholism treatments. A number of studies have suggested that time limited outpatient behavioral approaches may have similar efficacy to more intense and skill based therapies. A multisite study is proposed to compare the effectiveness of combining medications with therapies which differ in intensity and content. A primary main study and a pilot study are proposed in this application. In the primary study 240 alcohol dependent individuals, at our site, will be assigned to eight treatment cells formed by the combination of 4 medication (placebo, naltrexone, acamprosate, naltrexone plus acamprosate) and 2 behavioral (cognitive behavioral (CBT) and modified motivational enhancement [MET-light]) therapies. Subjects will receive behavioral therapy for 12 weeks and medication for 26 weeks and follow-up assessments will occur at 3, 6, 9 and 12 months post-therapy. Standard alcohol consumption instruments, biological markers, and collateral will be used. The measurement of medication compliance (riboflavin as a marker) will be emphasized. In the pilot study 40 alcohol dependent individuals will be assigned to four treatment cells formed by the combination of 2 medication (placebo or naltrexone plus acamprosate) and 2 behavioral (CBT or MET-light) treatments. Subjects will be assessed and treated as proposed for the primary study. However, half of the subjects will receive the medication packaged in blister packs and half in MEMS cap bottles to determine (based on riboflavin measurements) which method has better compliance monitoring capabilities. In addition, one of the subjects will receive "booster therapy" at week 20 with retention and medication compliance evaluated in both groups. Patient satisfaction and adverse medication effects will also be evaluated between the two medication groups. If the primary study occurs at eight sites, a total of 1920 subjects (240/treatment cell) should provide enough power to detect medication versus placebo effects in both therapies. ANTON RF MEDICAL UNIVERSITY OF SC, 171 ASHLEY AVE, CHARLESTON, SC 29425 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 29425 Crisp Data Base National Institutes Of Health alcoholism antagonist alcoholism /alcohol abuse therapy alcoholism /alcohol abuse chemotherapy naltrexone clinical trial packaging material cooperative study combination chemotherapy drug adverse effect human subject interview longitudinal human study coping motivation mental disorder diagnosis placebo therapy compliance human therapy evaluation cognitive behavior therapy combination therapy clinical research behavioral /social science research tag outcomes research CRISP RPROJ SOUTH CAROLINA G CRISP/99/AA11783-02 CRISP/99/AA11783-02 199904 eng PHARMACOLOGICAL AND BEHAVIORAL TREATMENTS WITH ALCOHOLICS Research RPROJ DESCRIPTION: Recent research has suggested that a number of medications hold promise for improving outcomes in alcohol treatment. In particular, naltrexone and acamprosate act at different receptor sites and have each demonstrated an impact on alcohol consumption in animal and clinical trials. However, these drugs are typically given as an adjunct to behavioral interventions, despite little research to guide the specific nature of possible interactions between behavioral and pharmacological interventions. The present application proposes both a pilot study and a larger scale study for inclusion in the NIAAA cooperative agreement focusing on combined use of pharmacotherapies and behavioral interventions in the treatment of alcohol problems. A preliminary randomized trial is proposed that would evaluate the acceptability, safety, and clinical outcomes of naltrexone and acamprosate used in combination. The preliminary study will monitor side effects, adverse consequences, medication compliance, treatment retention, and changes in drinking, and craving during a 3-month trial period. The larger study is designed as a double-blind, placebo-controlled clinical efficacy trial of six-month intervention investigating the relative efficacy of two medications, naltrexone and acamprosate, used in combination with two behavioral interventions, one basic Motivation and Compliance Enhancement (MCE) and one where specific relapse preventive skills-training is added, MCE plus Integrated Relapse Prevention (MCE/IRP) across a twelve|month post-intervention follow-up. Subjects will be randomly assigned to one of four conditions combining the medications 1) naltrexone/acamprosate, 2) naltrexone placebo, 3) placebo acamprosate, and 4) placebo/placebo. All subjects will also receive a behavioral intervention to enhance motivation for chance (based on Motivational Enhancement Therapy) and medication compliance. Participants will be randomized to either eight sessions of Motivation and Compliance Enhancement or fifteen sessions of Integrated Relapse Prevention. Aims for the large study include: a) comparing, main and interactive effects of naltrexone, acamprosate, and behavioral interventions on outcome, b) evaluating medication compliance, treatment retention, and therapist effects medication clinical outcomes, and c) identifying patient attributes associated with differential response to the intervention combinations, thus suggesting, possible client-treatment matches. DONOVAN DM UNIVERSITY OF WASHINGTON, 3937 15TH AVE N E, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 98195 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse alcoholism /alcohol abuse prevention alcoholism /alcohol abuse chemotherapy naltrexone taurine clinical trial cooperative study drug administration rate /duration drug adverse effect drug screening /evaluation human subject interview questionnaire statistics /biometry longitudinal human study motivation psychotherapy therapy compliance human therapy evaluation combination therapy clinical research behavioral /social science research tag outcomes research CRISP RPROJ WASHINGTON G CRISP/99/AA11799-02 CRISP/99/AA11799-02 199904 eng ETHANOL AND INTERLEUKIN 6 SIGNAL TRANSDUCTION Research RPROJ Alcoholic liver disease if the result of alcohol-induced hepatotoxicity coupled with impaired hepatic regenerative capacity. In animal models, acute or chronic exposure to ethanol impairs liver regeneration following partial hepatectomy or chemically induced liver injury, but the mechanisms by which ethanol inhibits liver regeneration are still unknown. Recent evidence obtained in 'knock-out' mice deficient in interleukin-6 (il-6) indicates that activation by IL-6 of the signal transducer and activation of transcription protein 3 (Stat3) is a critical step in liver regeneration. Preliminary experiments have shown that acute treatment with ethanol can block the activation of Stat3 in the rat liver, induced by IL- 6 in vitro or by partial hepatectomy in vivo. These findings suggest that the anti-regenerative effects of ethanol are mediated, at least in part, through blocking IL-6 induced Stat3 activation. The mechanism by which ethanol inhibits IL-6-induced Stat3 activation will be explored by analyzing the effects of acute ethanol treatment on the IL-6-induced signal transduction cascade, including the interaction of IL-6 with its receptor, the tyrosine phosphorylation of the gp130 protein and IL-6- induced activation of the JAK kinases. The effects of chronic ethanol on Stat3 activation induced by IL-6 or partial hepatectomy will be explored in rats maintained on a ethanol-containing liquid diet. Identification of the IL-signaling pathway modulated by ethanol will not only enhance our understanding of the pathogenesis of alcoholic-induced liver disease but may also shed light on the effects of ethanol on signal systems in other tissues such as the brain. GAO B VA COMMONWEALTH UNIVERSITY, P O BOX 980613, RICHMOND, VA 23298-0613 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol laboratory rat biological signal transduction pathologic process transcription factor interleukin 6 hepatotoxin liver regeneration hepatectomy cytokine receptor tissue /cell culture JAK kinase CRISP RPROJ VIRGINIA G CRISP/99/AA11823-01 CRISP/99/AA11823-01 199904 eng ALCOHOL METABOLISM GENES IN TRANSGENIC MICE Research RPROJ DESCRIPTION: Ethanol metabolism in the liver is mediated by class I alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1). Class I ADH in mice is encoded by the Adh-1 gene which is under complex hormonal and developmental control. Transgenic mice will be used to determine the role of intragenic and 5'- and 3'-flanking sequences in control of the total expression phenotype. A region extending to -10kb attached tot an ADH minigene controls proper expression in kidney, adrenal, and other tissues; however, other cis-acting elements are required for liver expression. Cosmid and lambda genomic clones containing all intragenic sequences and different 5'- and 3'-flanking distance are now isolated and will be used in transgenic analysis to identify sequences necessary for liver expression. The same transgenic approach, using alternative promoters and cosmid clones, will be used to produce transgenic mice over expressing ADH and/or CYP2E1. A unique approach to analysis will allow RNA and protein produced from the transgene to be distinguished from production from the endogenous gene. For Adh-1, the unique genetic variant to be used will enable proper expression at the level of cell type within a tissue to be determined. Over expressing mice will be use to test major hypotheses about ethanol-induced liver injury including free radical formation, lipid peroxidation and production of fatty-liver. In addition, a genetic variant in Adh-3 expression identified among inbred strains will be defined at the molecular level and will provide a mechanism for differential tissue expression of this gene. FELDER MR UNIV OF SOUTH CAROLINA, COLUMBIA, SC 29208 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 29208 Crisp Data Base National Institutes Of Health ethanol laboratory mouse transgenic animal gene expression genetic enhancer element cytochrome P450 liver metabolism alcohol dehydrogenase toxin metabolism CRISP RPROJ SOUTH CAROLINA G CRISP/99/AA11828-01 CRISP/99/AA11828-01 199904 eng MECHANISMS OF ETHANOL EFFECTS ON SYNAPTIC TRANSMISSION Research RPROJ Acute and chronic effects of ethanol on synaptic transmission are critically involved in the effects and neuropathologies such as induction and expression of severe alcohol-related intoxication, neurotoxicity, dependence and withdrawal. Evidence suggests that such effects of ethanol are at least in part dependent upon modification of information processing at the level of the synapse. Primary sites of ethanol action at the synaptic level include glutamate receptors and voltage-gated calcium channels (VGCCs). Recent evidence suggests complex roles for VGCCs in both presynaptic and postsynaptic components of glutamatergic synaptic transmission. preliminary data from this laboratory demonstrates ethanol modulation of both of these presynaptic and postsynaptic VGCC-mediated effects. We propose to investigate these interactions through the use of patch clamp recording of spontaneously-occurring synaptic events (mEPSCs) mediated by glutamate receptor activation (GLU) in brain slice preparations. Frequency and amplitude comparisons will be used to assess presynaptic and postsynaptic components of synaptic transmission. Initially, these studies will assess acute effects of ethanol on basal mEPSCs recorded under controlled conditions. Second, ethanol effects on mEPSCs enhanced by specific and selective measures to modify the involvement of presynaptic and postsynaptic VGCCs in glutamatergic transmission will be analyzed. Third, the modulation of these processes by chronic ethanol exposure in hippocampal explant cultures will be assessed. Finally, quantitative autoradiographic techniques will be used to measure subtypes of VGCCs following acute and chronic treatments. Taken together, such information will increase our understanding of the effects of ethanol at the synaptic level and also help define short and long-term synaptic alterations related too the development and expression of various neuropathologic effects of alcohol. MORRISETT RA UNIVERSITY OF TEXAS AT AUSTIN, AUSTIN, TX 78712-1074 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol hippocampus patch clamp synapse neural transmission calcium channel autoradiography glutamate receptor tissue /cell culture neurotoxicology CRISP RPROJ TEXAS G CRISP/99/AA11845-02 CRISP/99/AA11845-02 199904 eng AFRICAN AMERICAN NEONATE FACE MORPHOMETRY--ETHANOL RISK SCREENING Research HEADINGS V HOWARD UNIVERSITY, 520 W ST NW STE 3408, WASHINGTON, DC 20059 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 20059 Crisp Data Base National Institutes Of Health alcoholism /alcohol abuse ethanol biomarker face newborn human (0-6 weeks) teratogen mass screening diagnosis design /evaluation disease /disorder proneness /risk prognosis human subject morphology gestational age embryo /fetus toxicology fetal alcohol syndrome African American clinical research CRISP RPROJ DIST OF COL G CRISP/99/AA11898-020002 CRISP/99/AA11898-020002 199904 eng PRENATAL ALCOHOL--EFFECTS ON CENTRAL DOPAMINE RECEPTOR SUBTYPES Research SOBRIAN SK HOWARD UNIVERSITY, 520 W ST NW STE 3408, WASHINGTON, DC 20059 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 20059 Crisp Data Base National Institutes Of Health ethanol laboratory rat biological signal transduction brain mapping congenital nervous system disorder developmental neurobiology gestational age embryo /fetus toxicology fetal alcohol syndrome behavior test receptor binding receptor expression dopamine receptor sex difference neurotoxicology disease /disorder etiology CRISP RPROJ DIST OF COL G CRISP/99/AA11898-020003 CRISP/99/AA11898-020003 199904 eng EFFECTS OF ETHANOL ON CARDIAC NEUROENDOCRINE DEVELOPMENT Research MCKENZIE JC HOWARD UNIVERSITY, 520 W ST NW STE 3408, WASHINGTON, DC 20059 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 20059 Crisp Data Base National Institutes Of Health ethanol laboratory rat acetylcholine neural crest atrial natriuretic peptide immunocytochemistry radioimmunoassay heart innervation developmental neurobiology neuroendocrine system gestational age embryo /fetus toxicology CRISP RPROJ DIST OF COL G CRISP/99/AA11898-020004 CRISP/99/AA11898-020004 199904 eng SOCIOEMOTIONAL DEVELOPMENT OF CHILDREN WITH PRENATAL ALCOHOL EXPOSURE Research CALMES D CHARLES R DREW UNIV, 1774 E 118TH ST MP 19B, LOS ANGELES, CA 90059 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 90059 Crisp Data Base National Institutes Of Health alcoholic beverage consumption alcoholism /alcohol abuse child psychology child behavior disorder preschool child (1-5) cooperative study maternal behavior human subject developmental neurobiology embryo /fetus toxicology fetal alcohol syndrome emotional adjustment behavior test depression social behavior disorder socioeconomics low income clinical research behavioral /social science research tag CRISP RPROJ CALIFORNIA G CRISP/99/AA11899-020001 CRISP/99/AA11899-020001 199904 eng MAGNETOENCEPHALOGRAPHIC ASSAY OF FETAL ALCOHOL EXPOSURE Research RPROJ DESCRIPTION: The objective of this project is to develop a Magnetoencphaloic (MEG) assay of the effects of marginal or overt callosal hypoplasia associated with Fetal Alcohol Syndrome (FAS) and Prenatal Exposure to Alcohol (PEA). The long term goal is to develop, using MEG, a set of detection methods to identify ambiguous cases of FAS or PEA. The study will involve a well characterized population of FAS and PEA children (ages 11-16) already undergoing a long term study, including MR scans, an appropriate selection of age matched normal controls. The proposed project will involve MEG and simultaneous EEG, with subjects involved in a visual letter judgement task, which will compare single hemifield and two-hemifield letter presentions. Behavior performance differences and reaction times will be compared with EEG and MEG event related responses (latencies and distributions), for evidence of callosal agenesis or functional impairment. Mid-sagittal MR slices-will be evaluated to seek evidence of callosal hypoplasia. The same MEG data sets will be evaluated for changes in the distribution of spontaneous alpha band suppression in visual areas, associated with attention to the visual task. The data analysis will also involve MEG dipole modeling to localize evoked sources in visual cortex. These measures can then be applied as an instrument to assess abnormal function in correlated with cognitive performance and development of FAS/PEA subjects in longitudinal studies. SCHWARTZ BJ SCRIPPS RESEARCH INSTITUTE, 10550 NORTH TORREY PINES ROAD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM 92037 Crisp Data Base National Institutes Of Health biomedical equipment development corpus callosum visual cortex electroencephalography magnetoencephalography brain mapping adolescence (12-18) diagnosis design /evaluation noninvasive diagnosis visual stimulus human subject embryo /fetus toxicology fetal alcohol syndrome clinical research CRISP RPROJ CALIFORNIA G CRISP/99/AA11927-01 CRISP/99/AA11927-01 199904 eng ALCOHOL AND DEVELOPMENT--EFFECTS ON CEREBELLAR SYSTEMS Research RPROJ DESCRIPTION: (from applicant's abstract) Prenatal exposure to alcohol can damage the central nervous system (CNS) and produce life-long learning disabilities, but there is unexplained variability in the observed effects. Different patterns, durations, and timing of drinking episodes during pregnanc may have major influences on the extent of damage, but systematic analysis still is needed in animal models. There has never been integrated, systematic analysis of alcohol-induced effects on the structural integrity and functional plasticity within a defined neural system that has a known, essential role in mediating associative learning. Such an effort requires a well-defined animal model with control over the alcohol exposure, in which the damage involves a key CNS structure and models human outcomes. The behavioral responses and neuronal correlates of learning must be operationally defined and precisely measured, and the neural circuits mediating the associative learning must be known. The studies proposed in this application fulfill each of these requirements. Binge alcohol exposure of neonatal rats has been extensively characterized as a model of third trimester exposure. Dose-related loss of cerebellar neurons during an early neonatal period of enhanced vulnerability i now well established, and the structural damage is similar to effects seen in MRI studies of prenatally exposed children. Recent studies have found that neonatal binge exposure induces profound impairments in classical conditioning of eyeblink responses. Cerebellar mediation of eyeblink conditioning is one of the best-understood models of mammalian associative learning in neuroscience. The components of the cerebellar-brainstem circuit essential for learned eyeblink responses, i.e., the deep cerebellar nuclei, cerebellar cortex, inferior olive, and pontine nuclei, appear to be targets of alcohol neurotoxicity in development. Five specific aims are proposed to test this by evaluating alcohol-induced deficits in structure, functional plasticity, and behavior. Aim 1 will evaluate the deficits in eyeblink conditioning in juveniles and adults, including an assessment of threshold, dose-response, and will extend the analysis to complex motor learning for adults. Aim 2 will determine the extent of cell loss in the four populations, using the same rats tested in Aim 1. Aim 3 will systematically assess learning-related neuronal activity and plasticity in the four areas. Aim 4 will evaluate neonatal temporal windows of vulnerability to structural and behavioral effects. Aim 5 will test whether the cerebellar effects are observed with gestational exposur or whether exposure that extends into the hypothesized critical period of vulnerability is required for cerebellar damage. GOODLETT CR INDIANA UNIVERSITY-PUI, 402 N BLACKFORD STREET, INDIANAPOLIS, IN 46202-3275 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health newborn animal juvenile animal mature animal ethanol laboratory rat brain stem cerebellar cortex eye movement disease model neuron developmental neurobiology neural plasticity gestational age embryo /fetus drug adverse effect embryo /fetus toxicology fetal alcohol syndrome association learning cognition disorder learning disorder animal developmental psychology neurotoxicology behavioral /social science research tag CRISP RPROJ INDIANA G CRISP/99/AA11945-01 CRISP/99/AA11945-01 199904 eng SIGNALING SYSTEMS AND ALCOHOL IMPAIRED CELL PROLIFERATION Research RPROJ Our long-term objectives are to understand the mechanisms of alcohol impairment of neural cell proliferation in the developing brain and how cell-signaling systems mediate the ethanol action. Our general strategy is to focus on a well-characterized neurotransmitter-gated ion channel, GABAA receptor/C1 channel using a newly developed in vitro model of the telencephalic neuroepithelium (TNE) in the central nervous system (CNS). TNE cells can be maintained in division in culture and express ethanol- sensitive GABAA receptor/C1 channels, making the TNE cell culture a useful model of the CNS neuroepithelium to elucidate the cellular and molecular role of cell-signaling systems in ethanol-induced impairment of neural cell proliferation. Our hypothesis is 1) that proliferating TNE cells in culture express functional GABAA receptor/C1 channels; 2) that GABA depolarizes TNE cells via activation of GABAA/C1 channels, and subsequent membrane depolarization activates voltage-gated Ca2+ channels, which trigger an increase in cytoplasmic Ca2+ ([Ca2+]c) levels, and in turn, the elevated [Ca2+]c inhibits DNA synthesis; 3) that ethanol may trigger and potentiate the GABAA receptor/C1 channel signaling pathway and subsequently, inhibit cell proliferation. Three specific aims of this proposal are to: 1) characterize GABAA receptor/C1 channels in TNE cells; 2) assess the sensitivity of GABAA/C1 channels and TNE cell proliferation to ethanol; and 3) determine how the activation of GABAA/C1 channels and subsequent increase in [Ca2+]c are involved in ethanol-inhibited cell proliferation. The accomplishment of the three aims will advance our understanding of how the GABA- signaling system mediates ethanol action on brain cell proliferation. The pharmacological results of these studies may provide the basis for devising potential therapeutic strategy for treating Fetal Alcohol Syndrome. MA W GEORGE MASON UNIVERSITY, 4400 UNIVERSITY DRIVE, FAIRFAX, VA 22030-4444 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health ethanol brain cell calcium flux calcium indicator patch clamp immunocytochemistry chloride channel receptor sensitivity GABA receptor epithelium tissue /cell culture neurotoxicology cell proliferation CRISP RPROJ VIRGINIA G CRISP/99/AA12237-01 CRISP/99/AA12237-01 199904 eng NUTRITION ACIDS IN PREGNANT WOMEN/FETUSES AT RISK FOR ALCOHOL BIRTH DEFECTS Research RPROJ This contract will support the LMBB investigation of the role of nutrition in the EFA status of pregnant women whose fetuses are at risk for ARBD. Contract objectives include (a) recruitment of an appropriate sample of pregnant women and (b) collection of the mothers nutritional, medical, demographic, and alcohol and other drug use data as well as collection of data to characterize their newborns. Extensive information shall be collected concerning the alcohol consumption history. Tissues or fluid samples adequate to provide for an assessment of the EFA status of the women are required. Blood samples shall be withdrawn from the mother on at least two occasions as well as from the placenta at the time of birth. Biochemical assays of vitamin levels shall be performed on fresh blood samples by the contractor. Other samples may also be collected in order to assess the nutritional status of the mother and infant (e.g., adipose, placenta, umbilical cord or meconium). Intact samples shall be frozen and sent to the laboratory of Membrane Biochemistry and Biophysics (LMBB) within the NIAAA Intramural Program in Rockville, MD. The LMBB shall perform fatty acid analyses of the total lipid extracts and various lipid fractions for these specimens using established GC/FID and GC/MS or LC/MS methods, as appropriate. The Contractor shall insert clinical and nutritional data into spreadsheets and supply them to the LMBB. Both the LMBB and the Contractor shall perform statistical analyses of the data in their relationship as research collaborators. HANNIGAN J 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM Crisp Data Base National Institutes Of Health alcoholic beverage consumption blood chemistry newborn human (0-6 weeks) human subject human pregnant subject developmental nutrition diet essential fatty acid nutrition related tag embryo /fetus toxicology fetal alcohol syndrome clinical research CRISP RPROJ MICHIGAN C CRISP/99/AA83019-000 CRISP/99/AA83019-000 199904 eng CELL BIOLOGY OF MODELS FOR HUMAN BRAIN DISORDERS Research RPROJ Hippocampal neurons from the fetal trisomy 16 mouse (Ts16), a model for Down syndrome, showed increased high-voltage- activated calcium currents in comparison to control diploid neurons. Ts16 neurons also bound more L-type calcium ligand. NMDA evoked currents in Ts16 neurons did not differ from currents in normal diploid neurons. Thus, this trisomy condition affects ionic responses that could have an impact on mental retardation in Down syndrome. We also investigated long-term potentiation (LTP), a model for learning and memory, in a new genetic model of Down syndrome, trisomy 16Dn mice, trisomic only for the part of mouse chromosome 16 that corresponds to human chromosome 21. The mice survive into adulthood. In hippocampal slices from trisomy 65Dn mice, LTP, induced by a single tetanizing pulse train at 100 Hz, was reduced, consistent with their learning impairment. GALDZICKI Z NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory mouse hippocampus cell biology polymerase chain reaction Downs syndrome human tissue disease model neuron neuropharmacology calcium channel potassium channel sodium channel voltage gated channel tissue /cell culture saxitoxin CRISP RPROJ A CRISP/99/AG00132-14 CRISP/99/AG00132-14 199904 eng REGULATION OF PHYSIOLOGICAL FUNCTIONS DURING AGING--BEHAVIORAL BIOLOGY Research RPROJ Summary of work: The aim of this project is to assess the effects of aging at a behavioral level of analysis, to identify neurobiological mechanisms associated with these effects, and to evaluate interventions that might alter age-related performance decrements. Rodent models are tested in a battery of sensorimotor and learning/memory tasks. Neurochemical and neurohistological assays are conducted to determine neurobiological correlates of functional losses. Interventions include dietary restriction, various pharmacologic treatments, and gene transfer via adenoviral vectors. Multiple genotypes are examined to determine possible genetic involvement in the pattern of age-related behavioral impairment. We have identified various effective pharmacologic strategies for improving learning performance of aged rats using manipulations of the cholinergic and glutamatergic neurotransmitter systems of great current interest is the improved learning of aged rats treated with a combination of drugs stimulating glycine polyamine receptors. In addition, specific inhibitors of butylcholinesterase also appear to have cognitive enhancing effects. Use of nitric oxide generating compounds has also proven to be a successful strategy for cognitive enhancement in aged rats. We are also examining inflammatory responses in the aged brain and have noted enhanced cytokine responses in aged mice following endotoxin stress. We have begun neuromorphological analysis using unbiased stereology to count neurons, synapses, microglia and astrocytes in hippocampi of mice of different ages. Results indicate no significant age- related changes; thus, the enhanced cytokine response observed in aged mouse brain does not appear associated with glia proliferation nor were there any signficant age differences in numbers of hippocampal neurons or synapses, although the latter parameter was correlated with performance in a memory task. Regarding impaired motor performance, we have continued development of an adenoviral vector for gene transfer of the dopamine D2 receptor and confirmed that the vector can produce a functional response in rats. We are planning experiments to test in a D2 knock-out mouse. INGRAM DK NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory mouse laboratory rat erythrocyte vasodilator vasodilation acetylcholine glutamine exercise transfection neurotrophic factor cytokine endotoxin sensorimotor system neuropharmacologic agent neuropharmacology reducing diet nutrition related tag learning memory dopamine receptor Adenoviridae nitric oxide transfection vector dietary restriction CRISP RPROJ A CRISP/99/AG00302-15 CRISP/99/AG00302-15 199904 eng MODULATION OF DNA EXCISION REPAIR AT CELLULAR SENESCENCE Research DUKER NJ ALLEGHENY UNIV & HLTH SCI, 2900 QUEEN LANE, PHILADELPHIA, PA 19129 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 19129 Crisp Data Base National Institutes Of Health aging fibroblast enzyme mechanism human tissue DNA replication DNA repair nucleic acid sequence photostimulus gel electrophoresis cytosine thymine radiobiology tissue /cell culture WI 38 cell cell senescence CRISP RPROJ PENNSYLVANIA G CRISP/99/AG00378-260009 CRISP/99/AG00378-260009 199904 eng GUSTATORY AND OLFACTORY CHANGES WITH AGE Research RPROJ DESCRIPTION (Adapted from the Investigator's Abstract): An overview of human psychophysical studies of taste and smell indicates that there are significant chemosensory losses with age, and these losses can affect nutritional status and quality of life. Olfactory losses can also reduce the ability to detect fire, dangerous fumes, and polluted environments. Little is known about the role that medications play in these age-related losses of taste and smell. In 1988, Americans spent $27.1 billion on prescription medications. In that year, the elderly constituted 12 percent of the total population but accounted for 35 percent of the prescription drug expenditures. Epidemiological studies indicate that the mean number of medications used by community-dwelling elderly over 65 ranges from 2.9 to 3.7 medications, and this number increases significantly to as high as 7.2 or more for elderly in nursing homes. Most reports of taste and smell losses from medications are based on clinical reports rather than experimental data. The purpose of this proposal is to use quantitative experimental methods to measure the effect of medications used by the elderly on the senses of taste and smell. The effect of drugs on taste perception at the periphery will be assessed in young and elderly subjects by psychophysical techniques after direct topical application of the pharmaceutical compounds to the tongue. Experimental subjects in topical studies will be taking no medications. These studies will be performed because drugs are secreted into the saliva at concentrations that can modify taste transduction. Pharmaceutical compounds will be applied topically at concentrations from 0.1 M to 1 mM to determine the salivary concentration of a drug that produces a taste change. Exposure to a drug will never exceed 1/1000 of a dose. In addition, elderly patients will be tested for taste and smell acuity prior to administration of these drugs by their physicians as well as during the course of treatment. This will permit the development of a data base that will provide accurate data on frequency of taste and smell changes that result from medications. The effect of medications on taste and smell perception in the elderly will be evaluated according to: 1) the therapeutic class of the drug (e.g., antihypertensive, diuretic, anticholinesteremic), 2) the type of peripheral receptor or ion channel to which the drug binds (e.g., cholinergic or dopamine receptor; calcium channel); and/or 3) the physicochemical characteristics of the drug (e.g., lipid:water partition coefficients). The ultimate goal is to find methods to alleviate taste and smell losses that result from use of medications. SCHIFFMAN SS DUKE UNIVERSITY, BOX 3259, DURHAM, NC 27706-0086 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health human old age (65+) young adult human (19-34) aging topical drug application drug adverse effect human subject saliva receptor sensitivity drug receptor olfaction perceptual distortion perceptual masking taste taste threshold age difference clinical research CRISP RPROJ NORTH CAROLINA G CRISP/99/AG00443-25 CRISP/99/AG00443-25 199904 eng SIGNAL TRANSDUCTION PATHWAYS IN RESPONSE TO STRESS Research RPROJ Summary of work: This project focuses on signal transduction pathways mediating the molecular reponse to stress in normal and aged cells. Studies over the past year have concentrated on three topics. (1) Identification of upstream mediators in arsenite-triggered ERK cascade. Previously we demonstrated that arsenite can activate ERK, JNK and p38 MAP kinases. Recent studies have focused on the role of growth factor receptors in mediating ERK activation. We have shown that arsenite treatment results in the rapid activation of epidermal growth factor receptor (EGFR), tyrosine phosphorylation of the Shc adaptor, and the formation of EGFR-Shc-Grb2 complexes. These events, as well as activation of ERK, were all drastically reduced by down-regulation of EGFR activity. These results provide the first evidence that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that this tumor promoter acts largely by usurping this growth factor signaling pathway. (2) Structural basis of MAP kinase phosphatase-1(MKP-1). MAP kinase phosphatases are a group of dual specificity phosphatases induced by extracellular stimuli including growth factors and stress. They can exhibit selectivity towards different members of the MAP kinase family. The goal of this study is to understand the structural basis for MKP-1 substrate specificity. Various domains of MKP-1 are being swapped with corresponding regions of PAC-1 or MKP-3 to generate chimeras. Analyzing the binding and enzymatic specificities of these chimeras for different MAP kinases may provide important information about the structural basis for the substrate selectivity. (3) Age-associated alteration in signaling pathways in rat hepatocytes. Previously we demonstrated that aging is correlated with decreases in both ERK MAP kinase and p70 S6 kinase activities following growth factor treatment. A decline in the activities of both kinases suggests that aged cells may display an alteration in an early upstream event common to these pathways. We have compared the earliest signaling events which occur in response to EGF stimulation in young and aged cells. In young hepatocytes, EGF triggers rapid tyrosine phosphorylation of EGFR and Shc, and complex formation between them. Formation of this complex in response to EGF is significantly reduced in aged cells, although no difference in tyrosine-phosphorylation of either EGFR or Shc is observed. The alteration in the growth factor receptor complexes may contribute to the decline in proliferation capacity in aged cells. LIU Y NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging biological signal transduction mitogen epidermal growth factor liver cell arsenic peroxide phosphorylation protein kinase stress methane sulfonate enzyme activity mitogen activated protein kinase cell proliferation CRISP RPROJ G CRISP/99/G00510-02 CRISP/99/G00510-02 199904 eng LINKS BETWEEN PROTEOLYTIC PROCESSING AND BRAIN AGING Research LYNCH GS UNIVERSITY OF CALIFORNIA, IRVI, INST FOR BRAIN AGING, IRVINE, CA 92697-4540 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory rat hippocampus brain metabolism lysosome enzyme inhibitor protease inhibitor histopathology neural degeneration neurotoxin endopeptidase amyloid protein proteolysis chloroquine tissue /cell culture cytotoxicity age difference CRISP RPROJ CALIFORNIA G CRISP/99/AG00538-220009 CRISP/99/AG00538-220009 199904 eng FREE RADICAL MECHANISMS IN NEURAL INJURY IN VITRO Research RPROJ Free radicals have long been postulated to contribute to neuronal injury in acute central nervous system injury, including stroke and trauma, and in the more chronic neurodegenerative diseases, including Parkinsons disease, multi-infarct dementia, and Alzheimers disease. These conditions are fast becoming the most pressing health care issues for the coming decades. Globally, 15% of the population over age 65 is reported as having some form of dementia, and current estimates in the U.S. place the number of affected individuals at well over 4 million, a number which may double or triple in the next 20 years (NIA Special Report, 1991; Odenheimer, 1989). In addition, cerebral infarction (stroke) is a leading cause of neurologic disability in the elderly. Yet, to date, there is no effective therapy to prevent or treat a majority of these neurologic disease states. The goal of the proposed project is to characterize free radical processes initiated by glutamate receptor overactivation (excitotoxicity), and to assess the contribution of free radicals to the death of neurons excitotoxicity. Glutamate is the major excitatory neurotransmitter in the brain, and is felt to play a major role in learning and memory. However, both free radical injury and glutamate receptor-mediated excitotoxicity are believed to contribute to the death of neurons in acute and chronic neurologic diseases, including stroke and head trauma, Parkinsons Disease, Huntingtons Disease, and possibly Alzheimers dementia. Production of free radicals, in particular hydroxyl radical, may be one key mechanism by which excitotoxicity results in irreversible neuronal damage. The hypothesis we will test is whether glutamate receptor activation can initiate hydroxyl radical production in neurons, and whether hydroxyl radical in turn is a component of glutamate neurotoxicity. This hypothesis will be tested using state-of-the-art methods for detecting free radicals, such as Electron Spin Resonance and High Performance Liquid Chromatography, to measure hydroxyl radical production during excitotoxicity in mouse brain cell cultures, together with pharmacologic and molecular biology approaches to altering free radical production and clearance. Clarifying the sources of free radicals and the events that trigger their production may assist in the development of rational treatment in neurologic diseases, including stroke and many forms of neurodegenerative diseases. DUGAN LL WASHINGTON UNIVERSITY, 660 S EUCLID AVE., ST LOUIS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 63110 Crisp Data Base National Institutes Of Health laboratory mouse electron spin resonance spectroscopy free radical neural degeneration high performance liquid chromatography receptor expression glutamate receptor embryo /fetus cell culture oxidative stress neurotoxicology CRISP RPROJ MISSOURI G CRISP/99/AG00599-05 CRISP/99/AG00599-05 199904 eng TISSUE SPECIFIC EXPRESSION OF DAT & MOR TRANSGENES IN STUDY OF AGING DISORDER Research RPROJ The goal of this work is to examine the role of the dopamine transporter (DAT) and the Mu Opioid Receptor (MOR) in age related disorders (DAT: Parkinsons Disease, neurotoxicity; MOR: pain, immune challenge). The strategy is via tissue specific, conditionally expressed transgenic mouse models. The ability to conditionally express a gene in a tissue specific manner is essential because only then can a resultant phenotype be assigned to a finite group of cells. This work is broken into three parts. The first two parts work with the DAT gene to characterize the genetic elements responsible for dopamine (DA) cell specific gene expression and, the spectrum of gene products (mRNAs) expressed in DA cells. With these tools, and the genes encoding DA cell-specific mRNAs, we hope to better choose future candidate genes to be conditionally expressed (or knocked out), and to further characterize the DNA sequences required to drive the expression of these genes uniquely to DA cells. The approach is to create a mouse harboring a loxp- inserted MOR gene, and in conjunction with tissue specific Cre expressing transgenic mice to create several different MOR knockout lines of mice, with each line knocking out the MOR in a different tissue. The first tissue targeted is the dorsal root ganglion to study the contributions of the MOR in peripheral analgesia. DONOVAN DM NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory mouse transgenic animal gene expression disease model spinal ganglion Parkinson's disease opioid receptor analgesia pain dopamine transporter gene targeting neurotoxicology protein localization CRISP RPROJ G CRISP/99/G00620-01 CRISP/99/G00620-01 199904 eng ADJUSTMENT OF ESTROGEN DOSING ACCORDING TO BONE TURNOVER Research RPROJ The study proposes to examine whether dosage for estrogen replacement therapy should be individualized, as is done for thyroid hormone replacement therapy. We will examine whether estrogen dosage can be optimized based on an individual's bone turnover, anthropometric indices, or serum levels of estrogen metabolites. This question will be addressed in a randomized double-blinded, placebo-controlled study where bone density will be measured sequentially in postmenopausal women randomized to receive either 0.3, 0.6, or 1.2 mg of Premarin daily. We hypothesize that women who take standard doses of Premarin and have elevated biochemical markers of bone resorption that fall after increasing their dose from 0.625 to 1.25 mg/day will derive skeletal benefit from treatment with 1.25 mg/day of Premarin. Similarly, women who take Premarin whose biochemical markers of bone resorption remain suppressed after having their dose reduced from 0.625 to 0.3 mg will still derive maximal skeletal protection at the lower dose. Potential beneficial or adverse effects of unconventional doses of estrogen on cardiovascular risk factors, body composition, clotting factors, mood and sexual function will also be examined. Because the results of this study will help physicians to adjust estrogen dosing for millions of postmenopausal women taking estrogen replacement, this study has tremendous relevance to aging. ROSEN HN BETH ISRAEL DEACONESS MED CTR, RW-563, BOSTON, MA 02215 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02215 Crisp Data Base National Institutes Of Health biomarker clotting factor body composition body weight adipose tissue prognosis dosage pharmacokinetics drug adverse effect hormone therapy estrogen postmenopause human subject blood lipid longitudinal human study sex behavior emotion female osteoporosis pathologic bone resorption bone density bone metabolism physiologic bone resorption body height oxidative stress CRISP RPROJ MASSACHUSETTS G CRISP/99/AG00680-04 CRISP/99/AG00680-04 199904 eng IMPROVING THE USE OF ANTICOAGULANT THERAPY IN THE AGED Research RPROJ Anticoagulant therapy is effective in the prevention of many thromboembolic disorders, such as stroke, myocardial infarction, and venous thrombembolism. In addition to being the most rapidly growing segment of our population, older patients are also the most likely to have these common indications for anticoagulant therapy. Although anticoagulant therapy has proven benefit in these conditions, it also has an adverse effect--bleeding. Concerns about this adverse effect of anticoagulant therapy may make clinicians less willing to prescribe anticoagulant therapy to older patients who might benefit most from treatment. The main goal of this proposal is to improve the use of anticoagulant therapy in older patients; it will also serve as a model for the investigation of other drug-induced illnesses in older patients Past studies and my own preliminary work indicate that warfarin-related bleeding may be more common in older patients. Which older patients are at greatest risk, and why they are more likely to bleed, is not known. This may lead physicians to erroneously under- or over-estimate the risk of warfarin-related bleeding in older patients, resulting in the withholding of therapy to some older patients whose benefit may outweigh their risk. Additionally, the attitudes and beliefs of physicians and patients about the risks of warfarin-related bleeding may affect the decision to initiate therapy. Clarification of these issues will lead to improvement in the appropriate use of warfarin therapy in older patients. I have designed an incremental research plan with three specific aims. Aims 1 and 2 will address critical unresolved issues involving the safety of anticoagulant therapy in older patients. Aim 3 will extend my research into a new area central to improving the use of anticoagulant therapy in older patients with atrial fibrillation--i.e., patient and physician attitudes concerning the risks and benefits of anticoagulant therapy, and the relationship of their attitudes to the appropriate use of anticoagulant therapy. This research proposal will build directly on my training and current work, while taking advantage of the research environment provided by my sponsors and others at Case Western Reserve University. It will also provide me with the scientific methods and clinical strategies to investigate other drug therapies in older patients, as well as to develop preventive strategies that will lead to improvements in the health care of older persons. BEYTH RJ CASE WESTERN UNIVERISYT, 11100 EUCLID AVENUE, CLEVELAND, OH 44106-6033 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health human old age (65+) aging warfarin blood coagulation anticoagulant drug adverse effect drug screening /evaluation atrial fibrillation human subject questionnaire attitude belief human therapy evaluation comorbidity clinical research CRISP RPROJ OHIO G CRISP/99/AG00712-02 CRISP/99/AG00712-02 199904 eng REGULATION AND FUNCTION OF THE PUTATIVE TRANSCRIPTION FACTOR GADD153 Research RPROJ Summary of work: GADD153 is a highly conserved mammalian gene whose expression is increased in response to a variety of stresses including those associated with growth arrest and DNA damage. It has been implicated in both the induction of growth arrest and apoptosis, but its functional role in these processes is still far from clear. GADD153 is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. It cannot bind to consensus C/EBP binding sites, but can act as a negative regulator of these C/EBPs by virtue of its ability to dimerize with them and inhibit their binding to DNA. It has also recently been suggested that GADD153-C/EBPbeta dimers can act as positive regulators of transcription through interaction with novel sites. Studies in this project have focused on the regulation of GADD153 gene expression during stress and the function of the GADD153 protein during the cellular response to diverse stimuli. With respect to regulation, efforts over the past year have concentrated on the role of two CREB-family transcripiton factors in regulating GADD153 transcription during stress. We have provided evidence that CREB2 acts as a transcriptional activator, while ATF3 acts as a repressor of GADD153 expression following sodium arsenite treatment. Acting in a sequential manner these transcription factors bind to the C/EBP site in the GADD153 promoter and contribute to the biphasic induction of GADD153, emphasizing the importance of both positive and negative regulatory factors in controlling GADD153 expression during stress. Studies on the function of GADD153 have focused on its influence on cell survival during stress. Employing a model system in which GADD153 protein is constitutively expressed at high levels in myc-transformed Rat1 cells, we have found that GADD153 overexpression leads to enhanced sensitivity of a variety of cells to stressful treatments. This is correlated with reduced levels of Bcl-2 protein and current studies are exploring the mechanism whereby GADD153 represses Bcl-2 expression. Our findings are consistent with GADD153 serving a pro-apoptotic function. HOLBROOK NJ NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health chemical kinetics genetic promoter element gene induction /repression arsenic intermolecular interaction protein structure /function stress protein physiologic stressor tissue /cell culture environmental toxicology enhancer binding protein CRISP RPROJ G CRISP/99/G00720-07 CRISP/99/G00720-07 199904 eng GENE SPECIFIC DNA REPAIR Research RPROJ Summary of work: The nuclear matrix is a central structure in the cellular hierarchy. A number of processes have been shown to be located here, including transcription, replication and topological binding sites. There has also been documentation that DNA repair processes are preferentially located at this site. We have demonstrated that the early DNA repair processes of nucleotide excision and transcription coupled DNA repair take place here at the nuclear matrix. In response to UV irradiation of mammalian cells, the protein proliferating cell nuclear antigen (PCNA) forms an insoluble complex with nuclear substructures. This complex can be detected by immunofluorescence and western blot analysis within 30 min after UV irradiation. We have studied the role of nucleotide excision repair (NER) and its subcomponent, transcription coupled repair (TCR), in PCNA complex formation. PCNA complex formation was studied in genetically related hamster cell lines that differ only in their capacity to perform NER. The hamster cell lines UV5 and UV24, which are homologs of the human DNA repair mutants xeroderma pigmentosum (XP) groups D and B, are completely deficient in NER. The hamster cell line UV61 is deficient in TCR of UV induced pyrimidine dimers, and is homologous to the human DNA repair mutant Cockayne syndrome (CS) complementation group B (CS-B). In the NER deficient cells, UV5 and UV24 cells, the PCNA complex was not detectable within 30 min after UV. When the UV5 cells were transfected with the human XPD gene, the PCNA complex formation was restored to normal. In the TCR defective UV61cells, the rate of PCNA complex formation was intermediate between normal and NER deficient cells. This defect in UV61 cells was complemented by transfection of the human CSB gene. We conclude that efficient PCNA complex formation induced by UV irradiation is dependent on both the genome overall repair of 6-4 photoproducts and the TCR of pyrimidine dimers in hamster cells. The complex formation occurs only in non-replicating cells and is not affected by pretreatment with aphidicolin. We have investigated the DNA repair of photo lesions in the ribosomal DNA in relation to the RNA polymerase I transcription in the nucleoli using a combination of immunological and biochemical approaches. Labeling of DNA repair sites with BudR in UV treated cells at different post incubation times showed a lack of DNA repair in the nucleolar regions comprising rDNA genes. Immunofluorescent labeling of UV induced repair and of transcription sites in interphase nuclei of hamster cells indicates that the repair of photo lesions in the rDNA genes is not coupled to transcription. The nucleoli are completely deficient in repair despite the presence of abundant RNA pol I transcription foci. Gene specific repair assays showed that both cyclobutane pyrimi-dine dimers and 6-4 photoproducts are removed much less efficiently from the rDNA than from an endogenous essential gene. This obse BOHR VA NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health hamster cell growth regulation genetic regulation genetic transcription radiation genetics human tissue DNA repair ultraviolet radiation DNA damage programmed cell death CHO cell CRISP RPROJ G CRISP/99/G00724-06 CRISP/99/G00724-06 199904 eng MOLECULAR RISK FACTORS FOR CEREBRAL AMYLOID ANGIOPATHY Research RPROJ DESCRIPTION: (Adapted from the application) Cerebral amyloid angiopathy (CAA) is a major cause of primary intracerebral hemorrhage in the elderly and a disease of growing importance in the aging population. CAA also represents an intriguing model system for exploring the pathogenesis of Alzheimer's disease (AD). The research proposal at the core of this training program is a multidisciplinary epidemiologic study aimed at understanding the molecular pathogenesis of CAA. Several potential molecular risk factors, including the apolipoprotein E e4 allele, cerebrospinal fluid B-amyloid peptide, and serum cholesterol, will be assessed for their effects on the presence and progression of CAA. A major goal of this research program is to lay the groundwork for future interventional drug trials in CAA. Towards this end, the technique of gradient-echo MRI will be evaluated as a possible clinical marker for extent of disease. In addition, a preliminary pharmacologic study will be performed to determine whether activation of a specific signaling pathway might decrease production of B-amyloid peptide in patients with CAA. These research projects will be performed in collaboration with and under the supervision of Drs. John Growdon and Bradley Hyman, two distinguished investigators who have applied similar batteries of approaches to the understanding of AD. The quality of training will be further enhanced by the candidate's use of the diversity of clinical and scientific expertise available in the Harvard Medical School community. The proposed training program will include advanced epidemiologic and statistical coursework at the Harvard School of Public Health. Building on the candidate's strong background in molecular research and substantial early progress in clinical studies, the proposed training will assure the candidate's development into a fully independent clinical investigator. GREENBERG SM MASSACHUSETTS GENERAL HOSPITAL, FRUIT STREET, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02114 Crisp Data Base National Institutes Of Health aging warfarin cardiovascular pharmacology cerebrovascular disorder cerebral hemorrhage cholesterol disease /disorder proneness /risk pathologic process drug adverse effect human subject blood lipoprotein magnetic resonance imaging amyloid protein tissue /cell culture clinical research apolipoprotein E CRISP RPROJ MASSACHUSETTS G CRISP/99/AG00725-03 CRISP/99/AG00725-03 199904 eng A CONNECTION BETWEEN DNA REPAIR AND HIV RELATED IMMUNODEFICIENCY Research RPROJ Summary of work: DNA repair enzymes are activated in response to DNA damage. Alterations in DNA repair enzyme function and thus the rate and extent of repair contribute to malignant transformation. Transcription of the gene encoding DNA polymerizing enzyme beta- polymerase, a repair enzyme, is shown to be inhibited by the human T-cell leukemia virus type I (HTLV-I) transactivator protein Tax. The resultant inhibition of DNA repair may lead to unrepaired chromosomal damage. Severe combined immunodeficient (SCID) mice have a mutation in the catalytic subunit of the DNA binding protein kinase that is involved in repair of double-strand breaks in DNA. To deter- mine if the protein is involved in repair of other types of breaks, we examined the ability of SCID cells to repair lesions introduced by ultraviolet light and X-ray irradiation. After ultraviolet irradiation, murine SCID fibroblast cell lines had 50% less repair of photoproducts in chromosomal DNA compared to a wildtype cell line, as shown by impaired cellular survival and decreased unscheduled DNA synthesis. SCID cells also had 50% less repair of ultraviolet damage in the transcribed dihydrofolate reductase and c-myc genes than wildtype cells. After gamma irradiation, SCID fibroblasts had 30% less repair of single-strand breaks than wildtype cells in the genes encoding immunoglobulin heavy and light chains. BOHR VA NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health T lymphocyte fibroblast enzyme inhibitor genetic transcription transcription factor virus genetics radiation genetics HeLa cell DNA repair DNA directed DNA polymerase calmodulin dependent protein kinase virus protein ultraviolet radiation human immunodeficiency virus 1 human T cell lymphotropic virus type 1 recombinant DNA SCID mouse CRISP RPROJ G CRISP/99/G00734-02 CRISP/99/G00734-02 199904 eng PROCESSING OF OXIDATIVE STRESS IN ALZHEIMER'S DISEASE Research RPROJ Summary of work: Studies have demonstrated that several cellular markers of oxidative stress are higher in cells from Alzheimer disease (AD) patients as compared to normal age- matched controls. These markers include oxidative damage to lipids, proteins, and DNA in various tissues from AD patients. It has been proposed that AD cells may have a defect in the DNA repair processing of oxidative base lesion leading to accumulation of DNA damage in AD cells. We have investigated the repair of oxidative base lesions using whole cell extracts from cultured AD lymphoblasts. DNA substrates containing both pyrimidine and purine lesions were obtained by treatment of plasmid with either gamma irradiation or fluorescent light (FL). Plasmid DNA containing primarily thymine glycol or 8- hydroxyguanine was prepared by damaging DNA with either OsO4 or methylene blue plus light ,respectively. The DNA substrates were purified free of strand breaks and were used in DNA repair synthesis assays. FAD cells were proficient in repair of these substrates containing various oxidative base lesions. Extracts from FAD cells repaired the plasmids damaged by gamma or FL- irradiation with equal efficiency as extracts from unaffected individuals. Furthermore, DNA damaged with methylene blue plus light and OsO4 were repaired with greater efficiency using FAD extracts (approximately 0.5-fold increase) as compared to cells from unaffected individuals. Our data indicate that the DNA damages resulting from the oxidative stresses used here are repaired efficiently in FAD cells. It is possible that the repair of specific oxidative base damages, such as that seen for thymine glycol and 8-hydroxyguanine, may be upregulated in FAD due to chronic oxidative stress which has been previously implicated in this disease. Additionally, there have been reports of unusual accummulation of oxidative DNA damage in mitochondrial DNA from patients with AD, and we are have recently developed novel techniques to study DNA repair in these organelles. We thus plan to investigate the DNA repair efficiency in mitochondrai from individuals with AD. BOHR VA NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health Alzheimer's disease gas chromatography mass spectrometry fibroblast plasmid radiation genetics DNA repair fluorescence tissue /cell culture DNA damage family genetics oxidative stress CRISP RPROJ G CRISP/99/G00735-02 CRISP/99/G00735-02 199904 eng DNA REPAIR AS A MECHANISM OF THE ADAPTIVE RESPONSE Research RPROJ The adaptive response (AR) is a phenomenon whereby the harmful effects of high dose ionizing radiation or other genotoxic agents can be mitigated by prior exposure to a low dose of the same or similar genotoxic stress. The adapted cells show an increased survival, less chromosomal aberration and decreased mutagenesis termed the adaptive response. It is not clear which biologic pathways are involved in the AR, speculation centers on cell cycle controls, signal transduction, and DNA repair mechanisms. DNA repair mechanisms, once thought to be constitutive, have now been proven to be inducible. Wilson, Mitra, and others have shown that genes and gene products involved in base excision repair are induced after low doses of certain forms of DNA damaging agents. We are working on the hypothesis that induced DNA repair, both base excision and nucleotide excision repair are equally important as underlying mechanisms of AR. The components of the proximal limb of the p53 DNA damage response pathway likely are critical in the initiation and maintenance of the adaptive response. We believe that the most interesting hypotheses to construct center on understanding the role of p53 damage response pathways in the adaptive response. Specifically, we hypothesize that nucleotide excision repair induced by low doses of ionizing radiation occurs through induction or activation of p53 related genes. There is evidence that PARP and ATM are required for AR. However, the role of DNA-PK in concert with these two components and possible c-ABL is unclear. It is also not discerned how other p53 related genes such as interferon regulatory factors 1 and 2 (IRF1,2), GADD45, p21 are involved. GADD45 known to be induced by low-moderate doses of ionizing radiation and IRF 1 a tumor suppresser protein and transcription factor known to regulate responses to DNA damage by interacting with p53 and p21 have yet to be examined in the AR pathway. We speculate that these genes are critical elements in the AR because they are essential in the DNA damage recognition mechanisms, and cell cycle check points and may activate or induce nucleotide excision repair. Although Human AP Endonuclease 1 has been implicated as a possible gene involved in the induction of base excision repair after low doses of oxidative damage, it is not known which nucleotide excision repair related genes may play a comparable role. Our work to date has focused on evaluating the role of DNA-PK in AR. Using SCID mouse models with different mutations in DNA-PK, we are evaluating AR in terms of biological endpoints that include apoptosis, cell survival, and persistence of DNA damage measured by the Single Cell Gel Electrophoresis (COMET Assay) after gamma irradiation. Other mutant cell lines with defects in p53 related genes and nucleotide excision repair related genes will be used as well to dissect the pathways involved. We will also quantify the magnitude of nucleotide excision repair induced by low doses EVANS MK NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health environmental adaptation tumor suppressor gene transcription factor radiation genetics DNA repair gel electrophoresis ionizing radiation DNA damage cell line SCID mouse CRISP RPROJ G CRISP/99/G00736-01 CRISP/99/G00736-01 199904 eng AGE, ENERGY AND EXCITOTOXICITY Research RPROJ The successful development of a young investigator is determined by multiple factors. While intellect and curiosity play major roles, the environment in which the transition to independent investigator occurs can be of equal and potentially greater importance. An ideal setting has been created at the Ohio State University. Active expansion of laboratory based research is occurring in the College of Medicine primarily through nurturing the developing of young investigators. Recent growth in the Department of Neurology has produced a climate where basic research initiatives ar needed to complement established strengths in the clinic. With three faculty specializing in movement disorders, patientcare responsibilities can be limited providing substantial protected time in the laboratory. Finally, the assistance of an experienced scientific mentor greatly enhances the likelihood of successful development. The proposed investigation will examine of advancing age on mitochondrial energy metabolism, seeking insight into the mechanism(s) of senescent decline in function. Quantitative autoradiographic, histochemical and immunohistochemical methods will provide a comprehensive assessment of the respiratory chain with a degree of anatomic precision not previously achieved. Delineation of age effect on other metabolic compartment will clarify the role of respiratory chain enzyme alterations in senescent decent. As modification stress enhances excitatory amino acid receptor activation, the relationship, interaction of senescent modification of glutamate receptors and respiratory chain enzymes will be determined. Completion of the proposed research will require training in new methodologies: emulsion autoradiography and immunocytochemistry. In addition, research techniques in molecular biology will be learned and applied to future research endeavors. Frequent seminars and involvement in local and multicenter research groups will a source of continuing education. The opportunities for scientific development and maturation virtually ensure a successful career in neuroscience research. HIGGINS DS OHIO STATE UNIVERSITY, 1654 UPHAM DRIVE, COLUMBUS, OH 43210 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 43210 Crisp Data Base National Institutes Of Health aging laboratory rat bioenergetics brain metabolism ouabain mitochondria kainate immunocytochemistry neuron neurotoxin cytochrome oxidase glutamate dehydrogenase lactate dehydrogenase succinate dehydrogenase autoradiography glutamate receptor cellular respiration tissue /cell culture cell senescence dizocilpine excitatory aminoacid enzyme activity neurotoxicology CRISP RPROJ OHIO G CRISP/99/AG00751-02 CRISP/99/AG00751-02 199904 eng INTRACEREBROVENTRICULARLY ADMINISTERED CYTOKINES ON NEURODEGENERATION Research RPROJ Several hypotheses have been created to account for the neurodegeneration and subsequent cognitive deficits observed in Alzheimer's disease. One hypothesis, in particular, has focused on the effects of inflammation as a mediator of neurodegeneration. To address this possibility, we have initiated studies examining the direct and indirect effects that endotoxin and inflammatory cytokines may have on neural tissue and neuronal cell signaling. Initial studies have demonstrated that the intracerebral infusion of endotoxin produces a significant age-related increase in brain tumor necrosis factor- alpha (TNFa) levels, but does not effect the production of a number of other inflammatory cytokines such as interleukin-1 or interleukin-12. We are currently examining young and old rodent brain homogenates and plasma post endotoxin treatment for the presence of various inflammatory cytokines (e.g., interleukin-6, interleukin-4, interleukin-10) and chemokines (e.g., interleukin- 8, MCP-1). Moreover,animals are also being examined for the effects of direct cytokine administration on inducing brain inflammation, permeabilizing of the blood-brain barrier, and effects on leukocyte trafficking, CNS surface markers, neurodegeneration, and cognitive behavior. We believe that cytokine and chemokine infusion (icv and iv) in rodent brains will have significant biological and physiological effects on neural tissue and will ultimately reveal a relationship between neuroinflammation, age, and cognitive behavior. These studies have been initiated using intracerebral TNF-a infusion and we hope to soon follow these efforts with chemokine and interferon infusions. In addition, additional studies will also be performed to determine whether administration of signaling inhibitors and antagonists and/or through in vivo leukocyte depletion can ameliorate the biological and physiological effects of administered cytokines. It is our hope that these efforts will assist in our understanding of the contribution of cytokines and chemokines in the neuroinflammatory and neurodegenerative effects observed in various neurotrauma models and age-related disease states. TAUB DD NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory rat cerebral ventricle cell migration inflammation injection /infusion tumor necrosis factor alpha endotoxin neural degeneration age difference chemokine CRISP RPROJ G CRISP/99/G00759-01 CRISP/99/G00759-01 199904 eng VITAMIN K AND SKELETAL HEALTH Research RPROJ The proposed program will facilitate the development of Neil Binkley as an academic geriatrician and gerontologist at the University of Wisconsin, an institution with a long history of gerontologic research. It will provide the opportunity to further develop and apply basic science and clinical skills to his focused area of interest, osteoporosis, by protecting his time and allowing the acquisition of skills and experience needed to mature as an independent researcher. Skeletal fragility becomes common with advancing age with up to 40 percent of females sustaining an osteoporotic fracture. As at least three vitamin K dependent skeletal proteins exist, it is plausible that vitamin K plays a role in the maintenance of skeletal health. While vitamin K insufficiency has classically been defined in terms of coagulation abnormalities (prolongation of the prothrombin time), it has recently become appreciated that measurement of undercarboxylation of other proteins, notably osteocalcin, is a much more sensitive measure of adequacy. If defined as elevated serum levels of undercarboxylated osteocalcin, some reports find the majority of postmenopausal women to be vitamin K insufficient. Thus it is plausible that vitamin K insufficiency is common and contributes to the development of osteoporosis. However, prior studies of the effect of vitamin K upon the skeleton have yielded contradictory results. Some reports find that vitamin K insufficiency is associated with low bone mass or skeletal fragility, while others find no effect, or even an increase in bone mass. These data have lead to diametrically opposed suggestions, i.e., that vitamin K insufficiency may cause osteoporosis and conversely, that vitamin K antagonists may be useful to treat osteoporosis. To address these divergent hypotheses, the overall goal of the proposed research is to determine if vitamin K or the vitamin K antagonist warfarin have a role in determining skeletal health. In this regard, we hypothesize that: 1. Vitamin K insufficiency produced by therapeutic anticoagulation leads to bone loss. 2. The disparate animal studies may be the result of unrecognized toxic effects of warfarin on the skeleton. 3. Vitamin K insufficiency and resultant reduced bone osteocalcin content may contribute to skeletal fragility by reducing bone strength. 4. Vitamin K insufficiency accentuates estrogen depletion bone loss. To test these hypotheses, we will utilize the rhesus monkey as a model in which to assess the effect of therapeutic warfarin treatment upon the skeleton. In addition, utilizing adult rats, we will evaluate the effect of long-term dietary vitamin K insufficiency and warfarin regimens on serum and bone osteocalcin, markers of skeletal turnover and calcium homeostasis, bone density and biochemical strength. Subsequently, we will assess the effect of pre-existing and ongoing vitamin K insufficiency upon ovariectomy induced bone loss in female rats. Delineation of the role(s) of vitamin K in skeletal health may have implications for osteoporosis prevention and treatment. BINKLEY NC UNIVERSITY OF WISCONSIN, 2245 MSC, 1300 UNIVERSITY AVE, MADISON, WI 53706 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 53706 Crisp Data Base National Institutes Of Health aging Macaca mulatta laboratory rat warfarin biomechanics calcium metabolism drug adverse effect estrogen pathogenic diet nutrition related tag vitamin K deficiency tensile strength osteocalcin ovariectomy female osteoporosis bone density bone metabolism vitamin K CRISP RPROJ WISCONSIN G CRISP/99/AG00801-01A1 CRISP/99/AG00801-01A1 199904 eng BENZODIAZEPINE USE AND RISK OF DISABILITY IN THE ELDERLY Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The applicant states that her long-term career goal is to become an independent investigator, focusing on the adverse health outcomes of medication use in the elderly, by utilizing principles of pharmacoepidemology. She currently holds the rank of Assistant Professor in the School of Pharmacy at the applicant institution. She has completed a 2-year research pharmacy fellowship in geriatrics, has conducted research in the area of adverse drug effects in the elderly, and provides clinical services as part of geriatric team. To meet her career objectives, she is proposing to merg her clinical pharmacy background with expertise in epidemiologic methods over a 5-year period by: 1) completing course-work towards a master's degree in the School of Public and Community Health, University of Washington; and 2) conducting supervised research under the guidance of her sponser (David Buchner and co-sponsers (Andreas Stergachis and Andrea LaCroix) Some adverse health consequences of benzodiazepine use in elderly are established and represent a significant public health concern. Yet, according to the applicant, little is known about whether these agents contribute to the disability process. During the proposed award, the applicant plans to determine if benzodiazepine use is associated with the losss of mobility, loss of independence in activities of daily living (ADLs), use of health services, and mortality over a 3- year study period in community dwelling elderly. This research will use data from four large, federally-funded, population-based logitudinal studies of the elderly, containg data on approximately 17,000 individuals. Use of the following data sets is proposed: Established Populations for Epidemiologic Studies of the Elderly; Women's Health and Aging Study; Cardiovascular Health Study; and Group Health Cooperative Demonstration Project. The four outcomes will be evaluated within each dataset, and the data will be poooled for a meta-analysis. Beginning in the third year of the proposed award period, the applicant plans to submit additional competitive grants, and initiate research in other areas related to medication use and adverse health outcomes in the elderly, working closely with her sponsors. GRAY SL UNIVERSITY OF WASHINGTON, BOX 357630, SEATTLE, WA 98195-7630 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health human old age (65+) aging benzodiazepine disease /disorder proneness /risk disabling disease drug adverse effect health care service utilization epidemiology longitudinal human study functional ability human data meta analysis outcomes research CRISP RPROJ WASHINGTON G CRISP/99/AG00808-01 CRISP/99/AG00808-01 199904 eng ZINC AND CALCIUM ION PERMEABLE AMPA CHANNELS Research RPROJ DESCRIPTION: (Adapted from the application) Since starting at U.C. Irvine in late 1991, the applicant has established a laboratory studying cellular mechanisms of selective neurodegeneration of relevance to the aging brain. He has obtained independent funding (R01 and private grants), and achieved promotion to the rank of Associate professor, with tenure, in August, 1996. To address the sophisticated mechanistic questions which he intends will constitute the core of his research, it will be necessary to supplement toxicity and histology paradigms with techniques of fluorescent imaging and patch clamp electrophysiology, both powerful tools for studying behaviors of living neurons with which he presently has limited experience. Unfortunately, while experts on campus can provide technical assistance, his present heavy teaching, clinical and administrative duties leave insufficient time for mastery of these techniques. The salary support provided by an ISA, by assuring 80% protected time for research, would immeasurably aid the applicant s career development. The physiologic basis for the highly selective pattern of neurodegeneration seen in many diseases of the aging brain is largely unknown. Recent studies have revealed a potentially important clue: While most AMPA/kainate receptor-gated channels are Ca2+ impermeable, certain populations of neurons, including many types that preferentially degenerate in Alzheimer's disease or ischemia, express AMPA/kainate receptor gating channels with high direct Ca2+ permeability. The present proposal follows from our preliminary studies indicating that the endogenous synaptically released cation, Zn2+ appears to permeate Ca2+-permeable AMPA channels with particular rapidity selectively damaging neurons expressing these channels. Initial experiments will thus seek to examine the cellular and subcellular distribution of Zn2+ and Ca2+ permeable AMPA/kainate channel on hippocampal neurons. Histologic approaches and fluorescent imaging will be employed to localize these channels, and whole-cell patch-clamp techniques will be used to examine their permeabilities to Zn2+ and Ca2+. Subsequent experiments will examine consequences of Zn2+ permeation through these channels. Both neurotoxic and physiologic effects will be assessed, using histological, fluorescence imaging and electrophysiological techniques. It is hoped that these experiments will provide initial clues to physiological effects of Zn2+ that may be of importance to selective neurodegeneration in disease as well as to normal neuronal functioning. WEISS JH UNIVERSITY OF CALIFORNIA, IRVINE, CA 92697-4290 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory mouse membrane permeability calcium flux kainate fluorescent dye /probe patch clamp zinc neural degeneration neural plasticity membrane channel glutamate receptor embryo /fetus cell culture excitatory aminoacid neurotoxicology AMPA receptor CRISP RPROJ CALIFORNIA G CRISP/99/AG00836-01 CRISP/99/AG00836-01 199904 eng ION CHANNELS AND AMYLOID INDUCED CELL DEATH Research RPROJ DESCRIPTION: (Adapted from the application) Basal forebrain cholinergic neurons are prominently affected early in the course of Alzheimer's disease (AD). In rodent models, infusion of amyloid B peptide (AB, a candidate neurotoxin) into the basal forebrain affects cholinergic neurons, while sparing other neuronal populations. The mechanisms underlying this selective vulnerability of cholinergic neurons are presently unknown. To develop a model of selective cholinergic neuronal vulnerability, the applicants assayed the in vitro cytotoxicity of AB on a hybrid cholinergic septal cell line (SN56). SN56 cells, when differentiated with cAMP, express choline acetyltransferase (ChAT), and are killed by AB, whereas a related septal cell line (SN48), which does not express ChAT, is resistant to AB toxicity. In striking contrast to SN48 cells, the vulnerable SN56 cells express a tetraethylammonium (TEA)-sensitive delayed rectifier K+ current (IK), which is up-regulated by exposure to AB prior to cell death. Moreover,-TEA concentrations that block IK also protect SN56 cells from AB induced death, suggesting that alteration in this potassium current may underlie the vulnerability of these cholinergic cells to AB. This is relatively specific, as neither modulation of IK nor TEA protection was observed in SN56 cells after injury from hypoglycemia. These observations suggest that modulation of potassium currents may be a critical determinant of the selective vulnerability of cholinergic neurons to AB toxicity. However, the specific channels involved, the mechanisms underlying AB-induced IK enhancement, and the mechanisms linking IK enhancement with toxicity remain to be determined. To address these questions, the applicants propose to characterize IK in vulnerable SN56 cells, identify the K+ channel subunits underlying this current, and test whether susceptibility to AB may be conferred by the expression of specific K+ channel subunits. Furthermore, they will investigate the link between AB-induced IK enhancement, altered intracellular calcium concentration ([Ca2+]i), and AB toxicity. The applicants hypothesize that expression of specific K+ channels mediate the selective vulnerability of basal forebrain cholinergic neurons to candidate neurotoxins such as AB. The proposed experiments will test this novel hypothesis relating selective neuronal vulnerability in a neurodegenerative disorder to the expression of membrane ionic currents. Data from the proposed work will be critical to the development of specific strategies to modify cell vulnerability in patients with AD, and will be of great interest to investigators in a number of related fields, such as mechanisms of cell death, ion channel function, and other neurodegenerative illnesses. The candidate is committed to a research career. His background in septo-hippocampal cholinergic systems seems appropriate to understanding these structures' neurodegeneration in AD. The candidate's short-term goal is to understand the mechanisms underlying basal forebrain cholinergic cell vulnerability in AD. His long-term goal is to develop a strong independent program in AD research. The proposed studies will be carried out at the facilities of Baylor College of Medicine under the sponsorship of Stanley H. Appel, M.D., an internationally renowned investigator with long-standing interest in Alzheimer's disease and other neurodegenerative disorders. COLOM LV BAYLOR COLLEGE OF MEDICINE, ONE BAYLOR PLAZA, HOUSTON, TX 77030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 77030 Crisp Data Base National Institutes Of Health laboratory mouse prosencephalon calcium flux patch clamp electrophysiology transfection molecular cloning protein structure amyloid protein potassium channel receptor expression tissue /cell culture cytotoxicity cell line CRISP RPROJ TEXAS G CRISP/99/AG00850-01 CRISP/99/AG00850-01 199904 eng PATHWAYS RESPONSE TO GENOTOXIC STRESS IN NORMAL AND AGED CELLS Research RPROJ Summary of work: Genotoxic/oxidative stresses contribute to the development of degenerative diseases, and may underlie the aging process itself. Cells respond to such stresses with the induction of numerous gene products, but much remains to be learned concerning the signaling pathways mediating these effects or the functional significance of the induced gene products. This project encompasses studies related to these cellular responses. Efforts over the past year have been focused in several areas: 1) Activation of Akt Kinase by Oxidative Stress. We have demonstrated that hydrogen peroxide treatment results in the activation of a downstream mediator of PI-3 kinase signaling, Akt, in a variety of human cell types. Recent findings from several laboratories have implicated the PI-3 kinase/Akt pathway in enhancing survival in response to growth factor deprivation. In keeping with this view we found that inhibitors of PI-3 kinase (i.e. wortmannin) likewise prevented activation of Akt by hydrogen peroxide and enhanced the cytotoxicity seen in hydrogen peroxide-treated cells. These findings suggest an important survival role for this pathway during the cellular response to oxidant injury. 2) Role of Posttranscriptional Events in the Reglation of Cyclin D1 Expression Following Stress. Recent studies from our laboratory have demonstrated that cyclin D1 expression is greatly reduced following treatment with many stressful agents including the cyclopentenone prostaglandin PGA2. We have begun to explore the mechanism contributing to this down-regulation and have found that this occurs largely through posttranscriptional events leading to reduced stability of cyclin D1 mRNA. Actinomycin D treatment can prevent this loss, suggesting that a short-lived stress-inducible destabilizing protein is involved. Current studies are aimed at identifying this protein, and the mechanism through which it acts. 3) Role of c-Jun in Influencing Cell Survival Following Stress. We are employing cell lines harboring a dominant negative mutant form of c-jun (which can not be phosphorylated and activated) to test the role of this transcription factor in influencing cell surival in the gliobastoma cell line T98G following different stresses. Our results indicate that inhibition of c-jun activity sensitizes cells to DNA damaging stresses (UVC irradiation, x-irradiation, etoposide) but does not affect sensitivity to other non-DNA damaging stresses that induce apoptosis (tunicamycin, thapsigargin, taxol). Current studies are exploring the basis for this differential sensitivity. HOLBROOK NJ NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging mitogen prostaglandin liver cell arsenic hydrogen peroxide phenylacetate ultraviolet radiation growth factor receptor physiologic stressor methane sulfonate tissue /cell culture DNA damage enzyme activity mitogen activated protein kinase CRISP RPROJ G CRISP/99/G00902-05 CRISP/99/G00902-05 199904 eng EXENDIN-4 AS A TREATMENT FOR DIABETES MELLITUS Research RPROJ Beta cells of the pancreas, which make insulin, do not respond normally to glucose in type 2 diabetes. In conjunction with this, and despite all treatments currently available to treat diabetes, beta cell function continues to deteriorate over time. With the data now available from the United Kingdom Prospective Diabetes Study (Sept. 1998) this point was brought home even more forcefully. Despite continual monitoring of patients enrolled in the study euglycemia could not be maintained, even with intensive therapy, because of declining beta cell function. We have been working for some time with GLP-1, a naturally occurring peptide, produced and released from the gut in response to food. It augments the insulin-releasing capacity of beta cells in response to food. Of great interest is the finding that in type 2 diabetes pharmacological doses of GLP-1 can normalize blood sugars, ie, euglycemia is achieved. However, GLP-1 has a biological half-life of only 2-4 minutes and has to be given systemically. This means that it would have to be given continuously in order to maintain euglycemia. The Gila monster is a lizard whose natural habitat is in Arizona. It produces a venom in its saliva which is a homolog of GLP-1, called Exendin-4. When it is given systemically to rodents its biological half-life is 12-16 hours. We gave Exendin-4 intraperitoneally, once daily, to db/db mice and showed that the hemoglobin A1c, a marker of long-term control of blood glucose, was 5.7% in the treated animals vs 9.1% in the non treated animals. We are expanding this project to find the minimal amount of Exendin-4 that is anti-diabetogenic and we are looking at the mechanisms whereby Exendin-4 has such beneficial long-term effects. We have obtained antibodies to exendin-4 so as to develop assays in order to measure its concentrations in plasma. An Investigational IND from the FDA has just been granted to us so that we can begin to administer Exendin-4 to diabetic subjects in order to monitor its efficacy after subcutaneous administration at various concentrations. We plan to begin the studies in the next year. EGAN JM NIA, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory mouse obesity blood glucose diabetes mellitus therapy noninsulin dependent diabetes mellitus drug administration rate /duration drug administration route peptide hormone analog hormone therapy gastrointestinal hormone disease model reptile poison CRISP RPROJ G CRISP/99/G00905-02 CRISP/99/G00905-02 199904 eng SPECIFICITY OF OXYGEN DNA DAMAGE AND MUTAGENESIS Research LOEB LA UNIVERSITY OF WASHINGTON, BOX 357470, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 98195 Crisp Data Base National Institutes Of Health aging Aves laboratory mouse transgenic animal species difference congenital aplastic anemia lymphocyte flow cytometry polymerase chain reaction mutagen gene mutation human subject Werner's syndrome DNA repair free radical oxygen DNA damage oxidative stress clinical research CRISP RPROJ WASHINGTON G CRISP/99/AG01751-200007 CRISP/99/AG01751-200007 199904 eng HIPPOCAMPAL SYNAPTIC STRUCTURE DURING AGING Research RPROJ DESCRIPTION (Adapted from applicant's abstract): This is a proposal for a competitive continuation of a project on basic membrane and synaptic mechanisms of brain aging that has been ongoing for over 15 years. The past periods in this project have found two main electrophysiological alterations in rat hippocampal CAl neurons with aging: 1) impaired synaptic frequency potentiation (facilitation); and 2) an increase in voltage-gated Ca2+ influx. These results and others have contributed to the general Ca2+ hypothesis of brain aging and dementia. In the most recent period, the single channel patch clamp configuration was adapted for brain aging studies and identified an increase in the membrane density of available L-type Ca2+ channels as a potential molecular basis for the changes with aging seen at the cellular level. Here, the specific hypothesis that the increase in L-type Ca2+ channels is a key mechanism in both impaired function (synaptic potentiation or spike generation) and neuronal vulnerability to death of aged mammalian neurons will be tested. Studies will be conducted in rat hippocampal slices and long-term hippocampal cultures in which L-type Ca2+ channels are enriched. Effects of repetitive synaptic activation on the magnitude and topography of Ca2+ transients in hippocampal slice neurons of adult and aged rats will be studied, using a rapid UV-compatible confocal laser scanning microscope for Ca2+ imaging simultaneously with intracellular electrophysiological recording. Multiple specific channel blockers and kinase modulators will be used to define critical Ca2+ entry pathways. These studies will determine whether postsynaptic Ca2+ transients, particularly through L-channels, can modulate neuronal short-term synaptic plasticity and contribute to changes in aged brain neurons. In parallel studies of cell cultures, the role of time-dependent ion channel changes in cell death will be tested, by investigating differences in vulnerability to excitotoxicity in cells with different complements of L-type channels. Single channel recording, Ca2+ imaging, pharmacologic blockade and kinase modulation will be used to define Ca2+ sources critical for necrosis and apoptosis. LANDFIELD PW UNIVERSITY OF KENTUCKY, 800 ROSE STREET, LEXINGTON, KY 40536-0084 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory rat hippocampus brain electrical activity cell death patch clamp electrophysiology membrane potential membrane structure neural degeneration synapse synaptic vesicle neuron neurophysiology neural plasticity neurotoxin calcium channel tissue /cell culture confocal scanning microscopy calcium ion CRISP RPROJ KENTUCKY G CRISP/99/AG04542-12 CRISP/99/AG04542-12 199904 eng PREVENTION OF AGE-RELATED BONE LOSS WITH CALCIUM THERAPY Research RIGGS BL MAYO FOUNDATION ROCHESTER, 200 FIRST ST SW, ROCHESTER, MN 55905 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 55905 Crisp Data Base National Institutes Of Health human old age (65+) aging biomarker clinical trial drug adverse effect postmenopause human subject immunochemistry bone fracture diet therapy dietary calcium nutrition related tag dietary supplement parathyroid hormone longitudinal human study cyclic AMP female osteoporosis pathologic bone resorption bone density bone metabolism human therapy evaluation photon absorptiometry chemoprevention CRISP RPROJ MINNESOTA G CRISP/99/AG04875-150006 CRISP/99/AG04875-150006 199904 eng BETA AMYLOID FREE RADICAL PRODUCTION AND NEUROTOXICITY--RELEVANCE TO AD Research BUTTERFIELD DA UNIVERSITY OF KENTUCKY, SOUTH LIMESTONE STREET, LEXINGTON, KY 40536-0230 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health jird /gerbil laboratory rat Alzheimer's disease electron spin resonance spectroscopy pathologic process free radical neurotoxin nitrone protein structure /function amyloid protein oxidative stress CRISP RPROJ KENTUCKY G CRISP/99/AG05119-120001 CRISP/99/AG05119-120001 199904 eng PILOT--ESTABLISHMENT AND USE OF PERPETUAL HUMAN NEURONAL CULTURES Research RAY J UNIV OF CALIFORNIA, SAN DIEGO, 9500 GILMAN DRIVE - 0948, LA JOLLA, CA 92093-0948 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory rat cell differentiation cell cycle polymerase chain reaction glutamate gene expression genetic transduction fibroblast growth factor human embryo /fetus tissue immunocytochemistry neuron central nervous system neurotoxin protein metabolism amyloid protein tissue /cell culture CRISP RPROJ CALIFORNIA G CRISP/99/AG05131-150026 CRISP/99/AG05131-150026 199904 eng MODELING ALZHEIMER'S DISEASE--B AMYLOID AND APOE Research LE BOEUF R VA PUGET SOUND HLTH CARE SYSTE, 1660 SOUTH COLUMBIAN WAY, SEATTLE, WA 98108 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 98108 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal Alzheimer's disease molecular pathology transfection allele gene expression gene interaction immunocytochemistry amyloidosis model design /development disease model neurofibrillary tangle neurotoxin in situ hybridization amyloid protein animal breeding human genetic material tag protein isoform gene targeting apolipoprotein E CRISP RPROJ WASHINGTON G CRISP/99/AG05136-150017 CRISP/99/AG05136-150017 199904 eng CELLULAR SIGNALING AND ALZHEIMER-LIKE NEURODEGENERATION Research MATTSON MP UNIVERSITY OF KENTUCKY, 101 SANDERS-BROWN CTR ON AGING, LEXINGTON, KY 40536-0230 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory rat biological signal transduction hippocampus Alzheimer's disease cell death glutamate free radical enzyme induction /repression neurotrophic factor human tissue western blotting neural degeneration neurotoxin antisense nucleic acid antioxidant phosphorylation tau protein amyloid protein calcium transporting ATPase sodium potassium exchanging ATPase embryo /fetus cell culture cell cycle protein enzyme activity oxidative stress neuroprotectant mitogen activated protein kinase CRISP RPROJ KENTUCKY G CRISP/99/AG05144-150005 CRISP/99/AG05144-150005 199904 eng NEURONAL INSULT AND CYTOSKELETAL DISRUPTION Research GEDDES JW UNIVERSITY OF KENTUCKY, 101 SANDERS-BROWN CTR ON AGING, LEXINGTON, KY 40536-0230 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory rat hippocampus Alzheimer's disease calcium cytoskeleton cellular pathology free radical human tissue immunocytochemistry western blotting neurofibrillary tangle paired helical filament neurofilament neurotoxin phosphorylation calpain cytoskeletal protein microtubule associated protein tau protein tubulin embryo /fetus cell culture cell cycle protein enzyme activity oxidative stress transmission electron microscopy mitogen activated protein kinase CRISP RPROJ KENTUCKY G CRISP/99/AG05144-150010 CRISP/99/AG05144-150010 199904 eng CELLULAR ENGINEERING TO TREAT DISEASES OF AGING Research RPROJ We propose to continue long-term efforts to perfect bone marrow transplantation (BMT) as a means to prevent or treat many different diseases. This series of systematic experimental investigations will explore further the usefulness of bone marrow or fetal liver cell transplantation and ultimately stem cell transplantation as an experimental approach to analysis, prevention or treatment of a number of diseases and disorders not yet effectively treated. First we will explore BMT as an approach to prevention or to causation of senility in mice. We will try to prevent senility associated disease or disorders, including amyloid deposition and arthritis or arthroses, in senility accelerated mice [SAM(P)] mice. Alternatively, congenic or allogeneic BMT or congenic or allogeneic neonatal liver transplantation will be tested as a means of introducing from SAM(P) into the congenic resistant SAM(R) mice the senility accelerating propensity. Similarly, BMT will be tested as a means of causing, preventing or treating senility-associated changes and amyloidosis when BMT donors share the major histocompatibility complex (MHC) but are allogeneic to the SAM(P) mice, e.g. CBA/H or C3H/He. Alternatively, we will attempt to use donors that differ from SAM(P) at MHC, e.g. C57B1/6. BMT will also be tested as a means of preventing or treating two new models of non-inflammatory coronary vascular diseases or atherosclerosis in inbred mice. We will compare the immunological consequences of long-term BMT induced chimeric state where BMT have either been performed from autoimmune-resistant donors to mice of autoimmune-prone strains or to mice of autoimmune-resistant strains. We will also ascertain whether it is possible to treat effectively by BMT advanced, putatively irreversible kidney disease associated with autoimmunity states. Experiments will attempt to employ BMT from MHC-matched, MHC-mismatched or haploidentical donors to treat effectively advanced kidney disease by complete correction by BMT of the autoimmunity and immunologic dysregulation that led to the kidney disease in the first place. Finally, we will initiate experiments to evaluate the potential of stem cell preparations to reconstruct the entire hematopoietic and lymphoid systems and immunologic functions as an approach to correcting the genetically determined propensity to develop autoimmunities nad other diseases associated with aging and to analyze and influence the abnormal accumulations of Ly1+ B lymphocytes that occur in these autoimmune states. GOOD RA UNIVERSITY OF SOUTH FLORIDA, 4202 E. FOWLER AVENUE, TAMPA FL 33620 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 33620 Crisp Data Base National Institutes Of Health aging laboratory mouse genetic strain bone marrow transplantation hematopoietic stem cell B lymphocyte T lymphocyte atherosclerosis cellular pathology genetic manipulation radiation genetics leukocyte activation /transformation immunoregulation immunogenetics autoimmune disorder kidney disorder liver transplantation amyloidosis diet nutrition related tag tissue /cell culture histocompatibility tissue mosaicism CRISP RPROJ FLORIDA G CRISP/99/AG05628-14 CRISP/99/AG05628-14 199904 eng SEROTONERGIC NEUROPHARMACOLOGY OF THE AGING BRAIN Research RPROJ Serotonergic receptors are the focus of the proposed studies since alterations in this neurotransmitter system have been implicated in the etiology of age-related diseases, including anxiety and clinical depression. The 5-HT1A receptor has been chosen for examination as it has been implicated in each of these disorders. The pattern of age-related changes in 5-HT1A receptor function will be investigated using an appropriate model, male Fischer 344 rats (ages 3, 14 and 24 mo). The overall goal is to test the hypothesis that the functional dynamics of 5-HT1A receptors are significantly compromised with aging. The specific aims of this project include evaluation of age- related alterations in the: l) neuroanatomical, functional and molecular representation of the 5-HT1A receptor using radioligand binding, quantitative autoradiograph, and in situ hybridization techniques, 2) physiological responses of 5-HT1A receptors using in vivo and in vitro electrophysiological recording preparations and 3) neurochemical and physiological characterization of 5-HT1A spare receptor pools following 1-ethoxycarbonyl-2-ethoxy-1,2- dihydroquinoline (EEDQ) treatment. These data will provide the foundation for a better understanding of the neurochemical basis of behavioral processes mediated by serotonin in the elderly patient. Ultimately, this research will contribute new information for the development of more effective treatments for the management of depression and anxiety in the elderly. KECK BJ PENNSYLVANIA STATE UNIV, P O BOX 850, HERSHEY, PA 17033 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 17033 Crisp Data Base National Institutes Of Health aging laboratory rat serotonin electrophysiology neuroanatomy neuropharmacology neurotoxin in situ hybridization autoradiography serotonin receptor CRISP RPROJ PENNSYLVANIA G CRISP/99/AG05728-03 CRISP/99/AG05728-03 199904 eng MOLECULAR ANALYSIS OF AMYLOID BETA TOXICITY Research RPROJ The overall goal of this research is to identify genes involved in the toxicity of beta-amyloid (Abeta), a peptide associated with Alzheimer's disease (AD). Abeta is toxic to cultured neuronal cells, but the molecular details of the response are largely unknown. Alterations in gene expression of cells upon the addition of Abeta will be analyzed by differential display technique, which is one of the specific aims of this proposal. In addition, cells surviving long-term exposure to the peptide become totally resistant to Abeta, reflecting the induction of protective genes and/or the inhibition of sensitive genes. Comparison of gene expression between Abeta- resistant and sensitive cells using differential display technique has revealed a number of candidate genes involved in Abeta protection. The second specific aim of this proposal is to examine these candidate genes for the relevance of their activation or inhibition to the Abeta resistance. It is hoped that the research will lead to the identification of new genes involved in Abeta toxicity which will then help to devise new therapeutic means to attack AD. LI Y SALK INST BIOLOGICAL STUDIES, PO BOX 85800, SAN DIEGO, CA 92186-5800 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health Animalia Alzheimer's disease gene induction /repression neuron amyloid protein tissue /cell culture neurotoxicology CRISP RPROJ CALIFORNIA G CRISP/99/AG05769-02 CRISP/99/AG05769-02 199904 eng EFFECT OF SUPPRESSED BONE TURNOVER ON SKELETAL FRAGILITY Research BURR DB INDIANA UNIV SCHOOL OF MED, 545 BARNHILL DR, EM 421, INDIANAPOLIS, IN 46202-5124 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging dog biomarker calcification inhibitor dosage drug administration rate /duration drug adverse effect drug screening /evaluation histopathology bone fracture etidronate acid phosphatase alkaline phosphatase diphosphonate osteocalcin radiography osteoporosis pathologic bone resorption skeletal pharmacology bone density normal ossification bone metabolism physiologic bone resorption nonhuman therapy evaluation chemoprevention CRISP RPROJ INDIANA G CRISP/99/AG05793-140006 CRISP/99/AG05793-140006 199904 eng NEUROPROTECTIVE ACTIONS OF ESTROGEN IN CORTICAL EXPLANTS Research RPROJ NO ABSTRACT AVAILABLE WILSON ME UNIVERSITY OF KENTUCKY, 800 ROSE STREET, LEXINGTON, KY 40536 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 40536 Crisp Data Base National Institutes Of Health aging Animalia cerebral cortex glutamate pharmacokinetics estradiol estrogen gene expression insulinlike growth factor neural degeneration messenger RNA lactate dehydrogenase tissue /cell culture organ culture programmed cell death neuroprotectant neurotoxicology CRISP RPROJ KENTUCKY G CRISP/99/AG05818-01 CRISP/99/AG05818-01 199904 eng NEURONAL CA++ SEQUESTERING COMPARTMENTS PROTECTING ROLE Research RPROJ The principle research focus of this grant is to gain insight into the structure and function of the intracellular Ca2+ sequestering compartments of neurons. These compartments are of great interest because recent evidence has accumulated that they play a protective role in neurons exposed to glutamate, the major excitatory neurotransmitter in the CNS and the cause of "excitotoxicity". Excitotoxicity is a major cause of neuronal damage in stroke and trauma as well as a likely mediator of neuronal injury in age related neurodegenerative diseases, e.g. Huntington's, ALS. The specific aims are: 1. To localize the specific isoforms of CaATPase and calreticulin two, by components of neuronal Ca2+ sequestering compartments in brain and cultured neurons and neuroblastoma cells. 2. To investigate the biosynthetic route(s) by which the CaATPase and calreticulin follow to reach the Ca2+ sequestering compartments. 3. To study the effects of agents which raise cytosolic Ca2+ on the mRNA and protein level of CaATPase and calreticulin using neuroblastoma cells and cultured neurons. 4. To transfect neuroblastoma cells with cDNAs for CaATPase and calreticulin to determine if these proteins are protective against toxic effects of elevated cytosolic Ca2+ and 5. To employ intracellular dyes to directly measure cytosolic Ca2+ in neurons and neuroblastoma cells. We will employ biochemical, morphological and molecular techniques in these investigations. FINE RE BOSTON UNIVERSITY, 80 EAST CONCORD ST, BOSTON, MA 02118 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02118 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory rat intracellular transport ionophore calcium endoplasmic reticulum hybrid cell fluorescent dye /probe transfection genetic regulation immunofluorescence technique homeostasis glioma neuroblastoma neurophysiology neurotoxin in situ hybridization protein biosynthesis protein transport calcium transporting ATPase neoplastic cell culture for noncancer research neuroprotectant protein isoform calreticulin CRISP RPROJ MASSACHUSETTS G CRISP/99/AG05894-26 CRISP/99/AG05894-26 199904 eng CHOLINERGIC MECHANISMS IN AGING AND ALZHEIMERS DISEASE Research RPROJ DESCRIPTION: (Applicant's Abstract) This laboratory is interested in the development of new therapies for human brain diseases that can be controlled by drugs selective for m1, m2, m3, m4, or m5 muscarinic receptors. At present many groups are interested in the potential use of an m1 agonist for memory disorders, based largely on the prevalence of m1 receptors in the cortex and hippocampus, the amnesic effects of scopolamine, and the loss of acetylcholine in Alzheimer's disease (AD). In reality, we don't know how new selective agonists or antagonists for m1-m5 receptors might work, because we have not had these drugs to study. In fact, the field of muscarinic neurotransmission is still at the stage where the most pressing need is to understand how individual receptor subtypes regulate the functions of neurons, circuits and specific behaviors. This lab has discovered the only specific antagonists for m1 and m4 receptors, m1-toxin and m4-toxin. These toxins now permit, and we propose to carry out, precise and coordinated anatomical, physiological, biochemical and behavioral experiments to establish the cells and circuits at which new m4- and m1-selective drugs may be use to modify movement, memory and pain. Studies of the striatum begin with the premise that an m4 antagonist will be useful for hypokinetic disorders (e.g., Parkinson's disease) and an m4 agonist for hyperkinetic disorders (e.g., tardive dyskinesia), based on the exceptional prevalence of striatal m4 receptors, 5-fold lower levels elsewhere, and the effects of scopolamine on movement. Fluorescent toxins and laser scanning confocal microscopy (LSCM) will be used to test the idea that m4 receptors are located preferentially on rat striatal projection neurons in the direct pathway, plus or minus nigrostriatal lesions. Collaborative electrophysiological studies with both toxins will establish to cells and currents that can be regulated. Both toxins will be used in vivo to study the agonist-induced turning responses of rats having unequal dopaminergic or cholinergic receptor levels in the right and left striata. These studies should provide a rational basis for testing m4-selective drugs for the treatment of movement disorders. Studies of hippocampus are based on the exceptional prevalence of m1 receptors and the importance of acetylcholine for memory. LSCM will be used to test the idea that m1 and m4 receptors are on different neurons. Both toxins will be used in collaborative studies to establish how m1 and m4 activation modulate hippocampal excitation and inhibition. These studies should help validate the idea of using m1 agonists for memory, and disclose some potential clinical effects of m4-selective drugs. Studies of nociception are based on evidence that muscarinic agonists for m1 or m4 receptors diminish nociception in rats by a mechanism unaffected by naloxone. LSCM will be used to test the idea that the key receptors are m4 in the dorsal spinal cord and rostral vent POTTER LT UNIV OF MIAMI SCHOOL OF MED, 1600 N W 10 AVE, MIAMI, FL 33101 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 33101 Crisp Data Base National Institutes Of Health aging laboratory rat medulla oblongata corpus striatum hippocampus Alzheimer's disease acetylcholine spinal cord neuroanatomy anticholinergic agent cholinergic agent neuropharmacology neurotoxin affinity chromatography behavior test receptor expression muscarinic receptor dopamine receptor pain reptile poison confocal scanning microscopy CRISP RPROJ FLORIDA G CRISP/99/AG06170-13 CRISP/99/AG06170-13 199904 eng ALZHEIMER'S DISEASE PATIENT REGISTRY Research RPROJ The University of Washington, Group Health Cooperative Alzheimer's Disease patient Registry (ADPR) is a model incident case registry for dementia and Alzheimer&#176;s disease developed in 1986 in response to a National Institute of Aging request for proposals. This application is a competing continuation of the ADPR. The application proposes to continue follow-up of cases enrolled between January 1987 and may 1996, during which approximately 1,000 patients were enrolled. These cases have been and will continue to be part of ongoing studies of diagnostic markers, natural history and have served as sources of cases for other investigators at the University of Washington and throughout the country. During the last funding cycle, we established a cohort of 2,58 persons over age 65 from the Group Health enrollment. Based on biennial follow-up examinations, we are detecting all incident cases of Alzheimer&#176;s disease and related dementias from this cohort. This application proposes to continue our follow-up and maintenance of this cohort. Our goal is to estimate age-group specific incident rates of Alzheimer&#176;s disease and related dementias and to test environmental and genetic risk factors which have been previously identified in case-control studies. In addition, the methods of the cohort study have been standardized to companion cross-cultural studies of Japanese-Americans in Honolulu and Seattle, and Japanese in Hiroshima. Since these studies have similar design and data collection protocols, we propose to compare our results wit the results from these affiliated cross-cultural studies, comparing incidence rates, distribution of dementia subtypes, and distribution of risk factors in different sites. The cohort study design affords the opportunity to reduce bias in measurement of exposure and allows us to obtain truly incident dementia cases. The parallel cross- cultural studies offer a unique opportunity to take advantage of the natural variation in environmental exposures that occurs with population migration. Migration studies have traditionally been helpful in determining whether there are modifiable risk factors for prevention of important chronic diseases. LARSON EB UNIV OF WASHINGTON MED CTR, BOX 356330, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 98195 Crisp Data Base National Institutes Of Health Alzheimer's disease brain disorder diagnosis disease /disorder proneness /risk pathologic process drug adverse effect human population genetics patient /disease registry human subject mental health epidemiology mental health information system neurotoxin longitudinal human study neuropsychological test racial /ethnic difference clinical research CRISP RPROJ WASHINGTON G CRISP/99/AG06781-12 CRISP/99/AG06781-12 199904 eng INFLAMMATORY MECHANISMS IN ALZHEIMERS DISEASE Research RPROJ DESCRIPTION (Adapted from Applicant's Abstract): Inflammatory mediators have been widely reported in Alzheimer's disease (AD), and some 21 clinical studies to date have suggested that anti-inflammatory drugs may delay the onset or slow the progression of AD. The NIA is conducting a multicenter trial of an anti-inflammatory. Several major pharmaceutical and biotech firms are pursuing AD inflammation-related strategies. Much still remains to be done, however, to elucidate specific mechanisms. Moreover, how inflammation is sustained over a disease course of a decade or more, despite abundant evidence that inflammation-quenching mechanisms are upregulated in AD, is a question that virtually no one has addressed. In this application we put forward an overall hypothesis for how inflammation might arise, be sustained, and cause neurodegeneration in AD. This hypothesis is divided into three specific aims: induction of pro-inflammatory molecules by AD pathology; neurotoxic actions of the pro-inflammatory molecules; and completing the loop between the pro-inflammatory molecules and AD pathology. In the first specific aim we will evaluate and demonstrate mechanisms for the induction of three major inflammatory classes as the result of exposure to Abet and neurofibrillary tangle material (NFTs). In the second specific aim we will evaluate and demonstrate mechanisms for the neurotoxicity of these Abeta/NFT-mediated inflammatory processes. In the third specific aim we will evaluate and demonstrate mechanisms by which AD inflammation feeds back to further foment Abeta production, yielding the potential for a vicious cycle. Included in the preliminary data underlying these aims are three new findings: Abeta increases AD microglial expression of complement, cytokines, and apoE; NFTs activate complement in an antibody-independent fashion; and complement component C1q increases APP internalization and Abeta secretion by as much as 10-fold, putatively by blocking alpha-secretase cleavage. These novel interactions between pro-inflammatory molecules and AD pathology can help inform inflammation-related therapeutic approaches now being pursued by industry and, in particular, may provide insights to novel agents that might inhibit AD brain inflammation while leaving intact peripheral inflammatory processes (some of which are beneficial) (e.g., immune complex clearance). ROGERS JB SUN HEALTH RES INSTITUTE, 10515 W SANTA FE DR, BOX 1278, SUN CITY, AZ 85372 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 85372 Crisp Data Base National Institutes Of Health Alzheimer's disease pathologic process inflammation human tissue cytokine complement pathway neurofibrillary tangle neurotoxin amyloid protein tissue /cell culture CRISP RPROJ ARIZONA G CRISP/99/AG07367-11 CRISP/99/AG07367-11 199904 eng GENETIC DIFFERENCES IN ALZHEIMERS CASES AND CONTROLS Research RPROJ DESCRIPTION: (Adapted from Investigator's Abstract) The goal of this application is to conduct further analyses in the investigators' large, population-based case-control study. All subjects entered the study between 1987 and 1996. Alzheimer's disease (AD) cases were identified with new AD associated symptoms and then given a complete diagnostic evaluation by research clinical investigators. Control subjects were randomly selected from the same health maintenance organization (HMO) as cases. They were of similar age and sex, intact cognitive status, and were enrolled concurrently. All subjects have been followed annually to verify disease and vital status. The investigators state that the case subjects are likely to reflect sporadic AD occurrence in the general population. Subject accrual is complete; data and biological specimens have been obtained and stored; and the HMO computerized pharmacy database (est. 1977) contains medication history. Environmental and genetic factors may both play a role in the etiology of AD. Apolipoprotein E (e4-allele containing genotypes; APOE) has been shown to increase susceptibility to AD, i.e., it is a strong risk factor. The investigators will describe whether having an e4-containing genotype modifies the risk associated with occupational solvents, anti-inflammatory medications, estrogen replacement, and cigarette smoking. Polymorphic cytochrome p450 and glutathione-s-transferase (GST) genes biotransform environmental substances such as solvents and cigarette smoke. Depending on the polymorphic form of the gene, the original substance can be transformed to either a neutral or a risky state. The investigators will examine whether AD association with solvents or cigarette smoking is modified by polymorphic forms of CYP1A1, CYP2D6, CYP2E1, and GST-T1. Further, the alpha-1-antichymotrypsin gene (2-allele system) may act together with APOE to modify the APOE-AD association (Kamboh et al., 1995). The investigators will compare the strength of association between AD and APOE-e4 across alpha-1-antichymotrypsin genotypes (described as AA, AT, TT). KUKULL WA UNIVERSITY OF WASHINGTON, PO BOX 357236, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 98195 Crisp Data Base National Institutes Of Health Alzheimer's disease disease /disorder proneness /risk molecular pathology antiinflammatory agent tobacco abuse estrogen genetic marker gene expression genotype genetic polymorphism cytochrome P450 human subject nervous system disorder epidemiology nonwater solvent chymotrypsin environmental toxicology glutathione transferase gene /environment interaction smoking clinical research apolipoprotein E CRISP RPROJ WASHINGTON G CRISP/99/AG07584-10 CRISP/99/AG07584-10 199904 eng TRANSMITTER NEUROANATOMY IN ALZHEIMER'S DISEASE Research RPROJ DESCRIPTION (Applicant's Abstract): The proposed studies will address the issue of selective vulnerability of neurons in Alzheimer's disease. Underlying these studies is the hypothesis that excitotoxicity, particularly that which is mediated via stimulation of ionotropic glutamate receptors, contributes to the neuro-degeneration of Alzheimer's disease. Moreover, we predict that in Alzheimer's disease the perturbation of specific glutamate receptor subunits, particularly those involved in the gating of calcium, may contribute significantly to the viability of the cell. The current investigation focuses on the N-methyl- D-aspartate (NMDA) receptor. Notably, previous work of ours has focused on the distribution and expression of specific non-NMDA (i.e. AMPA) receptor subunits in the hippocampus of patients with Alzheimer's disease pathology. Collectively, these investigations attempt to provide a comprehensive understanding of the anatomy of the ionotropic glutamate receptor in the hippocampus of normal subjects and in subjects with Alzheimer's disease. In this application, we propose a series of highly correlated immunohistochemical (Specific Aims 1&2), biochemical (Specific Aim 3) and in situ hybridization studies investigating the distribution and level of expression of specific NMDA receptor subunits (e.g., NMDAR1, NR2A, NR2B, & NR2D). Studies will focus on the human hippocampus, in part, because this region is known to be affected very early within the progression of the disease. Subjects representing a broad range of neuropathologic severity (i.e. Braak stages I-VI) will be studied thus providing us with the opportunity of examining alterations in glutamate receptor expression throughout various stages of the disease. Moreover, the use of specific antibodies identifying early events in the evolution of neurofibrillary pathology provides an additional opportunity of correlating alterations in NMDA receptor subunit expression with initiating events of neurodegeneration. An understanding of the anatomical organization of NMDA and AMPA receptors and the mechanism underlying glutamate-mediated excitotoxicity is important before appropriate drugs aimed at halting Alzheimer's disease-associated neuronal degeneration can be developed. ARMSTRONG DM ALLEGHENY UNIV OF HEALTH SCI, 2900 QUEEN LANE, PHILADELPHIA, PA 19129 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 19129 Crisp Data Base National Institutes Of Health hippocampus Alzheimer's disease brain mapping human tissue immunocytochemistry western blotting neural degeneration in situ hybridization receptor expression NMDA receptor cytotoxicity neurotoxicology AMPA receptor protein localization CRISP RPROJ PENNSYLVANIA G CRISP/99/AG08206-10 CRISP/99/AG08206-10 199904 eng DOPAMINERGIC NEUROTOXINS AND AGING Research RPROJ DESCRIPTION: (Applicant's abstract) Parkinson's Disease (PD), a neurodegenerative disorder of unknown etiology which afflicts a large number of our elderly, is characterized by a massive loss of midbrain dopaminergic (DA) neurons and some loss of noradrenergic (NA) and serotonergic (5HT) neurons in the locus coreuleus and raphe nuclei, respectively. The goal of the proposed research is to investigate the potential neuroprotective role of synaptic vesicles in monoaminergic neurons. The hypothesis is that synaptic vesicles within various populations of monoaminergic neurons can provide a storage compartment for neurotoxins and thus reduce the amount of free toxin in the cytosol capable of producing damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpryidinium (MPP+) will be used because they are potent neurotoxins and because MPP+ is an excellent substrate for the synaptic vesicle monoamine transporter. There are marked differences in the susceptibility of rats and mice to MPTP-induced degeneration of DA neurons which is not associated with the amount of MPP+ to which the neurons are exposed. These findings suggest that there may be a much greater intracellular sequestration of MPP+ within DA neurons in rats as compared to mice. This species difference in neurotoxic response will be used to test the hypothesis. In other studies, the role of vesicular sequestration in NA and 5HT neurons and MPTP toxicity will be evaluated. A multidisciplinary approach will be utilized, including in vivo and in vitro studies, neurochemical and immunohistochemical studies. The specific aims of this project are: 1) to determine if differences in the capacity of vesicles obtained from the neostriata of rats or mice to accumulate MPP+ contribute to the species differences in the sensitivity of dopaminergic neurons to MPTP/MPP+-induced neurotoxicity; 2) to further examine in vivo the degeneration of monoaminergic neurons produced by the administration of MPTP to mice with dysfunctional vesicles; 3) to examine if reserpinization of animals affects MPTP biodistribution of MPP+ accumulation within the brain; 4) to compare dose-response curves for damage produced by intrastriatal infusions of MPP+ in mice and rats treated with or without a vesicular uptake inhibitor. The possibility that vesicles might provide a storage site for exogenous or endogenous neurotoxins is a novel and intriguing concept. This in turn raises the question as to whether defects in vesicular function of monoaminergic neurons might contribute to the neurodegeneration seen in PD. SONSALLA PK UMDNJ-ROBERT W JOHNSON MED SCH, 675 HOES LANE, PISCATAWAY, NJ 08854-5635 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging reserpine amine laboratory mouse laboratory rat serotonin species difference mesencephalon locus coeruleus immunocytochemistry western blotting synaptic vesicle Parkinson's disease neurotoxin dopamine norepinephrine phenylethylamine high performance liquid chromatography membrane transport protein methylphenyltetrahydropyridine microdialysis CRISP RPROJ NEW JERSEY G CRISP/99/AG08479-07 CRISP/99/AG08479-07 199904 eng GROWTH FACTORS IN THE ADULT AND AGING BRAIN Research RPROJ Parkinson's disease is characterized by the premature neurodegeneration of nigrostriatal dopamine neurons. In order to discover what may be causing this cellular loss or provide a treatment that may decrease the progression of the disease, studies in our laboratory have been focused on identifying factors that are important for the survival and plasticity of dopamine neurons. In order to carry out this goal, we have been utilizing three mouse models. The first, is the weaver mutant mouse in which abnormal development of the dopaminergic nigrostriatal fibers is first observed followed by degeneration of dopaminergic neurons in a similar pattern to what is found in Parkinson's disease, TGF-alpha, during the time of dopaminergic neuronal degeneration. In addition, we have found that these mice also have decreased levels of thyroid hormone, a potent regulator of brain development. Therefore, we have proposed studies aimed at discovering whether the decreases in either TGF-alpha or thyroid hormone are responsible for the neurodegeneration of dopamine neurons in the weaver mutant mouse. Recently, another mutant mouse has been discovered, waved-1, which has a deficiency in TGF-alpha expression. The availability of this second mouse mutant, allows us to specifically test whether a deficiency in TGF-alpha alone results in the degeneration of dopamine neurons or increase their sensitivity to the neurotoxin, 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). There is evidence that the more resilient mesolimbic dopaminergic neurons are able to undergo collateral axonal sprouting in response to degeneration of the nigrostrital neurons. Thus, these dopaminergic neurons represent a potential target to stimulate collateral axonal sprouting after neurodegeneration of dopamine neurons in the substantia nigra. However, there is an age-related loss in the capacity of these neurons to spontaneously sprout. Therefore, in the last mouse model system we investigate what endogenous factors may be responsible for lesion-induced plasticity of dopamine neurons in young animals, how their induction in response to injury is regulated, and whether lesion-induced activation becomes the limiting factor in the age-related loss of dopaminergic plasticity. The results of these studies may reveal therapeutic possibilities for enhancing the recovery of dopamine neurons in neurodegenerative diseases in which spontaneous recovery does not normally occur. BLUM MM MT SINAI SCHOOL OF MEDICINE, ONE GUSTAVE LEVY PLACE 106, NEW YORK, NY 10029 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 10029 Crisp Data Base National Institutes Of Health aging laboratory mouse genetic strain limbic system substantia nigra hormone regulation /control mechanism gene expression neurotrophic factor transforming growth factor disease model neural degeneration nervous system regeneration Parkinson's disease neural plasticity neurotoxin dopamine methylphenyltetrahydropyridine thyroid hormone tissue /cell culture age difference CRISP RPROJ NEW YORK G CRISP/99/AG08538-07 CRISP/99/AG08538-07 199904 eng HYPONATREMIC ENCEPHALOPATHY--ROLE OF AGE AND GENDER Research RPROJ Hyponatremia is the most common electrolyte abnormality seen in a general hospital population. Hyponatremic brain damage appears to represent a major but poorly recognized public health problem, with a projected morbidity in the United States of more than 5,000 per year. Age and gender are major factors predisposing to brain damage from hyponatremia. Plasma vasopressin levels are increased in virtually all hyponatremic patients and vasopressin has a number of deleterious effects upon brain homeostasis. The major questions to be addressed in this proposal relate to the effects of sex steroid hormones and neuropeptides on the susceptibility of the brain to injury from hyponatremia. Hyponatremic brain damage appears to involve an interaction between vasopressin (AVP) and gonadal steroid hormones, resulting in impaired cerebral microcirculation and cytotoxic brain edema. The integrity of the cerebral microcirculation will be quantitatively evaluated in hyponatremic rats, using perfusion sensitive MRI in vivo. Cytotoxic brain edema will be evaluated on the basis of changes in the distribution of cell water between intra- and extra-cellular compartments. Changes in this distribution will be evaluated by means of the apparent diffusion coefficient (ADC), which is determined using diffusion-weighted MRI. In addition to the osmotic effects of hyponatremia, it appears that sex steroid hormones affect brain adaptation at the level of volume regulatory mechanisms in neuronal and glial cell membranes, in particularly by impairing in ion transport through the Na+ -K+ ATPase system, the Na+/Ca2+ exchanger, and the Na+/H+ exchanger. These important pathways for brain cell volume regulation will be evaluated in vitro in synaptosomes from the brains of normonatremic and hyponatremic rats, untreated or pretreated with steroid hormones. The effect of sex steroid hormones on volume regulation in brain astrocytes and the most important pathway for regulation of cell volume in response to hyponatremia, the Na+ -K+ ATPase system, will be evaluated in vitro in astrocytes in culture. The sex steroid hormones will be administered in vivo to those groups of rats in which synaptosome studies will be carried out, while they will be administered in vitro (in the astrocyte culture medium) to the astrocytes in culture. Our clinical and experimental studies suggest that the pathogenesis of hyponatremic encephalopathy involves multiple interactive mechanisms and that permanent brain damage from hyponatremia results from a failure of brain adaptation, which is most severe in cyclic (menstruant) females and prepubertal individuals. In this proposal we will determine the pathophysiology of hyponatremic brain damage by determining the role of the interactive components, particularly endogenous neuropeptides and steroid hormones, impaired pathways for extrusion of sodium and potassium, reduction of cerebral perfusion, and redistribution of cell water between intra- and extra-cellular components. ARIEFF AI VETERANS AFFAIRS MED CENTER, 4150 CLEMENT ST (111G), SAN FRANCISCO, CA 94121-1598 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory rat ion transport secretion hyponatremia chronic brain damage brain edema brain visualization microcirculation cell volume cerebral circulation hormone regulation /control mechanism steroid hormone sex cycle synaptosome neuropeptide magnetic resonance imaging arginine vasopressin sodium potassium exchanging ATPase sex hormone sex difference tissue /cell culture age difference neurotoxicology CRISP RPROJ CALIFORNIA G CRISP/99/AG08575-06 CRISP/99/AG08575-06 199904 eng SELECTIVE VULNERABILITY OF SPORADIC NEURODEGENERATIVE DISEASE Research APPEL SH BAYLOR COLLEGE OF MEDICINE, ONE BAYLOR PLAZA, HOUSTON, TX 77030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 77030 Crisp Data Base National Institutes Of Health Alzheimer's disease calcium flux neural degeneration calcium binding protein amyloid protein calcium channel voltage gated channel tissue /cell culture cytotoxicity amyotrophic lateral sclerosis Xenopus oocyte CRISP RPROJ TEXAS G CRISP/99/AG08664-090008 CRISP/99/AG08664-090008 199904 eng NEUROIMMUNE REGULATION IN ALZHEIMERS DISEASE Research GIULIAN DJ BAYLOR COLLEGE OF MEDICINE, ONE BAYLOR PLAZA, HOUSTON, TX 77030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 77030 Crisp Data Base National Institutes Of Health Alzheimer's disease cell death cell type histopathology human tissue surface antigen gliosis neuritic plaque microglia neuroanatomy neurotoxin amyloid protein receptor expression tissue /cell culture cytotoxicity confocal scanning microscopy neuroimmunomodulation CRISP RPROJ TEXAS G CRISP/99/AG08664-090010 CRISP/99/AG08664-090010 199904 eng PILOT STUDY--AMYLOID MEDIATED TOXICITY Research SCHULZ P BAYLOR COLLEGE OF MEDICINE, ONE BAYLOR PLAZA, HOUSTON, TX 77030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 77030 Crisp Data Base National Institutes Of Health laboratory rat calcium flux patch clamp electrophysiology amyloid protein calcium channel voltage gated channel cytotoxicity CRISP RPROJ TEXAS G CRISP/99/AG08664-090012 CRISP/99/AG08664-090012 199904 eng NEUROPHARMACOLOGIC CHALLENGES IN DEMENTIA Research FOSTER NL UNIVERSITY OF MICHIGAN, ANN ARBOR, MI 48109-0316 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health human old age (65+) Alzheimer's disease ketamine glutamate dosage drug administration rate /duration pharmacokinetics drug adverse effect drug interaction drug tolerance human subject Huntington's disease neuropharmacology neurotoxin psychological test behavior test cognition psychomotor function psychopharmacology phenobarbital NMDA receptor neuroprotectant CRISP RPROJ MICHIGAN G CRISP/99/AG08671-100003 CRISP/99/AG08671-100003 199904 eng REPRODUCTIVE AGING AND THE HYPOTHALAMUS Research RPROJ Our overall objective is to characterize and understand the events that occur in the human central nervous system in response to the ovarian failure of menopause. In the previous funding period, significant progress was made in describing dramatic changes in the hypothalamus of postmenopausal women. There is increased hypothalamic tachykinin gene expression in postmenopausal women, accompanied by cellular hypertrophy. In addition, cellular levels of LHRH mRNA are increased within a subpoplation of neurons within the medial basal hypothalamus. We have hypothesized that these changes are secondary to ovarian failure and not due to age per se. The present proposal tests this hypothesis by determining the effects of hormone withdrawal and replacement on neuropeptide gene expression in the primate hypothalamus. In the first specific aim, we will determine if ovariectomy of young cynomolgus monkeys mimics the increase in LHRH and tachykinin gene expression that occurs in postmenopausal women. If so, this would provide compelling evidence that the changes we have observed in postmenopausal women are due to ovarian failure. In specific aims 2 and 3, we will examine the hypothalamus of young ovariectomized monkeys that have received steroid replacement therapy in treatment regimens designed to duplicate those prescribed to humans. In specific aim 4, we will examine the hypothalamus of postmenopausal women treated with hormone replacement therapy. In situ hybridization will be used to determine if hormone replacement reduces LHRH and tachykinin gene expression in the monkey and human hypothalamus as predicted from our previous studies. We will also determine if long-term continuous estrogen replacement produces signs of neurotoxicity in the human and monkey arcuate nucleus. These studies will provide a firm foundation for understanding the important events that occur in the hypothalamus of postmenopausal women and provide the first information on the neuroanatomic effects of hormone replacement therapy in the primate hypothalamus. Millions of women are currently receiving hormone replacement but little is known about the effects of these treatments on the central nervous system. RANCE NE UNIVERSITY OF ARIZONA, 1501 N CAMPBELL AVENUE, TUSCON, AZ 85724 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 85724 Crisp Data Base National Institutes Of Health hypothalamus postmortem hormone regulation /control mechanism hormone therapy estrogen postmenopause human tissue neuroanatomy neuroendocrine system tachykinin in situ hybridization hypothalamic pituitary axis hypothalamic hormone gonadotropin gonadotropin releasing factor pituitary gonadal axis ovariectomy female nonhuman therapy evaluation Macaca fascicularis neurotoxicology CRISP RPROJ ARIZONA G CRISP/99/AG09214-07 CRISP/99/AG09214-07 199904 eng OXIDATIVE STRESS CELL DEATH AND THE (AH) GENE BATTERY Research RPROJ The long-range goal of this research project is to study the relationship between oxidative stress and the [Ah] gene battery. It is becoming increasingly clear that we "age" because our genes undergo more and more damage by reactive oxygen metabolites (ROMs) as a function of time. When genes are damaged, several escape mechanisms"--including programmed cell death (apoptosis)--occur with increasing frequency. During the past 5 years, an oxidative stress signal transduction pathway has been defined, comprising more than 15 steps and initiated by ROMs by way of physical agents (ionizing and UV irradiation) as well as the metabolism of both endogenous and foreign chemicals. This laboratory has taken a genetic approach to study the role of oxidative stress in gene regulation and cell death. The 14CoS/14CoS mouse contains a 3,800-kb deletion on chromosome 7 and dies during the first 24 h post partum. We found that this mouse exhibits a constitutive oxidative stress response, in which expression of the NAD(P)H:menadione oxidoreductase (Nmol) and other [Ah] Phase II genes is increased. Recent work in other laboratories has shown that homozygous disruption of the fumarylacetoacetate hydrolase (Fah) gene--located in the 3,800-kb deleted region--completely mimics the 14CoS/14CoS mouse, due to ROMs generated by blockade of the tyrosine degradation pathway. Now that we understand more about the 14CoS/14CoS mouse, we can investigate directly the role of oxidative stress in aging through construction of transgenic mouse lines having defects in the control of redox homeostasis. In the next 5 years, we therefore propose to: [1] develop a conventional, as well as an inducible, knockout transgenic mouse having a homozygous disruption in the Fah gene, which will allow us to study ROM pharmacokinetics and cell type- and organ-specific responses of aging secondary to endogenous ROMs; [2] characterize in cells in culture, as well as in the intact animal, the mechanism(s) by which the intracellular levels of reduced glutathione (GSH) are regulated; and [3] develop a conventional, as well as an inducible, knockout transgenic mouse having a homozygous disruption in the gamma glutamylcysteine synthase (Gcs) gene. The GCS enzyme controls GSH production and thus affects at least two critical, distinct steps in the less than 15-step oxidative stress pathway. These studies will greatly enhance our understanding of the cellular responses, and consequences of the role of endogenous ROM-mediated oxidative stress, during the aging process. NEBERT DW UNIVERSITY OF CINCINNATI, PO BOX 670553, CINCINNATI, OH 45267-0553 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health glutamate ammonia ligase newborn animal aging laboratory mouse transgenic animal biological signal transduction cell death pharmacokinetics enzyme induction /repression molecular cloning gene expression genetic library transcription factor radiation genetics genetic model complementary DNA free radical oxygen glutathione DNA binding protein programmed cell death oxidative stress CRISP RPROJ OHIO G CRISP/99/AG09235-09 CRISP/99/AG09235-09 199904 eng INTERACTION OF HYPOXIC AND EXCITOTOXIC NEURONAL INJURY Research RPROJ DESCRIPTION: Injuries to the CNS caused by ischemia, anoxia, epilepsy, AIDS associated dementia, Huntington's disease, and trauma are thought to be mediated either directly or indirectly by excitotoxicity. Concomitant with increases in total intracellular calcium ([Ca2+]i), glutamate receptor overstimulation produces a triphasic fluctuation in intracellular hydrogen ion concentration, an initial transient acidification followed by a longer alkalinization and eventually a progressive acidification. The progressive acidification has been associated with glutamate-induced neurotoxicity. These changes in intracellular pH (pHi) may act in concordance with [Ca2+]i to promote excitotoxic neurodegenerative changes. The experiments proposed in this application are designed to increase understanding of the normal hydrogen ion regulatory processes in cultured hippocampal neurons and to determine how disruption of these processes and abnormal fluctuations in pHi contribute to neuronal damage from excitotoxic, hypoxic, and related insults. The long term objectives of the research are to understand the sequence of events underlying glutamate- and hypoxia-induced neuronal death by studying activation of membrane receptors and intracellular biochemical pathways. The specific aims for this project include 1) to test the hypothesis that the slow, progressive acidification observed several hours after glutamate receptor overstimulation directly mediates the ensuing toxicity. 2) To test the hypothesis that normal hippocampal neurons contain a minimum of 3 acid/base regulatory mechanisms, a Na+/H+ antiporter, a Na-dependent HCO3-/Cl- exchanger and a passive HCO3-/Cl- exchanger and do not contain a Na+/HCO3- symporter. 3) To test the hypothesis that of the cellular hydrogen ion regulatory mechanisms, operation of the Na+/H+ antiporter and the Na-dependent HCO3-/Cl-- exchanger are compromised following excitotoxic insult and the passive HCO3-/Cl- exchanger is not. And 4) to test the hypothesis that the Na+- dependent acid/base regulatory mechanisms are compromised following excitotoxic insult due to rundown of the Na gradient. DUBINSKY JM UNIVERSITY OF MINNESOTA, 435 DELAWARE ST SE, MINNEAPOLIS, MN 55455 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 55455 Crisp Data Base National Institutes Of Health acidity /alkalinity laboratory rat antiport hippocampus bicarbonate cerebral ischemia /hypoxia glutamate patch clamp chlorine neurotoxin membrane transport protein glutamate receptor sodium tissue /cell culture nerve injury CRISP RPROJ MINNESOTA G CRISP/99/AG10034-07 CRISP/99/AG10034-07 199904 eng KERATOCONJUNCTIVITIS SICCA--PREVALENCE AND RISK FACTORS Research SCHEIN O JOHNS HOPKINS HOSPITAL, 600 NORTH WOLFE STREET, BALTIMORE, MD 21287-9019 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health human old age (65+) aging Sjogren's syndrome disease /disorder proneness /risk drug adverse effect tear keratoconjunctivitis sicca vision disorder human subject autoimmune disorder epidemiology sex hormone oxidative stress smoking CRISP RPROJ MARYLAND G CRISP/99/AG10184-05S30004 CRISP/99/AG10184-05S30004 199904 eng NEUROPROTECTIVE ESTROGENS AND ALZHEIMER'S DISEASE Research SIMPKINS JW UNIVERSITY OF FLORIDA, BOX 100487, GAINESVILLE, FL 32610 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 32610 Crisp Data Base National Institutes Of Health aging laboratory rat Alzheimer's disease calcium flux chemical structure drug screening /evaluation hormone regulation /control mechanism estratriene compound estrogen gene expression western blotting oxidized lipid neuron antioxidant lactate dehydrogenase amyloid protein estrogen receptor NMDA receptor ovariectomy tissue /cell culture northern blotting neuroprotectant neurotoxicology cAMP response element binding protein CRISP RPROJ FLORIDA G CRISP/99/AG10485-080005 CRISP/99/AG10485-080005 199904 eng NICOTINIC AGONISTS AND ALZHEIMER'S DISEASE Research MEYER EM UNIVERSITY OF FLORIDA, BOX 100487, GAINESVILLE, FL 32610 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 32610 Crisp Data Base National Institutes Of Health aging laboratory rat hippocampus Alzheimer's disease calcium flux drug screening /evaluation auditory stimulus electrophysiology disease model neural degeneration neuron mecamylamine protein kinase C amyloid protein calcium channel voltage gated channel receptor expression nicotinic receptor NMDA receptor bungarotoxin programmed cell death enzyme activity PC12 cell neuroprotectant neurotoxicology CRISP RPROJ FLORIDA G CRISP/99/AG10485-080006 CRISP/99/AG10485-080006 199904 eng CORE--ANIMAL CARE AND BEHAVIORAL ASSESSMENT Research DEFIEBRE C UNIVERSITY OF FLORIDA, BOX 100487, GAINESVILLE, FL 32610 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 32610 Crisp Data Base National Institutes Of Health aging laboratory rat biomedical facility Alzheimer's disease neurochemistry neuropharmacology behavior test ethology animal breeding animal care neurotoxicology behavioral /social science research tag CRISP RPROJ FLORIDA G CRISP/99/AG10485-089003 CRISP/99/AG10485-089003 199904 eng AGING VULNERABILITY TO INFLAMMATORY PROCESSES Research RPROJ DESCRIPTION: The proposed studies will continue investigations into the nature of the selection process that leads to degenerative changes in the brains of humans with Alzheimer's Disease (AD). AD is recognized is one of the major public health and quality-of-life problems facing the world community. AD is characterized by a distinct set of neuropathological lesions, e.g. senile plaques,a nd the death of selected neural systems. Inflammatory processes play an as yet undefined role in the pathogenesis of AD. The studies will investigate the biochemical, histopathological and behavioral consequence of chronic exposure to inflammatory agents within the brain of normal rats and rats that over-express human beta-amyloid. The project will test the hypothesis that inflammatory processes are involved in the initial stages of cellular dysfunction that may alter cellular vulnerability to endogenous toxins; these changes may in turn lead to cell death. In addition, the Pi will investigate whether there is an age-associated increase in the vulnerability to these processes. Finally, because the cascade of biochemical processes that leads to neuronal degeneration may involve the activation of NMDA receptors, the production of nitric oxide or prostaglandins, or the activation of interleukin-1 receptors, the studies will also investigate whether it is possible to antagonize these processes and provide neuroprotection. WENK GL UNIVERSITY OF ARIZONA, PO BOX 245115-LIFE SCI N BLDG, TUCSON, AZ 85724-5115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory rat transgenic animal Alzheimer's disease experimental brain lesion inflammation inhibitor /antagonist histochemistry /cytochemistry interleukin 1 disease model neural degeneration neuropharmacology neurotoxin neurotransmitter lipopolysaccharide behavior test cytokine receptor NMDA receptor neuroprotectant nitric oxide synthase nonsteroidal antiinflammatory agent CRISP RPROJ ARIZONA G CRISP/99/AG10546-04A2 CRISP/99/AG10546-04A2 199904 eng GLIAL NEURONAL INTERACTIONS IN NEURODEGENERATION Research RPROJ This research program will investigate the relationship between neuron survival and intracellular Ca2+ homeostasis. We have found that both of these functions are defective in neurons from the trisomy 16 (Ts16) mouse. The Ts16 mouse has an extra copy of chromosome 16, the mouse homolog of human chromosome 21. Since patients with trisomy 21 (down syndrome) inevitable develop Alzeheimer's disease (AD), a neurodegenerative disorder characterized by neuronal death, understanding the mechanisms regulating Ts16 neuron survival may reveal abnormalities that contribute to AD. Using a novel in vitro assay for neuron survival, we have discovered that hippocampal neurons from the Ts16 mouse die 2-3 times faster than do normal (euploid) neurons. Our data demonstrate that survival of euploid neurons is promoted by micromolar concentrations of glutamate acting at kainate/AMPA receptors. Ts16 neurons lack this survival response to glutamate and this deficit can account for the accelerated death of Ts16 neurons. In contrast, both euploid and Ts16 neurons are rescued by peptide growth factors and killed by excitotoxic concentrations of added glutamate. Using computer-assisted fura-2 [Ca2+] imaging, we have also discovered that Ca2+ homeostasis is abnormal in both Ts16 neurons and astrocytes. We hypothesize that survival-promoting concentrations of glutamate maintain [Ca2+]cyt in an optimal range for euploid neuron survival and that this response is lacking in Ts16 neurons due to a genetically- determined defect in Ca2+ homeostasis. These hypotheses will be tested by correlating neuron survival with [Ca2+] in parallel experiments under conditions of varying degrees of survival. We propose experiments to determine the cellular mechanism underlying glutamate-promoted survival of normal neurons and the mechanistic basis for defective survival and Ca2+ homeostasis in Ts16 neurons. The Ts16 mouse is a naturally-occurring genetic defect that confers two discrete, but interested, deficits n normal cell physiology, viz., decreased neuronal survival and altered Ca2+ homeostasis. Both of these deficits may be masked in vivo by compensatory processes depending on cell type and cellular environment and, therefore, are most easily studied in vitro under well-controlled conditions. Deficits of this kind would be expedited to make cells vulnerable to toxic influences that accumulate with aging. Such vulnerability may play a role in the development of neurodegenerate disorders. KRUEGER BK UNIV OF MARYLAND SCH OF MED, 655 W BALTIMORE ST., BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 21201 Crisp Data Base National Institutes Of Health laboratory mouse genetic strain cerebral cortex hippocampus Alzheimer's disease calcium flux calcium indicator cell cell interaction cell death disease /disorder proneness /risk gene expression trisomy fibroblast growth factor neurotrophic factor disease model neural degeneration glia neuron glutamate receptor tissue /cell culture neurotoxicology CRISP RPROJ MARYLAND G CRISP/99/AG10686-06 CRISP/99/AG10686-06 199904 eng SUPPRESSION OF APP-MEDIATED, DIFFERENTIATION RELATED CYTOTOXICITY Research MARTIN GM UNIVERSITY OF WASHINGTON, ADRC XD-43, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 98195 Crisp Data Base National Institutes Of Health aging laboratory mouse transgenic animal brain cell cell differentiation postmortem yeast site directed mutagenesis linkage mapping molecular cloning gene expression lac operon genetic regulatory element structural gene genetic library human tissue immunocytochemistry western blotting kidney cell model design /development disease model genetic model substance P neurotoxin complementary DNA cathepsin B protein sequence amyloid protein tissue /cell culture cytotoxicity northern blotting southern blotting suppressor mutation transfection vector transmission electron microscopy CRISP RPROJ WASHINGTON G CRISP/99/AG10917-070001 CRISP/99/AG10917-070001 199904 eng ESTROGEN-INDUCED BRAIN AGING Research MILLER MA UNIVERSITY OF WASHINGTON, ADRC XD-43, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 98195 Crisp Data Base National Institutes Of Health aging dihydrotestosterone testosterone laboratory rat brain hypothalamus preoptic area amygdala hippocampus chronic brain damage blood chemistry hormone regulation /control mechanism estrogen gene expression neurobiology neuropeptide neurotoxin neurotransmitter dopamine hypothalamic pituitary axis neurotensin vasopressin progesterone ovariectomy female sex difference stress CRISP RPROJ WASHINGTON G CRISP/99/AG10917-070002 CRISP/99/AG10917-070002 199904 eng EFFECTS OF VENTRICULAR LEUPEPTIN INFUSION IN MONKEYS Research DUBACH MF UNIVERSITY OF WASHINGTON, ADRC XD-43, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 98195 Crisp Data Base National Institutes Of Health juvenile animal brain dementia Alzheimer's disease lysosome postmortem intravenous administration drug administration route protease inhibitor histopathology immunocytochemistry western blotting model design /development disease model neuritic plaque neurofibrillary tangle neurobiology neurotoxin tyrosine 3 monooxygenase oligopeptide cathepsin B endopeptidase amyloid protein proteolysis Macaca fascicularis entorhinal cortex CRISP RPROJ WASHINGTON G CRISP/99/AG10917-070005 CRISP/99/AG10917-070005 199904 eng GENETIC / ENVIRONMENTAL INFLUENCES--DEMENTIA IN PARKINSON'S DISEASE Research MARDER K COLUMBIA UNIVERSITY, 630 WEST 168TH STREET, NEW YORK, NY 10032 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 10032 Crisp Data Base National Institutes Of Health human old age (65+) dementia Alzheimer's disease blood chemistry disease /disorder proneness /risk pathologic process genetic disorder genetic promoter element gene mutation trisomy genetic polymorphism United States human subject data collection interview questionnaire statistics /biometry nervous system disorder epidemiology Parkinson's disease nucleic acid sequence amyloid protein ethnic group tissue /cell culture cell bank /registry environmental toxicology age difference family genetics chromosome 21 CRISP RPROJ NEW YORK G CRISP/99/AG10963-070002 CRISP/99/AG10963-070002 199904 eng ACTIVE LIFE EXPECTANCY IN THE OLDER POPULATION Research RPROJ DESCRIPTION: Using the Health and Retirement Survey, the proposed research will investigate the effects of socioeconomic status (SES) on chronic health problems and disability for a nationally representative sample of middle-aged and older persons. Specifically, the aims of the proposed research are to (1) determine the SES gradient across types of health problems defining the course of the disablement process and (2) investigate how SES differences in health changes are related to differential exposure to unhealthful and demanding work environments, risky health behaviors, and access to and utilization of medical care. CRIMMINS EM UNIV OF SOUTHERN CALIFORNIA, 3715 MCCLINTOCK AVE, GER 218, LOS ANGELES, CA 90089-0191 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health human middle age (35-64) human old age (65+) longevity person with disability marriage /marital status health care service utilization Medicare edicaid statistics /biometry occupational hazard retirement epidemiology health survey health behavior socioeconomics functional ability human data clinical research behavioral /social science research tag CRISP RPROJ CALIFORNIA G CRISP/99/AG11235-05 CRISP/99/AG11235-05 199904 eng MITOCHONDRIAL DYSFUNCTION IN NORMAL AGING AND AD Research BEAL MF MASSACHUSETTS GENERAL HOSPITAL, FRUIT STREET, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02114 Crisp Data Base National Institutes Of Health aging laboratory rat bioenergetics biomarker hippocampus Alzheimer's disease mitochondria electron transport postmortem point mutation histochemistry /cytochemistry histopathology human tissue disease model neurotoxin in situ hybridization mitochondrial DNA oxidative phosphorylation cellular respiration tissue /cell culture human genetic material tag hydrogen transporting ATP synthase entorhinal cortex enzyme activity oxidative stress CRISP RPROJ MASSACHUSETTS G CRISP/99/AG11337-050003 CRISP/99/AG11337-050003 199904 eng EXCITATORY TRANSMITTERS, MEMORY, AGING AND DEMENTIA Research RPROJ The title of this Program Projects Application "Excitatory Transmitters, Memory, Aging and Dementia" accurately identifies the several closely related themes that the proposed research addresses. The principal excitatory neurotransmitters in the mammalian CNS are glutamate (Glu) and acetylcholine (ACh). Both of these transmitters act at multiple receptor subtypes; either hyperstimulation or blockade of either Glu or ACh receptors is associated with neuropathological changes in animal brain and/or memory/cognitive disturbances in animals and humans. Within the framework of two cores and seven separate projects, the Principal Investigator, 11 Co-investigators and 7 Consultant/Collaborators will study mechanisms by which excitatory transmitters, either in early adulthood or old age, can contribute to normal memory/cognitive functions or to memory/cognitive impairment, neuronal degeneration and cell death. A major emphasis of the research will be on identifying mechanisms of neuronal degeneration that might help explain the pathophysiology of Alzheimer's disease. One project pertains to human subjects and will involve the measurement of excitotoxic amino acids (Glu, aspartate, glycine, cysteine) in cerebrospinal fluid and blood of clinically characterized AD patients in different stages of the illness. The remainder of the proposed research comprises either in vivo or in vitro animal experiments. A wide range of neurochemical, neuropharmacological, neurophysiological, neurobehavioral, neurohistological and molecular biological or immunobiological methods will be employed, including the use of nucleotide and immunological probes for studying subtypes of excitatory amino acid and muscarinic cholinergic receptors which have recently been cloned. OLNEY JW WASHINGTON UNIVERSITY, 4940 CHILDREN'S PLACE, ST LOUIS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 63110 Crisp Data Base National Institutes Of Health aging neural degeneration neurotoxin excitatory aminoacid CRISP RPROJ MISSOURI G CRISP/99/AG11355-04 CRISP/99/AG11355-04 199904 eng EXCITATORY NEURODEGENERATIVE MECHANISMS-I Research OLNEY JW WASHINGTON UNIVERSITY, 4940 CHILDREN'S PLACE, ST LOUIS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 63110 Crisp Data Base National Institutes Of Health aging chick embryo laboratory rat cerebral cortex Alzheimer's disease acetylcholine cellular pathology hormone regulation /control mechanism histopathology neural degeneration neuropharmacology neurotoxin phencyclidine pregnancy progesterone muscarinic receptor glutamate receptor NMDA receptor sex difference cysteine dizocilpine excitatory aminoacid cingulate gyrus age difference neuroprotectant CRISP RPROJ MISSOURI G CRISP/99/AG11355-040001 CRISP/99/AG11355-040001 199904 eng EXCITATORY NEURODEGENERATIVE MECHANISMS-II Research SESMA M WASHINGTON UNIVERSITY, 4940 CHILDREN'S PLACE, ST LOUIS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 63110 Crisp Data Base National Institutes Of Health gamma aminobutyrate aminoacid analog laboratory rat second messenger cerebral cortex acetylcholine cellular pathology histopathology immunocytochemistry neural degeneration synapse neuroanatomy neuropharmacology neurotoxin in situ hybridization stress protein autoradiography receptor expression muscarinic receptor glutamate receptor NMDA receptor dizocilpine excitatory aminoacid cingulate gyrus neuroprotectant CRISP RPROJ MISSOURI G CRISP/99/AG11355-040002 CRISP/99/AG11355-040002 199904 eng EXCITOTOXINS IN CEREBROSPINAL FLUID AND BLOOD OF ALZHEIMERS DISEASE PATIENTS Research CSERNANSKY JW WASHINGTON UNIVERSITY, 4940 CHILDREN'S PLACE, ST LOUIS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 63110 Crisp Data Base National Institutes Of Health glycine biomarker prosencephalon cerebral cortex hippocampus dementia Alzheimer's disease brain disorder diagnosis blood chemistry aspartate glutamate human subject neural degeneration cerebrospinal fluid neurotoxin cognition memory neuropsychological test psychometrics sulfate cysteine cystine excitatory aminoacid CRISP RPROJ MISSOURI G CRISP/99/AG11355-040003 CRISP/99/AG11355-040003 199904 eng GLUTAMATE RECEPTORS, MEMORY AND LONG TERM POTENTIATION Research WOZNIAK D WASHINGTON UNIVERSITY, 4940 CHILDREN'S PLACE, ST LOUIS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 63110 Crisp Data Base National Institutes Of Health aging laboratory rat hippocampus experimental brain lesion inhibitor /antagonist electrophysiology sensorimotor system anticholinergic agent neuropharmacology neurotoxin behavior test open field behavior memory reversal learning NMDA receptor chordate locomotion stereotaxic technique scopolamine dizocilpine long term potentiation cingulate gyrus age difference neuroprotectant CRISP RPROJ MISSOURI G CRISP/99/AG11355-040004 CRISP/99/AG11355-040004 199904 eng TRANSGENIC MODELS TO STUDY ALZHEIMERS DISEASE Research RPROJ DESCRIPTION Alzheimer's disease (AD) is characterized clinically by dementia and neuropathologically by amyloid plaques, amyloid angiopathy, dystrophic neurites, NFT's, gliosis, and loss of neuronal subpopulations and synapses. Increasing evidence suggests that the AB peptide derived from APP plays a central role in AD. The goals of this project are to characterize neuronal and glial products that contribute to amyloidogenesis and neurodegeneration and identify cortical AD-related pathogenetic pathways that could be targeted by therapeutic interventions. Mice expressing a PDGF-promoter driven, alternatively spliced hAPP minigene, (PDGF-APP mice) develop several aspects of AD neuropathology. This proposal is to utilize this and related models to study cerebral amyloidogenesis and amyloid-induced neurodegeneration in vivo with the following aims: 1) Determine what factors influence AB production and B-amyloid deposition in vivo.; 2) Determine whether development of neuronal/synaptic degeneration in PDGF-hAPP mice depends on B-amyloid formation; 3) Determine whether B-amyloid in the brain results in aberrant induction of neuronal apoptosis and whether this process involves excitotoxic mechanisms. MUCKE L THE J GLADSTONE INSTITUTES, PO BOX 419100, SAN FRANCISCO, CA 94141-9100 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal Alzheimer's disease genetic promoter element gene induction /repression platelet derived growth factor model design /development disease model neuron neuropharmacology neurotoxin nucleic acid sequence protein biosynthesis amyloid protein neuropsychological test tissue /cell culture human genetic material tag programmed cell death behavioral /social science research tag CRISP RPROJ CALIFORNIA G CRISP/99/AG11385-05A2 CRISP/99/AG11385-05A2 199904 eng NEUROFILAMENT KINASES Research RPROJ DESCRIPTION (Adapted from Applicant's Abstract): Neurofilaments (NF), the major cytoskeletal proteins expressed in mature myelinated axons, are intrinsic determinants of axonal diameter (caliber). Maintenance of axonal caliber is important since conduction velocity of nerve action potentials is a function of axonal diameter. NF modulation of axonal caliber appears to be determined not by NF number, per se, but by their phosphorylation state. The mechanisms controlling NF phosphorylation are not understood. However, abnormal phosphorylation and accumulation of NF proteins are features of Alzheimer's disease (AD) and the motor neuron disease amyotrophic lateral sclerosis (ALS). The applicant is studying protein kinases that phosphorylate NF using mouse as a model system. He has identified and characterized a novel 115 kDa NF-associated kinase from mouse brain extracts, which has been named NAK115. NAK115 is expressed in neurons and its activity is down-regulated during myelination induced in culture. A kinase related to NAK115 is found in human brain. In addition to NAK115 two other NF kinases have been discovered. One of these appears to be a novel cdc2-like kinase that is associated with NF in mouse brain. A kinases with similar immunological properties has also been found in humans and is associated with the paired helical filaments that comprise the neurofibrillary tangles of Alzheimer disease. A 35 kDa NF kinase whose activity is up-regulated by myelination has also been discovered. In order to fully understand the regulation NF phosphorylation by these kinases, the applicant proposes to characterize the kinases biochemically, immunologically and at the molecular level. In a consortium study, it will also be determined whether the three NF kinases are associated with NF proteins during axonal transport in nerve fibers. In these studies it will also be determined whether axonal transport of the kinases are impaired to the same extent as the NF proteins after systemic intoxication with IDPN (beta,beta'-iminodipropionitrile), a neurotoxin that selectively blocks NF transport without significantly altering the transport of other cytoskeletal proteins, such as actin or tubulin. Finally, the applicant proposes to study how expression of the NF kinases changes during peripheral nerve injury when the cytoskeleton is restructured. These studies should provide valuable information on the association and transport of kinases with NF proteins, and role of the kinases in modification of NF proteins in neuronal function during normal development, during nerve injury, and in human diseases in which NF are affected. MONTEIRO MJ UNIVERSITY OF MARYLAND, 725 W BALTIMORE ST, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 21201 Crisp Data Base National Institutes Of Health laboratory mouse Alzheimer's disease enzyme induction /repression neurofibrillary tangle myelination neurogenesis neurofilament neurotoxin protein kinase actin tubulin tissue /cell culture cell cycle protein enzyme activity neurotransmitter transport nerve injury CRISP RPROJ MARYLAND G CRISP/99/AG11386-06 CRISP/99/AG11386-06 199904 eng ACTIVE LIFE EXPECTANCY IN THE OLDER POPULATION Research RPROJ DESCRIPTION: Using the Health and Retirement Survey, the proposed research will investigate the effects of socioeconomic status (SES) on chronic health problems and disability for a nationally representative sample of middle-aged and older persons. Specifically, the aims of the proposed research are to (1) determine the SES gradient across types of health problems defining the course of the disablement process and (2) investigate how SES differences in health changes are related to differential exposure to unhealthful and demanding work environments, risky health behaviors, and access to and utilization of medical care. HAYWARD MD PENNSYLVANIA STATE UNIVERSITY, 110 TECHNOLOGY CENTER, UNIVERSITY PARK, PA 16802 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 16802 Crisp Data Base National Institutes Of Health human middle age (35-64) human old age (65+) longevity chronic disease /disorder person with disability health care service utilization occupational hazard health survey health behavior socioeconomics human data clinical research behavioral /social science research tag CRISP RPROJ PENNSYLVANIA G CRISP/99/AG11758-05 CRISP/99/AG11758-05 199904 eng TRANSGENIC C ELEGANS AS AMYLOID DISEASE MODEL Research RPROJ DESCRIPTION: The P.I. proposes to use the nematode C. elegans as a model system to study the pathophysiology underlying Alzheimer's disease. In his preliminary results he has expressed a fragment of human amyloid precursor protein (APP), called unc-54/b1-42, in the somatic musculature of the worm. Extracellular deposits of APP are most likely one of the underlying causes of Alzheimer's disease in humans, although it is unclear how they cause the widespread neuronal death typical of this disease. Expression of the APP fragment b1-42 leads to intracellular deposits in the musculature, although no extracellular secretion could be noted. However, vacuoles in the CNS developed which in other mutations are sign of cell death. The proposal entails the following four sets of experiments: 1. Analysis of the phenotype of the existing APP expressing lines in greater detail: TEM and specific markers will be used to establish in which cells of the CNS vacuoles appear; suspected are glia cells surrounding the neurons. Other experiments address the pathophysiological connection between b-peptide expression and vacuole formation. Intracellular Ca levels will be monitored by injection of the fluorochrome fura-2. b1-42 expressing animals will be reared in different ionic conditions to test the influence of these ions on vacuole formation. Sodium-channel blockers will be applied to test whether sodium channels are involved in vacuole formation. A number of mutations known to suppress vacuole formation during regular neuronal cell death will be tested as to their ability to also suppress vacuoles in b-peptide expressing animals. 2. Generation of novel lines in which other b1-42 variants and different regulatory elements will be used to achieve expression in other tissues. In particular, promoters of the mec-7, unc-4 and unc-119 genes will be used which should drive expression in subsets of neurons. To achieve secretion of the b1-42, several experiments will be carried out. It will be first established whether the too small size of the peptide is responsible, by fusing the peptide with green fluorescent protein (GFP) and thereby increasing its size. Alternatively, the signal sequence used in the unc-54/b1-42 construct may be faulty; signal sequences which successfully have lead to secretion of another peptide, transthyretin, will be used. Finally, a C. elegans gene, bli-4, encoding a pro-protease routed to the secretory pathway will be fused with b-peptide to ensure its secretion. Several new constructs in which selected residues of the b peptide are changed in vitro will be transformed into worms to test for increase or decrease of the phenotype caused. The P.I. is guided by vertebrate cell culture studies in which the corresponding residue changes were shown to modify the toxicity of the APP deposits in a certain way. 3. Coexpression of other human proteins suspected to modify the deposition of APP together with b1-42 peptide. Preliminary results had shown that co-expression of b1-42 and wild-type transthyretin reduces the number of amyloid-like deposits in transgenic worms. The interaction between the two proteins suggested by this result will be further tested by coexpressing a mutant form of transthyretin which in an in vitro assay does not interact with b-peptide. In another experiment, the gene encoding apolipoprotein E which according to clinical and in vitro data enhances the formation of amyloid, will be co-expressed with b1-42 to test whether the interaction can be replicated in the worm system. 4. Genetic screen for C. elegans genes modifying the phenotype caused by b1-42 expression is proposed. Three screens are proposed: A. b1-42 expressing animals will be treated with EMS; the F2 will be stained with the histological stain Congo Red to visualize whether increase or decrease in the amount of deposits has occurred. Animals of interest will be recovered and their progeny retested for altered deposits. Animals with altered deposits will be tested by ELISA whether the level of expression of b1-42 has not changed, since only mutations in genes which affect the ability of deposit formation are of interest. B. Screen for suppressors of the paralysis phenotype: it was observed that b-peptide expressing animals suffer from paralysis, possibly caused by intramuscular amyloid deposits. An EMS mutagenesis to obtain mutations in genes which affect the paralysis phenotype is proposed. C. Screen for suppressors of the vacuole phenotype. Since the vacuoles in b1-42 expressing animals are only infrequently observed, possibly due to the fragmentation of the degenerating cell, the ced-2 mutant which allows dead cells to persist will be introduced into the unc-54/b1-42 background. LINK CD UNIVERSITY OF COLORADO, BOULDER, CO 80309-0447 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health Caenorhabditis elegans transgenic animal Alzheimer's disease calcium flux calcium indicator vesicle /vacuole mutation gene mutation enzyme linked immunosorbent assay model design /development disease model glia neuron central nervous system neurotoxin thyroid hormone binding protein retinoid binding protein amyloid protein sodium channel transmission electron microscopy apolipoprotein E green fluorescent protein CRISP RPROJ COLORADO G CRISP/99/AG12423-03 CRISP/99/AG12423-03 199904 eng MITOCHONDRIAL FOCUS OF REPERFUSION INJURY IN AGING HEART Research RPROJ DESCRIPTION: (Adapted from the Investigator's Abstract) This application is to study the effect of aging on ischemia/reperfusion injury. Myocardial injury is increased following ischemia and reperfusion in the aging heart compared to adults, including elderly Fischer 344 rats (24 months old) compared to 6 month old adult controls. The elderly rats have an aging-related decrease in oxidative phosphorylation that is selective to the interfibrillar population of cardiac mitochondria (IFM). IFM from elderly rats have an aging-related decrease in complex III activity in the electron transport chain. The contents of the three catalytic centers of complex III remain unaltered, suggesting that damage or loss of non-catalytic subunit peptide(s) of complex III is the source of the defect. Depletion of non-catalytic subunits that contribute to cytochrome c1, is observed in IFM from the aging heart. The applicant's hypothesize that the aging defect creates a partial block in electron flow in the distal portion of complex III, resulting in greater reduction of more proximal redox centers in complex III, in turn leading to an increased production of reactive oxygen species by complex III in IFM of the aging heart. In addition to the aging defect, myocardial ischemia damages the iron-sulfur protein in the aging heart. The iron-sulfur protein, one of the catalytic centers in complex III, is located immediately proximal to the proposed site of the aging defect. Thus, during reperfusion, IFM in the aging heart have two defects in complex III. They hypothesize that these two defects, acting in concert, create an additional block in complex III, leading to a further reduction of proximal redox centers, setting the stage for additional increases in oxyradical production following the reintroduction of oxygen during reperfusion. The decrease tolerance of the aging heart represents a novel situation in which to examine the interaction of pre-existing aging-related metabolic defects and the added damage caused by subsequent ischemia to the impaired recovery and excess tissue damage present following ischemia and reperfusion in the senescent heart. LESNEFSKY EJ CLEVELAND VA MED CENTER, 10701 EAST BOULEVARD, CLEVELAND, OH 44106 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 44106 Crisp Data Base National Institutes Of Health animal old age aging laboratory rat reperfusion mitochondria electron transport molecular pathology enzyme complex myocardial ischemia /hypoxia cytochrome c oxidative phosphorylation cytotoxicity iron sulfur protein age difference enzyme activity oxidative stress CRISP RPROJ OHIO G CRISP/99/AG12447-05 CRISP/99/AG12447-05 199904 eng GLIA MEDIATED BRAIN INJURY IN ALZHEIMERS DISEASE Research RPROJ DESCRIPTION (from applicant's abstract) Alzheimer's Disease (AD) is a degenerative disorder associated with senile plaques. Although loss of cortical neurons, decreased synaptic connection, and marked reactive microgliosis are prominent features of AD, the mechanisms to account for these histologic abnormalities remain uncertain. The applicant believes that AD plaques elicit local microglial reactivity. Because of their size and chemical stability, these plaques, containing a complex of collection of constitutes including beta amyloid, persist as chronic irritants. The applicant's suggest that plaque-associated reactive microglia chronically release cytotoxic factors that contribute to the neuronal injury and synaptic loss resulting in AD dementia. The applicant's have identified and characterized a neurotoxin, produced by microglia when brought into contact with isolated plaques. This same toxic agent can be extracted from autopsied AD brain gray matter. The neurotoxic agent, a lipophilic amine, has been purified over 100,000 fold, is distinct from known mammalian neurotoxins, is highly potent, acts through the NMDA receptor, and demonstrated in vivo effects in animal brain. The applicants propose to elucidate the chemical structure of this toxin by mass spectrometric and NMR studies. In addition, they will conduct studies to understand the mechanisms by which beta-amyloid, the major constitute of plaques, elicit the adherence and neurotoxicity of microglia. They will also evaluate the roles of minor protein constituents of native senile plaques in these behaviors. Finally, they have also produced an animal model for the role of microglial neurotoxicity on AD, through direct infusion, of the toxin into rat hippocampus. The investigators will conduct in vivo investigations to evaluate a number of candidate NMDA receptor blocking agents for effect in preventing the neuronal death which results from this neurotoxin in the brain. If successful, this proposed research will uncover fundamentally important signals and events which regulate immune-mediated mechanisms of brain injury in AD. Uncovering such mechanisms may lead to the development of immunosuppressive strategies to treat AD dementia. GIULIAN DJ BAYLOR COLLEGE OF MEDICINE, ONE BAYLOR PLAZA, HOUSTON, TX 77030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 77030 Crisp Data Base National Institutes Of Health laboratory rat hippocampus Alzheimer's disease cell adhesion nuclear magnetic resonance spectroscopy human tissue immunocytochemistry neuritic plaque microglia neurotoxin high performance liquid chromatography protein structure /function amyloid protein NMDA receptor tissue /cell culture excitatory aminoacid CRISP RPROJ TEXAS G CRISP/99/AG12548-03 CRISP/99/AG12548-03 199904 eng MOLECULAR BIOLOGY OF ALZHEIMER DISEASE NEURODEGENERATION Research RPROJ The isolation by our laboratory and others of the gene for the precursor of the Alzheimer's disease amyloid polypeptide, has made it possible to begin dissecting at a molecular level the processes whereby this normal protein may become altered in Alzheimer's disease. The sequence of events which leads to the deposition of the small self-aggregating amyloid peptide in Alzheimer's disease is not known. A key question concerns the causal relationship of amyloid to the pathology of Alzheimer's disease. Projects are proposed to address these questions. In the original application, we proposed to do a detailed analysis of amyloid protein precursor (APP) transcription in defined regions of Alzheimer's disease and normal brains, to test the hypothesis that the development of pathology in Alzheimer's disease is related to changes in the expression of the APP mRNAs. This specific aim has been completed: we were able to show that the mRNA encoding APRP-563, an APP-related mRNA lacking the amyloid-encoding sequence, is specifically elevated in pathologically affected regions of Alzheimer's disease brain. We have shown, by the construction and characterization of recombinant transfected cell lines expressing particular domains of APP, that the carboxyterminal 105 amino acids of the human amyloid protein precursor is neurotoxic. Studies are proposed to examine the relationship of the neurotoxicity of this fragment (termed AB1) to Alzheimer's disease pathology, by analyzing the pattern of segregation of the microtubule associated protein tau in neurons treated with AB1, and by assaying for differential vulnerability of CA1 hippocampal neurons to AB1 neurotoxicity. Characterization of AB1 neurotoxicity will include definition of the mechanism of glial protection of neurons against AB1 toxicity, determination of the vulnerability of hippocampal neurons from rats of various ages to AB1 neurotoxicity, and assessment of the minimum exposure time of neurons to the AB1 conditioned medium that is necessary to cause their subsequent degeneration. We recently discovered that the neurotoxic AB1 fragment can, under the appropriate conditions, be trophic: experiments to quantify and characterize this trophic effect, and to relate it to the neurotoxicity, are described. Both genetic and pharmacologic methods will be utilized in an attempt to neutralize or block the neurotoxicity of the carboxyterminal 105 amino acids of AB1. The development of alternate methods of synthesis of this AB1 fragment will facilitate the performance of the proposed studies. Studies to follow up on our recent finding that the AB1 fragment binds specifically to the surface of NGF-differentiated PC12 cells but not to undifferentiated PC12 cells, are proposed. Finally, we show that mouse brains transplanted with AB1-transfected PC12 cells demonstrate progressive neurodegeneration in the region of the transplant, and show immunoreactivity with the Alz50 antibody. The possibility of developing these transplanted mice as an animal model for Alzheimer's disease is proposed. NEVE RL MC LEAN HOSPITAL, 115 MILL STREET, BELMONT, MA 02178 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02178 Crisp Data Base National Institutes Of Health hamster laboratory mouse laboratory rat transgenic animal hippocampus Alzheimer's disease molecular pathology transfection molecular cloning gene expression neurotrophic factor human tissue immunocytochemistry model design /development disease model neural degeneration neurogenesis glia neuron nervous system transplantation neuropharmacology neurotoxin in situ hybridization endopeptidase tau protein amyloid protein membrane protein radioassay tissue /cell culture PC12 cell CRISP RPROJ MASSACHUSETTS G CRISP/99/AG12954-08 CRISP/99/AG12954-08 199904 eng GLUCOCORTICOID RECEPTOR MECHANISMS STRESS AND AGING Research RPROJ DESCRIPTION (Investigator's Abstract): Regulation of glucocorticoid secretion is essential for maintenance of neuronal homeostasis. In normal physiology, these hormones bind to endogenous adrenocorticosteroid receptors (ACRs), through which they exert trophic actions on neurons (by way of the mineralocorticoid receptor [MR]) and serve to promote defensive responses to physiological or psychological stress (by way of the glucocorticoid receptor [GR]). The latter defensive responses are adaptive in the short run, serving to mobilize body resources. However, if release is prolonged, glucocorticoids can have multiple negative consequences for the animal. Included among these are neurotoxic effects on neurons in the hippocampus. In normal individuals, glucocorticoid release is tightly controlled, thereby maintaining subtoxic levels. Unfortunately, as a consequence of aging or Alzheimer's disease (AD), this control is lost, resulting in prolonged glucocorticoid release which has been linked with age-related hippocampal cell loss and memory deficits. Loss of the capacity to regulate glucocorticoids is a consequence of impaired stress regulation, and appears to prominently involve neuronal ACR imbalances. The present studies are designed to identify cellular mechanisms underlying ACR dysregulation in stress and aging, with the eventual goal of targeting specific pathways for prevention of glucocorticoid-related cell loss. Specific Aim 1 will address the hypothesis that stress and glucocorticoids affect ACR gene expression and protein synthesis by the same molecular mechanism, verifying the primacy of glucocorticoids in regulating receptor synthesis in vivo. Specific Aim 2 will characterize specific molecular pathways regulating GR and MR biosynthesis in neurons, identifying molecular targets for age-induced dysregulation. Specific Aim 3 will evaluate the hypothesis that stress and aging work by way of the same glucocorticoid-mediated pathway to disrupt ACR regulation. Finally, Specific Aim 4 will determine whether age- and stress-induced changes in ACR regulation specifically target the neurotrophins, glucocorticoid-responsive molecules involved in maintenance of neuronal cell viability. It is predicted that the results of this project will identify specific mechanisms responsible for impaired ACR regulation in aging and AD. HERMAN JP UNIVERSITY OF KENTUCKY, 800 ROSE ST224 UKMC, LEXINGTON, KY 40536-0084/RADIOTHERAPY 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health adrenalectomy aging laboratory rat hippocampus hormone regulation /control mechanism gene expression western blotting immunologic assay /test protein biosynthesis autoradiography radiotracer receptor expression corticosteroid receptor stress tissue /cell culture neurotoxicology CRISP RPROJ KENTUCKY G CRISP/99/AG12962-03 CRISP/99/AG12962-03 199904 eng OXYGEN RADICAL TOXICITY AND CHRONIC NEURONAL LOSS Research ROTHSTEIN JD MASSACHUSETTS GENERAL HOSPITAL, 149 13TH ST, RM 6627, CHARLESTOWN, MA 02129 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02129 Crisp Data Base National Institutes Of Health laboratory mouse laboratory rat transgenic animal carbonyl group glutamate protooncogene neurotrophic factor lipid peroxide neural degeneration spinal cord motor neuron neuropharmacology neurotoxin antisense nucleic acid antioxidant superoxide dismutase free radical oxygen peroxidation glutamate receptor organ culture cytotoxicity DNA damage programmed cell death oxidative stress neuroprotectant CRISP RPROJ MASSACHUSETTS G CRISP/99/AG12992-040002 CRISP/99/AG12992-040002 199904 eng TETANUS TOXIN C-FRAGMENT HYBRID AS A NEUROPROTECTANT Research FISHMAN PS MASSACHUSETTS GENERAL HOSPITAL, 149 13TH ST, RM 6627, CHARLESTOWN, MA 02129 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02129 Crisp Data Base National Institutes Of Health laboratory mouse laboratory rat glutamate drug administration route pharmacokinetics drug vehicle free radical human tissue active immunization tetanus toxoid antibacterial antibody tetanus toxin central nervous system neuropharmacology neurotoxin antioxidant superoxide dismutase chimeric protein tissue /cell culture embryo /fetus cell culture cytotoxicity enzyme activity oxidative stress neuroprotectant recombinant protein CRISP RPROJ MASSACHUSETTS G CRISP/99/AG12992-040003 CRISP/99/AG12992-040003 199904 eng CORE--MOLECULAR GENETICS Research BROWH RH MASSACHUSETTS GENERAL HOSPITAL, 149 13TH ST, RM 6627, CHARLESTOWN, MA 02129 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02129 Crisp Data Base National Institutes Of Health biomedical facility polymerase chain reaction site directed mutagenesis molecular cloning gene mutation human tissue tetanus toxin complementary DNA nucleic acid sequence superoxide dismutase chimeric protein human genetic material tag recombinant protein single strand conformation polymorphism CRISP RPROJ MASSACHUSETTS G CRISP/99/AG12992-049001 CRISP/99/AG12992-049001 199904 eng NEUROTOXICITY, NMDA RECEPTORS, AND FREE RADICALS Research MICHAELIS EK UNIVERSITY OF KANSAS, MALOTT HALL, LAWRENCE, KS 66045 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 66045 Crisp Data Base National Institutes Of Health laboratory rat glutamate neural degeneration neuron neurotoxin nitroso compound antisense nucleic acid oxidation superoxide dismutase free radical oxygen protein sequence protein structure /function receptor binding receptor expression NMDA receptor tissue /cell culture embryo /fetus cell culture nitric oxide northern blotting enzyme activity oxidative stress RNase protection assay granule cell neuroprotectant nitric oxide synthase CRISP RPROJ KANSAS G CRISP/99/AG12993-040005 CRISP/99/AG12993-040005 199904 eng MECHANISMS AND MOLECULAR PROFILES OF DEGENERATION IN ALZHEIMER'S DISEASE Research RPROJ DESCRIPTION: (adapted from Applicant's Abstract) Recent in vitro evidence by this group and others predicts that apoptosis may be one of the pathways leading to cell death in AD. The cell death program is initiated in response to several stimuli, such as beta-amyloid (Ab), related analogs, or conditions causing excessive oxidative injury to the cells. This proposal centers on employing Ab, select derivatives, and a newly developed hydrogen peroxide model to initiate the cell death program and study the mechanisms of cell death in response to those stimuli at the molecular level. An interactive in vitro/in vivo approach employing well defined cell culture techniques and well characterized postmortem tissues will be used. In this way, they can evaluate the factors and molecular pathways that lead to neuronal dysfunction and death in AD. The secondary hypothesis is that specific cascades of immediate early genes (IEGs) participate in the neuronal cell death program. They will examine the response and profile of immediate early genes (IEGs) elicited by Ab and related analogs. It is predicted that select analogs initiating apoptosis will induce select IEGs and that these IEGs will both mediate and serve as molecular signatures of apoptosis. They will compare the IEG response initiated by Ab to that elicited by excessive oxidative injury caused by hydrogen peroxide. COTMAN CW UNIVERSITY OF CALIFORNIA, 1305 BIOLOGICAL SCIENCES II, IRVINE, CA 92717-4550 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory mouse laboratory rat transgenic animal cerebellum hippocampus Alzheimer's disease postmortem molecular pathology regulatory gene gene induction /repression human tissue immunocytochemistry disease model neural degeneration neuron neurotoxin hydrogen peroxide protein structure /function amyloid protein tissue /cell culture cytotoxicity programmed cell death entorhinal cortex oxidative stress CRISP RPROJ CALIFORNIA G CRISP/99/AG13007-04 CRISP/99/AG13007-04 199904 eng WORKING LIVES AND MORTALITY IN AGING Research RPROJ DESCRIPTION: (Adapted from Investigator's Abstract) Work continues to be a central social aspect of a person's life providing meaning and identity throughout the lifecourse, ultimately influencing mortality. The proposed research will use data from the Panel Study of Income Dynamics (PSID), an ongoing longitudinal survey of U.S. Households begun in 1968 with mortality follow-up through 1992, to resolve several major issues regarding the relationship between psychosocial work conditions and mortality, and assess whether work life is causally related to mortality. The PSID data constitute an unprecedented opportunity to establish a 'data laboratory' for studying 25 consecutive years of work environment exposure history and how exposures over the life course relate to mortality in a national cohort including working women and blacks. The proposed research has three specific aims, as follow: (1) to examine how psychosocial work conditions influence survival; (2) to explore how the progression of a person's career, the arrangement of jobs within the context of an individual's worklife, influences survival; and (3) to describe how survival rates vary by race and gender. The four major hypotheses are as follow: (1) hazardous psychosocial work conditions will predict mortality after adjustment for hazardous physical and environmental conditions of work; (2) the progression of careers will influence mortality after adjustment for hazardous working conditions; (3) the relationship between working life (exposure to hazardous working conditions and the progression of careers) and mortality will vary by race and gender; and (4) non-work burdens (e.g., household work or stressful residential neighborhoods) and resources (e.g., social support) will differentially influence racial and gender survival rates. A sub-sample of approximately 4,500 adults age 25 to 65 who were working in 1968 and followed until 1992 comprise the cohort. About 1,000 deaths have occurred during the follow-up period. Data analysis will employ proportional hazard models with and without time-dependent covariates. To avoid potential biases of self-reported work conditions, exposures are imputed from three-digit occupational codes to establish up to 25 years of exposure history. Hazardous physical and environmental work conditions are also assigned to individuals based on the three-digit occupational code improving the precision of the estimate of the relationship of psychosocial work conditions to survival. With year-to-year determination of occupation, the bias introduced by retrospective assessment of occupation is avoided. The investigators state that the proposed research significantly improves on previous analyses by examining the dynamics of the relationship between hazardous work and health including the selection of workers out of the workforce due to work-related disability and the movement of individuals from more hazardous psychosocial work conditions to less hazardous conditions. In addition, they state they will compare, for the first time, the two methods used for describing psychosocial work conditions -- one based on job ratings in the Dictionary of Occupational Titles, and the other using aggregate measures of job stress from national quality of employment surveys. AMICK BC NEW ENGLAND MED CTR, 750 WASHINGTON ST BOX 345, BOSTON, MA 02111 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02111 Crisp Data Base National Institutes Of Health occupational hazard occupation epidemiology human morbidity occupational psychology sex difference racial /ethnic difference social support network socioeconomics occupational stressor quality of life human data clinical research behavioral /social science research tag CRISP RPROJ MASSACHUSETTS G CRISP/99/AG13036-03 CRISP/99/AG13036-03 199904 eng AGE DEPENDENT RESPONSE OF HIPPOCAMPAL NEURONS TO STRESS Research RPROJ DESCRIPTION (from the applicant's abstract) The most significant etiological factor in Alzheimer's disease (AD) is age. But how does age contribute to the cellular and molecular pathology of AD? It is hypothesized that hippocampal neurons from aged animals are more susceptible to Abeta than younger neurons due to a decreased ability to generate the energy needed to control ion fluxes in this region of the AD brain. Novel techniques for the culture of aged neurons in serum-free medium have been developed recently by the PI, enabling examination of age-related cellular responses. New data have been obtained that subtoxic levels of lactate acidosis alter APP processing resulting in deposition of more amyloidogenic fragments. Also, neurons from aged rats are more susceptible to Abeta, lactate acidosis, and glutamate toxicity and fail to increase ATP levels to meet demands of increased ionic fluxes. Using neurons cultured from adult and aged rats, the first aim is to compare responses to fibrillar Abeta in terms of intracellular calcium, pH, and viability. Also regional AD pathology will be examined by comparing responses to Abeta in hippocampal with cerebellar granule neurons. The second aim examines the impact of glutamate and lactate acidosis on Abeta toxicity. The third aim examines whether age-related changes in susceptibility to Abeta are accounted for by declines in energy production, free radical damage, or induction of apoptosis. These studies hope to provide a better understanding of the age-related responses of cultured neurons to stressors thought to be important in the AD brain. BREWER GJ SOUTHERN ILLINOIS UNIVERSITY, PO BOX 19626, SPRINGFIELD, IL 62794-9626 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory rat hippocampus calcium flux glutamate free radical lactate lactic acidosis neuron neurotoxin amyloid protein tissue /cell culture toxin metabolism programmed cell death oxidative stress granule cell neurotoxicology CRISP RPROJ ILLINOIS G CRISP/99/AG13435-02 CRISP/99/AG13435-02 199904 eng METABOTROPIC GLUTAMATE RECEPTORS IN NEURODEGENERATION Research RPROJ Toxic actions of the neurotransmitter, glutamic acid, have been implicated in a number of diseases that effect the brain including stroke, epilepsy, Huntington&#176;s disease, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. These toxic actions of glutamate are mediated through the same neurotransmitter receptors on nerve cells that ordinarily serve to convey glutamate's excitatory signal to nerve cells. Recent experimental evidence suggests that some subtypes of metabotropic glutamate receptors may attenuate glutamate excitotoxicity while other subtypes of metabotropic glutamate receptors aggravate excitotoxicity. In addition, drugs that specifically bind to the different subtypes of metabotropic receptors have been invented recently. This project will use molecular pharmacologic techniques to better define the role of metabotropic glutamate receptors in excitotoxic and metabolic death of nerve cells. A combination of stereotaxic lesions, pharmacological manipulations, quantitative morphometry and in situ hybridization histochemistry will be used to test the hypothesis that metabotropic receptors linked to the phosphatidyl inositol second messenger system aggravate while metabotropic receptors linked to the adenylate cyclase second messenger system attenuate excitotoxicity. Injections of antisense messenger RNA to specifically block synthesis of the different metabotropic receptors will be used to further define the roles of the different metabotropic receptors. YOUNG AB MASSACHUSETTS GENERAL HOSPITAL, FRUIT STREET, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02114 Crisp Data Base National Institutes Of Health laboratory rat corpus striatum decortication chemical binding molecular site kainate immunocytochemistry western blotting neural degeneration neuropharmacology neurotoxin in situ hybridization antisense nucleic acid complementary DNA nucleic acid probe oligonucleotide adenylate cyclase phosphatidylinositol cyclic AMP quinolinate receptor binding receptor coupling glutamate receptor stereotaxic technique excitatory aminoacid CRISP RPROJ MASSACHUSETTS G CRISP/99/AG13617-04 CRISP/99/AG13617-04 199904 eng AGING AND SLEEP--ROLE OF MELATONIN Research RPROJ DESCRIPTION (Adapted from Investigator's Abstract): Aging is associated with disturbances of both sleep and circadian rhythms. Nocturnal melatonin levels in the elderly are often substantially lower than in younger people. Studies have shown that high pharmacological doses of melatonin produce sleepiness and reduce sleep latency. The proposed studies will test the hypothesis that sleep disturbances of the elderly can be related to deficiencies in nocturnal melatonin secretion, and that restoration of rhythmic, high amplitude variations in circulating melatonin can improve sleep quality and quantity in the elderly without diminishing mood or performance the following morning. Elderly (60 -75 years) men and women participants (N=60) with and without symptomatic and polygraphically-confirmed insomnia will be tested under four conditions (placebo, 0.1, 0.2, 0.3 mg melatonin). Sleep quality and quantity will be polygraphically assessed along with measures of alertness, sleepiness, mood and performance. Parallel measures of cortisol and body temperature will be taken. WURTMAN RJ MASSACHUSETTS INST OF TECHNOLO, 77 MASSACHUSETTS AVENUE, CAMBRIDGE, MA 02139-4307 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health cortisol human old age (65+) young adult human (19-34) aging secretion circadian rhythm blood chemistry dosage drug adverse effect drug screening /evaluation human subject sleep disorder melatonin polygraphy attention sleep emotion performance female male sex difference body temperature oral administration age difference clinical research CRISP RPROJ MASSACHUSETTS G CRISP/99/AG13667-03 CRISP/99/AG13667-03 199904 eng SUPRAMOLECULAR A--GLIAL INTERACTIONS AND CELL RESPONSE Research RPROJ DESCRIPTION: (adapted from applicant abstract) The overall hypothesis is that the cellular responses to amyloid beta (A beta ) that culminate in the cell dysfunction and degeneration characteristic of Alzheimer disease (AD) are dependent on the specific structure of the supramolecular aggregates formed by the A beta peptides. The applicants propose that part of the process of maturation of the predominantly diffuse, non-neuritic amyloid plaques of "normal" aging into the neuritic plaques associated with dementia involves the generation of these specific A beta structures. Extending this hypothesis, the applicants further propose that the generation of "toxic" supramolecular structures of A beta aggregates can be influenced by the presence of other plaque components, particularly those components derived from glia, and that responses of glial cells contribute to an environment that facilitates and enhances the formation of these bioactive A beta forms. It is their contention that glia are not just passive bystanders, but are a part of the pathogenesis process. They postulate that A beta toxicity can be mediated through its effects on glia, that glia-derived proteins can influence the structure of A beta and its effects on neurons, and that these processes are relevant to the actual neuropathology seen in the AD brain. The questions being addressed in these studies are: What does A beta do to glia? What do glia do to A beta structure and activity? Are glia relevant to AD neuropathology? Three aims are proposed to address these questions. 1) They will evaluate the effects of A beta on glia. Specific A beta aggregates will be prepared, characterized by atomic force microscopy (AFM), and tested for activity on cultures of rat primary astrocytes. The investigators will examine the effects of A beta on the levels and activity of six relevant glial proteins: a 1-antichymotrypsin (ACT), apolipoprotein E (apoE), apoJ, butyrylcholinesterase (BChE), interleukin-1 (IL-1), and S100 beta . 2) They will evaluate the effects of glia on A beta structure and neurotoxicity. In co-aggregation experiments, they will evaluate AFM structure of A beta aggregates formed in the presence of the six glial proteins studied in aim 1. The investigators will also test A beta -evoked neurotoxicity in the presence of these six glial proteins and in glial-neuronal co-cultures. 3) They will correlate the presence and distribution of glial proteins with plaque type in AD brain. They will determine the regional distribution and immunoreactive density of the six glial proteins in AD and control brain tissue, and establish correlations with the distribution of non-neuritic vs neuritic plaques. This system examination should provide insight into how A beta affects glia, and how glia participate in amyloid plaque progression and development of neurotoxicity. VAN ELDIK LJ NORTHWESTERN UNIVERSITY, 303 EAST CHICAGO AVENUE, CHICAGO, IL 60611-3008 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory rat Alzheimer's disease cholinesterase interleukin 1 apolipoprotein intermolecular interaction glia neurotoxin protein structure /function amyloid protein tissue /cell culture enzyme activity atomic force microscopy CRISP RPROJ ILLINOIS G CRISP/99/AG13939-03 CRISP/99/AG13939-03 199904 eng INSOMNIA IN ELDERLY--PASSIVE BODY HEATING VS ZOLPIDEM Research RPROJ DESCRIPTION (Adapted from Investigator's Abstract): This application will examine sleep and sleep disturbance in the elderly, seeking to develop an alternative method for improving sleep quality in the elderly. Forty elderly female volunteers with sleep maintenance insomnia will participate in a 10-day/night protocol involving a comparison of the effects of Passive Body Heating (PBH) and a commonly used hypnotic (zolpidem) on sleep, temperature, growth hormone secretion, subjective sleepiness, and performance. Baseline actigraphic and sleep diary data will be collected for ten days prior to the study to evaluate sleep at home and to confirm insomnia. Subjects will be randomly assigned to one of four treatment conditions: PBH/placebo, PBH/zolpidem, PBH control/placebo, PBH control zolpidem. The PBH intervention will involve having the subjects sit in a tub filled with 40 C (active) or 37 C (control) water for 30 minutes. Zolpidem will be administered at 5mg. Objective measures of sleep will be quantified using standard sleep scoring criteria. Changes in sleep efficiency and architecture will be measured and amplitude and density of slow wave sleep will be quantified using power spectral analysis. Subjective measures of sleep will be taken using a post-sleep questionnaire and a visual analog scale. Core body temperature and motor activity will be recorded continuously using ambulatory monitoring systems. Growth hormone will be sampled every 30 minutes during one baseline and one treatment night and assayed using standard procedures. Measures of memory, reaction time and vigilance will be made during the day. The PBH technique may achieve the same beneficial effects on sleep as zolpidem, with the risk of side-effects associated with hypnotic medication. The investigators intend to determine whether the PBH procedure will be a useful non-pharmacologic method for treating insomnia in the elderly. DORSEY CM MCLEAN HOSPITAL, 115 MILL STREET, BELMONT, MA 02178 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02178 Crisp Data Base National Institutes Of Health human old age (65+) patient monitoring device circadian rhythm electroencephalography drug administration rate /duration drug adverse effect drug screening /evaluation electrophysiology electrocardiography human subject questionnaire statistics /biometry electromyography sleep disorder somatotropin wakefulness memory performance sedative /hypnotic respiratory airflow measurement female heat body temperature human therapy evaluation clinical research CRISP RPROJ MASSACHUSETTS G CRISP/99/AG13961-03 CRISP/99/AG13961-03 199904 eng TOXICANT OR AGING INDUCED OXIDATIVE STRESS IN PROSTATE Research RPROJ The long term objective of our research is to understand the basis of diethylstilbestrol (DES) and cadmium(Cd) toxicities in the ventral prostate (VP) of young and old testosterone (T)-supported rats. We and others have shown that the VP exhibits unique susceptibilities to these toxicants. Our preliminary data revealed that both T+DES- and Cd- treatment of rats induced severe oxidative stress (OS) as well as aberrant proliferative and apoptotic activities in this prostatic lobe. T+DES and Cd separately induced VP tumors in life-time studies, and synergistically early dysplastic changes. We found that the VP expresses minimal levels of mRNA of metallothioneins (MTs) which are heavy metal binding proteins believed to function as intracellular metal-ion chelators and/or antioxidants. We recently reported marked increases in lipid peroxidation status and drastic decline in the activities of reactive oxygen species (ROS) detoxification enzymes in the VPs of aged rats. Taken together, we hypothesize that T+DES and Cd are potent carcinogens for rat VP because, inter alia, they exacerbate oxidative stress (OS) in the aging VP. We thus postulate that 1) induction of OS and associated events, such as DNA single-strand breaks and apoptosis- cell proliferation imbalance, are early events leading to T+DES- and/or Cd-induced dysplasia and cancer development in rat VP, and 2) the VP of an aged rat is more susceptible to tumor induction by these toxicants than its younger littermates. Experiments have been designed to determine whether l) T+DES and/or Cd induce OS, DNA damage, and imbalance in cell proliferation-apoptosis in the VP and these events precede the development of dysplasia 2) these OS-inducers activate MT gene expression 3) the aged VPs have reduced ROS detoxification capability and thus are more sensitive to T+DES and/or Cd action 4) T+DES and Cd, given simultaneously to rats, have compounded effects on OS, dysplasia, and tumor induction in the VP and 5) T+DES with and without Cd can induce OS parameters in organ cultures of VP of young and aged rats and, if so, whether antioxidants added in vitro can reverse these damages. Results form this work will enhance our understanding of aging and OS in toxicant- induced carcinogenesis and the potential application of antioxidants as anti-cancer chemopreventive measures. HO S TUFTS UNIVERSITY, MEDFORD, MA 02155 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02155 Crisp Data Base National Institutes Of Health aging testosterone laboratory rat immunocytochemistry metallothionein cadmium hormone related neoplasm /cancer prostate preneoplastic state chemical carcinogenesis antioxidant toxicant interaction prostate male diethylstilbestrol DNA damage programmed cell death oxidative stress cell proliferation CRISP RPROJ MASSACHUSETTS G CRISP/99/AG13965-02 CRISP/99/AG13965-02 199904 eng PEROXYNITRITE AND NEURODEGENERATIVE DISEASES OF AGING Research RPROJ DESCRIPTION: Experiments evidenced have implicated oxidative stress as a contributing element in the neuropathology of degenerative disorders of late life such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Oxidative stress is the result of over production of reactive species which can overwhelm cellular neuroprotective mechanisms and inactivate critical processes for preserving cellular integrity in function. While much of the existing data has concentrated on the contribution of oxygen-derived reactive species, recent evidence has also implicated nitric oxide derived oxidants in the pathogenesis of neuronal injury. The toxicity of reactive oxidant species is coupled with the reactivity of nitric oxide. Since both nitric oxide and superoxide or free radicals they react in a near diffusion limited rate to form peroxynitrite. Recent data has revealed that peroxynitrite is a major oxidant generated in biological systems under pathologic conditions demonstrating that the proposed chemistry is possible in vivo. Furthermore, peroxynitrite reacts selectively with nitrogen residues of protein to form nitrotyrosine. Protein tyrosine nitration results in the inactivation of protein function and interferes with tyrosine phosphorylation a key event in cellular signal transduction. Previously this group of collaborative investigators has shown that exposure of PC12 cells to low levels of peroxynitrite resulted in significant and irreversible inhibition of DOPA synthesis. Preliminary data indicates that peroxynitrite is mediated nitration of tyrosine residues in the active site of tyrosine hydroxylase could account for the inhibition of DOPA synthesis. Moreover, tyrosine hydroxylase is detected after exposure of PC12 lysates to peroxynitrite or after induction of endogenous peroxynitrite production. The major goal of this proposed grant proposal is to elucidate the role of peroxynitrite-mediated tyrosine nitration in the inactivation of tyrosine hydroxylase in vitro as well as cellular and animal models of aging and Parkinson's disease. The investigators propose two major specific aims. 1) to determine if tyrosine nitration is responsible for the peroxynitrite inactivation of tyrosine hydroxylase. 2) characterize the peroxynitrite mediated modifications present in tyrosine hydroxylase in three models 1) PC12 cells stimulate to generate peroxynitrite, 2) brains of aged rats and 3) mouse brains following administration of MPTP. ISCHIROPOULOS H CHILDRENS HOSPITAL, 3400 CIVIC CENTER BLVD, ATTN: MR. ROBERT G. URIS 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory mouse laboratory rat tyrosine enzyme inhibitor nervous system disorder nitrite tyrosine 3 monooxygenase peroxide methylphenyltetrahydropyridine enzyme activity oxidative stress PC12 cell neurotoxicology CRISP RPROJ PENNSYLVANIA G CRISP/99/AG13966-02 CRISP/99/AG13966-02 199904 eng THE IMPACT OF GERIATRIC CARE ON DRUG-RELATED PROBLEMS Research RPROJ DESCRIPTION: This is a new application from Duke University Medical Center whose overall aim is to determine if a multi-disciplinary geriatric health service known as Geriatric Evaluation and Management (GEM) is effective in reducing drug-related problems in the elderly, including adverse drug events (ADEs) and adverse drug reactions (ADRs), inappropriate prescribing, polypharmacy, and undermedication. The specific aims are to determine if an inpatient GEM unit (GEMU) and outpatient GEM Clinic (GEMC) care, alone or in combination, can reduce the above drug-related problems in the elderly when compared with usual care. This proposed study capitalizes on the methods of an ongoing Veterans Administration cooperative study (Evaluation of GEM units and geriatric follow-up), a randomized controlled trial with a 2 X 2 factorial design which is studying the effects of the above combinations of GEM care on other study outcomes (health, quality of life, and mortality) at 10 VA medical centers. The proposed study thus incorporates the existing design, sample, interventions, and independent variables of the parent study. Patients are followed for one year following randomization to the study conditions. The proposed investigation will collect selected additional clinical data from medical records and computer files and will interview patients for suspected ADEs. Baseline and twelve-month follow-up measures will include previously used measures of inappropriate prescribing, polypharmacy, and undermedication. Data analysis will be conducted to determine the main and interactive effects of the GEM interventions on these outcomes. SCHMADER KE DUKE UNIVERSITY MEDICAL CTR, BOX 3003, DURHAM, NC 27710 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 27710 Crisp Data Base National Institutes Of Health human old age (65+) aging medication error drug adverse effect drug interaction health care service evaluation health care service planning human subject clinical research behavioral /social science research tag CRISP RPROJ NORTH CAROLINA G CRISP/99/AG14158-03 CRISP/99/AG14158-03 199904 eng MITOCHONDRIAL DNA MUTATIONS IN ALZHEIMER'S AND PARKINSON'S DISEASES Research PARKER DW UNIVERSITY OF VIRGINIA, PO BOX 394, HSC, CHARLOTTESVILLE, VA 22908 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 22908 Crisp Data Base National Institutes Of Health bioenergetics Alzheimer's disease hybrid cell electron transport gene mutation human tissue disease model Parkinson's disease mitochondrial DNA cytochrome oxidase free radical oxygen toxin metabolism cell line age difference enzyme activity CRISP RPROJ VIRGINIA G CRISP/99/AG14373-01A10001 CRISP/99/AG14373-01A10001 199904 eng MITOCHONDRIAL MOVEMENT IN CELLULAR MODELS OF ALZHEIMER'S AND PARKINSON'S D Research TRIMMER PA UNIVERSITY OF VIRGINIA, PO BOX 394, HSC, CHARLOTTESVILLE, VA 22908 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 22908 Crisp Data Base National Institutes Of Health laboratory rat azide intracellular transport mesencephalon hippocampus Alzheimer's disease cytoplasm mitochondria cell differentiation hybrid cell cellular pathology human subject disease model neural degeneration Parkinson's disease methylphenyltetrahydropyridine tissue /cell culture toxin metabolism clinical research cell component structure /function CRISP RPROJ VIRGINIA G CRISP/99/AG14373-01A10002 CRISP/99/AG14373-01A10002 199904 eng MITOCHONDRIAL DEFECTS IN GUAM PDC Research PARKER WD Wiederholt, Wigbert, C., Department of Neurosciences, LA JOLLA, CA 92093-0624 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory rat brain metabolism mitochondria electron transport genome myocardium heart metabolism human tissue muscle metabolism striated muscle Parkinson's disease neurotoxin mitochondrial DNA cellular respiration Asian acific Islander amyotrophic lateral sclerosis CRISP RPROJ CALIFORNIA G CRISP/99/AG14382-020006 CRISP/99/AG14382-020006 199904 eng TRANSGENIC MOUSE MODELS OF ALZHEIMERS DISEASE Research RPROJ DESCRIPTION This is an R29 proposal to use YAC based approaches to presenilin wild-type and mutant transgenics linked to FAD and to breed them to YAC FAD mutant APP transgenics already in hand and examine effects on alterations in cell biology of APP and PS-1, excitotoxicity, LTP and neuropathology. LAMB BT CASE WESTERN RESERVE UNIVERSIT, 10900 EUCLID AVE, CLEVELAND, OH 44106-4955 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal Alzheimer's disease model design /development disease model neurotoxin amyloid protein long term potentiation neuropathology CRISP RPROJ OHIO G CRISP/99/AG14451-02 CRISP/99/AG14451-02 199904 eng B-AMYLOID AND NEURONAL CALCIUM MISREGULATION Research RPROJ The long-term goal of this research is to gain an understanding of the cellular and molecular events that lead to neuronal damage and death in Alzheimer's disease (AD). This project tests the hypothesis that beta- amyloid destabilizes cellular calcium homeostasis and thereby renders neurons more vulnerable to environmental insults. A hippocampal cell culture system will be used to examine the cellular and molecular mechanisms whereby beta-amyloid destabilizes neuronal calcium homeostasis, and potentiates excitatory amino acid neurotoxicity. Immunolocalization studies of AD brains are designed to determine whether the neuropathology of AD is consistent with the calcium destabilization hypothesis of beta- amyloid neurotoxicity. The first aim will test the hypothesis that beta- amyloid affects specific cellular systems for calcium homeostasis (NMDA receptors, calcium channels, Na+/Ca2+ exchanger, calcium binding protein, and calcium ATPase). The second aim is to determine whether endogenous beta-amyloid contributes to selective neuronal vulnerability in cell culture. The third aim is to establish whether the cytoskeletal manifestations of neuronal degeneration induced by beta-amyloid resemble the neurofibrillary pathology of AD. The fourth aim is to determine the relationships of beat-amyloid, NMDA receptors, and calcium-regulating proteins in the histopathology of AD. These aims will be accomplished using the following technologies: immunocytochemistry to localize beta- amyloid and calcium-regulating proteins in cell cultures and in AD brains; light and confocal laser scanning microscopy in living neurons; electron microscopy; fluorescence ratio imaging of intracellular calcium levels. Each of these techniques will be applied to neurons at different stages in the progression of the neurodegenerative process. Taken together, these studies will: (1) Provide insight into the cellular and molecular mechanisms whereby beta-amyloid destabilizes neuronal calcium homeostasis. (2) Tell us whether the cellular pathology of AD is consistent, at the molecular level, with the calcium-destabilization hypothesis. (3) Generate information that can be used to develop new approaches to preventing and treating the neuronal damage that is responsible for the progression of AD. MATTSON MP UNIVERISTY OF KENTUCKY, 800 SOUTH LIMESTONE ST, LEXINGTON, KY 40536-0230 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory rat antiport brain hippocampus Alzheimer's disease calcium calcium flux cytoskeleton cell death histopathology human tissue immunocytochemistry immunofluorescence technique homeostasis neural degeneration neurofibrillary tangle neuron neurotoxin electron microscopy calcium binding protein amyloid protein calcium channel membrane transport protein calcium transporting ATPase NMDA receptor sodium tissue /cell culture confocal scanning microscopy excitatory aminoacid light microscopy CRISP RPROJ KENTUCKY G CRISP/99/AG14554-07 CRISP/99/AG14554-07 199904 eng EFFECTIVENESS OF RETROSPECTIVE DRUG UTILIZATION REVIEW Research RPROJ Improper prescribing of medications is an important public health problem. Because the aged are more likely to have concomitant illnesses and medications, they are at greatest risk for adverse outcomes. For example, one study conducted in the elderly found that 28% of hospital admissions were due to medication problems, and showed that Medicaid recipients were at an especially increased risk. Drug utilization review (DUR) is a procedure that seeks to reduce the consequences of improper prescribing. In part because of federal mandate, retrospective DUR is now being applied to millions of older Americans, including all Medicaid enrollees and seventy- five percent of all people with prescription insurance coverage. This is despite the complete absence of rigorous evidence indicating that DUR is effective in either altering drug therapy or improving health outcomes. Given the existing federal mandate, an experimental trial of DUR in the Medicaid population is infeasible. Therefore, a timely, well-controlled observational trial of DUR's effectiveness is badly needed. The University of Pennsylvania has a unique opportunity to conduct a rigorous observational trial of DUR's effects on prescribing patterns and on clinical outcomes. Using a large, unique data source, we intend to evaluate the effects of DUR on both process measures and clinical outcome measures. The methodology that we propose incorporates both pre-post intervention comparisons, and concurrent controls. This will enable us to measure a number of important effects of DUR, controlling for possible effects of differences between state programs and causally unrelated secular trends. We intend to employ three distinct, complementary sampling frames from which to pose the study questions. The study questions have been carefully selected, and address issues of practical and theoretical importance. The results of this study, whether positive or negative, will have wide-ranging health policy and scientific implications, especially with regard to the aged. The University of Pennsylvania has a great deal of experience in pharmaceutical outcomes research projects such as this, especially those using large claims databases. The investigators will be able to provide clear, meaningful answers to the study questions in a very short time-frame, and do so in a very cost-efficient manner. STROM BL UNIVERSITY OF PENNSYLVANIA, 824 BLOCKLEY HALL/423 GRDN DR, PHILADELPHIA, PA 19104-6021 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health warfarin hemorrhage antiinflammatory agent pharmacy drug adverse effect gastritis peptic ulcer health care service evaluation health care service utilization angina pectoris myocardial infarction hospital utilization asthma salicylate human data chronic obstructive pulmonary disease clinical research behavioral /social science research tag CRISP RPROJ PENNSYLVANIA G CRISP/99/AG14601-02 CRISP/99/AG14601-02 199904 eng EXCITOTOXIC INTERACTIONS Research RPROJ DESCRIPTION (Investigator's Abstract): this is a new R01 proposal to study mechanisms of excitotoxicity at the cellular level. The author proposes to - 1 - use confocal microscopy as well as specific intracellular dyes to dissect the involvement of calcium, mitochondria and the production of free radicals as related to neuronal death. The fundamental hypothesis is that there is functional and temporal coupling between an NMDA receptor stimulation and cytosolic calcium accumulation, intramitochondrial accumulation, production of free radicals, mitochondrial depolarization, opening of the mitochondrial transition pore and eventual neuronal death. The author proposes four specific aims. In specific aim 1 he will use primary cultures of neurons as well as laser scanning confocal microscopy to examine and correlate the above mentioned sequence temporally. In specific aim 2 he will test the hypothesis that activation of nonNMDA receptors or voltage dependent calcium channels will have less of an effect than an NMDA receptor activation on mitochondrial calcium, free radical production and membrane potential. Specific aim 3 will determine whether inhibition of the electron transport chain will impair the ability of mitochondria to accumulate calcium as well as to cause abnormal elevation of cytosolic calcium and to facilitate opening of the permeability transition pore. Lastly the author will examine lymphoblastoid cell lines derived from patients with known mitochondrial defects to determine whether these cells lines exhibit abnormal mitochondrial calcium accumulation, membrane potential and free radical production. Furthermore he will then transfect these lymphoblasts with an NMDA receptor to determine whether they are abnormally sensitive to receptor activation as compared to lymphoblastoid cell lines from normal controls. GREENAMYRE JT EMORY UNIVERSITY, 1639 PIERCE DRIVE, ATLANTA, GA 30322 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 30322 Crisp Data Base National Institutes Of Health Animalia membrane permeability calcium flux mitochondria fluorescent dye /probe free radical membrane potential transfection neural degeneration neurotoxin calcium channel NMDA receptor tissue /cell culture confocal scanning microscopy CRISP RPROJ GEORGIA G CRISP/99/AG14648-02 CRISP/99/AG14648-02 199904 eng ESTROGENIC STEROIDS--NEUROTROPHIC ACTION AND MECHANISM Research BRINTON R UNIV OF SOUTHERN CA CENTER, ETHEL PERCY ANDRUS GERONTOLOGY, LOS ANGELES, CA 90089-0191 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health laboratory rat cerebral cortex hippocampus glutamate hormone regulation /control mechanism estrogen gene induction /repression neurogenesis neuron hydrogen peroxide amyloid protein calcium channel receptor expression estrogen receptor glutamate receptor tissue /cell culture oxidative stress neuroprotectant neurotoxicology neurogenetics CRISP RPROJ CALIFORNIA G CRISP/99/AG14751-020002 CRISP/99/AG14751-020002 199904 eng CYCLOOXYGENASE IN NEURODEGENERATION Research RPROJ DESCRIPTION The epidemiological data showing NSAIDS reduce risk for AD demands further investigation at the animal model level. One major target of NSAIDs is Cox-2. Pasinetti and others have found that neuronal COX-2 is elevated in experimental neurodegeneration and in AD brain and that overexpression of Cox-2 potentiates Abeta-mediated oxidative stress in vitro. Based on this and other data, they propose to: Aim 1. Study the role of Cox-2 in neurodegeneration using experimental Abeta and kainic acid damage in transgenic mice with neuronal, human C0X-2 (NHC) overexpression and in COX-2 knockout mice. They hypothesize COX-2 overexpression will increase neurodegeneration. Aim 2. To identify the role of COX-2 in primary cultures of NHC transgenic and COX-2 KO mice in the response to Abeta and glutamate neurotoxicity evaluated by MTT and neuronal counts. This parallels aim 1. Aim 3. To identify mechanisms involved in COX-2-mediated potentiation of Abeta toxicity using organotypic hippocampal slice cultures derived from NHC transgenic and COX-2 KO mice. The hypothesis that COX-2 overexpression amplifies free radical mediated DCF fluorescence and lipid peroxidation will be tested. Aim 4. To study the role of COX-2 overexpression in bigenic NHC X APPsw (Hsiao mice) which are hypothesized to have accelerated neurodegeneration and cognitive impairment. PASINETTI GM MOUNT SINAI SCH OF MEDICINE, ONE GUSTAVE L LEVY PLACE, NEW YORK, NY 10029 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 10029 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal hippocampus Alzheimer's disease kainate glutamate enzyme induction /repression lipid peroxide neural degeneration prostaglandin endoperoxide synthase amyloid protein tissue /cell culture enzyme activity oxidative stress neurotoxicology CRISP RPROJ NEW YORK G CRISP/99/AG14766-01A1 CRISP/99/AG14766-01A1 199904 eng NEURONAL RESPONSES TO AB-DERIVED ACTIVE LIGANDS Research KLEIN W EVANSTON NORTHWESTERN HEALTHCA, 1033 UNIVERSITY PLACE, SUITE 1, EVANSTON, IL 60201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 60201 Crisp Data Base National Institutes Of Health laboratory mouse biological signal transduction Alzheimer's disease molecular shape conformation histochemistry /cytochemistry ligand nerve /myelin protein neural degeneration glia neurotoxin protein structure /function amyloid protein sectioning cell line neuropathology CRISP RPROJ ILLINOIS G CRISP/99/AG15501-020003 CRISP/99/AG15501-020003 199904 eng HUMANIZED ANIMAL MODEL OF OXYRADICAL PRODUCTION Research RPROJ The brain has a limited repertoire of response to injury and disease. Microglial activation is the cornerstone of most of the brains protective responses which are usually associated with inflammatory processes. Aberrant microglial activation in Multiple Sclerosis (MS), stroke, severe head trauma and Alzheimer's Disease (AD) can directly contribute to neurotoxicity and tissue destruction through release of reactive oxygen species, proteases, cytokines and excitatory neurotransmitters. As one of the reactive oxygen species released from microglia and macrophages, NITRIC OXIDE has been reported to be toxic in some, and protective in other experimental paradigms of CNS disease. In peripheral tissues, the induction of nitric oxide release appears to contribute to rheumatoid arthritis and inflammatory bowel disease. Of the 3 different Nitric Oxide Synthases, the inducible form of NOS, known as iNOS or NOS2, is mainly found in microglia and macrophages. The induction of iNOS activity to release nitric oxide is highly regulated and is also regulated in a species specific manner. We have confirmed and extended this by finding that the amyloid-beta peptide (Abeta) associated with AD can stimulate iNOS activity in mouse cells, but fails to stimulate human cells to release nitric oxide. We also find that apolipoprotein- E, another gene associated with Alzheimer s, does stimulate human cells to release nitric oxide. To develop a better animal model of oxyradical in a fashion similar to that found in humans, we specifically propose to make a "Humanized-NOS2" transgenic mouse expressing only human iNOS2 enzyme. We have recently isolated a PAC clone containing the human NOS2 gene and we propose to insert this piece of genomic DNA into fertilized mouse oocytes by standard injection techniques and mate the resulting human-NOS2 transgenic mice lacking the mouse-NOS2 gene (aka. NOS2-knockout mice, Jackson labs Bar Harbor, ME). The net result will be a transgenic mouse which express human-NOS2 enzyme and whose regulation of nitric release, will be tested for its similitary to the human-specific pattern. Such a mouse model will have broad utility to more accurately predict the role played by oxyradicals in the development of human disease. VITEK MP DUKE UNIVERSITY MEDICAL CENTER, BOX 2900, DURHAM, NC 27710 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 27710 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal macrophage inflammation injection /infusion egg /ovum molecular cloning genome gene expression genetic promoter element reporter gene genetic regulation model design /development disease model neural degeneration microglia transposon /insertion element free radical oxygen tissue /cell culture animal breeding human genetic material tag nitric oxide enzyme activity nitric oxide synthase neurotoxicology CRISP RPROJ NORTH CAROLINA G CRISP/99/AG15609-01 CRISP/99/AG15609-01 199904 eng LYSOPHOSPHATIDIC ACID--ASTROCYTES AND NEURONAL DEATH Research RPROJ Neurodegenerative disorders of aging result from cumulative neuronal damage due to direct toxic effects on neurons plus indirect glia- associated effects. Events common to many neurodegenerative processes are injury due to reactive oxygen species (ROS), such as nitric oxide, NO, and mitochondrial dysfunction. The lipid biomediator lysophosphatidic acid (LPA) is likely to participate in neurodegenerative diseases since: i) LPA causes neuronal death, ii) ROS including NO, and mitochondrial dysfunction are important in LPA-induced neurotoxicity, iii) LPA-induced astrocyte responses can enhance neuronal death, e.g., LPA stimulates astrocyte expression of inflammatory cytokines, and iv) the synthesis of LPA is stimulated by neurodegeneration-associated events. Based on these published and preliminary findings, it is proposed that the production of LPA is stimulated by neurotoxic and inflammatory events, and, in turn, LPA is neurotoxic and inflammatory. To evaluate this hypothesis, the specific aims are to: 1. characterize the increase in LPA levels in neurons, PC12 cells and astrocytes in response to injury-related signals, and 2. define the LPA-induced increase in astrocyte production of cytokines and determine the effect of LPA on astrocyte NO synthesis. The stimulation of LPA synthesis by H2O2 treated PC12 cells, glutamate or H2O2 treated neurons and cytokine treated astrocytes will be characterized by metabolic labeling of cells with 32Pi, treatment of cells and then lipid extraction, chromatography and quantification. The LPA-mediated stimulation of astrocyte production of cytokines will be determined at the RNA (reverse transcription-polymerase chain reaction) and protein (ELISA assays) levels. The potential stimulation of NO synthesis in astrocytes by LPA will be assessed by nitrite analysis (Griess reaction). These studies should i) define neuronal injury-associated events resulting in LPA synthesis and LPA-induced astrocyte responses which can contribute to neuronal death, and ii) serve as foundation for future studies, including the elucidation the in vivo roles of LPA in neurodegenerative disorders. STEINER MR UNIVERSITY OF KENTUCKY, MS415 MEDICAL CENTER, LEXINGTON, KY 40536-0084 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory rat cell death polymerase chain reaction glutamate gene expression cytokine interleukin 1 interleukin 6 tumor necrosis factor alpha enzyme linked immunosorbent assay lipid biosynthesis neural degeneration astrocyte neuron hydrogen peroxide lysophospholipid phosphatidate phorbol nitric oxide PC12 cell neurotoxicology neuropathology CRISP RPROJ KENTUCKY G CRISP/99/AG15642-01 CRISP/99/AG15642-01 199904 eng PROSTATE CANCER--EXPOSURE AND DNA ADDUCTS Research RPROJ NO ABSTRACT AVAILABLE LANG NP UNIVERSITY OF ARKANSAS MED SCI, 4301 WEST MARKHAM, SLOT 725, LITTLE ROCK, AR 72205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 72205 Crisp Data Base National Institutes Of Health aminoacyltransferase mutagen genetic polymorphism cytochrome P450 human subject neoplasm /cancer genetics prostate neoplasm neoplasm /cancer epidemiology cancer risk chemical carcinogen male glutathione transferase sulfotransferase DNA damage human genetic material tag clinical research disease /disorder etiology testosterone 5 alpha reductase CRISP RPROJ ARKANSAS G CRISP/99/AG15722-02 CRISP/99/AG15722-02 199904 eng RISK PREDICTION MODELS AND APPLICATIONS TO BREAST CANCER Research RPROJ The long-term objective of our research is to develop statistical framework that will be used to translate the research findings to cancer prevention, diagnosis and management practices. The specific objective of this proposal is to develop a general framework for constructing risk prediction models (RPMs) for breast cancer that incorporates both environmental and genetic factors as well as family history, in the absence of genetic markers. The proposed framework can be used to develop RPMs for other cancers and possible other chronic diseases. This proposal has four specific aims: 1) to develop a general framework for constructing RPMs by incorporating known risk factors such as candidate genes, other biological markers, demographic variables, birth history, diet, history of medications, medical history and other lifestyle variables, which can be either time-independent or time-dependent; 2) to develop general framework for constructing RPMs by incorporating, (in addition to known risk factors described above), family history data as well as family data collected from family members; 3) to outline protocols on how to use such programs and to develop computer programs for all these new methodologies with a "user friendly" interface, which will be disseminated to the scientific community via the Internet; 4) to develop RPMs for breast cancer based on the data sets collected in the Breast Cancer Detection Demonstration Program and Cancer Steroid Hormone study (among other available data sets in the Fred Hutchinson Cancer Research Center), to compare the RPMs that are constructed on different dat a sets and to validate them across study populations. Further, we will attempt to validate the refined RPM for breast cancer via ongoing studies in the Center. This development is rooted in recent advances made in statistics, epidemiology, genetics and genetic epidemiology. Since most of the risk factors are varying with age and outcomes are generally ages of onset, the proportional hazard model has been generalized to include the birth history and two-stage models, and is chosen as a basic model for constructing RPMs. ZHAO LP FRED HUTCHINSON CANCER RES CTR, 1100 FAIRVIEW AVENUE NORTH, SEATTLE, WA 98109-1024 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health computer human interaction computer program /software case history statistics /biometry mathematical model environment related neoplasm /cancer neoplasm /cancer genetics breast neoplasm neoplasm /cancer epidemiology cancer risk family genetics human data Internet clinical research CRISP RPROJ WASHINGTON G CRISP/99/AG15749-02 CRISP/99/AG15749-02 199904 eng BENEFITS OF TESTOSTERONE ON STRENGTH IN ELDERLY MEN Research RPROJ DESCRIPTION (Adapted from the Applicant's Abstract): In this proposal, the effects of testosterone administration alone, or, in combination with exercise, on muscle function in elderly, sedentary men with testosterone deficiency will be investigated. Aging is associated with skeletal muscle sarcopenia and diminished muscle performance, which lead to significant disability from falls and loss of independence. One factor that may lead to sarcopenia is the presence of testosterone deficiency. Serum testosterone levels decrease progressively with age and the majority of elderly men have serum testosterone and free-testosterone levels below the mean for young men. Because testosterone deficiency may lead to alterations in body composition that include decreases in lean mass and strength, it is hypothesized that administration of testosterone therapy to older men with testosterone deficiency (relative to young men) may lead to improvements in muscle function and overall performance. In this proposal, 96 physically inactive elderly men with relative testosterone deficiency will be randomized in a double blind, placebo controlled fashion to receive testosterone enanthate therapy as intramuscular injections at a dose of 200 mg given every 2 weeks versus placebo for a total of 12 weeks. This study will test the hypothesis that administration of gonadal steroids will have a beneficial effect on lean muscle mass, muscle strength, and overall function. These men will be randomized further either to maintain the same level of physical inactivity or to undergo a graded resistance exercise program. This part of the protocol will test the hypothesis that the combined modalities of testosterone and exercise will have additive beneficial effects on muscle mass and function. The goal of these studies will be to determine the efficacy and safety of testosterone administration and, additionally, a home-based exercise program on muscle function in older men with relative testosterone insufficiency. KATZNELSON L MASSACHUSETTS GENERAL HOSPITAL, 55 FRUIT STREET, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 02114 Crisp Data Base National Institutes Of Health human old age (65+) testosterone clinical trial drug adverse effect hormone regulation /control mechanism hormone therapy exercise human subject muscle function male placebo human therapy evaluation age difference muscle strength clinical research CRISP RPROJ MASSACHUSETTS G CRISP/99/AG15882-01 CRISP/99/AG15882-01 199904 eng ALZHEIMERS DISEASE PREVENTION TRIAL WITH ESTROGENS Research RPROJ This is a randomized, double-blind, placebo-controlled clinical trial to determine if estrogen can delay the onset of Alzheimer's disease(AD) and reduce memory decline. 900 healthy, non-demented, women, 65 years of age or older, with a family history of AD will be recruited in 18 months from 3 different cities (New York, NY, Baltimore, MD and Jacksonville, FL) over an 18 month period using 4 distinct methods: 1) healthy non-demented female relatives of patients with AD seen at each of the 3 participating AD centers; 2) women with a family history of AD contacted through community service providers surrounding each medical center, 3) women with a family history of AD identified through advertisement using local media (newspaper, television and radio) and; 4) women with a family history of AD identified from a regional sample of female Medicare recipients provided by the Health Care Finance Administration (HCFA). At study entry, family history of AD in a first degree relative will be confirmed and each participant will have a physical, neurological, neuropsychological and functional assessment to insure the absence of dementia, any other degenerative neurological disease or potentially fatal disorder. Exclusions include a history of breast, uterine or ovarian cancer, history of arterial or deep vein thrombosis, a history of breast cancer in a first-degree relative. Randomization to estrogen, estrogen with progesterone or identical placebo among eligible women will be stratified by site and hysterectomy status (hysterectomized women will be randomized to unopposed estrogen or placebo; non-hysterectomized women to opposed estrogen or placebo); non-hysterectomized women to opposed estrogen or placebo). Analyses will combine opposed and unopposed estrogen treatments into a single group and compare them to placebo. Patients will be followed over a 3 year (36-month) period, and will be examined at 6 month intervals to assess compliance, adverse events and general health status. Annual complete medical, gynecological, neuropsychological and functional assessments will occur during follow-up. Outcome measures will include incident dementia and memory decline. We will use an intent-to-treat analysis from the primary analysis. Secondary analysis will examine potential co-variates. Safety evaluations will be based on finding from annual assessments and reported adverse events. Participants who become demented will be informed of standard-of-care treatment and will continue to be followed at annual intervals for the length of the study. SANO M COLUMBIA UNIV HEALTH SCIENCES, 630 WEST 168TH STREET, NEW YORK, NY 10032 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 10032 Crisp Data Base National Institutes Of Health human old age (65+) Alzheimer's disease clinical trial combination chemotherapy drug adverse effect hormone regulation /control mechanism hormone therapy estrogen electrocardiography human subject mental disorder prevention mammography longitudinal human study progesterone memory disorder mental disorder chemotherapy psychometrics hysterectomy female placebo therapy compliance human therapy evaluation family genetics clinical research outcomes research CRISP RPROJ NEW YORK G CRISP/99/AG15922-01 CRISP/99/AG15922-01 199904 eng ADVERSE DRUG EVENTS IN THE AMBULATORY GERIATRIC SETTING Research RPROJ The prescribing of a medication is the most common intervention employed by physicians in the care of geriatric patients in the office setting. However, older patients are at increased risk for drug-related iatrogenic injury due to age-related pharmacologic changes, the physiologic declines associated with aging, and the increased burden of chronic illness that characterizes the elderly patient population. In this prospective study, the occurrence of adverse drug events (ADEs) will be measured over a one- year period of observations in a large population of persons 65 years of age or older,, who are enrolled in the Fallon Community Health Plan, the first health maintenance organization in the United States to enroll Medicare beneficiaries and to accept reimbursement from HCFA on a prospective per capita basis. This study the emphasize the identification of preventable ADEs. Information on ADEs will be ascertained through computerized surveillance of pharmacy records and laboratory data,. reporting of drug-related events by healthcare providers, review of ambulatory and hospital medical records, and systematic interviews with physicians, nurse practitioners, nurses and pharmacists. This study will identify patient and healthcare provider factors associated with the occurrence of ADEs, as well as the underlying causes and systems failures that lead to their occurrences. Resource utilizations and costs associated with ADEs will also be assessed utilizing a nested case-control study design. The research will employ a multi-method approach which will include descriptive statistics and open-ended qualitative key informant interviews, utilizing structured interview instruments, to evaluate and prioritize systems failures that lead to preventable ADEs. The research design allows for the examination of factors relating to the occurrence of ADEs at the individual group, and organization level to gain insight into the causes of preventable events among ambulatory elderly patients. Information derived from this study will provide essential information to inform the design of interventions to reduce the risk for the occurrence of preventable ADEs in this vulnerable population. GURWITZ JH UNIV OF MASSACHUSETTS MED CTR, 55 LAKE AVENUE NORTH, WORCESTER, MA 01655 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 01655 Crisp Data Base National Institutes Of Health human old age (65+) aging drug adverse effect outpatient care patient professional relations health care service utilization health care cost /financing human subject longitudinal human study clinical research health services research tag CRISP RPROJ MASSACHUSETTS G CRISP/99/AG15979-01 CRISP/99/AG15979-01 199904 eng CHRONIC HEALTH PROBLEMS AND RETIREMENT Research RPROJ Chronic health problems play a fundamental role in individuals' retirement decisions and serve as the impetus for health-mandated disability exits. Despite the volume of empirical evidence on this association, almost nothing is known about the specific types of health conditions that prompt retirement and differentiate retirement from disability. Current understanding of the retirement-health relationship is based almost exclusively on global measures of health assessment such as "does health limit the amount or kind of work." Much less attention has been given to the nature of disease conditions that combine or progress to interfere with older workers' desire and ability to work. The proposed study is designed to redress this shortcoming. Our project has two primary aims. First, we propose to investigate how specific chronic health conditions, across a range of health domains, prompt retirement and disability exits. The analysis will focus on the effects of major fatal chronic diseases and other nonfatal diseases, conditions, impairments, functioning difficulties, and symptoms. Second, we propose to investigate how characteristics of the work environment accommodate or constrain the work ability of persons with chronic health conditions. Almost no research has evaluated how workers' retirement responses to specific chronic conditions differ according to the nature of their work. The theoretical framework takes into account the voluntary and involuntary responses (i.e., retirement and disability) that may be motivated by chronic health conditions. Workers with the same chronic conditions, but varying job demands, are presumed to face different obstacles to continued work activity. Some jobs will place few demands on workers with health problems, permitting them to continue working should they so desire. Other jobs will pose demands that exceed the individual's abilities to carry out the requisite tasks. As the job demands challenge and then exceed workers' abilities, we anticipate that workers' responses to chronic health problems will shift from retirement to disability. Panel data from three waves (covering four years) of the Health and Retirement Study will be used to estimate hazard models of the effects of chronic health conditions on the retirement and disability experiences of retirement-aged workers. PIENTA AM WAYNE STATE UNIVERSITY, 87 E FERRY, 224 KNAPP BLDG, DETROIT, MI 48202 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 48202 Crisp Data Base National Institutes Of Health chronic disease /disorder disabling disease occupational hazard retirement decision making sex difference functional ability work site human data CRISP RPROJ MICHIGAN G CRISP/99/AG16127-01 CRISP/99/AG16127-01 199904 eng AGING AND VULNERABILITY TO 6HYDROXYDOPAMINE Research RPROJ This application is from a new investigator and is aimed at research topic 16 - sensory and motor processing. The nigrostriatal dopamine (DA) system of the brain has been shown to play a major role in the control of movement. A gradual loss of nigrostriatal dopamine neurons occurs during normal aging in humans, and many elderly persons display one or more of the signs of Parkinson's disease without having the disease. This may indicate that older people that have parkinsonian signs may have a greater than normal loss of nigrostriatal DA. Although the cause for the normally occurring loss of midbrain DA cells in the elderly is not known, animal studies have indicated that the DA neurons of older animals may be more vulnerable to the effects of various neurotoxins. The purpose of the experiments in the present application is too determine if the nigrostriatal DA system of older animals is more sensitive to the neurotoxic effects of the dopaminergic toxin 6- hydroxydopamine (6-OHDA), and to begin to develop a model of the aging brain that is partially depleted of DA. It is hypothesized that the nigrostriatal DA system in older rats will be more sensitive to the neurotoxic effects of 6-OHDA than the nigrostriatal DA system of younger rats. The initial set of experiments will be to generate a dose- response curve in young adult animals for the DA-depleting effects of 6-OHDA. Male Fischer-344 rats (4 months old) will be given a single, unilateral injection of 6-OHDA into the right lateral ventricle. The doses examined will be 0, 50, 100, 150, and 200 mug of 6-OHDA per injection. Three weeks later, DA and metabolite levels will be measured in the striatum, nucleus accumbens, substantia nigra, and medial prefrontal cortex. Based on the results of these experiments, a dose of 6-OHDA that reduces striatal DA levels by approximately 50 percent in young adult rats will given to rats of three ages; 4 months, 18 months, and 24 months old. Three weeks later the rats will be anesthetized and in vivo electrochemistry experiments will be carried out on both sides of the brain to map out potassium-evoked overflow of DA, and subsequent clearance of DA, in the striatum. Following the experiments, post-mortem levels of DA and metabolites will be measured to determine the extent of whole tissue depletion by the 6-OHDA. The results of these experiments will help determine if the nigrostriatal DA system of the aging rat brain is more susceptible to the neurotoxic effects of 6-OHDA, and will begin to determine possible differences in presynaptic dopaminergic functioning in the aging versus younger brain in response to a neurotoxic insult. CASS WA UNIVERSITY OF KENTUCKY, 800 ROSE STREET, LEXINGTON KY 40536-0084 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING Crisp Data Base National Institutes Of Health aging laboratory rat nucleus accumbens substantia nigra corpus striatum brain metabolism drug administration rate /duration model design /development disease model developmental neurobiology dopamine 6 hydroxydopamine high performance liquid chromatography potassium channel neurotoxicology prefrontal lobe /cortex CRISP RPROJ KENTUCKY G CRISP/99/AG16368-01 CRISP/99/AG16368-01 199904 eng ISSUES OF SURVIVORSHIP AMONG BREAST CANCER SURVIVORS Research RPROJ This study proposes to development and evaluate a treatment protocol for- Shaping Cognitive Representations for Hypertensive Women. The high incidence of uncontrolled blood pressure (BP) in middle-aged African American women (AAW) who are being treated suggests chronic non-compliance with medical regimens. For some people, compliance is extremely difficult due to knowledge deficits, denial of need for treatment, inadequate access to health care and problems with interacting with health care providers. Studies have shown that individuals use enduring memories or cognitive representations (CRs) of beliefs, attitudes and intentions for behavior to structure knowledge for guiding decisions for behavior. CRs involve integration of perceptual stimuli for the purpose of appraising, coping, adapting, and learning. Research has not thoroughly examined whether cognitive representations of lifestyle behavior (CRLB) may be linked to non-compliance with hypertension (HTN) treatment. Preliminary data demonstrated that AAW manifested higher BP and more maladaptive cognitive representations of medication behavior (CRMB) than White American Women (WAW), and a theoretically-derived cognitive-behavioral intervention (CBI) may be effective in decreasing non-compliance and uncontrolled BP. The purpose of this proposed research is to test the effectiveness of a CBI in the management and modification of lifestyle behavior to improve responsiveness for HTN treatment among diverse populations. This pilot study addresses the following specific aims with measures at baseline and three months post intervention: 1) to evaluate the impact of a CBI on (a) compliance and (b) ambulatory BP measures (ABPM); 2) to evaluate the impact of a CBI on CRLB; 3) to assess subjects' perceived usefulness of a messages; and 4) to test whether the CBI has a greater impact on HTN treatment in AAW than WAW. A Solomon-four group design will be used to randomly assign women (N=28), stratified by racial identity, ages 55-70, with Stages I and II HTN recruited from ambulatory clinics to a CBI or standard medical therapy group. The CBI will require participants to perform a self-monitored in-home-learning program over 90 days. Outcomes that include compliance with life style behavior, CRLB, and ABPM will be measured at baseline and three months post intervention. Data will be analyzed using repeated measures ANOVA. Descriptive, parametric and non-parametric statistical procedures will be used to analyze the results and research hypothesis. PASKETT ED WAKE FOREST UNIVERSITY, MEDICAL CENTER BLVD, WINSTON-SALEM, NC 27157 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE ON AGING 27157 Crisp Data Base National Institutes Of Health telemedicine lymphedema postoperative complication menopause health care service utilization cardiotoxin human subject telecommunication interview breast neoplasm longitudinal human study psychosocial separation female reproductive system disorder female osteoporosis socioeconomics counseling design /development quality of life women's health clinical research long term survivor CRISP RPROJ NORTH CAROLINA G CRISP/99/AG16602-01 CRISP/99/AG16602-01 199904 eng T CELL ACTIVATION Research RPROJ Over the past several years, we have been studying two phenomena in cloned populations of CD4+ T lymphocytes referred to as costimulation and anergy. Both affect the production of the T cell growth factor interleukin-2 (IL-2) produced by these cells. Costimulation entails a 30 to 100-fold enhancement of IL-2 production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Anergy is an anti-proliferative state that the T cell enters when it only receives a signal through the antigen- specific receptor. In this case, subsequent stimulation of IL-2 production is inhibited 20-50-fold. Our goals are to try and understand the molecular mechanisms behind these two phenomena and to explore their relevance in vivo. During the past year we have made progress in four areas: 1. We discovered that the drug rapamycin, which inhibits certain signals transmitted through the IL-2 receptor, cannot only inhibit T cell proliferation but lead to a state of anergy, even in the presence of costimulation. This state resembles the anergy induced by T cell receptor occupancy in the absence of costimulation except that the inhibition of the production of other cytokines, such as IFN-g and IL-3, is more profound. 2. We have previously identified the ?180 region of the IL-2 enhancer as critical for an anergy effect on IL-2 production. We have now shown that three different transcription factor complexes from nuclear extracts of EL4 cells (a T cell tumor) can bind to this region: ATF2/cJun; CREB-1/CREM; and cJun/OCT. A fourth band detected on electrophoretic mobility shift assays was not identified. When we examined nuclear extracts from normal T cell clones, the dominant binding complex was CREB/CREM. This complex showed increased levels of binding after T cell receptor stimulation and most interestingly showed increased binding with extracts from anergic T cell clones even in their resting state. 3. We and others have previously shown that part of the effect of CD28 signaling that enhances IL-2 production is a stabilization of IL-2 mRNA. We have now shown using sequenced-tagged constructs containing deletions, that the 3? untranslated region of the mRNA is required for this effect. The CD28-induced stability is lost when the 3? UTR is deleted. It is also abolished if a nonsense codon is introduced into the first exon of the reporter. Finally removal of the 3? UTR revealed a small CD28-destabilizing effect on the mRNA which was controlled by sequences mapping between the middle of the third exon of the gene and the stop codon. 4. We previously set up an in vivo tolerance model in which a T cell receptor transgenic mouse on a RAG2 knock-out background (to eliminate endogenous T cell receptors) was injected three times with the superantigen staphylococcal enterotoxin A (SEA). We now have identified two separate mechanisms that are responsible for the inhibition of IL-2 production and T SCHWARTZ RH NIAID, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal T lymphocyte genetic enhancer element transcription factor leukocyte activation /transformation interleukin 2 staphylococcal enterotoxin T cell receptor clone cell anergy gel mobility shift assay superantigen CD28 molecule CRISP RPROJ A CRISP/99/AI00485-12 CRISP/99/AI00485-12 199904 eng TREATMENT OF CHRONIC HEPATITIS B VIRUS INFECTION Research RPROJ Recent advances in our understanding of the replicative mechanism of HBV, and the development of potent nucleoside analogues as clinically effective inhibitors of the HIV reverse transcriptase or herpesvirus polymerases has opened a new era in the treatment of chronic HBV infection. There is now a logical basis for combination therapy, because of the clear need for prolonged treatment and the associated possibility that drug resistant strains will emerge with monotherapy, but the choice of agents to combine and the regimens in which they should be employed remain uncertain. We have performed a preliminary study to assess the safety and to obtain preliminary information on the efficacy of the combination of alpha interferon with famciclovir. In this study, we treated patients with chronic HBV infection who had failed previous interferon alfa therapy, with an overlapping regimen of interferon alfa and famciclovir therapy totaling 20 weeks. This study is now closed. We are in the final stages of planning a multicenter, double blind, placebo-controlled study comparing the combination of famciclovir with lamivudine with lamivudine alone. The objectives of this study are to determine the safety, tolerance, pharmacokinetics, and preliminary efficacy of combination therapy over 48 weeks in a total of 40 patients with chronic HBV infection. This study is being developed in collaboration with the NIAID Collaborative Antiviral Study Group. STRAUS SE NIAID, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health clinical trial antiviral agent chronic disease /disorder drug administration rate /duration drug adverse effect drug screening /evaluation human subject interferon hepatitis B antigen HIV infection hepatitis B liver disorder chemotherapy virus DNA nucleoside analog longitudinal human study human therapy evaluation cytotoxicity hepatitis B virus group cytomegalovirus combination therapy clinical research CRISP RPROJ G CRISP/99/I00650-07 CRISP/99/I00650-07 199904 eng MOLECULAR AND CELL BIOLOGY OF PATHOGENIC MYCOBACTERIA Research RPROJ M. marinum The objectives of the reasearch described in this project are to use M. marinum as a model for identifying genes required for survival and replication of M. tuberculosis (MTB) within the macrophage as well as for identifying genes required for MTB mediated cytotoxicity In previous studies, we established that M. marinum like M. tuberculosis survives in a unique phagosomal compartment which does not fuse with the lysosome. In order to determine how mycobacteria are able to survive in macrophages, we devised a novel strategy for isolating gfp fusions to mycobacterial genes expressed differentially in the phagosomal environment. In this method, infected cells were lysed and the released phagosomes FACS-sorted to obtain bacteria containing fusions to genes expressed intracellularly. These were then assayed for expression on solid media to obtain populations of fusions differentially expressed intracellularly. Using this methodology we identified 12 fusions to genes which are differentially expressed in macrophages. DNA sequence adjacent to GFP showed homology with genes in M. tuberculosis.. A further analysis of these genes and identification of their products should provide insight into how mycobacteria survive within the macrophage. During the process of isolating GFP fusions, we identified a construct , GFP13, which contained a promoter expressed constitutively at least 5 fold better than HSP60, the mycobacterial heat shock promoter.. The identification of this very strong promoter could provide a very useful tool for achieving high level expression of mycobacterial genes. M. ulcerans. Infection with M. ulcerans, the causative agent of Buruli ulcer , results in a severe necrotizing skin lesion with very little acute inflammatory response. In order to understand how M. ulcerans causes disease, we have purified and characterized a toxin, MULT, from M. ulcerans,. Structural studies of MULT reveal that it is a complex polyketide, a 12- membered ring macrolide. Complex polyketides include a large number of potent bioactive molecules such as antibiotics (erythromycin), immunosuppressants (FK506), antifungals (amphotericin) and cytostatins (Bafilomycin). Although complex polyketides are common among Streptomyces species, MULT is the first complex polyketide, and first macrolide isolated from Mycobacteria species. We have characterized the biological activities of MULT using both in vitro tissue culture and in vivo studies. In pg amounts, MULT causes mouse fibroblasts to arrest in G1 of the cell cycle. More remarkably, intradermal injection of MULT into a guinea pig produces lesions pathologically identical to those of Buruli ulcer. SMALL PL NIAID, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health guinea pig Mycobacterium bovis Mycobacterium Mycobacterium tuberculosis systematic biology species difference host organism interaction gene expression bacterial genetics bacterial toxin virulence tissue /cell culture confocal scanning microscopy CRISP RPROJ G CRISP/99/I00671-06 CRISP/99/I00671-06 199904 eng ANTIGEN PROCESSING AND PRESENTATION IN THE INTESTINE Research RPROJ This project focuses on how antigens are processed in the intestine of mice. While it is clear that the outcome of oral antigen exposure can be either positive, i.e., the development of mucosal IgA responses, and in some cases the induction of systemic immunity as well, or negative, i.e., the induction of oral tolerance, the details of why one or the other outcome occurs is complex and poorly understood. While it is known that the antigen formulation, the presence of adjuvants, and the antigen dose, as well as genetic factors, can affect mucosal immune responses, how these act to influence immunity has never been established. Prior studies have established the presence of different antigen- presenting cell populations in the Peyer?s patch and lamina propria of the intestine. We have described the presence of at least two populations of dendritic cells (DCs) in the Peyer?s patch, which is the primary inductive site for mucosal immune responses. One population of DCs appears to be immature and poised for capture of antigens transported from the intestinal lumen into the Peyer?s patch as it is present in a dense layer just beneath the intestinal epithelium overlying the Peyer?s patch follicle in a region referred to as the subepithelial dome (SED). A second population of more mature cells is present in the major T cell regions of the Peyer?s patch, the interfollicular regions (IFR). These findings suggest that oral antigens are taken up by immature DCs and following migration and differentiation (or activation), these cells present antigens to T cells in the IFR. Another possibility is that such antigen-loaded DCs migrate to draining mesenteric lymph nodes where they act to induce primary T cell responses. A third population of DCs appears to be of the less mature phenotype and is present in the B cell follicles of the Peyer?s patch. How these different DCs act to present intestinal antigens is the focus of this project. The studies will entail the isolation and further characterization of DCs from the Peyer?s patch, mesenteric lymph nodes, and lamina propria. This will involve studies of surface phenotype by flow cytometry, cytokine production by RT-PCR or isolated and stimulated cells, and the ability to induce T cell differentiation in vivo. Finally, the ability of mucosal adjuvants such as cholera toxin, and different immunization regimens to affect DC antigen presentation, will be explored. Findings with DCs will be compared to antigen loading and phenotypic changes in other antigen presenting cell populations, such as B cells and monocyte/macrophages from intestinal lymphoid tissue. KELSALL B NIAID, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse dendritic cell Peyer's patches T lymphocyte cell differentiation flow cytometry polymerase chain reaction intestine cytokine antigen presentation cholera toxin immunomodulator mucosal immunity oral tolerance CRISP RPROJ G CRISP/99/I00833-01 CRISP/99/I00833-01 199904 eng NASAL STEROIDS VS IMMUNOTHERAPY IN ALLERGIC RHINITIS Research RPROJ DESCRIPTION: The project is based on the hypothesis that immunotherapy is a more potent treatment than intranasal steroids for allergic rhinitis because it can more profoundly alter the immune system and reach sites beyond the nose. A double blind, placebo-controlled study is proposed. The effects of immunotherapy and intranasal steroids on the nasal response to allergen will be compared by studying mast cell activation and distribution, cellular inflammation, and allergen-induced hyperresponsiveness. In addition, immunotherapy will be compared with intranasal steroids with regard to controlling all the symptoms of allergic rhinitis such as eye irritation and sinus and pulmonary problems. The candidate will also test the hypothesis that immunotherapy alters the T cell response of the nasal mucosa to allergen from the Th2 to the Th1 profile. BAROODY FM UNIVERSITY OF CHICAGO, 5841 SOUTH MARYLAND AVE MC1035, CHICAGO, IL 60637 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 60637 Crisp Data Base National Institutes Of Health pyroglyphid helper T lymphocyte mast cell inhalation drug administration drug adverse effect hormone therapy human subject airborne allergen respiratory hypersensitivity histamine leukocyte activation /transformation interleukin 2 immunotherapy immunocytochemistry rhinitis albumin computed axial tomography steroid human therapy evaluation interferon gamma clinical research CRISP RPROJ ILLINOIS G CRISP/99/AI01236-03 CRISP/99/AI01236-03 199904 eng VIBRIO CHOLERAE AS A VECTOR FOR INDUCING MUCOSAL IMMUNITY Research RPROJ This application details a two-part, five-year research career development program in mucosal immunity and mucosal vaccinology. The program will be conducted under the mentorship of Dr. Stephen B. Calderwood, Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA. The candidate is trained in Infectious Diseases and Tropical Medicine and has the long-term career goals of gaining expertise in vaccinology with special emphasis on mucosal immunity. The program will include sessions of instruction in laboratory techniques and courses in microbiology, molecular genetics and vaccinology at Harvard University. The research component of the program will focus on the development and evaluation of live, attenuated Vibrio cholerae to act as a delivery vehicle for heterologous antigens presented at mucosal surfaces. Three specific aims will be addressed: optimization of the in vivo expression of heterologous antigens by live, attenuated V. cholerae utilizing both constitutive and in vivo-regulated promoters; evaluation of the potential of attenuated, detoxified heat-labile enterotoxin of Escherichia coli (LT), expressed by V. cholerae vector strains, to act as an immunoadjuvant; and evaluation of the ability of live, attenuated V. cholerae vector strains to deliver heterologous antigens derived from specific microbial pathogens to mucosal surfaces. The applicant's progress and research will be overseen by the mentor and by an advisory committee of scientists at the Harvard Medical School and Massachusetts General Hospital. The program will allow the candidate to obtain the skills, knowledge and experience to become an independent biomedical researcher in mucosal immunity and mucosal vaccinology. RYAN ET MASSACHUSETTS GENERAL HOSPITAL, FRUIT STREET, GRAY 5, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02114 Crisp Data Base National Institutes Of Health laboratory rabbit hamster laboratory mouse Escherichia coli Vibrio cholerae active immunization immunologic preparation enterotoxin immunomodulator attenuated microorganism bacterial vaccine live vaccine mucosal immunity vaccine development vector vaccine CRISP RPROJ MASSACHUSETTS G CRISP/99/AI01332-03 CRISP/99/AI01332-03 199904 eng DIFFERENTIAL INDUCTION OF MUCOSAL IMMUNE RESPONSES Research RPROJ Mucosal infections, especially those inducing diarrheal diseases, are common causes of morbidity and death worldwide. We hypothesize that the pattern of expression of key cytokines and costimulatory molecules during the initial interaction between pathogen or vaccine and the mucosal immune system is a critical determinant of healing and immunizing outcomes. The hypothesis will be tested in a swine model of mucosal immunity. The use of swine in this model is especially relevant since swine health is important to protection of the human food supply, and because of interest in pigs as donors for human xenotransplantation. The hypothesis will be tested in two parts. In part I, two types of mucosal adjuvants, cholera toxin (CT) and avirulent Salmonella (AS), will be used in combination with an orally administered model antigen, keyhole limpet hemocyanin (KLH) [1] to determine the pattern of expression of cytokines and costimulatory molecules induced in gut associated lymphoid tissues (GALT) following oral administration of CT or AS; and [2] to characterize the immune response to KLH delivered alone or coadministered with CT or AS. In part II, the casual relationship between the initial cytokine environment and immune outcome will be investigated [3] to characterize the patterns of type 1 and type 2 cytokines produced by T cells isolated from pigs immunized with KLH alone or with CT and AS; and [4] to determine if the differential expression of IL-10,IL-12 and CD 80/86 in GALT macrophages induces type 1 or type 2 responses in cocultured T cells. The PI holds a B.S. degree in Veterinary Science and a D.V.M. from the University of Minnesota, College of Veterinary Medicine. After 10 yrs in veterinary practice, he now is in his 4th yr of the Ph.D. program in the Department of Veterinary PathoBiology, University of Minnesota. The proposal will support further training and research in the areas of molecular biology and immunology leading to a career in biomedical research. It will be conducted in the Department of Veterinary PathoBiology, University of Minnesota. The graduate program has an emphasis on molecular mechanisms of disease pathogenesis and protective immunity. Members of the department and advisory committee will provide a wealth of expertise relative to the successful completion of the proposed research. FOSS DL UNIVERSITY OF MINNESOTA, 1100 WASHINGTON AVE SUITE 20, MINNEAPOLIS, MN 55415-1226 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health swine Salmonella macrophage plasma cell T lymphocyte polymerase chain reaction histology delayed hypersensitivity cytokine interleukin 4 immunoregulation antibody formation cholera toxin hemocyanin biological model protein biosynthesis tissue /cell culture lymphocyte proliferation oral administration interferon gamma mucosal immunity interleukin 10 interleukin 12 CRISP RPROJ MINNESOTA G CRISP/99/AI01396-03 CRISP/99/AI01396-03 199904 eng GASTROINTESTINAL PATHOGENESIS OF HELICOBACTER PULLORUM Research RPROJ DESCRIPTION: The long term objectives of this project are to investigate the role Helicobacter pullorum in human gastrointestinal disease, to provide comprehensive training in molecular biology and in vivo models of pathogenesis for the candidate, and to prepare the candidate to become an independent investigator. H. pullorum is a newly described Helicobacter species which has been isolated from poultry and human patients with gastroenteritis. H. pullorum produces a novel cytotoxin originally discovered and characterized in H. hepaticus, which is distinct from the previously described vacuolating cytotoxin (VAC) found in other Helicobacter species, including H. pylori. Preliminary work indicates that this toxin may be in part responsible for the pathogenesis of H. hepaticus and by analogy, H. pullorum. The candidate proposes to purify and characterize the cytotoxin produced by H. pullorum. He then plans to isolate and characterize the gene encoding the cytotoxin from a library made from genomic H. pullorum DNA. Furthermore, the candidate proposes to develop a murine model for H. pullorum infection and to characterize the nature of the pathogenic changes caused by this infection. Through use of this animal model, the candidate plans to elucidate the role cytotoxin has in pathogenesis as well as determine other factors that contribute to the pathogenesis of H. pullorum. YOUNG VB MASSACHUSETTS GENERAL HOSPITAL, GRAY 5, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02114 Crisp Data Base National Institutes Of Health laboratory rabbit athymic mouse laboratory mouse bacterial cytopathogenic effect cellular pathology molecular pathology gastritis genetic library bacterial toxin disease model protein purification tissue /cell culture Helicobacter CRISP RPROJ MASSACHUSETTS G CRISP/99/AI01398-03 CRISP/99/AI01398-03 199904 eng ASTROCYTES AND HIV1 NEUROPATHOGENESIS Research RPROJ The candidate has extensive research experience and is now completing a fellowship in infectious Diseases at the University of Rochester during which he has undertaken research in the neuropathogenesis of HIV-1 infection in the sponsor's laboratory. The MCSDA will allow him to devote >90% FTE to realize his immediate goal of expanding the scope of his research in this area, under guidance of the sponsor and collaborators, and become expert in the field. It will be a crucial step towards the long-term goal of becoming a successful, independent, full time researcher in the field of HIV-1 neuropathogenesis. The career development plan includes mechanisms for acquiring the necessary knowledge base, critical thinking, technical, presentation, and mentoring skills, ethics, and development and extension of a research project, through the proposed research, seminar series, lab and national meetings and through collaboration with Dr. Gelbard. The research will be done in the laboratory of the sponsor, Dr. Stephen Dewhurst, who maintains a vibrant research environment with adequate laboratory space and equipment and easy access to all required facilities, within a single building at the University of Rochester Medical Center. Between one- and two-thirds of children infected with HIV-1 will develop manifestations of central nervous system (CNS) disease which is accompanied by neuronal apoptosis and decreased neuronal density in discrete areas of the brain. Since HIV-1 rarely, if ever, infects neurons directly, indirect mechanisms must be involved in neuronal death. It is proposed that changes in the neuroprotective and neurotrophic properties of astrocytes during HIV-1 infection contribute to neuronal death. SPECIFIC AIM 1 of this application is to test the effects of monocyte derived factors on neuroprotective functions of astrocytes. This will involve adding conditioned media (CM) from activated, HIV-1 infected macrophages or specific candidate neurotoxins, to fetal astrocyte cultures and testing astrocyte neuroprotective functions such as neurotrophin release and glutamate transport as well as toxin production such as nitric oxide (NO) synthesis. In addition, In vivo evidence suggests that astrocytes undergo persistent "restricted" infection with HIV-1 in which HIV-1 Nef is expressed in the absence of structural proteins. SPECIFIC AIM #2 is to explore the effect of "restricted" HIV-1 infection of astrocytes on astrocyte neuroprotective functions. Primary human astrocytes will be infected with HIV-1 or transfected with Nef expressing constructs and glutamate transport, release of neurotrophic factors, or NO will be measured. Understanding the role of astrocytes in HIV-1 neuropathogenesis, specifically, what changes occur in their neuroprotective functions, may suggest interventions that could prevent or treat CNS manifestations of HIV-1 infection. FINE SM UNIVERSITY OF ROCHESTER MED CT, 601 ELMWOOD AVE, BOX 689, ROCHESTER, NY 14642 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 14642 Crisp Data Base National Institutes Of Health aminoacid transport macrophage monocyte glutamate transfection neurotrophic factor HIV infection astrocyte neurotoxin embryo /fetus cell culture human immunodeficiency virus 1 nitric oxide neuroprotectant CRISP RPROJ NEW YORK G CRISP/99/AI01419-03 CRISP/99/AI01419-03 199904 eng MOLECULAR PATHOLOGY OF PHAGOCYTOSIS IN SEPSIS Research RPROJ DESCRIPTION: a. Career Development Plan: The candidate will spend 80% of her time performing research as outlined in the proposal. A formal course in Macromolecular and Cellular Structure and Chemistry is proposed. In addition, the candidate will attend seminars at Scripps, and weekly lab meetings where she will give about 4 presentations a year. b. Research Plan: Septic shock is a tragic complication of Gram-negative bacterial infections. Myeloid cells clear the body of hazardous invading Gram-negative bacteria and bacterial endotoxin (LPS). CD14, a 55 kD glycophosphatidylinositol (GPI)-anchored plasma membrane protein, found on all myeloid lineage cells is an important mediator of responses to LPS. Secreted LPS-binding protein (LBP) binds to LPS with high affinity and serves as a carrier protein that enables efficient binding of LPS to CD14. The long term goal of the proposal is to understand how CD14 participates in LPS uptake and the phagocytosis of Gram-negative bacteria by myeloid cells. Preliminary studies suggest that the GPI-anchoring of CD14 confers special properties to this receptor that determine uptake of LPS and phagocytosis by myeloid cells. One of the primary goals is to determine the contribution of the CD14/LBP-dependent pathway to LPS uptake and phagocytosis by myeloid cells using flow cytometry. In addition, the role, if any, of the GPI-anchor of CD14 in mediating the signal(s) necessary for LPS uptake and phagocytosis of LPS-bearing particles will be determined. Finally, the localization of GPI-anchored CD14 in the plasma membrane will be characterized by biochemical and immunological approaches. Information obtained from these studies will help answer important questions pertaining to CD14 and its role in host defense, and may contribute to the development of new strategies for the treatment of Gram-negative infections. SCHIFF DE SCRIPPS RESEARCH INSTITUTE, 10550 NORTH TORREY PINES ROAD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 92037 Crisp Data Base National Institutes Of Health gram negative bacteria myeloid stem cell calcium flux cell membrane phagocytosis flow cytometry electroporation transfection human tissue immunocytochemistry antigen antibody reaction CD antigen endotoxin lipopolysaccharide binding protein actin cell line glycosylphosphatidylinositol CRISP RPROJ CALIFORNIA G CRISP/99/AI01442-02 CRISP/99/AI01442-02 199904 eng MOLECULAR BASIS OF LUNG INJURY IN GBS PNEUMONIA Research RPROJ DESCRIPTION: This is an application by Victor Nizet, M.D. for a KO8 award for mentored studies under the direction of Craig Rubens, M.D., in the Department of Pediatrics, Washington University and the Children's Hospital and Medical Center, Seattle, WA. Dr. Nizet is an acting instructor in the Department of Pediatrics, Children s Hospital, Seattle, Washington. Dr. Nizet obtained his M.D. in 1989 from Stanford University. Dr. Craig Rubens will act as mentor. Dr. Rubens is Associate Professor of Pediatrics. He obtained a Ph.D. in microbiology from the Medical University of South Carolina in 1978 and M.D. from the University of Washington in 1982. Dr. Nizet will examine the role of beta hemolysin in group B streptococcal infections and its ability to cause cellular damage in the lung. His studies will focus on the identification of the genes involved in the regulation and structure of this lysin, and in vitro and in vivo pathogenesis studies using group B streptococci with knock-outs in these genes. The proposed studies are based on successful preliminary studies in which Dr. Nizet has made TN916, TN917, and chemical mutagenesis mutants that resulted in elevated and diminished levels of hemolysin. He has also prepared site directed mutants in group B streptococci to test the possibility that a known group B hemolysin might be the major group B hemolysin. He found that it was not. His "training plan" appears to primarily constitute protection of 85% of this time for research training. He proposes to develop molecular, genetic, and pathogenesis skills required for his studies. In particular he will learn techniques involved in cloning and sequencing DNA, preparing knock out mutants, conducting rigorous evaluation of the validity of molecular and genetic techniques, and applying them to in vitro and in vivo models of infection. NIZET VF UNIV. OF CALIFORNIA DIEGO, 9500 GILMAN DRIVE, DEPT 0672 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health newborn animal laboratory rat Streptococcus agalactiae hemolysin electroporation molecular cloning gene expression structural gene bacterial genetics mutant virulence disease model transposon /insertion element nucleic acid sequence protein purification bacterial pneumonia tissue /cell culture lung injury gene targeting CRISP RPROJ CALIFORNIA G CRISP/99/AI01451-03 CRISP/99/AI01451-03 199904 eng MOLECULAR ANALYSIS OF STAPHYLOCOCCAL EXFOLIATIVE TOXIN A Research RPROJ The research plan is to study Staphylococcal scalded skin syndrome (SSSS) caused by infection with exotoxin producing Staphylococcus. PLANO LR PEDIATRICS DEPT, MIAMI, FL 33136 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 33136 Crisp Data Base National Institutes Of Health laboratory mouse Staphylococcus aureus Staphylococcus infection B lymphocyte T lymphocyte cell type chemical structure /function disease /disorder proneness /risk electroporation host organism interaction cellular immunity cytokine western blotting staphylococcal exotoxin nucleic acid sequence receptor binding tissue /cell culture toxin metabolism age difference enzyme activity CRISP RPROJ FLORIDA G CRISP/99/AI01466-02 CRISP/99/AI01466-02 199904 eng BIOCHEMISTRY OF TOXIN A MEDIATED RHO GLUCOSYLATION Research RPROJ I am applying for this K08 award to help me fulfill my long-term career objective of become an independent investigator in the field of Infectious Disease. The K08 award will allow me the time to obtain, in a mentored environment, the knowledge base and experience I will need to realistically be able to make the difficult transition from clinician to physician-scientist. My interest in infectious diseases, pathophysiology, and biochemistry has lead me to the project I am proposing for this grant and my fellowship. Clostridium difficile is the major cause of nosocomial diarrhea in the United States. The disease appears to be the results of the action of one toxin animal models, toxin A. Toxin A's mechanisms of action are not entirely understood, but recent studies have identified the Rho family of small GTPases as intracellular targets of toxin A. Rho proteins are involved in regulation of the actin cytoskeleton and gene transcription. Toxin A glucosylates these proteins at a crucial threonine residue, thus inactivating them. The biochemistry of this reaction and the exact role it plays in the pathogenesis of the disease is unclear.We propose the following aims for this grant: 1) develop an in vitro assay for the toxin A-mediated Rho glucosylation; 2) characterize the biochemistry of the reaction; 3) develop inhibitors for the reaction. The inhibitors would then be used to probe the effect of blocking the glucosylation of the known sequella of toxin A exposure in culture cells and eventually could serve as a novel, non-antibiotic therapy for the disease. The environment at the University of Virginia and in the lab of my sponsor is ideal for my development as an independent researcher. My sponsor has expertise in small GTPases and toxin A research; there also are renowned researchers in diarrheal pathogenesis in the division, and through collaborations with outside researchers, from whom advice can be sought. There are numerous opportunities for course work to expand my knowledge base and excellent research facilities with all the necessary equipment needed to conduct my work. Most importantly, the division of Infectious Diseases and the university have a long tradition of fostering the environment necessary to develop competent academic physicians. CIESLA WP UNIVERSITY OF VIRGINIA, BOX 485, HSC, CHARLOTTESVILLE, VA 22908 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 22908 Crisp Data Base National Institutes Of Health glucose metabolism polymerase chain reaction enzyme inhibitor molecular cloning glycosylation glycosyltransferase cytokine bacterial toxin guanosinetriphosphatase glycoprotein biosynthesis posttranslational modification tissue /cell culture programmed cell death enzyme activity Clostridium difficile CRISP RPROJ VIRGINIA G CRISP/99/AI01494-01 CRISP/99/AI01494-01 199904 eng HERPES SIMPLEX VIRUS INHIBITION OF CTL INDUCED APOPTOSIS Research RPROJ The herpes simplex viruses (HSV) establish lifelong infection in their host. These viruses are thought to have evolved in parallel with their hosts, and therefore have developed intricate strategies for co-existing with the immune response. In preliminary studies for this proposal, I have demonstrated that HSV-1 inhibits the oligonucleosomal DNA fragmentation characteristic of apoptosis, including apoptosis induced by CTL. Since recent reports suggest that cells undergoing apoptosis are not suitable for viral replication, the induction of apoptosis may be a critical function for CTL control of viral infection. Inhibition of apoptosis would therefore promote viral replication. In contrast to its inhibition of DNA fragmentation, HSV has no effect on the membrane manifestations of apoptosis, such as phosphatidylserine exposure. In this proposal, the ability of HSV-2 to inhibit apoptosis will be evaluated. The HSV-1 and -2 genes mediating the anti-apoptotic effect will be identified, using compounds limiting HSV gene expression to individual transcriptional classes, followed by analysis of HSV deletion mutants. The cellular targets of each HSV anti-apoptotic gene will be identified using the yeast two-hybrid system. Finally, the ability of HSV to interfere with different apoptosis-inducing mechanisms of CTL will be determined using anti-Fas antibody and isolated perforin and cytotoxic granule components. The results of these studies will improve our understanding of HSV evasion of the immune response, and may suggest therapeutic strategies to circumvent this evasion. In addition, these studies will provide new probes and insight into the cascade of intracellular events following induction of apoptosis, especially the terminal effector events, since HSV inhibits nuclear but not membrane events of apoptosis. The work will also provide insights into how the manifestations of apoptosis vary depending on the inducing stimulus. JEROME KR FRED HTCHINSON CANCER RES CTR, 1124 COLUMBIA ST M-115, SEATTLE, WA 98104 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 98104 Crisp Data Base National Institutes Of Health genetic strain cytotoxic T lymphocyte host organism interaction genetic mapping molecular cloning genome gene expression virus genetics pore forming protein tissue /cell culture cytolysin herpes simplex virus 1 herpes simplex virus 2 programmed cell death yeast two hybrid system CD95 molecule CRISP RPROJ WASHINGTON G CRISP/99/AI01504-01 CRISP/99/AI01504-01 199904 eng TYPE III SECRETION IN PSEUDOMONAS Research RPROJ Pseudomonas aeruginosa is a leading cause of nosocomial infections and responsible for significant morbidity and mortality amongst hospitalized patients. Virulence of this bacterium in an animal model of acute pneumonia correlates with in vitro cytotoxicity towards polarized Madin- Darby canine kidney (MDCK) cells. Preliminary results suggest that the newly described type III secretion system of Pseudomonas is necessary for this cytotoxicity and that this system secretes three proteins in addition to exoenzymes S and T: a 73 kDa protein named PepA, a 40 kDa protein, and a 32 kDa protein. We hypothesize that these proteins are virulence factors and will further characterize them. The gene encoding PepA has already been sequenced, and the genes encoding the 40 kDa and 32 kDa proteins will likewise be cloned and sequenced. Mutagenesis studies will be performed to confirm that these proteins are indeed secreted via the type III pathway. Enzymatic assays and confocal immunofluorescence microscopy will be used to determine which, if any, of these proteins are translocated to the cytoplasmic compartment of MDCK cells. Mutants will be made using allelic replacement techniques and used to demonstrate which proteins are necessary for cytotoxicity and virulence in a mouse model of acute pneumonia. Examination of clinical isolates will be performed to determine which of these genes are variable traits and whether their presence correlates with virulence. Promoter fusions and immunoblots will be used to identify environmental conditions that induce expression of type III system genes and activate this secretion pathway. Finally, the role of pili in type III secretion will be examined. Together, these studies will aid in the understanding of the Pseudomonas type III secretion system and suggest targets for therapeutic interventions. Dr. Alan Hauser is in his 22nd month as a post-doctoral fellow in Dr. Joanne Engel's laboratory. He is dedicated to developing into as independent investigator in the field of bacterial pathogenesis. The experiments on Pseudomonas outlined in this proposal will complement his past work on Streptococcus and increase his exposure to numerous experimental techniques. Drs. Engel and Mostov have much experience in the fields of bacterial pathogenesis and cell biology. UCSF is one of the premier biological research centers in the country. HAUSER AR UNIV OF CALIFORNIA FRANCIS, 521 PARNASSUS AVE BOX 0654, SAN FRANCISCO, CA 94143 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 94143 Crisp Data Base National Institutes Of Health laboratory mouse Pseudomonas aeruginosa bacterial cytopathogenic effect secretion pilus molecular cloning fusion gene genetic regulation bacterial genetics gene mutation western blotting virulence nucleic acid sequence protein structure /function bacterial protein cytotoxicity confocal scanning microscopy MDCK cell CRISP RPROJ CALIFORNIA G CRISP/99/AI01524-01 CRISP/99/AI01524-01 199904 eng EXPANDING PREVENTING THERAPY FOR TUBERCULOUS INFECTION Research RPROJ NO ABSTRACT AVAILABLE JASMER RM SAN FRANCISCO GENERAL HOSPITAL, 1001 POTRERO AVENUE, SAN FRANCISCO, CA 94110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 94110 Crisp Data Base National Institutes Of Health rifamycin isoniazid Mycobacterium tuberculosis tuberculosis latent bacterial disease clinical trial communicable disease control antitubercular agent disease /disorder proneness /risk drug adverse effect drug screening /evaluation restriction fragment length polymorphism self care human subject longitudinal human study respiratory disorder chemotherapy human therapy evaluation chemoprevention clinical research CRISP RPROJ CALIFORNIA G CRISP/99/AI01549-01 CRISP/99/AI01549-01 199904 eng VECTOR COMPETENCE FOR LA CROSSE VIRUS IN AEDES Research RPROJ Aedes triseriatus, the treehole mosquito, is the primary vector of La Crosse virus (LAC) cause of the most common form of encephalitis in the midwest. Both control and monitoring of this mosquito are exceptionally difficult. Continued genetic and ecological research on vector competence are proposed, with the hope than an "Achilles heel" can be exploited. Research includes a sibling species, Ae. hendersoni (non-vector of LAC), Ae. brelandi, Ae. zoosophus and Ae. atropalpus. (1) Ability to transmit LAC differs greatly in different populations (10-90%). Oral, transovarial and venereal transmission will be analyzed genetically; virus-refactory strains with genetic markers will be developed. Studies on pathology (EM, FA), salivation rate, biting behavior and fecundity will be used to assess the cost of viral infection to the mosquito. Effect of environmental stress on transmission rate will be determined. (2) Studies on genetic structure involve further isolation of biochemical and morphological mutants, as well as improvement of linkage maps. Physiological traits (barriers to LAC infection, inability to diapause in the egg. resistance to drying) will be located on the linkage map. Electrophoretic analysis of clinical variation and sibling species differences will be compared via isozyme allele frequency. Effect of breeding system will be determined in inbred lines (F10 + of brother-sister mating). (3) Ecological studies will concentrate on population dynamics and productivity of mosquitoes that breed in discarded tires. Strains with rare isozyme alleles will be used to trace field dispersal. Since virtually nothing is known of field bionomics of Ae. hendersoni, we will study this species in the forest canopy via high towers. Tire productivity and adult dispersal of Ae. atropalpus will be followed. Finally, we will try to develop improved capture methods for adults. (5) Biological control laboratory experiments and field trials will be continued with predatory mosquitoes, Toxorhynchites sp. and Anopheles barberi. Control potential of a gregarine protozoan parasite of the larval midgut, Ascogrenarina, will be determined. Larval competition experiments will be undertaken with a female-sterile mutant of Aedes aegypti; this mutant absorbs the available food, causing Ae. triseriatus to starve. (5) Our Stock Center will continue to maintain 40 species and 180 strains and to furnish them to other laboratories. Finally Ae. hendersoni will be colonized (without forced-mating) for the first time. GRIMSTAD P UNIVERSITY OF NOTRE DAME, VECTOR BIOLOGY LABORATORY, NOTRE DAME, IN 46556 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 46556 Crisp Data Base National Institutes Of Health laboratory mouse biological polymorphism genetic strain circadian rhythm disease vector pheromone invertebrate endocrinology humidity season animal ecology host organism interaction isozyme sex sterilization field study genetic marker linkage mapping genetic registry /referral center mutation chromosome translocation animal population genetics radiation genetics Africa Central America North America diapause immunofluorescence technique electron microscopy filariasis malaria pest control insect control nonvisual photosensitivity electrophoresis epizootiology psychobiology reproduction virus replication Bunyaviridae Aedes Anopheles CRISP RPROJ INDIANA G CRISP/99/AI02753-39 CRISP/99/AI02753-39 199904 eng SECRETIONS OF ARTHROPODS Research RPROJ The study deals with the chemical mechanisms by which insects interact with organisms in their environment. It is aimed at elucidating the chemical structure and biological function of substances that insects use for defense against enemies, communication with mates, and protection of offspring. Molecular characterization will be effected by modern analytical techniques (GC- and HPL-chromatography; mass-and nuclear- magnetic spectrometry); biological studies will involve field and laboratory bioassay, structural and ultrastructural studies of glands, and various techniques for elucidation of physiological functions. Insects are major vectors of disease. An understanding of how insects depend on natural products for regulation of their biotic interactions is of broad applied significance to both medicine and agriculture. EISNER T CORNELL UNIVERSITY, ITHACA, NY 14853-1301 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Aves laboratory rat Insecta chemical structure /function mass spectrometry nuclear magnetic resonance spectroscopy pheromone animal ecology communication behavior physical /chemical interaction gas chromatography high performance liquid chromatography behavior sex behavior ethology predation arthropod poison insect poison behavioral /social science research tag CRISP RPROJ NEW YORK G CRISP/99/AI02908-40 CRISP/99/AI02908-40 199904 eng IN VITRO APPROACH TO PROBLEMS OF CLINICAL ALLERGY Research RPROJ DESCRIPTION (Adapted from Investigator's abstract): Over the last two decades this grant has supported studies showing that the then current therapy for insect allergy, whole body extracts, was not effective but that venom immunotherapy was effective. Venom immunotherapy became accepted in the United States (US) and worldwide. In the US, all adult patients with a history of anaphylaxis on insect sting and a positive venom skin test are treated. Recently, investigators in Holland found that if they sting challenge history positive, skin test positive patients, more than 70 percent have no reaction and, they contend, need no therapy. US studies found fewer non-reactors on re-sting. Neither the US nor the Dutch investigators have developed diagnostic parameters to predict which patients will or will not react on stings. The current application is based on the hypothesis that the Dutch study is, in part, correct and that it is unnecessary to treat as many patients as we do now. The investigators will repeat their work and address a flaw in that study - whether a single sting predicts the reaction to subsequent stings. It is known that in the early years of immunotherapy, IgG anti-venom antibodies correlate with clinical protection. It is hypothesized that this is an association, but not a causal one, and that clinical protection results from some other aspect of immunization. Using Fel d 1 peptides to immunize cat allergic individuals leads to a lesser immediate allergic response on exposure to cats. The investigators will immunize patients with peptides from antigen 5. Standard immunotherapy with venom leads to significant side-effects: approximately 50 percent of those treated have reactions ranging from a large local response to systemic anaphylaxis. Peptide immunotherapy does not cause immediate reactions, but in about 50 percent of patients, there is a late "anaphylactic-like" response, significantly milder in severity than the reactions to the allergen. The investigators hypothesize that these reactions are due to a HRF-like cytokine or one with direct anaphylactogenic properties. They have developed a protocol to ascertain the nature of this response. Finally, the major goal of these studies is to use the stings of more than 200 patients to develop parameters that predict which individuals will have a reaction to a sting. While this has not been accomplished before, this was due to stinging few unprotected individuals and the measurement of a limited number of parameters. It is now planned to study not only immunoglobulin levels but inflammatory cell activation, cytokine production, blood and urine histamine levels, and plasma tryptase and fibrinogen levels. LICHTENSTEIN LM JOHNS HOPKINS ASTHMA & ALRY CT, 5501 HOPKINS BAYVIEW CIRCLE, BALTIMORE, MD 21224 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21224 Crisp Data Base National Institutes Of Health lymphocyte fibrinogen blood chemistry urinalysis inflammation human subject hypersensitivity histamine leukocyte activation /transformation cytokine immunotherapy active immunization antibody formation antivenin protein biosynthesis insect poison tryptase CRISP RPROJ MARYLAND G CRISP/99/AI08270-31 CRISP/99/AI08270-31 199904 eng SHIGELLA FLEXNERI ENTRY INTO EUKARYOTIC CELLS Research RPROJ NO ABSTRACT AVAILABLE BLOCKER AJ EMBL, CELL BIO PROGRAMME, MEYERHOFSTRASSE 1, 69117 HEIDELBERG, GERMANY 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Shigella bacterial cytopathogenic effect host organism interaction bacterial toxin HeLa cell electron microscopy fluorescence microscopy gel electrophoresis actin cell line video microscopy CRISP RPROJ FRANCE G CRISP/99/AI09768-02 CRISP/99/AI09768-02 199904 eng REGULATION OF THE HEMOLYSIN GENES OF HAEMOPHILUS DUCREYI Research RPROJ Haemophilus ducreyi is the etiologic agent of chancroid. The current understanding of the pathogenesis of chancroid is limited. Recently, the genes for the hemolytic cytotocin of H. ducreyi have been cloned. Further, the hemolytic cytotoxin has been shown to be responsible for the cytopathic effect observed on human foreskin fibroblasts. The aim of this study is to address the factors responsible for the regulation of the hemolysin. In our preliminary studies, we have demonstrated that hemolysin is elaborated during log phase growth and is dependent on the concentration of heme in the medium. To more conveniently characterize the regulation of hemolysin expression, a H. ducreyi mutant will be constructed that contains a reporter gene, lacZ, under the control of the hemolysin promoter. Such a construct will allow a more efficient measurement of the transcription of the hemolysin gene cluster and will aid in defining what environmental factors regulate hemolytic activity. The hemolysin will be further characterized at the transcriptional level. mRNA will be measured to determined if the hemolysin genes are transcribed as an operon or as two separate transcripts. Furthermore, the start site of the single or both mRNA fragments will be mapped. Since virulence in chancroid is undoubtly a multifactorial process, an understanding of eh regulation of hemoloysin expression will lead to the identification of other co-regulated virulence determinants. BOZUE JA CHILDREN'S HOSPITAL RES FDN, 700 CHILDREN'S DRIVE, COLUMBUS, OH 43205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 43205 Crisp Data Base National Institutes Of Health Haemophilus hemolysin molecular cloning gene expression genetic regulation bacterial genetics mutant messenger RNA northern blotting southern blotting RNase protection assay CRISP RPROJ OHIO G CRISP/99/AI09813-02 CRISP/99/AI09813-02 199904 eng ANION BINDING BY DIPHTHERIA TOXIN REPRESSOR Research RPROJ Diphtheria toxin (DT) expression from corynebacteriophage beta is regulated in an iron dependent fashion by the chromosomally encoded diphtheria toxin repressor (DtxR) in C. diphtheriae. Under high iron conditions the DtxR homodimer binds to iron-regulated promoters (IRPs) and represses transcription, thus inhibiting expression of DT and corynebacterial siderophore. Homologs to DtxR have been identified in other Gram-positive bacteria, including Mycobacterium tuberculosis, indicating that DtxR-related repressors may also be involved in iron- dependent regulation of virulence factors in some other Gram positive organisms. Mutational and crystallographic studies showed that there are two metal binding sites in each DtxR monomer. X-ray diffraction data indicate that metal binding site 1 binds both a divalent cation and an anion such as sulfate or phosphate. A model based on these findings is that phosphate may serve as a co-corepressor for DtxR. Preliminary mutagenesis experiments indicate that substitution of an amino acid involved directly in coordination of the anion dramatically decreases the repressor activity of DtxR as measured by expression of beta-galactosidase from a tox operator/promoter-lacZ reporter construct in E. coli. The studies outlined in this proposal are designed to analyze the role of the anion both in metal binding by DtxR and in the signaling pathway for activating DtxR. This will be accomplished by mutagenesis studies of the anion ligands combined with DNA- and metal- binding assays and footprinting analyses with the purified mutant forms of DtxR. X-ray crystallography of the mutant DtxR molecules will be performed in collaboration with Dr. Wim Hol to determine the structural consequences of specific amino acid substitutions that alter DtxR function. These structural, genetic and biochemical analyses will provide new insights into the role of the cation-anion binding site in the activity of DtxR and its homologs as iron-activated global regulators in C. diphtheriae and other pathogens such as M. tuberculosis. SIEKIERKE JG UNIV OF COLORADO HLTH SCI CTR, 4200 E NINTH AVE, BOX B175, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80262 Crisp Data Base National Institutes Of Health Corynebacterium diphtheriae Escherichia coli chemical binding X ray crystallography site directed mutagenesis mutant diphtheria toxin anion DNA footprinting gel mobility shift assay CRISP RPROJ COLORADO G CRISP/99/AI10038-01 CRISP/99/AI10038-01 199904 eng NEMATODE MODEL FOR AERUGINOSA PATHOGENESIS Research RPROJ The bacterium, Pseudomonas aeruginosa, is capable of causing diseases in a wide range of plant and animal hosts. In particular, it is an opportunistic pathogen for humans and causes serious infections in individuals with Cystic Fibrosis, and those with other immunological deficiencies. Recent studies have shown that P. aeruginosa also behaves as a pathogen of the nematode, Caenorhabditis elegans. Specifically, the bacterium produces a toxin that quickly paralyzes the worm, leading to its death. Mutant worms can be isolated that are resistant to the toxin, and such mutations confer neuromuscular defects. The long-term objective of this study is to develop a model system for the study of host-pathogen interactions at the molecular genetic level. The proposed research addresses the following specific aims. 1) Two genetic screens are proposed to identify genes encoding the P. aeruginosa paralytic toxin. 2) The toxin will be purified and characterized by standard biochemical techniques. 3) The role of the Xcp protein secretion pathway in toxin production will be characterized. 4) Known C. elegans mutants with a variety of neuromuscular defects will be screened for resistance to the paralytic toxin. Such a screen should help elucidate the toxin's target and mechanism of action. COSMA CL PRINCETON UNIVERSITY, PRINCETON, NJ 08544 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 08544 Crisp Data Base National Institutes Of Health Caenorhabditis elegans Pseudomonas aeruginosa bacterial cytopathogenic effect mass spectrometry host organism interaction bacterial toxin disease model gel filtration chromatography protein purification bacteria infection mechanism CRISP RPROJ WASHINGTON G CRISP/99/AI10094-01 CRISP/99/AI10094-01 199904 eng IMMUNOBIOLOGY OF LY+ T-CELL SUBCLASSES Research RPROJ The proposed research is aimed at defining fundamental mechanisms responsible for the development and function of the CD4 T-cell subset. We propose studies to define the mechanisms of intrathymic commitment to the CD4 lineage, alternative signalling pathways coupled to TCR ligation of CD4 cells by superantigen or conventional antigen and the role of the CD4 molecule in regulating the response of these cells to TCR ligation. <SA1- Our approach to defining the mechanism of CD4 commitment uses thymocytes from mice deficient in a class II-dependent instructional signal. We propose experiments to test the hypothesis that committed CD4 transitional thymocytes are generated stochastically and require a CD4- dependent signal for further development. <SA2,3- We have obtained considerable evidence that two functionally distinct signal transduction pathways are coupled to the TCR using dual- reactive (superantigen/peptide) CD4 clones. We propose to further define the biochemical elements of the two pathways and to delineate the interaction between the TCR and the two types of ligand that may be coupled to the two signalling pathways. <SA4- We propose studies to extend our analyses of unresponsiveness in dual-reactive CD4 clones to the more physiological response of primary CD4 T-cells. One approach uses primary CD4 cells which express the 2B4 TCR transgene. These cells display the differential response phenotype after stimulation in vitro by superantigen (anergy) and peptide antigen (responsiveness) noted in earlier studies of dual-reactive CD4 clones. We will use this system to define the cellular and molecular mechanism of superantigen-induced unresponsiveness in primary CD4 cells. A second set of studies examines the mechanism of negative signalling by the CD4 molecule in primary CD4 T cells. We propose to define the contribution of the cytoplasmic (CYT) domain of the CD4 molecule to negative signalling in experiments which compare the regulatory effects of CD4- derived CYT domains with CD8-derived CYT domains. <SA5- Recognition of Staph enterotoxins (SEs) by CD4 T cells is usually associated with presentation of these ligands by class II products. We have recently identified a subgroup of SEs which represent exceptions to this rule. We propose studies to distinguish the potential antigen-presenting role of non-class II SEC/SEE receptors from the role of direct TCR ligation by these SEs. CANTOR HI DANA-FARBER CANCER INSTITUTE, 44 BINNEY STREET, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health laboratory mouse biological signal transduction thymus T lymphocyte cell differentiation polymerase chain reaction leukocyte activation /transformation monoclonal antibody antigen presentation CD4 molecule staphylococcal enterotoxin receptor binding antigen receptor T cell receptor tissue /cell culture MHC class I antigen MHC class II antigen anergy autoimmunity animal genetic material tag superantigen CRISP RPROJ MASSACHUSETTS G CRISP/99/AI13600-20 CRISP/99/AI13600-20 199904 eng CONTROL OF BACTERIAL TOXINS BY VIRUSES AND PLASMIDS Research RPROJ Our long term objectives are to understand molecular determinants of virulence in pathogenic bacteria, using both structural and genetic approaches to explore functions and regulation of representative bacterial toxins. We will study diphtheria toxin (DT) and heat-labile enterotoxins (LTs), which are major virulence factors of Corynebacterium diphtheriae and enterotoxigenic Escherichia coli (ETEC), respectively. Synthesis of DT is controlled by the diphtheria toxin repressor (DtxR), the prototype for a new class of bacterial global regulatory proteins that includes a recently discovered functional homolog of DtxR in mycobacteria. Information from such studies provides a basis for rational design of improved vaccines, immunotoxins, hybrid toxins, related biologics, and chemotherapeutic agents. Our specific aims for the next grant cycle are to: 1) Characterize the diphtheria toxin repressor. We will determine the crystal structure of DtxR by X-ray diffraction, characterize its functional domains, construct a defined DtxR-negative mutant of C. diphtheriae, and screen for DNA sequences homologous with dtxR in coryneform bacteria and other related bacteria. 2) Characterize DtxR- regulated promoter/ operator regions and genes of C. diphtheriae. We will clone a set of DtxR-regulated promoter/operators from C. diphtheriae, identify conserved features, and use site-directed mutagenesis to probe operator function. We will clone and characterize genes of C. diphtheriae that are regulated by these promoter/operators to identify the proteins, pathways, and functions included in the DtxR regulon. 3) Characterize the mycobacterial homolog of DtxR. We will purify the functional DtxR homolog from M. tuberculosis, determine its metal ion-binding and DNA-binding specificities, crystallize it, and determine its structure. We will use molecular modeling to design potential new chemotherapeutic agents that will enhance activity of the DtxR homolog and inhibit growth of M. tuberculosis and other pathogenic mycobacteria under low-iron conditions in vivo. 4) Characterize type II heat-labile enterotoxins of E. coli. We will determine crystal structures of LT-IIa and LT-IIb, compare their toxicities in several animal models, and test their immunogenicity and adjuvanticity in mice. We will develop improved vectors for construction of holotoxin-like chimeras and for efficient secretion of foreign proteins to the periplasm in E. coli and explore the use of these vectors and type II enterotoxins for development of new oral vaccines against pathogenic microbes. HOLMES RK UNIVERSITY OF COLORADO HLTH SC, 4200 EAST 9TH AVENUE, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80262 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Mycobacterium tuberculosis Corynebacterium diphtheriae Escherichia coli X ray crystallography site directed mutagenesis plasmid genetic operator element genetic promoter element bacterial genetics virus genetics bacterial toxin diphtheria toxin enterotoxin immunotoxicity virulence nucleic acid hybridization DNA footprinting nucleic acid sequence gel electrophoresis protein purification protein structure /function bacterial protein DNA binding protein CRISP RPROJ COLORADO G CRISP/99/AI14107-23 CRISP/99/AI14107-23 199904 eng CLINICAL TRIALS TO EVALUATE THERAPIES OF HIV DISEASE Research RPROJ The Clinical Research Program and the Treatment Research Operations Program of DAIDS conduct clinical trials to evaluate potentially effective therapeutic interventions against HIV infection and its sequelae. These trials are conducted through an academic-based consortium of medical institutions called the AIDS Clinical Trials Group (ACTG), as well as a community-based consortium called the Community Programs for Clinical Research on AIDS (CPCRA). The purpose of this contract will be to provide DAIDS with a third clinical trials mechanism, separate and distinct from both the ACTG and the CPCRA, which will enable DAIDS to rapidly address critical questions about therapeutic agents or innovative intervention approaches and to evaluate potentially effective therapies, which may fall outside the immediate priorities of the existing consortia. This contract specifically will not replicate the structure, functions, or research conducted through the ACTG and CPCRA systems; rather, it is expected to complement those important research activities. The contractor will provide personnel, materials, equipment and facilities to accomplish the following tasks: a. Provision of medical and statistical expertise for study design and interpretation b. Protocol development and review c. Provision of clinical trial sites and laboratory services d. Provision of an investigational drug repository and distribution service e. Site monitoring, site registration, preparation of INDs and other tasks required for regulatory compliance f. Provision of a biospecimen repository g. Implementation of clinical trials h. Data management and analysis services DURAKO S 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health clinical trial computer data analysis drug /agent drug adverse effect human subject AIDS AIDS therapy HIV infection antiAIDS agent statistics /biometry human therapy evaluation tissue resource /registry CRISP RPROJ MARYLAND C CRISP/99/AI15123-032 CRISP/99/AI15123-032 199904 eng MOLECULAR PATHOLOGY OF LPS INDUCED INJURY Research RPROJ It is estimated that septic shock causes 175,000 deaths annually in the United States. Gram-negative sepsis is a major cause of septic shock; the endotoxin (LPS) of Gram-negative bacteria is believed to be responsible for initiating the cellular responses leading to septic shock. The long term goal of the research proposed here is to define the pathogenic mechanisms of septic shock at the molecular level thereby identifying potential new targets for therapeutics. Cells of the myeloid lineage play a key role in the pathogenesis of septic shock because these cells are directly stimulated by LPS to express a multiplicity of new genes encoding proteins that include cytokines and other secreted proteins, cell surface proteins and enzymes that regulate the synthesis of small molecules participating in systemic inflammation. Our discoveries of LPS binding protein (LBP), of membrane-bound CD14 as a receptor on myeloid cells for LPS-LBP complexes and of a newly recognized MAP kinase family member, p38, activated after LPS binds to CD14 provides a new basis for understanding how LPS-induced myeloid cell activation occurs. Here we will test two hypotheses stemming from these discoveries; HYPOTHESIS 1: That the LPS receptor of myeloid cells is comprised of CD14 and (an) unidentified transmembrane protein(s) that after binding LPS (LBP-LPS complexes) enables cell stimulation by the activation of intracellular kinase cascades. HYPOTHESIS 2: That LPS binding to CD14 is a key event in vivo leading the production of mediators that cause septic shock. The first hypothesis will be addressed by in vitro experiments to identify the independent structural domains of mCD14 that are involved in binding LPS or in steps involved in cell activation after LPS binds to CD14 at the cell surface, to identify and characterize the function of additional proteins of the LPS receptor and to identify components the kinase cascade activated after LPS binds to CD14. These experiments will utilize approaches relying on immunologic, biochemical and recombinant DNA techniques. The second hypothesis will be tested by using anti-CD14 antibodies to evaluate the importance of LPS-CD14 interactions in several animal models of LPS-induced cellular and organ injury with direct relevance to septic shock. Implicit in all of these studies is the potential for development of new therapies to intervene in septic shock in man. ULEVITCH RJ SCRIPPS RESEARCH INSTITUTES, 10550 NORTH TORREY PINES ROAD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 92037 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse goat gram positive bacteria bacterial disease biological signal transduction blood toxicology chemical binding molecular pathology molecular cloning gene expression genetic library immunocytochemistry monoclonal antibody CD antigen in situ hybridization bacterial polysaccharide lipopolysaccharide protein structure /function binding protein proteolysis receptor binding septic shock CRISP RPROJ CALIFORNIA G CRISP/99/AI15136-20 CRISP/99/AI15136-20 199904 eng MOLECULAR BASIS FOR MICROBICIDAL ACTION IN LEUKOCYTES Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Research is proposed to: 1) use specifically designed probes to identify the toxins generated by stimulated phagocytes and to monitor their intracellular reaction. 2) characterize their oxidation-reduction chemistry in sufficient detail that their potential role in cellular reactions can be properly evaluated, and 3) investigate the microbicidal mechanisms of putative phagocyte-generated toxins on representative test organisms. The oxidants include HOCl, H2O2, ONO2- and secondary oxidants derived from them. Experiments will use stopped-flow kinetics, esr spin trapping and Raman spectroscopy to identify the reaction products and to characterize their behavior toward biological materials. Recent studies have demonstrated that lethal reactions of very reactive oxidants cannot be accurately assessed by in vitro microbicidal assay systems. Fluorescein-conjugated polyacrylamide beads which are capable of discriminating between chlorine-based oxidants, ONO2- ion, and carbon-based oxidants will be used to probe events in the phagocytic vacuole. The bactericidal mechanisms of these oxidants will be examined by the protocols used for HOCl-inflicted cell damage. The studies will focus on metabolic functions associated with the bacterial plasm membrane. Cellular studies will use control neutrophils, a macrophage cell line, and MPO-deficient and CGD neutrophils. HURST JK WASHINGTON STATE UNIVERSITY, PULLMAN, WA 99164-4630 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Escherichia coli bioenergetics leukocyte neutrophil glucose metabolism carbonate cell membrane vesicle /vacuole phagocytosis Raman spectrometry membrane potential Candida albicans Saccharomyces cerevisiae chlorine bactericidal immunity cellular immunity oxidizing agent oxidation /reduction free radical oxygen hydrogen peroxide stop flow technique tissue /cell culture toxin nitric oxide hydroxyl radical CRISP RPROJ WASHINGTON G CRISP/99/AI15834-16 CRISP/99/AI15834-16 199904 eng IRON AND O2 IN THE REGULATION OF P AERUGINOSA VIRULENCE Research RPROJ DESCRIPTION: Pseudomonas aeruginosa is a malicious opportunistic pathogen both in terms of the severity and the outcome of infections it causes. A significant proportion of patients with cystic fibrosis (CF) are coloized at an early age (1-2 yrs.) and many ultimately succumb to a chronic lung infection from P. aeruginosa. The reason for the extraordinary pathogenicity of P. aeruginosa in these patients, as compared to that of other Pseudomonas spp. is not clear. The myriad of virulence factors, alone or in combination, to even the simplest of P. aeruginosa infections has not yet been elucidated. Expression of virtually all the identified major virulence determinants in this organism is regulated by a variety of environmental conditions which the organism encounters at some point in its journey through the infected host. For example, iron levels undoubtedly play a role in the pathogenesis of P. aeruginosa infections. Production of specific virulence factors of this organism are induced in response to limiting amounts of iron a natural occurring environment in mammalian hosts. Some of these factors are crucial to acquisition of iron from the host. On the other hand P. aeruginosa must also limit its intake of iron to avoid oxidative damage from the iron catalyzed production of hydroxyl radicals. It is not completely clear how this intricate balance is maintained. In P. aeruginosa endocarditis, O2 tension has a significant influence on the outcome of this disease. On the right side of the heart O2 tension is 40 mm Hg lower than is on the left side. This variance contributes to the milder outcome of right side disease as compared to the much poorer prognosis associated with left-sided disease. In contrast to iron concentration, much less is known about the effects of O2 on the pathogenic potential of P. aeruginosa. Consequently further studies on this regulatory mechanism are clearly warranted. Finally, because O2 tension and iron levels in a given environment (e.g. human host) are exquisitely interrelated, analyses of their effects require integrated approaches. A more comprehensive understanding of iron and O2 response genes in the expression of virulence by this opportunistic pathogen could aid in the rational development of effective measures for the prevention and therapy of P. aeruginosa infections. VASIL ML UNIV OF COLORADO HLTH SCIS CTR, 4200 E NINTH AVENUE, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80262 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Pseudomonas aeruginosa opportunistic infection gene expression genetic regulation bacterial genetics gene mutation bacterial endocarditis exotoxin iron virulence disease model oxygen tension CRISP RPROJ COLORADO G CRISP/99/AI15940-19 CRISP/99/AI15940-19 199904 eng SHIGA FAMILY TOXINS-- RECEPTORS, ACTIONS AND VACCINES Research RPROJ In this proposal, the role and significance of the Shiga family of toxins in diseases due to Shigella (dysenteriae Type 1 and Escherichia coli producing the related "Shiga-like" toxins (or SLT's) will be studied. The health relatedness of this research is clear: Shiga family toxins play an important role in the intestinal manifestations of S. dysenteriae Type l and probably SLT-producing E. coli. They are the only common explanation of the strong association of these pathogens with severe and fatal hemolytic-uremic syndrome (HUS). In the U.S., HUS is primarily associated with SLT-producing E. coli acquired from hamburgers at fast-food restaurants, and is the most common cause of acute renal failure in children. There are three specific aims: 1) to study how toxin is internalized by susceptible cells and the relationship of this process to the glycolipid toxin receptor, globotriaosylceramide, using a intestinal cell lines in culture, 2) to study the impact of toxin, with or without lipopolysaccharide endotoxin, on endothelial cells in culture, or with in vitro reconstituted blood vessels designed to resemble the target organ, the renal glomerulus, on the production of biological mediators relevant to initiation and propagation of HUS; and 3) to use the techniques of molecular genetics to engineer a vaccine candidate based on development of neutralizing antibody to the non-toxic binding (B) subunit of the toxin. The last aim includes development of vaccines to deliver a protective antigen, and the study of an experimental oral cholera vaccine strain and a totally new approach to utilize the spore of Bacillus subtilis (a nonpathogenic heat and environmentally resistant microbial form) as vehicles to deliver antigen. The research utilizes cell biology to study toxin-receptor interactions and uptake mechanisms; cell physiology to study responses of endothelial cells and vascular smooth muscle cells to toxin and endotoxin challenge; and molecular genetics to clone and express toxin B-subunit in selected oral delivery systems for use as a vaccine. We will correlate in vitro and in vivo data to guide the research. At the end of the project we will know more about the mechanisms of toxin interactions with intestinal epithelial cells and vascular tissue and the pathophysiology of HUS, and we will develop strategies to block impending HUS and a vaccine candidate to prevent this problem. KEUSCH GT NEW ENGLAND MED CTR HOSPS INC, 750 WASHINGTON STREET, BOSTON, MA 02111 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02111 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Escherichia coli Shigella dysenteriae bacterial cytopathogenic effect membrane permeability hemolysis vascular smooth muscle cellular pathology drug design /synthesis /production host organism interaction bacillary dysentery colitis intestinal mucosa glycolipid immunocytochemistry antigen antibody reaction endotoxin exotoxin membrane structure virulence receptor binding tissue /cell culture bacterial vaccine vaccine development CRISP RPROJ MASSACHUSETTS G CRISP/99/AI16242-17 CRISP/99/AI16242-17 199904 eng LEISHMANIASIS--ALLOPURINOL TREATMENT Research MAGUIRE JH HARVARD SCHOOL PUBLIC HEALTH, 665 HUNTINGTON AVE, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health clinical trial antiprotozoal agent communicable disease chemotherapy allopurinol relapse /recurrence injection /infusion combination chemotherapy drug adverse effect drug screening /evaluation Brazil human subject antimony leishmaniasis Leishmania human therapy evaluation tropical medicine oral administration CRISP RPROJ MASSACHUSETTS G CRISP/99/AI16305-200016 CRISP/99/AI16305-200016 199904 eng DIPHTHERIA TOXIN RECEPTOR Research RPROJ DESCRIPTION (Adapted from the Applicants Abstract): Diphtheria toxin(DT) is the major virulence factor of the disease diphtheria. The recent reappearance of this disease in a number of countries of Europe, Asia, Africa, and South America, underscores it's continuing significance as a worldwide health problem. The long-term goals of this proposal are to characterize fully the cell surface receptor which DT utilizes to gain illicit entry into toxin-sensitive host cells and to understand completely the interaction between the toxin and its receptor.The investigators earlier discovered that DT binds to the transmembrane/precursor form of heparin-binding EGF-like growth factor (HB-EGF). They have recently identified the EGF-like domain of the cell surface of HB-EGF precursor as the region where the toxin binds and have shown that the soluble/mature form of HB-EGF inhibits binding of the toxin to the native toxin receptor on cells. The first specific aim is to determine the role of the cytoplasmic domain of the toxin receptor. The second specific aim is to characterize the functional interactions of the extracellular domain of the receptor with the toxin and with other receptor-associated proteins. The third specific aim is to characterize the toxin-binding site of the receptor employing a combination of site-directed mutagenesis and X-ray crystallography. The fourth specific aim is to produce transgenic mice bearing the HB-EGF precursor in order to determine whether this precursor can act an a DT receptor in vivo and to test the ability of recombinant mature HB-EGF to ameliorate intoxication by DT in this novel DT-sensitive experimental animal model. The results of this proposed project, in addition to extending our knowledge on toxin:receptor interactions, may provide a new kind of therapy for clinical diphtheria in which mature HB-EGF would replace equine horse anti-DT serum as the antidote of choice. EIDELS L UNIV TEXAS SOUTHWESTERN MED CT, 6000 HARRY HINES BLVD, DALLAS, TX 75235-9048 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal Corynebacterium diphtheriae X ray crystallography site directed mutagenesis transfection epidermal growth factor immunochemistry monoclonal antibody CD antigen diphtheria toxin microorganism immunology protein purification receptor binding receptor expression receptor bacterial toxicology toxin metabolism MDCK cell structural biology yeast two hybrid system CRISP RPROJ TEXAS G CRISP/99/AI16805-18 CRISP/99/AI16805-18 199904 eng SYNTHESIS OF ANTIINFECTIVE AGENTS Research RPROJ DESCRIPTION: (Principal Investigator's) The overall goals of this program are: (i) The development of advances in strategy and the discovery of enabling reactions of value in organic synthesis and (ii) the application of these findings to advance human health. While we continue our long term interest in the synthesis of naturally occurring antiinfectious agents, we also include cytotoxic antitumor agents, particularly where a novel mechanism of action has been inferred. Moreover, in this program we continue our quest to develop and evaluate new vaccine constructs to foster immunity against tumorgenesis and metastasis. Among the specific goal structures that we will attempt to synthesize are (i) class of marine derived natural products which can be classified as the eleuthesides (including eleutherobin, sarcodictyn and valdivone A) (eleutherobin has a demonstrated taxol like effect on the rate of microtubulin depolymerization), (ii) heliquinomycin and related structures such as rubromycin and purpuromycin (heliquinomycin is the first reported inhibitor of DNA helicase), (iii) saframycin B, an antibiotic with potent action against gram positive bacteria, (iv) yaoundamine B which is a potent antimalarial agen and (v) frondosin which is a potent PKC inhibitor and an inhibitor of IL-8 receptors. In addition, (vi) we continue our work on the design, synthesis and evaluation of new second and third generation constructs for evaluation as anticancer vaccines. The scope of this inquiry includes lipid conjugates to replace (or augment) KLH, clustered presentations to mimic antigen as presentations in mucins, and antigen analogs in the hope of triggering more aggressive immune responses. DANISHEFSKY SJ SLOAN-KETTERING INSTITUTE, 1275 YORK AVENUE, NEW YORK, NY 10021 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 10021 Crisp Data Base National Institutes Of Health antineoplastic antibiotic antimitotic biological product stereochemistry antiinfective agent antifungal agent drug design /synthesis /production immunosuppressive neoplasm /cancer vaccine CRISP RPROJ NEW YORK G CRISP/99/AI16943-20 CRISP/99/AI16943-20 199904 eng IMMUNOCHEMICAL STUDIES OF INSECT VENOM ALLERGENS Research RPROJ The most common type of insect sting allergy is caused by honey bees followed by vespids which include hornets, yellowjackets and wasps. Vespid venom contains three major allergens: hyaluronidase, phospholipase A1 and an antigen 5 of as yet unknown biological function. The cDNA and the protein sequences of homologous antigen 5s and phospholipases A1 from several vespids are known and the sequences of homologous hyaluronidases are being investigated. Having the sequence data of families of homologous vespid venom proteins can facilitate the structure-function analysis of these allergen molecule. The first aspect of the proposed work is to map the major B and T cell epitopes of vespid venom allergens. The epitopes will be mapped by testing various fragments of venom allergens ford their binding of venom-specific antibodies, and for their stimulation of venom-specific T cells. The necessary fragments will be prepared by recombinant technology and/or chemical degradation of natural or recombinant venom proteins. The second aspect of the proposed work is to study whether there is antigenic cross reactivity of vespid antigen 5s with mammalian testis proteins and vespid phospholipases with mammalian lipases as these proteins are found to have sequence similarity.The third aspect of the proposed work is to study tolerance induction in mice with T and B cell epitope peptides of venom allergens. These studies will be made first with the bee venom allergen melittin as this 26-residue peptide has one major epitope each for the T and B cells. Preliminary findings suggest that one melittin analog can suppress antibody response to melittin in mice. A knowledge of the B and T cell epitopes of vespid venom allergens will help to clarify the common clinical observation of multiple sensitivity of insect allergic patients which can be due to exposure to different vespids and/or cross reactivity of vespid allergens. Data on epitopes of different allergens can help our understanding of what makes an allergen. If tolerance can be induced efficiently in vivo with T and B cell epitope peptides, this will be a useful alternative to the standard immunotherapy with allergen extracts. KING TP ROCKEFELLER UNIVERSITY, 1230 YORK AVENUE, NEW YORK, NY 10021 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 10021 Crisp Data Base National Institutes Of Health laboratory mouse laboratory rat Hymenoptera B lymphocyte helper T lymphocyte polymerase chain reaction hypersensitivity allergen cross immunity immune tolerance /unresponsiveness immunochemistry monoclonal antibody epitope mapping lipase phospholipase A2 protein sequence T cell receptor tissue /cell culture MHC class II antigen insect poison lymphocyte proliferation recombinant protein CRISP RPROJ NEW YORK G CRISP/99/AI17021-18 CRISP/99/AI17021-18 199904 eng BORDETELLA CYCLASE--STRUCTURE AND BIOLOGICAL ACTIVITY Research RPROJ DESCRIPTION: Adenylate cyclase (AC) toxin is a novel protein bacterial toxin produced by Bordetalla pertussis, the causative agent of whooping cough. The sequence of this toxin is known and palimiolyation of a single Lys residue has been demonstrated to be essential for toxin activities. Nevertheless, little is known about mechanisms for toxin-mediated processes or their functional interrelationships. This proposal will focus on the individual steps involved in AC toxin action. Assays will be adapted or developed to enable the isolation and characterization of each individual step in the pathway. The methodologic approaches will include use of intrinsic tryptophan fluorescence fluoresecence energy transfer, lipid bilayer and whole cell patch-clamp conductance, electron microscopy and x-ray diffraction. The functional studies will be complemented with th use of random and site-specific mutagenesis to generate mutants of AC toxin that are defective in toxin and/or hemolysin activities. Similarly, an in vitro acylation will provide a mechanism for preparation of toxins with other than palmitoyl groups on LYs983, in order to determine the role of acylation. Additional structural data will be collected with CD spectroscopy an x-ray diffraction studies to develop a better understanding of the actions of AC toxin. HEWLETT EL UNIVERSITY OF VIRGINIA, BOX 419, SCHOOL OF MEDICINE, CHARLOTTESVILLE, VA 22908 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 22908 Crisp Data Base National Institutes Of Health acylation laboratory mouse Bordetella pertussis low angle X ray diffraction analysis patch clamp host organism interaction enzyme mechanism site directed mutagenesis bacterial toxin virulence adenylate cyclase electron microscopy protein structure /function CRISP RPROJ VIRGINIA G CRISP/99/AI18000-17 CRISP/99/AI18000-17 199904 eng GENETIC ANALYSIS OF TOXINOGENESIS IN VIBRIO CHOLERAE Research RPROJ The primary goal of the proposed investigation is the detailed elucidation of the molecular aspects of toxin regulation in V. cholerae. Biochemical and genetic methods will be used to locate the DNA sites involved in formation of the cholera toxin operon (ctx) promoter and control sites. We will attempt to define the precise molecular mechanism by which the positive regulatory gene toxR, activates ctx transcription. The regulatory role played by the repetitive sequence TTTTGAT will be studied by localized and mismatch primer mutagenesis. The nutritional and physical factors which regulate toxin production will be correlated with specific transcriptional effects on ctx or toxR. We will continue to study ctx amplification as a genetic mechanism controlling toxin production in E1 Tor strains of V. cholerae. In this regard, we will test the repetitive sequence RS1 for its ability to mediate various recombinational events including transposition, duplication and site-specific recombination. The knowledge gained from all the above studies will be used to devise methods with which to study toxin regulation and ctx amplification in vivo. Specifically, we will study how the intestine provides a selective environment for increased toxin expression by defining which genes (ctxA, ctxB or toxR) are necessary for in vivo selection of genetic reversion and amplification events. We will apply what we have learned about ctx regulation and expression to the development of improved live and dead, oral vaccines against cholera. Mutations will be constructed in the cloned recA gene of V. cholerae and then recombined back in place of the rec+ gene of the prototype live oral vaccine strain 0395-N1. In addition to recA, streptomycin-dependent, DAP- and ga1E mutations will also be incorporated into 0395-N1 derivatives as a means of limiting the persistence of this strain in humans and the environment. Finally, derivatives of 0395-N1 which are suitable for use as a dead, oral cholera vaccines will be constructed which hyperproduce the B subunit of the toxin in both a secreted and nonsecreted form. MEKALANOS JJ MICRO AND MOLECULAR GENETICS, 200 LONGWOOD AVENUE, D1-515, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Escherichia coli Salmonella typhimurium Vibrio cholerae biological signal transduction chemotaxis genetic manipulation site directed mutagenesis molecular cloning genetic promoter element genetic transcription bacterial genetics mutant cholera toxin nucleic acid repetitive sequence DNA directed RNA polymerase protein sequence protein structure /function DNA binding protein chimeric protein cholera vaccine recombinase CRISP RPROJ MASSACHUSETTS G CRISP/99/AI18045-18 CRISP/99/AI18045-18 199904 eng INTERACTIONS OF ENDOTOXIN WITH PLASMA AND CELLS Research RPROJ Endotoxins (gram-negative bacterial lipopolysaccharides, or LPS) play a major role in the pathogenesis of invasive gram-negative bacterial diseases; the inflammatory reaction elicited by these molecules may produce great morbidity and even death. Little is known about host mechanisms for recognizing and modulating the toxic signal information in LPS. During the past decade we have purified, characterized, and cloned a human leukocyte enzyme, acyloxyacyl hydrolase (AOAH), that selectively removes certain acyl chains from the lipid A region of lipopolysaccharides, thereby detoxifying the LPS. Although LPS detoxification is the likely function of the enzyme in animals, recent findings suggest alternative roles such as LPS signal transduction and phospholipid turnover. We now would like to build on our previous work to determine the biological role(s0 of acyloxyacyl hydrolase in human neutrophils and macrophages, and to lay groundwork for subsequent in vivo uses of the enzyme. The proposed research will (1) use site-directed mutagenesis to determine structure function relationships for AOAH and to produce mutated enzyme to use in the subsequent studies, (2) determine the functions of AOAH in human macrophages and optimize the uptake of exogenous AOAH by these cells, (30 determine the fate and LPS-deacylating ability of exogenous recombinant AOAH in mice, (4) localize AOAH in neutrophils and macrophages and evaluate its intracellular movement in response to various stimuli, and (5) clone rabbit AOAH CDNA to use for in vivo studies of AOAH synthesis and regulation. These studies should greatly clarify the roles played by this novel enzyme and provide a solid scientific foundation for its possible uses in the prevention and therapy of gram-negative bacterial diseases (endotoxic shock). Useful new information will also be generated about cellular uptake mechanisms and the intracellular fate of LPS, a bacterial toxin that is encountered continuously by animals, including man. MUNFORD RS U TEXAS SW MED CTR AT DALLAS, 5323 HARRY HINES BOULEVARD, DALLAS, TX 75235-9113 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse macrophage neutrophil site directed mutagenesis molecular cloning hydrolase hydroxyl group endotoxin microorganism immunology phospholipase A2 lysophospholipase lysophospholipid lipopolysaccharide detoxification acyltransferase enzyme activity CRISP RPROJ TEXAS G CRISP/99/AI18188-18 CRISP/99/AI18188-18 199904 eng VIRULENCE FACTORS IN THE PATHOGENESIS OF SALMONELLOSIS Research RPROJ The overall objective continues to be the probing of the pathogenic mechanism of experimental salmonellosis to define the role of Salmonella enterotoxin (Stn) as a selected virulence factor in the intestinal phase of Salmonella infection. Our past research has enabled us to clone a fragment of Salmonella chromosomal DNA, encoding the stn gene. When expressed with the T7 RNA promoter/polymerase system, cell-free lysates of E. coil, containing Stn, evoked enterotoxic responses in rabbit intestinal loops. We determined the molecular structure of cloned Stn, based on predictions from the nucleotide sequence, which allowed us to localize the stn gene at approximately 90 min on the Salmonella chromosome opposite the hydHG operon. We used site-directed mutagenesis to identify the stn initiation codon, which was a "TTG" rather than the typical "ATG." Further, the unusually high pI of the protein (11.7) confers a strong negative charge on the toxin's surface, causing it to bind to positively charged molecules at pH 7.0, a property that has complicated Stn purification. Our Specific Aims seek to construct an isogenic stn derivative of a virulent Salmonella strain(s) (e.g., TML- R66), for use in determining the precise role of Stn in the pathogenic mechanism (i.e., secretion and/or invasion). Precautions will be taken to avoid secondary alterations in hydHG function. Further, we are striving to improve gene expression and Stn solubility by subcloning the stn gene into selected fusion protein vectors. Total Stn antigen was, increased 64 fold by preparing two fusion proteins [glutathione S- transferase::Stn (Gst::Stn) and thioredoxin A::Stn (TrxA::Stn], and TrxA::Stn increased Stn solubility by 50 fold. With improvements in gene expression of Stn, we anticipate that purification to homogeneity is achievable. The purified protein will be used in experiments to study the molecular mechanism of the enterotoxin in elevating cAMP and PGE2 levels in intestinal cells, as well as to generate a polyclonal antiserum to Stn. Finally, stn genes from selected Salmonella isolates, that differ in intestinal virulence (e.g., fluid accumulation), will be amplified by PCR so that the nucleotide sequence of each can be examined and compared. The proposed studies should provide new and helpful information about the pathogenesis of salmonellosis. PETERSON JW UNIV OF TEXAS MEDICAL BRANCH, GALVESTON, TX 77555-1019 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory rabbit Escherichia coli Salmonella Salmonella typhimurium genetic strain polymerase chain reaction molecular pathology pathologic process prostaglandin E salmonellosis site directed mutagenesis molecular cloning fusion gene gene expression ADP ribosylation antibacterial antibody bacterial antigen enterotoxin virulence bacterial DNA nucleic acid sequence protein purification G protein cyclic AMP glutathione transferase CRISP RPROJ TEXAS G CRISP/99/AI18401-15 CRISP/99/AI18401-15 199904 eng CHARACTERISTICS OF T-CELL RECEPTORS Research RPROJ This application is designed to study the factors which control the repertoire of alpha/beta T cell receptor (TCR) bearing T cells in mice. Contact with self antigen causes the death of immature T cell precursors. Contact with antigen can have several outcomes for mature T cells, however, including death, inactivation, or full blown response. The experiments in this application will investigate what determines which of these outcomes occurs. Variables to be studied will include receptor occupancy and the strength of the T cell response. Although positive selection, the fact that T cell precursors are selected to mature only if their receptors will be able to recognize antigenic peptides in association with self major histocompatibility complex (MHC) proteins has been known to occur for some years, the mechanisms whereby this phenomenon operates are not understood. It is thought that positive selection involves low affinity reactions between TCRs on developing T cells and MHC on thymus cortical epithelial cells. The experiments in this application will test this hypothesis and will also examine the idea that non-MHC, non-TCR background gene products help raise the avidity of the reaction between the developing T cells and its TCR and MHC on target thymus epithelial cells. One reason why progress in understanding of positive selection has been slow in that there is no easily manipulable experimental system for study of the problem. A cell line capable of inducing positive selection would, for example, be extremely useful. So far cell lines of this type have not been identified in in vitro tests, perhaps because no single cell line can provide all the factors needed for thymocyte growth and maturation. Experiments described in this application will test thymus epithelial cell lines for their ability to participate in positive selection in whole thymuses. Many autoimmune diseases are driven by autoimmune T cells, cells which have somehow inadvertently broken through the processes of self tolerance induction. The studies in this application are designed to test why this might occur, and what can be done about it. MARRACK PC NATL JEWISH CTR FOR IMMUNOLOGY, 1400 JACKSON STREET, DENVER, CO 80206 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80206 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal thymus T lymphocyte flow cytometry antigen presenting cell immune tolerance /unresponsiveness antigen presentation staphylococcal enterotoxin receptor expression T cell receptor tissue /cell culture lymphocyte proliferation programmed cell death CRISP RPROJ COLORADO G CRISP/99/AI18785-17 CRISP/99/AI18785-17 199904 eng CELLULAR AND MOLECULAR MECHANISMS FOR MUCOSAL IMMUNITY Research RPROJ DESCRIPTION (Adapted from Investigator's abstract): This investigator has recently provided evidence that oral delivery of protein antigens to mice, in concert with the adjuvant cholera toxin (CT), induced T helper type 2 (Th2) cells in Peyer's patches and in spleen, and resulted in mucosal IgA and serum IgG1 and IgE antibody isotypes characteristic of Th2-type responses. A major goal of this renewal application will be to define the molecular basis for Th cell-mediated and humoral B-cell responses to well defined oral immunogens. Despite intensive recent study, the relative contributions of Th1-type and Th2-type cells and derived cytokines for mucosal IgA and systemic antibody or cell-mediated immune (CMI) responses are not well understood. This is due in part to our incomplete understanding of the contributions of major antigen-presenting cells in the Peyer's patches, e.g., macrophages (MO), dendritic cells (DC), and class II+ B-cells for the induction of Th1/Th2 cells for IgA responses. Further, the underlying mechanisms for signal transduction pathways in mucosal Th1 and Th2 cells has never been addressed. In this renewal application, the first specific aim will use the adjuvants CT and E. coli labile toxin (LT) versus rIL-12 and rSalmonella expressing proteins to establish the precise role of Th2 and Th1 cells for mucosal immune responses, respectively. Unique CD4+ Th cell clones will be derived from both normal and cytokine knockout (IFN-g -/-, IL-4 -/-, IL-6 -/- and IL-10 -/-) mice for study. The relative roles for MO, DC or class II+, surface IgA- positive (sIgA+) B-cells in Peyer's patches, for induction of CD4+ Th cells and the role of second signals in T-cell activation will be given emphasis in the second aim. Construction of an antigen-specific sIgA+ B-cell hybridoma expressing class II and development of IgG-versus IgA B-cell anti-ovalbumin transgenic mice will facilitate this effort. The third aim will define the signal transduction pathways involved in mucosal activation of Th1-type versus Th2-type cells. The fourth aim will focus on cytokine-specific gene promoters and their mRNA expression in Th cell subsets which result from an adjuvant-induced mucosal response. The final specific aim will focus on the role of Th2 cells and their cytokines for regulation of mucosal immunity in vitro and in vivo. These studies will take advantage of alpha/beta TCR -/- mice, which lack mucosal IgA plasma cells, as adoptive hosts for CD4+ Th cell clones for analysis of antigen-specific mucosal IgA responses. MCGHEE JR UNIV OF ALABAMA, 845 19TH ST SOUTH, BIRMINGHAM, AL 35294-2170 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal biological signal transduction dendritic cell lymphatic tissue Peyer's patches macrophage B lymphocyte helper T lymphocyte hybridoma flow cytometry polymerase chain reaction genetic promoter element immunoglobulin A cellular immunity leukocyte activation /transformation cytokine humoral immunity bacterial toxin tissue /cell culture MHC class II antigen mucosal immunity CRISP RPROJ ALABAMA G CRISP/99/AI18958-16 CRISP/99/AI18958-16 199904 eng DEVELOPMENT OF A LIVE ORAL CHOLERA VACCINE Research RPROJ DESCRIPTION (Adapted from applicant's abstract): An ideal vaccine for the prevention of cholera is not yet available. Previous work by the investigator resulted in development of an attenuated live oral cholera vaccine, V. cholerae CVD103-HgR. This vaccine confers strong protective immunity against experimental challenge with virulent V. cholerae O1 after a single dose. However, this vaccine is limited in that it does not protect against the El Tor biotype as well as it does against the classical biotype and it offers no protection against the recently emerged V. cholerae O139 serogroup. Additional vaccine candidates have been developed by deleting genes encoding cholera toxin and other toxins of V. cholerae but these other strains produce varying amounts of diarrhea and non-diarrheal symptoms such as headache, fever, abdominal cramps, and malaise in many individuals. The proposed project will develop additional vaccine candidates for O139 V. cholerae by deletion of specific genes encoding toxins and other virulence factors from a toxigenic O139 parent strain and by transferring genes encoding important antigens of O139 to CVD 103-HgR. In addition, the investigator will study previously uncharacterized virulence factors that may be involved in the reactogenicity of vaccine candidates other than CVD 103-HgR. Additional vaccine candidates for O139 V. cholerae will be constructed by deletion of specific genes encoding toxins and other virulence factors from a toxigenic O139 parent strain and by transferring genes encoding important antigens of O139 to CVD 103-HgR. In addition, previously uncharacterized virulence factors that may be involved in the reactogenicity of vaccine candidates other than CVD 103-HgR will be studied. The investigator has recently cloned the genes for the OmpU outer membrane protein and the phospholipase of V. cholerae. He has shown that OmpU has adhesive properties and that altered expression of this protein may be responsible for the diminished intestinal colonization which is associated with diminished vaccine reactogenicity. The phospholipase may be involved in a recently recognized inflammatory response to V. cholerae infection. The phospholipase may act on epithelial cells and break down membrane phospholipids, thereby releasing chemoattractants to trigger an inflammatory response that may be involved in vaccine reactogenicity. KAPER JB UNIV OF MARYLAND SCH OF MEDICI, 685 W BALTIMORE STREET, RM. 48, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health laboratory mouse Bacillus subtilis Vibrio cholerae drug screening /evaluation gastrointestinal epithelium molecular cloning gene deletion mutation human tissue active immunization immunogenetics antibacterial antibody bacterial toxin cholera toxin virulence disease model nucleic acid sequence phospholipase A2 bacterial protein membrane protein cholera vaccine live vaccine oral administration southern blotting vaccine development CRISP RPROJ MARYLAND G CRISP/99/AI19716-16 CRISP/99/AI19716-16 199904 eng MECHANISM OF ACTION OF C PERFRINGENS ENTEROTOXIN Research RPROJ Clostridium perfringens enterotoxin (CPE) is emerging as one of the most important bacterial virulence factors for gastrointestinal (G.I.) disease. There is compelling epidemiologic evidence for the involvement of CPE in one of the most common human food poisonings; recent studies also link CPE-positive C. perfringens to other serious human gastrointestinal illnesses and to Sudden Infant Death Syndrome. CPE has a unique action on mammalian membranes which is distinguishable from all other known toxins. Every step of CPE action involves close co-participation between CPE and mammalian membrane proteins. The long-term objective of this project is to understand the role of CPE (and CPE-positive isolates) in human disease; these studies may ultimately identify strategies to prevent or treat CPE-associated illnesses. To accomplish this objective, the following specific aims are proposed: 1) to test a recent hypothesis that a 50kDa membrane protein serves as a functional CPE receptor (the binding of CPE to the CPE receptor is essential for cytotoxicity) using "anti-receptor antibody" CPE binding inhibition studies and cloning of the CPE receptor, 2) to further clarify CPE action by a) determining the topologic orientation of CPE in membranes and b) dissecting individual events in CPE action using both biochemical and ultrastructural techniques, 3) to high-resolution map CPE functional regions (and eventually combine this information with CPE structural data) to identify how the CPE molecule participates in cytotoxicity and 4) to dissect the molecular pathogenesis of CPE-positive isolates by studying the importance of CPE (and possibly other toxins) in the virulence of these isolates. These important studies are expected to i) increase the knowledge of how CPE participates in disease, ii) broaden perspectives of pathophysiologic mechanisms of intestinal diseases, iii) allow use of CPE as a defined probe of plasma membrane structure/function relationships and iv) provide a model for understanding sporulation-associated virulence factor regulation in the biomedically important genus Clostridia. These findings should have significance for numerous biomedical disciplines seeking to understand intestinal physiology under normal and disease conditions and how enteric pathogens cause G.I. disease. MC CLANE BA UNIV OF PITTSBURG SCH OF MED, SCHOOL OF MEDICINE, PITTSBURGH, PA 15261 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 15261 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Clostridium liposome cell membrane molecular pathology host organism interaction gastrointestinal disorder gastrointestinal toxin absorption molecular cloning bacterial genetics western blotting enterotoxin membrane activity virulence nucleic acid sequence peptidase protein biosynthesis protein purification protein structure /function membrane protein receptor binding tissue /cell culture northern blotting CRISP RPROJ PENNSYLVANIA G CRISP/99/AI19844-15 CRISP/99/AI19844-15 199904 eng CELL BIOLOGY OF LOCOMOTION AND CHEMOTAXIS OF LEUKOCYTES Research RPROJ Our long term goal is to understand at a fundamental level the mechanism of chemotaxis by the polymorphonuclear leukocyte (PMN). The PMN protects the host against pathogenic bacteria. To do so, the cell detects chemical signals emanating from a site of infection, crawls toward the site, and upon reaching it, ingests and kills the bacteria. To understand the mechanism underlying this behavior is of obvious relevance to health. Earlier we studied the PMN's ability to detect a gradient of chemoattractant; we now are investigating the molecular basis for the PMN's oriented locomotion. Locomotion induced by chemoattractant is an exceedingly complex phenomenon involving many signaling pathways and many proteins. Here we focus on actin whose polymerization from the globular (G-actin) to the filamentous (F-actin) form is essential for locomotion of all cell types. F-actin levels in the PMN are regulated by chemoattractant and the induced changes in F-actin levels are sufficiently large to permit quantitative analysis. Our calculations based on the speed and magnitude of the changes indicate that there must be separate mechanisms to regulate both polymerization and depolymerization. Therefore, our goals are, to: 1) define the rates of actin turnover in vivo ; 2) localize the F-actin turnover and translocation processes in the intact cell; 3) study the factors regulating this turnover. For goal 1, we will measure the F-actin flux in resting and stimulated cells using a non-perturbing assay (exchange of 3H-adenine labeled ATP into F-actin). We will also identify factors that determine the resting F- actin level. For goal 2, we will define the rate of F-actin turnover and translocation at various positions in a locomoting cell through injection of cells with fluorescent or photoactivatable actin. We will also examine the interactions between F-actin's turnover and translocation. For goal 3, we will determine the contributions of various factors that regulate F- actin levels and dynamics. These studies will use permeabilized cells which retain their ability to increase F-actin upon stimulation with GTP- gamma-S and to decrease F-actin levels upon addition of cytochalasin. Thus, we have a functional assay which can be used to identify factors that regulate F-actin levels and turnover. Once identified, the factors will be purified from cell extracts and characterized in vitro. We anticipate that quantitative information about the rates and locations of the F-actin dynamics and identification of the molecular functions being regulated will yield a coherent mechanistic model of the PMN's chemotactic behavior. ZIGMOND SH UNIVERSITY OF PENNSYLVANIA, 415 SOUTH UNIVERSITY AVENUE, PHILADELPHIA, PA 19104-6018 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory rabbit neutrophil cell motility cell type microinjection chemotaxis human subject chemoattractant antibody pertussis toxin polymerization nucleotide high performance liquid chromatography protein metabolism G protein actin guanosine triphosphate cytochalasin CRISP RPROJ PENNSYLVANIA G CRISP/99/AI19883-15 CRISP/99/AI19883-15 199904 eng PATHOGENICITY OF SHIGA TOXIN PRODUCING E COLI (STEC) Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Shiga toxin (Stx)-producing Escherichia coli (STEC) express Stx1 and/or Stx2 (or a variant of Stx2) and harbor a 90kb plasmid. STEC 0157:H7, which also express the adhesin intimin from the eae locus, are food- and water-borne pathogens that are the most common infectious cause of bloody diarrhea in the U. S. Moreover, the hemolytic uremic syndrome (HUS), as a sequela of 0157:H7 infection, is the most frequent basis for acute kidney failure in U.S. children. The long term goals of this project are to define the pathogenic mechanisms by which STEC cause disease and to develop strategies for the prevention and treatment of STEC-mediated HUS. The specific aims are to: 1) examine the reason for the striking virulence of eae- STEC 091:H21 strain B2F1 for streptomycin (str)-treated, orally-infected CD-1 mice by assessing whether virulence correlates with intestinal mucus activation of Stx2d2 produced by B2F1 and related STEC, by identifying the Stx2d2-activating substance in mucus, and by characterizing poorly adherent or mouse-virulence attenuated mini-TnphoA mutants of B2F1; 2) investigate the basis for differences in toxicity among Stx family members by creating various Stx1/Stx2 hybrid molecules and by defining both the structural changes that occur in Stx2d2 after activation and the amino acids in the Stx2d2 A2 domain required for activation; 3) analyze stx2d2 regulation by characterizing a gene encoding a repressor of Stx2d2 expression, inactivating the repressor gene in B2F1 and B2Flstx2d2::cat, and measuring the effect of that mutation on toxin production or chloramphenicol acetyl transferase (CAT) expression in vitro and on CAT levels expressed from the mutants in str-treated mice; 4) characterize the interaction between 86-24 intimin and the host cell by identifying a fragment of 86-24 intimin that can elicit anti-EHEC and anti-Enteropathogenic E. coli (EPEC) adherence blocking antibodies, defining environmental signals that regulate 86-24 intimin expression, and attempting to isolate the mammalian cell receptor for 86-24 intimin-mediated adherence; and, 5) develop potential vaccine candidates that will elicit antibodies that inhibit colonization of the host by EHEC 0157:H7 and EPEC and neutralize Stx, Stx1, and Stx2, e.g., intimin or intimin fragments combined with toxoids. OBRIEN AD UNIFORMED SERV UNIV HLTH SCI, 4301 JONES BRIDGE ROAD, BETHESDA, MD 20814-4799 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Escherichia coli Shigella dysenteriae bacterial cytopathogenic effect Escherichia coli infection molecular pathology host organism interaction molecular cloning fusion gene gene expression genetic regulation gene induction /repression bacterial genetics gene mutation enterotoxin virulence affinity chromatography protein purification receptor binding toxin metabolism chloramphenicol acetyltransferase yeast two hybrid system CRISP RPROJ MARYLAND G CRISP/99/AI20148-16 CRISP/99/AI20148-16 199904 eng E COLI HEMOLYSIN AND RELATED TOXINS Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The hemolysin (Hly) of uropathogenic E. coli is the prototype for the RTX family of hemolysins/leukotoxin produced by many different important human and animal pathogens. The putative role for RTX exotoxins in pathogenesis is the inhibition and killing of phagocytic cells. This occurs by formation of cation-selective pores in plasma membranes that leads to a metabolically disruptive Ca++ influx and eventual cell lysis at high toxin doses. The RTX exotoxin structure, mechanism, and sequence of events responsible for cytotoxicity are unknown. In contrast to the RTX leukotoxin, the E. coli hemolysin is cytotoxic to many cell types from different hosts. It contains a cysteineless, Ca++-bound, acylated, 1023 amino acid hemolysin polypeptide. The native size of hemolysin varies from 300 to > 1,000 kDa. There is conflicting evidence about the multimeric state of HlyA and if HlyA is associated with lipopolysaccharide (LPS). The proposal's specific aims are: 1) to derive models for the structure and action of hemolysin in soluble, host-membrane bound, and lytic-complex states based on different methods of composition and structural analysis. These methods involve CD spectroscopy, mass spectrometry, tryptophan fluorescence spectrometry, FACS analysis of hemolysin-MAb reactivities, and site-specific-fluorescein-labeled hemolysin. 2) to perform genetic and biochemical tests of hypothetical hemolysin structure and function models. The following types of mutants will be sought: gain of function by Hly A mutants affected in acylation and wild type Hly A expressed in LPS deep rough backgrounds, dominant negative Hly A mutants, altered pore-size Hly mutants and superhemolytic Hly A mutants. Mutants isolated from these screens will be tested for altered structural and functional changes by the appropriate methods covered in aims #1. Together the work in aims #1 and 2 will assess the critical residues and structures necessary for target cell binding, acylation, membrane insertion, multimerization, LPS interactions, and host-cell specificity of RTX toxins. 3) to produce mg quantities of Hly. Hly will be provided to a X-ray crystallographer with the long term, collaborative goal of the solution of a high resolution Hly structure. Increased knowledge of the structure and function of the prototype for this large family of exotoxins may lead the identification of novel RTX toxin inhibitors and prophylactic strategies. WELCH RA UNIVERSITY OF WISCONSIN, 1300 UNIVERSITY AVENUE, MADISON, WI 53706 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 53706 Crisp Data Base National Institutes Of Health acylation Escherichia coli hemolysin flow cytometry chemical structure /function fluorescence spectrometry mass spectrometry X ray crystallography host organism interaction site directed mutagenesis molecular cloning mutant monoclonal antibody immunoprecipitation exotoxin structural model circular dichroism lipopolysaccharide cytotoxicity toxin metabolism structural biology CRISP RPROJ WISCONSIN G CRISP/99/AI20323-15 CRISP/99/AI20323-15 199904 eng DUST MITE, COCKROACH AND CAT ALLERGENS IN ASTHMA Research RPROJ The objective of these studies is to improve understanding of the role that indoor allergens play in asthma and to establish effective regimes for treating the disease by reducing exposure to allergens in homes. Asthma affects at least 8 million people in N. America, and the disease appears to be increasing both in prevalence and severity. Case control studies on emergency room patients suggest that greater or equal to 40% of adult asthma and as much as 75% of asthma among children is related to sensitization to indoor allergens. These studies utilize rapid sensitive monoclonal antibody based immunoassays for allergens derived from dust mites of the genus Dermatophagoides, the cat Felis domesticus and cockroaches, eg. Blatella germanica. In order to understand the relevance of exposure in childhood, tbe prevalence of asthma among chfldren age 12-14 will be assessed and related to the allergen levels in houses in different climatic areas. These studies will be designed to answer whether exposure in houses influences the sensitization of children and also whether high levels of exposure can increase the prevalence of asthma. The results should also allow a reevaluation of the proposed threshold levels that are associated with increased risk of sensitization to mite or cat allergens and with the development of asthma. The present experiments will also focus on techniques for reducing exposure to dust mite and cat allergens. In particular, the effects of both physical measures (e.g. covering mattresses, hot washing bedding, removing carpets) and chemical measures (e.g. acaricides or denaturing agents applied to carpets and sofas) on airborne dust mite allergen will be investigated. Similarly, detailed measures for controlling exposure to cat allergens will be studied (e.g. air filtration, removing carpets and washing the cat). Finally, controlled trials of allergen avoidance will be carried out on children admitted to hospital with asthma, patients presenting to clinics with asthma, and also on mite allergic patients with atopic dermatitis. The long term objective is to develop effective and realistic methods for reducing exposure to allergens and to answer whether allergen avoidance should be a first line form of treatment for these patients. PLATTS-MILLS TA UNIVERSITY OF VIRGINIA, HEALTH SCIENCE CENTER, BOX 225, CHARLOTTESVILLE, VA 22908 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 22908 Crisp Data Base National Institutes Of Health cat laboratory rabbit laboratory mouse pyroglyphid cockroach adolescence (12-18) clinical trial climate environmental contamination disease /disorder prevention /control human subject airborne allergen asthma immunochemistry monoclonal antibody pest control pollution related respiratory disorder respiratory disorder epidemiology atopic dermatitis CRISP RPROJ VIRGINIA G CRISP/99/AI20565-15 CRISP/99/AI20565-15 199904 eng MOLECULAR MODE OF ACTION OF BACTERIAL ENTEROTOXINS Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The applicant's overall research objective is to define essential components in the mechanism of action of cholera toxin (CT) on water and electrolyte secretion in the small intestine. Various mediator responses to CT in the rabbit and porcine models were characterized in the last project period, which enhanced our understanding of the mechanism of cholera. The proposed research is targeted toward CT-induced effects on cell proteins, as they impact intestinal physiology. Antimetabolites (e.g., actinomycin D and cycloheximide) and nonsteroidal anti-inflammatory drugs (e.g., indomethacin) block both CT- induced secretion and eicosanoid synthesis. One CT-induced acute-phase reactant, known as phospholipase A2- activating protein (PLAP), has been identified by the investigator in cultured cells and mouse intestinal tissue. His hypothesis is that cell proteins, such as PLAP, participate in the pathogenesis of cholera. The pathognomonic role of PLAP in stimulating eicosanoid synthesis by activating PLA2 will be assessed with PLAP antisense DNA and PLAP antibodies. In addition, other cell proteins affected by CT, such as those phosphorylated by CT-induced protein kinase activity, will be characterized. Using immunocytochemistry, the investigator will determine the intestinal cell types that synthesize these proteins and other mediators following luminal challenge with CT, Vibrio cholerae, and Salmonella typhimurium. Mutant CT proteins will be used to determine whether the active site of the CT-A subunit that catalyzes the ADP- ribosylation of GS alpha is involved in the induction of PLAP synthesis. The effects of CT on eicosanoid synthesis will be evaluated in a cyc- (GS alpha-deficient) mutant S49 cell line (compared to wild-type control cells), to separate CT effects on the cAMP system from those on arachidonic acid (AA) metabolism. Rapid induction of PLAP synthesis suggests the involvement of transcriptional activators that could enable CT to signal the rapid de novo synthesis of PLAP. Consequently, the mechanism by which CT rapidly signals the synthesis of PLAP mRNA, which, in turn, stimulates AA release from membrane phospholipids and leads to increased eicosanoid synthesis, will be defined. The DNA sequence 5' to the plap gene will be examined for conserved transcription factor- binding motifs, and a DNA mobility shift assay with transcription factor-binding [32P]- deoxyoligonucleotides will be used to test nuclear extracts from CT-treated cells for transcription factor activity. Studies on the role of Ca++ in the mechanism of action of CT have also been proposed, because CT-induced secretion is diminished by calcium channel blockers and CT elicits the synthesis of LTC4, a Ca++ channel activator. Knowledge about the molecular events in cholera should improve future strategies to control the hypersecretion of water and electrolytes in cholera and other diarrheal diseases. PETERSON JW UNIV OF TEXAS MEDICAL BRANCH, GALVESTON, TX 77555-1070 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Vibrio cholerae secretion calcium flux leukotriene prostaglandin eicosanoid metabolism diarrhea gastrointestinal epithelium small intestine gene expression genetic regulatory element transcription factor immunocytochemistry acute phase protein cholera toxin antisense nucleic acid adenylate cyclase phospholipase A2 phosphorylation protein tyrosine kinase protein biosynthesis cyclic AMP enzyme activity gel mobility shift assay CRISP RPROJ TEXAS G CRISP/99/AI21463-09 CRISP/99/AI21463-09 199904 eng DIPHTHERIA TOX REPRESSOR--GENETIC AND STRUCTURAL ANALYSIS Research RPROJ Iron is an essential nutrient for most all living cells. Since iron has an extraordinarily low solubility at neutral pH (Ca., 10(-17 M), a variety of complex systems have evolved to facilitate the uptake of iron. The favorable redox potential and coordination chemistry of iron allows this element to be used intracellularly both as a cofactor for a variety of structural and metabolic functions. In addition, the regulation of virulence determinants (e. g., toxin, colonization factors, etc.) in most pathogenic microorganisms is also controlled by iron-activated regulatory factors. For example, the diphtheria tox repressor, DtxR, is activated in the presence of iron and other heavy metal ions and functions as a negative regulatory element in the control of diphtheria toxin expression, as well as other iron-sensitive genes in lysogenic toxigenic strains of Corynebacterium diphtheriae. Upon depletion of exogenous iron from the culture medium, the Fe/DtxR/DNA complex rapidly disassociates resulting in derepression of all iron-sensitive genes. Since conjugation, transfection, and transformation are not well developed in C. diphtheriae, we have used molecular cloning of dtxR and its target sequences in recombinant Escherichia coli in order to study the molecular biology and genetics of this iron-activated regulatory factor. The long term goals of this research are to develop a detailed understanding of the role played DtxR in the regulation of gene expression in C. diphtheriae. Other than the recent demonstration that DtxR controls the expression of siderophore genes in C. diphtheriae, virtually nothing is known about the global nature of DtxR-mediated regulation of iron sensitive gene expression. This proposal seeks continued support for the study of the molecular genetic analysis of DtxR and the determination of the role that this regulatory factor plays in the molecular pathogenesis of C. diphtheriae. In addition, we propose to solve the X-ray crystallographic structure of apoDtxR, the metal ion/DtxR complex, and the metal ion/DtxR/DNA complex. To the best of our knowledge DtxR is the first iron-activated regulatory element to be crystallized. The solution of these structures will provide the paradigm for the mechanism of action of other iron-activated regulatory elements in pathogenic microorganisms. MURPHY JR BOSTON MEDICAL CENTER, 88 EAST NEWTON STREET, BOSTON, MA 02118 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02118 Crisp Data Base National Institutes Of Health Corynebacterium diphtheriae Escherichia coli chemical binding polymerase chain reaction X ray crystallography molecular cloning gene expression regulatory gene genetic operator element genetic regulation transcription factor bacterial genetics radioimmunoassay diphtheria toxin iron intermolecular interaction messenger RNA nucleic acid sequence protein purification protein structure /function tissue /cell culture northern blotting gel mobility shift assay CRISP RPROJ MASSACHUSETTS G CRISP/99/AI21628-13 CRISP/99/AI21628-13 199904 eng MOLECULAR GENETICS OF ENTEROPATHOGENIC E COLI ADHESION Research RPROJ Enteropathogenic E. coli (EPEC) are an important cause of diarrhea in infants. The long-term objectives of this project are to understand the pathogenesis of disease due to this organism and to develop diagnostic reagents and vaccine candidates for prevention of disease due of disease. There are also many similarities between the pathogenesis of EPEC and the pathogenesis of E. coli 0157:H7, the enterohemorrhagic E. coli (EHEC) which have been responsible for recent large outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) in the U.S. due to ingestion of contaminated beef, water, apple cider and other vehicles. We have shown that there is genetic similarity between some virulence factors of EPEC and EHEC and information resulting from the proposed studies will also yield insights into the pathogenesis of EHEC. We have proposed a three stage model of EPEC pathogenesis and identified genes for virulence factors of each stage. The first stage consists of non-intimate adherence of the bacteria to epithelial cells which is mediated by a type IV pilus, the Bundle-Forming Pilus (BFP). The second stage involves a signal transduction event between the bacteria and the epithelial cell resulting marked cytoskeletal changes, dissolution of the microvilli, increase in intracellular calcium, and tyrosine phosphorylation of a 90 kDa epithelial cell protein. The third stage involves an intimate attachment between the bacteria and the epithelial cell with a concentration of polymerized actin and other cellular components accumulating at the site of bacterial attachment. There are three broad aims for the proposed experiments: l) Mutagenize and characterize a large (ca. 35 kb) region of the chromosome which contains multiple genes encoding virulence factors of EPEC. 2) Determine the extent of a putative virulence regulon in which the transcriptional activator PerA regulates a variety of EPEC virulence genes. 3) Characterize the effect of EPEC on intestinal epithelial cells using polarized intestinal cell lines and rabbit models. The proposed experimental approach will use a combination of molecular genetics, cell biology, and animal studies to achieve a full understanding of how EPEC infects epithelial cells and effects the ion secretion which presumably is responsible for diarrhea caused by EPEC. KAPER JB UNIV OF MARYLAND, BALTIMORE, 655 WEST BALTIMORE ST, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health laboratory rabbit enteric bacteria Escherichia coli Escherichia coli infection biological signal transduction genetic strain cell adhesion molecular pathology diarrhea gastrointestinal infection gastrointestinal epithelium genetic mapping molecular cloning fusion gene bacterial genetics mutant enterotoxin brush border membrane virulence nucleic acid probe nucleic acid sequence tissue /cell culture bacterial vaccine interleukin 8 CRISP RPROJ MARYLAND G CRISP/99/AI21657-13 CRISP/99/AI21657-13 199904 eng MOLECULAR MECHANISMS OF BACTERIAL PATHOGENESIS Research RPROJ The long range goals of this project are to understand structure- function relationships of the ADP -ribosylating exotoxins at the level of 3-dimensional crystallographic structure. The work described is focused on diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (ETA), two related toxins that block protein synthesis by ADP-ribosylation of Elongation Factor-2 (EF- 2). With respect to DT, studies will be performed: (i) to characterize the active site, by combining site-directed mutagenesis with various biochemical and biophysical methods; (ii) to characterize the interaction of fragment A with EF-2 by new approaches, including the use of diphthamide synthesized by organic chemistry, and new assays for detecting interaction of EF-2 with active toxin fragments; (iii) to select toxin mutants that are defective in membrane-insertion and/or channel- formation at acidic pH, by means of a new positive selection procedure in E. coli; (iv) to explore a new method to study translocation of the enzymically active fragment A moiety of DT to the trans face of planar lipid bilayers; and (v) to determine the 3-dimensional structures of monomeric native DT, an enzymically active fragment (fragment A), and mutant forms of particular interest. With respect to ETA, studies are proposed which largely parallel those describe above for DT. Results will be interpreted in terms of a 3.0 A crystallographic structure of ETA determined recently, and an emerging 3-dimensional structure of DT-dimer at similar resolution. These studies are pertinent to a variety of problems in biomedical science, including mechanisms by which proteins penetrate membranes, routes to new vaccines, and mechanisms of directing toxic proteins to specific cells. COLLIER RJ HARVARD MEDICAL SCHOOL, 200 LONGWOOD AVENUE, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health aminoacid analog guinea pig laboratory mouse Pseudomonas aeruginosa bacterial disease bioassay chemical structure /function chemical synthesis X ray crystallography histidine molecular pathology enzyme mechanism site directed mutagenesis translation factor bacterial genetics point mutation ADP ribosylation diphtheria toxin exotoxin membrane activity transposon /insertion element chromatography adenosine diphosphate CRISP RPROJ MASSACHUSETTS G CRISP/99/AI22021-15 CRISP/99/AI22021-15 199904 eng MOLECULAR POPULATION GENETICS OF PATHOGENIC BACTERIA Research RPROJ The primary objectives of the proposed research are (1) to analyze the multi-locus genetic structure of natural populations of Salmonella and certain other human-pathogenic bacteria through the application of several methods of indirectly and directly detecting nucleotide substitutions in chromosomal genes, combined with the mathematical theory and statistics of molecular population genetics; (2) to develop basic evolutionary genetic frameworks within which to analyze and interpret data on the distribution of cell-surface antigens, plasmids, and other characters that have been implicated as determinants of pathogenicity and disease specificity; and (3) to develop powerful new genetic markers for epidemiological studies of Salmonella and other important human pathogenic bacteria. Proposed studies include: (A) Continuation of an analysis of electrophoretically demonstrable allelic variation in 23 chromosomal enzyme genes to assess clonal diversity and relationships in the medically and economically most important serotypes of Salmonella, with special reference to variation in antigenic profile and the evolutionary origins of typhi and other organisms causing human typhoid fever. (B) Determination of the genetic diversity, genomic relatedness, and extent of clonal host specificity among strains of serotypes that are commonly recovered from both humans and domesticated animals. (C) Analysis, by nucleotide sequencing, of the molecular and population genetic bases for the occurrence of the same somatic (O) and flagellar (H) cell-surface antigen profile in strains belonging to distantly related phylogenetic lineages. (D) Development of a molecular epidemiological marker system for discrimination among isolates of the predominant clones of the Salmonella serotypes enteritidis and typhimurium. (E) Selection and characterization of a standard set of Salmonella reference strains (SARC) that are representative of the full span of genotypic diversity within the genus for general use in microbiological research. Other research will be concerned with the molecular genetic correlates of phylogenetic differentiation of populations of Escherichia and Salmonella. SELANDER RK PENNSYLVANIA STATE UNIVERSITY, 516 MUELLER LABORATORY, UNIVERSITY PARK, PA 16802-100 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Haemophilus influenzae Escherichia coli Salmonella Legionella Neisseria gonorrhoeae Escherichia coli infection gonorrhea legionellosis biochemical evolution genetic strain cell adhesion communicable disease enzyme structure isozyme genetic marker linkage mapping molecular genetics plasmid allele gene amplification structural gene genotype microorganism genetics bacterial genetics population genetics surface antigen bacterial antigen bacterial toxin serology /serodiagnosis microorganism classification virulence nucleic acid hybridization complementary DNA nucleic acid sequence gel electrophoresis bacterial protein CRISP RPROJ PENNSYLVANIA G CRISP/99/AI22144-14 CRISP/99/AI22144-14 199904 eng MOLECULAR BIOLOGY OF TOXIC SHOCK SYNDROME TOXIN-1 & OTHER SUPERANTIGEN TOXINS Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): This is a competitive renewal of a collaborative project between the Novick and Schlievert laboratories aimed toward understanding the molecular genetics and pathobiology of one prototypical superantigen, toxic shock syndrome (TSST 1). Their results and the results of others suggest that the interaction of TSST 1 with the TCR and MHC Class II receptors is responsible for their superantigen activity. However, these investigators have provided evidence that its interaction with different types of receptors on other cells is responsible for its lethality. They have recently determined the structure of TSST 1 and several derivatives by X ray crystallography and have constructed a series of mutants with properties that have led to one hypothesis of this proposal, namely that lethality and superantigenicity are separately determined. The first two aims will focus on a definitive test of this hypothesis through confirmation of their preliminary results which suggest separable functions. This part of the project will investigate the pathobiology of TSST 1 by identifying and characterizing tissue specific receptors and determining the mechanism of intracellular cytotoxicity with reference to activities of mutant forms of the toxin. Aims 3 and 4 will be a continuing investigation into the molecular genetics of S. aureus toxin production. Aim 3 will focus on extending their preliminary genetic data suggesting that TSST 1 is encoded by a transposon, Tn557. This part of the project will determine the structure and function of Tn557 and also of closely linked heterologous insertions containing determinants of SEB and certain resistances. Aim 4 will attempt to develop an explanation for the mutual exclusion of TSST 1 positive and alpha-hemolysin positive phenotypes in S. aureus, despite the fact that many strains have both genes. A similar situation exists in group A streptococci with two superantigens (SPEA and SPEB). They have obtained preliminary results suggestive of an explicit regulatory antagonism between TSST 1 and alpha hemolysin. They propose to determine whether other exoproteins are involved and what is the basis of this antagonism. They will perform similar studies with SPEA and SPEB. Aim 5 will attempt to elucidate the mechanism of action for glycerol monolaurate effects on eukaryotic cells and on S. aureus. In a series of unrelated experiments, they have observed that GML inhibits the synthesis of TSST 1 and other exoproteins and also of the induction of resistance to several antibiotics, probably by interfering with signal transduction. Furthermore, they showed that GML is mitogenic for T lymphocytes but not for myoblasts. Although the study of GML was initially focused on TSST 1 they now propose to continue this investigation on a broader basis. They propose to identify the putative signal signalling pathway(s) in bacteria that are sensitive to GML inhibition, to confirm that signal transduction is the actual target, and to determine the putative signalling pathway used by GML to stimulate lymphocytes. The PI's expect that this work will lead to a comprehensive understanding of the structure function, genetics, regulation and pathobiology of TSST 1 and will help develop certain diagnostic and therapeutic applications plus a general method of immunization against superantigen toxins. NOVICK RP NEW YORK UNIV MEDICAL CENTER, 540 FIRST AVE, NEW YORK, NY 10016 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 10016 Crisp Data Base National Institutes Of Health Staphylococcus aureus Streptococcus pyogenes toxic shock syndrome biological signal transduction polymerase chain reaction molecular cloning bacterial genetics bacterial antigen bacterial toxin staphylococcal exotoxin glyceride virulence transposon /insertion element protein structure /function receptor binding T cell receptor chemoreceptor MHC class II antigen superantigen CRISP RPROJ NEW YORK G CRISP/99/AI22159-15 CRISP/99/AI22159-15 199904 eng BORDETELLA PERTUSSIS TRACHEAL CYTOTOXIN Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Of the various toxins and virulence-related factors produced by Bordetella pertussis, only one has been demonstrated to reproduce the specific respiratory tract cytopathology of the pertussis syndrome. That molecule is tracheal cytotoxin (TCT),a 921 dalton peptidoglycan fragment released by Bordetella pertussis during normal growth. During the past 3 years of funding by this NIH grant, most of the research effort has centered on understanding TCT structure-function relationships, defining TCT target cells, and elucidating TCT mechanism of action. This renewal application is focused on experiments to describe further the TCT toxicity pathway, target cell specificity, and the molecular basis for TCT production. Five years are requested to explore the following specific aims: I. Define more precisely the role of interleukin-1 (IL-1) and nitric oxide (NO ) in the mechanism of TCT action. Experiments will evaluate whether IL-1 is indeed an essential step in the TCT toxicity pathway and will define the specific respiratory epithelial cells that respond to TCT (and Bordetella pertussis infection) by synthesizing IL-1 and/or NO ; similar studies will be designed to examine the molecular basis for species-specific responsiveness to TCT. II. Compare TCT's toxicity for neutrophils to the known biochemistry and biology of TCT's respiratory epithelial effects. The potent effects of TCT on neutrophils will be examined in experiments that parallel our previous work with respiratory epithelial cells: structure-activity relationships, evidence for binding to a surface receptor, the potential involvement of IL-1 and NO , and the possibility of synergy with endotoxin. III. Identify the genetic and biological basis of TCT release by Bordetella pertussis. These experiments will test the hypothesis that Bordetella pertussis release of TCT is largely due to a defective or missing membrane transport protein. AmpG, that is critical for peptidoglycan recycling. In addition, a novel mutant screen will be used to identify other gene(s) that may be involved in production of TCT. GOLDMAN WE WASHINGTON UNIV SCHOOL OF MED, BOX 8230, 660 SOUTH EUCLID AVE, ST LOUIS, MO 63110-1093 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health hamster laboratory rat Bordetella pertussis pertussis neutrophil chemical binding molecular pathology bacterial genetics mutant histopathology interleukin 1 endotoxin pertussis toxin disease model peptide analog protein structure /function membrane transport protein tritium radiotracer respiratory epithelium trachea tissue /cell culture organ culture nitric oxide CRISP RPROJ MISSOURI G CRISP/99/AI22243-11 CRISP/99/AI22243-11 199904 eng LIFE AND DEATH OF ANTIGEN ACTIVATED T CELLS IN VIVO Research MARRACK P NATL JEWISH CTR FOR IMMUNOLOGY, 1400 JACKSON STREET, DENVER, CO 80206 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80206 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal T lymphocyte cell death flow cytometry polymerase chain reaction leukocyte activation /transformation interleukin 2 tumor necrosis factor alpha staphylococcal enterotoxin lipopolysaccharide T cell receptor growth factor receptor tissue /cell culture tissue mosaicism CRISP RPROJ COLORADO G CRISP/99/AI22295-130013 CRISP/99/AI22295-130013 199904 eng POLYSACCHARIDE VACCINE FOR PSEUDOMONAS AERUGINOSA Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The long term objectives of this proposal are to understand mechanisms of immunity against lipopolysaccharide (LPS)-smooth strains of P. aeruginosa and develop effective immunotherapies that would augment the treatment of infections. P. aeruginosa is a leading nosocomial pathogen and LPS- smooth strains are the initial colonizers among patients with cystic fibrosis, an additional potential target for vaccine intervention. This proposal seeks to provide a better understanding of the diversity of protective epitopes found on the O-side chains of clinically important strains in order to formulate a comprehensive vaccine. The vaccine candidates are called high molecular weight polysaccharides and represent large, hence immunogenic, forms of the O-side chain structures. Although a limited number of serogroups of P. aeruginosa (7-10) have been implicated as pathogens, within some of these serogroups there are slight variations in the basic oligosacchaaride repeat unit that gives rise to antigenic variants that are the basis for expression of subtype epitopes. The need to include representative subtype epitopes in a comprehensive vaccine is undefined. The first aim of this proposal will look at the role of serogroup and subtype-specific epitopes in protective immunity. These studies will include: an evaluation of the immunogenicity of serogroup and subtype epitopes expressed on different preparations of high molecular weight polysaccharide; evaluation of the protective capacity, in mice, of antibodies specific to serogroup and subtype determinants, and production of a multivalent vaccine designed to elicit immunity to relevant serogroup and subtype epitopes. The second aim will investigate the local and systemic correlates of protective immunity against P. aeruginosa colonization and infection using recombinant Salmonella strains, systemically administered polysaccharide vaccines, or passively delivered antibodies as inducers or effectors of immunity. The serum and mucosal antibody response to P. aeruginosa LPS will be measured following oral vaccination with recombinant Salmonella strains or systemically delivered high molecular weight polysaccharides. These vaccines will be used to compare oral and systemic immunization as a strategy to promote P. aeruginosa clearance from the GI tract, prevent dissemination from the GI tract during neutropenia, and prevent infections at a distant mucosal site, the eye. Confirmation of the role of antibody will be accomplished by producing isotype disparate, variable-region identical monoclonal IgM, IgG and IgA antibodies against P. aeruginosa LPS O-side chains that will be used in evaluating the role of different antibody isotypes using passive protection studies in the various animal models. Overall this work should provide the formula for a comprehensive P. aeruginosa O-antigen- specific vaccine, provide important information about protective epitopes o PIER GB CHANNING LABORATORY, 180 LONGWOOD AVENUE, BOSTON, MA 02115-5899 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Pseudomonas aeruginosa genetic strain T lymphocyte mutant immunoglobulin A immunoglobulin G immunoglobulin M immunoglobulin isotype human subject bactericidal immunity cross immunity active immunization passive immunization immunoconjugate monoclonal antibody enzyme linked immunosorbent assay epitope mapping antibacterial antibody antigen antibody reaction bacterial antigen exotoxin serotyping bacterial polysaccharide bacterial vaccine CRISP RPROJ MASSACHUSETTS G CRISP/99/AI22535-14 CRISP/99/AI22535-14 199904 eng RECOMBINANT TOXOIDS OF BACTERIAL EXOTOXINS Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The major goal of the proposed studies is to apply the current wealth of knowledge about the molecular structure and function of diphtheria toxin (DT) to create genetically inactivated cross- reactive mutant forms of the toxin (CRMs) that will serve as ideal components of future vaccines. The crystallographic structure of DT was solved recently, revealing the topography of the 3 functionally distant domains-the C (catalytic), T (transmembrane), and R (receptor-binding) domains. Using known substitution and deletion mutations that selectively abrogate individual functions, we will seek to define a limited set of CRMs that are appropriate for developing various types of vaccines. First, as an alternative to standard formal intoxoid, the investigators will seek to develop a holotoxin CRM with negligible biologic activity and with minimal alteration in antigenicity and immunogenicity. Second, toxin fragments corresponding to the 3 domains and selected inter- and intra-domain peptide sequences will be investigated as potential immunogens. Third, the investigators will investigate the hypothesis that disruption of the receptor-binding or membrane-translocation functions may alter the immunogenicity of the molecule. Fourth, to create a new vehicle for polysaccharides in conjugate vaccines, the investigators will introduce specific functional residues within CRMs to generate coupling sites for polysaccharides. Fifth, to identify mutant forms of DT appropriate for incorporation into live-vectored vaccines, the investigators will prepare and test multiply mutated CRMs that have suitably low probabilities of reversion. These studies will enhance understanding of the role of each of the critical vaccine antigens which will be: biologically inactive and stable without chemical toxoiding, highly consistent from batch to batch, inexpensively purified by simple affinity chromatography methods, suitable for chemical coupling to bacterial polysaccharide antigens in defined locations, and suitable for insertion into live vectors or for creation of chimeric proteins. The approaches developed in these studies may be applicable to other important vaccine antigens, such as tetanus toxin, pertussis toxin, and other pertussis antigens. COLLIER RJ HARVARD MEDICAL SCHOOL, 200 LONGWOOD AVENUE, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health Escherichia coli Salmonella biological product biotechnology genetic manipulation site directed mutagenesis gene deletion mutation mutant toxoid diphtheria toxin exotoxin affinity chromatography protein structure /function receptor binding bacterial vaccine synthetic vaccine recombinant DNA CRISP RPROJ MASSACHUSETTS G CRISP/99/AI22848-13 CRISP/99/AI22848-13 199904 eng IMMUNOCHEMISTRY OF ENDOTOXIN-UNRESPONSIVE C3H-HEJ MICE Research RPROJ Bacterial endotoxic lipopolysaccharides (LPS) can initiate fever, profound hypotension (shock), disseminated intravascular coagulation, multisystem organ failure and death. There is substantial evidence to support endotoxin-induced host responses as a major contributing factor to the more than 25,000 deaths estimated to occur annually in the United States as a result of gram negative bacterial sepsis. Estimates of mortality in patients with profound shock due to endotoxin are 40-50 percent. Recent evidence has implicated cytokines released from endotoxin- stimulated lymphoreticular cells as major factors in the pathogenesis of endotoxin shock. The long range objective of this research project is to define the mechanism(s) by which LPS triggers the activation of lymphoreticular cells with specific focus on endotoxin receptors. For many of these studies, advantage will be taken of the mutant inbred C3H/HeJ mouse strain, whose lymphoreticular cells are refractory to stimulation with many LPS preparations. In this renewal application experiments are described to provide molecular, biochemical and immunologic characterization of a recently identified 80 kDa LPS-specific binding protein expressed on lymphoreticular cells of many species. We will investigate the hypothesis that this 80 kDA candidate LPS receptor serves an important biochemical function in cell activation. Monoclonal antibodies to this protein will be utilized to dissect specific LPS-receptor interactions, to follow the intracellular fate of the receptor and as affinity reagents to purify this molecule. Biochemical characterization and molecular cloning of the structural gene for this protein will be employed to understand further the function of this protein. Experiments to dissect the molecular basis for R-chemotype LPS activation of C3H/HeJ lymphoreticular cells will continue. It is suggested that the successful completion of these studies will contribute to our understanding of molecular interactions of LPS with host cells, and provide information of value in the design of therapeutically effective reagents to treat endotoxin shock. MORRISON DC UNIV OF KANSAS MEDICAL CTR, 3901 RAINBOW BLVD, KANSAS CITY, KS 66160-7832 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory rabbit hamster laboratory mouse B lymphocyte crosslink transfection molecular cloning immune tolerance /unresponsiveness immunochemistry monoclonal antibody antiidiotype antibody endotoxin immunotoxicity lipopolysaccharide protein purification binding protein receptor CRISP RPROJ KANSAS G CRISP/99/AI23447-13 CRISP/99/AI23447-13 199904 eng TRANSLOCATING BACTERIA--ROLE IN SURGERY AND TRAUMA Research RPROJ DESCRIPTION: The working hypothesis is that in situations of altered intestinal permeability, which would include hemorrhagic shock and ischemia-reperfusion injury, there is an increased exposure of the lateral surface of the enterocyte. This in turn leads to paracellular adherence (via adhesion molecules) and invasion of normally non-invasive enteric flora. Her design model incorporates an organized and logical approach that utilizes in vitro techniques of cell cultures (Caco-2 and HT-29 cells) and then applies the results in an in vivo model, the mouse. Initially the applicant will attempt to clarify the relationship between enterocyte adhesion molecules (E-AM), bacterial adherence, internalization and intracellular survival, and then investigate the effects of an iatrogenically altered epithelial permeability on E-AM expression and bacteria-enterocyte interactions. Finally she will attempt to clarify the association between E-AM, epithelial permeability, PMN transmigration and bacteria-enterocyte interaction. Adhesion molecules (AM) are felt to play a major role in bacteria-enterocyte interaction. Three families of AM are specifically interesting in this regard: ICAM-1 (modulates PMN migration and decreases epithelial barrier function), E-cadherin (calcium dependent and essential in cell-cell adhesion), and integrins (are located on basolateral surfaces and trigger internalization). Dr. Wells will show the binding pattern, spatial relationship, and effect of monoclonal Ab (mAb) to AM on adherence and internalization in the cultured cells and in the in vivo model. Next she will then investigate the effects of an altered epithelial membrane on these binding patterns using double fluorochrome labeling, also using both the culture line and mouse model. Finally the association between the enterocyte AM, bacteria, and the migrating PMN will be investigated. WELLS CL UNIVERSITY OF MINNESOTA, 420 DELAWARE ST SE BOX 609, ST PAUL, MN 55455 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 55455 Crisp Data Base National Institutes Of Health muscarine laboratory mouse laboratory rat enteric bacteria bacterial disease membrane permeability neutrophil cell migration carbachol electrical conductance gastrointestinal epithelium gene expression genetic regulation cytokine monoclonal antibody bacterial toxin trauma binding protein cell adhesion molecule actin integrin postoperative state tissue /cell culture transmission electron microscopy cadherin CRISP RPROJ MINNESOTA G CRISP/99/AI23484-13 CRISP/99/AI23484-13 199904 eng EXPRESSION OF ANTIBODY DIVERSITY DURING IMMUNE RESPONSES Research RPROJ DESCRIPTION: The investigator will study the process of antigen selection and hypermatutation, and how they contribute to B cell memory, in the mouse response to phenylarsonate (Ars). This project involves the identification of clones in the Ars response at different stages of their differentiation, and the development of a correlation between this maturation stage and position in the spleen relative to the putative sites of antigen selection and somatic mutation. The Ars response is a well-characterized system by many laboratories. Two broad aims are proposed that are subdivided into several subaims. First, the investigator will characterize the anti-Ars response of A/J mice via the determination of spatial organization of anti-Ars-specific clones with the location of antibody forming cells (AFC) and germinal centers (GC). The first subaim will address the appearance and timing of Ars-specific clones by the use of an anti-Ars V gene antibody and PCR analysis. The investigator will distinguish between two principle models for antigen-driven B cell maturation. A second subaim will employ the same methodologies to evaluate the spatial organization of memory anti-Ars clones. The second broad aim will utilize the investigator s transgene system for study. In the previous period, the investigator has developed a transgene model where an anti-Ars specific rearranged IgVh gene is able to be recruited into the anti-Ars response (and undergo somatic mutation). Using these transgenic mice, they will study the organization of the transgene-encoded anti-Ars clones and B cell maturation. In the first subaim of this section, the investigator will compare the transgene-encoded clonotype (high affinity to Ars) with endogenously emerging clones (low affinity to Ars) for their population of spatial sites of B cell maturation, AFC and GC. For this study an additional antibody reagent will be employed that is more specific for the transgene clonotype. In the second subaim of this section, using the same general methodologies, they will characterize the relationship between affinity for Ars by these two antibody types and the somatic mutation levels of these genes. In a third subaim they will determine whether there is a positive relationship between sites of apoptosis in a GC and the relative incidence of affinity maturation. MANSER TL THOMAS JEFFERSON UNIV MED COLL, 233 S 10TH STREET, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 19107 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal B lymphocyte cell differentiation polymerase chain reaction molecular cloning gene mutation immunoglobulin idiotype immunoglobulin structure leukocyte activation /transformation immunologic memory immunocytochemistry monoclonal antibody antibody formation antibody specificity arsenic sulfonate CRISP RPROJ PENNSYLVANIA G CRISP/99/AI23739-10 CRISP/99/AI23739-10 199904 eng ROLE OF CD14 AND OTHER LPS RECEPTORS IN ENDOTOXIN SHOCK Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The ability to reproduce endotoxin shock in mice provides a unique opportunity to study the biology of the disease and its consequences. The studies proposed here take advantage of the ease of doing experiments in mice and the newest genetic technology. Dr. Goyert has recently produced mice in which the CD14 gene has been disrupted by homologous recombination. These mice provide a unique opportunity to ask several fundamental questions about the nature of endotoxin shock, particularly as regards CD14- dependent and CD14-independent pathways to endotoxin shock. In addition, this animal model will allow Dr. Goyert to evaluate the relative contribution of endothelial cell responses to sCD14- LPS complexes in endotoxin shock.This is the first time that such a model has been established for delineating the effects of these three mechanisms. She will also continue development of a new animal model that will respond to endotoxin in a CD14-dependent manner through the human CD14 protein. This model will be used to evaluate the efficacy of reagents (anti-human CD14 mAb, recombinant soluble human CD14) on the endotoxin response of mice expressing a human CD14 gene and lacking expression of the murine CD14 gene. The following questions will be addressed: 1. How does the lack of CD14 affect the response of CD14-knockout mice to LPS, Gram-negative bacteria, Gram-positive bacteria, and complexes of LPS and soluble CD14? 2. How effective are soluble human CD14 and anti-human CD14 mAb in preventing endotoxin shock in mice expressing human but not murine CD14? Furthermore, can efficacious, unique CD14-specific mAbs be generated in CD14-deficient mice? 3. What is the three-dimensional structure of CD14? 4. What is the nature of the endothelial cell receptor for the complex of LPS-soluble CD14 and is it present on other cell types that also respond to this complex? GOYERT SM NORTH SHORE UNIV HOSPITAL, 300 COMMUNITY DRIVE, MANHASSET, NY 11030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 11030 Crisp Data Base National Institutes Of Health laboratory mouse gram negative bacteria gram positive bacteria molecular pathology genetic manipulation gene expression antireceptor antibody CD antigen endotoxin microorganism immunology disease model lipopolysaccharide protein structure antigen receptor tissue /cell culture septic shock CRISP RPROJ NEW YORK G CRISP/99/AI23859-13 CRISP/99/AI23859-13 199904 eng SYNGENEIC GRAFT-VERSUS-HOST DISEASE Research RPROJ DESCRIPTION (Adapted from Investigator's abstract): Cyclosporine (CsA) is a potent immunosuppressive drug that paradoxically elicits a T-lymphocyte dependent autoimmune disease when administered after syngeneic/autologous bone marrow transplantation (BMT). This autoaggression syndrome which develops after withdrawal of CsA treatment was termed syngeneic graft-vs-host disease (SGVHD) because of its similarity (histologically, target organs) to GVHD occurring after allogeneic BMT. Two important factors responsible for the pathogenesis of this autoimmune syndrome have been identified: the enhanced production of CD4+ and CD8+ MHC class II specific autoreactive T cells by the thymus and the elimination of a T-lymphocyte dependent peripheral host resistance mechanism. Both CD8+ and CD4+ SGVHD autoreactive T lymphocytes promiscuously recognize MHC class II antigens and express the V beta 8.5 T-cell receptor determinant. Preliminary studies reveal that the peptide from the invariant chain plays a critical role in the recognition of MHC class II antigens in SGVHD. The studies proposed in this application plan to characterize the autoreactive and regulatory effector T cells. The T-cell receptor repertoire with respect to a Valpha and Vbeta gene usage and the cytokine profile of the autoreactive T-cell subsets will be assessed in vitro by the reverse transcriptase polymerase chain reaction in clones from limiting dilution cultures. These results will be confirmed by in situ hybridization and immunoperoxidase analysis of the target tissue and by adoptive transfer studies with established autoclones. Structure function studies with modified CLIP peptides will be evaluated in order to identify the regions that allow autoimmune recognition in SGVHD. Thirdly, mechanisms by which CsA inhibits the clonal deletion or promotes selection of these autoreactive T cells in the thymus will be defined assessing if CsA inhibits clonal deletion of the MHC class II reactive T cells. Finally, studies will be performed to evaluate the mechanism by which the regulatory T cells recognize and inactivate the autoreactive T cells. Specifically, the ability of the autoregulatory T cells to recognize V beta T-cell receptor peptides presented by MHC class II determinants will be assessed. These studies will define some of the immunobiological mechanisms involved in this unique autoaggression syndrome. HESS AD JOHNS HOPKINS ONCOLOGY CENTER, 600 N WOLFE STREET, BALTIMORE, MD 21287-8985 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory rat cyclosporine bone marrow transplantation T lymphocyte polymerase chain reaction drug adverse effect leukocyte activation /transformation cytokine passive immunization immunoperoxidase autoantigen autoimmune disorder in situ hybridization T cell receptor clone cell graft versus host disease MHC class II antigen autologous transplantation invariant chain CRISP RPROJ MARYLAND G CRISP/99/AI24319-12 CRISP/99/AI24319-12 199904 eng SHIGA TOXIN MODE OF ACTION IN BACTERIAL DISEASE Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Hemolytic uremic syndrome (HUS) is a vascular disease with primary damage of the kidneys in which glomerular microcapillaries become occluded with fibrin and platelets. Escherichia coli 0157:H7 is an "emerging infectious disease" responsible for outbreaks of food-borne disease and is the leading cause of HUS and acute renal failure in young children. Most new information on HUS has come from studies related to the E. coli Shiga-like toxin (Stx)- producing dysentery bacteria and the subsequent appearance of HUS those individuals. The long-term goal is to describe, in biochemical terms, the mechanisms by which Stx's and host factors elicit the HUS disease state and to use this knowledge to develop effective preventative or therapeutic intervention modalities. The goal of the present study is to delineate the role of Stx's at the vascular endothelial cell level in the development of HUS. These studies utilize the human renal microvascular endothelial cell type which is believed to be the primary target of Stx's during the development of HUS. The research plan seeks to answer why some endothelial cell types are more sensitive to the Stx's than are others and why human renal endothelial cells are particularly sensitive to Stx2, as they recently demonstrated. The plan consists of four parts: 1) to examine the isoforms of Stx receptor (Gb3) produced by different endothelial cells, 2) to determine the nature of internalization and processing of Stx's by endothelial cells, 3) to study the biochemical pathways utilized by other bacterial and host factors (TNF-alpha & IL 1-beta) to sensitize endothelial cells to the E. coli Stx's, and 4) to identify those endothelial factors that contribute to coagulation in HUS. In summary, this research combines the areas of infectious disease and vascular physiology and has as its primary goal to provide an understanding of the mechanisms underlying the development of renal vascular disease in HUS. OBRIG TG UNIVERSITY OF VIRGINIA, BOX 133 HSC, CHARLOTTESVILLE, VA 22908 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 22908 Crisp Data Base National Institutes Of Health Escherichia coli Shigella bacterial disease bacterial cytopathogenic effect biological signal transduction hemolytic anemia vascular endothelium cellular pathology interleukin 1 tumor necrosis factor alpha enzyme linked immunosorbent assay bacterial toxin kidney circulation disorder renal failure lipopolysaccharide receptor expression antigen receptor tissue /cell culture toxin metabolism thrombomodulin CRISP RPROJ VIRGINIA G CRISP/99/AI24431-13 CRISP/99/AI24431-13 199904 eng LIPOOLIGOSACCHARIDE BIOSYNTHESIS IN NEISSERIACEAE Research RPROJ Neisseria gonorrhoeae is responsible for over l million cases of gonorrhea each year, and the total health care costs associated with treating gonorrhea and complications that arise from infections caused by this organism exceed l billion dollars per year. A closely related pathogen, N. meningitidis, causes significant morbidity and mortality worldwide. If effective vaccines to prevent these diseases were available, health care costs would be dramatically reduced. The gonococcus possesses a wide variety of surface structures that can serve as virulence determinants for pathogenesis. Lipooligosaccharide (LOS) is the least understood cell surface antigen, both chemically and genetically, even though these molecules mediate most of the toxic damage in fallopian tubes, are responsible for complement activation on the cell surface, and serve as the target for bactericidal antibody. Furthermore, antibody directed against lipopolysaccharide is protective for a variety of Gram-negative infections. A vaccine that stimulated an anti-LOS response might be protective for both gonococcal and meningococcal infections. Since gonococcal and meningococcal LOS share many epitopes, and all of the genes that have been identified that are involved in LOS biosynthesis are conserved in both species, information gleaned about LOS biosynthesis in one species will be directly applicable to the other. This proposal will focus on LOS biosynthesis in the gonococcus. It will identify by gene cloning and genetic complementation techniques, genes required for LOS biosynthesis, characterize the genes by DNA sequence analysis, define the biochemical properties of the gene products and determine the mechanisms by which the gonococcus regulates their synthesis. If LOS is to be exploited as a vaccine candidate, we need to have strains of the gonococcus that stably produce the desired epitopes. Since the gonococcus can vary its LOS, depending on the host, and local environment, studies on the genetic regulation of its synthesis are warranted. Well defined, stable LOS producing strains are needed if we are to dissect the role of LOS in the disease process. By understanding how the gonococcus makes LOS, it will be possible to construct strains with defined LOS. These strains will be the basis of future studies into the role of LOS in the disease process. STEIN DC UNIVERSITY OF MARYLAND, COLLEGE PARK, MD 20742 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 20742 Crisp Data Base National Institutes Of Health Escherichia coli genetic strain carbohydrate biosynthesis drug resistance molecular cloning plasmid gene complementation genetic regulation bacterial genetics gene deletion mutation mutant galactose bacterial antigen endotoxin microorganism metabolism bacterial DNA transposon /insertion element oligosaccharide lipopolysaccharide protein purification recombinant DNA SDS polyacrylamide gel electrophoresis sialyltransferase CRISP RPROJ MARYLAND G CRISP/99/AI24452-09 CRISP/99/AI24452-09 199904 eng GENETIC DETERMINATION OF VIRULENCE OF VIBRIO CHOLERAE Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The long range goal of the proposed work is to define the molecular components and mechanisms mediating V. cholerae colonization and virulence protein secretion to the point where there is sufficient knowledge to intelligently incorporate this information into improved cholera vaccine strategies and antimicrobial therapies designed to inhibit these events. Most of the proposal involves analysis of the molecular mechanisms by which TCP (toxin correlated pilus) is formed and mediates intestinal colonization. TCP is the major factor required for colonization by V. cholerae, with tcpA mutants of all epidemic serogroups and biotypes showing a 5 log decrease in colonization ability. A number of properties attributable to TCP that potentially contribute to colonization will be examined. These include a collaborative effort to solve the pilin crystallographic structure, study of the role of bacterial aggregates formed upon autoagglutination, search for specific receptor functions, study of the mechanisms of TcpA-mediated serum resistance, and further definition of potential immunogenic domains of TcpA. The practical application of incorporating this knowledge into the use of TcpA peptide vaccines will also be tested. In addition, because of the discovery that the cholera toxin genes are located on a lysogenic transducing phage that utilizes TCP as a receptor, the PI will seek tcpA mutations for incorporation and improved safety in live, oral vaccine strains that still allow colonization functions of TCP, but abolish the phage receptor or uptake function. Some steps in the process by which the pilus is built are also linked and related to the process of toxin and other virulence determinant secretion and represent potential targets for antimicrobial intervention. The PI will further examine two aspects of pilus biogenesis in detail; that of the processing of prepilin to mature pilin by the TcpJ type 4 prepilin peptidase, and the mechanism by which the TcpG (DsbA) periplasmic disulfide bond oxidoreductase interacts with its target substrates. These studies will involve mutational analyses to further define active sites involved in these events in conjunction with continuing collaborative efforts that have led to solving the crystallographic structure of TcpG, and will now include structures incorporating peptide substrates. Lastly, the use of the MshA pilin for surface antigen display in V. cholerae heterologous carrier vaccine strains will be examined. This pilus is immunogenic, yet dispensable for V. cholerae colonization, thus making it a candidate for pilin replacement by other type 4 pilins or partial substitution with heterologous epitopes. TAYLOR RK DARTMOUTH MEDICAL SCHOOL, VAIL BLDG, HANOVER, NH 03755-3842 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Vibrio cholerae genetic strain nuclear magnetic resonance spectroscopy X ray crystallography host organism interaction cholera site directed mutagenesis gene complementation structural gene bacterial genetics monoclonal antibody hemagglutinin cholera toxin virulence nucleic acid sequence immunoelectron microscopy oxidoreductase protein biosynthesis protein structure /function pilin active site suppressor mutation CRISP RPROJ NEW HAMPSHIRE G CRISP/99/AI25096-13 CRISP/99/AI25096-13 199904 eng SYNTHESIS AND BIOTRANSFORMATION OF ANTIHIV PRODRUGS Research RPROJ DESCRIPTION (Adapted from Applicant's Abstract): Despite the effectiveness of triple drug combinations in anti-HIV chemotherapy, there are still several critical issues remaining to be resolved in HIV chemotherapy. The first issue is the viral reservoir. Triple combination therapy (two nucleoside reverse transcriptase inhibitors plus one protease inhibitor) can virtually eliminate the HIV viral load to undetectable level in the serum in many HIV patients who are undergoing therapy. There are at least two other major compartments which should be considered: the central nervous and the lymphatic system. In this application, Dr. Chu will continue to study these two systems with some proposed prodrugs to improve their pharmacokinetics/ pharmacodynamic profiles. If any prodrugs are found to be promising during these studies, the focus will be on them to determine their potential as clinical candidates. Studies will include pharmacokinetic/pharmacodynamic studies in mice, rats and rhesus monkeys. In view of the number of children born with HIV infection, another area of focus is the maternal-fetal drug delivery issue which will be investigated using a rat model. Additionally, as drug interactions have become an increasingly important issue in HIV chemotherapy this will also be studied in vitro and in animal models to provide information on cellular, virological, pharmacological and toxicological interactions. CHU CK UNIVERSITY OF GEORGIA, 374 MEDICINAL CHEMISTRY, ATHENS, GA 30602-2352 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health adenosine deaminase Macaca mulatta laboratory mouse laboratory rat blood brain barrier mass spectrometry nuclear magnetic resonance spectroscopy combination chemotherapy pharmacokinetics prodrug drug adverse effect drug interaction drug design /synthesis /production AIDS AIDS dementia complex AIDS therapy antiAIDS agent biotransformation biological model placental transfer tissue /cell culture human immunodeficiency virus 2'3' dideoxynucleoside 2'3' dideoxyinosine CRISP RPROJ GEORGIA G CRISP/99/AI25899-12 CRISP/99/AI25899-12 199904 eng STAPHYLOCOCCAL ENTEROTOXINS--SUPERANTIGEN BRMS Research RPROJ Superantigens may play an important role in cancer, acquired immune deficiency syndrome (AIDS), and arthritis, and staphylococcal enterotoxins are prototype superantigens. The objective of this proposal is to determine the structural basis for staphylococcal enterotoxin superantigen activation of lymphocytes. In particular, the P.I. proposes to determine the structural basis for staphylococcal enterotoxin interaction with both major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and T-cell receptors (TCR) on responding T lymphocytes. This will entail determination of the following structures: certain staphylococcal enterotoxin superantigens, superantigen sites which interact with receptors, and receptor sites which are bound by these superantigens. These studies should help identify structural motifs which convey the property of superantigenicity, and provide insights into how to modulate such activity. The P.I. proposes to achieve the objective through the specific aims which follow: (1) Identify the binding site(s) on staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin-1 (TSST-1) for MHC class II molecules and TCR using peptides, recombinant native and mutant molecules, and their antibodies; (2) Determine the structures of SEA and TSST-1 peptides as well as recombinant native and mutant molecules to facilitate identification of structural motifs which convey the property of superantigenicity for possible identification of other superantigens in a variety of systems; (3) Determine the agonist / antagonist properties of SEA and TSST-1 receptor binding peptides and recombinant mutant molecules for enhancement of these properties through peptide engineering; (4) Identify the site(s) on MHC class II molecules and TCR that bind SEA and TSST-1; (5) Determine the modulating effect of enterotoxin superantigens on autoimmune diseases such as experimental allergic encephalomyelitis (EAE); and (6) Determine the structural basis for the interaction of the minor lymphocyte stimulating (Mls) superantigens with MHC class II molecules and TCR and its possible relationship to staphylococcal enterotoxin binding to receptors. JOHNSON HM UNIVERSITY OF FLORIDA, BLDG 981, RM 1052, GAINEVILLE, FL 32611 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 32611 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse biological response modifier toxic shock syndrome T lymphocyte polymerase chain reaction gene mutation human tissue leukocyte activation /transformation antireceptor antibody staphylococcal enterotoxin experimental allergic encephalomyelitis circular dichroism synthetic peptide high performance liquid chromatography protein structure /function receptor binding T cell receptor tissue /cell culture histocompatibility antigen MHC class II antigen recombinant protein superantigen CRISP RPROJ FLORIDA G CRISP/99/AI25904-11 CRISP/99/AI25904-11 199904 eng MUCOSAL IMMUNITY AND PROTECTION Research BOSCH ML UNIVERSITY OF WASHINGTON, BOX 357330, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 98195 Crisp Data Base National Institutes Of Health laboratory mouse Escherichia coli flow cytometry postmortem humoral immunity cell mediated lymphocytolysis test enzyme linked immunosorbent assay bacterial antigen enterotoxin virus antigen immunomodulator HIV infection nonhuman therapy evaluation live vaccine AIDS vaccine vaccinia virus human immunodeficiency virus 1 simian immunodeficiency virus Macaca nemestrina recombinant protein HIV envelope protein gp160 HIV envelope protein mucosal immunity recombinant virus transcytosis vector vaccine CRISP RPROJ WASHINGTON G CRISP/99/AI26503-100006 CRISP/99/AI26503-100006 199904 eng LISTERIA HEMOLYSIN AND ESCAPE FROM A VACUOLE Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Listeria monocytogenes is a model facultative intracellular pathogen which primarily infects pregnant women and immunocompromised individuals. A primary determinant of L. monocytogenes pathogenesis is a secreted pore-forming protein referred to as listeriolysin O (LLO). LLO is largely responsible for rupture of the host vacuole which results from phagocytosis. Perfringolysin O (PFO) is a related pore-forming protein which is involved in the pathogenesis of infections by an extracellular pathogen. When expressed by L. monocytogenes, PFO mediates escape from the phagocytic vacuole, but is toxic to the cell. Normally, LLO is continually expressed during infection, but in contrast to PFO, it is proteolytically degraded in the cytosol and presented on the cell surface in association with MHC class I molecules. It is hypothesized that pH optimum and cytosolic processing are essential LLO-encoded determinants which distinguish it from PFO. The focus of the current proposal is to define the precise structural and mechanistic features of LLO which differentiate it from other members of the family of thiol-activated cytolysins, facilitate its intravacuolar activity, and direct its fate in the cytosol. In Aim I, protein sequences responsible for LLO's acidic pH optimum and processing in the host cytosol will be identified. This will be accomplished by domain and sub-domain swapping between LLO and PFO, and modified charged-to-alanine scanning mutagenesis. The LLO/PFO chimeras will be purified from E. coli and characterized biochemically. Next, the chimeras will be introduced into L. monocytogenes and characterized in tissue culture models of infection., In Aim II, the pathway of LLO processing in the host cytosol will be fully evaluated. LLO will be identified by metabolic labeling of L. monocytogenes within infected host cells, followed by immunoprecipitation. Precursor/product relationships will be determined by pulse-chase experiments. Specific inhibitors will be used to evaluate the role of the proteosome and other proteases in degradation. The role of LLO phosphorylation will be evaluated biochemically and genetically. In Aim III, the precise nature of the L. monocytogenes phagosome will be characterized with regard to pH, time of perforation, and markers of the endosome/lysosome pathway of maturation. The role of pH optimum and the L. monocytogenes phospholipases will be evaluated by using mutant and chimeric strains. In Aim IV, the investigators will evaluate the feasibility of using E. coli K12 expressing LLO and a recombinant protein as a novel system to introduce foreign proteins into the mammalian cytosol for antigen presentation. PORTNOY DA UNIVERSITY OF CALIFORNIA, BERKELEY, CA 94720-3202 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Listeria Escherichia coli Listeria infection macrophage hemolysin acid base balance vesicle /vacuole phagocytosis host organism interaction site directed mutagenesis molecular cloning plasmid antigen presentation virulence intracellular parasitism posttranslational modification protein sequence pore forming protein tissue /cell culture transfection vector recombinant protein CRISP RPROJ CALIFORNIA G CRISP/99/AI27655-11 CRISP/99/AI27655-11 199904 eng MECHANISMS OF COMPLEMENT RESISTANCE BY NAEGLERIA Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Naegleria fowleri is the causative agent of Primary Amebic Meningoencephalitis a rapidly fatal disease of the central nervous system. Pathogenic and nonpathogenic species have been described, but the determinants of pathogenicity and virulence are unknown. Dr. Marciano have demonstrated that an important determinant of virulence is the ability of the ameba to resist complement-mediated lysis. Therefore, the goal of this study is to define the mechanisms by which highly-pathogenic strains of N. fowleri develop resistance to complement.In conducting these studies, N. fowleri LEEmp, a highly-pathogenic strain and N. gruberi, a non-pathogenic strain will be employed. The rationale for using theses strains is that both pathogenic and nonpathogenic ameba activate the complement cascade, but pathogenic strains are complement-resistant while nonpathogenic strains are complement-sensitive.The applicant has determined that complement-resistant Naegleria have developed at least two mechanisms for rendering the membrane attack complex nonfunctional. Under conditions of low serum concentrations, complement-resistant Naegleria remove the assembled membrane attack complex from their surface by vesiculation. Alternatively, in the presence of high concentrations of serum, complement regulatory proteins may play a more prominent role in resistance to complement-mediated lysis. The involvement of surface proteins in resistance to complement has been demonstrated in experiments in which treatment of N. fowleri trophozoites with trypsin, papain, endoglycosidase H or tunicamycin, converted complement-resistant amebae to complement-sensitive organisms. Also, antiserum to CD59, a complement regulatory protein on erythrocytes and platelets, cross- reacts with a Naegleria protein of approximate molecular mass of 42-44 kDa. Their data suggest that surface membrane proteins, especially glycoproteins, of highly pathogenic amebae play a role in resistance to complement-mediated lysis by inhibiting the lytic membrane attack complex. They propose to test the hypothesis that cell surface membrane modifications of N. fowleri account for their resistance to complement lysis. In order to accomplish these objectives the following specific aims will serve as guidelines to the research: 1. To determine the cellular events that regulate ameba membrane vesiculation in response to complement which lysis to protection from complement-mediated lysis. The role of calcium ions, the activity of protein kinases, and cytoskeletal changes within Naegleria will be determined since these may play an important role in vesiculation. 2. To purify the Naegleria proteins GP28, GP42-44, GP49/50, GP62, GP65, and P95 which have been shown to be upregulated in Naegleria during the transformation from a state of complement-sensitive to that of complement-resistant. The basic biochemical properties of these p MARCIANO-CABRAL F VIRGINIA COMMONWEALTH UNIVERSI, BOX 980678, RICHMOND, VA 23298-0678 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health genetic strain calcium flux vesicle /vacuole polymerase chain reaction host organism interaction heme protozoal antigen complement complement inhibitor complement pathway virulence protein kinase C high performance liquid chromatography glycoprotein Sarcodina cytochalasin CRISP RPROJ VIRGINIA G CRISP/99/AI27807-04 CRISP/99/AI27807-04 199904 eng MOLECULAR ANALYSIS OF STAPHYLOCOCCAL TYPE C ENTEROTOXINS Research RPROJ Staphylococcal enterotoxins (SE) are unusual bacterial exotoxins in that they have the ability to cause two distinct human illnesses, food poisoning and toxic shock syndrome (TSS). SE belong to a family of pyrogenic toxins (PT) produced by Staphylococcus aureus and Streptococcus pyogenes. The PT family, which contains prototypic microbial superantigens, shares a set of immunomodulating biological properties that appear to contribute to the pathogenesis of TSS. SE association with good poisoning results from a separate ability to induce emesis when ingested. We formed several hypotheses to analyze the structural and functional organization of PT using the Type C SE (SEC) group as a model. The first three aims will determine residues necessary for emesis or T cell receptor and Class II MHC binding by mutation of SEC1 nd SEC3, subtypes on which we have the most detailed structure-function data. Information made available by our solution of the SEC3 crystal structure has allowed us to prioritize regions of SEC molecules and provided a logical approach to focus our investigation. A unique aspect of this proposal is our ability to address whether altered biological activity is due to changes in conformation rather than modification of critical residues. This will be accomplished through crystallographic analysis in addition to conventional methods. Since the SEC3 structure has already been solved, refinement to higher resolution (Aim 5) and structural determination of mutants can be done in a timely manner. As part of the mapping of functional molecular regions, we will also identify immunogenic regions on the three classical SEC subtypes, in addition to five novel SEC variants that we recently identified, as well as their ability to induce a protective immune response. Information on the topology of functional regions will contribute to understanding the molecular and immunological mechanisms by which these toxins mediate disease. Potential practical applications such as toxoid and immunotherapeutic agent development requires a dissection of critical toxic and immunogenic regions. BOHACH GA UNIVERSITY OF IDAHO, AND BIOLOGY AND BIOCHEMISTRY, MOSCOW, ID 83844-3052 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health aminoacid laboratory rabbit laboratory mouse Staphylococcus aureus toxic shock syndrome X ray crystallography bacterial food poisoning emesis molecular cloning mutant human subject cytokine epitope mapping bacterial antigen staphylococcal enterotoxin zinc virulence synthetic peptide protein purification protein sequence protein structure /function T cell receptor pyrogen MHC class II antigen Macaca nemestrina CRISP RPROJ IDAHO G CRISP/99/AI28401-09 CRISP/99/AI28401-09 199904 eng SELECTABLE MARKERS AND MOSQUITO TRANSFORMATION Research FFRENCH-CONSTANT RH UNIVERSITY OF WISCONSIN, 1655 LINDEN DRIVE, MADISON, WI 53706 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 53706 Crisp Data Base National Institutes Of Health acetylcholinesterase disease /disorder proneness /risk host organism interaction genetic manipulation genetic marker genetic promoter element gene mutation genetic recombination genetic transduction dihydrofolate reductase pesticide resistance clone cell Aedes CRISP RPROJ WISCONSIN G CRISP/99/AI28781-090007 CRISP/99/AI28781-090007 199904 eng VIRUS EXPRESSION SYSTEMS IN MOSQUITO BIOLOGY Research BEATTY BJ UNIVERSITY OF WISCONSIN, 1655 LINDEN DRIVE, MADISON, WI 53706 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 53706 Crisp Data Base National Institutes Of Health larva laboratory mouse transgenic animal polymerase chain reaction drug delivery system juvenile hormone host organism interaction esterase genetic manipulation gene expression genetic transduction western blotting in situ hybridization arthropod nonpollutant control insecticide Togaviridae Sindbis virus insect virus serine proteinase Aedes transfection vector CRISP RPROJ WISCONSIN G CRISP/99/AI28781-090008 CRISP/99/AI28781-090008 199904 eng FUNCTIONAL DOMAINS OF BACILLUS THURINGIENSIS ENDOTOXIN Research RPROJ Bacillus thuringiensis is a microbial insecticide that is widely used to control numerous insects, including agricultural pests; and, mosquitoes and blackflies, vectors of human diseases. The active components are the insecticidal crystal proteins or Cry toxins. These insecticidal proteins may be manipulated by genetic and protein engineering to alter and improve their activity. The overall goal of this project is to investigate in detail the binding of several Cry toxins to a known receptor and improve the insecticidal activity by improving the binding to the receptor. The specific aims are to identify the amino acid residues on the Cry toxins that interact with the receptor and to experimentally alter these residues to identify which substituted residues enhance binding and toxicity. This will be accomplished by the genetic technique of site-directed mutagenesis. The components on the receptor that are responsible for interaction with the toxin will also be determined biochemically. The proposed research employs a model system consisting of a cry toxins of known structure as a well characterized receptor. When the basic understanding of toxin-receptor interaction is obtained, this may used to improve Cry toxins against the mosquito. DEAN DH OHIO STATE UNIVERSITY, 484 WEST 12TH AVENUE, COLMBUS, OH 43210 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 43210 Crisp Data Base National Institutes Of Health aminoacid Lepidoptera bioassay mass spectrometry patch clamp site directed mutagenesis endotoxin pest control insecticide biological effect pesticide residue pesticide interaction affinity chromatography high performance liquid chromatography ion exchange chromatography aminopeptidase protein structure /function bacterial protein receptor binding tissue /cell culture cytotoxicity SDS polyacrylamide gel electrophoresis CRISP RPROJ OHIO G CRISP/99/AI29092-08 CRISP/99/AI29092-08 199904 eng BIOCHEMISTRY AND FUNCTION OF NOVEL MONOKINES Research RPROJ DESCRIPTION (Adapted from Investigator's Abstract): The overall goal of this project is to characterize the role played by MIP-1a and MIP-1b in host defense against bacterial, viral and parasite invasion. Despite medical advances, the activation of host defenses is an important determining factor in the outcome of infection. Effective antimicrobial host defense requires the generation of a vigorous inflammatory response that involves the recruitment and activation of specific leukocyte populations. These processes depend on chemotactic signals, largely supplied by a class of cytokines termed chemokines. An aim, since discovering MIP-1a and MIP-1b, two members of the chemokine family, has been to characterize the role each plays in host defense against bacterial, viral, and parasite invasion. Progress made in the prior two funding periods of this grant, in combination with the work of others in the field of chemokine biology, has revealed that b-chemokine expression is upregulated in response to many inflammatory stimuli, and that there is differential control of these molecules in several animal disease models. This control is critical because inflammatory reactions that are disproportionate to the magnitude of the immune challenge can be harmful, or even fatal, to the host. The new objective is to better define the molecular mechanisms by which the b-chemokines work together, and in opposition, to modulate the cellular and systemic responses of the host to invasion, and in particular we plan to evaluate their role, pathogenic versus protective, in three specific disease states (septic shock, HIV infection, and malaria infection) where we have shown them to be specifically upregulated. With regard to sepsis, this will be addressed by passive immunization of mice with MIP-1a versus MIP-1b specific antibodies, and by direct administration of recombinant chemokines in experimental endotoxemia. It will also be determined whether the protective effects of MIP-1b in sepsis are related to its antagonism of MIP-1a through either competition for, or downregulation of, specific chemokine receptors, or whether other mechanisms are at work. With regard to HIV infection, it will be established which molecular mechanisms lead from HIV infection to chemokine production in macrophages, and addressed why b-chemokines are not protective in macrophages, as they have been recently shown to be in T cells. With regard to malaria, where MIP-1a exerts a beneficial effect, the course of infection in MIP-1a knockout mice and wild-type mice is to be compared, as well as in mice passively administered b-chemokine-specific antibodies, in order to determine whether MIP-1b positively or negatively impacts the course of infection. Hopefully, these studies will eventually lead to the development of intervention strategies to benefit the host in times of invasion. SHERRY BA THE PICOWER INST FOR MED RES, 350 COMMUNITY DRIVE, MANHASSET, NY 11030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 11030 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal macrophage polymerase chain reaction inflammation host organism interaction histology human subject cellular immunity passive immunization immunocytochemistry HIV infection disease model malaria protein structure /function receptor expression cytokine receptor tissue /cell culture measles virus human immunodeficiency virus 1 infection nitric oxide recombinant protein gene targeting septic shock chemokine macrophage inflammatory protein CRISP RPROJ NEW YORK G CRISP/99/AI29110-10 CRISP/99/AI29110-10 199904 eng MECHANISMS OF SELECTIVE ACTIONS OF ANTIVIRAL AGENTS Research RPROJ DESCRIPTION (Adapted from the Investigator's Abstract): The long term goal of the proposed research in this application is to determine the selective basis of host cell response (sensitivity or resistance) to antiviral dideoxynucleosides (ddN). Prolonged therapy of AIDS patients with anti-HIV ddN, in addition to selecting drug resistant HIV-1 variants, may induce cellular resistance because of their dependence on the host cells' activating enzymes. The research effort in this area remains extremely limited. The Principal Investigator observed that chronic exposure to 0.5 ZM dideoxycytidine (ddC) in vitro induced drug resistance H9 lymphocytic cells (H9-ddC cells). These cells compensated for impaired mitochondrial functions and had lower deoxycytidine kinase (dC kinase) activity and ddCTP levels. The aim of this proposal is to study biochemical basis of the ddC resistance and whether such a mechanism operates in the patients undergoing ddC therapy. Therefore, the following specific aims will be investigated (i) mechanisms of the reduced dC kinase activity and ddCTP levels; (ii) compensatory mechanisms for impaired mitochondrial functions in the resistant cells; (iii); differences in ddC resistance from that of its analog arabinosyl-cytosine (ara-C); and (iv) development of cellular resistance in the patients treated with ddC. These goals will be achieved by studying kinetics and genetic expression of dC kinase gene, cellular nucleotide pool, cross resistance to other nucleoside analogs, and mitochondrial DNA content structure and number per cell H9, H9-ddC, CEM, CEM-araC cells and the lymphocytes and autopsy specimens from the patients who had been treated with ddC. It is expected that these studies will enhance understanding of the host cell determinants responsible for ddC antiviral activity and may lead to strategies for improved chemotherapy. AGARWAL RP UNIVERSITY OF MIAMI SCH OF MED, 1550 NW 10TH AVE (M862), MIAMI, FL 33136 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 33136 Crisp Data Base National Institutes Of Health mitochondria antiviral agent drug metabolism drug adverse effect drug resistance drug screening /evaluation human tissue AIDS therapy zidovudine thymidine kinase cytosine arabinoside tissue /cell culture human immunodeficiency virus 1 alcohol phosphotransferase 2'3' dideoxynucleoside 2'3' dideoxycytidine enzyme activity CRISP RPROJ FLORIDA G CRISP/99/AI29155-09 CRISP/99/AI29155-09 199904 eng RECOMBINANT AND LIVE ORAL SALMONELLA TYPHI HYBRID VACCINES Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The overall goal of the proposed research is to develop, through appropriate manipulations of a suitable attenuated Salmonella typhi live vector, a mucosally-administered multivalent vaccine to prevent diphtheria, pertussis, and tetanus (i.e., a mucosal DTP vaccine). To accomplish this, the PI will initially successfully express within attenuate S. typhi appropriate protective antigens from C. diphtheriae, B. pertussis, and C. tetani and confirm, using the murine intranasal model of immunogenicity, that such constructs elicit the relevant types of immune responses. The PI and his colleagues will then attempt to improve such immune responses in several ways: (1) relevant proteins will be expressed in attenuated S. typhi strains in which expression of the Vi capsular polysaccharide has either been removed or expression has been made constitutive. (2) A fusion protein consisting of fragment C of tetanus toxin fused to the truncated S1 subunit of pertussis toxin, used as a test antigen, will be secreted extracellularly using the E. coli hemolysin and secretion apparatus. (3) Proteins will be expressed from stabilized plasmids which encode a critical enzyme necessary for survival of the attenuated S. typhi carrier strain. (4) The fragment C-S1 fusion protein will be expressed in S. typhi live vector strains which will co-express either a mutant heat-labile enterotoxin of E. coli (the K63 LT holotoxoid) that functions as a powerful adjuvant yet does not cause intestinal secretion or will co-express the IL-4 cytokine (which has the effect of enhancing antibody responses). The PI and his colleagues will make amino acid substitutions in the NAD-binding region of diphtheria toxin, aiming to construct a stable mutant that lacks enzymatic (i.e., toxic) activity, but retains the ability to stimulate neutralizing antitoxin. It is expected that by the end of the research plan, all the individual constructs necessary to stimulate protective immune responses will have been constructed, their immunogenicity established in the mouse intranasal immunization model and modifications selected to enhance the specific immune responses. This will set the stage for a future effort that would examine the immunogenicity of a prototype multivalent DTP vaccine consisting of a mixture of the optimized CVD908-htrA constructs expressing diphtheria, pertussis and tetanus antigens. LEVINE MM UNIV OF MARYLAND, 685 WEST BALTIMORE ST, RM 480, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health laboratory mouse Clostridium Bordetella pertussis Corynebacterium diphtheriae Salmonella typhi clostridial tetanus hemolysin molecular cloning gene expression bactericidal immunity interleukin 4 active immunization bacterial antigen bacterial toxin attenuated microorganism protein engineering chimeric protein Salmonella vaccine oral administration mucosal immunity vaccine development vector vaccine CRISP RPROJ MARYLAND G CRISP/99/AI29471-08A1 CRISP/99/AI29471-08A1 199904 eng SUPERANTIGENS AND THE MHC--STRUCTURE AND FUNCTION Research RPROJ We propose to continue our studies of the superantigen (Sag) exotoxins produced by pathogenetic strains of Staphylococcus aureus and Streptococcus pyogenes. We will focus primarily on four members of this family, the staphylococcal enterotoxins SEA, SEB and SEE and the related streptococcal superantigen SSA, which we isolated and characterized during the initial period of support. These Sag are chosen for study because of functional and structural properties that make them particularly suitable for answering the questions posed and because of the valuable library of molecular variants of the relevant genes and their products that we have developed. Our overall objective is to enhance understanding of the interaction between bacterial parasites and mammalian hosts and the mechanisms whereby Sag alter this relationship. Five specific aims are proposed: 1) Definition of those elements of the staphylococcal enterotoxins SEA and SEE and of T cell receptors that determine their interactions with one another; 2) Characterization of class II-independent Sag presentation, including definition of the responsible cell surface ligand; 3) Comparative studies of SEB with SSA and its alleles and mutants, based on structural resolution and functional analysis in order to explore the relationship between Sag potency, MHC class II and TCR interactions; 4) Analysis of the mechanism of interaction between SEA and the affinity ligand red dye A to determine whether definition of the biochemical specificity of this interaction can provide insight into biological activities of bacterial Sag and to extend use of this strategy for the identification of additional novel Sag, e.g., from M. tuberculosis; and 5) Use of SEA insertion and deletion mutants of S. aureus to assess the role of SEA in S. aureus pathogenesis in mice and to determine effects of infection with an enterotoxin-producing strain on host immune responses. We believe that the experiments proposed are conceptually novel and the experiments designed to answer them general decisive. They can be expected to improve understanding of fundamental aspects of mammalian immune response to Sag and Sag-producing bacterial pathogens and to provide insight into pathogenetic mechanisms of Sag-mediated human disease. RICH RR BAYLOR COLLEGE OF MEDICINE, ONE BAYLOR PLAZA CTR, HOUSTON, TX 77030-3498 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Mycobacterium tuberculosis Streptococcus T lymphocyte dye human tissue humoral immunity immunologic memory antigen presentation bacterial antigen staphylococcal enterotoxin protein structure /function receptor binding T cell receptor tissue /cell culture MHC class II antigen superantigen CRISP RPROJ TEXAS G CRISP/99/AI30036-08 CRISP/99/AI30036-08 199904 eng CELLULAR INVASION BY GROUP B STREPTOCOCCUS Research RPROJ Group B streptococci (GBS) are the most common cause of bacterial sepsis and meningitIs in the newborn infant. Although an extensive literature exists describing the immunology and epidemiology of GBS infections in neonates, very little information exists on the specific mechanisms this organism uses to induce disease. We had previously demonstrated, in an in utero model of neonatal sepsis induced by GBS in subhuman primates, that this organism is capable of invading alveolar respiratory epithelial cells. Based on this data, we developed in vitro assays for investigating epithelial cell invasion by GBS. During the first two years of the funded proposal, we demonstrated that GBS were capable of invading respiratory epithelial cells and identified some of the important bacterial and cellular processes necessary for invasion. During the current year, we have investigated the ability of GBS to traverse an intact polar epithelial monolayer in vitro. In addition, we have begun to generate isogenic invasion mutants by transposon mutagenesis in order to define the molecular pathogenesis of cellular invasion by this organism. To extend our previous observations, we propose to continue to use transposon mutagenesis to derive isogenic mutant strains of GBS with altered abilities to invade epithelial cells in vitro. Mutants which invade poorly relative to the parent strain, remain as adherent to epithelial cells and are otherwise unchanged in their phenotype, will be analyzed further. The genes important for invasion determinants, identified by transposon mutagenesis or genomic cloning into B. subtilis, will be analyzed by nucleotide sequencing, and the gene products identified using standardized gene expression assays. The ability of the wild type invasion genes to complement these mutants will be determined by using cloning vectors and transformation procedures we have recently adapted for GBS. The virulence of well characterized invasion mutants will be tested for their ability to induce GBS infections in neonatal rat GBS infection models compared to the parent strain, confirming the relevance of our in vitro findings. Effects of the mutations on GBS surface composition or secretion of extracellular products, the ability of surface extracts of GBS to augment invasion, and the induction of proteins from GBS during the intracellular state, will be investigated in pilot experiments for future studies. We anticipate these studies should begin to identify the bacterial traits important in the early steps in the pathogenesis of neonatal infections caused by group B streptococci. RUBENS CE CHILDREN'S HOSPITAL & MED CTR, 4800 SAND POINT WAY NE, SEATTLE, WA 98105-0371 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health newborn animal laboratory rat Bacillus subtilis Streptococcus agalactiae Streptococcus infection blood toxicology congenital infection pathologic process site directed mutagenesis molecular cloning open reading frame gene mutation mutant histopathology western blotting virulence disease model transposon /insertion element nucleic acid sequence pregnancy infection CRISP RPROJ WASHINGTON G CRISP/99/AI30068-08 CRISP/99/AI30068-08 199904 eng MOLECULAR GENETICS OF EXOTOXIN REGULATION IN S AUREUS Research RPROJ We have previously identified, cloned, sequenced and preliminarily characterized a complex polycistronic regulatory element, agr, that controls the post-exponential phase synthesis of virulence factors and other exoproteins in Staphylococcus aureus. The 3.3 kb chromosomal agr locus consists of two divergent operons transcribed from promoters P2 and P3. The P2 operon contains four genes, agrA, B, C and D. AgrA and AgrC resemble the two components of the bacterial signal transduction systems and respond to a metabolic signal early in exponential phase by activating P2 and P3, making the system autocatalytic. The P3 operon encodes an agr- regulated exoprotein delta-hemolysin, and its 0.5 kb transcript, RNAIII, is the agr-generated regulator of other, unlinked genes. We have shown that in some strains agr is activated early in exponential phase but that the target genes do not respond until some 2 h later and that a second agr-independent post-exponential phase signal (PEPS) is also required. Protein synthesis-inhibitors such as erythromycin and chloramphenicol cause the immediate activation of target gene transcription, bypassing both agr and the PEPS, suggesting that labile regulatory proteins are involved. We have also observed that the 5' end of RNAIII is required for translation of alpha-hemolysin and that delta- hemolysin is not translated until one h after the appearance of RNAIII. On the basis of these and other findings we have developed a working model in which five distinct stages are discerned in the exoprotein regulatory pathway. A broad based continuation of these studies is proposed, focussing on the different stages in the activation pathway and the mechanisms underlying the activation steps at each stage. Particular attention will continue to be paid to the structure and mechanism of action of the remarkable RNA molecule, RNAIII, that is the intracellular effector of the exoprotein response. We have discovered an external activator and an external inhibitor of the response and studies are proposed to test these for possible therapeutic use in staphylococcal disease. NOVICK RP NEW YORK UNIVERSITY MEDICAL CT, SKIRBALL INSTITUTE, 2ND FLOOR 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Staphylococcus aureus biological signal transduction hemolysin polymerase chain reaction molecular cloning open reading frame operon regulatory gene genetic regulatory element genetic promoter element genetic transcription transcription factor gene induction /repression bacterial genetics gene mutation microorganism culture exotoxin RNA DNA footprinting nucleic acid structure gel electrophoresis protein structure /function bacterial toxicology northern blotting southern blotting CRISP RPROJ NEW YORK G CRISP/99/AI30138-09 CRISP/99/AI30138-09 199904 eng PHYSIOLOGIC PHOSPHOLIPASE A2 ACTIVATION IN NEUTROPHILS Research RPROJ Platelet activating factor (PAF) and leukotriene B4 (LTB4) formation by human polymorphonuclear neutrophils (PMN) is an important aspect of normal PMN function but overproduction of these lipid mediators has been correlated with tissue damage occurring during acute and chronic inflammation. Activation of phospholipase A2 (PLA2) is the initial step in production of LTB4 and PAF and demonstration that PMN contain two calcium-dependent PLA2/s, a "cytosolic" enzyme, cPLA2, and a "secretory" enzyme, sPLA2, has led to intense interest in determining whether one or both are responsible for their formation. This proposal will utilize a permeabilized cell model where cPLA2 is specifically activated in the absence of sPLA2 activity and where both PAF and LTB4 formation occur to determine the actual mechanisms involved in cPLA2 activation. While progress has been made in understanding cPLA2 activation, the question of what mechanism links cell stimulation to cPLA2 activation is still unanswered. In the permeabilized cell model, phospholipase D (PLD) catalyzed production of phosphatidic acid (PA) and diglyceride (DG) are always present when cPLA2 activation occurs, inhibiting PLD activity blocks cPLA2 activation, and adding PA and DG, individually, back to permeabilized cells partially restores cPLA2 activity while a combination of both reconstitutes enzymatic activity. Enhanced PAF and LTB4 formation can be induced in vitro by pretreating PMN with one agonist before stimulation with a second agent by a process called "primed-stimulation". In the permeabilized cell model increased LTB4 and PAF formation occur when cells are primed with the cytokine, tumor necrosis factor, before stimulation. The increased cPLA2 activity is paralleled by enhanced PA and DG formation and blocking PLD activity in primed cells leads to a parallel decrease in LTB4 and PAF production. This proposal will test the hypothesis that PA and DG function as second messengers in both direct activation of cPLA2 and in enhanced cPLA2 activity occurring in primed-stimulation of human PMN. Studies will utilize PMN permeabilized with Staphylococcus aureus alpha-toxin where cPLA2 is maintained in situ and enzyme activation is induced by addition of calcium, guanine nucleotides, and stimulation with a physiologic agonist. In this model, each agent can be added individually or in combination and effects on enzyme activation monitored. Specific Aim 1 will focus on determining the interrelationship between cPLA2 activation and PLD activity, will determine the relative importance of PA and DG in inducing cPLA2 activation and will define the molecular species of PA and DG that effect cPLA2 activity. In Specific Aim 2, requirements for translocation of cPLA2 from cytosol to membrane, for phosphorylation of cPLA2, for activation of the MAP kinase cascade and for activation of protein kinase C will first be determined in the permeabilized PMN, then in cells where PLD activity is blocked and finally in cells where enzyme activity is reconstituted with PA and DG to determine which steps in the signal transduction pathway are effected by PA and DG. This may identify new targets for therapies to combat excessive LTB4 and PAF production occurring during acute and chronic inflammation. BAULDRY SA BOWMAN GRAY SCHOOL OF MEDICINE, MEDICAL CENTER BLVD, WINSTON-SALEM, NC 27157-1054 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health biological signal transduction second messenger membrane permeability neutrophil platelet activating factor calcium indicator gas chromatography mass spectrometry inflammation enzyme induction /repression enzyme mechanism leukotriene human subject leukocyte activation /transformation western blotting bacterial toxin diacylglycerol phospholipase A2 phospholipase D phosphatidate protein kinase C liquid chromatography gel electrophoresis radiotracer enzyme activity mitogen activated protein kinase CRISP RPROJ NORTH CAROLINA G CRISP/99/AI30142-08 CRISP/99/AI30142-08 199904 eng ACELLULAR VACCINES AGAINST BACTERIAL PATHOGENS Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The goal of this proposal is to contribute to the generation of effective acellular vaccines to prevent pertussis and Pseudomonas aeruginosa infections. The focus of this research is to define functional residues and domains of pertussis toxin and Pseudomonas exoenzyme S which will lead to the generation of noncatalytic toxoids that can be tested as vaccine candidates. These studies will also determine the mechanism by which pertussis toxin intoxicates eukaryotic cells and presents strategies for engineering a trivalent a cellular vaccine to prevent diphtheria, pertussis and tetanus. The specific aims of this proposal are to: (i) define the kinetic constants of mutants of the S1 subunit of PT that express reduced enzymatic activity. These studies will define residues that contribute to NAD binding, G protein binding, and catalysis; (ii) determine how pertussistoxin ADP-ribosylates both plasma membrane- and Golgi- associated G(i) proteins. Experiments will define the rate of ADP- ribosylation of plasma membrane- and Golgi-associated G(i) proteins and correlate this data with the physical localization of S1 during the entry of PT into sensitive cells. These studies will also determine the contribution of the ADP-ribosylation of plasma membrane- and Golgi-associated G proteins in the pathogenesis of pertussis toxin; (iii) identify the minimal amino acid sequence of S1 required for association with B oligomer; (iv) engineer a recombinant trivalent diphtheria-pertussis-tetanus vaccine composed of non-toxic deletion peptides of each respective toxin. These fusion proteins will be analyzed for the elicitation of a neutralizing immune response against their respective toxin; and (v) define the kinetic constants of recombinant Pseudomonas exoenzyme S, which ADP-ribosylates the Ras protooncogene product, and identify residues that contribute to the catalysis of exoenzyme S. Mapping functional residues and domains of pertussis toxin and exoenzyme S will define rational approaches for the production of acellular pertussis and Pseudomonas vaccines. Parallel studies on pertussis toxin and exoenzyme S will define relationships between these two members of the family of bacterial ADP-ribosylating exotoxins and may identify new strategies for engineering effective toxoids against other bacterial pathogens. BARBIERI JT MEDICAL COLLEGE OF WISCONSIN, 8701 WATERTOWN PLANK ROAD, MILWAUKEE, WI 53226 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 53226 Crisp Data Base National Institutes Of Health laboratory rabbit guinea pig laboratory mouse Bordetella pertussis Pseudomonas aeruginosa pertussis cell membrane Golgi apparatus chemical association enzyme structure exoenzyme ADP ribosylation adenine phosphoribosyltransferase immunologic preparation diphtheria toxin exotoxin pertussis toxin tetanus toxin protein sequence G protein chimeric protein proteolysis pertussis vaccine CHO cell enzyme activity vaccine development CRISP RPROJ WISCONSIN G CRISP/99/AI30162-07 CRISP/99/AI30162-07 199904 eng IMMUNE RESPONSE TO S PNEUMONIAE CAPSULAR POLYSACCHARIDE Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Streptococcus pneumoniae with capsular polysaccharide (PS) especially those with 6A and 6B serotype capsule, are significant human pathogens and often antibiotic resistant. Vaccines against S. pneumoniae are being developed using capsular PS, which can elicit protective antibodies (AB) against infection with elicit Ab to 6A capsular PS in humans. Current vaccines as well as the conjugate vaccines under development contain only serotype 6B PS. They have found that 10 to 25 percent of vaccines may produce Ab which poorly opsonize S. pneumoniae 6A serotype. Furthermore, by inducing immune memory for ineffective Ab these vaccines may alter one's subsequent immune response to S. pneumoniae 6A ( original antigenic sin ). They have also found that anti-6A Ab are often derived from two V lambda 2 family genes, products of which express 8.12 idiotype, a marker for nephritogenic Ab to dsDNA. Pneumococcal infections may prime B cells for production of anti dsDNA Ab. Specific aims of their study are to: A) Study clinical relevance of Ab poorly opsonizing S. pneumoniae 6A serotype, by determining their in vivo protective activity and frequency of individuals with poorly opsonic (to 6A) Ab among infant and elderly vaccines. B) Study pneumococcal vaccination for potential induction of immune memory for Ab poorly opsonizing S. pneumoniae 6A serotype, including the impact on immune memory development (antigenic sin) in SCID mice. C) Study pneumococcal vaccination for potential priming of B cells which later produce anti dsDNA Ab. A key assumption in formulating S. pneumoniae vaccines is that cross reactive Ab are functional. So far, the function of cross reactive Ab has not been critically examined. If their study finds that 6B PS often induces Ab with little protective function against S. pneumoniae 6A, then the vaccine formulation may need to be modified. PS protein conjugate vaccines are promising approaches for immunizing children against many PS antigens from bacterial pathogens. Yet their immunobiology is still poorly understood. The investigator's long term research goal is to study immunobiology of PS and PS protein conjugate vaccines. NAHM MH UNIVERSITY OF ROCHESTER, 601 ELMWOOD AVENUE, ROCHESTER, N Y 14642 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 14642 Crisp Data Base National Institutes Of Health adult human (19+) laboratory mouse Streptococcus pneumoniae infant human (0-1 year) preschool child (1-5) drug adverse effect immunoglobulin G human subject bactericidal immunity immunologic memory active immunization immunoconjugate enzyme linked immunosorbent assay opsonin antibody formation antibody specificity antigen antibody reaction serotyping bacterial polysaccharide human therapy evaluation Streptococcus pneumoniae vaccine clinical research CRISP RPROJ NEW YORK G CRISP/99/AI31473-05 CRISP/99/AI31473-05 199904 eng REGULATION OF PSEUDOMONAS AERUGINOSA VIRULENCE FACTOS BY VFR Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Pseudomonas aeruginosa is an opportunistic pathogen that primarily infects injured, immunodeficient or otherwise compromised patients, especially those with cystic fibrosis. The investigator has recently identified a global regulator of gene expression in P. aeruginosa, designated vfr for virulence factor regulator, which is 67% identical to E. coli cyclic AMP receptor protein, CRP. CRP, when complexed with the effector molecule cAMP, functions either as an activator or repressor of numerous genes including, but not limited to those involved in nutrient assimilation, flagellum synthesis, enterotoxin production and the heat shock response. The investigator has found that Vfr, in P. aeruginosa, directly or indirectly regulated the production of numerous proteins, including the virulence factors exotoxin A, elastase, alkaline protease, non-hemolytic phospholipase C, and perhaps others. Thus, the investigator hypothesizes that Vfr plays a central role in the ability of P. aeruginosa to cause disease. Additionally, the identification of the genes whose expression Vfr regulates and characterization of the mechanism which controls Vfr expression will enhance the understanding of how P. aeruginosa causes disease. The investigator will determine whether Vfr is required for virulence of P. aeruginosa using several animal infection models. The investigator will also identify additional genes, including potential virulence factors, which are regulated by Vfr. For these studies, the investigator will use a powerful DNA-binding/antibody capture/PCR amplification technique and random transposon mutagenesis. The P. aeruginosa effector molecule has not been identified. Therefore, the investigator will identify the effector molecule which is required for Vfr activity. The investigator will also characterize the structure/function relationships of Vfr. The investigator proposes that the information gained from this study could lead to the identification and/or development of novel therapeutic approaches against P. aeruginosa. This knowledge is especially important because of the decreasing utility of antibiotics. WEST SH UNIVERSITY OF WISCONSIN, 2015 LINDEN DRIVE WEST, MADISON, WI 53706 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 53706 Crisp Data Base National Institutes Of Health laboratory mouse Escherichia coli Pseudomonas aeruginosa bacterial disease molecular cloning gene expression regulatory gene genetic regulation bacterial genetics gene mutation western blotting antibody exotoxin virulence disease model nucleic acid sequence adenylate cyclase CRISP RPROJ WISCONSIN G CRISP/99/AI31477-07 CRISP/99/AI31477-07 199904 eng ROLE OF E7 IN HPV INFECTIONS Research LAIMINS LA Indiana University, 545 Barnhill Drive 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health life cycle cell differentiation cell growth regulation cooperative study pathologic process plasmid gene expression virus genetics mutagen mutant western blotting viral carcinogenesis virus protein virus replication human papillomavirus DNA damage northern blotting cell line cyclin recombinant virus oncoprotein p21 CRISP RPROJ INDIANA G CRISP/99/AI31494-080008 CRISP/99/AI31494-080008 199904 eng BACTERIA INDUCED CYTOKINE PRODUCTION IN THE GUT Research RPROJ DESCRIPTION (Adapted from the applicants abstract): The mechanism(s) involved in the intestinal tract response to S. typhimurium infection is not completely understood. We have recently shown that the interaction(s) of stem cell factor (SCF) with its receptor, c-kit, may be important in the intestinal tract response to S. typhimurium infection. The overall goal of this proposal is to test the following hypothesis: The production of SCF and its subsequent interaction(s) with its receptor, c-kit, are important events in the intestinal tract response to S. typhimurium infection. To test this hypothesis, we will address the following Specific Aims: 1) Determine the role(s) of SCF production and c-kit/SCF-positive cells in the susceptibility of mice to oral Salmonella challenge. 2) Determine if SCF:c-kit interaction(s) play an important role in the in vivo production of IFN( and/or TNF-( following Salmonella infection. 3) Characterize the cells in the intestinal tract and/or gut-associated lymphoid tissue which are producing elevated levels of SCF and/or c-kit following in vivo challenge with S. typhimurium. To address the above aims, we have proposed experiments to answer the following questions: 1) Do intestinal intraepithelial lymphocytes (IEL), and/or IEL-mast cell interactions play a role in the enhanced susceptibility of WWv mice to oral Salmonella challenge? 2) Can in vivo SCF supplementation of S1/S1d mice or normal mice alter their respective susceptibilities to oral Salmonella challenge? 3) Do normal mice treated in vivo with SCF or antibody to SCF and/or c-kit have altered intestinal tract or mucosal-associated lymphoid tissues in response to Salmonella infection, and if so, does bone marrow transplantation and/or in vivo SCF supplementation change such altered cytokine production? The results from these studies will be important for future development of vaccines which use Salmonella, and for further understanding the role of SCF plays in the pathophysiology associated with enteric bacterial infections. KLIMPEL GR UNIV TEXAS MEDICAL BRANCH, GALVESTON, TX 77555-1019 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse enteric bacteria Salmonella typhimurium bone marrow transplantation hematopoietic growth factor lymphocyte mast cell polymerase chain reaction host organism interaction salmonellosis gastrointestinal infection intestine protooncogene bactericidal immunity cytokine interferon tumor necrosis factor alpha immunocytochemistry in situ hybridization cell population study receptor binding growth factor receptor gut associated lymphoid tissue CRISP RPROJ TEXAS G CRISP/99/AI31519-06A2 CRISP/99/AI31519-06A2 199904 eng ROLE OF TOXT IN REGULATION OF VIBRIO CHOLERAE VIRULENCE Research RPROJ Coordinate regulation of virulence determinants in the agent of human cholera, Vibrio cholerae, is directed by the ToxR protein. ToxR is proposed to act by controlling expression of another regulator protein, encoded by the toxT gene, which activates genes required for virulence. The goals of this research proposal are to determine how ToxR controls toxT expression and to understand the role of ToxT in this regulatory cascade. The nucleotide sequence of toxT will be determined and its product will be characterized by biochemical and immunochemical means. The mechanism of ToxR control over toxT expression will be defined by identifying and analyzing the toxT transcript and its promoter using genetic and biochemical methods. A toxT mutant strain of V. cholerae will be constructed and characterized in vitro and in animal models. Two dimensional gel and reporter gene fusion analyses will be used to determine if there are proteins regulated by ToxT that are independent of ToxR, and vice versa. The promoter of a ToxT activated gene, tagA, will be identified and characterized by mutation analysis and DNA binding experiments. The role of ToxT in temperature control of virulence genes will be determined by a combination of in vitro and in vivo experiments. Recent evidence suggests that regulatory cascades may act in virulence gene expression in other bacteria, although components of the cascades in these systems have yet to be identified. Thus, the ToxR-ToxT system in V. cholerae, represents a model for how a regulatory circuit operates in pathogenesis. In addition, knowledge obtained by studying regulation in V. cholerae is essential to the development of good live, oral vaccines against cholera. DIRITA VJ UNIV OF MICHIGAN MED SCH, 018 ANIMAL RESEARCH FACILITY, ANN ARBOR, MI 48109-0614 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Vibrio cholerae genetic strain pilus electroporation host organism interaction cholera gene expression genetic promoter element reporter gene genetic library genetic regulation transcription factor bacterial genetics point mutation mutant cholera toxin virulence nucleic acid sequence protein structure /function bacterial protein DNA binding protein CRISP RPROJ MICHIGAN G CRISP/99/AI31645-08 CRISP/99/AI31645-08 199904 eng EXOENZYME S EXPRESSION AND PSEUDOMONAS VIRULENCE Research RPROJ My long-term research goals are to identify and understand the mechanisms of bacterial gene expression in response to changes in the environment. The objective is to determine which gene products of pathogenic organisms allow survival, growth, and reproduction within a hostile host environment. The model system that we study involves an organism that normally inhabits the soil and water, Pseudomonas aeruginosa. Immunocompromised individuals, cancer patients, cystic fibrosis patients, and persons with extensive burns can suffer serious and sometimes fatal infections from P. aeruginosa. In my laboratory we have focused on one of the extracellular virulence determinants produced by P. aeruginosa, exoenzyme S. Exoenzyme S is a member of the ADP-ribosyltransferase family of bacterial toxins with target specificities that include eukaryotic cytoskeletal proteins and small GTP-binding proteins that may mediate vesicle trafficking within cells. The molecular events that include production of exoenzyme S in vivo, intoxication of cells, and the events that damage cells are not understood. However, in animal infection and tissue culture model systems it is clear that exoenzyme S production correlates with epithelial damage and dissemination of Pseudomonas to the bloodstream. The specific aims of this study are to: 1. Analyze the mechanisms regarding exoenzyme S production; 2. Identify the mechanism of exoenzyme S export; and 3. Determine the requirement of exoenzyme S in P. aeruginosa pathogenesis and dissemination. To achieve these aims, my laboratory utilizes both genetic and biochemical approaches to clone, characterize, express, and purify proteins involved in the exoenzyme S pathway. With these strategies we have isolated two structural genes for exoenzyme S, a regulatory locus, and two loci that we propose encode proteins required for exoenzyme S export. This application focuses on the regulatory pathway and the mechanisms that govern exoenzyme S expression, the export pathway as it relates to exoenzyme S regulation, and the role that exoenzyme S plays in P. aeruginosa pathogenesis. The proteins controlling exoenzyme S synthesis and secretion are highly homologous to proteins controlling the expression of virulence in Yersiniae. Thus, the study of the exoenzyme S pathway may be the key to the discovery of novel Pseudomonas extracellular or membrane proteins coordinately regulated with exoenzyme S production that (i) contribute to epithelial damage, (ii) alter host immune response, or (iii) may be candidates for vaccine production. FRANK DW MEDICAL COLLEGE OF WISCONSIN, 8701 WATERTOWN PLANK RD, MILWAUKEE, WI 53226 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 53226 Crisp Data Base National Institutes Of Health Pseudomonas aeruginosa secretion enzyme mechanism exoenzyme enzyme biosynthesis molecular cloning gene expression structural gene bacterial genetics bacterial toxin virulence protein purification DNA binding protein CRISP RPROJ WISCONSIN G CRISP/99/AI31665-08 CRISP/99/AI31665-08 199904 eng TARGETS AND MECHANISMS OF ACTION FOR PARASITICAL AGENTS Research ROOS DS YALE UNIV SCHOOL OF MED, 333 CEDAR STREET, NEW HAVEN, CT 06520-8022 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse chloramphenicol clindamycin macrolide antibiotic microtubule organelle ribosome cell cycle mitotic spindle antiprotozoal agent pharmacokinetics drug resistance drug screening /evaluation molecular cloning extrachromosomal DNA microorganism genetics gene deletion mutation monoclonal antibody in situ hybridization circular DNA nucleic acid sequence herbicide gel electrophoresis protein biosynthesis Toxoplasma gondii CRISP RPROJ CONNECTICUT G CRISP/99/AI31808-080002 CRISP/99/AI31808-080002 199904 eng STUDIES OF ALLERGIC DRUG REACTIONS Research ADKINSON NF JOHNS HOPKINS UNIVERSITY, 5501 HOPKINS BAYVIEW CIRCLE, BALTIMORE, MD 21224 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21224 Crisp Data Base National Institutes Of Health clinical trial cooperative study diagnosis quality /standard diagnosis procedure safety diagnostic test diagnosis design /evaluation immunoglobulin E immunoglobulin G human subject allergen airborne allergen anaphylaxis atopy chemical hypersensitivity immediate hypersensitivity hypersensitivity test histamine release western blotting serology /serodiagnosis immunopathology diagnosis plant extract rubber epidemiology SDS polyacrylamide gel electrophoresis dermatitis CRISP RPROJ MARYLAND G CRISP/99/AI31867-080013 CRISP/99/AI31867-080013 199904 eng ASTHMA RISK FACTORS AND MANAGEMENT IN INNER CITY ADOLESCENTS Research TOGIAS A JOHNS HOPKINS UNIVERSITY, 5501 HOPKINS BAYVIEW CIRCLE, BALTIMORE, MD 21224 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21224 Crisp Data Base National Institutes Of Health air pollution adolescence (12-18) methacholine cooperative study disease /disorder proneness /risk health education environmental health patient care management health care service utilization human subject allergen asthma hypersensitivity test questionnaire longitudinal human study human morbidity diagnostic respiratory lavage respiratory airflow measurement spirometry respiratory airway pressure respiratory airway volume African American caucasian American socioeconomics urban poverty area behavioral /social science research tag CRISP RPROJ MARYLAND G CRISP/99/AI31867-080014 CRISP/99/AI31867-080014 199904 eng GENETIC ANALYSIS OF CHOLERA TOXIN STRUCTURE AND FUNCTION Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Cholea is a pandemic disease caused by V. cholerae in which cholera toxin (CT) cause life-threatening diarrhea. Enterotoxigenic E. coli and other enteroxic enteric bacteria cause diarrheal disease closely related to cholera. Highly effective vaccines against these diseases are not yet available. Our long range goals are to understand the structure and function of cholera toxin (CT) at the molecular level and to exploit the remarkable properties of holotoxin diseases. Specific Aim 1 will use biochemical, genetic, cell biological, and structural methods to characterize features of CT-A and CT-B that determine the biological actives of CT. These studies will analyze interactions between CT-A and CT-B that are essential for assembly of CT, determine structure of conformation-dependent epitopes of CT-B that elicit neutralizing antibodies, investigate the activation pathway for CT-A and determine the structure of active CT-A1, explore the role of specific gangliosides in intracellular trafficking of CT and related enterotoxins, and develop assays for translocation of CT-A across intracellular membranes. Specific Aim 2 will evaluate holotxin-like chimeras, in which microbial protective antigens replace the A1 domain of CT, as model oral factors of Vibrio Cholerae, will be tested for ability to induce protective anti-toxic and anti-bacterial immunity against cholera; and chimeras that incorporate Streptococcus pneumoniae PspA, which express conserved protective epitopes, will be tested for their ability to induce cross-protective immunity against pneumococci of multiple capsular serotypes. HOLMES RK UNIVERSITY OF COLORADO HLTH SC, 4200 EAST 9TH AVENUE B-175, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80262 Crisp Data Base National Institutes Of Health laboratory mouse Vibrio cholerae Streptococcus pneumoniae intracellular transport X ray crystallography host organism interaction site directed mutagenesis molecular cloning bacterial genetics mutant ganglioside bactericidal immunity monoclonal antibody bacterial antigen cholera toxin protein engineering protein structure /function protein transport chimeric protein CRISP RPROJ COLORADO G CRISP/99/AI31940-08 CRISP/99/AI31940-08 199904 eng MOLECULAR PATHOGENESIS OF CHANCROID Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Chancroid, an ulcerogenital disease caused by the fastidious gram-negative bacterium Haemophilus ducreyi, is one of the least understood sexually transmitted diseases. The association between genital ulcer disease and transmission of HIV makes control and prevention of chancroid a public health concern. The ultimate goals of this research project is to identify bacterial products that could serve in a vaccine. The applicant has developed model systems for studying virulence of H. ducreyi, including a temperature-dependent rabbit model for studying dermal lesions and a human foreskin fibroblast (HFF) for cell culture studies. Moreover, the applicant has developed a generalized mutagenesis system for this pathogen and interesting mutants have been isolated and characterized. The applicant proposes to use the mutagenesis system to identify and characterize mutants that are unable to adhere and/or form microcolonies on HFF cells. The applicant will also investigate a cytolethal distending-like toxin and other extracellular proteins for their role in virulence. Mutants with altered lipooligosaccharide will be isolated and characterized and examined for their virulence properties. Selected mutants will be compared to the parental strain in a human infection model. HANSEN EJ U OF TEXAS SW MED CTR, 6000 HARRY HINES BLVD, DALLAS, TX 75235-9048 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory rabbit Haemophilus bacterial disease bacterial cytopathogenic effect sexually transmitted disease electroporation site directed mutagenesis genetic library bacterial genetics mutant glycolipid human subject bacterial toxin virulence disease model bacterial DNA bacterial protein tissue /cell culture bacterial vaccine phenotype clinical research CRISP RPROJ TEXAS G CRISP/99/AI32011-07 CRISP/99/AI32011-07 199904 eng MOLECULAR DETERMINANTS OF LPS HOST PROTEIN COMPLEXES Research RPROJ This is a proposal to further examine the biochemistry, cell biology and pathogenesis of lipopolysaccharide binding protein (LBP) Dr. Tobias' thesis is that LBP assists in the clearance and detoxification of LPS and other microbial products. The following specific aims are listed. Specific Aim 1: to characterize the mechanism by which LBP enhances cellular uptake of LPS. This will be done by determining which pathways and molecules mediate cellular internalization of LPS. Specific Aim 2: to reinvestigate the role of LPB in the association of LPS with lipoproteins in normal and acute phase sera. This will be done by identifying and quantitating the interactions of LPS with the proteins of normal and LBP deficient sera and by studying the binding of LBP to normal and acute phase lipoproteins. Specific Aim 3: To characterize the role of LBP in clearance of LPS from the circulation of intact animals. This will be done by assessing the role of LBP in tissue and cellular targeting of LPS in normal and LBP knockout mice. Specific Aim 4: To establish the structural features of LBP important for formation of complexes with LPS and CD14. This will be done using LBP mutants, peptides derived from LPS, and anti-peptide antibodies. TOBIAS PS SCRIPPS RESEARCH INSTITUTE, 10550 NORTH TORREY PINES RD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 92037 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal gram negative bacteria bacterial cytopathogenic effect chemical binding host organism interaction site directed mutagenesis antibacterial antibody immune complex CD antigen endotoxin nonblood lipoprotein lipopolysaccharide protein structure /function binding protein CHO cell immunoaffinity chromatography CRISP RPROJ CALIFORNIA G CRISP/99/AI32021-07 CRISP/99/AI32021-07 199904 eng PLACENTA--ANTIHIV THERAPY Research RPROJ DESCRIPTION: (Adapted from Applicant's Abstract) This proposal will concentrate on how the intact human placental villous becomes infected with different strains of HIV. Whether there is a differential ability of syncytial-inducing and non-syncytial-inducing HIV strains to infect the placenta during the first/third trimesters will be determined. Specific infection of placental cell types - syncytiotrophoblast, cytotrophoblast, stromal, endothelial, and Hofbauer cells - will be determined by in situ PCR/hybridization. A model that simulates cellular conditions as noted in utero and allows definition of the nature and localization of the HIV infection and viral infectivity for placentae from the first/third trimesters will be used. How anti-HIV therapy either alone or in combination can reduce the infectivity of the non-in utero infected placentae will be resolved, while examining if there is any significant cellular/placental toxicity being produced by the mono/multiple therapy. How the in utero infected placentae responds to anti-HIV therapy in vitro will be determined. Two placental populations will be studied. a) Infected placentae from mothers who are currently receiving anti-HIV therapy. In these placentae, which multiple anti-HIV therapy benefit the fetus in reducing the infection of the placenta and transmission of HIV to the fetus will be established. The in vitro data will be correlated with the clinical information. b) The infected placentae from HIV-positive mothers, who have never been treated during pregnancy with any anti-HIV therapy, will be evaluated for mono/multiple therapy concurrently and compared with those placentae treated in utero. The pharmaco-kinetics/toxicity of mono/multiple anti-HIV therapy will be examined under extended (16-24 hr) dual human term placental perfusion conditions in a multiple-dosing manner. It is anticipated that these studies will establish how different HIV strains infect the human placenta from different gestational ages, which placental cells are being infected, and establish how mono/multiple anti-HIV therapy can be metabolized/ transported and reduce the HIV infection in the human placenta. MILLER RK UNIVERSITY OF ROCHESTER, 601 ELMWOOD AVENUE, ROCHESTER, NY 14642-8668 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health polymerase chain reaction perfusion combination chemotherapy pharmacokinetics drug adverse effect human tissue AIDS AIDS therapy antiAIDS agent in situ hybridization zidovudine gestational age placenta placental transfer placental hormone tissue /cell culture virus infection mechanism human immunodeficiency virus 2'3' dideoxynucleoside 2'3' dideoxyinosine vertical transmission CRISP RPROJ NEW YORK G CRISP/99/AI32319-07 CRISP/99/AI32319-07 199904 eng TRANSCRIPTIONAL REGULATION OF THE IMMUNE RESPONSE Research RPROJ The initiation and amplification of an inflammatory response depend on the action of transcription factors which induce the genes for adhesion and chemotactic molecules and cytokines. A growing number of transcription factor families, including NF-kappaB, C/EBP, and AP-1 are known to be vital targets of signal transduction pathways that lead to proinflammatory gene activation. In the current funding period, we isolated and characterized a transcription factor, ATF-2 (mXBP) which bound to a CRE- element in the MHC class II A-alpha promoter. By targeted gene disruption, we then produced mice lacking ATF-2. Studies of ATF-2-deficient mice now reveal that this member of the ATF/CREB family of transcription factors is also an important proinflammatory molecule. ATF-2 has widespread effects on induction of immediate early genes, adhesion molecules, chemokines, and cytokines, with potentially important effects on the inflammatory response found in joints and internal organs in infectious and rheumatic diseases. In this renewal application we propose to perform in vitro studies to delineate the molecular mechanism for ATF2's proinflammatory effect. These studies use molecular approaches to establish the role of ATF-2 in each step of the cascade of cytokine induction. We will also directly test the role of ATF-2 in the in vivo inflammatory response. Three models of inflammation will be studied: endotoxin-D-gal-septic shock, carditis induced by the Lyme agent, B. burgdorferi and Coxsackievirus B myocarditis. Finally, we will assess the role of ATF-2 in three rheumatic syndromes: HTLV-1-related synovitis and Sjogren's syndrome, and TNF-alpha induced polyarthritis. This work will identify potential targets for the blockade of ATF-2 action, and could lead to new therapies for inflammatory diseases. GLIMCHER LH HARVARD SCHOOL OF PUBLIC HEALT, 665 HUNTINGTON AVENUE, BOSTON, MA 02115-6023 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Borrelia Sjogren's syndrome inflammation genetic transcription heart disorder myocarditis interleukin 1 interleukin 6 tumor necrosis factor alpha immunoregulation disease model lipopolysaccharide protein biosynthesis polyarthritis synovitis tissue /cell culture Coxsackievirus human T cell lymphotropic virus type 1 gel mobility shift assay interleukin 8 septic shock cAMP response element binding protein CRISP RPROJ MASSACHUSETTS G CRISP/99/AI32412-13 CRISP/99/AI32412-13 199904 eng STRUCTURE AND FUNCTION OF THE E. COLI STB ENTEROTOXIN Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Diarrheal diseases contribute to serious morbidity and mortality in humans and domestic animals world-wide. Enterotoxigenic strains of Escherichia coli (ETEC) are significant agents of watery diarrheal disease and induce illness by the elaboration of polypeptide toxins which alter the normal fluid and electrolyte balance in the gut. The ETEC heat-stable enterotoxins (STs) are peptides which mediate fluid and electrolyte loss by inducing hormone-like secretory pathways in the gut. Two STs (STa and STb) are currently recognized. STa is an 18- or 19-mer acidic peptide which binds to and activates guanylate cyclase C (GCC), a unique intestinal form of membrane guanylate cyclase. The resulting accumulation of cGMP results in chloride secretion via activation of the CFTR chloride channel. In contrast to STa, STb is a 48-mer basic peptide which causes intestinal secretion in the absence of elevated cyclic nucleotides, a hallmark of the other E. coli secretory toxins. Work in the previous funding period demonstrated that STb causes release of intestinal 5- hydroxytryptamine (serotonin, 5-HT) and formation of prostaglandin E2 (PGE2), two mediators also known to be involved in cholera toxin (CT) mediated secretion. Unlike CT, however, the cellular action of STb causes activation of a pertussis toxin-sensitive heterotrimeric G protein of the Gi subtype. Under appropriate experimental conditions, STb-mediated Gi activation causes an elevation of cytosolic calcium ions in treated cells. STb also induces G-protein dependent exocytosis from a cultured mast-like cell line resulting in release of 5-HT in vivo. Recent structural studies indicate that STb is an alpha-helical amphipathic peptide with a positively-charged polar face formed by one helix and a non-polar face formed by a second alpha helix. The STb structure and action are consistent with a broad group of compounds which directly activate G proteins following insertion or penetration of cell membranes. In this application the PI proposes to: 1) determine the specific association of STb with target cells by employing biochemical, biophysical and ultrastructural techniques; 2) determine the cellular signaling mechanism of STb and the role of G protein activation in intestinal secretion; 3) investigate the cascade of intestinal secretagogues evoked by STb action and their specific role in secretion, and 4) determine the structural features responsible for the cellular and molecular action of STb. The elucidation of the cellular and molecular action of STb, a unique bacterial toxin, will undoubtedly increase our understanding not only of bacterial enterotoxin action, but also, the means by which the intestine maintains normal fluid and electrolyte balance. The result will be the identification of new approaches to therapeutic intervention of intestinal secretory diseases. DREYFUS LA UNIVERSITY OF MISSOURI, 5100 ROCKHILL ROAD, BSB 109, KANSAS CITY, MO 64110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 64110 Crisp Data Base National Institutes Of Health laboratory rat Escherichia coli biological signal transduction cell membrane cell type nuclear magnetic resonance spectroscopy enzyme inhibitor prostaglandin E gastrointestinal toxin absorption gastrointestinal epithelium site directed mutagenesis immunofluorescence technique enterotoxin secretory immune system immunoelectron microscopy G protein receptor binding chemoreceptor tissue /cell culture CRISP RPROJ MISSOURI G CRISP/99/AI32736-08 CRISP/99/AI32736-08 199904 eng MECHANISMS OF HEMATOLOGICAL TOXICITY OF ANTIAIDS DRUGS Research RPROJ DESCRIPTION (adapted from Abstract): The objectives of this competing renewal application is to continue to investigate the mechanisms associated with drug-induced hematological toxicity in AIDS therapy. The hematological toxicity of zidovudine (AZT) remains a limiting factor in clinical management of AIDS and is often the single most important complication ultimately requiring cessation of therapy. In addition, impaired hematopoiesis is also one of the major abnormalities associated with HIV-1 infection. The preliminary data included in this application would suggest that the trans-activator protein (Tat) of HIV-1 may down regulate gamma-globin gene expression in hematopoietic progenitor cells and also may enhance AZT-induced inhibition of erythroid differentiation. The Principal Investigator, therefore, hypothesizes that a combination of cellular and molecular effects caused by HIV-1 TAT together with drug- induced effects may lead to an enhanced hematopoietic toxicity. To test this hypothesis, he proposes: (1) to study the role of Tat-induced myelosuppression in enhancing the AZT and other 2',3'-dideoxynucleoside (ddN)-induced inhibition of hematopoietic differentiation in human hematopoietic cell line (K-562) and in human bone marrow CD34+ cells; (2) to examine the effect of Tat on transmembrane signalling by erythropoietin (EPO)-EPO receptor complex involving tyrosine phosphorylation and protein kinase C during proliferation and differentiation of erythroid progenitor cells; (3) to determine the role of TAT-induced cytokines, TNF alpha and beta and TGF beta, in the microenvironment of human mixed bone marrow cells, in modulating differentiation in the presence and absence of AZT and other ddNs; (4) to determine the role of Tat on the expression of proto-oncogenes c-myc, c-fos, and c-raf in the progenitor cells in the presence and absence of AZT; (5) to examine the effects of Tat and/or AZT on apoptosis in progenitor cells by monitoring DNA fragmentation and utilizing video imaging techniques; and 6) to develop new therapeutic approaches to overcome the HIV-1 Tat and AZT-induced bone marrow toxicity based upon the underlying mechanisms. In completing these specific aims, the Investigator to gain an in-depth understanding of the mechanisms associated with hematological toxicity in AIDS patients, and a basis for development of effective therapeutic approaches to overcome the hematological suppression. AGRAWAL KC TULANE UNIVERSITY SCHOOL OF ME, 1430 TULANE AVE, NEW ORLEANS, LA 70112 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 70112 Crisp Data Base National Institutes Of Health laboratory mouse biological signal transduction hematopoiesis erythropoietin erythroid stem cell blood toxicology drug adverse effect protooncogene transcription factor transforming growth factor human tissue colony stimulating factor interleukin 3 tumor necrosis factor alpha antiAIDS agent zidovudine protein kinase C virus protein cytosine growth factor receptor tissue /cell culture human immunodeficiency virus 1 2'3' dideoxynucleoside programmed cell death CRISP RPROJ LOUISIANA G CRISP/99/AI32893-06 CRISP/99/AI32893-06 199904 eng TOXINS OF BACTERIOIDES FRAGILES Research RPROJ Bacteroides fragilis is a species of anaerobic bacteria which lives in the colon of humans and animals. It makes up only about 1% of the human colonic flora, however, it is the most commonly isolated anaerobe from abscesses and soft tissue infections. Recent reports have shown that certain strains of B. fragilis produce an enterotoxin, suggesting a new role for this organism as an enteropathogen as well as evidence for a new virulence factor involved in its pathogenicity in tissues. This toxin has been implicated as a cause of diarrhea in several species of farm animals and more recently in humans, particularly infants. In preliminary studies, it appears that about 10% of isolates produce the toxin, suggesting that it is present in the colon in many individuals over long periods of time. The activity of the toxin was first discovered in the mid-1980's using the ligated ileal loop assay in lambs. Then, in 1990, it was shown that the activity could be detected by tissue culture assay using HT-29 colon carcinoma cells. We have now purified the toxin to homogeneity and showed that the enterotoxic activity observed in the lamb ligated loop assay and the cytotoxic activity (i.e., rounding of HT-29 cells) is due to the same protein. We have characterized the toxin, which has an M of about 20,000 and determined a partial N-terminal amino acid sequence. We are now cloning and sequencing the toxin gene, and studying its receptors and mechanism of action. We are also making specific polyclonal and monoclonal antibodies against the toxin to develop specific ELISA assays for detecting the toxin in feces and other clinical samples. This will establish a firm base for future collaborations with clinical investigators to determine the role of this toxin in etiology of diarrheal and infectious diseases and to determine approaches to diagnosis, therapy and treatment. WILKINS TD VIRGINIA POLYTECH INST, 301 BURRUSS HALL, BLACKSBURG, VA 24061-0249 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Bacteroides bacterial cytopathogenic effect second messenger enzyme mechanism diarrhea molecular cloning open reading frame bacterial genetics immunocytochemistry western blotting immunofluorescence technique enterotoxin basement membrane brush border membrane posttranslational modification bacterial protein cytoskeletal protein collagen laminin membrane protein proteolysis receptor tissue /cell culture tight junction metalloendopeptidase CRISP RPROJ VIRGINIA G CRISP/99/AI32940-06 CRISP/99/AI32940-06 199904 eng STREPTOCOCCUS PYOGENES--CLONAL ANALYSIS OF VIRULENCE Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Streptococcal pyrogenic exotoxin B (SpeB) is highly conserved among the group A streptococci (i.e., Streptococcus pyogenes), it has been shown that SpeB is cysteine protease and that it is important virulence factor in streptococcal pathogenesis (including data provided by the P.I. describing the relative virulence of isogenic pairs of SpeB producing and non-producing strains). Three studies will further address the role of SpeB in streptococcal virulence: 1) generation of site-specific mutants of speB, production of the altered SpeB proteins and determination of the enzymatic properties of the SpeB variants to continue ongoing structure-function analyses; 2) using purified SpeB variants (both naturally occurring and mutationally derived, in tissue culture experiments (using human umbilical vein epithelial cells) to probe the molecular pathophysiological processes induced by SpeB and including potential synergies between SpeB and the cytolytic toxin streptolysin O; 3) using transmission electron microscopy to determine if group A streptococci are internalized in culture and in episodes of human invasive disease. The critical data motivating these studies, and produced by the P.I.'s laboratory, is that SpeB can degrade host extracellular matrix proteins like fibronectin and vitronectin, cleaves and activates interleukin-1 beta and that immunization of mice with SpeB protects in intraperitoneal challenge experiments. The P.I. also proposes to explore in depth his own finding, recently published, that SpeB cleaves and activates a matrix metallo-protease produced by human endothelial cells. MUSSER JM BAYLOR COLLEGE OF MEDICINE, ONE BAYLOR PLAZA, HOUSTON, TX 77030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 77030 Crisp Data Base National Institutes Of Health Streptococcus pyogenes bacterial cytopathogenic effect Streptococcus infection genetic strain polymerase chain reaction site directed mutagenesis bacterial genetics gene mutation microorganism culture exotoxin virulence microorganism population study endopeptidase protein structure /function bacterial protein tissue /cell culture transmission electron microscopy CRISP RPROJ TEXAS G CRISP/99/AI33119-06 CRISP/99/AI33119-06 199904 eng MECHANISMS OF NUCLEOSIDE INDUCED TOXICITY AND ACTIVITY Research RPROJ DESCRIPTION (Adapted from the Investigator's Abstract): The long term objectives of this renewal application are to investigate and extend ongoing studies on the cellular and molecular mechanism(s) by which nucleoside analogs selectively target the reverse transcriptase of the human immunodeficiency virus (HIV). Extensive studies on the interaction of these drugs with the host cell have been shown in the past funding period to be critical in our understanding of the underlying mechanism(s) of drug action and toxicity. The synthesis of novel L-purine nucleoside analogs and the PI's recent discovery that a new series of L-dideoxyadenosine analogs are potent inhibitors of HIV and HBV replication demonstrates for the first time that development of L-purine derivative should also be vigorously pursued. These data provide a strong rationale for elucidation of their mechanisms of action and metabolism in target cells of these viral infections. This project includes three major aims: (I) Identification of the metabolic pathways and mechanism of action of L-purine analogs with particular emphasis on L-dideoxyadenosine derivatives. This proposal will evaluate the cellular metabolism (anabolism and catabolism) and compartmentalization of L-ddA, L-d4A and derivative in established cell lines and in human primary cells which are infectable by HIV, including lymphocytes and monocyte-macrophages. Selective inhibitors and mutant genetically defective (dCK-, AK-, etc.) cells will be used to elucidate activating kinase enzymes involved in the activation processes of these nucleosides. Nucleoside kinases responsible for nucleoside anabolism will be purified and kinetic constants for the various nucleosides will be determined. Effect of structural alterations on substrate specificities targeted toward maximization of antiviral activity will be assessed. The impact of stereoisomerism of the beta-enantomers of purine analogs on their cellular metabolism as well as their interaction with the antiviral target and/or hypothesized toxicity sites will be examined, including the assessment of potential combination therapies through metabolic interactions. (II) Evaluation of rationally designed pronucleotides incorporating enzyme-mediated bioreversible protection groups allowing the direct intracellular delivery of b-L-nucleoside-5'-monophosphate. (III) Investigations of the potential for HIV-1 resistance development to L-purine nucleoside analogs by phenotypic and genotypic characterization; confirmation of the role of specific mutations by site-specific mutational analysis; examination of potential cross-resistance to clinically approved nucleoside analogs; impact of L-purine nucleoside resistant mutation of HIV-RT on substrate/inhibitor recognition by beta-L-5'-triphosphate derivatives. SOMMADOSSI JC UNIVERSITY OF ALABAMA AT BIRMI, 1670 UNIV BLVD, BOX 600, BIRMINGHAM, AL 35294 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 35294 Crisp Data Base National Institutes Of Health stereoisomer drug metabolism drug adverse effect enzyme inhibitor site directed mutagenesis human tissue antiAIDS agent nucleoside analog RNA directed DNA polymerase protein kinase protein purification human immunodeficiency virus 2'3' dideoxynucleoside cell line enzyme activity CRISP RPROJ ALABAMA G CRISP/99/AI33239-06 CRISP/99/AI33239-06 199904 eng ANTI-OPPORTUNISTIC INFECTION AGENTS--DICATIONIC HETEROCYCLES AND PRODRUGS Research BOYKIN UNIVERSITY OF NORTH CAROLINA C, CHAPEL HILL, NC 27599-7525 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health amidine chemical synthesis antiinfective agent opportunistic infection cooperative study heterocyclic compound pharmacokinetics prodrug drug adverse effect drug design /synthesis /production gastrointestinal absorption /transport antiAIDS agent divalent cation dimer furan pyrimidine analog oral administration CRISP RPROJ NORTH CAROLINA G CRISP/99/AI33363-070002 CRISP/99/AI33363-070002 199904 eng TOXIN A SYNTHESIS IN PSEUDOMONAS AERUGINOSA Research RPROJ DESCRIPTION (adapted from the applicant's abstract): Pseudomonas aeruginosa is a gram negative opportunistic pathogen which causes serious infections in severely burned patients, immuno-compromised hosts, and Cystic Fibrosis patients. Toxin A is one of the most toxic virulence factors produced by P. aeruginosa. The synthesis of toxin A in P. aeruginosa is highly regulated by different environmental factors especially iron. Despite several studies, the exact mechanism of this regulation is not completely understood. The long term goal of this proposal is to determine the mechanisms of regulating toxin A synthesis in P. aeruginosa and the factors involved in this regulation. Recently, a new toxA positive regulatory gene, ptxR, was isolated. Analysis of ptxR showed that it enhances transcription of both toxA and the previously described toxA regulatory gene, regAB. Nucleotide sequence analysis revealed the presence of an open reading frame which codes for a 34 kDa predicted protein. Computer analysis revealed the presence of an adjacent gene which interferes with ptxR function. An open reading frame (ORF2) which is divergently transcribed from ptxR (from the minus strand) and codes for a 30 kDa protein was identified. This predicted protein has a significant homology to several proteins of the GalR family of repressors. The specific aims of this proposal are: 1) to examine the mechanism of ptxR function; 2) to determine the transcriptional regulation of ptxR; and 3) to examine the effect of ORF2 on ptxR function. Examining the mechanism of ptxR function will include the purification of ptxR production, DNA/protein binding experiments (gel retardation experiments), phosphorylation experiments, two dimensional gel experiments, and the construction of a PAO1 isogenic mutant in ptxR. Regulatory studies will include transcriptional analysis of ptxR, regA, and toxA. Analysis of ORF2 function will involved in vivo and in vitro transcription experiments, co-immunoprecipitation experiments, and the isolation of an ORF2 isogenic mutant. HAMOOD AN TEXAS TECH UNIV HEALTH SCI CTR, 3601 4TH ST, LUBBOCK, TX 79430 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 79430 Crisp Data Base National Institutes Of Health enteric bacteria Pseudomonas aeruginosa nuclear runoff assay site directed mutagenesis open reading frame regulatory gene genetic regulation genetic transcription transcription factor bacterial genetics mutant exotoxin iron DNA footprinting protein biosynthesis northern blotting gel mobility shift assay CRISP RPROJ TEXAS G CRISP/99/AI33386-03 CRISP/99/AI33386-03 199904 eng CYTOLYSINS OF HAEMOPHILUS DUCREYI Research RPROJ Haemophilus ducreyi is the causative agent of chancroid, a disease characterized by genital ulcers and, in 50% of the cases, inflammation of the regional lymph nodes. The occurrence of chancroid outbreaks in the United States coupled with its association with the heterosexual transmission of HIV in Africa makes understanding the pathogenesis of this disease imperative so that rational intervention strategies can be devised. Little is known about the pathogenesis of H. ducreyi but the necrosis seen in genital ulcers suggests that these organisms produce toxins. Potential targets of these toxins are both epithelial and inflammatory cells, resulting in destruction of epithelial tissues and impairment of the immune response. The primary goal of these proposed studies is the identification of cytolysins produced by H. ducreyi and elucidation of their role in the pathogenesis of chancroid. I have detected a cytolytic activity in H. ducreyi by its ability to lyse red blood cells. Further characterization of this activity revealed that it is expressed optimally in late logarithmic growth phase, is inhibited by phosphate and EGTA, and is enhanced by calcium. Transposon mutagenesis of cytolytic strain 35000 resulted in nine transposon mutants with three phenotypes: two with no cytolysin expression, one with reduced cytolysin expression, and six with enhanced cytolysin expression. Using Southern blots of whole cell DNA from strain 35000 and radiolabelled probes, I showed homology with both the RTX class of hemolysins and a Serratia-like hemolysin. The restriction fragment containing the Serratia-like toxin is disrupted in the cytolysin- negative mutants but not in the mutants with reduced or enhanced expression of cytolysin. These results suggest that the mutation is in the structural gene for a Serratia-like cytolysin in the cytolysin- negative mutants, and in genes that are regulators or modulators of cytolysin expression in the other mutants. In the studies outlined in this proposal, I plan to characterize the H. ducreyi cytolysin using genetic and biochemical techniques. The contribution of the cytolysin to the pathogenesis of chancroid will be determined using rabbit and primate models for chancroid. The range of cells the toxin affects in vivo will be studied by analyzing the toxin's effect on different cell types, including inflammatory cells and primary cultures of human foreskin epithelial and fibroblast cells. These studies will provide a better understanding of the virulence determinants of H. ducreyi and will provide a groundwork on which to base future strategies for disease intervention. TOTTEN PA HARBORVIEW MEDICAL CENTER, 325 NINTH AVE, BOX 359779, SEATTLE, WA 98104-2499 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory rabbit Primate Haemophilus fibroblast molecular pathology pathologic process molecular cloning regulatory gene structural gene bacterial genetics gene mutation cell mediated lymphocytolysis test virulence disease model tissue /cell culture cytolysin southern blotting CRISP RPROJ WASHINGTON G CRISP/99/AI33522-04 CRISP/99/AI33522-04 199904 eng VIRULENCE GENE EXPRESSION BY BACILLUS ANTHRACIS Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): To be successful pathogens, bacteria must possess mechanisms for sensing specific host environments, processing changes, and making appropriate adaptations. In many bacteria, expression of disparate virulence factors is controlled by a common regulatory system. Virulence gene expression in Bacillus anthracis, the causative agent of anthrax, is a unique example of a coordinately regulated response to a specific host-related signal. Virulent Bacillus anthracis produce two known virulence factors, a tripartite toxin, composed of edema factor, lethal factor, and protective antigen, and a poly-D-glutamic acid capsule. The toxin and capsule genes are located on plasmids pXO1 (185 kb) and pXO2 (95 kb), respectively. Synthesis of these virulence factors is enhanced when B. anthracis is grown in elevated levels of carbon dioxide. CO2 is postulated to be a physiologically significant signal during anthrax infection. Concentrations of bicarbonate and CO2 in mammalian tissues are comparable to those that activate toxin and capsule synthesis during in vitro growth. The long term goal of these studies is to elucidate the molecular basis for virulence gene expression in B. anthracis. The PI has determined that the trans-acting regulatory gene atxA is required for CO2-induced transcription of all three toxin genes during growth in vitro. AtxA also activates toxin expression in vivo; atxA mutants are avirulent in mice and mice infected with atxA- strains show a decreased immunological response to the toxin proteins. Another gene, acpA, has been implicated in CO2-induced capsule gene expression. In this study, the PI will further probe regulation of toxin and capsule synthesis and investigate whether B. anthracis harbors additional virulence genes. The specific aims are to: 1) identify atxA-regulated non-toxin genes and test the effect of these genes on virulence; 2) identify and characterize additional regulatory genes that affect toxin expression, 3) investigate the physiological significance of acpA expression in cells harboring atxA. These studies will provide information relevant to the pathogenesis of anthrax disease and increase knowledge concerning host-parasite relationships and signal transduction. KOEHLER TM UNIV OF TEX HLTH SCI CTR, 6431 FANNIN, JFB 1 765, HOUSTON, TX 77030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 77030 Crisp Data Base National Institutes Of Health Bacillus bacterial capsule host organism interaction site directed mutagenesis molecular cloning gene expression regulatory gene genetic regulatory element genetic regulation bacterial genetics gene mutation bacterial toxin virulence transposon /insertion element protein biosynthesis carbon dioxide CRISP RPROJ TEXAS G CRISP/99/AI33537-06A1 CRISP/99/AI33537-06A1 199904 eng ENHANCING THE IMMUNOGENICITY OF HUMAN ROTAVIRUS VACCINES Research RPROJ Rotavirus (RV) is the major cause of severe gastroenteritis in infants and young children worldwide. The failure of rotavirus vaccines to consistently induce protection against disease in infants and animals underscores a need for more effective vaccines. Our goal is an improved understanding of disease pathogenesis and mucosal immune responses to human rotaviruses (HRV) and identification of correlates of protective immunity in the gnotobiotic (Gn) pig model. The Gn pig is an ideal animal model because of its susceptibility to infection and disease (including intestinal lesions) with HRV, a lack of previous exposure or maternal antibodies to RV and its similarity to human infants in size, milk diet, gastrointestinal physiology and ontogeny of mucosal immune responses. To study disease pathogenesis, the putative role of the RV nonstructural protein, NSP4 as a viral enterotoxin and virulence determinant or target for induction of protective immune responses will be investigated in Gn pigs inoculated with recombinant NSP4 and NSP 4 peptides from virulent and attenuated pairs of RV. Expression of NSP4 and induction of immune responses to NSP4 will be further analyzed in Gn pigs infected with the respective virulent and attenuated pairs of HRV. In previous studies, the magnitude of the intestinal igA antibody secreting cell and lymphoproliferative immune responses coincided with the degree of RV replication and diarrhea induction, and were positively correlated with the level of protection induced. Hence we will further explore the role that viral replication and virulence (lesions and/or diarrhea) play in induction of mucosal immunity and protection against HRV. To assess which viral proteins are targets of protective immunity, B cell responses to RV and to RV structural and nonstructural proteins will be measured by virus neutralization, ELISA and ELISPOT assays and T cell responses will be examined by lymphoproliferative assays and analysis of cytokine profiles (RT- PCR,ELISA,ELISPOT). The type, magnitude, kinetics and correlation with protection of these mucosal immune responses will be compared in Gn pigs inoculated orally with virulent HRV (induces diarrhea and lesions and mimics natural infection) or candidate HRV vaccines including: attenuated HRV (no diarrhea or lesions); or the viral subunits, NSP4 (from virulent RV, induces diarrhea; attenuated RV, no diarrhea) and rotavirus-like particles (VLP) (no diarrhea or lesions), and challenged with virulent homotypic HRV. Finally we will examine if selected immunoenhancers (microencapsulation, mucosal adjuvants) can augment the mucosal immune responses to the attenuated and subunit HRV vaccines and increase vaccine efficacy in the Gn pig model. SAIF LJ OHIO STATE UNIV, OHIO AGRICULT RESEARCH DEV CTR, WOOSTER, OH 44691 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 44691 Crisp Data Base National Institutes Of Health swine pathologic process intestinal mucosa cytokine active immunization enzyme linked immunosorbent assay western blotting antigen antibody reaction enterotoxin immunomodulator attenuated microorganism virulence virus protein rotavirus vaccine lymphocyte proliferation oral administration mucosal immunity vaccine development vector vaccine CRISP RPROJ OHIO G CRISP/99/AI33561-05 CRISP/99/AI33561-05 199904 eng BACTERIAL AND LIPOSOMAL ANTIGEN PROCESSING Research RPROJ Class I MHC (MHC-I) molecules primarily present endogenous antigens, i.e. antigens that are present in the cytosol and are subject to the cytosolic processing mechanisms that comprise the conventional MHC-I processing pathway. However, exogenous antigens can also be presented by MHC-I molecules under certain circumstances, particularly in the case of particulate antigens. Recently, considerable attention has been focused on mechanisms that may contribute to alternate MHC-I processing pathways. Divergent results in several different systems have suggested that more than one alternate processing mechanism may exist. In some cases, MHC-I molecules present vacuolar antigens via alternate MHC-I processing mechanisms that are quite distinct from the conventional MHC- I processing pathway. These mechanisms may play important roles in generating CD8 T cell responses, especially to antigens expressed by vacuolar microorganisms and tumor cells. This project will define the mechanisms involved in the alternate MHC-I processing of several different types of exogenous antigens (intravacuolar bacteria and particulate vaccines). These studies may elucidate the basis of CD8 T cell responses to intravacuolar pathogens, such as Leishmania, Salmonella, and Toxoplasma species, and Mycobacterium tuberculosis. These studies also apply to tumor immunology and the role of alternate MHC-I processing mechanisms in the genesis of anti-tumor CD8 T cell responses. In addition, these studies will provide basic information that may be applied to develop strategies of therapeutic immunization to achieve protective CD8 T cell responses with non-viable vaccine preparations, in the absence of the endogenous antigen synthesis that is provided by live viral vaccine preparations. The use of non-viable particulate vaccines would be a safer way to elicit CD8 immunity in immunocompromised patients. HARDING CV CASE WESTERN RESERVE UNIVERSIT, 2085 ADELBERT ROAD, CLEVELAND, OH 44106 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 44106 Crisp Data Base National Institutes Of Health laboratory mouse Escherichia coli Salmonella typhimurium intracellular transport macrophage cytotoxic T lymphocyte vesicle /vacuole flow cytometry antigen presentation bacterial antigen cholera toxin tissue /cell culture MHC class I antigen CRISP RPROJ OHIO G CRISP/99/AI34343-05 CRISP/99/AI34343-05 199904 eng SALMOLYSIN MEDIATED VIRULENCE IN SALMONELLA Research RPROJ The long term goal of this proposal is to understand the role salmolysin, a cytolysin identified in Salmonella, plays in Salmonella virulence. The sly gene encodes the salmolysin protein which possesses both hemolytic and cytolytic activity. The sly gene has been cloned and sequenced, and salmolysin has been partially purified and biochemically characterized. The sly gene is conserved among Salmonella serotypes examined. Inactivation of the sly gene profoundly attenuates Salmonella typhimurium for virulence by the intraperitoneal, oral, and intravenous routes of infection. Salmolysin mutants (sly-) are unable to survive and replicate in murine macrophages in vitro. The central hypothesis of this proposal is that sly is one of the major genes involved in the characteristic ability of Salmonella to survive and replicate within the cells of the reticuloendothelial system. In this proposal, the survival and replication of a sly mutant of Salmonella typhimurium will be examined in murine macrophages from different sources. To determine the biological role of salmolysin in the ability of Salmonella to survive and replicate in macrophages, various macrophage functions will be assayed following infection with wild type and a sly mutant of Salmonella. The regulatory gene(s) that control the expression of salmolysin will be identified by transposon mutagenesis of the Salmonella typhimurium chromosome. Reporter gene fusions will be used to determine if salmolysin gene expression is induced in the presence of macrophages. The following Specific Aims are proposed: 1) to determine how salmolysin is regulated using chromosomal reporter gene fusions and transposon mutagenesis. 2) to determine the cellular location of salmolysin. 3) to identify macrophage bactericidal functions which may be altered in the presence or absence of a functional sly gene. 4) to determine the expression of the sly gene in macrophages using reporter gene fusions. LIBBY SJ NORTH CAROLINA STATE UNIVERSIT, RALEIGH, NC 27695-7615 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Salmonella macrophage phagocytosis polymerase chain reaction site directed mutagenesis plasmid fusion gene gene expression regulatory gene reporter gene genetic regulation bacterial genetics mutant Kupffer's cell virulence transposon /insertion element hydrogen peroxide cytolysin southern blotting CRISP RPROJ NORTH CAROLINA G CRISP/99/AI34397-05 CRISP/99/AI34397-05 199904 eng OUTER MEMBRANE PROTEINS OF PATHOGENIC LEPTOSPIRA SPECIES Research RPROJ The objective of this proposal is to identify leptospiral outer membrane proteins (OMPs) that have the capacity to serve as protective immunogens. Leptospirosis is an important, global human and veterinary health problem. Commercially available whole cell vaccines are highly toxic, produce only short-term immunity, and provide little cross- protection against infection with many of the 170 different leptospiral serovars. Whole cell vaccines have been shown to be ineffective in protection of cattle from infection by serovar hardjo, a prevalent North American leptospiral serovar. For these reasons, there is a critical need for development of new vaccine strategies for prevention of leptospirosis. The genes encoding four surface-exposed leptospiral OMPs have been cloned and sequenced: OmpL1, OmpL2, OmpL36, and LipL41. Specific Aim #1 is to evaluate the capacity of recombinant OMPs, alone or in combination, to serve as protective immunogens. Two complementary animal models will be used: fulminant leptospirosis in hamsters, and sublethal infection of BALB/c mice resulting in the carrier state. Animals will be challenged with virulent Leptospira by two routes: intraperitoneal and conjunctival inoculation. Sublethal infection will be detected by four methods: culture, antibody response, immunohistochemistry, and by the polymerase chain reaction. We have developed a novel method of purifying the leptospiral outer membrane in the form of unilamellar vesicles. The outer membrane vesicles contain four additional OMPs with molecular masses of 20-, 46-, 51-,, and 59-kDa. Specific Aim #2 is to clone and sequence their genes, express them as recombinant proteins, generate specific antisera, and determine whether they have surface-exposed epitopes by surface immunoprecipitation and immunoelectron microscopy. Specific Aim #3 is to determine whether protective immunogens are expressed during infection by evaluating the humoral immune response of hamsters infected with host-adapted Leptospira, and by immunohistochemistry of Leptospira found in host tissues during infection. Specific Aim #4 is to study how OMPs serve as protective immunogens. Passive immunization will be performed to assess the contribution of humoral immunity. The ability of immune serum and complement to kill Leptospira will be used to determine if OMPs are targets of bactericidal antibody. Immune serum will also be used to block leptospiral adherence to eucaryotic cells in tissue culture. It is anticipated that these studies will improve the diagnosis and prevention of leptospirosis. HAAKE DA WEST LOS ANGELES VA MED CTR, 11301 WILSHIRE BLVD, LOS ANGELES, CA 90073 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 90073 Crisp Data Base National Institutes Of Health hamster laboratory mouse Spirochaetales spirochetes disease polymerase chain reaction molecular cloning microorganism culture bactericidal immunity cell mediated cytotoxicity humoral immunity active immunization immunocytochemistry agglutination reaction epitope mapping antibacterial antibody bacterial antigen bacterial toxin bacterial DNA bacterial protein membrane protein nonhuman therapy evaluation bacterial vaccine recombinant protein CRISP RPROJ CALIFORNIA G CRISP/99/AI34431-03 CRISP/99/AI34431-03 199904 eng CRYSTALLOGRAPHY OF CHOLERA AND HEAT LABILE ENTEROTOXINS Research RPROJ DESCRIPTION (Adapted from abstract): This project encompasses a range of structural studies on two secreted bacterial toxins, Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT). The severe diarrheal disease cause by cholera toxin may result in death within hours. The milder infectious diarrhea produced by LT is rarely life-threatening in the developed world, but is a major cause of infant death in the third world. Specific aims of this proposal include x-ray crystallographic investigation of the fully active ADP-ribosylating A subunit to reveal catalytic mechanism and substrate binding modes, or engineered variants of the toxins, of the native toxins complexed with candidate inhibitors developed through structure-based drug design, and of hybrid molecular assemblies which use the native toxin structure as a scaffold for vaccine design. Long-term goals of this project are: (1) to investigate fundamental biological questions including the catalytic mechanism of ADP- ribosylation, structural determinants of toxin assembly, and the structural basis for recognition of complex sacharides. (2) to guide design of drugs effective against enterotoxigenic disease by providing a structural explanation for the biological function and activity of these toxins. LT and CT are 80% identical in sequence, exhibit similar subunit assembly, are immunologically cross-reactive, bind specifically to the same cell surface receptor, and share a common catalytic activity. Three aspects targeted for structure-based drug design are toxin assembly, receptor binding, and catalytic activity. (3) to use the remarkable ability of LT and CT to stimulate the mucosal immune system by designing prototype vaccines which retain the desirable immunological properties of the toxin while minimizing or abolishing the toxic activity. This work comprises structural study of engineered variants of the native toxins and of hybrid protein assemblies which incorporate foreign epitopes into the toxin structure. HOL WG UNIVERSITY OF WASHINGTON, BOX 357420, SEATTLE, WA 98195 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 98195 Crisp Data Base National Institutes Of Health biotechnology conformation X ray crystallography computer simulation drug design /synthesis /production enzyme inhibitor enzyme mechanism enzyme substrate analog ganglioside ADP ribosylation galactose cholera toxin enterotoxin intermolecular interaction crystallization protein engineering protein structure /function chimeric protein receptor binding thermostability active site enzyme activity structural biology vaccine development CRISP RPROJ WASHINGTON G CRISP/99/AI34501-06 CRISP/99/AI34501-06 199904 eng PATHOGENIC MECHANISMS OF SHIGA AND SHIGA-LIKE TOXINS Research RPROJ Infections with Shiga toxin-producing Shigella dysenteriae type I or Shiga-like toxin-producing E. coli cause outbreaks of dysenteric disease which continue to be a major cause of morbidity and mortality in maid parts of the world. Patients infected with these toxin-producing bacteria are at increased risk for developing life-threatening complications involving extensive damage of blood vessels serving the renal and central nervous systems. We have previously shown that purified Shiga-like toxins (SLTs) only manifest direct cytotoxicity for human vascular endothelial cells when present at high doses. We hypothesized that both SLTs and cytokines produced in response to the toxins or other bacterial products were necessary to cause the profound vascular lesions characteristic of the toxin-associated diseases. We have shown that murine peritoneal macrophages are essentially refractory to the cytotoxic actions of SLTs but respond to toxins by producing proinflammatory cytokines (TNF-alpha, IL-1alpha, IL-6) in vitro. We now wish to extend our examination of SLT- mediated cytokine production to human peripheral blood monocytes, human macrophage-like cell lines, and human renal epithelial cells. Use of macrophage-like cell lines allows us to treat the cells with differentiation factors to examine the relationship of cell maturation with toxin sensitivity, toxin receptor expression, and cytokine production. We will examine the SLT holotoxin structural component(s) and biological activity(ies) necessary to mediate cytokine production. We will use a series of inhibitors of transmembrane signalling pathways to determine the mechanism(s) of SLT-induced cytokine expression. We will utilize a well-defined mouse model of SLT-mediated kidney damage to measure in vivo cytokine mRNA and product expression in response to challenge with SLT-producing | coli or injection of purified SLTs. The levels of cytokines will then be modulated in toxin-treated animals to examine the role of cytokines in kidney damage and death. Finally, we will infuse purified SLTs into baboons to define, clinically and histopathologically, a large animal model of SLT-mediated diarrhea and acute renal failure. TESH VL TEXAS A&M HEALTH SCIENCE CENTE, 407 REYNOLDS MEDICAL BLDG, COLLEGE STATION, TX 77843-111 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Escherichia coli Shigella dysenteriae Escherichia coli infection bioassay biological signal transduction macrophage monocyte pathologic process cytokine enzyme linked immunosorbent assay bacterial toxin endotoxin kidney disorder disease model protein structure /function receptor expression epithelium clone cell enzyme activity CRISP RPROJ TEXAS G CRISP/99/AI34530-04 CRISP/99/AI34530-04 199904 eng INITIAL EVENTS IN A GONOCOCCAL INFECTION Research RPROJ Gonococci adhere to and enter certain cell types, aided by several surface proteins. They elaborate one or more toxins to compromise ciliary function and aid colonization. They encounter a variety of environmental conditions as they progress through the various stages of a mucosal infection, and are likely to adapt to these changes by turning various genes on or off. In the last several years, we have laid the groundwork for studying at the molecular level several aspects of GC/host interactions. We propose to continue these studies, which we believe will help us understand key elements of GC biology. A portion of these studies may also help us identify steps for the intervention of cytopathology in gonorrhea, and thereby help prevent a major cause of sterility in women. SO MH OREGON HEALTH SCIENCES UNIV, 3181 SW SAM JACKSON PARK RD, L, PORTLAND, OR 97201-3098 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory rabbit Neisseria gonorrhoeae gonorrhea polymerase chain reaction molecular pathology pathologic process host organism interaction open reading frame genetic promoter element genetic regulation mutant histopathology human tissue immunoprecipitation bacterial toxin electron microscopy fallopian tube southern blotting CRISP RPROJ OREGON G CRISP/99/AI34560-04 CRISP/99/AI34560-04 199904 eng XENOBIOTICS AND ALLERGIC INFLAMMATION Research RPROJ Description (adapted from the applicant's Abstract): The Project Leader's laboratory was the first to describe the inducible PGS2 cDNA, protein, and gene. Subsequent work demonstrated that non-steroidal anti-inflammatory drugs exert much of their anti-inflammatory effects by inhibiting PGS2 enzyme activity. The applicant also made the first inroads into the apparent paradox presented by the presence, in the same cell, of the constitutive prostaglandin synthase (PGS1) and inducible prostaglandin synthase (PGS2). The applicant and his colleagues showed that arachidonic acid generated by phospholipase (PL) activation in response to mitogens, inflammatory stimuli, and other agents is often sequestered from the constitutive PGS1 enzyme present in cells and can only be converted to prostaglandins via the PGS2 enzyme induced in response to these stimuli. In work performed in this AAIDCRC, Dr. Herschman and his co-workers made the singular discovery that prostaglandin production in activated mast cells differs from prostaglandin production in nearly all other cells. Specifically, production of prostaglandin D2 production in activated mast cells occurs in two phases, an early phase that couples a distinct PL to PGS1 and a late phase in which a second PL is coupled to PGS2. The applicant now plans the following: (1) to identify the phospholipase A2 (PLA2) enzymes that provide arachidonate to PGS1 and PGS2, (2) to determine the subcellular localizatio of the PGS1, PGS2, type V soluble (s)PLA2 and type IV cytoplasmic (c)PLA2 proteins in mast cells, to understand the basis for arachidonic acid channeling in prostaglandin synthesis, (3) to determine the amino acid sequences of PGS1 and PGS2 that are responsible for differential temporal accessibility to arachidonic acid and PGS subcellular localization in mast cells, and (4) to determine the cis-acting elements, transcription factors, and signal transduction pathways that mediate PGS2 expression in mast cells. The applicants also discovered that phenanthrenes profoundly inhibits prostaglandin production in activated mast cells and in endotoxin-stimulated macrophages. The investigators now plan (5) to determine how phenanthrene inhibits prostaglandin induction in mast cells and macrophages, and (6) to determine whether phenanthrene enhances leukotriene synthesis in activated mast cells following IgE receptor aggregation. The applicants work will (1) provide new insight into fundamental mechanisms of prostaglandin synthesis in cells that mediate allergic response; (2) identify new targets for pharmacologic modulation of prostaglandin synthesis; and (3) investigate mechanisms by which PAH may modulate airway inflammation. SAXON A UNIVERSITY OF CALIFORNIA, 10833 LE CONTE AVENUE, LOS ANGELES, CA 90024-1680 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health cooperative study carbopolycyclic compound inflammation respiratory hypersensitivity pollutant interaction CRISP RPROJ CALIFORNIA G CRISP/99/AI34567-06 CRISP/99/AI34567-06 199904 eng XENOBIOTIC EFFECTS ON ALLERGIC RESPONSES VIA MACROPHAGES Research NEL AE UNIVERSITY OF CALIFORNIA, 10833 LE CONTE AVENUE, LOS ANGELES, CA 90024-1680 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health exhaust laboratory mouse macrophage helper T lymphocyte cooperative study carbopolycyclic compound inflammation immunoglobulin E human subject respiratory hypersensitivity leukocyte activation /transformation cytokine antibody formation CD antigen protein kinase pollutant interaction quinone tissue /cell culture oxidative stress chemokine CRISP RPROJ CALIFORNIA G CRISP/99/AI34567-060002 CRISP/99/AI34567-060002 199904 eng MODULATION OF THE HUMAN IGE RESPONSE BY XENOBIOTICS Research SAXON A UNIVERSITY OF CALIFORNIA, 10833 LE CONTE AVENUE, LOS ANGELES, CA 90024-1680 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health exhaust pyroglyphid cooperative study carbopolycyclic compound inflammation gene rearrangement immunoglobulin E human subject pollen respiratory hypersensitivity cytokine immunoglobulin gene antibody formation hemocyanin pollutant interaction tissue /cell culture mucosal immunity CRISP RPROJ CALIFORNIA G CRISP/99/AI34567-060003 CRISP/99/AI34567-060003 199904 eng DEP INDUCTION OF ALLERGIC INFLAMMATION AND IDENTIFICATION OF GENES INVOLVED Research HANKINSON O UNIVERSITY OF CALIFORNIA, 10833 LE CONTE AVENUE, LOS ANGELES, CA 90024-1680 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health exhaust laboratory mouse T lymphocyte cooperative study carbopolycyclic compound inflammation enzyme induction /repression immunoglobulin E human subject respiratory hypersensitivity antibody formation mucosal immunity CRISP RPROJ CALIFORNIA G CRISP/99/AI34567-060006 CRISP/99/AI34567-060006 199904 eng DEMONSTRATION AND EDUCATION--ASTHMA MANAGEMENT FOR INNER CITY CHILDREN Research WEDNER HJ WASHINGTON UNIVERSITY, 660 SOUTH EUCLID AVENUE, ST LOUS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 63110 Crisp Data Base National Institutes Of Health child (0-11) cooperative study health education environmental contamination home health care patient care management health service demonstration project human subject asthma African American Hispanic American urban poverty area behavioral /social science research tag health services research tag CRISP RPROJ MISSOURI G CRISP/99/AI34580-060006 CRISP/99/AI34580-060006 199904 eng EFFICACY OF TB CHEMOPROPHYLAXIS IN PPD NEGATIVE, HIV POSITIVE ADULTS Research RPROJ Individuals co-infected with HIV and Mycobacterium tuberculosis have an increased risk of developing tuberculosis (TB). Skin testing with purified protein derivative (PPD) is normally used to identify people co-infected with HIV and M. tuberculosis who could benefit from TB chemoprophylaxis; however, the immune deficit induced by HIV may suppress the immune response to PPD, preventing identification of co-infected individuals. A series of skin tests to other common antigens, including mumps and Candida, can be applied to PPD negative, HIV seropositive adults to help determine whether the individuals are anergic or possibly not infected with M. tuberculosis. People who are anergic to PPD, and the battery of other test antigens a?e presumably infected with the TB bacillus but unresponsive to PPD because of, the HIV induced immune deficit. These people might, therefore, benefit from TB chemoprophylaxis; however, the efficacy of chemoprophylaxis in HIV seropositive, PPD negative, anergic adults has not been evaluated thoroughly enough to justify providing it to all such individuals. We propose to conduct an unmasked, randomized clinical trial in Cite Soleil, Haiti to determine the efficacy of two short course TB chemoprophylactic regimens in HIV seropositive, PPD negative, anergic adults with normal chest radiographs. Eligible residents of Cite Soleil (n=345) will be randomized to receive isoniazid and pyridoxine (B6) daily for six months, rifampin and B6 daily for three months followed by daily B6 for three months, or B6 daily for six months. Participants will be followed for 12-54 months and monitored closely for development of active TB, mortality, and progression of HIV infection. Participants with active TB will be treated with the WHO recommended therapeutic regimen. We will determine the efficacy of the chemoprophylactic regimens and evaluate the effect of chemoprophylaxis on survival. We will also evaluate the acceptability and toxicity of the two regimens; identify patient characteristics associated with compliance; identify risk factors for failure of chemoprophylaxis; and determine the prevalence of anergy in HIV seropositive, PPD negative adults with normal chest radiographs in this high risk setting. The results of this study will help establish the efficacy and effectiveness of TB chemoprophylaxis in HIV seropositive, PPD negative, anergic adults in areas where TB is highly endemic. COBERLY JS JOHNS HOPKINS UNIVERSITY, 615 NORTH WOLFE ST, BALTIMORE, MD 21205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21205 Crisp Data Base National Institutes Of Health adult human (19+) rifamycin isoniazid Mycobacterium tuberculosis tuberculosis clinical trial communicable disease control communicable disease chemotherapy communicable disease diagnosis disease /disorder proneness /risk drug adverse effect Caribbean island human subject immunologic skin test HIV infection radiography African Caribbean therapy compliance human therapy evaluation human immunodeficiency virus pyridoxine CRISP RPROJ MARYLAND G CRISP/99/AI34848-05 CRISP/99/AI34848-05 199904 eng WOMEN AND INFANTS TRANSMISSION STUDY (WITS II) Research RPROJ DESCRIPTION: The Women and Infants Transmission Study (WITS) enrolls HIV-infected pregnant women and their infants into a multicenter, epidemiologic cohort study whose objectives are to answer critical questions relevant to vertical transmission, and to HIV disease during pregnancy and infancy. In addition, research issues relevant to implementation of therapeutic intervention in this population will be addressed. The specific aims of WITS are to 1) Determine mechanisms of perinatal HIV transmission and maternal cofactors related to transmission including the role of maternal drug use, the role of the placenta, the role and characteristics of HIV in the birth canal, and of maternal coinfections; 2) Evaluate factors related to successful perinatal HIV prevention strategies, and intensely assess transmission cases that occur despite perinatal use of antiretrovirals; 3) Determine the impact of pregnancy on disease progression of HIV infection among women through the postpartum period, including immunologic and virologic changes in pregnancy; 4) Characterize acute HIV infection among HIV-infected children in light of antiretroviral, prophylactic and immune-based therapy; 5) Determine factors predicting pediatric disease progression among the cohort of HIV infected children in WITS. A Boston/Worcester consortium of investigators seeks to continue participation in WITS, utilizing the same experienced investigators and field staff that have successfully accrued over 300 pregnant women and over 250 HIV-at risk infants to date. The applicants propose to expand on the considerable expertise of the co-investigators by the addition of basic science consultants as well as two specific scientific collaborations. They propose 1) The utilization of new and sensitive techniques to perform a detailed analysis of CTL responses to pediatric HIV infection that will be helpful in understanding the pathogenesis and progression of perinatal HIV infection as well as insights to guide immunotherapeutic intervention; and 2) To explore the pathogenesis of vertical transmission in relation to both ontogeny of expression of CC chemokine receptor genes and also to receptor - tropism of particular strains of transmitted virus. TUOMALA RE BRIGHAM & WOMEN'S HOSPITAL, 75 FRANCIS STREET, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health child physical development infant human (0-1 year) cooperative study disease /disorder proneness /risk AIDS education /prevention human subject AIDS AIDS therapy HIV infection longitudinal human study pregnancy embryo /fetus toxicology placental transfer pregnancy infection receptor expression cytokine receptor vagina female human therapy evaluation tissue resource /registry pediatric AIDS quality of life vertical transmission chemokine clinical research CRISP RPROJ MASSACHUSETTS G CRISP/99/AI34856-06 CRISP/99/AI34856-06 199904 eng MOLECULAR PROPERTIES AND MODULATION OF BACTERIAL PORINS Research RPROJ Porins are large channels of the outer membrane of Gram-negative bacteria, which represent the major entry pathway for hydrophilic solutes into these organisms. Porins have classically been considered as permanently open pores, and little is known about their regulation and mode of action. The objectives of this proposal are to study, at the molecular level, the function and modulation of these proteins in Escherichia coli. Bacteria are one of leading agents in human infectious diseases. Because of their abundance and location at the external surface of the bacterial cells, porins may play a role in the host- pathogen interactions involved in bacterial infection and have been shown to be targets for immune responses. Changes in porin expression patterns have also been observed in virulent strains of E. coli causing urinary tract infections. The significance of this project resides in the study of the control of the bacterial outer membrane permeability which plays an vital role in microorganism's survival. The disruption of the normal functional state of the outer membrane by drug-mediated modulation of porin activity can be conceived as a potential strategy for controlling bacterial infection. The patch clamp technique, widely used in the description of eukaryotic channels, will be applied for the real-time measurement, in the sub- millisecond range, of electrical fluctuations across the membrane during the course of activity of a single or small number of channels. Channel activities will be studied in giant spheroplast or giant cells of E.coli, and in outer membrane fractions reconstituted into giant liposomes. Experiments will be conducted to investigate the molecular mechanisms underlying voltage sensitivity and cooperativity of the major porins expressed by the ompF and ompC genes. OmpC activity can be regulated by membrane-derived oligosaccharides (MDOs), a family of piroplasmic sugar polymers synthesized at high osmolarity. The nature of the molecular events taking place at the level of the channel protein during regulation will be studied. We will also determine the class of MDOs which is responsible for the modulation and define the location of the binding site. A search for other regulatory substances will be conducted, in particular form molecules which promote channel opening and inhibition. Some of these should ultimately be relevant to therapeutic approaches. To fully understand the molecular mechanisms underlying the observed ion channels properties, the relationship between structure and function will be explored by the used of mutant channels. A variety of spontaneous mutants will be used first to map the general regions relevant to specific channels functions. Ultimately, site-directed mutagenesis will be implemented to refine these locations. This is an approach which is widely used in the structure/function relationship studies of eukaryotic channels. The advantages of the bacterial system are that mutant channels are to be studied in their natural environment without the need for injection into foreign expression systems, and porins are the first channels for which an X-ray crystallographic structure has been published. This information on the three-dimensional structure of porins will be extremely valuable for the design of genetically engineered channels and the meaningful interpretation of the data. DELCOUR AH UNIVERSITY OF HOUSTON, 4800 CALHOUN, HOUSTON, TX 77204-5513 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Escherichia coli membrane permeability lysosome protoplast /spheroplast X ray crystallography patch clamp site directed mutagenesis gene expression mutant bacterial toxin membrane reconstitution /synthesis oligosaccharide protein structure /function bacterial protein pore forming protein membrane channel CRISP RPROJ TEXAS G CRISP/99/AI34905-05 CRISP/99/AI34905-05 199904 eng INDUCTION OF MUCOSAL IMMUNITY TO HIV IMMUNOGENS Research PALKER TJ DUKE MEDICAL CENTER, RESEARCH DRIVE, BOX 3258, DURHAM, NC 27710 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 27710 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse secretion serum cytotoxic T lymphocyte cooperative study immunoglobulin A immunoglobulin G human subject cholera toxin virus antigen complement HIV infection disease model synthetic peptide chimeric protein genital secretion AIDS vaccine human immunodeficiency virus 1 HIV envelope protein gp120 Macaca fascicularis microcapsule neutralizing antibody mucosal immunity CRISP RPROJ NORTH CAROLINA G CRISP/99/AI35351-04S10001 CRISP/99/AI35351-04S10001 199904 eng NOVEL FORMULATIONS AND DELIVERY TECHNOLOGIES FOR HIV PEPTIDE IMMUNOGENS Research PARADISO PR DUKE MEDICAL CENTER, RESEARCH DRIVE, BOX 3258, DURHAM, NC 27710 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 27710 Crisp Data Base National Institutes Of Health laboratory mouse Salmonella Salmonella typhimurium B lymphocyte cytotoxic T lymphocyte helper T lymphocyte lysosome cooperative study drug delivery system drug design /synthesis /production gastrointestinal system gene induction /repression antiviral antibody antibody formation diphtheria toxin pertussis toxin virus antigen synthetic peptide chimeric protein AIDS vaccine Rotavirus human immunodeficiency virus neutralizing antibody mucosal immunity vaccine development CRISP RPROJ NORTH CAROLINA G CRISP/99/AI35351-04S10004 CRISP/99/AI35351-04S10004 199904 eng PROTECTIVE EFFICACY OF LIVE ATTENUATED SIV VACCINES Research DESROSIERS RC HARVARD UNIVERSITY, ONE PINE HILL DR BOX 9102, SOUTHBOROUGH, MA 01772-9102 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Macaca mulatta genetic strain drug adverse effect drug screening /evaluation glycosylation active immunization antigen antibody reaction virus antigen attenuated microorganism virus DNA nucleic acid sequence live vaccine AIDS vaccine vaccinia virus human immunodeficiency virus 1 simian immunodeficiency virus neutralizing antibody method development recombinant virus vaccine development CRISP RPROJ MASSACHUSETTS G CRISP/99/AI35365-050004 CRISP/99/AI35365-050004 199904 eng KILLING OF CD4+ T CELLS BY MACROPHAGE TROPIC, NSI HIV-1 Research RPROJ DESCRIPTION: (Adapted from investigator's abstract) The aim of this study is to continue and extend recent observations of the author that certain macrophage-tropic, non-syncytium inducing (NSI) HIV, like T cell tropic SI HIV, could induce direct killing of primary CD4+ T lymphocytes. Although amino acid variations in the V3 loop of gp120 greatly influenced the killing of CD4+ T cells by T cell tropic SI viruses, regions outside the loop appear to be more important for the killing of CD4+ T cells by macrophage-tropic, NSI viruses. Chimeric viruses will be constructed to determine regions of the HIV genome that are important for the killing of CD4+ T cells by macrophage- tropic, NSI HIV. Fine mapping of specific amino acids that are critical for this phenomenon will be performed by site-directed mutagenesis. The mechanism of CD4+ T cell killing by these viruses will also be studied. The goals of this study are to obtain a more complete understanding of HIV pathogenesis, to identify additional genetic determinants for cytopathogenicity of HIV and markers for the prognosis of AIDS. YU X JOHNS HOPKINS UNIV SCH PUB HLT, 615 N WOLFE ST, BALTIMORE, MD 21205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21205 Crisp Data Base National Institutes Of Health helper T lymphocyte pathologic process host organism interaction site directed mutagenesis molecular cloning fusion gene epitope mapping AIDS tissue /cell culture cytotoxicity human immunodeficiency virus CRISP RPROJ MARYLAND G CRISP/99/AI35525-05 CRISP/99/AI35525-05 199904 eng ACCESSORY CHOLERA ENTEROTOXIN, ACE, MECHANISM OF ACTION Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Three toxins of V. cholerae that increase short circuit current in Ussing chambers have been identified. They include cholera toxin (CT), Zot (zonula occludens toxin) which acts by disrupting tight junctions, and Ace (accessory cholera enterotoxin), the subject of this proposal. The investigator has identified, cloned and sequenced the ace gene. Preliminary studies using crude toxin extracts in animal models indicated that Ace acts by increasing transcellular ion transport. Ace increased short- circuit current in Ussing chambers and caused fluid secretion in ligated rabbit ileal loops, characteristic of a classic enterotoxin. The predicted protein sequence of Ace shows striking similarity to the product of the cystic fibrosis bene. Based on activity and structural correlates, the investigator proposes to examine two hypotheses for the mechanism of action of Ace. The "second messenger" model is that Ace binds to a receptor on the epithelial cell membrane and activates a second messenger which increases chloride secretion via an endogenous channel. The "pore-forming" model is that Ace forms a new channel by inserting into the epithelial cell membrane. The specific aims are to: 1) compare native and recombinant Ace by purifying and characterizing both proteins: 2) determine the presence and protective function of an immune response to Ace: 3) study the effects of Ace on cellular function to distinguish between the "second messenger" and "pore- forming" hypotheses of Ace activity; 4) identify protein domains contributing to Ace activity; and 5) study the role of each of the V. cholerae toxins in virulence by constructing isogenic chromosomal mutants. The investigator will use bacterial and molecular genetic and cell physiology methods to examine the mechanism(s) of action of Ace on the gastrointestinal epithelial cell. The long term objectives of the proposal are to enhance understanding of the role of Ace in cholera pathogenesis, to identify possible new mechanisms of action of toxins, and to determine how the toxins of V. cholerae interact to cause disease. TRUCKSIS MM UNIVERSITY OF MARYLAND, 685 W. BALTIMORE STREET, RM 48, BALTIMORE, MD 21201-1509 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health second messenger ion transport cholera gastrointestinal epithelium mutant microorganism culture bactericidal immunity cholera toxin enterotoxin virulence protein purification protein sequence bacterial protein membrane channel SDS polyacrylamide gel electrophoresis gel mobility shift assay recombinant protein CRISP RPROJ MARYLAND G CRISP/99/AI35717-04 CRISP/99/AI35717-04 199904 eng ENTEROTOXIN FACTORS ELABORATED BY SHIGELLA FLEXNERI A2 Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The long term objective of the proposed project is to characterize new enterotoxic determinants potentially involved in the pathogenesis of Shigella flexneri diarrhea. The attenuated S. flexneri vaccine candidates tested so far have shown various degrees of reactogenicity, particularly watery diarrhea. Studies of the PI have resulted in the discovery of enterotoxins elaborated by S. flexneri that have not previously been described. These toxins may be responsible for the residual diarrhea observed during Shigella vaccine trials. The PI proposes to investigate these findings further in a blend of basic and applied studies which will yield fundamental insights into the pathogenesis of S. flexneri diarrhea. These findings will also provide crucial information that can be applied to engineer improved attenuated vaccines. Specific aims are: AIM 1: To purify a new enterotoxic factor of S. flexneri 2a which induces secretion both in vivo and in vitro in animal models. The PI has already partially purified this toxin, termed ShET1 for Shigella enterotoxin 1, and now intends to purify this moiety further and to raise specific monoclonal antibodies to it. AIM 2. To study the genetic regulation of ShET1 and to construct mutants of the set1 genes. The PI has identified, cloned, and sequenced the chromosomal region encoding this factor. He will now characterize these genes and their mechanisms of regulation. AIM 3.To establish the role of ShET1 in the pathogenesis of shigellosis and to study its mechanism of action. The PI will perform intestinal perfusion studies in human volunteers and, using an animal model, will determine ShET1 interaction with enterocyte receptors, its effects on transepithelial water and electrolyte transport, and the intracellular mediator(s) of its enterotoxic effect. AIM 4. To determine whether ShET1 and enteroinvasive Escherichia coli enterotoxin (EIET/ShET2), a second enterotoxin recently described by this group, share any functional similarities. FASANO A UNIV OF MARYLAND, 685 W BALTIMORE STREET, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Escherichia coli Shigella perfusion diarrhea gastrointestinal absorption /transport jejunum gene expression genetic promoter element genetic regulation genetic transcription genetic translation bacterial genetics gene mutation human subject monoclonal antibody enzyme linked immunosorbent assay enterotoxin brush border membrane virulence protein purification receptor binding receptor clinical research CRISP RPROJ MARYLAND G CRISP/99/AI35740-03 CRISP/99/AI35740-03 199904 eng V(D)J RECOMBINATION AND DNA REPAIR MUTANTS--"SEXI" Research RPROJ Our objective is to gain an understanding of the molecular and biochemical mechanisms of lymphoid V(D)J recombination and DNA double strand break (DSB) repair. These two pathways share at least three common factors, SCID (severe combined immune deficiency), XRS-6 (x-ray sensitive-6), XR-1 (x- ray-1) and probably share many others. In particular, in this grant application we describe a novel and powerful strategy using retroviral mutagenesis for the creation of mammalian cell mutants. This strategy has already allowed us to isolate and characterize three mutants that are defective in these two pathways. The use of retroviral insertional mutagenesis to simultaneously mutate and molecularly "tag" in cis genes will expedite the cloning of these and additional genes involved in recombination and repair. The relatedness of V(D)J recombination and DSB repair is underscored by the existence of several human immune deficiency/chromosome breakage syndromes, such as Blooms' syndrome, ataxia telangiectasia and Fanconi's anemia. Individuals affected by these diseases are characterized by having impaired immune capabilities as well as being cancer prone. We intend to characterize these two important biological pathways using 6 lines of experimentation: 1. Creation and isolation of mammalian cell mutants mutants which are sensitive to x-irradiation (sexi mutants). 2. Characterization of the DNA repair capacity of the sexi mutants. 3. Characterization of the V(D)J recombinational capacity of the sexi mutants. 4. Characterization of homologous and illegitimate recombinational capacities of the sexi mutants. 5. Cloning of the SEXI genes. 6. Molecular characterization of the SEXI genes. The ultimate goal of these studies will be to use the sexi mutations as tools to understand the molecular mechanisms of lymphoid gene rearrangement and DNA repair. HENDRICKSON EA BROWN UNIVERSITY, 69 BROWN ST BOX G-J1, PROVIDENCE, RI 02912 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02912 Crisp Data Base National Institutes Of Health cell cycle transfection molecular cloning gene expression gene complementation gene rearrangement structural gene genetic library gene mutation mutant immunoglobulin gene DNA repair X ray radiation sensitivity tissue /cell culture Retroviridae southern blotting animal genetic material tag CRISP RPROJ RHODE ISLAND G CRISP/99/AI35763-05 CRISP/99/AI35763-05 199904 eng THE EPIDEMIOLOGY OF HOME ALLERGENS AND ASTHMA Research RPROJ Childhood asthma causes substantial morbidity and health care costs. Of the 18 million asthmatics in the United States, roughly 10 million are children under the age of 16. Asthma is the most common admitting diagnosis to pediatric hospitals in the United States, and asthma accounts for 2.8 million office visits per year in the United States. It is estimated that the cost of asthma care in children accounts for over $6 billion in health care costs per year. We propose to conduct a prospective longitudinal cohort study to examine the role of allergen exposure in the home on the development of two main outcomes: 1) asthma/wheeze during infancy and early childhood; 2) allergic sensitization, as measured by skin test reactivity (during early childhood). The primary allergen exposures of interest include house dust mite (der p I, der f I), fungi, cockroach (bla g I), and cat (fel d I) antigens. Additional exposures will be examined that may impact the association between allergens and the two main outcomes. These exposures include: heredity, assessed via family history of allergy and/or asthma, and via cord blood IgE; in utero and postnatal tobacco smoke exposure; acute lower respiratory illness; gender; race; socioeconomic status; and perinatal factors. If allergen exposure proves to be important in the prediction of sensitization and the development of asthma/wheeze in early life, it is a finding of great public health significance, as control of exposure measures could be instituted which could potentially minimize sensitization and exposure and, hence, the development of the disease. GOLD DR CHANNING LABORATORY, 181 LONGWOOD AVE, BOSTON, MA 02115-5804 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health cat mite cockroach blood chemistry infant human (0-1 year) preschool child (1-5) disease /disorder proneness /risk passive smoking environmental contamination Fungi human subject allergen airborne allergen asthma hypersensitivity desensitization hypersensitivity test enzyme linked immunosorbent assay questionnaire dust longitudinal human study respiratory disorder epidemiology sex difference racial /ethnic difference socioeconomics family genetics CRISP RPROJ MASSACHUSETTS G CRISP/99/AI35786-05 CRISP/99/AI35786-05 199904 eng CYCLOSPORA--A NEW HUMAN PATHOGEN Research STERLING CR JOHNS HOPKINS HYGIENE & PUB HL, 615 N WOLFE STREET, #5515, BALTIMORE, MD 21205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21205 Crisp Data Base National Institutes Of Health laboratory mouse life cycle genetic strain feces analysis child (0-11) clinical trial communicable disease transmission cooperative study disease /disorder proneness /risk field study diarrhea Peru human subject serology /serodiagnosis microorganism classification coccidiosis parasitic disease chemotherapy parasitic disease diagnosis epidemiology Sporozea protozoal cyst trimethoprim sulfamethoxazole human therapy evaluation water sampling /testing CRISP RPROJ MARYLAND G CRISP/99/AI35894-050002 CRISP/99/AI35894-050002 199904 eng ORAL IMMUNIZATION AGAINST ENTERIC INFECTIONS IN CHILE Research RPROJ We propose a collaborative research program to carry out field studies of bacterial enteric vaccine candidates in Santiago, Chile in conjunction with the Servicio de Salud Metropolitano Norte, the Universidad de Chile, Roberto del Rio Children's Hospital and the Ministry of Health. The studies will attempt to optimize oral immunization so that young children can be successfully immunized with a single dose of vaccine. Projected studies would investigate the clinical acceptability and immunogenicity of single-dose live oral recombinant cholera vaccine strain CVD 103-HgR in toddlers and infants. If a single dose of this oral vaccine is indeed well-tolerated and immunogenic in young infants, this would provide support for the concept of the Children's Vaccine Initiative. A number of oral vaccines (e.g., attenuated poliovirus vaccine) exhibit diminished immunogenicity in low socioeconomic level children in less-developed countries. Using CVD 103-HgR as a model, we will investigate the hypothesis that such diminished immunogenicity is in part due to small bowel bacterial overgrowth (environmental enteropathy). We will perform non-invasive breath H2 tests in Santiago schoolchildren to screen for small bowel bacterial overgrowth, prior to administering a dose of CVD 103-HgR vaccine to the children. The magnitude of the vibriocidal antibody response will be compared in children with and without small bowel overgrowth. Studies will be carried out in young children to establish the safety and immunogenicity of multivalent vaccines against Shigella and enterotoxigenic Escherichia coli (ETEC), important pathogens of diarrhea disease among children in developing countries. The ETEC vaccine will be a live vector vaccine in which ETEC colonization factor fimbriae and B subunit of heat-labile enterotoxin are expressed in attenuated live vectors. By maintaining prospective surveillance at two health centers that serve large populations of low socioeconomic level, we shall prepare a field site in which we propose to carry out a large-scale field trial of efficacy of a multivalent vaccine against Shigella or ETEC. The groups of U.S. and Chilean investigators and their institutions have a long history of productive and harmonious collaboration. LEVINE MM UNIV OF MARYLAND BALTIMORE, 685 W BALTIMORE ST, ROOM 480, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health enteric bacteria Escherichia coli typhoid pilus child (0-11) infant human (0-1 year) clinical trial cooperative study breath test drug design /synthesis /production cholera diarrhea South America human subject immunization enterotoxin secretory immune system nucleic acid probe epidemiology bacterial vaccine cholera vaccine Salmonella vaccine Shigella vaccine oral administration recombinant DNA vector vaccine CRISP RPROJ MARYLAND G CRISP/99/AI35948-05 CRISP/99/AI35948-05 199904 eng IMMUNE RECONSTITUTION AND GENE TRANSFER IN SCID-HU MICE Research RPROJ DESCRIPTION: The current proposal will focus on the mechanisms of hematopoietic reconstitution following drug treatment, and utilize combination anti-retroviral therapy as an adjunct to assess the efficacy of various stem cell genetic therapeutic approaches to combat HIV-induced pathology. The specific aims include: (1) optimize and further define the loss and subsequent reconstitution of thymopoiesis seen following anti- retroviral treatment of HIV-infected Thy/Liv implants; (2) optimize and further define the mechanisms responsible for loss and subsequent reconstitution of non-lymphoid hematopoietic precursor activity in vivo; and (3) optimize in vivo efficacy of anti-viral gene therapeutic approaches in conjunction with pharmacologic anti-retroviral therapy. ZACK JA UCLA SCH OF MEDICINE, 11-934 FACTOR, LOS ANGELES, CA 90095-1678 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health hematopoiesis thymus cell differentiation flow cytometry drug adverse effect gene therapy human subject AIDS AIDS therapy HIV infection antiAIDS agent disease model nonhuman therapy evaluation tissue mosaicism human immunodeficiency virus 1 combination therapy SCID mouse clinical research CRISP RPROJ CALIFORNIA G CRISP/99/AI36554-05 CRISP/99/AI36554-05 199904 eng MATERNAL IMMUNITY BY CHIMERIC IG AND NAKED DNA Research RPROJ Advances in biotechnology have ushered in a new era of vaccine development These developments are based on the utilization of peptides corresponding to protective epitopes, recombinant proteins, viral or bacteria vectors etc. At present, there are many difficulties in developing safe vaccines that can protect children against infectious agents. Two promising avenues of research are maternal immunization which may confer protection to infants and neonatal immunization which may confer protection to children. During the past years, we have used engineered immunoglobulins as a delivery system for viral B and T cell epitopes. The overall goal of our proposal is to use the influenza virus system to evaluate the effects of maternal and neonatal immunization of two types of new vaccine antigenized Ig and naked DNA. We choose the influenza virus system because it provides a convenient experimental model with which we have extensive experience. Our aims are to understand the basic immune mechanisms of maternal and neonataI immunization with antigenized Ig and naked DNA which may lead to protective response against influenza virus. Thus, the specific aims are: 1. To construct a doubly antigenized lg molecule bearing influenza virus hemagglutinin B and T cell epitopes. The B cell epitope will be expressed in CDR2 loop and T cell epitope in CDR3 loop. 2. To study the effect of neonatal immunization with doubly antigenized Ig with respect to anti-HA humoraI and cellular responses and protection against influenza virus infection. Based on information obtained, we propose to study the eventual enhancement of responses by adjuvants, to determine the isotypes of protective antibodies and the pattern of HA- specific B cell clonotypes. The potential side effects will also be evaluated. 3. To study the effect of neonatal immunization with naked DNA. We will use in this study a plasmid containing influenza virus HA gene. Our studies are designated to determine the half life of plasmids injected in neonates, HA specific antibody and CTL response and the protection against virus. The potential side effects will also be studied. 4. To evaluate the effects of maternal immunization with antigenized Ig and naked DNA on the protection of offspring against influenza virus infection. BONA CA MOUNT SINAI SCHOOL OF MEDICINE, 1 GUSTAVE L. LEVY PLACE, NEW YORK, NY 10029-6574 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health newborn animal laboratory mouse B lymphocyte T lymphocyte antigen presenting cell drug delivery system drug adverse effect drug screening /evaluation drug design /synthesis /production plasmid immunoglobulin cellular immunity humoral immunity immunization radioimmunoassay hemagglutinin antibody formation virus antigen virus DNA placental transfer pregnancy immunology influenza vaccine vaccine development CRISP RPROJ NEW YORK G CRISP/99/AI37115-05 CRISP/99/AI37115-05 199904 eng ENHANCED EFFICACY AND SAFETY OF RECOMBINANT VACCINES Research RPROJ The long-term objective of this proposal is to increase the safety and efficacy of live recombinant vaccines, for use on control of human diseases. The immediate objective of this proposal is to enhance the safety and efficacy of vaccinia virus (VV) recombinant vaccines for humans and other animals. The principals that we address in this proposal using VV as a model would have application for other live recombinant vaccines. We propose improving the safety and enhancing the immunogenicity by co- expressing cytokines and immunogenic antigens in a modified VV vector that has had specific immune-modulating genes inactivated or deleted. The efficacy of these alterations in the VV vector will be tested using a vesicular stomatitis (VSV) model. This will enable us to evaluate our vaccines for both systemic and localized disease conditions, using mice and cattle, respectively, as hosts. We have demonstrated the adjuvant effects of interferon-gamma (IFN-gamma) in cattle and mice immunized with a mixture of the cytokine and the glycoprotein of vesicular stomatitis virus. Accordingly, we co-expressed cytokine genes with other immunogenic foreign genes in VV in an effort to increase efficacy of these vaccines. Although dramatic attenuation of VV was demonstrated, no immune enhancement to VV or co-expressed foreign proteins was detected. Our hypothesis is that safety and immunogenicity of recombinant VV (rVV) vaccines can be improved by co-expressing the lymphokine, interferon-gamma (IFN-gamma) with an immunogenic protein, and inactivating one or more VV immune-modulating genes. Such a vaccine will combine the effectiveness of live attenuated vaccines with the safety of subunit vaccines and will have several distinct advantages: 1) Antigens expressed by rVV are effective in inducing both cytotoxic T-lymphocyte (CTL) and humoral immune responses; 2) IFN-gamma attenuates VV virulence; 3) IFN-gamma will favor a Type 1 immune response, an important component of the immune response to virus infection to antigens; and 4) the inactivation of immune-modulating genes will increase the safety of rVVs. YILMA TD UNIVERSITY OF CALIFORNIA, 1126 HARING HALL, DAVIS, CA 95616 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 95616 Crisp Data Base National Institutes Of Health athymic mouse laboratory mouse cow cytotoxic T lymphocyte drug adverse effect drug screening /evaluation drug design /synthesis /production gene induction /repression cellular immunity cytokine humoral immunity immunization immunogenetics virus antigen immunomodulator virulence disease model stomatitis nonhuman therapy evaluation viral vaccine recombinant DNA interferon gamma vaccine development vector vaccine CRISP RPROJ CALIFORNIA G CRISP/99/AI37182-05 CRISP/99/AI37182-05 199904 eng SALMONELLA VACCINES THAT DELIVER BOTH ANTIGEN AND CYTOKINE Research RPROJ Attenuated strains of Salmonella have been used as carriers of heterologous antigens for human and animal vaccination. in mice, S. typhimurium normally infects macrophages in the Peyer's patches of the gut and also cells of the liver and the spleen. We will compare the immune response of mice orally- inoculated with S. typhimurium which expresses one of two model antigens: (l) Simian immunodeficiency virus gag protein (SIV gag) and (2) the B subunits of Shiga-like toxin type l (SLT-l) and SLT-ll from Enterohemorragic E. coli. One of these strains, designed to enhance a cellular immune response to an antigen, will deliver DNA encoding the IL-1 2 cytokine and the SIV gag protein directly to macrophages which will then appropriately transcribe, translate, and process these proteins. To do this, the Salmonella strain will contain two sets of prokaryotic genes under a tightly regulated promoter that after phagocytosis will 1) liberate the bacteria from the phagosome into the cytoplasm of a macrophage, and then 2) lyse the bacterium thereby releasing a prokaryotic/eukaryotic shuttle vector carrying cytokine and viral DNA into the cytoplasm of the macrophage. Those macrophages infected with the Salmonella carrier strains will process the antigen and co-express specific cytokines thus resulting in a targeted and specific enhanced immune response. One of the other strains, designed to augment a mucosal immunity, will co-express IL-5, a cytokine that augments slgA production, together with the SLT protective antigens. finally, we will use the Salmonella vaccine system to evaluate the type of T cell response generated to a heterologous antigen that is delivered via a vaccine strain directly to the cytoplasm of an antigen presenting cell. The results from these studies have direct application to the production of effective vaccines that can specifically modulate the immune system to develop a protective response against intracellular and extracellular pathogens. HEFFRON FL OREGON HLTH SCI UNIV, 3181 SW SAM JACKSON PARK RD, PORTLAND, OR 97201-3098 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Escherichia coli Salmonella typhimurium bacteriolysis cytotoxic T lymphocyte phagocytosis antigen presenting cell polymerase chain reaction drug delivery system plasmid cellular immunity cytokine interleukin 5 humoral immunity major histocompatibility complex enzyme linked immunosorbent assay bacterial toxin virus antigen secretory immune system mucosa complementary DNA nonhuman therapy evaluation Salmonella vaccine simian immunodeficiency virus CRISP RPROJ OREGON G CRISP/99/AI37201-05 CRISP/99/AI37201-05 199904 eng DOMAIN MAPPING CLOSTRIDIUM PERFRINGENS THETA TOXIN Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Many bacterial cytolytic toxins exhibit an amphimorphic nature in which they are produced as soluble monomers, but ultimately end up as membrane-associated oligomers. These toxins lack obvious transmembrane domains and in fact have relatively hydrophilic structures. The mechanism by which these proteins carry out the transition from a soluble protein to a membrane associated complex has not been identified. Unlike many bacterial toxins, such as diphtheria toxin or the colicins, pH does not act as a trigger for the formation of a "molten globule" or insertion intermediate. Perfringolysin O (PFO), a cytolysin (Mr 54,000) produced and secreted by Clostridium perfringens, belongs to a family of related cytolysins collectively called the "thiol-activated cytolysins" that are produced by a variety of gram positive pathogenic bacterial species. PFO typifies a cytolytic toxin which has a hydrophilic primary structure but forms a cytolytic membrane complex. After binding to the target membrane, PFO monomers oligomerize into supramolecular complexes and lyse the cell. How membrane insertion and pore formation by PFO is accomplished remains unknown. This proposal is designed to identify the location (i.e., protein-aqueous, protein-membrane, or protein-protein) of various domains of PFO before and after its interaction with target membranes. The specific aims of this proposal are: 1) generation of single-site cysteine substitutions in PFOala, 2) identification of PFO-membrane interactions; 3) identification of regions of PFO exposed to the aqueous medium at various stages of the cytolytic mechanism; 4) identification of residues located at the interfacial domains of the monomeric subunit of the PFO oligomer; and 5) determination of the extent of PFO insertion into the membrane at various stages of the cytolytic process. Unique cysteines will be placed into the primary structure of PFO to act as specific attachment sites for fluorescent dyes such as NBD whose emission is sensitive to the polarity of their environment. The fluorescence lifetime intensity and collisional quenching of the single dye attached to PFO will be monitored to determine if a specific residue remains is the aqueous phase, moves into the lipid bilayer, or forms part of the interfacial domains that are in contact in the oligomerized PFO. Since oligomerization only occurs above 10C, whereas binding occurs at all temperatures, insertion of a probe labeled residue into the bilayer can be assigned to either the binding event or the oligomerization event. TWETEN RK UNIV OF OKLAHOMA HLTH SCIS CTR, 940 STANTON L YOUNG BLVD, OKLAHOMA CITY, OK 73190 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 73190 Crisp Data Base National Institutes Of Health Clostridium chemical binding crosslink fluorescence spectrometry fluorescent dye /probe site directed mutagenesis mutant tetanus toxin intermolecular interaction posttranslational modification protein sequence cysteine cytotoxicity cytolysin toxin metabolism CRISP RPROJ OKLAHOMA G CRISP/99/AI37657-02 CRISP/99/AI37657-02 199904 eng EXTRACELLULAR SECRETION IN VIBRIO CHOLERAE Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Cholera toxin (CT), an enterotoxin expressed by Vibrio cholerae that is the virulence factor responsible for eliciting severe diarrheal symptoms in humans is released from the bacterium by a secretory mechanism encoded, in part, by chromosomal genes in the eps (extracellular protein secretion) cluster. Only CT and a few other periplasmic proteins (protease and chitinase) are secreted by the eps system, suggesting that specific transport signals recognized by the secretory apparatus are located in each protein. The PI and others have shown that V. cholerae secretes CT and LT-IIa, an enterotoxin produced by strains of Escherichia coli, with equal efficiency. Since the B polypeptides of CT and LT-IIa that are essential for extracellular transport share little, if any, amino acid homology, the transport signals on these toxins must be conserved structural motifs. Studies on the molecular aspects of toxin secretion by V. cholerae are hampered by the lack of a cloned secretion complex. Eps C, D, E, F, G, H, I, J, K, L, M, and N have been cloned, but the gene cluster does not confer upon E. coli the ability to secrete CT. These data suggest that additional genes are required to encode the secretion machinery. It is also likely that one or more of the Eps proteins interacts with CT on a physical level during transport. The PI's goals are: 1) to investigate the molecular structure of the extracellular transport signals in CT and LT-IIa; 2) to characterize the genetic lesion in CC9453, a mutant of V. cholerae that is deficient in extracellular secretion; and 3) to determine which of the Eps proteins have specific CT-binding activity. There is no longterm, effective vaccine for V. cholera. Until a vaccine is available, alternative strategies to ameliorate the symptoms of the disease are potentially very important. A better understanding of the process of extracellular secretion by V. cholerae is a necessary first step in the subsequent design of therapeutic strategies to ameliorate cholera symptoms by inhibiting release of CT. CONNELL TD STATE UNIV OF NEW YORK, 3435 MAIN STREET, BUFFALO, NY 14214 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 14214 Crisp Data Base National Institutes Of Health aminoacid Escherichia coli Vibrio cholerae biological signal transduction extracellular site directed mutagenesis gene expression gene complementation bacterial genetics gene mutation mutant enzyme linked immunosorbent assay immunoprecipitation antibacterial antibody cholera toxin enterotoxin protein biosynthesis protein engineering protein structure /function protein transport chimeric protein CRISP RPROJ NEW YORK G CRISP/99/AI37817-03 CRISP/99/AI37817-03 199904 eng ANALYSIS OF MICROBICIDES IN MODEL SYSTEMS Research HEYNER S PENNSYLVANIA STATE UNIVERSITY, PO BOX 850, DEPT OF PATHOLOGY, HERSHEY, PA 17033 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 17033 Crisp Data Base National Institutes Of Health acidity /alkalinity Neisseria gonorrhoeae mucus cell cycle Chlamydia trachomatis antiinfective agent drug adverse effect drug screening /evaluation semen sperm human tissue disease model fluorescence microscopy female reproductive system epithelium tissue /cell culture CRISP RPROJ PENNSYLVANIA G CRISP/99/AI37829-040008 CRISP/99/AI37829-040008 199904 eng FUNCTION OF CD8T CELLS IN TUBERCULOSIS Research RPROJ The bacterial pathogen, Mycobacterium tuberculosis, is responsible for 8 million new cases of tuberculosis and 2.9 million deaths per year, worldwide. The host immune responses necessary for protection against disease are not clearly defined. Cell-mediated immunity is required for protection, and recent work has confirmed a role for both CD4 and CD8 T cells in the immune response to this pathogen. Here, experiments designed to examine the exact nature of the contribution of CD8 T cells to the protective immune response are proposed. We have outlined a strategy for determining the actual function of CD8 T cells, either as cytotoxic T cells or IFN-gamma producing cells, or both, in M. tuberculosis infection. This involves testing for the presence of mycobacterial specific CD8 CTLs in various mice, establishing CTL lines, and testing these lines for protective capacity in vivo. A systematic approach to this problem is proposed, including the use of various alternative target cells and sources for CD8 T cells. As an alternative hypothesis, the role of IFN-gamma produced by CD8 T cells will be examined. Using in vitro methods and immunological complementation in gene-disrupted mice, we will determine the role of CD8 T cells in protection against tuberculosis in mice. These studies on the actual role of CD8 T cells will increase our understanding of the host immune responses necessary for protection against M. tuberculosis. Information obtained from the results of the proposed experiments will be of value in designing new immunization strategies for tuberculosis and may suggest potential pathogenic mechanisms by which mycobacteria evade the necessary immune responses, and unveil possible virulence factors for further study. FLYNN JL UNIV OF PITTSBURGH SCH OF MED, PITTSBURGH, PA 15261 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 15261 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal Mycobacterium tuberculosis tuberculosis cytotoxic T lymphocyte host organism interaction bactericidal immunity cellular immunity cytokine cell mediated lymphocytolysis test virulence pore forming protein tissue /cell culture MHC class II antigen cytolysin interferon gamma interleukin 12 CRISP RPROJ PENNSYLVANIA G CRISP/99/AI37859-02 CRISP/99/AI37859-02 199904 eng CORE--TOXICOLOGY Research REISING SF CHILDREN'S HOSPITAL RESEARCH F, 3333 BURNET AVE, CINCINNATI, OH 45229-3039 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory rat biomedical facility drug adverse effect drug screening /evaluation fertility spermicide histopathology enzyme linked immunosorbent assay immunotoxicity mucosa cytotoxicity CRISP RPROJ OHIO G CRISP/99/AI37940-049001 CRISP/99/AI37940-049001 199904 eng TOPICAL PROTEGRINS TO PREVENT CHLAMYDIAL SEXUALLY TRANSMITTED DISEASES Research WAGAR E UNIV OF CALIFORNIA, LOS ANGELE, 10833 LECONTE AVE, LOS ANGELES, CA 90095-1690 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse genetic strain Chlamydia communicable disease control antibacterial agent local antiinfective agent sexually transmitted disease topical drug application drug adverse effect drug screening /evaluation drug vehicle histopathology disease model peptide analog nonhuman therapy evaluation CRISP RPROJ CALIFORNIA G CRISP/99/AI37945-040002 CRISP/99/AI37945-040002 199904 eng TARGETED MUCOSAL VACCINES AGAINST HIV-1 Research RPROJ The principal goal of this project is to enhance antibody and T cell responses to HIV-1 mucosal subunit vaccines. To achieve this goal, we are using the Escherichia coil heat-labile enterotoxin (LT) to target chimeric HIV-1 proteins to mucosal surfaces. Because there is no single accepted correlate of protective immunity, our studies will pursue both cellular and humoral responses that follow from mucosal immunization. To augment T cell responses, we have developed systems to replace segments of LT with epitopes of HIV-1 proteins in order to selectively deliver these determinants to the cytoplasmic compartment that intersects with the Class I MHC antigen processing pathway. The abilities of these chimeric proteins to induce and elicit responses in this pathway will be evaluated in murine and monkey models, with special emphasis being placed on responses that call up T cells that secrete beta-chemokines. In addition, we have developed a novel eukaryotic expression system that allows the proper folding of LT-Env chimeras that are expected to elicit potent neutralizing antibody responses after mucosal immunization. These chimeras will be evaluated in murine and monkey models with emphasis on the induction of antibodies that neutralize primary isolates of HIV-1. LEWIS GK UNIV OF MARYLAND, 655 WEST BALTIMORE STREET, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health laboratory mouse polymerase chain reaction transfection gene mutation humoral immunity active immunization enzyme linked immunosorbent assay CD4 molecule enterotoxin protein engineering protein folding chimeric protein tissue /cell culture AIDS vaccine human immunodeficiency virus 1 HIV envelope protein gp120 neutralizing antibody mucosal immunity CRISP RPROJ MARYLAND G CRISP/99/AI38192-03 CRISP/99/AI38192-03 199904 eng RHO MODIFYING CYTOTOXIC NECROTIZING FACTOR OF E COLI Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Escherichia coli is the most common cause of urinary tract infections (UTIs) in otherwise healthy individuals. The uropathogenic E. coli isolates that cause these infections are characteristically of certain serotypes and express P fimbriae, alpha- hemolysin, and aerobactin. Recent findings from Europe indicate that the cytotoxic necrotizing factor type 1 (CNF1), a toxin little studied in the U.S., is also frequently produced by uropathogenic E. coli. CNF1 and the immunologically related CNF2 are 110-115 kDa polypeptides that induce multinucleation and actin polymerization in eukaryotic cells and necrosis and death of some animals. We recently cloned and sequenced cnf2 and found that the N-terminal half of CNF2 is homologous to the dermonecrotic toxin of Pasteurella multocida, a toxin that mediates the pathology of progressive rhinitis in pigs. We also observed that CNF2 modifies a small GTP-binding protein designated Rho that is involved in stress fiber assembly. The long term goals of this proposal are to examine the role that CNF1 plays in the virulence of uropathogenic E. coli and to determine the precise mechanism by which CNF1 modifies Rho. The specific aims are designed to achieve these goals are to: 1) analyze the role of CNF1 in the pathogenicity of E. coli strain with J96 by constructing a cnf1-negative derivative of that strain and comparing the mutant with the wild-type for virulence in a mouse model of ascending UTI and in human kidney and bladder cells; 2) investigate the nature of the chemical modification of Rho by CNF1 and clarify how that modification leads to actin polymerization; 3) dissect the relationship between the structure of CNF1 and its function by generating a set of mutant CNF1s through deletion, regionally-directed, and site-specific mutagenesis and preparing monoclonal and monospecific antibodies as probes for toxin structural integrity; 4) attempt to identify the functional receptor for CNF1. OBRIEN AD UNIFORMED SERV UNIV HLTH SCI, 4301 JONES BRIDGE ROAD, BETHESDA, MD 20814-4799 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Escherichia coli Escherichia coli infection pathologic process site directed mutagenesis gene deletion mutation monoclonal antibody bacterial toxin kidney cell virulence disease model polymerization actin cytotoxicity urinary bladder urinary tract infection CRISP RPROJ MARYLAND G CRISP/99/AI38281-04 CRISP/99/AI38281-04 199904 eng ADP RIBOSYLATION OF A POTENT INDUCER OF PROLIFERATION Research RPROJ DESCRIPTION (Adapted from Investigator's abstract): The broad goal of the proposed study is to improve understanding of signal transduction pathways involved in B cell activation. This knowledge will provide important understanding of both normal physiology and unregulated cell growth. Abnormalities of signal transduction proteins have been identified in both cancer and immunodeficiencies. Bacterial toxins have been useful tools to dissect the function of G proteins, a group of signal transduction proteins. Using NAD as a source, bacterial toxins add adenosine diphosphate ribose (ADP-ribose) to proteins, a process called ADP-ribosylation. This modification can alter the function of signal transduction proteins in a manner that mimics their physiologic function. Based on his previous work, the applicant hypothesizes that cholera toxin ADP-ribosylates a non-Gs protein that regulates B cell activation. The goals are to identify this protein and to determine its physiologic function. In Specific Aim 1, the ability of cholera toxin to ADP-ribosylate proteins in intact B cells will be used to block subsequent ADP-ribosylation of these proteins in vitro in the presence of [32P]NAD. While this reverse labeling technique has identified a single, non-Gs protein that is ADP-ribosylated by cholera toxin, other candidate proteins will be sought. Specific Aim 2 is designed to develop the necessary tools to study the physiologic function of the ADP-ribosylated proteins. Following microsequencing, the sequence information will be used to clone the corresponding cDNA. At each step, sequence information will be compared to known amino acid and nucleic acid data bases 1) to help identify the protein or any functional motifs and 2) to determine which proteins to pursue for further analysis. Finally, antibodies will be generated. Specific Aim 3 will begin to address the physiologic function of these proteins. Using antibodies, the investigator will determine whether and how these proteins are physiologically modulated by B cell stimulants, such as anti-Ig. In particular, he will determine whether these proteins are phosphorylated or dephosphorylated. If more than one candidate protein remains, these studies will establish which is the target protein. Conversely, selective activation of this protein by cholera toxin will permit him to explore the downstream effects that are specifically mediated by this protein. FRANCIS ML WAKE FOREST UNIVERSITY SCHOOL, MEDICAL CENTER BLVD, WINSTON SALEM, NC 27157-1052 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse biological signal transduction B lymphocyte molecular cloning ADP ribosylation leukocyte activation /transformation antibody cholera toxin protein structure /function protein lymphocyte proliferation CRISP RPROJ NORTH CAROLINA G CRISP/99/AI38308-01A2 CRISP/99/AI38308-01A2 199904 eng IN VIVO EFFECTS OF BACTERIAL SUPERANTIGENS Research RPROJ Bacterial superantigens have profound in vivo effects and have been implicated in human diseases such as toxic shock syndrome, adult respiratory distress syndrome (ARDS), and autoimmune disease. In addition, superantigen-secreting strains of Staphylococcus aureus have been associated with unexpected death resulting from influenza virus. A murine model has been developed to study the interplay between the bacterial superantigen, Staphylococcus enterotoxin B (SEB) and influenza infection. Injection of SEB into normal mice has numerous effects, including activation of T cells bearing the relevant T cell receptor (TCR) Vbeta elements, non-specific immune suppression, anergy, apoptosis, and massive cytokine secretion, both by T cells and MHC class II+ presenting cells. Despite these dramatic in vivo effects, bacterial superantigens are not lethal in mice. However, injection of SEB into mice 7 days after non- lethal infection with influenza virus results in the rapid onset of death. The experiments proposed here are designed to understand the role of viral infection in sensitizing mice to the lethal effects of SEB, and, in a broader sense, to determine the effects of superantigen on a concurrent immune response to infectious virus. This is of clinical relevance, because secondary bacterial infections are commonly associated with viruses that cause pathology in the respiratory tract. BLACKMAN MA ST JUDE CHILDRENS RES HOSPITAL, 332 N LAUDERDALE, MEMPHIS, TN 38105 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 38105 Crisp Data Base National Institutes Of Health laboratory mouse Staphylococcus aureus Staphylococcus infection B lymphocyte cytotoxic T lymphocyte cell cycle secondary infection cytokine immunologic memory antibody formation staphylococcal enterotoxin microorganism immunology microorganism interaction disease model receptor expression T cell receptor tissue /cell culture influenza influenzavirus A programmed cell death superantigen CRISP RPROJ TENNESSEE G CRISP/99/AI38349-03 CRISP/99/AI38349-03 199904 eng PASTEURELLA MULTOCIDA TOXIN--STRUCTURE AND ACTIVITY Research RPROJ Pasteurella multocida is a pathogenic bacterium associated with the agricultural diseases pasteurellosis, hemorrhagic septicemia, dermonecrosis, and progressive atrophic rhinitis. P. multocida can cause severe complications in human infections from animal bites or scratches, respiratory infections, and exposure to animals during pregnancy. P. multocida toxin is an important virulence factor of P. multocida, and purified PMT alone is sufficient to experimentally induce progressive atrophic rhinitis. PMT appears to enter cells via receptor-mediated endocytosis and causes activation of signal transduction events and DNA synthesis. Recent studies from our laboratory have identified G alpha protein as the primary target of PMT action that activates the phosphatidylinositol-specific phospholipase C-beta 1 and the inositol triphosphate pathway in Xenopus oocytes. Studies from our laboratory have also shown that the N-terminus of PMT is important for this activity, and we have proposed a model for PMT's intracellular action. We have cloned the entire toxA gene (1285 residues) from P. multocida and have generated a number of deletion mutants, encoding residues 1-73, 1-293, 1-506, 506- 1285, and 1059-1285. Our long range goals are to use these recombinant proteins to understand the structure and mechanism of action of PMT at the molecular and biochemical level, both to facilitate future therapeutic intervention in the bacterial pathogenesis of P. multocida , as well as to provide insight into the molecular signalling events involved in the control of cell growth and differentiation. In particular, we hope to demonstrate the utility of PMT as a new biochemical tool for studying intracellular signalling pathways involving the Gq family of regulatory proteins. To achieve our goals, we propose the following: (1) To define the functional domains of the protein, so as to determine which of the toxin's domains are responsible for (1) binding to the eukaryotic cell receptors and (2) stimulating the intracellular signal transduction pathways. (2) To elucidate the molecular mechanism by which PMT activates Gq- protein, by determining whether PMT's activation of Gq-protein is caused by a covalent modification or by noncovalent interaction. (3) To test the hypothesis that PMT uncouples the ligand-regulated interaction between receptor and Gq-protein, using the Xenopus oocyte system overexpressing exogenous 5-HT2 receptor and Gqalpha-protein. WILSON BA WRIGHT STATE UNIVERSITY, 3640 COLONEL GLENN HIGHWAY, DAYTON, OH 45435 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 45435 Crisp Data Base National Institutes Of Health Pasteurella multocida biological signal transduction covalent bond polymerase chain reaction microinjection transfection gene deletion mutation western blotting bacterial toxin protein sequence protein structure /function bacterial protein G protein receptor binding receptor coupling bacterial toxicology SDS polyacrylamide gel electrophoresis 3T3 cell Xenopus oocyte recombinant protein CRISP RPROJ OHIO G CRISP/99/AI38396-03 CRISP/99/AI38396-03 199904 eng BORDETELLA VIRULENCE REGULATION IN VITRO AND IN VIVO Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Nearly all of the known virulence factors synthesized by Bordetella pertussis, the causative agent of human whooping cough, and Bordetella bronchiseptica, which causes respiratory disease in other animals, are positively regulated by the Bvg signal transduction system. Bvg also negatively regulates production of a class of outer membrane proteins in B. pertussis and flagella synthesis and motility in B. bronchiseptica. The ability of Bordetella species to alternate between distinct phenotypic phases in response to environmental conditions has been recognized for many years, yet the role of Bvg- mediated signal transduction in the infectious cycle is unknown. The investigators' experiments will focus on the following questions: 1) How and why are Bordetella virulence genes regulated by Bvg, and 2) What are the functions of the regulated gene products? To address these they will use a comparative approach by conducting parallel studies with the human pathogen B. pertussis and the closely related animal pathogen B. bronchiseptica. They have established in vitro assays for studying the biochemistry of signal transduction, genetic methods for manipulating the virulence regulon, and natural host- animal models for characterizing respiratory tract infection. These form the foundation for this proposal. The research plan begins with an examination of molecular aspects of signal transduction using purified proteins and measurements of BvgA and BvgS function in E. coli. They will also address mechanisms of signal recognition by BvgS and these basic studies will facilitate an in vivo analysis. The effect of alterations in Bvg signal transduction pathways will then be assessed using animal models for B. bronchiseptica and B. pertussis. Studies with defined mutants will be accompanied by the development of a method for directly determining the phase of Bordetella populations in respiratory tissue. The relationship between the virulence regulons of B. pertussis and B. bronchiseptica will be investigated next, with specific emphasis on loci that are negatively regulated by Bvg. Finally, they will assess the functions of putative adhesins and toxins during infection of natural hosts by B. bronchiseptica. A novel approach using "ectopic expression" will be used for this purpose. By considering the Bvg regulon from a broad perspective they hope to discover its role in pathogenesis. MILLER JF UNIVERSITY OF CALIFORNIA-LA, 10833 LE CONTE AVENUE 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Brucellaceae Bordetella pertussis biological signal transduction cilium /flagellum motility flagellum genetic regulatory element genetic regulation bacterial genetics gene deletion mutation mutant bacterial toxin virulence disease model bacterial protein adhesin membrane protein CRISP RPROJ CALIFORNIA G CRISP/99/AI38417-04 CRISP/99/AI38417-04 199904 eng GENETICS AND CELLULAR BASIS OF H PYLORI PATHOGENESIS Research RPROJ Helicobacter pylori infects nearly half of the world's population, especially in developing countries paralleled by rates of gastric cancers, a leading cause of malignancy in some countries, and peptic ulcer disease. Significant epidemiological and biological evidence suggests that H. pylori infection is a necessary risk factor for the development of gastric cancer and peptic ulcers. Little is known about the bacterial factors that permit H. pylori to infect gastric tissue or those gene products that bring about the state of chronic, persistent inflammation that accompanies infection and gastric diseases. As well, the response of host cells to H. pylori infection has not been defined. We propose two broad specific aims to elucidate the nature of the interaction between the pathogen and the host: 1) to define the contribution of H. pylori virulence factors to the molecular and cell biology of the interaction with host cells; and 2) to define the biological consequences to the host cell associated with the attachment and subsequent stable infection by H. pylori to cellular targets. We will utilize unique methods of video and confocal microscopy to study Intracellular trafficking and response of host cells to Infection, and we will exploit novel molecular methods to identify new bacterial genes that are environmentally regulated and expressed in vivo. In addition, we propose to utilize a molecular method known as differential display to study the effect of viable bacteria and bacterial factors on eukaryotic cells. Previous studies in our laboratories on H. pylori and other enteric pathogens utilizing these approaches have provided the relevant experience and have produced exciting insights into the biological consequences of the host-pathogen interaction. Thus, we expect that a similar approach to understand the pathogenesis of Helicobacter infection will be fruitful. TOMPKINS LS STANFORD UNIVERSITY, SCHOOL OF MEDICINE, STANFORD, CA 94305-5402 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health urease intracellular transport genetic strain hemolysin polymerase chain reaction cellular pathology molecular pathology host organism interaction genetic marker genotype microorganism genetics mutant virulence tissue /cell culture confocal scanning microscopy Helicobacter video microscopy CRISP RPROJ CALIFORNIA G CRISP/99/AI38459-03 CRISP/99/AI38459-03 199904 eng THE EFFECT OF COCKROACH ALLERGIES IN CHILDREN WITH ASTHMA Research WILLIAMS LW DUKE UNIVERSITY MEDICAL CENTER, BOX 2898, DURHAM, NC 27710 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 27710 Crisp Data Base National Institutes Of Health cockroach child (0-11) cooperative study human subject allergen asthma enzyme linked immunosorbent assay arthropod nonpollutant control insect control insecticide dust housing human therapy evaluation immunopathology therapy CRISP RPROJ NORTH CAROLINA G CRISP/99/AI38550-040004 CRISP/99/AI38550-040004 199904 eng PRODUCING SEPSIS ANTIDOTE PROTEINS IN VIVO Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The systemic host inflammatory response to microbial invasion ("sepsis") can damage the infected host, leading to multiple organ failure, coagulopathy, shock, and death. Despite much effort, recent attempts to improve the outcome of severe sepsis in humans have been unsuccessful. In particular, using clinical criteria to select the best candidates for anti-cytokine therapy has proven very difficult: it is not possible to determine whether a patient will benefit most from none, a little, or a lot of a given antidote. The research proposed here takes a new approach to this problem. The goal of this approach is to limit (damp) the inflammatory response without interfering with the normal, beneficial defense against invading microbes. DNA constructs will be engineered so that sepsis antidote proteins (anti-endotoxins or anti-cytokines) will be expressed downstream of promoters that respond to inflammatory cytokines (e.g., IL- 1, IL-6, TNF-alpha). These constructs, introduced into mice using adenoviral vectors or DNA-liposome complexes, will be tested for their ability to protect the animals from gram-negative bacterial infection or endotoxin challenge. Since production of the antidote proteins in vivo will be controlled by the intensity of the animal's own inflammatory response, dangerous inflammatory excess may be prevented without blocking the beneficial components of the host response. In effect, each animal will determine its own dose of antidote protein(s). Potential clinical uses include the prophylaxis and therapy of sepsis and other systemic inflammatory disorders, as well as certain tissue-specific applications. MUNFORD RS U TEXAS SW MED CTR AT DALLAS, 5323 HARRY HINES BOULEVARD, DALLAS, TX 75235-9113 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse bacterial disease blood toxicology inflammation transfection bactericidal immunity cytokine immunoregulation immunotherapy acute phase protein protein biosynthesis CRISP RPROJ TEXAS G CRISP/99/AI38596-04 CRISP/99/AI38596-04 199904 eng HIV PATHOGENESIS IN NEW SCID MOUSE MODELS Research RPROJ The study of the pathogenesis of HIV, and the evaluation of intervention modalities, continues to be limited by the lack of a reliable small animal model of HIV infectivity. We propose to address this deficiency by utilizing genetically altered strains of NOD/LtSz-scid/scid mice to develop a relevant model for the study of HIV-1 infection in human lymphohematopoietic cells. This model will be used for the study of viral pathogenesis and intervention strategies, including in vivo evaluation of antibodies, antiviral drugs, and CTL. To accomplish our objective, we propose an integrated approach involving 3 laboratories with expertise in mammalian genetics, in lymphocyte development and function, and in HIV pathogenesis and therapy. Specific Aim #1 will evaluate the engraftment and immune function of human lymphocytes in new genetic stocks of NOD/LtSz-scid/scid mice. We have shown that the genetic background of the NOD/Lt mouse with defects in innate immunity allows improved engraftment and HIV infection of human PBMC in scid/scid mice. We will test the ability of NOD/LtSz-scid/scid mice deficient in NK cell activity, MHC class I, and MHC class II antigens to support engraftment with human lymphocytes and to generate primary and secondary immune responses. Specific Aim #2 will determine levels of HIV infection and pathogenesis in human lymphohematopoietic cell engrafted NOD/LtSz-scid/scid mice. Studies of HIV infection in these mice will include evaluation of virus load, and effects of different viral phenotypes on engrafted cells. Rapidly growing cytopathic viruses, slow-growing viruses with low cytopathic potential, and primary viral isolates obtained from long-term nonprogressors with novel genetic defects will be tested. Infection with nef-deleted viruses on immature and mature human lymphocytes will be studied. Specific Aim #3 is to evaluate therapeutic modalities aimed at preventing or interrupting HIV-1 replication in human lymphocyte engrafted NOD/LtSz-scid/scid mice. We will evaluate interruption of HIV-l replication using anti-retroviral therapies (AZT, Nevirapine, etc.), HIV-1 specific antibodies, and HIV-1 specific cytotoxic T cells. Accomplishment of the work in this proposal will provide a reliable small animal model system that allows the rapid evaluation of HIV therapeutics, vaccines, and gene therapy for studies aimed at interrupting HIV- 1 pathogenesis. HESSELTON RA UNIV OF MASS MEDICAL SCHOOL, 373 PLANTATION STREET, WORCESTER, MA 01605 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 01605 Crisp Data Base National Institutes Of Health hematopoietic tissue transplantation lymphocyte cytotoxic T lymphocyte pathologic process genetic manipulation allele beta globulin human tissue antiviral antibody AIDS therapy HIV infection antiAIDS agent model design /development disease model nonhuman therapy evaluation MHC class II antigen tissue mosaicism cytolysin virus replication human immunodeficiency virus 1 viremia SCID mouse CRISP RPROJ MASSACHUSETTS G CRISP/99/AI38757-03 CRISP/99/AI38757-03 199904 eng NEUROGENIC INFLAMMATION IN ASTHMA AND OZONE LUNG INJURY Research RPROJ Asthma is characterized by episodic bronhoconstriction, chronic lung inflammation, and airway hyperreactivity to constricting stimuli. Airway inflammation in asthma is thought to be a critical factor in the pathogenesis of the disease, but the molecular mechanisms underlying the inflammation that develops in asthma are not known. Airborne pollutants, including ozone, have been reported to worsen asthma and associated inflammation, but the mechanisms by which pollutants contribute to the pathology of asthma are not known. Since there are many potential inflammatory mediator involved, it has not been possible to establish in vivo which molecules are playing a mechanistic role in the development of the disease. The studies proposed here will examine sensory neuropeptides known a tachykinins that can act as inflammatory mediators. Tachykinins include the neuropeptides substance P and neurokinin A, which exert their effects by binding to neurokinin receptors. Levels of the tachykinin substance P are increased in asthmatic lung, and substance P is released in humans after allergen or ozone exposure. The central hypothesis to be tested here is that tachykinin neuropeptides released from sensory nerve endings are key mediators in the development of allergen- and ozone-induced airway inflammation. The hypothesis will be tested by using transgenic and gene knock-out technology to manipulate the amount of sensory nerve fibers that innervate the lungs of mice. The inflammation that develops in the lungs of these genetically altered mice after exposure to allergen or ozone, or a combination of the two agents, will be measured by histological, biochemical, and cytological methods. Our hypothesis predicts that mice releasing increased amounts of tachykinins will be unusually susceptible to allergen-and ozone-induced airway inflammation and that mice releasing reduced amounts of tachykinins will be resistant tot he development of inflammation. The propose experiments will allow a determination of whether ozone and allergen act synergistically to produce inflammation and whether tachykinins are involved in the action of these agents, either individually or in combination. In addition, the molecules through which tachykinins exert their effects will be examined in the genetically manipulated mice. For example, neurokinin receptor antagonists will be used to characterize receptor subtypes mediating neurogenic inflammation in mice that release increased amounts of tachykinins. Molecular biological and biochemical methods will also be used to determine whether expression of cytokine mRNAs and protein products are altered in mice releasing increased or reduced amounts of tachykinins. This novel approach employing genetically manipulated mice will provide valuable mechanistic information concerning inflammation induced by tachykinins in airway disease. HOYLE GW TULANE UNIVERSITY, 1430 TULANE AVE, SL9, NEW ORLEANS, LA 70112 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 70112 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal polymerase chain reaction disease /disorder proneness /risk inflammation inhibitor /antagonist genetic manipulation gene mutation neurotrophic factor allergen asthma cytokine colony stimulating factor enzyme linked immunosorbent assay immunologic assay /test disease model nerve ending innervation neuron substance P tachykinin ozone neuropeptide receptor southern blotting lung injury CRISP RPROJ LOUISIANA G CRISP/99/AI39023-03 CRISP/99/AI39023-03 199904 eng MECHANISMS OF UREASE SURFACE LOCALIZATION IN H PYLORI Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and plays important roles in peptic ulcer disease, gastric carcinoma and gastric lymphoma. It is estimated that 30% of the adult US population is infected with the bacterium resulting in widespread gastro-intestinal disease and huge health care costs. H. pylori expresses significant urease activity which is an essential virulence factor. Recombinant urease apoenzyme is being tested as a vaccine (with the support of NIH) in human clinical trials, due in large part, because a significant fraction of this urease activity is located on the surface of the bacterium. The mechanism by which urease and HspB (a GroEL, heat shock protein homologue) become surface associated is not understood. As preliminary data for this proposal, "altruistic autolysis" was shown as the mechanism responsible for surface localization of H. pylori urease and other cytoplasmic proteins. The primary objective of this proposal is to elucidate the mechanisms by which urease becomes surface associated in H. pylori. The long term objective of this proposal is to understand the pathogenic contribution of this novel surface localization mechanism in pathogenesis by producing specific defective mutant strain(s) and testing them in animal models. Ultimately, such studies should allow development of effective therapies, including vaccines for prevention of H. pylori-associated disease. The specific aims of this proposal are: 1) Confirm and extend the investigators' observations, using molecular technology, that autolysis followed by surface-adsorption is the principle mechanism for translocation of urease onto the surface of H. pylori. 2) Confirm and extend their observations that urease and HspB are associated with the outer membrane of H. pylori in human gastric biopsies and determine whether these proteins are associated with the outer membrane of the related bacterium, H. felis, in gastric tissue from experimentally infected mice. 3) Define the pathogenic role of urease released by bacterial autolysis. 4) Elucidate the biochemical mechanisms of autolysis in H. pylori. 5) Elucidate the genetic mechanisms of autolysis in H. pylori. DUNN BE MEDICAL COLLEGE OF WISCONSIN, 8701 WATERTOWN PLANK ROAD, MILWAUKEE, WI 53226-0509 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health urease laboratory mouse gastritis peptic ulcer human subject bacterial toxin virulence protein metabolism bacterial protein membrane protein pore forming protein stress protein Helicobacter clinical research CRISP RPROJ WISCONSIN G CRISP/99/AI39045-02 CRISP/99/AI39045-02 199904 eng PULMONARY IMMUNE RESPONSE TO INHALED PARTICULATES Research RPROJ DESCRIPTION (Adapted from the applicant's abstract and specific aims): The gas-exchange surface of the lung is continuously challenged by inhaled particulate antigens, however, the generation of a T-cell mediated immune response within the lung is uncommon. The development of a pulmonary immune response reflects the net effects of proinflammatory and suppressive activities by immune cells in the lung. In this regard, alveolar macrophages (AM) play a pivotal role in the response to particulates by ingesting and sequestering them away from immune cells in the lung interstitium. Activated AM also secrete factors that effectively suppress the activities of antigen presenting cells (APC) and the proliferation of T-cells. Dendritic cells (DC) distributed in the airway and pulmonary interstitium entrap inhaled soluble antigens and present them to T-cells in draining lymph nodes and lung. Although DC show limited phagocytosis, they can present particulate antigens to T-cells in vitro. The broad goal of this project is to understand how macrophages interact with DC and T-cells during a particulate antigen challenge to suppress the immune response in vivo. This project will address the following questions: Aim 1. How do soluble and particulate antigens differ in their abilities to promote NO production by AM? Aim 2. How does NO suppress the activities of lung DC and T-cells? Aim 3. How do phagocytosis and NO production by AM in vivo contribute to the development of immune "tolerance" to particulate antigens? KRADIN RL MASSACHUSETTS GENERAL HOSPITAL, FRUIT STREET, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02114 Crisp Data Base National Institutes Of Health air pollution laboratory rat dendritic cell phagocytosis flow cytometry leukocyte activation /transformation immune tolerance /unresponsiveness antigen presentation particle alveolar macrophage nitric oxide CRISP RPROJ MASSACHUSETTS G CRISP/99/AI39054-02 CRISP/99/AI39054-02 199904 eng INTESTINAL TRANSPORT AND RESPONSE TO SHIGA-LIKE TOXINS Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The objective of this proposal is to address the pathogenesis of gastrointestinal disease associated with Shiga-like toxin (SLT) producing E. coli, which colonize the gastrointestinal tract. SLTs are considered to be responsible for both local and systemic disease. This proposal will focus on the interactions of the SLTs with intestinal epithelial cells (IECs) and endothelial cells, address how SLTs cross IECs, what effect SLTs have on IECs and how the combination of cytokines, SLTs and lipopolysaccharides (LPS) contribute to endothelial cell damage resulting in thrombotic microangiopathy (TMA) in the gut. Three specific aims are proposed. The first will determine how SLTs cross IECs using in vitro models. The effect of SLTs on the production of cytokines and the role of the bacteria in these interactions. The second specific aim is directed toward understanding how SLTs interact with, and cross IECs in vivo from bacterial infection within the gut. The third aim will study the effect of SLTs, cytokines from IECs and LPS on endothelial cell markers important in TMA. The methods to be used involve in vitro experiments using IECs in tissue culture and intestinal tissue in Ussing chambers to better understand SLT movement. Cytokine expression will be determined using RT-PCR and immunoassays. The in vivo experiments will be undertaken using SLT-producing bacteria and isogenic controls known to colonize either mice or rabbits to study both toxin interaction and cytokine production. Endothelial cell experiments will involve both tissue culture and in vivo studies and determination of TMA-associated factors in response to SLTs, cytokines and LPS. Shiga-like toxins produced in the gastrointestinal tract are associated with bloody and non-bloody diarrhea, as well as systemic disease (hemolytic uremic syndrome). It is anticipated that by gaining an understanding of SLT translocation and interaction with endothelial cells, considered to be the main target of this disease, it will ultimately allow the development of directed therapy to prevent SLT translocation and damage in the intestinal tract, and thereby reduce the clinical severity of the infection with SLT-producing E. coli. ACHESON DW NEW ENGLAND MEDICAL CTR, BOX 041, 750 WASHINGTON STREET, BOSTON, MA 02111 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02111 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Escherichia coli Shigella dysenteriae bacterial cytopathogenic effect Escherichia coli infection butyrate bacillary dysentery gastrointestinal toxin absorption gastrointestinal infection gastrointestinal epithelium cytokine immunologic assay /test enterotoxin electron microscopy fluorescence microscopy lipopolysaccharide tissue /cell culture CRISP RPROJ MASSACHUSETTS G CRISP/99/AI39067-03 CRISP/99/AI39067-03 199904 eng EPIDEMIOLOGY & ECOLOGY OF VIBRIO CHOLERAE IN BANGLADESH Research RPROJ DESCRIPTION: (Adapted from Applicant's Abstract). Cholera, the most severe of all diarrheal diseases, caused by Vibrio cholerae 01 (and most recently also by serogroup 0139) is one of the few pandemic infections of man. Its known history includes waves of severe, often fatal, disease covering the entire globe over decades of time, usually having originated from the cholera endemic area of the Ganges delta. In the heavily endemic regions of southeast Asia, cholera also exhibits a regular twice-yearly periodicity. Unfortunately, the mechanisms controlling the periodicity and pandemicity of cholera are not known. No studies to date have been able to explain this epidemiologic behavior of cholera. The primary objective of this proposal is to test the hypothesis that environmental factors involving surface waters are responsible for the observed epidemiology of cholera. We postulate that plankton living in surface waters are the reservoir for cholera vibrios, and that their growth and life cycle(s) control vibrio populations in surface water, and thus determine how vibrios are spread and when outbreaks of disease will occur. This information could 1) lead to an improved warning system for prediction of cholera outbreaks, both in endemic areas, and in areas into which a cholera pandemic is threatening, and possibly lead to new strategies for controlling the disease through environmental interventions directed toward growth of plankton. This joint project, involving also investigators from the International Center for Diarrheal Disease Research, Bangladesh, (ICDDR,B), the University of Maryland and Emory University, involves establishing four sentinel surveillance sites in bangladesh, three of which are known to exhibit cholera periodicity, and one that is usually free of cholera. A combined year-round effort will monitor 1) the clinical cases/infections with V.cholerae, 2) the occurrence and density of V. cholerae in the surface waters of these sites, including those attached to plankton, both as viable and as non-culturable forms, using sensitive sampling techniques (including PCR), 3) the growth of phytoplankton and zooplankton in these areas, using direct sampling and identification techniques as well as remote sensing by satellite, and 4) the genetic and phenotypic markers of isolates to determine whether V.cholerae isolated from surface waters are identical with those isolated from patients with cholera in the same vicinity. Based on these data, a model of cholera epidemiology will be developed which may be useful in predicting outbreaks of cholera, thereby allowing early mobilization of preventive and treatment measures. Solving this problem would be an enormous step toward our understanding of "emerging" infectious diseases on the planet. SACK RB JOHNS HOPKINS UNIVERSITY, 615 N WOLFE STREET, BALTIMORE, MD 21205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21205 Crisp Data Base National Institutes Of Health laboratory mouse Vibrio cholerae bioperiodicity polymerase chain reaction communicable disease transmission disease outbreak disease reservoir water microbiology cholera gastrointestinal disorder diagnosis Bangladesh public health human subject monoclonal antibody serology /serodiagnosis epidemiology aquatic organism water sampling /testing clinical research CRISP RPROJ MARYLAND G CRISP/99/AI39129-03 CRISP/99/AI39129-03 199904 eng LIPOPOLYSACCHARIDE ENDOTOXIN RESPONSE GENE IN CELLS Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The interaction of the outer membrane components of bacteria with host cells in one form or another has been investigated extensively over the past decades. The lipopolysaccharide endotoxin (LPS) of Gram-negative bacteria is an example of an essential element of the outer membrane of these organisms that as amphipathic molecule has the property of binding to and stimulating various types of mammalian cells. The results of this interaction are multiple pathophysiological, pharmacological and immunological responses by the host. Over the preceding years, our long term objective has been to gain a better understanding of how LPS endotoxin affects cells of the host. The knowledge that host responses are under genetic control is key to this understanding. Consequently, we have pursued the goal of isolating the LPS gene by the use of the C3H/HeJ mouse strain whose cells possess a specific defect for LPS. By employing a differential functional screening approach, we have isolated a cDNA from splenic B cells of C3H/OuJ responder mice, and its expression in splenic B cells of C3H/OuJ responder mice, and its expression in splenic B cells of C3H/HeJ non-responder mice resulted in polyclonal B cell activation in response to LPS stimulation. In this proposal, our specific aims, which are a logical consequence of this finding, are as follows: 1) to complete the confirmatory analysis that expression of the cDNA we isolated can functionally reconstitute a macrophage cell line derived from a C3H/HeH mouse, 2) to refine the mapping analysis indicating that the gene is located on mouse chromosome four, 3) to define the nature of the Lpsd gene we isolated from C3H/HeH mice, 4) to investigate its genomic organization and to verify two single-base substitutions found in C3H/HeJ cDNA, 5) to introduce the Lpsn gene into various primary cells of the C3H/HeJ non-responder mouse and measure their responsiveness after exposure to LPS, 6) to introduce the Lpsn gene into C3H/HeJ non-responder mice using a retrovirus vector and measure the key parameters of endotoxin responsiveness in these mice including B cell mitogenesis, macrophage activation and cytokine elaboration, and resistance to endotoxemia or lethal shock, and 7) to assess the role of Lpsn in resistance to Gram negative bacterial infection. WONG PM TEMPLE UNIVERSITY, 3420 NORTH BROAD STREET, PHILADELPHIA, PA 19140 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 19140 Crisp Data Base National Institutes Of Health laboratory mouse macrophage host organism interaction genetic mapping bacterial genetics immune response gene endotoxin lipopolysaccharide G protein Retroviridae cell line fluorescent in situ hybridization CRISP RPROJ PENNSYLVANIA G CRISP/99/AI39159-02 CRISP/99/AI39159-02 199904 eng RECOMBINANT ANTIBODIES FOR INFANT PROTECTION Research RPROJ In the current grant we propose to expand our studies of genetically engineered antibody molecules, focusing our attention on the goal of providing more effective immunity to the fetus and neonate. In one series of experiments we will identify and produce antibodies with the optimal combination of functional properties to provide protection at the mucosal surface following oral administration or transport from the mother via the placenta or mammary gland. Human IgA is the antibody normally found in the exocrine secretions. IgA, domain exchange proteins between human IgA and IgG, and site directed mutants will be characterized with respect to their stability, half-life, and biodistribution when administered either orally or intravenously. They will be characterized with respect to their ability to bind and be transported by the epithelial receptors. They also will be assessed for their ability to activate the complement cascade and to bind to Fc receptors specific for either IgG or IgA on phagocytes. We will also attempt to develop a cell which expresses antibody with attached secretory component (SC). One goal will be to produce recombinant antibodies which provide treatment or protection against enteric infections, a significant health problem world wide. We will assay the most promising recombinant antibodies in the suckling mouse model for enterotoxigenic Escherichia coli (ETEC) using antibodies specific for the F41 antigen. Antibodies to F4l have proven efficacy and recombinant antibodies administered orally to pups or intravenously to dams will be assessed for their ability to prevent lethality and to decrease colonization. In vitro assays of adherence, agglutination and complement mediated cytotoxicity will address the mechanism(s) of protection. If recombinant antibodies are to be broadly applied, an inexpensive expression system must be available and we will focus our attention on the development of the transgenic chicken as such an expression system. The chicken is inexpensive to maintain and targets large amounts of antibody to the egg yolk. Intravenous injection of iodinated antibodies into laying hens will be used to determine which antibodies will be transported to the yolk. Vectors tested for Ig expression using chicken lymphoid cell lines will be transfected into Stage X blastoderm cells and these transfected cells used to make chimeric embryos. The resulting chickens will be tested for the presence of chimeric lg in their serum and in the eggs of laying hens. MORRISON SL UNIV OF CALIFORNIA, LOS ANGELE, 405 HILGARD AVE, LOS ANGELES, CA 90095-1489 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health chicken laboratory mouse transgenic animal Escherichia coli Escherichia coli infection cell adhesion phagocyte communicable disease chemotherapy intravenous administration pharmacokinetics immunoglobulin A immunoglobulin G cell mediated cytotoxicity monoclonal antibody agglutination reaction antibacterial antibody enterotoxin complement antibody receptor nonhuman therapy evaluation neutralizing antibody oral administration secretory protein recombinant protein mucosal immunity CRISP RPROJ CALIFORNIA G CRISP/99/AI39187-04 CRISP/99/AI39187-04 199904 eng REGULATION OF PSEUDOMONAS AERUGINOSA GENES DURING INFECTION Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Pseudomonas aeruginosa, an opportunistic pathogen, is the major causative agent of mortality and morbidity in immunocompromised patients. It is also the most important pathogen of patients with cystic fibrosis. During chronic infection, numerous changes have been identified including repression of specific virulence genes and surface changes. In order to identify virulence genes of P. aeruginosa that are important in the infection of CF patients, Dr. Jin developed a selection system similar to the in vivo expression technology first described in Salmonella. Using this system, he has identified three genetic loci that are inducible by tracheal-bronchial mucus from CF patients. These loci include the pyochelin receptor, the migA/B locus which is homologous to genes involved in LPS synthesis and is controlled by a DNA rearrangement switch, and regA a positive regulator of exotoxin A synthesis. In this application, Dr. Jin proposes to focus on the migA/B locus and test the hypothesis that the DNA rearrangement in the migA/B locus is responsible for the LPS and exotoxin A phase variation observed in chronic infection. Experiments are designed to (i) define the function of the migA/B gene, (ii) characterize the molecular mechanism of the migA/B DNA rearrangement, and (iii) assess the importance of the DNA rearrangement in chronic infection of hosts. JIN S UNIV ARKANSAS FOR MED SCIENCES, 4301 WEST MARKHAM SLOT 511, LITTLE ROCK, AR 72205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 72205 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal Pseudomonas aeruginosa opportunistic infection host organism interaction molecular cloning gene expression gene rearrangement genetic regulation bacterial genetics human tissue exotoxin cystic fibrosis virulence restriction mapping lipopolysaccharide gene targeting CRISP RPROJ ARKANSAS G CRISP/99/AI39524-03 CRISP/99/AI39524-03 199904 eng MECHANISMS OF NITRIC OXIDE CYTOTOXICITY IN SALMONELLOSIS Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Nitric oxide (NO) is a biological mediator with remarkably broad-spectrum antimicrobial activity NO can exist in different redox forms, each with distinctive stability, reactivity, and biological actions. Although NO has been investigated in numerous animal models of infection, the effects of specific redox forms of NO on microbes have not been extensively investigated at a molecular level. This proposal analyzes the molecular mechanism and biological relevance of NO antimicrobial activity in a murine salmonellosis model. Preliminary data are presented suggesting that S-nitrosothiols are important endogenous antimicrobial redox forms of NO, and that microbial homocysteine antagonizes the cytostatic effects of S-nitrosothiols. The specific aims of this proposal are the following: A) Identification of Salmonella genes which determine susceptibility to different redox forms of nitric oxide in vitro; B) Identification of nitric oxide-regulated Salmonella genes; C) Correlation of Salmonella susceptibility to in vitro nitric oxide donors with susceptibility to cell-derived nitric oxide and virulence. S-nitrosoglutathione, SIN-1, and DETA/NO will be used as donors of nitrosonium (NO+), peroxynitrite (OONO-), and NO radical (NO-), respectively. Bacterial genes which influence NO susceptibility or whose regulation is influenced by NO will be mutated, mapped, cloned, sequenced, and complemented. In preliminary work, a considerable number of isogenic mutants with altered susceptibility to specific redox forms of NO have already been identified and characterized. The susceptibility of mutants to cell-derived NO and virulence of these strains in mice will be examined to define the biological significance of specific redox forms of NO. Genetic manipulation of intracellular homocysteine levels and the use of homocysteine-regulated gene fusions will be employed to test the provocative thesis that microbial homocysteine synthesis antagonizes the intraphagosomal antimicrobial activity of NO. Although this project exploits the advanced knowledge of Salmonella genetics in utilizing this specific model system, the implications of this work are by no means limited to salmonellosis. The preliminary findings have already identified novel pathways used by endogenous antimicrobial mediators, and novel mechanisms of microbial resistance to these mediators. The proposed studies will provide further insights into the molecular basis of NO cytotoxicity and the potential importance of this activity in a wide range of infections, particularly those caused by intracellular pathogens. FANG FC UNIV OF COLORADO, 4200 E 9TH AVENUE, B168, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80262 Crisp Data Base National Institutes Of Health laboratory mouse Salmonella typhimurium antibacterial agent drug resistance drug screening /evaluation salmonellosis molecular cloning bacterial genetics gene mutation virulence nucleic acid sequence oxidation /reduction homocysteine cytotoxicity nitric oxide oxidative stress CRISP RPROJ COLORADO G CRISP/99/AI39557-02 CRISP/99/AI39557-02 199904 eng CYTOTOXIC T CELL MEDIATED IMMUNITY TO CHLAMYDIA Research RPROJ NO ABSTRACT AVAILABLE STARNBACH MN HARVARD MEDICAL SCHOOL, 200 LONGWOOD AVE, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal genetic strain cytotoxic T lymphocyte cytolysis Chlamydia trachomatis chlamydial disease molecular cloning genetic library bacterial genetics bactericidal immunity cellular immunity passive immunization epitope mapping bacterial antigen bacterial protein pore forming protein cytolysin bacterial vaccine vaccinia virus transfection vector interferon gamma vector vaccine CRISP RPROJ MASSACHUSETTS G CRISP/99/AI39558-03 CRISP/99/AI39558-03 199904 eng MOLECULAR MECHANISMS FOR VASCULAR CELL DEACTIVATION Research RPROJ DESCRIPTION (Adapted from Investigator's Abstract): Overt activation of monocyte/macrophages (Mphage) and endothelial cells is a common feature of several diseases including sepsis, acute respiratory distress syndrome (ARDS) and atherosclerosis. During the studies in the pathogenesis of leishmaniasis, an intracellular parasitic disease, a major surface molecule of this parasite, namely, lipophosphoglycan (LPG) was found to deactivate Mphage. LPG suppress human Mphage expression of pro-inflammatory cytokine genes and nitric oxide synthase activity but had no effect on control gene in preliminary studies. Furthermore, LPG inhibits gene expression via transcription mechanisms and possibly by inducing a repressor protein(s). Since these factors are required for killing the parasite and T cell-dependent immunity, LPG effectively deactivates Mphage and alters T cell immunity resulting in parasite survival. The findings that LPG suppressed lipopolysaccharide (endotoxin) and cytokine activation of Mphage led to investigations to determine whether this molecule has broader applications. Human vascular endothelial cells are studied because the parasite at cutaneous site of entry traverses vascular endothelium to infect distant Mphage. LPG rapidly binds to endothelial cell and deactivates endothelial cell functions: thrombosis and leukocyte adherence but had no effect on HLA class I expression. Because HIV-1 replication also requires cell activation, we evaluated the effect of LPG on cells chronically infected with HIV. LPG suppressed agonist induction of HIV. The application shows evidence that the inhibitory activity of LPG is not due to low level endotoxin contamination of LPG and excludes a non-specific global effect because neither cell viability nor protein and DNA synthesis were affected by LPG. These findings in murine and human vascular cells suggest that LPG affects fundamental events in cell activation signaling. The application suggests that LPG is a ligand mimetic for undefined host factor(s) and that LPG triggers cell deactivation by utilizing existing host pathways. The mechanisms by which LPG deactivates vascular cells are undefined. It is hypothesized that LPG deactivates vascular cells by binding to membrane receptor(s), triggering specific intracellular and nuclear events, and thus, regulating transcription of multiple genes. The proposed studies will: 1) characterize the biologic effects of LPG on monocytes, 2) determine the specific membrane and intracellular signaling events of LPG, and 3) define the transcription regulation of IL-1B by LPG. Both LPG and endotoxin are evolutionary perfected molecules. LPG deactivates agonist induced cell signaling while endotoxin is a potent inflammatory molecule. By analogy to endotoxin, studies on LPG will identify fundamental mechanisms for vascular cell deactivation and likely have novel applications for several diseases of overt vascular cell activation accounting HO JL CORNELL UNIV MEDICAL COLLEGE, 1300 YORK AVENUE, RM A-431, NEW YORK, NY 10021 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 10021 Crisp Data Base National Institutes Of Health macrophage thromboplastin thrombosis vascular endothelium chemical structure /function cellular pathology site directed mutagenesis genetic promoter element transcription factor glycosphingolipid human tissue leukocyte activation /transformation interleukin 1 endotoxin DNA footprinting Leishmania receptor binding gel mobility shift assay nitric oxide synthase CRISP RPROJ NEW YORK G CRISP/99/AI39606-03 CRISP/99/AI39606-03 199904 eng CAP18-IGG FUSION PROTEIN--A NOVEL TREATMENT FOR SEPSIS Research RPROJ DESCRIPTION: (Adapted from the applicant's abstract) Gram-negative infections continue to cause substantial morbidity and mortality. A major part of the toxicity from these infections is believed to be caused by lipopolysaccharide (LPS) release from dying bacteria, that interacts with host systems leading to secondary inflammation. The availability of an agent with a long half-life that is capable of both killing Gram-negative bacteria and neutralizing its endotoxin would be a major advance which may prove to be therapeutically advantageous. Chemical conjugates of an 18 kD cationic protein present in human granulocytes (CAP18) coupled to human IgG appears to be such an agent. CAP18-IgG fusion proteins are believed to function by binding LPS that is present on or is released from the bacterial cell wall. Bacterial killing is presumed to occur by intercalation and disruption of the outer cell membrane. In preliminary studies CAP18-IgG fusion proteins appear to kill a wide variety of Gram-negative bacteria as well as neutralize bacterial LPS. This study seeks to develop and evaluate the efficacy of fusion proteins of CAP18 conjugated with the human heavy chain IgG in gram negative infections. In order to accomplish this goal the following series of experiments are proposed: 1) Design, construct, express, and purify fusion proteins of CAP18-IgG and CAP18104-135-IgG. 2) Characterize and assess the in vitro ability of these CAP18-IgG fusion proteins to bind and neutralize Gram-negative bacteria and LPS, assess their bactericidal activity, promote opsinophagocytosis of bacteria and bacterial membrane, and determine stability in blood over time. 3) Evaluate these CAP18-IgG fusion proteins in vivo using rabbits and rats to determine their pharmacokinetics, their functional stability in the bloodstream over time and ability to clear radiolabeled LPS and bacteria from the blood. 4) Study the protection by the best CAP18-IgG fusion proteins in two models of Gram-negative sepsis: a) Gram-negative infection in rats made neutropenic with cyclophosphamide, and b) a rat model of peritoneal sepsis. WARREN HS MASSACHUSETTS GENERAL HOSPITAL, 149 13TH ST, 1495234, CHARLSTOWN, MA 02129 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02129 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory rat gram negative bacteria bacterial disease erythrocyte phagocytosis chemical conjugate chemical stability antibacterial agent communicable disease chemotherapy pharmacokinetics drug screening /evaluation drug design /synthesis /production genetic manipulation immunoglobulin G cyclophosphamide endotoxin disease model bacteriorhodopsin lipopolysaccharide protein purification binding protein chimeric protein nonhuman therapy evaluation tissue /cell culture CRISP RPROJ MASSACHUSETTS G CRISP/99/AI39617-03 CRISP/99/AI39617-03 199904 eng HIERARCHY WITHIN ENVIRONMENTAL REGULONS Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The goal of this research is to discern mechanisms by which the environment within the human host are responsible for inducing expression of specific genes of infecting bacteria. The model system to be analyzed is the Vibrio cholerae ToxR regulon, for which a number of parameters that induce gene expression as well as many of the genes involved have been identified. ToxR can be thought of as an integral membrane protein positioned centrally in a regulatory scheme where it receives signals perhaps via small molecules and through protein-protein interactions that allow ultimate control of gene expression through a regulatory cascade. ToxR spans the bacterial cytoplasmic membrane and in this configuration is able to directly activate expression of a number of host specific genes. Some of these gene products are directly involved in allowing the bacteria to colonize and express an ADP-ribosylating exotoxin that stimulates the activity of host membrane adenylate cyclase. Another gene that ToxR activates, toxT, also encodes an activator protein of the AraC family that is cytoplasmically located. In turn, this protein activates additional genes including itself and colonization factors required for maintenance of an infected state. Thus, there is a hierarchy of gene expression that responds to the host to initiate and maintain infection. TAYLOR RK DARTMOUTH COLLEGE, VAIL BLDG, HANOVER, NH 03755-3842 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Salmonella typhimurium Vibrio cholerae host organism interaction gene expression open reading frame genetic regulatory element genetic promoter element transcription factor bacterial genetics mutant cholera toxin exotoxin virulence transposon /insertion element nucleic acid sequence bacterial protein DNA binding protein membrane protein bacteriophage P22 gel mobility shift assay CRISP RPROJ NEW HAMPSHIRE G CRISP/99/AI39654-03 CRISP/99/AI39654-03 199904 eng STRUCTURE AND REGULATION OF H PYLORI CYTOTOXIN Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Helicobacter pylori is a Gram-negative bacterium that is present in the stomach of at least half of the world's human population. In this niche, it persists for decades unless treated, and consistently induces gastric inflammation. Infection with this organism is a significant risk factor for the development of peptic ulcer disease, gastric carcinoma, and gastric lymphoproliferative diseases. The long-term objective of this project is to elucidate pathogenic mechanisms whereby H. pylori causes human disease, and to develop effective means for prevention and treatment of infection. In an effort to accomplish these goals, this proposal focuses investigation upon an important virulence determinant of H. pylori: the vacuolating cytotoxin. Although nearly all isolates contain a gene (vacA) encoding the cytotoxin, only about 50% produce vacuolating cytotoxin activity in vitro. Infection with cytotoxin-producing (tox+) strains is associated with an increased risk for peptic ulcer disease; moreover, administration of the purified cytotoxin intragastrically to mice induces gastric ulceration. In preliminary studies, it has been demonstrated that vacA structural gene sequences in tox+ strains differ substantially from those in tox- strains. In addition, it has been demonstrated that levels of vacA transcription are increased in tox+ strains compared to tox- strains. Finally, it has been demonstrated that the cytotoxin binds to epithelial cells and is internalized prior to inducing cytoplasmic vacuolation. The hypotheses of this proposal are (i) that the tox+ phenotype is dependent upon the presence of specific vacA structural gene domains which are absent from vacA homologs in tox- strains, (ii) that vacA transcription is regulated differently in tox+ and tox- strains, and (iii) that specific vacA domains or subunits are required for binding of the toxin to cells, intracellular trafficking, and induction of vacuolation. The specific aims are: to determine the basis for differences among H. pylori strains in levels of vacuolating cytotoxin activity, and to identify antigenic and functionally important domains of the cytotoxin. To accomplish the first objective, a series of chimeric vacA genes, derived in part from a tox+ strain and in part from a tox- strain, will be constructed and analyzed. In addition, a trans- acting vacA regulatory element will be sought. To accomplish the second objective, a series of recombinant vacA peptides will be synthesized, and polyclonal antisera prepared for these peptides. The antisera will be tested for the capacity to block binding and intracellular trafficking of the cytotoxin, as well as vacuole formation. In addition, the products of a series of vacA deletion mutants will be tested for functional activity. Finally, the regions of vacA that elicit an antibody response in humans will be determined by testing human sera for reactivity with the p COVER TL VANDERBILT UNIV SCH OF MEDICIN, A3310 MEDICAL CENTER NORTH, NASHVILLE, TN 37232-2605 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health intracellular transport genetic strain vesicle /vacuole chemical structure molecular pathology reporter gene structural gene genetic regulation genetic transcription gene deletion mutation epitope mapping antiserum bacterial toxin virulence disease model cytotoxicity confocal scanning microscopy Helicobacter recombinant protein CRISP RPROJ TENNESSEE G CRISP/99/AI39657-03 CRISP/99/AI39657-03 199904 eng THYMUS TRANSPLANTATION Research SACHS DH MASSACHUSETTS GENERAL HOSPITAL, MGH-EAST BLDG 149-9019, BOSTON, MA 02129 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02129 Crisp Data Base National Institutes Of Health newborn animal Macaca mulatta miniature swine hematopoiesis leukopoiesis thymectomy thymus transplantation hematopoietic stem cell T lymphocyte flow cytometry antigen presenting cell embryo /fetus cell /tissue cytokine humoral immunity immune tolerance /unresponsiveness radiation immunosuppression major histocompatibility complex skin transplantation nonhuman therapy evaluation mixed lymphocyte reaction test heterologous transplantation lymphocyte proliferation transplantation immunology cell transplantation embryo /fetus tissue transplantation CRISP RPROJ MASSACHUSETTS G CRISP/99/AI39755-030002 CRISP/99/AI39755-030002 199904 eng NUCLEAR FACTOR KAPPA BETA DEPENDENT GENES AND TRANSPLANT ARTERIOSCLEROSIS Research HANCOCK WW BRIGHAM & WOMEN'S HOSPITAL, BRIGHAM AND WOMEN'S HOSPITAL, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal tetracycline blood toxicology arteriosclerosis vascular smooth muscle vascular endothelium flow cytometry polymerase chain reaction pathologic process gene expression immunoglobulin G heart transplantation cytokine endotoxin biological model tissue /cell culture artificial immunosuppression transplant rejection nuclear factor kappa beta CRISP RPROJ MASSACHUSETTS G CRISP/99/AI40152-030003 CRISP/99/AI40152-030003 199904 eng COOPERATIVE CLINICAL TRIALS IN ADULT TRANSPLANTATION Research RPROJ DESCRIPTION: A randomized clinical trial and a pilot study are proposed. The objectives of the randomized clinical trial are to determine if antibodies to T cells and/or switching maintenance immunosuppression, when administered at the time of a biopsy-proven primary rejection episode, will significantly decrease the incidence of a second rejection and chronic rejection in kidney transplant recipients. The investigators will modify immunosuppression at the time of the first rejection episode by substituting OKT3 for steroid therapy and/or substituting mycophenolate for azathioprine or FK506 for Cyclosporine-A. The objective of the pilot study is to determine if early steroid withdrawal is tolerated by recipients of living, related donor transplants who are receiving Cyclosporine-A, mycophenolate, and Prednisone and have not had a rejection episode. Steroids will be withdrawn over a two week period three months after transplant. The primary endpoint will be the incidence of biopsy-proven rejection within six months of steroid withdrawal. If early steroid withdrawal is tolerated in this pilot study, a second pilot study will test the feasibility of withdrawing steroids at four weeks, and if this pilot is successful a third pilot will test the feasibility of administering steroids only during the perioperative period. If this series of pilot studies shows that steroids can be withdrawn with no increase in biopsy-proven rejection the consortium members plan to conduct a prospective randomized clinical trial. If any of the pilots fail, the consortium will proceed with a prospective randomized trial of the previous pilot. MATAS AJ UNIVERSITY OF MINNESOTA, 515 DELAWARE ST SE RM 11-136, MINNEAPOLIS, MN 55455 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 55455 Crisp Data Base National Institutes Of Health adult human (19+) cyclosporine mycophenolic acid clinical chemistry clinical trial cooperative study prognosis drug adverse effect drug withdrawal human subject immunotherapy antibody CD3 molecule kidney transplantation longitudinal human study human morbidity postoperative state human therapy evaluation artificial immunosuppression transplant rejection clinical research CRISP RPROJ MINNESOTA G CRISP/99/AI40166-03 CRISP/99/AI40166-03 199904 eng IMMUNOPATHOGENESIS OF GROUP A STREPTOCOCCAL INFECTIONS Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The reason for the recrudescence of severe invasive group A streptococcal (GAS) infections remains a mystery. Has the bacteria acquired new virulence, or has the host immune defense mechanism been altered? These mutually non-exclusive possibilities can be examined by investigating specific host and pathogen factors that are likely to contribute to disease pathogenesis. Streptococcal pyrogenic exotoxins (Spes), which are superantigens (SAgs), induce potent inflammatory responses, and are believed to play a pivotal role in these diseases. However, the ability of the same GAS strain to cause disease of varying severity in different hosts, suggests that host factors play a crucial role in modulating disease severity. Individuals with invasive GAS infections can be classified into a group with severe (hypotension & organ failure) and nonsevere (no hypotension and no organ failure) invasive disease. Differences in disease presentation may relate to different phenotypic expression of Spes/SAgs in genotypically identical strains, to differences in prior exposure of the host to Spes/SAgs (indicated by the presence of protective immunity), or to inborn variation in responsiveness when exposed to Spes/SAgs. Our hypothesis is that the interaction of pathogen and host factors contribute to the severity of invasive GAS infections. Our main objective is to identify immunogenetic host factors that are important in modulating the severity of symptoms in patients with invasive disease. In order to be able to identify these host factors, it is necessary to study a cohort of patients with invasive disease that was caused by same strain of GAS. To achieve this goal we will: (1) Identify a cohort of severe and nonsevere invasive cases caused by genotypically identical M1T1 strains; (2) Test the hypothesis that genotypically identical M1T1 strains isolated from severe or nonsevere invasive cases differ in the in vitro expression of specific virulence factors that are likely to contribute to the systemic manifestation of the disease; (3) Determine whether the in vivo inflammatory response differs in patients with severe and nonsevere invasive infections caused by identical strains; and (4) Determine whether the following host factors are important in modulating disease severity: serum neutralizing antibodies, HLA class II allotype, and baseline TCR V beta frequency distributions affecting cytokine responses to the infecting isolate. Genotypically identical M1T1 strains (from severe and nonsevere cases) will be analyzed for differences in phenotypic production of immunogenic factors, namely Spe/SAgs. Severe and nonsevere invasive cases who were infected with these isolates will be investigated to determine differences in their in vivo inflammatory cytokine responses during the infection, and identify immunogenetic factors that may be responsible for regulating the magnitude of the inflammatory re KOTB M UNIVERSITY OF TENNESSEE, 956 COURT AVENUE, MEMPHIS, TN 38163 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 38163 Crisp Data Base National Institutes Of Health Streptococcus pyogenes bacterial cytopathogenic effect lymphocyte polymerase chain reaction pathologic process host organism interaction restriction fragment length polymorphism human subject cytokine immunocytochemistry exotoxin immunopathology T cell receptor MHC class II antigen neutralizing antibody superantigen clinical research CRISP RPROJ TENNESSEE G CRISP/99/AI40198-01A2 CRISP/99/AI40198-01A2 199904 eng LYSIS OF TRYPANOSOMA BRUCEI BY HUMAN SERUM Research RPROJ Trypanosoma brucei brucei, unlike the human pathogens Tau beta rhodesiense and Tau gambiense, is lysed by human serum. Sensitivity to human serum is the only method which allows distinction between the cattle and human African trypanosomes. We have identified two distinct trypanolytic fractions in normal human serum (NHS) which differ physically, biochemically and with regard to their inhibition by serum factors. Significantly, an HDL-like trypanolytic factor (TLF1) described by others is not active in NHS, and only becomes lytic upon inhibitor removal during isolation procedures. It is proposed to characterize the more physiologically relevant lytic factor in human serum, and to determine the mechanism(s) by which parasites are killed by both factors. Data indicates that a mechanism of lysis may involve oxidative stress and subsequent programmed cell death, and these possibilities will be tested directly. This work may open new perspectives for the chemotherapy of trypanosomiasis. Understanding of the mechanisms of cytotoxicity and the affected metabolic pathways may provide new approaches for development of specific drugs. TOMLINSON S NEW YORK UNIVERSITY School of, 550 FIRST AVENUE, NEW YORK, NY 10016 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 10016 Crisp Data Base National Institutes Of Health laboratory mouse serum cytolysis gene expression human tissue interleukin 1 immunoprecipitation high density lipoprotein electron microscopy trypanosomiasis peroxidase peroxidation affinity chromatography gel electrophoresis protein structure /function binding protein Trypanosoma Trypanosoma rhodesiense receptor sensitivity protozoal toxin SDS polyacrylamide gel electrophoresis programmed cell death enzyme activity oxidative stress CRISP RPROJ NEW YORK G CRISP/99/AI40206-01A2 CRISP/99/AI40206-01A2 199904 eng CD8 ANTI VBETA T CELLS AND AUTOIMMUNITY Research RPROJ DESCRIPTION: CD8+ T-cells control immune responses, and recent studies suggest that this regulation is, in part, specifically directed towards T-cell receptor (TCR) structures expressed by CD4+ cells. To further study the function of CD8+ T-cells in immune regulation, the investigators studied the role of CD8+ T-cells in regulating superantigen induced CD4+ T-cell responses. They determined that the delayed deletion of CD4+Vbeta8+ T-cells following SEB injection is prevented by treatment of animals with anti-CD8 antibody and is not observed in beta2 M-/-mice. CD8+ T-cells obtained from SEB treated animals and stimulated in vitro with CD4+Vbeta8+ cells preferentially kill activated CD4+Vbeta8+ but not CD4+Vbeta-T-cells. CD8 anti-Vbeta cytotoxicity is dependent on beta2 microglobulin and is inhibited by antisera to Qa-1 but not by antibody to MHC class I-a. Furthermore, in recent studies, the investigators show that CD8 anti-Vbeta8 T-cells kill T hybridomas transfected with Vbeta8 cDNAs but not other VbetacDNAs. These data demonstrate that immunoregulatory CD8+ T-cells recongnize TCR Vbeta determinants and Qa-1 molecules expressed on antigen activated CD4+ T-cells. The investigators envision that Vbeta specific CD8+ T-cells of this type may regulate immune responses by direct interaction with antigen activated CD4+ cells. In this application, they will extend their analysis of CD8 anti-Vbeta T-cells in ways designed to further elucidate their normal biology and their potential role in autoimmunity and superantigen-mediated diseases. In Aim 1, they will investigate the ability of CD8 anti-Vbeta T-cells to regulate CD4+ T-cells in mice with defective Fas and FasL molecules and will examine the ability of normal or FasL mutant CD8 anti- Vbeta T-cells to kill CD4+ T-cells from normal or Fas-mutant mice. Furthermore, in adoptive transfer experiments, they will determine if wild-type CD8+ cells correct the defective SEB-induced deletion of Vbeta cells in FasLgld mice. In Aim 2, they will identify specific Vbeta and Jbeta sequences used by CD8 anti-Vbeta T cells and the length of these TCRbeta CDR3 regions and use this information to follow the development of CD8 anti-Vbeta8 T cells in vivo both in superantigen induced responses and during EAE. In Aim 3, the invesitgators will determine if CD8 anti-Vbeta T-cells differentially regulate the emergence of TH1 and TH2 cells following exposure to SEB and if CD8 anti-Vbeta T cells differ in their ability to be induced by or to kill TH1 versus TH2 cells. Moreover, based on observations that EAE is mediated by TH1 cells and that clinical EAE is moderated by CD8 T-cells, they will determine if CD8 T-cells regulate TH subset differentiation in EAE. CHESS L COLUMBIA UNIVERSITY HLTH SCIS, 630 WEST 168TH STREET, NEW YORK, NY 10032 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 10032 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal cytotoxic T lymphocyte helper T lymphocyte cellular immunity leukocyte activation /transformation immunoregulation immunoglobulin gene antiidiotype antibody CD8 molecule experimental allergic encephalomyelitis nucleic acid sequence T cell receptor cytolysin autoimmunity superantigen CRISP RPROJ NEW YORK G CRISP/99/AI40226-02 CRISP/99/AI40226-02 199904 eng DEVELOPMENT OF A VACCINE FOR CLOSTRIDIUM DIFFICILE Research RPROJ The goal of the proposed research is to develop a vaccine and/or antibody preparation useful for prevention and treatment of Clostridium difficile associated diarrhea (CDAD). Parenteral vaccination with C. difficile toxoid in animal models of a)AD has demonstrated protection from challenge. Furthermore, parenterally administered toxin neutralizing antibodies can protect animals from CDAD. Antigen production methods have been developed and will be used to manufacture vaccine for clinical testing. Plasma donors will be immunized with the vaccine to produce hyperimmune globulin preparations for use in treating and preventing CDAD in clinical trials. Nosocomial infection of the elderly with C. difficile often results in significant morbidity and increased length of hospital stays in spite of effective therapies. The health care costs due to prolonged hospitalization, diagnostic testing and specific C. difficile therapy are substantial. Immunotherapy of patients infected with C. difficile or at risk would offer a cost-effective preventative strategy to control C. difficile infection. Passive immunization can rapidly protect individuals at risk and will pave the way for active vaccine development by defining protective levels of antitoxin antibodies in humans. The goal of rapid and long term protection could be achieved using immune globulin strategies along with the vaccine. PROPOSED COMMERCIAL APPLICATION: Clostridium difficile infection is one of the leading nosocomial diseases in the U.S. As the primary etiological agent of antibiotic-associated diarrhea, this organism is responsible for considerable discomfort and increased health care costs. C. difficile disease is vaccine preventable. Immunotherapy provides a cost-effective strategy that would result in substantial savings in medical costs and reduced morbidity. THOMAS WD ORAVAX INC, 38 SIDNEY STREET, CAMBRIDGE, MA 02139 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02139 Crisp Data Base National Institutes Of Health hamster drug screening /evaluation drug design /synthesis /production diarrhea immunoglobulin nosocomial infection control human subject hyperimmunization passive immunization toxoid bacterial toxin antitoxin bacterial vaccine neutralizing antibody Clostridium difficile mucosal immunity clinical research vaccine development CRISP RPROJ MASSACHUSETTS G CRISP/99/AI40397-03 CRISP/99/AI40397-03 199904 eng INTERFERON Y MEDIATES ANTI E COLI DEFENSES AND SEPSIS Research RPROJ Infections caused by Gram-negative bacteria may be complicated by shock, multi-organ failure and death. Since bacterial lipopolysaccharide (LPS) is considered an important initiator of sepsis, clinical trials have been conducted with agents designed to neutralize its biologic activity or to modify the cytokines induced by LPS. These studies reported a decreased mortality in those patients most ill, but an increased mortality in those least ill. This proposal addresses this apparent paradox. We suggest that the host response to LPS is involved not only in the development of sepsis, but also in host anti-bacterial defenses, and that interferon gamma (IFN gamma) plays a central role in both the beneficial and deleterious responses to LPS. To test this hypothesis, in Aim Ia we will determine if the induction of IFN gamma is essential to the efficacy of treatments (exogenous tumor necrosis factor alpha [TNF] and IL-1; BCG; or LPS) previously shown to restore or enhance anti-bacterial protection in mice. In Aim Ib we will examine whether a threshold exists at which the beneficial enhancement of antibacterial defenses by LPS stimulation gives way to lethal sepsis, and what role IFN gamma may play in this transition. We hypothesize that a synergistic interaction of IFN gamma with either TNF alpha or IL-1 occurs as lethal doses of LPS are approached. Using SCID mice deficient in B and T lymphocytes or monoclonal antibody (MAb) depletion of cells in normal mice, we will determine if NK and/or gamma delta T cells are important sources of IFN gamma in these early responses to LPS. Since host anti-bacterial defenses can be adoptively transferred to naive mice with spleen cells, in Aim II we will determine the mechanisms by which splenocytes generate LPS-stimulated anti-bacterial defenses. We hypothesize that LPS induces a paracrine loop between macrophages, NK and/or gamma delta T cells that leads to the rapid production of IFN gamma, and that IL-12, IL-15 and IL-18 regulate this early induction of IFN gamma. We will pre-treat mice in vivo or spleen cells in vitro with either LPS or cytokines, adoptively transfer spleen cell subpopulations to naive mice and determine their ability to resist lethal bacterial challenge. In Aim III we will determine the mechanisms by which the macrophage response to LPS-induced cytokines is converted from a limited, effective response that kills bacteria to a septic one capable of collateral tissue damage and/or host death. We postulate that IFN gamma stimulation enhances the magnitude and kinetics of LPS-induced early gene activation and TNF alpha secretion by macrophages through interactions of signal transduction molecules. The mechanisms of these synergistic interactions will be determined in a macrophage cell line and in macrophages from Stat1 knockout (IFN gamma non-responsive) and C3H/HeJ (LPS-hyporesponsive) mice.. The demonstration of a spectrum of responses to LPS in these studies may identi CROSS AS UNIVERSITY OF MARYLAND, 10 S PINE STREET, RM 9-00, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health laboratory mouse Escherichia coli bacterial disease Escherichia coli infection biological signal transduction macrophage spleen natural killer cell blood toxicology inflammation gene expression bactericidal immunity cytokine interleukin 1 tumor necrosis factor alpha endotoxin lipopolysaccharide posttranscriptional RNA processing interferon gamma SCID mouse septic shock CRISP RPROJ MARYLAND G CRISP/99/AI40568-01A2 CRISP/99/AI40568-01A2 199904 eng TRANSPLANTATION TOLERANCE USING IMMUNOTOXIN Research RPROJ Organ transplantation in humans achieved a considerable degree of success over the past 30 years, primarily through technical improvements and the development of better immunosuppressive drugs. Nevertheless, all organ transplants currently performed rely on chronic pharmacological immunosuppression of a nonspecific nature to prevent rejection of allografts. Because of the nonspecific nature of these drugs, and the need for ongoing therapy, patients pay a substantial price for chronic immunosuppression in the way of both toxic side effects of the individual drugs and infections and malignancy. About 4 - 6% or organ transplants are lost annually due to rejection despite these drugs. A method has been developed by the Principal Investigator to use an immunotoxin, anti-CD3-CRM9, which given in three doses prior to the transplant permits immunologic tolerance to an organ transplant without any subsequent immunosuppressive drugs. The recipients appear to be normal in every respect and able to resist environmental pathogens. The immunotoxin, developed by Dr. David Neville, a collaborator at the NIH, is a potent means of transiently killing t- lymphocytes while permitting their gradual reconstitution over a period of about 3 - 6 weeks. When allogeneic kidney transplants are performed during this window of T-cell depletion, especially in combination with thymic manipulation, long term allograft tolerance develops. The objective of this proposal is to define the mechanism of tolerance induced by this immunotoxin. Specifically, we will seek to define the degree of T-cell depletion required to produce tolerance, the role of the thymus in this phenomenon, the impact of age and thymic involution on tolerance induced with immunotoxin, and the impact of such treatment of the immune repertoire of treated recipients. Importantly, this drug developed at the NIH has the potential to replace the current strategies of immunosuppression used in organ transplantation and to render organ transplant recipients tolerant, without the need for ongoing drug therapy. The goal of this proposal would be to develop adequate preclinical data to permit phase I and II clinical trials in organ transplant recipients. KNECHTLE SJ UNIVERSITY OF WISCONSIN, 600 HIGHLAND AVENUE, MADISON, WI 53792 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 53792 Crisp Data Base National Institutes Of Health Macaca mulatta thymus T lymphocyte depletion therapy flow cytometry polymerase chain reaction drug screening /evaluation immune tolerance /unresponsiveness immunoregulation enzyme linked immunosorbent assay antiantibody CD3 molecule diphtheria toxin immunotoxicity electrofocusing longitudinal animal study antibody receptor preoperative state nonhuman therapy evaluation tissue /cell culture MHC class II antigen heterologous transplantation transplantation immunology anergy artificial immunosuppression transplant rejection animal genetic material tag CRISP RPROJ WISCONSIN G CRISP/99/AI40597-02 CRISP/99/AI40597-02 199904 eng PMN ENHANCEMENT OF ENDOTHELIAL RESPONSE TO CANDIDA Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The incidence of bloodstream infections caused by Candida species has increased dramatically, so that these organisms now account for 10% of all bloodstream isolates. During the process of hematogenous dissemination, it is likely that blood-borne organisms must adhere to and penetrate the endothelial lining of the vasculature to invade the tissue parenchyma. Thus, a potential method to prevent or treat hematogenously disseminated candidal infections is to augment the combined response of neutrophils and endothelial cells against this organism while it is within the intravascular compartment. We have shown that adding neutrophils to Candida-infected endothelium prevents endothelial cell injury in vitro. Also, we have found that Candida albicans by itself can stimulate endothelial cells to express leukocyte adhesion molecules and proinflammatory cytokines. The expression of these factors is greatly increased when neutrophils are added to endothelium infected with C. albicans. Together, these results suggest that there is a two-way exchange of signals between endothelial cells and neutrophils during their response to intravascular infection. The experiments outlined in this proposal are designed to elucidate the mechanisms that mediate this neutrophil amplification of the endothelial cell proinflammatory response to C. albicans. The influence of the microbial target (C. albicans) on the neutrophil enhancement of the endothelial cell response will be evaluated first. Based on these results, the immunomodulatory substances that mediate this neutrophil amplification will be identified. The expression of the leukocyte adhesion molecules, E-selectin and VCAM-1, will be used as a marker of endothelial cell activation in these experiments. Next, the activities of the immunomodulatory substances identified by the above experiments will be inhibited to determine if theses substances also influence the ability of neutrophils to kill C. albicans and protect endothelial cells from candidal injury. Finally, the results of these in vitro experiments will be evaluated in vivo. Both immunocompetent and neutropenic mice will be infected with C. albicans and immunohistochemistry will be used to detect the local expression of leukocyte adhesion molecules and cytokines at sites of candidal infection. Investigating the interactions between endothelial cells, neutrophils and C. albicans will enable us to determine the mechanism by which endothelial cells are activated in response to infection. The long-range goal of these studies is to devise endothelial cell-based strategies to enhance the host inflammatory response to blood-borne microbial pathogens. FILLER SG HARBOR UCLA RES & EDUC INST, 1024 W CARSON ST, RB-2, TORRANCE, CA 90502 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 90502 Crisp Data Base National Institutes Of Health laboratory mouse neutrophil blood toxicology vascular endothelium host organism interaction Candida albicans leukocyte activation /transformation cytokine immunocytochemistry leukocyte adhesion molecule immunomodulator mycosis cell adhesion molecule tissue /cell culture selectin CRISP RPROJ CALIFORNIA G CRISP/99/AI40636-02 CRISP/99/AI40636-02 199904 eng RATIONAL DESIGN OF NOVEL, NON NEUROTOXIC ANTIMALARIALS Research RPROJ DESCRIPTION: There is a pressing need to develop new antimalarials. Drug resistant strains continue to compromise the effectiveness of known agents and relative to other worldwide diseases the international community has devoted less attention to developing a broad arsenal of drugs to treat malaria. The long term objectives of the proposed research program are to create nontoxic antimalarial drugs based on the natural product artemisinin. The Avery laboratory has been engaged in the study of artemisinin and its derivatives for a number of years. The great deal of preliminary research and thought reflected in this proposal is of an excellent quality. A wide range of structural types based on the artemisinin skeleton have been or will be prepared. In fact, a complete study of the effect of substituents at each of the sites of this skeleton is outlined. Application of computer aided modeling techniques to explore the structure-toxicity relationship is also suggested. Data on the issue of the neurotoxicity associated with the artemisinin skeleton has been obtained. Comparison of toxicity models with activity models should ultimately aid in the design of simplified structures with low predicted toxicity, and high predicted antimalarial activity. A high level of significance can be placed on this project because the mode of action of the artemisinin class appears to be unique in comparison to other antimalarials. AVERY MA UNIVERSITY OF MISSISSIPPI, 417 FASER HALL, UNIVERSITY, MS 38677 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 38677 Crisp Data Base National Institutes Of Health bioassay biological product chemical substitution chemical synthesis antimalarial agent computer simulation drug design /synthesis /production chemical model high performance liquid chromatography sesquiterpene neurotoxicology CRISP RPROJ MISSISSIPPI G CRISP/99/AI40641-02 CRISP/99/AI40641-02 199904 eng MACROPHAGE RESPONSES TO BACTERIAL TOXINS Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The proposed studies will examine the causes of misregulation of macrophage inflammatory responses. Pathogen-phagocytes interactions are critical in many infectious diseases. A newly recognized bacterial pathogen strategy is the expression of toxins which upregulate inflammatory functions which directly mediate acute pathologies and symptoms. Hyperstimulation of important macrophage functions by toxin was first described using the anthrax system. Anthrax lethal toxin mimics all the symptoms of systemic anthrax including death. Upon entering the macrophage, the toxin induces an extreme oxidative burst producing oxygen radicals (ROI) which rupture cell membranes. Lethal toxin also induces overexpression of TNFö and IL-1ö which directly cause symptoms and death in BALB/c mice. Macrophage-depleted mice are toxin-resistant. This well-defined model will be used to uncover patterns of key regulatory points of the lethal inflammatory process. The first goal is to identify macrophage target proteins for the lethal toxin protease activity using combinatorial chemistry techniques and to understand their role in mediating inflammation. The second major aim is to elucidate the role of the anthrax toxin receptor in this process. It is proposed to clone the receptor gene from ögt11 macrophage cDNA libraries and to determine sequence, receptor number and natural functions. The third aim will examine the signaling relationship between ROIs and initiation of immuno-specific macrophage gene expression. NFöB will be examined as a signaling element inking the two events. It is becoming apparent that highly targeted disruption of the normal regulation of immune cell antimicrobial functions by bacterial factors induce overexpression of host mediators of septic shock, acute respiratory distress syndrome and other pathologies seen during infections. Activation of the same systems contribute to autoimmune and chronic inflammatory disorders. This proposal examines key molecular aspects of these regulated pathways. HANNA PC DUKE UNIVERSITY MED CENTER, BOX 3020, DURHAM, NC 27710 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 27710 Crisp Data Base National Institutes Of Health Bacillus biological signal transduction macrophage phagocyte inflammation molecular cloning genetic library cytokine tumor necrosis factor alpha immunoregulation bacterial toxin nucleic acid sequence free radical oxygen endopeptidase receptor binding receptor expression toxin metabolism enzyme activity nuclear factor kappa beta CRISP RPROJ NORTH CAROLINA G CRISP/99/AI40664-02 CRISP/99/AI40664-02 199904 eng CHOLERA TOXIN--A MUCOSAL ADJUVANT, MECHANISMS & IMMUNITY Research RPROJ DESCRIPTION (Adapted from the Applicant's abstract): Cholera toxin and the similar heat labile toxin of E. coli are among the most effective adjuvants known for enhancing mucosal immune responses. These toxins have been shown to enhance immune responses after both oral and intranasal immunization. The mechanism(s) which these molecules use to enhance immune responses are not well established, however, and their inherent toxicity is a major problem which makes their use in humans impractical. As probes into the mechanism(s) by which these toxin molecules enhance immune responses, the investigators have assembled a panel of genetically engineered toxin derivatives, many with reduced or absent toxicity. Using both model antigens and infectious challenge models in mice, they will investigate the mechanisms by which native and altered toxins enhance oral and intranasal immune responses. The investigators will determine how native and engineered toxin molecules affect uptake of model antigens into mucosal tissues such as Peyer's patches and enterocytes and whether antigen presentation and intracellular antigen processing is up regulated. They will also study the direct effects of cholera toxin/heat labile toxin and engineered derivatives upon antigen responsive B lymphocytes and the effects upon patterns of cytokine secretion by T lymphocytes. Ultimately, if non-toxic derivatives of cholera toxin or heat labile toxin which still stimulate mucosal immune responses are identified, they may find use as adjuvants for mucosal vaccines versus pathogenic organisms or even dietary carcinogens. Thus, they will also investigate the effectiveness of non-toxic-engineered cholera toxin/heat labile toxin derivatives in promoting protective immune responses in two infectious challenge models. The models which the investigators have chosen are intranasal Sendai virus infection and oral Helicobacter felis infection. Sendai virus infects the respiratory tract and is a murine parainfluenza virus. Helicobacter infections of gastric tissue have been implicated as causes of gastroduodenal ulcers and gastric cancer. NEDRUD JG CASE WESTERN RESERVE UNIVERSIT, 10900 EUCLID AVE, CLEVELAND, OH 44106-4943 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health urease laboratory rabbit laboratory mouse B lymphocyte T lymphocyte gene rearrangement adenine phosphoribosyltransferase cytokine immunoglobulin gene immunologic assay /test antibody formation antigen presentation cholera toxin immunomodulator protein biosynthesis ovalbumin tissue /cell culture parainfluenza virus type 1 enzyme activity Helicobacter recombinant protein mucosal immunity CRISP RPROJ OHIO G CRISP/99/AI40701-02 CRISP/99/AI40701-02 199904 eng OCCUPATIONAL AFLATOXICOSIS Research RPROJ DESCRIPTION (Adapted from the Applicant's Abstract): Surveys have shown that AFB1 is frequently found in respirable grain dust (up to 43,700 ppb) and that the serum of occupational workers exposed to contaminated dust contains AFB1-albumin adducts. The investigators' data demonstrate that respiratory exposure of rodents to AFB1 impairs the alveolar macrophage (AM) phagocytic defense system. The results indicate that respiratory exposure to AFB1 should be considered a human health risk is associated with diseases resulting from impaired lung defenses where exposure to AFB1-laden dusts is common. Indeed, through its effect on lowered lung resistance, AFB1 exposure poses a more wide ranging and insidious health hazard than its genotoxic endpoint. It is well established that oral exposure to AFB1 impairs a wide spectrum of systemic host defenses however, the mechanism of this effect have not been addressed. The observation that oral AFB1 Impairs the antibody response to a T-dependent antigen but not to a T-independent antigen provides a clue to the mechanism by indicating that AFB1 has an effect on T-lymphocytes. Critical to the evaluation of the magnitude of human risk from inhalation of AFB1-contaminated grain dust is an experimental data base. However, the full biologic effect of respiratory exposure to AFB1 on lung defenses is not known nor are the mechanisms involved. The goal of the proposed research is to define the cellular mechanisms of AFB1-induced suppression of pulmonary defenses and elucidate the consequences of such impairments. The overall hypothesis of this application is that respiratory AFB1 exposure regulates the innate and acquired immune responses though alterations of the functional capacity of the antigen presenting cells, and the T-lymphocyte repertoire of the immune response. Also, data-response relationships between inhalation to AFB1- laden grain dust that estimate occupational exposure and the most sensitive parameters of altered immune response will define the no effect level. These studies will establish the experimental data base required for the development of a risk assessment model of human populations exposed to AFB1-contaminated grain dust. This data base can then be used as a guide to focus epidemiologic studies toward a more complete assessment of the health hazard of occupational inhalation exposure to AFB1-laden grain dust. JAKAB GJ JOHNS HOPKINS UNIVERSITY, 615 NORTH WOLFE STREET, BALTIMORE, MD 21205 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21205 Crisp Data Base National Institutes Of Health laboratory rat Haemophilus influenzae Listeria Klebsiella pneumoniae T lymphocyte antigen presenting cell cellular immunity immunosuppression immunotoxicity occupational hazard dust grain respiratory infection aflatoxin lung injury CRISP RPROJ MARYLAND G CRISP/99/AI40711-03 CRISP/99/AI40711-03 199904 eng MUCOSAL IMMUNITY TO ANTIGENS EXPRESSED BY V CHOLERAE Research RPROJ Microbial pathogenes which infect mucosal surfaces of the body continue to cause significant human morbidity and mortality. Current parenteral vaccination strategies for these infections are frequently ineffective. Antigens in the lumen of the gastrointestinal tract bind to specialized M cells in the epithelium and are presented to underlying cells of the immune system, leading to proliferation and differentiation of IgA- committed, antigen-specific lymphocytes which circulate to the lamina propria of diverse mucosal surfaces. This dissemination of a mucosal immune response from an individual inductive site to diverse effector sites has been termed the common mucosal immune system. Vibrio cholerae is an excellent model for studying mucosal immunity and stimulation of a common mucosal immune response. V. cholerae selectively adheres to M cells of the gastrointestinal tract and natural infection with V. cholerae is followed by long-lasting, systemic and mucosal immune responses. V. cholerae also has many advantages as a vector system for delivering antigens from heterologous organisms to the mucosal immune system. The long-term goal of these studies is to develop V. cholerae as a live, oral, attenuated vaccine vector strain for delivery of heterologous antigens to the mucosal immune system. There are four SPECIFIC AIMS in the present proposal: (1) optimizing in vivo expression of heterologous antigens by live, oral, attenuated vector strains of V. cholerae, using the B subunit of cholera toxin (CTxB) as a model antigen; (2) analysis of a fusion protein between the serine rich protein of Entamoeba histolytica (SREHP) and CtxB, expressed by a live, oral, attenuated V. cholerae vector, for inducing anti-SREHP mucosal immunity in an animal model; (3) examining the role of mutant LT-I as an immunoadjuvant, when expressed by live, oral, attenuated V. cholerae vector strains; and (4) use of live, oral, attenuated V. cholerae vector strains to deliver epitopes of toxin A of Clostridium difficile: application of general principles to a specific mucosal infection and assessment of the role of antigenic context in the mucosal immune response. CALDERWOOD SB MASSACHUSETTS GENERAL HOSPITAL, FRUIT STREET DISEAS, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02114 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Vibrio cholerae genetic strain host organism interaction gene expression structural gene bacterial genetics antibody neutralization test antigen presentation bacterial antigen cholera toxin protein engineering chimeric protein bacterial vaccine recombinant DNA Clostridium difficile mucosal immunity CRISP RPROJ MASSACHUSETTS G CRISP/99/AI40725-02 CRISP/99/AI40725-02 199904 eng NEISSERIAL PORINS AND ANTIGEN PRESENTING CELL Research RPROJ DESCRIPTION (Adapted from the applicants abstract): The overall aim of this proposal is to characterize and define the mechanism behind the adjuvant activity of Neisserial porins by extending our work regarding the effect of the porins on antigen presenting cells. The Neisserial porins to be used are protein IA (PIA) and protein IB (PIB) from the gonococcus and class 1 or 3 proteins from the meningococcus (C1 and C3 respectively). Neisserial porins have been investigated as adjuvants in vaccines using pneumococcal polysaccharide, Hemophilus polysaccharide (currently in use), malarial peptides, human gangliosides (as anti-melanoma vaccines), etc. as antigens. Understanding the mechanism behind the Neisserial porins immuno-potentiating ability will aid in the more effective use as adjuvants. Neisserial porins can increase the expression of the ligand B7-2 on B lymphocytes, which is an important mediator of T lymphocyte costimulation, and induce B cells to proliferate and to secrete immunoglobulin nonspecifially. To aid in investigating the mechanism of the porins immuno-potentiating ability and to connect the effect of the porins on antigen presenting cells to their adjuvant activity, this proposal with the following aims is submitted. The first aim will be to bridge the in vitro effect of the porins on B cells, as characterized by increased B7-2 expression, to the porins' adjuvant activity. This will be accomplished by using the porins as adjuvants in immunizations of B7-2 knockout mice, nude mice of CD3 transgenic mice. The second aim will be to discern if the porins can increase specific in vitro antibody towards bacterial capsular polysaccharides, again bridging the finding that porins can activate B cells to the porins's adjuvant activity. In addition, this aim will define the role of cytokines in the porins' adjuvant ability. The third aim will be to continue our studies on the ability of porins to stimulate murine B cells and discern if they affect other murine antigen presenting cells (macrophages/monocytes and dendritic cells) in a similar manner. The final aim will be to investigate whether Neisserial porins have a similar manner. The final aim will be to investigate whether Neisserial porins have a similar effect on human antigen presenting cells. If these aims are accomplished, the understanding of the Neisserial porins' effects on lymphocytes and antigen presenting cells will be greatly enhanced, and the use of the porins and other microbial substances as adjuvants can be improved. WETZLER LM THE MAXWELL FINLAND LAB FOR IN, BOSTON MEDICAL CENTER, BOSTON, MA 02118 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02118 Crisp Data Base National Institutes Of Health athymic mouse laboratory mouse transgenic animal Neisseriaceae Neisseria gonorrhoeae Neisseria meningitidis B lymphocyte antigen presenting cell cytokine enzyme linked immunosorbent assay bacterial toxin bacterial polysaccharide pore forming protein toxin metabolism CRISP RPROJ MASSACHUSETTS G CRISP/99/AI40944-02 CRISP/99/AI40944-02 199904 eng ANOPHELES STEPHENSI NO SYNTHASE--IMPACT ON PLASMODIUM Research RPROJ Vertebrate nitric oxide synthase (NOS) isoforms catalyze nitric oxide (NO) production for neuronal signalling, vasodilation, and destruction of pathogens, including liver-invading sporozoites of Plasmodium spp., the causative agents of malaria. Recent results from this laboratory indicate that Anopheles stephensi, a natural vector of Plasmodium, possesses a NOS gene (AsNOS) with striking homology to the recently described Drosophila NOS gene. Reverse transcriptase-polymerase chain reaction assays indicate that AsNOS is synthesized during P. falciparum and P. berghei infections, suggesting that anorpheline mosquitoes may possess an analogous NO killing mechanism for Plasmodium. Two objectives have been proposed for the characterization of AsNOS: (1) clone, sequence, and analyze enzyme activity of AsNOS, and (2) demonstrate NO toxicity to mosquito stage Plasmodium in vitro and in vivo. Sequence of full-length AsNOS and associated upstream regions will confirm the identity of AsNOS, provide insight into regulation of its expression, and facilitate construction of an expression system for enzyme analysis. Catalysis of NO production by AsNOS in vitro will confirm its behavior as a NOS isoform. Characterization of AsNOS in vitro will confirm its behavior as a NOS isoform. Characterization of AsNOS and its activity will provide new insights into mosquito-parasite biology, provide tools with which to examine NOS in other insect vectors, and perhaps provide a novel basis for engineering Plasmodium-refractory mosquitoes to interrupt the transmission of human malaria parasites. LUCKHART S VA POLYTECHNIC INST & STATE UN, 111 ENGEL HALL, BLACKSBURG, VA 24061-0308 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health catalyst polymerase chain reaction communicable disease control disease vector arthropod borne communicable disease host organism interaction enzyme biosynthesis molecular cloning gene expression enzyme linked immunosorbent assay computer assisted sequence analysis nucleic acid sequence malaria chimeric protein Plasmodium toxin northern blotting southern blotting enzyme activity gel mobility shift assay nitric oxide synthase Anopheles CRISP RPROJ VIRGINIA G CRISP/99/AI41027-03 CRISP/99/AI41027-03 199904 eng NOVEL ENTEROCOCCAL TOXIN--REGULATION OF EXPRESSION Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Many serious enterococcal infections are no longer treatable. Vancomycin resistance was the last antibiotic to which all enterococci were susceptible. However, vancomycin resistant isolates began to emerge in the late '80s, and now represent approximately 1 in 7 strains obtained from ICUs. The only therapeutic option available for treating these infections is supportive care, essentially as practiced in the pre-antibiotic era. The emergence of enterococcal strain that are completely resistant to all currently approved antibiotic combinations is an issue of great concern in medicine, to the scientific community, and to the general public. Direction in the search for new therapeutic approaches for treating enterococcal infection will come through a clearer understanding of the factors involved in the pathogenesis of enterococcal disease. One of the few enterococcal factors that has been shown to be associated with isolates from sites of infection, associated with severity of disease outcome, and directly shown to contribute to disease severity in several models of infection, is the cytolysin of Enterococcus faecalis. Although an understanding of the molecular nature of the cytolysin is reaching a sophisticated level, essentially nothing is known about the environmental cues that trigger its expression. Common to 1) the in situ production of a toxin associated with severity of infection, 2) the careful expression of an otherwise lethal substance by a producing organism, and 3) the ability of a cytolysin producing organism to transfer the plasmid encoding cytolysin production and resistance to a sensitive recipient, is the regulation of cytolysin expression. The goal of this proposal is therefore to characterize the molecular basis for regulation of the expression of the cytolysin and its auxiliary functions, including those for toxin maturation, secretion, activation, and immunity. GILMORE MS UNIV OF OKLAHOMA HLTH SCI CTR, PO BOX 26901, OKLAHOMA CITY, OK 73190 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 73190 Crisp Data Base National Institutes Of Health Streptococcus enterococcus group drug resistance genetic mapping molecular cloning gene expression genetic regulation genetic transcription gene deletion mutation microorganism growth bacterial toxin virulence transposon /insertion element nucleic acid sequence protein structure /function cytolysin toxin metabolism northern blotting CRISP RPROJ OKLAHOMA G CRISP/99/AI41108-01A1 CRISP/99/AI41108-01A1 199904 eng NOVEL PROTEINS SECRETED BY E COLI 0157:H7 Research RPROJ DESCRIPTION The major virulence factor of enterohemorrhagic Escherichia coli 0157:H7 is a family of potent cytotoxins known as Shiga toxin, Shiga-like toxin or verocytotoxin. Data from a variety of in vitro, animal, and human studies support the concept that Shiga toxins are crucial for the development of the HUS and hemorrhagic colitis caused by this pathogen. However, there are few data concerning other potential virulence factors expressed by E. coli 0157:H7. Previous work in this laboratory has identified an outer membrane protein known as intimin, encoded by the eaeA gene, which is involved in intestinal colonization by this pathogen. We have recently discovered several bacterial proteins that are secreted into the culture supernatant. These proteins, which are not Shiga toxins and are not produced by non-pathogenic E. coli, produce a strong serum antibody response in patients with HUS. We hypothesize that these proteins play an important role in the pathogenesis of disease due to E. coli 0157:H7. A multi-faceted approach is proposed to investigate these proteins. The genes encoding these proteins will be cloned and sequenced and the distribution of these genes among other Shiga toxin-producing E. coli will be studied. An ELISA assay will be developed for serodiagnosis and seroepidemiology studies and a large collection of well-characterized patient sera will be screened with this ELISA. Correlations of protein secretion patterns, patient immune response patterns and clinical outcome will be sought to study whether these proteins or the response to them can be used as a marker for clinical outcome. Using purified proteins and isogenic E. coli 0157:H7 strains specifically mutated in genes encoding these proteins, the effect of these proteins on cultured intestinal and renal cells will be studied and potential interactions with Shiga toxin will be examined in these in vitro systems. Isogenic strains mutated in these genes will also be studied in animal models to examine the contribution of these proteins to intestinal and renal manifestations of disease due to E. coli 0157:H7. Together, these studies should yield a more complete picture of disease due to EHEC and offer the possibility of new insights into pathogenesis, improved serodiagnostic methodologies, and identification of potential protective antigens that may be used for development of vaccines or immunotherapeutic agents. KAPER JB UNIV OF MARYLAND SCH OF MED, 511 W LOMBARD STREET, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health Escherichia coli host organism interaction gastrointestinal epithelium molecular cloning microorganism genetics gene mutation enzyme linked immunosorbent assay exotoxin kidney cell intermolecular interaction nucleic acid sequence bacterial protein membrane protein tissue /cell culture CRISP RPROJ MARYLAND G CRISP/99/AI41325-03 CRISP/99/AI41325-03 199904 eng SITE SPECIFIC HU MONOCLONAL ANTIBODIES--HUS PROPHYLAXIS Research RPROJ DESCRIPTION The objective of this application is to produce a panel of SLT-specific human monoclonal antibodies (Hu/mAbs), that can be used safely and effectively to protect children at risk of developing HUS; in particular those presenting with bloody diarrhea, confirmed infection with SLT-producing bacteria, or sibling and other contact individuals. We have firm data which show that SLT-specific antibodies when given to piglets, well after oral challenge with E. coli 0157:H7, are highly protective against development of systemic complications and death. The recent development by GenPharm International, our collaborators, of a transgenic mouse that in response to immunization produces stable human IgM- and IgG-secreting hybridomas, makes it possible to produce SLT specific human mAbs. After production at GenPharm, the Hu/mAbs will be fully characterized at the Tufts laboratory where we have extensive experience in the derivation and characterization of SLT-specific murine mAbs. A panel of the most effective neutralizing SLT-specific Hu/mAbs will then be evaluated for their prophylactic efficacy in gnotobiotic piglets orally challenged with E. coli 0157 H7. The Specific Aims of the application are: 1. production of Hu/mAbs against SLT-II and SLT-I in HuMab transgenic mice, 2. laboratory characterization of the SLT-II and SLT-I specific Hu/mAbs, and 3. evaluation of SLT specific Hu/mAbs in piglets challenged with E. coli 0157:H7, or SLT. This is a straightforward, low-risk project requiring no new technical development, with a simple hypothesis; that a panel of highly effective SLT-specific human mAbs will much improve the clinical outcome of HUS. We predict that at the end of the support period we will have a fully characterized panel of SLT-specific human mAbs ready to be tested for safety and reactogenicity in human volunteers. TZIPORI SV TUFTS UNIVERSITY SCH OF VET ME, 200 WESTBORO RD, NORTH GRAFTON, MA 01536 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 01536 Crisp Data Base National Institutes Of Health laboratory mouse swine transgenic animal Escherichia coli Escherichia coli infection hemolytic anemia immunotherapy monoclonal antibody exotoxin antitoxin renal failure nonhuman therapy evaluation CRISP RPROJ MASSACHUSETTS G CRISP/99/AI41326-03 CRISP/99/AI41326-03 199904 eng INTERVENTION IN E COLI VEROTOXEMIA--SWINE MODEL Research RPROJ DESCRIPTION The principal cause of HUS is Escherichia coli which produce verotoxin, e.g., Shiga-like toxin (SLT). Prior to the onset of HUS, SLT producing E.coli (SLTEC) colonize the intestine and produce SLT which is then absorbed systemically and binds to specific receptors in renal glomeruli causing vascular damage leading to kidney failure and HUS. It is thought that cytokines such as tumor neurosis factor (TNF) predispose to and enhance the vascular damage. This research will utilize a naturally occurring animal model of systemic SLT induced vascular damage (edema disease of swine) to define early (pre-clinical, pre-renal, and pre-vascular) stages in the pathogenesis of SLTEC infections. Specifically, the research will define the times from infection to SLT production, from SLT production to its appearance in blood, to increased TNF production, to initial vascular damage and to the first signs of clinical disease. It will determine if antibodies against SLT can induce protection when given after SLT is produced, but before it is absorbed, or if given at the time SLT is first detectable in blood, or at the time of the first clinical signs. It will determine if tests for the concentration of SLT in blood or intestine have predictive value as to the severity of the resulting vascular damage and clinical disease. The work will determine if programmed cell death is a mechanism of vascular damage. The knowledge gained will be useful in developing rational strategies for the prevention of HUS by detection of and intervention in SLTEC infection before SLT becomes systemic, binds to glomerular capillaries or causes irreversible vascular damage. Field trials, utilizing an antitoxin vaccine, will be conducted to determine if SLTEC infection causes low level vascular damage and impaired growth (subclinical disease) in animals that become infected, but do not develop clinical disease. MOON HW IOWA STATE UNIVERSITY, 1802 ELWOOD DR, AMES, IA 50011 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 50011 Crisp Data Base National Institutes Of Health swine Escherichia coli Escherichia coli infection hemolytic anemia blood vessel disorder vascular endothelium tumor necrosis factor alpha passive immunization exotoxin antitoxin renal failure nonhuman therapy evaluation programmed cell death CRISP RPROJ IOWA G CRISP/99/AI41328-03 CRISP/99/AI41328-03 199904 eng METABOLIC BASIS OF SULFONAMIDE TOXICITY IN AIDS PATIENTS Research RPROJ The use of sulfamethoxazole-trimethoprim (TMP-SMX) is associated with idiosyncratic/hypersensitivity reactions which significantly limit its use, and the frequency of these reactions is increased in HIV infected patients. Numerous studies support the hypothesis that bioactivation of sulfonamides is an initial step in the cascade of events which result in adverse drug reactions (ADR). The long-term goal of this project is to develop strategies which either prevent or minimize ADR to TMP-SMX, thereby maximizing the number of patients able to continue therapy with the most effective anti-pneumocystis agent. The objective of the present proposal is to test the hypothesis that the increased incidence of ADR in HIV infected patients is secondary to alterations in xenobiotic biotransformation. The specific aims are to determine: 1. IF ARYLAMINE N-ACETYLATION ACTIVITY IS REDUCED IN HIV INFECTED PATIENTS. If oxidation to form a hydroxylamine is the critical step in the bioactivation pathway, the rate of acetylation (an alternative elimination route) may be a determinant in the incidence of ADR to sulfonamides. The hypothesis that HIV patients with an acute infection exhibit a reduced rate of acetylation will be tested by combined phenotype/genotype determination. SMX acetylation clearance in HIV infected patients with acute bacterial/fungal infection, those without and acute infection and normal volunteers will also be determine. 2. IF IN VITRO CYTOTOXICITY OF HYDROXYLAMINE METABOLITES IS A VALID PREDICTOR OF PREDISPOSITION TO HYPERSENSITIVITY REACTIONS IN HIV- INFECTED PATIENTS. Several studies have demonstrated that patients experiencing an ADR to TMP-SMX exhibit a greater in vitro cytotoxicity towards peripheral blood mononuclear cells (PBMC) by metabolites of SMX than patients without hypersensitivity. To test the validity of this in vitro test as a surrogate marker for hypersensitivity, the ability of the test to discriminate between patient populations with different risks for ADR will be examined. 3. IF SERUM FROM HIV-INFECTED PATIENTS EXPERIENCING HYPERSENSITIVITY REACTIONS TO TMP-SMX POSSESS ANTIBODIES DIRECTED TOWARDS NEOANTIGENS IN PBMC. The hypothesis that the association between in vitro cytotoxicity and in vivo hypersensitivity is due to the development of neoantigens which, in turn, elicit an immune response will be tested. SVENSSON CK WAYNE STATE UNIVERSITY, DETROIT, MI 48202 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 48202 Crisp Data Base National Institutes Of Health acetylation opportunistic infection communicable disease chemotherapy drug metabolism drug adverse effect human subject AIDS trimethoprim Pneumocystis pneumonia sulfamethoxazole sulfonamide Pneumocystis carinii clinical research CRISP RPROJ MICHIGAN G CRISP/99/AI41395-02 CRISP/99/AI41395-02 199904 eng MAMMALIAN CELL CYCLE BLOCK BY A C JEJUNI CYTOTOXIN Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Campylobacter jejuni is the most common bacterial cause of diarrheal disease of humans in the United States. It has recently also been implicated as one of the most common infections to precede the development of Guillain-Barre syndrome. The study of C. jejuni pathogenesis has not yet identified many major virulence factors; in particular, almost nothing is known about the contribution of C. jejuni to disease. The broad long term goal of this proposal are to characterize the interactions of a C. jejuni toxin, called cytolethal distending toxin (CDT) with eukaryotic cells, and to determine the role of CDT in human disease. To date, CDT is the only toxin produced by C. jejuni which has actually been proven to exist. CDT blocks eukaryotic cells in the G2 phase of the cell cycle, an action unlike that of any other known bacterial toxin; and, the predicted amino acid sequence of CDT proteins share no homology with the amino acid sequences of any proteins in the database. The specific aims of the proposal are designed to test the hypothesis that CDT is a virulence factor in C. jejuni diarrheal disease by virtue of its ability to block eukaryotic cells in the G2 phase of the cell cycle. Under aim 1, the ability of the CDT to cause a G2 block in cultured cell line derived from intestinal epithelial cells will be tested. In addition, Dr. Pickett will test whether CDT is active against the cell lines when they are relatively undifferentiated or still proliferating. Under aim 2, the initial interactions of CDT with HeLa cells will be characterized and experiments will be performed to determine if all or a portion of CDT enter the cells. In aim 3, the nature of the G2 block caused by CDT will be examined in greater detail. Under aim 4, the ability of CDT to contribute to C. jejuni diarrheal disease in a piglet model will be tested. PICKETT CL UNIV OF KENTUCKY MED CTR, 800 ROSE ST, LEXINGTON, KY 40536-0084 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health swine bacterial disease cell growth regulation host organism interaction gastritis gastrointestinal epithelium enterotoxin virulence HeLa cell bacterial toxicology Helicobacter CRISP RPROJ KENTUCKY G CRISP/99/AI41477-02 CRISP/99/AI41477-02 199904 eng USE OF ANTHRAX TOXIN FUSIONS TO STIMULATE CTL IMMUNITY Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor to the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (Lfn; 255 amino acids) is devoid of toxic activity and binds LF to PA. Heterologous proteins fused to Lfn are delivered into the cytoplasm of host cells in the presence of PA. The investigators have fused a nine-residue cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to Lfn and have demonstrated the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a protective CTL response against the epitope in BALB/c mice. They propose to expand these studies to determine if anthrax toxin can be used as a system for priming a variety of CTL responses. They propose the following experiments: 1) They will immunize with an anthrax toxin fusion containing three separate CTL epitopes in an attempt to induce immunity similar to that seen following sublethal infection. 2) They will investigate the use of the anthrax toxin to deliver CTL epitopes from L. Monocytogenes presented by H-2 M3. Immunization with these epitopes, which contain n-formyl methionine, may be protective in mice of most MHC haplotypes. It has proven difficult to immunize with these epitopes using existing technologies. 3) They will determine if a single anthrax toxin fusion with LCMV NP is able to stimulate CTL and protect mice of different murine haplotypes against LCMV infection. Success of these experiments would suggest that the anthrax toxin system may be useful in immunizing genetically diverse populations. 4) They will incorporate epitopes from both LCMV and L.monocytogenes in the same anthrax toxin fusion to determine if a protective CTL response can be primed against multiple pathogens following immunization with a single fusion protein and 5) They will determine if addition of polycationic sequences can replace Lfn in these fusions, allowing the delivery of CTL epitopes by PA in vivo and stimulating protective CTL responses. This would greatly expand the ease with which this system could be used. The research in this proposal will allow the investigators to further establish whether anthrax toxin may be useful as a general CTL-peptide delivery system for research and medical applications. STARNBACH MN HARVARD MEDICAL SCHOOL, 200 LONGWOOD AVE, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health laboratory mouse Bacillus Listeria cytotoxic T lymphocyte cellular immunity bacterial antigen bacterial toxin chimeric protein virus protein MHC class I antigen lymphocytic choriomeningitis virus CRISP RPROJ MASSACHUSETTS G CRISP/99/AI41526-01A1 CRISP/99/AI41526-01A1 199904 eng IMMUNOPATHOGENESIS OF ACUTE HIV-1 INFECTION Research RPROJ Primary HIV-1 infection is a period of explosive viral growth in which critical lymphoid organs are seeded and in which the initial movement toward viral diversity is established. The mergence of a multifaceted humoral and cellular immune response is associated with a rapid decline in plasma levels of HIV-1 RNA but virus is not eliminated from the host. Initial events in the virus/host interactions clearly play a critical role in limiting viral replication and in determining the subsequent natural history f the illness. Viral and/or immunologic factors that permit the virus to evade elimination have not yet been delineated. Emerging data suggest that antiretroviral intervention during primary infection may have profound implications for the virus/host interaction. It has been suggested that this interval during which viral diversity is relatively limited and in which viral seeding of lymphoid organs is still incomplete may be a time during which potent antiretroviral chemotherapy might even eliminate virus from the host. We have assembled a group of laboratory-based and clinical investigators who wish to intensively characterize viral and immunologic events during primary infection in the context of therapeutic interventions with maximally active antiretroviral regimens. These investigations will provide additional insights into the immunopathogenesis of HIV-1 infection and will have important implications for designing antiretroviral chemotherapeutic strategies and for AIDS vaccine development. SCHOOLEY RT UNIV OF COLORADO HLTH SCIS CTR, 4200 EAST 9TH AVENUE, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80262 Crisp Data Base National Institutes Of Health lymph node cytotoxic T lymphocyte acute disease /disorder human subject microorganism immunology HIV infection pore forming protein tissue /cell culture cytolysin human immunodeficiency virus 1 mucosal immunity clinical research CRISP RPROJ COLORADO G CRISP/99/AI41536-02 CRISP/99/AI41536-02 199904 eng NEW MECHANISMS FOR REGULATING VIRULENCE GENE EXPRESSION Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Virulence gene expression in many pathogenic bacteria is controlled by environmental stimuli both inside and outside of the host. For the human pathogen Vibrio cholerae, the coordinate expression of cholera toxin (CT), toxin-coregulated pilus (TCP), and a number of other virulence factors is strongly influenced by environmental parameters such as temperature, pH, osmolarity, and oxygen tension. To elucidate the nature of this regulation, a transcriptional fusion between the promoter of the CT operon (ctx) and the Escherichia coli b-galactosidase (lacZ) gene in the V. cholerae chromosome was used as a screen to identify genes that influence the expression of the fusion in response to various environmental stimuli. Using this screen, two V. cholerae genes have been identified, cya and crp, that negatively regulate virulence gene expression under particular growth conditions. The product of the cya gene, adenylate cyclase, catalyzes the synthesis of cyclic AMP (cAMP), one of the most important and ubiquitous intracellular regulatory molecules. Together with the crp product, cAMP-receptor protein (CRP), this system serves as a global regulator of gene expression in enteric bacteria by controlling the utilization of carbon and energy sources in the environment. The observation that crp mutants in both the classical and El Tor biotypes of V. cholerae O1 are derepressed for the expression of CT and TCP under a variety of growth conditions in vitro and are defective for pathogenesis in the infant mouse cholera model is the first clear demonstration that cAMP-CRP plays a role in these processes. The primary focus of the proposed work is to elucidate the molecular and physiological mechanisms by which cAMP-CRP controls the expression of CT and TCP in V. cholerae. This will be accomplished in SPECIFIC AIM #1 by determining which regulatory genes are influenced by cAMP-CRP and by identifying the site(s) to which CRP binds, and in SPECIFIC AIM #2 by examining the relationship between the intracellular levels of cAMP under various growth conditions and the expression of CT and TCP. Since analysis of the crp mutants suggests that in addition to cAMP-CRP, other as yet unknown factors also influence the environmental control of virulence gene expression, the third SPECIFIC AIM of this proposal is to again exploit the ctx-lacZ fusion screen to identify these genes in both biotype strains and to shed light on their mechanisms of action. The long-term objectives of this work are to ultimately understand how stimuli from the environment are transmitted to the level of gene expression in V. cholerae so as to facilitate the development of new strategies to control and prevent bacterial diseases. SKORUPSKI KA DARTMOUTH MEDICAL SCHOOL, HANOVER, NH 03755-3842 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Vibrio cholerae chemical binding molecular site genetic regulatory element gene induction /repression bacterial genetics microorganism culture cholera toxin virulence nucleic acid sequence protein structure /function cyclic AMP cyclic AMP receptor CRISP RPROJ NEW HAMPSHIRE G CRISP/99/AI41558-02 CRISP/99/AI41558-02 199904 eng CYTOLYTIC ENTEROTOXIN OF AEROMONAS HYDROPHILA Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The long term objective of this proposal is determine the role of a cytolytic enterotoxin (Act) of A. hydrophila in the pathogenesis of disease. The investigators have isolated a 52 kDa polypeptide from a diarrheal isolate of A. hydrophila (SSU) that is associated with hemolytic, cytotoxic and enterotoxic activities and is lethal for mice. The toxin gene has been cloned, expressed and sequenced by the investigators. Site directed mutagenesis studies recently completed suggest different loci coding for the three biological activities associated with the toxin. The suggestion that Act plays a significant role in the infectious process is supported by the lack of virulence of an Act minus isolate of A. hydrophila and transposon mutants with reduced biological activity. More recent studies with an isogenic mutant of strain SSU that is without in vitro hemolytic and cytotoxic activity also supports the belief that this toxin is an important virulence factor. Characterization of the role of the toxin will proceed using the toxin negative mutant recently produced. Probing the antigenic and biologically active domains of the toxin will be accomplished following hyperexpression of the Act gene with a multi-host range vector. The functional domains of the toxin molecule will be mapped by generating anti-peptide antibodies and monoclonal antibodies. Site directed mutagenesis will be used to refine the role of amino acid residues in the biological activities of the toxin. The investigators also propose to study the regulation of the Act gene either by fusing it with a reporter gene or by mutagenesis of the chromosome of Aeromonas using a transposon which imparts antibiotic resistance to screen for toxin negative mutants. Finally, the investigators propose to study the mechanism of action of the toxin by probing the mechanism of cell damage and by examining the toxin receptor on the cell surface. HOUSTON CW UNIV OF TEXAS MEDICAL BRANCH, GALVESTON, TX 77555-1019 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Enterobacteriaceae disease pathologic process diarrhea site directed mutagenesis molecular cloning fusion gene gene expression monoclonal antibody bacterial toxin enterotoxin virulence transposon /insertion element nucleic acid sequence synthetic peptide toxicant interaction cytotoxicity Vibrionaceae CRISP RPROJ TEXAS G CRISP/99/AI41611-02 CRISP/99/AI41611-02 199904 eng PENETRATION OF MACROMOLECULES INTO MAMMALIAN CELLS Research RPROJ The goal of this proposal is to use new knowledge on endocytosis to optimize the cellular effects of anti-cancer immunotoxins. Once targeted to the surface of tumor cells, immunotoxins must be internalized and undergo an intracellular activation process that includes the reduction of their interchain disulfide bond. Almost nothing is known about reductive cleavage of endocytosed disulfides. The key importance of this step is demonstrated by the fact that immunotoxins lose effectiveness when their disulfide is replaced by a thioether bond. Biochemical measurements of this reductive cleavage have eluded many efforts, in part because of the lack of suitable probes that could be used as models for disulfide linked toxins. We have developed such probes by linking an efficiently endocytosed and proteolytically undegradable polymer (poly(D-lysine)), through disulfide linkage either to an anticancer drug (methotrexate) to obtain a cytocidal conjugate (MTX-SS-PDL) or to 125I-tyramine to obtain a labeled analogue (125I-tyn-SS-PDL) used in a simple cleavage assay. Disulfide cleavage in CHO cells pulse labeled with 125I-tyn-SS-PDL begins without lag and continues for several hours. Initial cleavage at the cell surface (first 15-30 minutes) is followed by intracellular cleavage which, according to our preliminary data on cell fractionation, occurs in the Golgi apparatus. Our experimental approach will be: 1) to clarify the functional significance of the surface cleavage; 2) to confirm the reductive function of the Golgi apparatus; 3) to study the mechanisms of reductive cleavage, using among others: blockers of surface sulfhydryls, antibodies against enzymes suspected to catalyze the disulfide reduction, inhibitors of golgi function and intracellular transport, an in vitro assay of Golgi function, mutants of CHO cells defective in their ability to cleave endocytosed disulfide bonds and mutants hypersensitive to ricin; factors known to increase ricin cytotoxicity. cystamine, which we found to be a strong enhancer of ricin cytotoxicity will be studied with particular attention. An improved biochemical assay and a new functional assay of cell-mediated ricin cleavage are being developed and will be used to correlate cleavage and cytotoxicity, to pinpoint the steps of intracellular processing (uptake, routing, activation, translocation) that insure optimal ricin toxicity, and to determine which of these steps are subject to experimental modulation. Two immunotoxins in which ricin A is disulfide- linked to monoclonal antibodies against cell surface proteins of CHO cells will be prepared. Since their surface targets are internalized by different forms of endocytosis (receptor-mediated versus non-receptor- mediated) the comparison will clarify which of the two insures more efficient delivery of activated ricin to its cellular target. Correlating the data obtained with the two poly(lysine) conjugates, ricin and two ricin immunotoxins, is exp RYSER HJ BOSTON UNIVERSITY SCH OF MED, 80 EAST CONCORD STREET, BOSTON, MA 02118 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02118 Crisp Data Base National Institutes Of Health aminothiol antineoplastic intracellular transport Golgi apparatus chemical cleavage disulfide bond chemical kinetics drug delivery system methotrexate iodine immunoconjugate monoclonal antibody immunotoxicity polymer macromolecule oxidoreductase glutathione radionuclide radiotracer sulfhydryl reagent sulfide tissue /cell culture cytotoxicity toxin animal tissue endocytosis CHO cell ricin CRISP RPROJ MASSACHUSETTS G CRISP/99/AI41758-22 CRISP/99/AI41758-22 199904 eng MACROMOLECULAR TRANSPORT IN ASEXUAL MALARIA PARASITES Research RPROJ The long term objective of this proposal is to characterize transport pathways in Plasmodium-infected erythrocytes. During the erythrocytic cycle, parasites actively remodel the host cell cytosol and plasma membrane to meet their metabolic requirements and to insure their long term survival. The pathways and processes that maintain the complex host cell-parasite relationship are poorly understood at the biochemical level. There is evidence that blood-stage parasites internalize membrane impermeable molecules from the external medium through a pathway that bypasses the erythrocyte cytosol. The identification and characterization of this new pathway has stimulated investigators to pursue new areas in malaria research, including parasite transfection, antisense RNA and Chemotherapy using membrane-impermeable drugs. Characterization of essential transport pathways will provide the opportunity to design strategies to disrupt (or utilize) these processes to compromise parasite viability. Ribosome inactivating proteins from plants will be used to investigate macromolecular transport pathways and evaluated for their antimalarial efficacy. These proteins were selected since they are biochemically well characterized and produce specific antiviral and antitumor cytotoxicity in vitro. Cytotoxicity appears to be best in situations where the organisms/cells are rapidly replicating as would be the case for Plasmodium and other infectious diseases. The intraparasitic targets of ribotoxins and basis of cytotoxicity will be investigated. Effects on parasite protein and nucleic acid syntheses and DNA stability and replication will be investigated. Depending on their mode of action, combined with the known replication schemes of other pathogens, the results from these studies may reveal opportunities whereby ribotoxins may be applied for the treatment of malaria and other infectious diseases. TARASCHI TF THOMAS JEFFERSON UNIVERSITY, 1020 LOCUST STREET, PHILADELPHIA, PA 19107 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 19107 Crisp Data Base National Institutes Of Health intracellular transport erythrocyte antimalarial agent drug screening /evaluation host organism interaction gene expression human tissue immunofluorescence technique membrane activity macromolecule deoxyribonuclease I nucleic acid biosynthesis DNA replication nucleic acid sequence electron microscopy intracellular parasitism malaria thin layer chromatography protein biosynthesis protein transport plant protein Plasmodium falciparum radionuclide tissue /cell culture cytotoxicity confocal scanning microscopy CRISP RPROJ PENNSYLVANIA G CRISP/99/AI41761-02 CRISP/99/AI41761-02 199904 eng NATURAL IGM IN IMMUNITY AND AUTOIMMUNITY Research RPROJ "Natural" antibodies are antibodies produced in the absence of apparent stimulation by specific antigens. Most of these antibodies are of IgM class. Natural IgM is thought to function in the innate immune response, in immune regulation, and in autoimmune processes because of its natural presence, polyreactivities with high avidities, and the exquisite ability to activate complement. However, the physiological role of natural IgM in these processes is not completely understood due to the lack of a suitable model system. To overcome this limitation, we have constructed a novel mouse strain that is completely deficient in circulating IgM. But this mouse strain still maintains a normal preimmune B cell compartment that can initiate an antibody response by secreting other IG isotypes. Utilizing this new mouse strain, this proposal outlines experiments aimed at elucidating the role of natural IgM in the immediate immune response against bacterial infection, in enhancing the ensuing antibody response, and in autoantibody production and autoimmune pathogenesis. We propose to: (1) determine the susceptibility of mutant mice to challenges by exogenous bacteria such as group B streptococcus (GBS), endogenous intestinal bacteria, or bacterial endotoxin; (2) measure the antibody response in mutant mice immunized with T-dependent and T-independent antigens and determine the effect of the absence of IgM on b cell activation, somatic hypermutation, antibody affinity maturation, and memory b cell differentiation and maintenance; (3) introduce the targeted mutation onto the lymphoproliferation (lpr) background and determine the effect of the absence of IgM on autoantibody production and autoimmune-mediated glomerulonephritis and vasculitis. GBS is the leading cause of bacterial sepsis and meningitis among newborns in the United States and endotoxin shock is an urgent medical problem. Elucidation of the role of natural IgM in protection against these pathogens may provide a basis for the development of IgM antibody for their treatment. Autoantibodies, including IgM rheumatoid factor, are often associated with human rheumatoid disease. Clarification of the role of autoreactive IgM in autoimmune responses and pathogenesis will help to reveal the etiology of human rheumatoid diseases. CHEN J MASSACHUSETTS INSTITUTE OF TEC, 77 MASSACHUSETTS AVE, CAMBRIDGE, MA 02139-4307 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse enteric bacteria Streptococcus agalactiae B lymphocyte flow cytometry gene mutation immunoglobulin M leukocyte activation /transformation immunocytochemistry immunoglobulin gene antibody formation endotoxin microorganism immunology autoantibody autoimmune disorder CRISP RPROJ MASSACHUSETTS G CRISP/99/AI41762-02 CRISP/99/AI41762-02 199904 eng NOVEL ASSAY FOR MEASUREMENT OF ENDOTOXIN Research RPROJ Currently, the only test commercially available to measure endotoxin in biological fluids, the Limulus amebocyte lysate (LAL) assay, is FDA approved for the detection of endotoxin in human and animal parenteral drugs, biological products, and medical devices. This test is not FDA approved as a clinical diagnostic to detect endotoxin in the blood of patients at risk of gram negative septicemia because a number of interfering substances are present in blood that enhance or inhibit the LAL. In the United States, septicemia is the 13th leading cause of death, the leading cause of death, the leading cause of death in the intensive care unit, and accounts for $5 - 10 billion in annual health care expenditures. The incidence of septicemia in academic medical centers is 2 cases per 100 hospital admissions and the 28 day and 5 month mortality rate is 34% and 45%, respectively. Moreover, over 600,000 cases of septicemia were reported in 1995 and approximately 60% of these cases were caused by gram negative bacteria. Following its release from gram negative bacteria, circulating endotoxin induces a cascade of cellular and chemical events that result in organ damage, shock, and death. Following the confirmation that a ligand binding assay (LBA) is a sensitive and specific test to detect endotoxin in biological fluids (specific aim 1), using this LBA, clinical studies will be conducted to demonstrate that the detection of endotoxin in human blood is an early sensitive and specific predictor of organ dysfunction (specific aim 2). The data obtained from these studies will be used to support commercialization of a LBA as a clinical diagnostic for detection of endotoxin. The data obtained from these studies will serve as a platform for the design of clinical trials for the use of an antiendotoxin therapy for the prevention and early treatment of endotoxin-induced-organ damage in Phase III of this project to be funded by a commercial entity, strategic alliance. PROPOSED COMMERCIAL APPLICATION: Currently, there is no reliable clinical diagnostic to detect endotoxin in the blood of patients. Based on a market analysis it is estimated that the potential commercial value for this clinical diagnostic is $300 - 500 million a year. NEELY CF LINK TECHNOLOGY INC, 2 DAVIS DRIVE, RES. TRI. PARK, NC 27709-2076 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health gram negative bacteria bacterial disease blood toxicology communicable disease diagnosis diagnostic test diagnosis design /evaluation human subject endotoxin serology /serodiagnosis ligand toxicant screening method development clinical research CRISP RPROJ NORTH CAROLINA G CRISP/99/AI41832-02 CRISP/99/AI41832-02 199904 eng CHEMOKINES AND INFLAMMATORY RESPONSES IN CYSTICERCOSIS Research RPROJ This project's long-term aim is to gain understanding of immune responses in cysticercosis, with a view to develop novel immunotherapies. Cysticercosis, due to invasion of the pork tapeworm larvae Taenia solium, is an emerging disease in wealthier nations and a major cause of morbidity in endemic areas including South America and India. Epilepsy is the most prominent clinical manifestation and is due to parasitic cysticerci in the central nervous system (CNS). In the USA, cysticercosis is increasingly common in part because of the influx of immigrants. The initial pathological response in cysticercosis is the formation of eosinophil-rich inflammatory granulomas. Drug treatment may eliminates cysts but is associated with transient leukocyte influx into the CNS leading to clinical deterioration and sometimes death. In order to develop novel therapeutic strategies and to limit cellular influx, information is required about immune responses to this infection. There have been very few previous studies. On the basis of preliminary observations, the aim of this project is to collect in vivo data from patients (plus controls) on the presence of proinflammatory and chemotactic cytokines in infected patients. Such cytokines are critical in initiating successful immunity and in controlling cellular influx to sites of infection. We shall measure by ELISA, cytokine concentrations in plasma and cerebrospinal fluid. The ability of patient leukocytes to express genes for (northern analysis) and secrete such cytokines will be determined. Patterns of cytokine secretion during treatment will be examined to determine if anti- cytokine therapies are a real possibility. Finally, pilot work will develop an in vitro model of infection examining interactions between cysticerci and macrophages. The information will provide essential data needed to understand immunity to cysticercosis and for development of new treatments. FRIEDLAND JS HAMMERSMITH HOSPITAL, DU CANE RD, LONDON, ENGLAND W12 ONN 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Cestoda biological signal transduction secretion anthelmintic drug adverse effect gene expression genetic transcription human subject cytokine interleukin 6 tumor necrosis factor alpha enzyme linked immunosorbent assay immunology helminthic antigen cerebrospinal fluid parasitic disease chemotherapy protein biosynthesis tissue /cell culture helminthiasis northern blotting chemokine clinical research CRISP RPROJ UNITED KINGDOM G CRISP/99/AI42037-01 CRISP/99/AI42037-01 199904 eng DELIVERY OF PROTEINS INTO CYTOSOL USING LLO-LIPOSOMES Research RPROJ DESCRIPTION (Adapted from Investigator's Abstract): The cytosolic space of cells is an important target for drug delivery systems since many therapeutic agents are aimed at intervening or modifying specific molecular events occurring in the cytoplasm of cells. Many of these therapeutic agents, particularly the ones discovered or designed in recent years, are intrinsically membrane-permeant due to their charge or high molecular weight. The long term objective of this proposal is to develop strategies and methods that can deliver molecules in this category into the cytosolic space of cells. The main idea of this proposal is to import cell biology and biochemistry of Listeria into pharmaceutics, i.e., to take a minimal component from a facultative intracellular bacterium, Listeria monocytogenes, and construct a liposome-based cytosolic delivery vehicle. Listeriolysin O (LLO), which is necessary and sufficient in the escape of Listeria into cytosol, will be purified and incorporated into liposomes. The LLO-containing liposomes, which mimic how Listeria deliver themselves into the cytosol where they grow, will be characterized in terms of their ability to delivery macromolecules into the cytosol. Although the proposed strategy should be applicable to the delivery of other types of macromolecules, the current proposal will focus on the delivery of proteinaceous macromolecules. Two types of proteins that show effect when delivered into the cytosol will be tested: (1) an antigenic protein that induces activation of specific cytotoxic T lymphocytes (CTL); and (2) toxins that can inhibit protein synthesis. The amount of cytosolic delivery will be measured by a newly developed biochemical assay which monitors the percent of cytosolically delivered molecules out of the total cell-associated amount. In vitro antigen presentation assays will also be employed to measure relative efficiency of delivery into the cytosolic pathway of antigen presentation. This has direct implications as a vaccine delivery vehicle, and will therefore be extended to a well-established mouse model of lymphocytic choriomeningitis virus (LCMV) infection. Induction of CTL, specific against nucleoprotein (NP) of LCMV, by NP-containing LLO-liposomes and subsequent protection from viral challenge will be monitored in mice. In addition to delivering antigenic proteins, a membrane-impermeant toxin, gelonin, will be encapsulated inside LLO-liposomes and tested in the cell growth inhibition assays. The liposome formations that have a long circulation time will be made with LLO, and their efficiency of cytosolic delivery will also be tested. Concurrently, the mechanism of endosome lysis by LLO via pore formation will be studied using biophysical and molecular biological methods. LEE K UNIVERSITY OF MICHIGAN, 428 CHURCH STREET, ANN ARBOR, MI 48109-1065 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse Listeria liposome membrane permeability cytotoxic T lymphocyte hemolysin cytoplasm drug vehicle active immunization immunocytochemistry cell mediated lymphocytolysis test antigen presentation macromolecule nucleic acid inhibitor light scattering phosphatidylethanolamine protein nucleoprotein cytotoxicity lymphocytic choriomeningitis virus method development polyethylene glycol fluorescence resonance energy transfer protein localization CRISP RPROJ MICHIGAN G CRISP/99/AI42084-01 CRISP/99/AI42084-01 199904 eng BIO-ORGANIC CHEMISTRY OF ANTI-BACTERIALS Research RPROJ The long term goal of this research is to develop novel anti-infective disease agents using rational drug design and combinatorial chemistry. The broad objectives of this program are the development of novel antibacterial agents that will address issues of drug resistance. The narrow objectives of the several projects are: discovery of inhibitors of the microbial histidine-aspartate kinase two-component signaling system that is responsible for vancomycin resistance; discovery of inhibitors of a metallopeptidase involved in lipid A biosynthesis; discovery of inhibitors of a 3-arabinosyl transferase involve din the biosynthesis of arabinan segments of mycobacterial cell wall. These molecular targets for antibacterial action come from both gram-negative and gram-positive bacteria. One project will also investigate the total synthesis on sold phase of molecules that exhibit strong anti-infective activity, butt whose molecular targets are not known. Methods to prepare combinatorial libraries of these materials for the discovery of more potent or different antibacterial actions will then be developed. An additional project will involved a "generic" combinatorial library that can be used to map space in active sites of novel protein targets derived from microbial pathogens, allowed integration of combinatorial techniques and rational design principles. One particularly valuable aspect of this work will be the development of a new combinatorial library tagging methodology that permits rapid identification of molecules by NMR. This project will utilize the methods of organic synthesis on the solid phase, combinatorial chemistry, and antibacterial screening. This research will be significant because of the great difficulty medicine now perceives with pathogens that are resistant to conventional antibacterial drugs. Molecules that can modulate signaling events associated with histidine-aspartate kinases would be of great value in both basic scientific investigation and in potential control of microbial physiology and growth. An ancillary benefit of this project will be the development of novel techniques in solid phase synthesis through the total synthesis of natural products. PIRRUNG MC DUKE UNIVERSITY, BOX 90346, DURHAM, NC 27708 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 27708 Crisp Data Base National Institutes Of Health ethambutol vancomycin biochemistry chemical registry /resource chemical structure /function chemical synthesis drug resistance drug design /synthesis /production microorganism culture alginate endotoxin naphthoquinone arabinose protein kinase transferase enzyme activity metalloendopeptidase CRISP RPROJ NORTH CAROLINA G CRISP/99/AI42151-01 CRISP/99/AI42151-01 199904 eng REGULATION OF BOTULINUM NEUROTOXIN Research RPROJ DESCRIPTION (Adapted from applicant's abstract): The primary goal of this research project is to reveal and analyze genes involved in the regulation of neurotoxin (BoNT) production in Clostridium botulinum type A strain 62A. Nontoxigenic (tox-) mutants and mutants with significantly decreased BoNT production, as well as mutants affected in synthesis of nontoxic components of the type A toxin complex, will be isolated after insertion mutagenesis with the conjugative transposon Tn916. Mutants affected in toxinogenesis will be identified by screening colonies by an immunoblot assay and by ELISA in microtiter plates. Detailed Southern hybridization analyses of the parent and mutant strains will be performed to locate regulatory genes on the clostridial chromosome, and to reveal which genes are regulated. The clostridial chromosomal fragments containing regulatory regions will be cloned, their sequence determined and analyzed. The role of the virulence genes will be determined by complementation of the mutant strain with the wild-type allele containing the region of interest isolated from the parent strain. Transcription and translation patterns of BoNT and nontoxic components of the toxin complex will be analyzed in C. botulinum 62A by Northern analyses, by Western blots, and by ELISA. Knowledge gained in these studies will have practical value in the development of improved botulinum toxin complexes as pharmaceuticals and for understanding the capacity of C. botulinum to cause foodborne disease and human and animal infections. JOHNSON EA UNIVERSITY OF WISCONSIN, 1925 WILLOW DRIVE, MADISON, WI 53706 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 53706 Crisp Data Base National Institutes Of Health laboratory mouse Clostridium genetic strain genetic manipulation molecular cloning plasmid gene expression gene complementation regulatory gene reporter gene genetic regulation genetic transcription genetic translation gene mutation mutant enzyme linked immunosorbent assay western blotting botulinum toxin neurotoxin nucleic acid sequence biosynthesis northern blotting southern blotting CRISP RPROJ WISCONSIN G CRISP/99/AI42226-01A1 CRISP/99/AI42226-01A1 199904 eng DRUG RESISTANCE IN MALARIA Research RPROJ DESCRIPTION (Adapted from the Applicant's Abstract): Dr. Sibley and coworkers have expressed both dihydrofolate reductase and dihydropteroate synthase from Plasmodium falciparum in the budding yeast, Saccharomyces cerevisiae and shown that one can study the parasite enzymes in this simple system. When completed their experiments will provide a detailed understanding of the mutations that modify function of the Pf-DHFR and Pf-DHPS enzymes or sensitivity of the enzymes to antifolate drugs. SIBLEY CH UNIVERSITY OF WASHINGTON, BOX 357360, SEATTLE, WA 98195-7360 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health antimalarial agent combination chemotherapy drug interaction drug hypersensitivity drug resistance drug screening /evaluation enzyme inhibitor enzyme structure field study Saccharomyces cerevisiae gene expression point mutation human tissue dihydrofolate reductase malaria protein structure /function Plasmodium falciparum sulfur compound thymidylate synthase CRISP RPROJ WASHINGTON G CRISP/99/AI42321-01A1 CRISP/99/AI42321-01A1 199904 eng MOLECULAR BIOLOGY AND VIRULENCE OF CTX PHAGE Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Cholera toxin is the principal virulence factor of Vibrio cholerae, the Gram-negative bacterium that causes the severe diarrheal disease cholera. The investigators recently discovered that this potent enterotoxin is encoded by a novel filamentous bacteriophage designated CTX. The CTX phage is the first filamentous phage known to result in the lysogenic conversion of a host bacterium. The CTX phage can integrate into the V. cholerae chromosome and form stable lysogens or, after induction, excise from the chromosome and replicate as a plasmid. During this replicative stage of the phage life-cycle, cholera toxin can be expressed independently of the factors which were believed to be essential for its expression. Our demonstration of the induction of CTX phage from V. cholerae lysogens within the host gastrointestinal tract suggests the possibility that in vivo CTX phage induction plays a significant role in the virulence of V. cholerae. The objectives of the proposed studies are to understand the life-cycle of the CTX phage at the molecular level and to assess the significance of this bacteriophage in the pathogenesis of cholera. These studies will establish the molecular biology of a mechanism of horizontal transfer of virulence genes and thereby further our understanding of the emergence of pathogens. The study of the intraintestinal induction of CTX phage from lysogens, could establish a new paradigm for understanding the regulation of the expression of phage encoded virulence factors in a variety of bacterial pathogens that are lysogenized with converting phage. This work will also have important ramifications for the design of safer live attenuated V. cholerae vaccine strains. WALDOR MK NEW ENGLAND MEDICAL CENTER, 750 WASHINGTON ST BOX 041, BOSTON, MA 02111 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02111 Crisp Data Base National Institutes Of Health laboratory mouse Vibrio cholerae life cycle polymerase chain reaction cholera gene expression gene induction /repression lysogeny virus genetics cholera toxin virulence molecular biology protein purification bacterial virus bacteriophage M13 CRISP RPROJ MASSACHUSETTS G CRISP/99/AI42347-01 CRISP/99/AI42347-01 199904 eng HEME OXYGENASE-1 AND SEPSIS USING TRANSGENIC MICE Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Heme oxygenase (HO) catalyzes the first and rate-limiting step in the oxidative degradation of heme to bilirubin. While HO-2 is constitutively expressed, HO-1 is highly induced by heme, metal ions, cytokines, and agents causing oxidative stress such as LPS during gram negative sepsis. The PI's laboratory has shown that HO-1 induction may play a role in protection against oxidative stress in an in vivo model of septic shock and MOSF. In this proposal the investigators propose to utilize HO-1 transgenic and knockout mice to test their hypothesis that HO-1 plays a critical role in providing protection against oxidative stress. Specifically, they propose to 1) generate and identify transgenic mice overexpressing HO-1 selectively in the endothelial and vascular smooth muscle cells in the vascular wall, 2) determine the physiological, biochemical and cellular responses of HO-1 overexpressing transgenic mice in a murine model of septic shock and MOSF, 3) apply HO-1 knockout mice to this sepsis model, and 4) determine the mechanism(s) by which HO-1 induction protects against oxidative stress. CHOI AM YALE UNIVERSITY SCHOOL OF MEDI, SECTION OF PULMONARY MEDICINE, NEW HAVEN, CT 06520-8057 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal blood toxicology vascular smooth muscle vascular endothelium enzyme induction /repression disease model multiple organ failure oxidative stress septic shock heme oxygenase CRISP RPROJ CONNECTICUT G CRISP/99/AI42365-02 CRISP/99/AI42365-02 199904 eng EVOLUTION OF DURABILITY IN EMERGING BACTERIAL PATHOGENS Research RPROJ A crucial step in the emergence of new waterborne and foodborne pathogens in developed countries is the evolution of durability traits that allow bacteria to survive in mass water and food distribution systems. For Escherichia coli O157:H7, a foodborne pathogen and growing public health problem, the ability to tolerate extreme acidity enables this organism to endure in foods and infect susceptible human in very low doses. In the research proposed here, the role of natural selection for acid tolerance and other aspects of durability in the external environment will be tested in an experimental system of bacterial evolution. The design simulates the transition between the two principal habitats of an enteric pathogen; the primary habitat in the intestine and the secondary habitat in the water, soil, and food. The conditions of the secondary environment will be varied to impart different selection pressures against which pathogenic strains can evolve. The environments range from relatively benign favoring growth, to a harsh environment favoring improved durability. The specific aims are: (1) to experimentally determine how variables which influence survival in the external environment affect the direction and rate of evolution of durability; (2) to investigate the evolution of the components of durability (acid, salt, and heat tolerance, resistance to desiccation) and their correlated change with selection for single resistance factors; (3) to quantify variation in durability among E. coli from natural environments and assess the potential for emergence of new pathogenic strains; and (4) to elucidate the genetic basis of evolutionary change in durability by characterizing mutations in genes known to influence acid tolerance and other protection mechanisms. The evolved strains will be compared to the ancestral (original) strains in their durability and the costs (in terms of reduced fitness) of adaptation to the secondary environment will be assessed. In addition, the durability of E. coli from natural environments and outbreaks of disease will be compared to the evolved levels of durability. The molecular basis of evolved durability will be investigated through subtractive hybridization to identify genes that contribute to protection and cross protection against environmental challenges. WHITTAM TS PENNSYLVANIA STATE UNIVERSITY, 208 MUELLER LABORATORY, UNIVERSITY PARK, PA 16802 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 16802 Crisp Data Base National Institutes Of Health acidity /alkalinity Escherichia coli bacterial cytopathogenic effect natural selection biochemical evolution genetic strain polymerase chain reaction environmental adaptation soil microbiology bacterial food poisoning gastrointestinal infection bacterial genetics gene mutation microorganism culture microorganism interaction virulence gel electrophoresis microorganism population study water environment southern blotting subtraction hybridization CRISP RPROJ PENNSYLVANIA G CRISP/99/AI42391-01 CRISP/99/AI42391-01 199904 eng BIOLOGICAL BASIS FOR HIV1 NEUROVIRULENCE Research RPROJ DESCRIPTION (adapted from the Abstract): The neuropathogenesis of HIV encephalitis and HIV dementia is associated with sustained viral replication in brain macrophages and microglia. Viral penetration of the BBB and productive infection of brain macrophages appears necessary but not sufficient for the development of neurological abnormalities. Therefore, the hypothesis of interest is that specific viral neurotropic strains may produce qualitative changes in brain function resulting in macrophage effector molecule neurotoxin release. In addition, different strains of HIV-1 could produce qualitatively distinct central nervous system (CNS) abnormalities that affect monocyte transendothelial migration and production of toxins. To assess the role of specific HIV-1 strains in viral neuropathogenesis, the Investigator plans to study virologic, cellular immune, and functional outcomes of virus-macrophage interactions. Utilizing a blood/brain barrier system in the SCID mouse model of HIV encephalitis, he and his associates will propagate large numbers of microglia and blood monocytes to perform interactive experiments that could validate the hypothesis that different HIV-1 strains could produce distinct CNS pathological lesions. These studies will provide a rationale to address scientifically whether specific HIV-1 strains induce changes, qualitative and quantitative, in macrophage function that affect CNS neuroimmune interactions leading to progressive neurological disease in AIDS. PERSIDSKY Y UNIVERSITY OF NEBRASKA MED CTR, 600 SOUTH 42ND STREET, OMAHA, NE 68198-5215 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health genetic strain macrophage monocyte cell migration blood brain barrier polymerase chain reaction human tissue immunocytochemistry AIDS dementia complex virulence encephalitis microglia neurotoxin virus replication virus cytopathogenic effect human immunodeficiency virus 1 SCID mouse AIDS neuropathy CRISP RPROJ NEBRASKA G CRISP/99/AI42404-02 CRISP/99/AI42404-02 199904 eng WET VACUUM UNITS FOR SAMPLING FOOD BORNE BACTERIA Research RPROJ Development of an innovative bacterial sampling vacuum unit is proposed, which could save millions of dollars each year for consumers, and for meat and food-preparation industries. This unit will provide a rapid method for more convenient and safer sample collection from larger surface areas of meat carcasses and other food-preparation surfaces. Verotoxigenic and other E. coli or similar pathogens need to be more rapidly located and identified in the U.S. food supply to meet the needs of improved public health and HACCP. Current methods for bacterial detection are hindered by time consuming bacterial collection and concentration techniques. Phase I research will demonstrate the feasibility of a hand-held wet-vacuum sampling unit utilizing hydrophilic and hydrophobic filters to collect bacteria. The proposed design, using .45 microns filters, increases vacuum and filter capacity allowing sufficient vacuum to lift and collect bacteria from meat carcasses or food-preparation surfaces. Recyclable, single-use units will increase safety for QA personnel while decreasing labor and preparation time for specimen collection and lab processing. Future development may allow more direct interfacing of the proposed unit and rapid bacterial detection methods (ELISA/PCR) to further expedite lab results and decrease human disease risks and costs. PROPOSED COMMERCIAL APPLICATION: Successful development of the proposed sampling unit will result in a more convenient and safer wet-vacuum bacterial collection device for use by thousands of meat and other food-preparation industries. This unit will significantly decrease costs in labor and time involved in food sampling, processing, and analyzing for pathogens, while increasing public safety. BRADLEY BJ ROCKY MOUNTAIN RESOURCE LABS, 801 NORTH LINCOLN, JEROME, ID 83338 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 83338 Crisp Data Base National Institutes Of Health bacteria Escherichia coli biomedical equipment development meat bacterial food poisoning food processing /preparation food quality /standard food sanitation evaluation /testing CRISP RPROJ IDAHO G CRISP/99/AI42435-01 CRISP/99/AI42435-01 199904 eng BACTERIAL TOXINS AND ANTI HIV1 IMMUNITY Research RPROJ DESCRIPTION (adapted from applicant's abstract): Infection with HIV-1 worldwide occurs mostly through mucosal exposure. Therefore first line defense against HIV- 1 must include mechanisms that act at the mucosal surface. In addition it is desirable that cytotoxic T Lymphocytes against the virus are induced in order to combat cells that become infected by virus that escaped from this first line of defense. An ideal HIV-1 vaccine would therefore induce both mucosal antibodies and CTL. Cholera Toxin (CT) and E. cold heat-labile enterotoxin (LT) have the capacity to do both. It is unlikely that immune responses to a single HIV-1 gene product will provide full protection from infection. It has been demonstrated that vaccine strategies for SIV that incorporate responses to multiple SIV gene products provide better and broader protection from subsequent challenges. A vaccine formulation that could be flexibly adapted to induce immune responses to multiple gene products is therefore preferable over one that is inherently limited. Such Flexibility would also provide the opportunity to incorporate multiple antigenic variants which seems necessary in the face of the extreme variability of HIV-1. 1. Chemical cross linking of CT or LT to antigenic peptides of HIV-1 in theory would provide the following possibilities: - oral, rectal, or nasal administration of the immunogen - induction of cytotoxic T Lymphocytes to the peptides - induction of mucosal and systemic antibodies to the peptides and to the HIV-1 proteins - combination of multiple peptides from different HIV genes and from different variants in one immunogen. We have established a successful procedure to crosslink peptides to CT. We will use this procedure to pursue the following aims using mice as experimental animals: AIM 1 Determine whether HIV-1 peptides crosslinked to the CT holotoxin confer enhanced immunogenicity when compared to CT mixed with either free peptides or HIV-1 gp120 proteins both systemically and at the mucosal surfaces when administered intrarectally AIM 2 Determine whether different routes of administration (including intragastrically, intranasally and intrarecatally) of HIV- 1 peptides crosslinked to or mixed with LT mutants induce anti HIV- 1 antibodies and cytotoxic T Imphocytes AIM 3 Determine whether the system of crosslinking HIV - 1 peptides to CT or LT mutants allows for simultaneous immunization with multiple epitopes by using multiple antigenic peptides from both allelic variants of the same gene and of different HIV-1 genes. BOSCH ML UNIVERSITY OF WASHINGTON, BOX 357238, SEATTLE, WA 98195-7238 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse cytotoxic T lymphocyte crosslink microorganism culture leukocyte activation /transformation immunoregulation antiviral antibody cholera toxin enterotoxin microorganism immunology AIDS vaccine human immunodeficiency virus 1 HIV envelope protein gp120 neutralizing antibody vaccine development CRISP RPROJ WASHINGTON G CRISP/99/AI42505-01 CRISP/99/AI42505-01 199904 eng MUCOSAL IMMUNIZATION STRATEGIES FOR PREVENTION OF AIDS Research RPROJ A number of vaccine strategies for prevention of AIDS have been proposed, including use of attenuated bacterial and viral vectors expressing various epitopes from HIV, hybrid hepatitis particles expressing a VC loop peptide, DNA vaccines expressing gp120, and synthetic peptides containing B- and T-cell epitopes of HIV as immunogens. HIV subunits (gp120, gp160) and whole killed HIV have been tested in humans and non-human primates. None of these has been effective. A major problem limiting the development of an effective HIV vaccine is that the immune correlates of protection against HIV are not known. In this proposal, the applicants address a new strategy for prevention of AIDS by vaccination. They have developed a novel mucosal adjuvant which has been shown in numerous animal studies to induce protective immunity when coadministered with whole inactivated bacteria or viruses, or subunits or relevant virulence determinants from these pathogens. This adjuvant promotes the development of both antigen- specific humoral(antibody) and cell-mediated immune responses against the pathogen in both the systematic and mucosal compartments. In addition, the adjuvant has recently undergone two Phase I clinical studies in humans and has been shown to be safe and nontoxic at adjuvant-effective doses. The proposed studies will focus on the use of this adjuvant for production of humoral and cell-mediated immune responses against a model of HIV antigen - gp120. The main goal of this study is to characterize the humoral and cellular response against HIV gp120 when administered with the adjuvant, and to determine whether the nature of the humoral or cellular immune responses to this antigen when delivered in the presence or absence of this adjuvant will be influence by the route of immunization, or the number of doses administered. Since the immune protective correlates to HIV infection are unknown, the type of immune response induced by vaccination will be characterized in depth. Serum and mucosal anti-gp120 antibodies will be determined and characterized with respect to antigen-specific Ig isotypes and distribution in serum and mucosal secretions by ELISA and Western blot, and the ability to neutralize HIV infectivity in vitro. Cellular studies will be applied to determine the type of T helper cell response induced and the cytokine profile during the effector phases of the immune response, with special regard to development of TH1 and TH1 type response, as well as CTL activity. Clements JD TULANE UNIV MED SCH, 1430 TULANE AVENUE, NEW ORLEANS, LA 70112 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 70112 Crisp Data Base National Institutes Of Health laboratory mouse cytotoxic T lymphocyte helper T lymphocyte polymerase chain reaction dosage drug administration route AIDS education /prevention immunoglobulin idiotype cytokine humoral immunity active immunization passive immunization enzyme linked immunosorbent assay western blotting antiviral antibody antigen antibody reaction enterotoxin immunomodulator messenger RNA parenteral feeding AIDS vaccine HIV envelope protein gp120 neutralizing antibody mucosal immunity vaccine development CRISP RPROJ LOUISIANA G CRISP/99/AI42601-02 CRISP/99/AI42601-02 199904 eng STIMULATION OF HIV SPECIFIC CTL WITH TOXIN FUSIONS Research RPROJ Although most HIV vaccine approaches investigated in the past have focused on generating neutralizing antibodies against the virus, there is increasing evidence to suggest that the key to success of this endeavor lies in strategies for eliciting cellular antiviral immunity. The applicants have proposed experiments aimed at developing a peptide vaccine with the capacity to prime a cytotoxic T- lymphocyte (CTL) response in vivo aginst HIV-1 epitomes. The nef and gag proteins have been identified as the sites of potentially important epitomes and will be the primary focus of their work. In order to prime a CTL response, a CTL epitome-containing protein must be delivered to the cytosol of mammalian cells. There, it is processed to form peptides which are then complexed with MH class I molecules and presented at the cell surface. Recent studies have revealed how the intra cellularly acting toxin, such as the anthrax toxin (AT), may be modified to ablate its toxicity and enable it to deliver heterologous proteins to the mammalian cell cytosol. As a ramification of these findings, the applicants have demonstrated that AT fusion proteins containing selected epitomes from Listeria monocytogenes and lymphocytic choriomenigitis virus may be used to prime protective CTL responses in mice against these pathogens. In the research proposed, the applicants intend to prepare similar AT fusions continuing the nef and gag proteins and test these constructs for ability to prime an antigen-specific CTL response in mice. Furthermore, the applicants will also attempt to determine whether human cells treated invitro with these fusions can induce proliferation of gag- and nef-specific CTL from peripheral blood ofpatients infected with HIV. Finally, the researchers will investigate whether patient-derived gag- and nef-specific CTL can lyse haplotype-matched target cells treated with the fusion proteins. These experiments will allow the utility of the AT-based vaccine strategy to generate a CTL response against HIV antigens to be evaluated and, if positive indications are obtained, will set the stage for safety and efficacy studies of AT-based vaccines in primate animal models. COLLIER RJ HARVARD MEDICAL SCHOOL, 200 LONGWOOD AVENUE, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health laboratory mouse laboratory rat Bacillus cytotoxic T lymphocyte human tissue leukocyte activation /transformation immunoconjugate bacterial toxin AIDS vaccine human immunodeficiency virus 1 vaccine development CRISP RPROJ MASSACHUSETTS G CRISP/99/AI42671-02 CRISP/99/AI42671-02 199904 eng BACTERIAL TOXINS AS CTL VACCINE VECTOR FOR HIV Research RPROJ The long-term goal of this project is to develop an HIV vaccine or vaccine component using genetically detoxified bacterial toxins as vector molecules for induction of CD8+ T cell responses, including cytolytic T lymphocytes (CTL). The candidate vaccine molecules will consist of genetic fusions of HIV antigens with the toxins, to stimulate HIV-specific CTL activity and other T-cell responses in vivo. Bacterial toxins are suitable vector molecules because they can enter eukaryotic cells and, therefore, deliver antigens for processing and presentation by MHC class I molecules, a complex which is recognized by CTL. This property of the toxins is important because only internalized (cytosolic) antigens are efficiently targeted to the MHC class I pathway. The toxins to be used are genetically detoxified derivatives of pertussis toxin (PT) and choler toxin (CT), PT-9K/129G and CT-K63, respectively, which are completely devoid of toxicity but retain other properties. The fusion molecules will be tested in model systems in vitro and in vivo to assess their MHC class I targeting capacity and their immunogenicity. The specific aims of the proposed research are as follows: (1) to construct detoxified toxin- HIV/SIV antigen fusions that are assembled and secreted efficiently by the bacterial hosts, and to purify these fusion proteins; (2) to demonstrate the CTL-stimulatory capacity of such fusion proteins in vitro, by infection of target antigen presenting cells and CTL lysis assays, and in vivo, by immunization of mice and rhesus macaques, and assay for antigen- specific CTL and other immune responses. one advantage of such strategy is that it utilizes a vector that, in one case, is already a vaccine molecule (PT-9K/129G, which is in use as a pertussis vaccine component), and in the other case (CY), is widely used as an adjuvant, particularly for enhancement of mucosal immune responses. These fusions may, therefore, represent powerful mucosal and systemic vaccine molecules for HIV. CARBONETTI NH UNIVERSITY OF MARYLAND, 655 W BALTIMORE STREET, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health Macaca mulatta laboratory mouse cytotoxic T lymphocyte cellular immunity immunization immunoconjugate major histocompatibility complex bacterial antigen cholera toxin pertussis toxin chimeric protein AIDS vaccine human immunodeficiency virus simian immunodeficiency virus vaccine development CRISP RPROJ MARYLAND G CRISP/99/AI42681-02 CRISP/99/AI42681-02 199904 eng CD8+ T CELL EFFECTOR MECHANISMS AGAINST L MONOCYTOGENES Research RPROJ CD8+ T cells are important mediators of immunity against many viral, protozoan and bacterial pathogens of humans. Activated CD8+ T cells respond to pathogen infected cells by elaborating several distinct effector mechanisms, including cytolysis, IFN-gamma and TNF-alpha production, with the potential to resist infection. In order to design successful vaccines against infectious agents it critical to understand which effector mechanisms and cells provide the most potent immunity. Given the complex biology of intracellular pathogens and the variety of strategies they use to avoid host immune responses it is unlikely that identical CD8+ T cell effector mechanisms will be required for resistance to all types of intracellular pathogens. Substantial research effort has been made to understand the CD8+ T cell effector mechanisms that provide immunity to viral infections. However, similar studies of CD8+ T cell effector mechanisms in resistance to protozoan and bacterial pathogens are lacking. The long term objectives of this research are to understand the CD8+ T cell effector mechanisms that provide immunity against intracellular bacteria. Studies in this proposal will use a well characterized mouse model of infection with the human enteric pathogen Listeria monocytogenes to test the hypothesis that at least one of the major CD8+ T cell effector mechanisms (cytolysis, IFN-gamma or TNF-alpha production) is required for CD8+ T cell mediated immunity to L. monocytogenes infection. The following specific aims will be addressed. Specific Aim-1. Determine the requirement for perforin dependent cytolysis in CD8+ T mediated immunity against L. monocytogenes infection. Specific Aim-2. Determine the requirement for CD8+ T cell derived IFN-gamma in immunity to L. monocytogenes infection in the absence of perforin. Specific Aim-3. Determine the requirement for TNF- alpha in: a) secondary resistance to L. monocytogenes infection and b) as a CD8+ T cell effector mechanism in the presence and absence of perforin and INF-gamma. Our experimental approach to these aims combines genetically manipulated L. monocytogenes strains, specific gene knockout mice, antigen specific CD8+ T cells and neutralizing anti- cytokine antibodies. These reagents provide sophisticated probes for identification of the CD8+ T cell effector mechanisms required for immunity to intracellular bacteria. The results of these studies will provide important information for vaccine design strategies and emerging approaches to the immunotherapy of human infectious diseases. HARTY JT UNIV OF IOWA, 3-512 BSB, IOWA CITY, IA 52242 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 52242 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal Listeria genetic strain cytotoxic T lymphocyte cytolysis host organism interaction cellular immunity leukocyte activation /transformation tumor necrosis factor alpha pore forming protein cytolysin interferon gamma gene targeting CRISP RPROJ IOWA G CRISP/99/AI42767-01 CRISP/99/AI42767-01 199904 eng MECHANISM OF CHOLERA TOXIN AND E COLI LT ADJUVANTICITY Research RPROJ The WHO report of Infectious Disease deaths for 1995 indicated that there were more than 13 million deaths world-wide during that year. The majority of those deaths were caused by organisms that first make contact with and then either colonize or cross mucosal surfaces to infect the host. A number of strategies have been developed to facilitate mucosal immunization to prevent these diseases, including addition of bacterial products with known adjuvant properties. The two bacterial products with the greatest potential to function as mucosal adjuvants are cholera toxin (T), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. A number of mutants of CT and LT have been developed in an attempt to dissociate the desirable adjuvant properties of these molecules from their toxic effects. Both active-site and protease-site mutants have been constructed and evaluated in a variety of animal models with different antigens. Important questions regarding the adjuvanticity of CT and CT and mutants of these toxins remain to be answered. Some of these questions are practical and the answers will impact the immediate and short term use of these molecules in human vaccines. Other questions address the underlying mechanisms associated with adjuvanticity and the answers will have their greatest impact in the design of future adjuvants and vaccine strategies and in the development of a better understanding of vaccine induced immunity. The proposal includes a series of Specific Aims designed to directly address these issues. One of the most important aspects of the proposed study is a side-by-side comparison of CT, LT, active-site mutants, protease-site mutants, and recombinant B-subunits for the ability to induce specific, targeted immunologic outcomes as a function of route of immunization and nature of the co-administered antigen. With the information obtained in the proposed studies, future vaccine strategies can be designed employing the optimum adjuvant/antigen formulation and route of administration for a variety of bacterial and viral pathogens. This proposal also examines the underlying cellular and intracellular signaling pathways activated by these different molecules to better understand the mechanisms of adjuvanticity at the cellular level. CLEMENTS JD TULANE UNIV MED SCH, 1430 TULANE AVENUE, NEW ORLEANS, LA 70112 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 70112 Crisp Data Base National Institutes Of Health laboratory mouse Listeria Escherichia coli Salmonella Vibrio cholerae biological signal transduction Candida site directed mutagenesis transfection mutant immunization antigen antibody reaction cholera toxin enterotoxin immunomodulator endopeptidase toxin metabolism active site HIV envelope protein gp120 cell line mucosal immunity CRISP RPROJ LOUISIANA G CRISP/99/AI42777-01 CRISP/99/AI42777-01 199904 eng MECHANISM OF PSEUDOMONAS MEDIATED EPITHELIAL CELL DAMAGE Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): P. aeruginosa is one of the most virulent opportunistic pathogens of man. The morbidity of P. aeruginosa infections results from the ability of the bacterium to colonize previously injured or disrupted epithelial cell layers, cause further epithelial cell damage, and in many cases, gain access to other tissues or the blood stream. To dissect these steps, the investigators make use of a recently developed in vitro and a recently developed animal model of acute pneumonia in which localized cytotoxicity in a polarized epithelial cell line grown in vitro correlates with virulence in this animal model of acute pneumonia. These events do not appear to depend on previously identified virulence factors with the possible exception of exoenzyme S. In preliminary work, they carried out a genetic screen of transposon mutants of a cytotoxic and animal-virulent human isolate of P. aeruginosa (PA103) to identify genes that mediate this localized cytotoxicity. In this grant, the investigators will explore the role revealed by each of these mutants in localized cytotoxicity in an in vitro epithelial cell model of P. aeruginosa-mediated epithelial cell damage and will test them in an animal model of acute pneumonia. Specifically, (i) They will investigate the site of action, function and structure/function relationship, regulation, cellular substrates, and virulence in an animal model of acute pneumonia of the putative novel cytotoxin (PepA) they have discovered. (ii) They hypothesize that either invasion is necessary for and precedes cytotoxicity or that these two events are mutually exclusive. They will test these hypotheses by genetic and pharmacological approaches. (iii) Their initial results demonstrate that while functional pili are necessary for P. aeruginosa-mediated epithelial cell damage in vitro, this function is independent of their ability to bind to the apical surface of MDCK cells. They propose experiments to test further confirm these results. (iv) They postulate that the role of pili in function in cytotoxicity is to allow bacteria to aggregate before binding the apical surface, to move along the surface of epithelial cells by twitching motility, or to transduce the signal that initiates contact-mediated secretion of the putative effectors of cytotoxicity by type III secretion. To test these hypotheses, they will employ time-lapse videomicroscopy to examine the ability of the bacteria to aggregate prior to mediating cell injury, they will use genetic approaches to determine whether twitching motility can be separated from cytotoxicity, and they will test whether pili function to transmit the signal that host cell binding has occurred, leading to contact-mediated type III secretion of PepA. From these studies will come novel targets for anti-Pseudomonal therapies and a better understanding how this bacterium colonizes and injures epithelial cells. In addition, the stud ENGEL JN UNIV OF CALIFORNIA, 521 PARNASSUSS AVE, RM C430, SAN FRANCISCO, CA 94143-0654 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health Pseudomonas aeruginosa bacterial cytopathogenic effect cell adhesion pilus bacterial genetics immunofluorescence technique virulence protein structure /function epithelium cytotoxicity MDCK cell video microscopy bacteria infection mechanism cell component structure /function CRISP RPROJ CALIFORNIA G CRISP/99/AI42806-01 CRISP/99/AI42806-01 199904 eng GLYCOLIPIDS AND HEMOLYTIC UREMIC SYNDROME Research RPROJ DESCRIPTION (Adapted from applicant's abstract): Hemolytic uremic syndrome (HUS) is characterized by verotoxin-mediated damage to endothelial cells that results in hemolytic anemia, throbocytopenia, and multisystemic complications including renal failure. In almost all cases, verotoxins (shiga-like toxins) released by enterohemorrhagic E. coli bind to glycolipids on the endothelial cells, are then routed to the endoplasmic reticulum, and thereby inactivate the 28S ribosomes and halt protein synthesis. Although glycolipid receptors are required for VT susceptibility, the degree of susceptibility does not correlate directly with the quantity of receptor. The clinical correlative of these observations is that children and adults differ in their susceptibility to HUS despite expressing similar quantities of VT-receptor and glycolipids in renal cells. The principal investigator therefore hypothesizes that differences in docking to verotoxin receptors or distinct pathways controlling intracellular trafficking of VT and its glycolipid receptors underlie these differences. In preliminary studies VT-susceptible Vero cells were transfected with the cDNA encoding Forssman synthetase (FS). FS-transfected cells were highly resistant to VT, yet still demonstrated toxin binding. Ligand blotting demonstrated the presence of two glycolipid receptors (R1 and R2) whereas only R2, a novel receptor not previously identified, was present in FS-transfected cells. In Specific Aim #1, the principal investigator will identify glycolipid receptor R2, purify this receptor from VT-resistant cell extracts, confirm its identity by mass spectroscopy, and add purified R2 glycolipid exogenously to other cell types with a priori resistance to verotoxin. Tandem experiments with receptor R1 will serve as a control. In Specific Aim #2, the principal investigator will analyze internalization and intracellular trafficking of verotoxin and cholera toxin receptor glycolipids in FS-transfected cells and wild-type cells. Immunogold electron microscopy will be used to track the intracellular fate of labeled verotoxin in FS-transfected and WT-cells. Intracellular trafficking of cholera toxin, which binds an altogether different glycolipid (GM1) will be examined in this same system. An inducible promoter will be used to titrate expression of FS to understand whether a minimal amount of the enzyme is required for VT-resistance. In Specific Aim #3, the principal investigator will use his newly developed transgenic mouse model for overexpression of FS to characterize mRNA expression of this enzyme and alterations in glycolipid expression in various tissues. A murine model for HUS, which fails to mimic precisely the effects of VT because mice express a spectrum of glycolipids different from that in humans, will be adapted using FS transgenic mice and littermate controls to determine whether altered glycolipid expression results in VT resistance in vivo. HASLAM DB WASHINGTON UNIV SCH OF MEDICIN, ONE CHILDREN'S PLACE, ST LOUIS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 63110 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal hemolytic anemia endoplasmic reticulum mass spectrometry transfection genetic promoter element glycolipid bacterial toxin cholera toxin renal failure immunoelectron microscopy protein purification protein transport receptor expression Vero cell CRISP RPROJ MISSOURI G CRISP/99/AI42817-01 CRISP/99/AI42817-01 199904 eng PHYSICAL BASIS OF T CELL ACTIVATION BY SUPERANTIGENS Research RPROJ In autoimmune disease, a breakdown of self-tolerance leads to the generation of an immune response to a specific target antigen or antigens. Clinical and epidemiological evidence indicates that infections play an important role in the induction of autoimmune disorders such as autoimmune myocarditis and diabetes mellitus. One mechanism by which this may occur is through the activation of autoreactive T cells by epitopes on microbial antigens that cross-react with antigens on target organs. It has also been proposed that so- called 'superantigens' (SAGs) from bacteria, mycoplasma, or viruses may initiate autoimmune disease by activating specific anti-self T cell clones. Superantigens are extremely potent T cell stimulants that act by cross- linking T cell antigen receptors (TCRs) and major histocompatibility complex (MHC) molecules. Our goal is to elucidate the physical basis of T cell activation by microbial SAGs through determination of the three-dimensional structure of TCR-SAG complexes by X-ray crystallographic techniques and to correlate this information with affinity measurements of TCR-SAG and SAG-MHC interactions. Thus, we have determined the crystal structure, to 3.5 A resolution, of a TCR beta chain complexed with a bacterial SAG, staphylococcal enterotoxin C3 (SEC3). This work enabled us to understand, for the first time, how SAGs circumvent the normal mechanism for T cell activation by specific peptide/MHC complexes. We now propose to extend our crystallographic studies of the TCR beta- SEC3 complex to a complex between the beta chain and SEB, for which crystals which diffract to high resolution (2.5 A) have been obtained. We also propose to define the relative contributions of TCR-SAG and SAG- MHC interactions to T cell stimulation by engineering a panel of mutants of SEB and SEC3 with both higher and lower affinities for TCR and MHC than the wild type toxin. We will investigate the possibility of cooperativity in the binding of SAGs to TCR and MHC by generating covalently linked SAG-MHC complexes. Finally, we propose to express a recombinant form of the Mycoplasma arthritidis SAG MAM, to measure its binding to TCR and MHC, and to determine the three-dimensional structure of MAM bound to the TCR beta chain and to MHC class II. The results of these studies will define the rules governing T cell activation by microbial SAGs and help lay the groundwork for understanding how SAGs can activate autoreactive T cell clones in autoimmune disease. MARIUZZA RA CTR FOR ADVANCED BIOTECHNOLOGY, 9600 GUDELSKY DRIVE, ROCKVILLE, MD 20850 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 20850 Crisp Data Base National Institutes Of Health T lymphocyte X ray crystallography leukocyte activation /transformation staphylococcal enterotoxin protein structure receptor binding T cell receptor MHC class II antigen recombinant protein superantigen CRISP RPROJ MARYLAND G CRISP/99/AI42937-01 CRISP/99/AI42937-01 199904 eng CALCIUM SIGNALING AND KILLING BY CYTOTOXIC T LYMPHOCYTES Research RPROJ DESCRIPTION: The long term goal of this proposal is to understand the role of intracellular calcium ([Ca2+]i) signals in the function of cytotoxic T lymphocytes (CTLs). These critical effectors of the immune system kill virally infected cells and cancer cells, and play a major role in the immune response to transplanted tissues; inappropriate killing can cause autoimmune diseases such as Lupus, certain forms of diabetes, and arthritis. CTL function is therefore of critical relevance to health and disease, and detailed knowledge of how CTLs kill is of key importance to understanding the etiology of viral diseases such as AIDS, cancer autoimmune disorders, and to preventing rejectio of transplanted organs. Therapeutic strategies may be suggested by detailed knowledge of CTL function. One of the two mechanisms CTLs use to kill is the perforin pathway, which involves the exocytotic release of pore-forming peptides and hydrolytic enzyme contained in specialized CTL lytic granules into an area of close apposition formed with the target. Granule exocytosis is a two step process, involving relocation of lytic granules followed by their fusion, and is known to require increased [Ca2+]i caused by influx across the plasma membrane. However, the specific role(s) of Ca2+ in driving granule exocytosis is unknown. Critically, granule exocytosis has not been studied using the powerful physiological approaches that have advanced our understanding of exocytosis in other cell types. The specific aims of this proposal are to use patch clamp recording, capacitance measurements and digital video imaging techniques to: 1) determine the mechanism of Ca2+ influx in CTLs; 2) investigate the idea that Ca2+ channels are localized to create functional Ca2+ gradients; and 3) determine the role(s) played by Ca2+ in granule exocytosis. These specific aims will establish a firm physiological foundation for understanding the mechanism of this critical exocytotic event. ZWEIFACH A UNIV COLORADO HLTH SCIS CTR, BOX C-240, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 80262 Crisp Data Base National Institutes Of Health cytotoxic T lymphocyte calcium channel blocker calcium flux granule exocytosis patch clamp cell mediated cytotoxicity neuropharmacology pore forming protein calcium channel voltage gated channel tissue /cell culture cytolysin CD95 molecule CRISP RPROJ COLORADO G CRISP/99/AI42964-01 CRISP/99/AI42964-01 199904 eng BACTERIAL TOXINS AS CTL VACCINE VECTORS FOR HIV Research CARBONETTI NH MED BIOTECHNOLOGY CENTER RES, 725 WEST LOMBARD STREET, BALTIMORE, MD 21201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21201 Crisp Data Base National Institutes Of Health Macaca mulatta cytotoxic T lymphocyte bacterial toxin cholera toxin pertussis toxin virus antigen HIV infection chimeric protein AIDS vaccine vaccine development vector vaccine CRISP RPROJ MARYLAND G CRISP/99/AI43046-010002 CRISP/99/AI43046-010002 199904 eng POLYETHYLENE GLYCOL LINKED ARTEMISININ ANTIMALARIALS Research RPROJ An urgent need exists for new and more effective antimalarials. Preliminary studies indicate that when the Chinese traditional medicinal drug, artemisinin, is covalently bonded through an ester linkage to poly(ethylene glycol) (PEG), the resulting molecule has much higher antimalarial activity than artemisinin. With the long-range goal of developing PEG-artemisinins as useful antimalarial drugs, the following initial experiments will be conducted during Phase I of this project: (a) the possibility of improving antimalarial activity will be evaluated in in vitro Plasmodium falciparum assays using branched PEG and pendant PEG ester derivatives of artemisinin to increase drug loading and by optimizing the molecular weight of the attached PEGs to increase drug loading and by optimizing the molecular weight of the attached PEGs (b) the stability of PEG linked artemisinins will be evaluated by hydrolysis rate measurements in serum and in rats of several PEG derivatives. (c) uptake of PEG-linked artemisinin in P. falciparum cells will be assessed using double-labeled PEG-artemisinin esters and (d) neurotoxicity of PEG-artemisinin in rats will be evaluated. Plans for moving closer to a viable PEG-linked antimalarial in Phase II include studies in animals of antimalarial drug effectiveness, pharmacokinetics, and metabolism. PROPOSED COMMERCIAL APPLICATION Poly(ethylene glycol) derivatives of artemisinin have high potential for use as drugs for the treatment of malaria. HARRIS JM SHEARWATER POLYMERS INC, 2305 SPRING BRANCH RD, HUNTSVILLE, AL 35801 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 35801 Crisp Data Base National Institutes Of Health laboratory rat bioassay chemical synthesis antimalarial agent pharmacokinetics drug screening /evaluation drug design /synthesis /production Plasmodium falciparum sesquiterpene neurotoxicology polyethylene glycol CRISP RPROJ ALABAMA G CRISP/99/AI43130-01 CRISP/99/AI43130-01 199904 eng SYSTEM FOR SENSITIVE DETECTION OF BACTERIAL PATHOGENS Research RPROJ The detection and measurement of pathogenic bacteria is an extremely important function in the diagnosis of disease, evaluation of contamination of the environment and foods, and in epidemiological surveillance. We have developed an assay amplification system which can be used to detect the antigens and DNA specific for infectious organisms at very low concentrations, and to report the results in a color-test format. In this proposal, we suggest that we will use this system for the measurement of several Salmonella species (Salmonella typhi, Salmonella paratyphi A) and the SLT (shiga-like toxin)-producing Escherichia coli strains especially E. coli O157/H7. The approach to be used is to develop ELISA assays based on the known specific antigens of these bacteria and DNA hybridization assays based on characteristic sequences for Salmonella sp in general (invA-invE), for Salmonella typhi and Salmonella paratyphi A (tyvelose synthetase gen) and for SLT-I and SLT-II genes. These tools will be assembled in protocols useful in evaluating patients thought to be infected with these organisms, including a new approach to amplify and recover viable bacteria from ELISA plates after detection. The protocols will be tested in a laboratory which routinely monitors the presence of infection by these organisms (in Bangalore, India) and compared with the results obtained using more traditional microbiological identification procedures. At the end of this effort, we would have produced a relatively inexpensive approach which would permit the application of this technology to the identification of infectious organisms, and in the Phase II effort we would extend the range of infectious organisms which could be measured using this technology. PROPOSED COMMERCIAL APPLICATION Detection of specific pathogens is an important component of disease detection, food safety and epidemiological surveillance. If the technology developed in this work results in the production of convenient, field-deployable kits for the detection of these pathogens, they will be widely used. BEARD GA ELCATECH, INC, 4291 LANTERN DRIVE, WINSTON-SALEM, NC 27106 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 27106 Crisp Data Base National Institutes Of Health Escherichia coli Salmonella Salmonella typhi communicable disease diagnosis diagnosis design /evaluation bacterial genetics enzyme linked immunosorbent assay surface antigen bacterial antigen bacterial toxin method development CRISP RPROJ NORTH CAROLINA G CRISP/99/AI43147-01 CRISP/99/AI43147-01 199904 eng MOLECULAR ADJUVANTS FOR MALT BASED IMMUNITY TO SIV Research RPROJ DESCRIPTION: This proposal will explore cellular and molecular mechanisms to induce mucosal and systemic immune responses specific for SIV and HIV through the nasal-associated lymphoreticular tissue (NALT). They will employ non-toxic cholera-toxin mutants as adjuvants in combination with SIV gp130 or HIV gp160 delivered intranasally as a means to understand the induction pathways in both mice and humans. In a separate proposal, they aim to conduct similar studies SIV/macaque model. In Aim 1, the applicant will characterize murine NALT and associated mucosal tissues after nasal immunization with SIV gp130 (or HIV gp160) with mCT and develop an in vitro human NALT system to assess mCT-induced antigen uptake. In Aim 2, signal transduction pathways of NALT antigen-specific CD4+ T cells will be analyzed following nasal immunization. In Aim 3, they will develop and characterize chimeric mCTs which can mimic induction of antigen-specific Th1 or Th2 type responses following nasal immunization. In Aim 4, they will assess the effects of mCTs on antigen-presenting cells from human tonsils and adenoids for potentiation of Th1-or Th2-type responses. In Aim 5, they will determine the safety and immunogenicity of a combined gp160 and mCT nasal vaccine in humans in a phase I vaccine trial. MCGHEE JR UNIVERSITY OF ALABAMA, 845 19TH STREET SOUTH, BIRMINGHAM, AL 35294-2170 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse biological signal transduction lymphatic tissue helper T lymphocyte antigen presenting cell human subject leukocyte activation /transformation cholera toxin immunomodulator virus protein AIDS vaccine human immunodeficiency virus 1 simian immunodeficiency virus HIV envelope protein gp160 mucosal immunity clinical research vaccine development CRISP RPROJ ALABAMA G CRISP/99/AI43197-01 CRISP/99/AI43197-01 199904 eng HIV CHIMERIC VIRUS PARTICLES AS A MUCOSAL VACCINE Research RPROJ DESCRIPTION (adapted from applicant's abstract): The applicants propose to test the hypothesis that a fusion protein made between a fragment of gp160 and the L1 capsid protein of human papilloma virus (HPV) will form a chimeric viral like particle (CVLP) that will be useful as a new delivery system for mucosal immunization against HIV-1. This hypothesis will be tested by fusing a segment of gp160 that harbors HLA-0201 restricted epitopes with L1 to produce a HIVgp160CVLP. This chimera will be used as the principal immunogen in HLA-0201 transgenic mice to induce mucosal and systemic CTL responses specific for these epitopes. There are 3 specific aims: 1) to determine whether immunization with HIVgp160CVLP will induce HIV-specific mucosal and systemic responses via the subcutaneous, intranasal, or intrarectal routes; 2) to determine whether CVLP that contains enriched HLA-A0201 epitopes can induce CTL responses to these epitopes; and 3) and to determine the ability of adjuvants (Cholera toxin (CT) and IL-12) to enhance a mucosal response. QIAO L LOYOLA UNIV MEDICAL CENTER, 2160 S FIRST AVE, MAYWOOD, IL 60153 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 60153 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal cytotoxic T lymphocyte helper T lymphocyte leukocyte activation /transformation active immunization cholera toxin immunomodulator virus protein AIDS vaccine human papillomavirus human immunodeficiency virus 1 recombinant protein mucosal immunity interleukin 12 vaccine development CRISP RPROJ ILLINOIS G CRISP/99/AI43214-01 CRISP/99/AI43214-01 199904 eng GENETIC ANALYSIS OF LETHAL FACTOR SENSITIVITY Research RPROJ The pathogenesis of anthrax, a severe disease of humans and numerous livestock species, involves host sensitivity to various exotoxins secreted by the causative agent of anthrax, Bacillus anthracis. One of these toxins, called Lethal Factor (LF), is a mononuclear phagocyte specific toxin, causing cytolysis of these very important immune system cells. The overall goal of this project is to identify genes in the mouse that play a role in the poorly understood LF intoxication process. Monocytic cells isolated from various inbred mouse strains exhibit differential susceptibility to cytolysis caused by LF. In at least one resistant/susceptible strain pair comparison, C57BL/6J X C3H/HeJ, the trait differences segregates as a single gene character that maps to chromosome 11. One of the main goals of this project is to isolate the gene on chromosome 11, called Ltx1, that is responsible for the difference in LF sensitivity. A high resolution genetic map of the Ltx1 region will be constructed using a (C57BL/6J X C3H/HeJ) X C57BL/6J cross, and regional mapped polymorphisms will be used to construct a physical map of YAC, BAC, and P1 clones. The search for Ltx1 in this physical map will consist of typical positional cloning strategies, including genomic sequencing, exon trapping, and identification of cross species conserved probes. Any genes identified will be analyzed to determine if they are involved in LF intoxication using functional complementation of the trait in tissue culture assays. Isolation of differentially expressed genes and genes whose proteins interact with LF will be used as a complement to the positional cloning strategies for isolating genes involved in LF intoxication. Specifically, differential display PCR or subtractive hybridization techniques will be used to search for genes whose transcription is altered upon LF intoxication. In addition, screens for LF interacting proteins utilizing 2-hybrid methods and co-immunoprecipitation will be undertaken. DIETRICH WF HARVARD MEDICAL SCHOOL, 200 LONGWOOD AVE, BOSTON, MA 02115 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02115 Crisp Data Base National Institutes Of Health laboratory mouse Bacillus bacterial disease genetic strain species difference polymerase chain reaction pathologic process genetic mapping molecular cloning gene expression lethal gene bacterial genetics immunoprecipitation bacterial toxin exotoxin in situ hybridization nucleic acid probe toxicant interaction receptor sensitivity tissue /cell culture northern blotting subtraction hybridization yeast artificial chromosome single strand conformation polymorphism yeast two hybrid system CRISP RPROJ MASSACHUSETTS G CRISP/99/AI43321-01 CRISP/99/AI43321-01 199904 eng GRANULYSIN, A NOVEL CYTOLYTIC MOLECULE Research RPROJ DESCRIPTION (Adapted from the Investigator's Abstract): Granulysin is a newly discovered cytolytic molecule co-expressed with granzymes and perforin in granules of CTL and NK cells. This molecule is expressed constitutively in NK cells but is expressed 3-5 days after activation of T cells. It is released from cytolytic granule upon stimulation via the TCR. Recombinant granulysin lyses tumor targets and bacteria, but does not lyse RBC. The purpose of this proposal is to better define the mechanism of action of granulysin, its target specificity, and expression in disease. The specific aims are as follows: (1) Characterize the mechanism of action of granulysin, including the lytic activity of truncated or mutated forms of granulysin, formation of pores in membranes, association with lipids, ability to induce apoptosis, localization in target cells, and synergy with other molecules. (2) Better define the clinical relevance of granulysin by: (a) evaluating the specificity of lysis on panels of virally infected, microbial, and tumor target cells; and (b) evaluating expression of granulysin in disease sites, focusing on infection and cancer. (3) Generate a granulysin knock-out (KO) mouse in order to confirm its functional importance in vivo and to better characterize its mechanism of action, target specificity, and role in disease. Together these studies should provide a greater understanding of granulysin function and may permit application of this information to the design of new therapies of immune-mediate diseases. KRENSKY AM STANFORD UNIVERSITY, 300 PASTEUR DR, STANFORD, CA 94305-5208 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal Mycobacterium secretion natural killer cell cytotoxic T lymphocyte granule cytolysis microorganism immunology neoplasm /cancer immunology lymphoma electron microscopy toxin programmed cell death gene targeting protein localization CRISP RPROJ CALIFORNIA G CRISP/99/AI43348-01 CRISP/99/AI43348-01 199904 eng 1998 GORDON CONFERENCE-MICROBIAL TOXINS & PATHOGENESIS Research RPROJ DESCRIPTION: (adapted from application abstract) Since its genesis in 1972, the Gordon Conference on Microbial Toxins and Pathogenesis has been a primary source of information gathering and exchange for scientists in the field of microbial pathogenesis. The phenomenal advances over the last decade in understanding the mode of action of known toxins at the molecular level, the identification of common and novel virulence determinants in a variety of bacterial pathogens, and the application of new molecular, cellular, and immunological tools to address basic questions of how microbes cause disease have moved this field into the forefront of modern biology. The 1998 Conference will include 8 plenary sessions on the following topics: 1) In vivo apoptosis; 2) technology; 3) regulation of gene expression; 4) microbe-host signaling; 5) new toxins/mechanisms; 6) genomics; 7) Structural biology of toxins/virulence factors; 8) secretion of toxins/virulence factors. An additional 9th session is planned but the topic and speakers have not been decided at the time of this grant submission. This ninth session will provide the flexibility to schedule the very latest advances for the meeting so as not to "lock into" a scientific program that is out-of-date by the time the meeting actually occurs. Speakers for these sessions have been chosen to provide a mixture of senior investigators and junior investigators who may not have previously presented at a Gordon Conference. Poster sessions will supplement the plenary sessions and there will be ample opportunity for informal discussion among participants. HEWLETT EL UNIVERSITY OF VIRGINIA, BOX 419, SCHOOL OF MEDICINE, CHARLOTTESVILLE, VA 22908 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 22908 Crisp Data Base National Institutes Of Health communicable disease pathologic process microorganism genetics microorganism growth microorganism toxin meeting /conference /symposium travel CRISP RPROJ RHODE ISLAND G CRISP/99/AI43386-01 CRISP/99/AI43386-01 199904 eng MUCOSAL IMMUNITY TO CHOLERA-VECTORED EHEC ANTIGENS Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): The principal investigator proposes to use Vibrio cholerae, the causative agent of cholera, as a live oral attenuated vaccine vector to deliver immunogenic antigens of enterohemmorrhagic Escherichia coli (EHEC) to the intestine to stimulate a common mucosal immune response. EHEC, a bacterium that causes hemorrhagic colitis and hemolytic uremic syndrome, is an important pathogen of humans which, like V, cholerae, acts by adhering to the mucosal surface of the intestine and producing toxin. The investigators hypothesize that oral EHEC may lead to protective immunity against EHEC infection in the gastrointestinal tract. Examination of the immune response to infection with vector strains may aid in elucidating the mechanisms of protective immunity to EHEC and may further the understanding of protective immunity at the mucosal surface. The cloned genes encoding EaeA and EspB from RDEC-1 and StxB1 from EHEC will be placed within the E. coli hemolysis secretion system for export from Vibrio cholerae vaccine strains. The genes will be amplified and cloned into an internal deletion of hlyA such that the gene is fused in frame to the carboxy-terminus of the E. coli hemolysin gene. The resulting constructs will be confirmed by restriction digests and sequence analysis, and placed into V. cholerae strain 0395-NT by electroporation. Localization of EaeA, EspB, and StxB1 in cellular compartments will be performed by ELISA or Western blot analysis using polyclonal anti-EaeA, EspB, and StxB1 antibodies. A rabbit model of Vibrio cholerae colonization will be used to examine the immune response to EaeA, EspB, and StxB1 expressed by the vector strains. Serum and mucosal secretions will be collected from rabbits that have been orally inoculated with the vector strains, and examined for the presence of specific antibody produced in response to immunization. The RDEC-H19A oral challenge model will be used to evaluate protection from the disease induced by immunization with the vaccine strains. Immunized rabbits that develop an immune response to EaeA, EspB, or StxB1 expressed by the vector strains will be orally challenge with the rabbit pathogen RDEC-H19A, and the induction of protective immunity will be evaluated by examining the animals for clinical disease and for histological lesions in the intestine. The proposal is divided into three specific aims: 1) Construct fusions of eaeA, espB, and stxB1 to the E. coli hemolysin secretion signal; and analyze the expression and localization of the hybrid productions. 2) Use the rabbit model to examine the immune responses to EaeA, EspB, and StxB1 expressed by the vector strains. 3) Use the RDEC-H19A rabbit challenge model to assess protection conferred by the immunization. BUTTERTON JR MASSACHUSETTS GENERAL HOSPITAL, 55 FRUIT STREET, BOSTON, MA 02114 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 02114 Crisp Data Base National Institutes Of Health laboratory rabbit Escherichia coli Vibrio cholerae Escherichia coli infection hemolysin molecular cloning fusion gene gene expression bacterial genetics histology bacterial antigen attenuated microorganism bacterial vaccine live vaccine oral administration mucosal immunity vaccine development vector vaccine CRISP RPROJ MASSACHUSETTS G CRISP/99/AI43547-01 CRISP/99/AI43547-01 199904 eng DNA SEQUENCE OF STAPHYLOCOCCUS AUREUS 8325 GENOME Research RPROJ In 1995, Fleischman et al. (40) determined the nucleotide sequence of Haemophilus influenzae Rd without prior restriction site mapping and cosmid cloning. This was a landmark event, signaling that automated DNA sequencing and analysis have matured into robust, highly efficient technologies promising to revolutionize the biological sciences. An important breakthrough that will greatly facilitate closure and proof- reading of the sequence which was reported by Dr. Mike Hunkapiller from ABI and Dr. Roe at the recent September Hilton Head DNA Sequencing conference was the results of experiments in which we successfully obtained sequence data in excess of 450 bases directly from several bacterial genomes. The genomic sequence of several other procaryotes have or will be completed shortly, including our own genomic sequencing of the important human pathogens Neisseria gonorrhoeae and Streptococcus pyogenes. Collectively, these studies will considerably enhance our understanding of procaryotic molecular biological processes, and provide new avenues for gene discovery and comparative genetics. From a practical standpoint, procaryotic genome sequencing will enhance our ability to understand processes occurring during the pathogenesis of infectious disease. These studies should provide new approaches for drug discovery, a necessity of increasing importance as microbial antibiotic resistance threatens our ability to treat bacterial infections. In this application, we propose to determine the nucleotide sequence of the genome of Staphylococcus aureus. This organism is a potent pathogen widely found in the human and animal environment. It is capable of producing upwards of 34 different extracellular proteins most of which have been shown to play a role in the pathogenicity of the organism or to enhance virulence. Although the incidence of infection in the general population is not well documented, it is generally accepted that S. aureus accounts for up to one-third of all nosocomial bacteremia. Other than the details of abscess formation, very little is actually of the pathogenesis of staphylococcal disease. Understanding the circuits (agr, sar and probably others) which control the expression of virulence related genes is paramount to understanding the genetic response of the organism to host generated signals. We propose that acquisition of the sequence of the genome of the Staphylococcus aureus strain 8325 will greatly facilitate understanding the mechanism of disease produced by this organism and closely related species. Therefore, as a single specific aim, we will sequence and annotate the 2.8 Mb genome of this important pathogen. IANDOLO JJ OKLAHOMA UNIV HLTH SCIS CTR, PO BOX 26901, OKLAMHOMA CITY, OK 73190 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 73190 Crisp Data Base National Institutes Of Health Staphylococcus aureus Staphylococcus infection genetic strain fluorescent dye /probe host organism interaction genetic mapping genome bacterial genetics staphylococcal exotoxin virulence computer assisted sequence analysis bacterial DNA nucleic acid sequence SDS polyacrylamide gel electrophoresis bacteria infection mechanism CRISP RPROJ OKLAHOMA G CRISP/99/AI43568-01 CRISP/99/AI43568-01 199904 eng IMMUNOTHERAPY FOR LATEX RUBBER ALLERGY Research RPROJ DESCRIPTION (Adapted from Investigator's abstract): Allergic sensitivity to plant proteins in the sap of the rubber tree has become a major occupational hazard of health care workers and some patients with frequent latex rubber exposures. Between 2.2 and 10.7% of health care workers and 1-2% of laboratory personnel are at risk for systemic reactions to rubber products, mainly latex gloves. Systemic latex reactions are predominantly of inhalant origin because cornstarch particles used to lubricate surgical gloves serve as a respirable carrier for the protein allergens. Since complete avoidance is difficult in most medical environments, latex allergy can necessitate involuntary career changes with substantial economic and emotional consequences. Using a latex allergen extract now being standardized for diagnostic purposes, this project proposes to develop further a natural exposure model of respiratory latex allergy and to use this model in clinical studies to demonstrate the safety and efficacy of immunotherapy for IgE-dependent latex allergy. The specific aims are: (1) to validate a prototype hooded environmental chamber technique as a provocational challenge model of respiratory allergy to natural rubber latex; (2) to conduct a Phase 1 clinical trial of immunotherapy with a candidate extract of non-ammoniated latex; and (3) to determine in a randomized, placebo-control clinical trial the safety and clinical efficacy of immunotherapy with a candidate vaccine in patients with disabling occupational allergy to latex proteins. Subjects will be health professionals with clinically significant respiratory or anaphylactic reactions to latex and documented failure of attempts at avoidance. The primary endpoint will be sensitivity to latex as determined by the hooded chamber graded exposure technique. Secondary endpoints for the clinical trial will include immunologic changes (IgG and IgE fluxes), skin test sensitivity, adverse reaction rates, and where possible assessments of work-related symptoms. The long-range objective of this research plan is to develop a useful therapeutic alternative for latex-sensitive health care workers and selected patients who have failed avoidance. Potential benefits include a reduction in professional disability, prevention of chronic disease, expanded occupational choices, and increased work productivity for those affected. ADKINSON NF JOHNS HOPKINS ASTHMA & ALL CTR, 5501 HOPKINS BAYVIEW CIRCLE, BALTIMORE, MD 21224 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 21224 Crisp Data Base National Institutes Of Health controlled environment chamber immunoglobulin E immunoglobulin G health care personnel human subject hypersensitivity airborne allergen anaphylaxis respiratory hypersensitivity hypersensitivity desensitization immunotherapy immunologic skin test occupational hazard rubber human therapy evaluation clinical trial phase I clinical research CRISP RPROJ MARYLAND G CRISP/99/AI43654-01 CRISP/99/AI43654-01 199904 eng IN VITRO DIAGNOSIS AND DETECTION OF BOTULINUM NEUROTOXIN Research RPROJ Botulism, a neuroparalytic disease caused by toxins from C. botulinum bacteria, affects more than half a million people worldwide per year. The goal of this project is to develop a rapid, sensitive and specific in vitro assay test for the diagnosis and detection of botulinum neurotoxins that can be used for effective monitoring of the food supply and diagnosis of infected patients. This assay test is meant to replace the currently used "mouse lethality test". The botulinum and tetanus toxins have recently been identified as zinc metalloproteases which cleave specific proteins in the neuron. Thus, we plan to exploit the proteolytic activity of these toxins as a method of toxin detection. Specific synthetic fluorogenic and/or chromogenic substrates for these toxins will be developed for use in a commercially viable, in vitro detection kit. In Phase I, four steps will be taken toward this goal : (l) reference substrates will be developed (2) suitable reporter groups will be developed for incorporation into the substrates (3) the ability to inhibit the proteolytic activity of toxin in vitro with specific antisera will be demonstrated, and (4) potential chemistries for the immobilization of substrates on various surfaces will be examined. PROPOSED COMMERCIAL APPLICATIONS: In vitro detection and diagnosis of botulinum toxin has widespread application in food safety and medical diagnosis. Food manufacturers worldwide must monitor all products for the presence of C. botulinum based on the neurotoxin it elaborates. The "mouse lethality test" in current use is slow, costly, and socially undesirable. Thus manufacturers are looking for an alternative, in vitro assay for botulinum toxin. For these same reasons, medical diagnosticians are also seeking alternative detection methods. WOLZ RL COMMONWEALTH BIOTECHNOLOGIES, 911 EAST LEIGH ST, SUITE G-19, RICHMOND, VA 23219 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 23219 Crisp Data Base National Institutes Of Health diagnosis quality /standard diagnosis design /evaluation rapid diagnosis food contamination immunologic assay /test botulinum toxin neurotoxin synthetic peptide toxicant interaction peptidase protein sequence toxicant screening enzyme activity method development CRISP RPROJ VIRGINIA G CRISP/99/AI43713-01 CRISP/99/AI43713-01 199904 eng AN RRE-LINKED MUCOSAL BARRIER TO LENTIVIRUS INFECTION Research RPROJ Feline immunodeficiency virus, a lentivirus of cats, provides a natural model for human immunodeficiency virus (HIV) infection and disease. This proposal is designed to further develop this model by exploring the feasibility of a gene therapy approach for preventing vaginal transmission of lentivirus infections. If successful in the FIV model, this approach will be applicable to the prevention of vaginally transmitted HIV infections in women. The FIV model is used to test the hypothesis that lentivirus infections can be prevented at the mucosal site of entry by introduction into cells of the vaginal mucosa of lentiviral Rev-responsive element (RRE) linked to a toxin (suicide) gene. Infection of cells harboring this RRE/suicide gene construct will result in expression of the toxin and death of the infected cells. The interaction of the Rev protein with the RRE is a critical step in the replication cycle of both FIV and HIV. This interaction can be exploited by linking a suicide gene (diphtheria toxin A) to the FIV-RRE in an expression plasmid. The gene will not be expressed unless the FIV Rev protein is present. In theory, once a cell containing the construct is infected with FIV, the plasmid's RRE will interact with the viral Rev, allowing expression of the toxin and resulting in death of the cell before virus can be disseminated beyond the mucosal surface. The objectives of the work detailed in this proposal are: 1) to employ and enhance the FIV model for vaginal transmission of HIV and mucosal defenses against HIV, and 2) to determine if an RRE/toxin construct can be used effectively to terminate lentivirus infection, in vitro and in vivo. These objectives will be achieved by concentrating on the following three specific aims: 1) To determine the effects on tissue- culture cells of an FIV-RRE/diphtheria toxin A expression plasmid, in the presence or absence of infectious FIV, by transfection of the FIV- RRE/toxin plasmid into cultured cells, infection of transfected cells with FIV, and determination of cell viability and virus production; 2) To introduce the FIV-RRE/toxin construct into the cells of the vaginal mucosa of cats and to identify the cells that are transfected, by exposure of the vaginal mucosa of female cats to endotoxin free plasmid DNA and cytologic examination of mucosal cells for the presence of plasmid DNA; and 3) To determine the ability of the FIV-RRE/toxin construct to protect against vaginal transmission of FIV, by exposure of the vaginal mucosa of female cats to the FIV-RRE/toxin plasmid DNA, vaginal introduction of infectious FIV, and examination of cats for evidence of FIV infection. BARR MC SCRIPPS RESEARCH INSTITUTE, 10550 NORTH TORREY PINES ROAD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 92037 Crisp Data Base National Institutes Of Health cat communicable disease transmission gene therapy transfection fusion gene diphtheria toxin HIV infection disease model virus RNA vagina female tissue /cell culture human immunodeficiency virus feline immunodeficiency virus mucosal immunity CRISP RPROJ CALIFORNIA G CRISP/99/AI43736-01 CRISP/99/AI43736-01 199904 eng IDENTIFICATION OF LPS ANTAGONISTS USING IN VITRO ASSAY Research RPROJ The use of LPS antagonists to reduce the pathophysiological effects of LPS released during gram negative sepsis is a promising approach to the treatment of septic shock for which current therapies have very limited clinical impact. In order to identify novel LPS antagonists we are developing a rapid microtiter plate-based assay for use in the primary screening of such compounds. This assay relies on the use of the human inducible nitric oxide synthase promoter (iNOS) to drive the expression of the reporter green fluorescent protein (GFP) in a monocytic cell line to detect LPS antagonist. This iNOS/GFP reporter construct will produce GFP fluorescence in response to LPS, which induces macrophage activation. These attributes of the assay will facilitate the identification of LPS antagonists with desirable therapeutic properties. PROPOSED COMMERCIAL APPLICATIONS: In collaboration with pharmaceutical companies we intend to use this assay as high-throughput screen on libraries of compounds for novel LPS antagonists. In addition to natural products which have been a traditional source of LPS antagonists, synthetic compound, combinatorial oligonucleotide or peptide, and phage display libraries will be screened with this assay. SHOREIBAH MG GLYCOBIOTICS INC, 1180 E BROAD ST, SUITE 2209, ATHENS, GA 30601 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 30601 Crisp Data Base National Institutes Of Health laboratory mouse bacterial disease bioassay blood disorder chemotherapy blood toxicology antibacterial agent inhibitor /antagonist drug screening /evaluation lipopolysaccharide method development septic shock green fluorescent protein CRISP RPROJ GEORGIA G CRISP/99/AI43767-01 CRISP/99/AI43767-01 199904 eng MICROBIAL DECONTAMINATION OF FOOD USING AQUEOUS OZONE Research RPROJ The extent of microbial contamination of food and development of methods to control foodborne pathogens have been the targets of increasing public concern and regulatory control. The proposed work will evaluate the feasibility of using aqueous ozone, produced using a novel, high concentration electrochemical method, for the reduction of pathogenic bacteria from food surfaces using fresh beef surface tissue as a model. Significant amounts of Escherichia Coli 0157:H7 and Salmonella typhimurium remain on beef carcasses after processing and can contaminate ground beef or processed beef products. Ozone has been demonstrated to be an effective reagent to reduce numbers of microbes that contaminate food surfaces. This disinfectant is a powerful oxidizing agent which has recently been given GRAS status for use in food processing and because it readily decomposes into oxygen, there are no chemical residues that could contaminate the food product or the environment. In phase I, the optimal delivery of ozone to meat surfaces inoculated with E. Coli and Salmonella will be developed and evaluated microbiologically. Phase II will involve construction of an ozone rinse cabinet, on-site evaluation of diverse foods in a processing facility and incorporation into the existing HACCP program at that site. PROPOSED COMMERCIAL APPLICATIONS: It is envisaged that the electrochemically-generated ozone food rinse cabinet will be designed to be utilized at food processing facilities without significant modifications of existing equipment. This environmentally friendly rinse system will l) reduce risk associated with consumption of food susceptible to pathogenic surface contamination, 2) be automated, easily operated and inexpensively incorporated into conventional facilities and 3) allow for control of existing, unidentified or emerging pathogens (i.e. B. Coli 0l57:H7, Salmonella, Listeria, Camphylobacter, antibiotic-resistant bacteria, Cryptosporidium, Hepatitis A virus). ROGERS TD LYNNTECH, INC, 7610 EASTMARK DRIVE, SUITE 105, COLLEGE STATION, TX 77840 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 77840 Crisp Data Base National Institutes Of Health Escherichia coli Salmonella typhimurium bioengineering /biomedical engineering biomedical equipment development biomedical automation electrochemistry meat food quality /standard food sanitation food contamination microorganism culture ozone water solution method development CRISP RPROJ TEXAS G CRISP/99/AI43784-01 CRISP/99/AI43784-01 199904 eng ULTRASENSITIVE METHOD FOR FOOD QUALITY TESTING Research RPROJ DESCRIPTION: We propose to develop an integrated diagnostics system that provides ultrasensitive, low cost and very rapid food quality diagnostics. This new methodology will be implemented initially as tests for contamination by pathogenic strains of E. coli. We will apply Multi Photon Detection (MPD) technology to food diagnostics to accomplish the following: A fast and very sensitive MPD enhanced quantitative PCR; An ultra fast and very sensitive MPD enhanced direct detection (i.e., non- amplification based) DNA assay for food contaminants; and Validation of this new methodology by comparison of MPD enhanced assays with conventional microbiological diagnostic methods. The main goal is to develop methodologies that translate the superior sensitivity of MPD into speed and reliability of food tests. These new methods are generic and can be used for all important food contaminants. Ultra-sensitive MPD already has been used to quantitate sub-zeptomole amounts (less than 10-21 M) of biological macromolecules such as DNA, RNA and proteins. Our prototype MPD Imager has enabled the development of super-sensitive tests using both immuno techniques and DNA probes. As an example, we developed a rapid diagnostic assay that permits reliable quantitation of a few copies of DNA in a few milliliters of sample. PROPOSED COMMERCIAL APPLICATION: If successful, this project will lead to the development of a good quality diagnostics system which provides rapid, high sensitivity and economical testing for major food borne pathogens, This system will be designed to operate in a food processing plant environment and will offer the ability to detect as little as a single viable pathogenic organism in a food sample in less that six hours. This unique capability will enable the BioTraces food pathogen system to capture a significant share of the market for in-plant testing for food borne pathogens, which now totals over 25 million tests per year with double digit growth rates. | DRUKIER AK BIOTRACES INC, 10517-A WEST DRIVE, FAIRFAX, VA 22030 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 22030 Crisp Data Base National Institutes Of Health Escherichia coli Salmonella typhimurium bioassay polymerase chain reaction diagnosis design /evaluation rapid diagnosis meat food quality /standard food contamination microorganism classification microbiology method development CRISP RPROJ VIRGINIA G CRISP/99/AI43785-01 CRISP/99/AI43785-01 199904 eng CONTROL OF FOOD PATHOGENS Research RPROJ Numerous investigations have shown that many poultry products at point of sale contain high levels of virulent pathogens such as Salmonella, Campylobacter, E. Coli, and Listeria. Using technology developed to reduce infection levels in medical patients, we have developed a packaging system, using a polymer, that drastically reduces free fluid and pathogen levels in processed poultry. A prime advantage of our approach is that it introduces no vector to which pathogens can evolve into a more virulent form. In our method, pathogens are encapsulated, deprived of any nutrient, and starved, leaving no potentially toxic substance for human consumption. Preliminary experimental data are presented. Two factorial experiments are proposed, one on whole body fryers and another on chicken parts. The chickens to be tested will be purchased locally and come from a large commercial poultry processor. Comparisons will be made between a control group of commercially processed chickens and two groups of chickens with our packaging, differing in applications of the technology. A similar experiment will be done on chicken parts. We expect to demonstrate drastic reductions in (1) levels of bacteria, both virulent and non-virulent and (2) elimination of free fluids within our packaging.PROPOSED COMMERCIAL APPLICATION: Applications exist for this packaging in almost all commercially produced foodstuffs--poultry, fish, meat, fruits and vegetables. Technology already exists to develop commercially viable forms of the material for packaging. The approach insures that pathogens cannot evolve or develop a tolerance for the material. Also, the material is effective at the point of sale to the consumer, minimizing or negating other, more expensive pathogen control measures suggested for processing plants. EASTMAN D EASTMAN MEDICAL PRODUCTS INC, 2000 POWELL ST, SUITE 1540, EMERYVILLE, CA 94608 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 94608 Crisp Data Base National Institutes Of Health chicken bacteria Listeria Escherichia coli Salmonella packaging material meat poultry product food quality /standard food sanitation food contamination polymer Helicobacter method development CRISP RPROJ CALIFORNIA G CRISP/99/AI43795-01 CRISP/99/AI43795-01 199904 eng METHODS FOR RAPID DETECTION OF MULTIPLEX PCR PRODUCTS Research RPROJ In the United States, 24-81 million people are infected with foodborne bacteria and 9,000 people die each year from such infections. Outbreaks of E. coli 0157:H7 have brought attention to the problem of contaminated foods, resulting in several new regulations. To ensure safe food supplies, rapid, new methods of detecting foodborne pathogenic bacteria are needed. PCR amplification of nucleic acids has been used to detect numerous bacterial specifies and is rapid and extremely sensitive. Modifications of PCR exist which permit simultaneous detection of several bacterial species. Using one such modification, multiplex PCR, nucleic acids from several bacterial species (e.g., Salmonella enteritidis, S. typhimurium, E. coli) can be amplified in a single reaction. One problem with multiplex PCR is the lack of rapid methods for detecting the various DNA products. This proposal aims to demonstrate the feasibility of a biosensor for rapid, one-step identification of all products produced by multiplex PCR. The process is amenable to full automation. This proprietary biosensor will open the door to rapid, simultaneous detection of multiple bacterial species. PROPOSED COMMERCIAL APPLICATION: PCR technology has wide spread applications in clinical, environmental, agrifood and other industrial areas. Multiplex PCR technologies permit simultaneous amplification of multiple nucleic acids and would be commercially valuable if rapid methods for identifying the multiple products existed. RUDOLPH DB SYMBIOTECH, INC, 8 FAIRFIELD BLVD, WALLINGFORD, CT 06492 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 06492 Crisp Data Base National Institutes Of Health Escherichia coli Salmonella typhimurium Salmonella infection biomedical automation biosensor polymerase chain reaction diagnosis quality /standard rapid diagnosis bacterial food poisoning microorganism culture microorganism classification bacterial DNA method development CRISP RPROJ CONNECTICUT G CRISP/99/AI43800-01 CRISP/99/AI43800-01 199904 eng BIOSENSOR DETECTION OF WATER BORNE CRYPTOSPORIDIUM Research RPROJ The proposed Phase I study will establish the feasibility of using an innovative, immunoassay-based biosensor for the detection and quatitation of Cryptosporidium oocysts in drinking water. Current method for detection and quantitation of this pathogen frequently are characterized as technically, demanding, time-consuming and labor intense, leading to poor recoveries and false positives and negatives. The propose study will determine the feasibility of using an immunoassay-based biosensor to quantitate the level of Cryptosporidium oocysts present in source and finished waters. The effort will focus on comparing the performance of the biosensor with intact versus extracted oocysts. The goal in Phase I will be to maximize the sensitivity of the assay to detect oocysts. The immunoassay itself should take less than 15 minutes to complete. The availability of more rapid, quantitative methods for detecting water-borne pathogens will help to identify and manage the presence of these organisms for community water supplies, thereby minimizing the exposure of the public. Proposed commercial applications: The proposed biosensor will help address the current deficiency in technology to monitor water-borne pathogens in drinking water. The availability of a rapid, low-cost and quantitative means to test for organisms like Cryptosporidium should help in the effort to minimize exposure for the public to contaminated water. SAND TT DISAN, INC, PO BOX 500948, SAN DIEGO, CA 92150-0948 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health bioengineering /biomedical engineering biosensor communicable disease control diagnosis design /evaluation rapid diagnosis public health immunologic assay /test microorganism classification cryptosporidiosis Cryptosporidium water pollution water quality water sampling /testing CRISP RPROJ CALIFORNIA G CRISP/99/AI43806-01 CRISP/99/AI43806-01 199904 eng POLYMERS FOR AUGMENTING R BOTULINUM VACCINE EFFICACY Research RPROJ DESCRIPTION (Adapted from the applicant's abstract): Botulism is a neuromuscular poisoning caused by clostridial neurotoxins. Humans are exposed to neurotoxins produced by Clostridial botulinum through food poisoning. Incidences of wound botulism and a colonizing infection of neonates known as infant botulism are rare. Although the infective microorganism can be treated by antibiotics, there exists no specific drug therapy for botulinum toxins. Toxoids, as in formalin-inactivated vaccines, can be prepared for active immunization of individuals who are likely to be exposed to C. Botulinum or its toxins. However, problems associated with such immunization, including the need for more than a single dose of expensive, multivalent toxoids, and concerns about reversion of the toxoids to toxins created a need for single-dose immunization with a safe vaccine. Recent availability of recombinant fragment C botulinum (r-FCBt) vaccine and absorbable gel-forming carriers justified pursuing the proposed program. The objective of Phase I is to determine the feasibility of using absorbable gel-forming and micro-spherical polyester carriers in developing a r-FCBt vaccine for intranasal and subcutaneous immunization. Phase I entails (1) preparing and characterizing the polymeric carriers; (2) formulating and evaluating, in mice, candidate controlled release systems using Type E and F r-FCBt vaccines; (3) determining the antibody response; and (4) conducting challenge studies with individual homologous serotypes. Phase I results will be used to design Phase II plans that include (1) studying immunization and protection of all r-FCBt vaccines (A, B, C, D, E, F, and G) against highest dose of homologous and possibly heterologous toxin serotypes; (2) developing, scaling-up, and evaluating a multivalent r-FCBt vaccine; and (3) conducting a brief safety study on the multivalent vaccine. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE SHALABY SW POLY-MED INC, 6309 HIGHWAY 187, ANDERSON, SC 29625 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 29625 Crisp Data Base National Institutes Of Health laboratory mouse Clostridium molecular cloning microorganism culture botulinum toxin microorganism immunology polymer nonhuman therapy evaluation bacterial vaccine recombinant protein vaccine development CRISP RPROJ SOUTH CAROLINA G CRISP/99/AI43816-01 CRISP/99/AI43816-01 199904 eng STRUCTURE OF AN ION MOTIVE ATPASE Research RPROJ DESCRIPTION: (Adapted from applicant's Abstract) The arsenical resistance (ars operon of the Escherichia coli plasmid R773 encodes a system for the active extrusion from cells of the toxic oxyanions arsenite (As(III)O21-) and antimonite (Sb(III)O21-) via an ATP-driven pump. The arsA and the arsB genes o the operon encode, respectively, the catalytic subunit (ATPase) and the membrane subunit of the pump. The arsC gene codes for a reductase that convert arsenate (As(V)O43) to arsenite and appears to channel it into the ArsA-ArsB pump, thus extending bacterial resistance also to the pentavalent state of arsenic. Crystals diffracting at high resolution (2.0 Angstrom) were obtained for both the catalytic subunit of the pump (ArsA) and for the reductase (ArsC) and native data sets have been collected. Structural studies are proposed to identify ArsA and ArsC binding sites for substrates and/or for allosteric effectors, and the regions of interaction with other proteins (e.g., ArsA with ArsB, ArsC and ArsA). Since expression of the arsB gene is highly toxic in E. coli, production of the protein in other hosts (Archaebacteria, yeast) will be pursued to obtain a large amount of pure protein for crystallization. Arsenical resistance is a useful model for the study of multiple drug resistance in both eukaryotic and prokaryotic cells. The ArsA-ArsB pump exhibits structural and functional similarity to the P-glycoprotein: both are efflux pumps for toxic compounds, have two nucleotide binding sites, are substrate-dependent ATPases, have 12 membrane spanning alpha-helices, and are each able to detoxify structurally distinct drugs. The latter point is illustrated for the ArsA-ArsB pump by the fact that while arsenate and arsenit are both oxyanions of arsenic, they are chemically dissimilar. The ArsA protein is also the only other ion-motive ATPase, besides the mitochondrial F1 ATPase, for which three-dimensional crystals have been obtained. Analysis of the similarities and differences between these two enzymes will further our understanding of how ions are transported across biological membranes. GATTI DL WAYNE STATE UNIVERSITY, 540 E CONFIELD AVE, DETROIT, MI 48201 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 48201 Crisp Data Base National Institutes Of Health Escherichia coli X ray crystallography drug resistance enzyme model yeast plasmid operon arsenic multidrug resistance oxidoreductase adenosinetriphosphatase crystallization protein structure /function membrane transport protein detoxification active site enzyme activity CRISP RPROJ MICHIGAN G CRISP/99/AI43918-01 CRISP/99/AI43918-01 199904 eng PULSE THERAPY IN SYSTEMIC JUVENILE RHEUMATOID ARTHRITIS Research RPROJ DESCRIPTION (adapted from applicant's abstract): Systemic juvenile rheumatoid arthritis (sJRA) is associated with significant long term morbidity and mortality. Current therapies including methotrexate are considered ameliorative rather than remission inducing or curative. There have been anecdotal reports suggesting that pulse therapy with intravenous corticosteroids, cyclophosphamide, and methotrexate may induce prolonged remissions in sJRA. Therefore, the objectives of this study are to determine and compare the ability of two pulse therapy regimens to induce remission in sJRA of less than 12 months duration during a 9-month, open-label, randomized, actively controlled clinical trial. The first pulse therapy regimen is composed of intravenous methylprednisalone 30 mg per kg per day (1 gram max) for three consecutive days, intravenous cyclophosphamide 0.4 grams per meter2 BSA on the third day, and up to 20 mg per meter2 per week of methotrexate. The second pulse therapy regimen is identical to the first, except no cyclophosphamide is given. Up to five cycles of these regimens may be given over a 9-month period. Patients in both groups may also receive background medications including a non-steroidal, anti-inflammatory drug and up to 0.5 mg/kg/d of oral prednizone. The primary outcome to measure the effect of this therapy will be the proportion of patients who achieve clinical remission according to the ACR criteria for remission in rheumatoid arthritis. Secondary outcome measures of effectiveness include proportion of patients who demonstrate clinical response according to the preliminary definition of improvement for JRA. In addition, time per remission and the duration of remission will be compared between the two groups among patients who do remit. Specific Aim 2 is to determine and compare the short and intermediate term (18 months) safety profiles of the pulse therapy regimens as defined in Specific Aim 1. Long term goals of the project are to determine and compare the longer-term safety profiles of this treatment regimen. This will involve analysis of patient/parent-derived data obtained by mail or phone follow-up to detect significant medical problems and reproductive or neoplastic complications. LOVELL DJ CHILDREN'S HOSPITAL MEDICAL CT, 3333 BURNET AVENUE, CINCINNATI, OH 45229-3039 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health methylprednisolone prednisone child (0-11) adolescence (12-18) clinical trial remission /regression combination chemotherapy pharmacokinetics drug adverse effect methotrexate cyclophosphamide human subject juvenile rheumatoid arthritis skeletal disorder chemotherapy human therapy evaluation clinical research nonsteroidal antiinflammatory agent CRISP RPROJ OHIO G CRISP/99/AI44046-01 CRISP/99/AI44046-01 199904 eng REGULATION AND IMMUNOTHERAPY OF IDDM Research RPROJ DESCRIPTION (adapted from applicant's abstract): The current proposal is a logical extension of previous research using the RIP-LCMV model for virus-induced autoimmune diabetes to analyze pathogenic mechanisms and define novel immuno-therapeutic approaches for IDDM. The most promising areas were identified and form the specific aims of this application: The first goal is to precisely define which effect cytokines produced in the islets have at different times during the development of diabetes. Recent reports have described controversial effects for TNF-(alpha), IL-10, TGF-(beta) and others. In the majority of these transgenic models it has been impossible to control, when and at what level the cytokine is produced. The applicant proposes to use novel transgenic mice that express cytokines under a tetracycline sensitive regulatory promoter and cross these with the RIP-LCMV model. Further, the role of stat-4 (IL-12) and stat-6 (IL-4) pathways that represent two key cytokines implicated in the pathogenesis of IDDM will be analyzed by mating stat-4 or -6 deficient mice to RIP-LCMV transgenics. The second aim is to test and improve immunotherapies for autoimmune diabetes. Promising preliminary results were obtained by treating RIP-LCMV mice with antibodies to B7.1, B7.2 or CD40L systemically, which abrogated development of IDDM without affecting primary systemic activation of autoreactive (LCMV-specific) lymphocytes. Further, treatment with anti-CD3Fa/b(2) will be attempted, because regulatory cells can be induced preferentially by anti-CD3. Lastly, it will be attempted to increase the efficacy of "oral tolerance" by administration of insulin coupled to cholera toxin B subunit. Preliminary studies look encouraging indicating more than 10-fold lower antigenic requirements. Since the oral effect of the insulin maps to the B-chain and the likely mechanism is induction of regulatory Lymphocytes that act as "bystander suppressors". Preliminary studies also indicate that the systemic application of plasmids expressing the insulin B-chain should have a therapeutic effect, which is reflected by the preliminary data. It is felt that RlP-LCMV model is an ideal tool for addressing these issues, because the initialing self-antigen is known, the time-point for triggering IDDM can be experimentally chosen and the kinetics of islet infiltration/destruction have been precisely mapped. This allows a segregation of autoreactive lymphocytes from bystander regulatory cells and precise tracking of self (LCMV) reactive T-lymphocytes. VON HERRATH MG SCRIPPS RESEARCH INSTITUTE, 10550 N TORREY PINES RD, LA JOLLA, CA 92037 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 92037 Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal biological signal transduction T lymphocyte diabetes mellitus therapy insulin dependent diabetes mellitus transforming growth factor cytokine interleukin 4 tumor necrosis factor alpha immunotherapy antibody antigen presentation cholera toxin insulin pancreatic islet protein biosynthesis tissue /cell culture oral tolerance interleukin 10 interleukin 12 CRISP RPROJ CALIFORNIA G CRISP/99/AI44451-01 CRISP/99/AI44451-01 199904 eng REGULATORY ROLE OF VIRUS SPECIFIC CD8 T LYMPHOCYTES Research RPROJ Although several risk factors for asthma have been defined the etiology for childhood asthma remains poorly understood. In particular, the role of viral respiratory tract infection in the development of this disease remains controversial. Infection with respiratory viruses, such as Respiratory Syncytial Virus (RSV), has been reported to predispose children to the subsequent development of asthma. In contrast, some epidemiologic studies have suggested that viral infection of the respiratory tract may have a protective effect against the development of this disease. In a major of asthmatic patients, particularly children, sensitizing and exposure to allergens is a major risk factor in the development of asthma. Recently, a critical a critical role of CD4+ T cells in the development of allergic responses has become clear. In particular, CD4+ T cells which are capable of producing IL-4 and IL-5 (Th2 T cells) are required for establishing an allergic response. Recent evidence from human and animal studies have also demonstrated the importance of this subset of CD4+ T cells in viral infections. In a murine model of RSV infection, sensitization to the RSV-G (attachment) glycoprotein results in a strong Th2 response and predisposes the animals to the development of allergic lung inflammation. Insights into the factors regulating the differentiation and activation of this subset of subset of CD4+ T cells is therefore extremely important to the understanding of the role of viral infections in the development of asthma. Recently, a study has demonstrated that the strong induction of a Th2 response by RSV-G glycoprotein is linked to the lack of memory of CD8+ T cell response specific to this antigen. This suggests that virus-specific CD8+ T cell may play an important role in the regulation of Th2 responses. The studies outlined in this proposal are designed to examine the role of virus-specific CD8+ T cells on the differentiation of CD4+ T cells of Th2 phenotype. Specifically, studies will focus on 1) the role of CD8+ T cells in regulating CD4+ T cell differentiation and the impact of this regulation on the kinetic of CD4+ T cell differentiation, 2) the molecular mechanisms employed by CD8+ T cells in this regulation, and 3) the impact of this regulation on allergic inflammation in an animal model of RSV infection. Studies will be carried out to examine the role of respiratory viral infection on allergen-specific T cell responses in human. Theses studies will lead to new insights into the role of virus-specific CD8+ T cells in the development of allergic lung diseases. SRIKIATKHACHORN A CHILDREN'S HOSP RES FDN, 3333 BURNET AVE, CINCINNATI OHIO 45229-3039 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Crisp Data Base National Institutes Of Health adult human (19+) leukopoiesis cytotoxic T lymphocyte suppressor T lymphocyte adolescence (12-18) host organism interaction human subject allergen leukocyte activation /transformation passive immunization CD8 molecule microorganism immunology rhinitis respiratory infection cytolysin virus disease rhinovirus vaccinia virus respiratory syncytial virus interferon gamma SCID mouse recombinant virus clinical research CRISP RPROJ OHIO G CRISP/99/AI45221-01 CRISP/99/AI45221-01 199904 eng MECHANISMS OF DRUG INDUCED LUPUS Research RPROJ The application proposes funding with the specific intent of developing an independent research program by the principal investigator. For the past three and a half years, the applicant has been pursuing his interest in basic science research in the areas of T cell immunology and pathogenesis of lupus. Both the clinical and basic science training have prepared the applicant for an independent career in academic rheumatology/immunology in the near future. The proposal is a natural extension of the applicant&#176;s current research. The applicant has shown that murine Th2 cells overexpress LFA-1 (CD11a/CD18) and become autoreactive following treatment with two distinct DNA hypomethylating agents. Adoptive transfer of these autoreactive cells will also induce a lupus-like disease in syngeneic mice. This proposal will first examine the relationship between different lupus-inducing drugs and T cell DNA hypomethylation. The role of LFA-1 in T cell autoreactivity and in vivo autoimmunity will be determined by overexpressing LFA-1 on T cells directly through transfection of murine LFA-1 constructs, and the use of ICAM-1 deficient mice in the murine system. The role of Th1 and Th2 cytokines in T cell autoreactivity in vitro and autoimmunity in vivo will also be examined. Finally, attempts will be made to knockout the murine T cell DNA methyltransferase gene by homologous recombination to definitively determine the role of the gene in the proposed hypothesis of environmental agents inhibiting T cell DNA methyltransferase , leading to DNA hypomethylation, LFA-1 overexpression, and T cell autoreactivity and in vivo autoimmunity. The murine model is likely to have relevance to human disease, as abnormal T cell DNA methylation, LFA-1 overexpression, and increased production of Th2 cytokines such as IL-6 have all been reported in human lupus patients. The application is now in the position to develop his own independent research program as a junior faculty member in the Department of Internal Medicine. It is expected that the applicant will be promoted to a tenure track position during the time of the award. The sponsor, Dr. Bruce Richardson, who is also head of the division of Rheumatology at the Ann Arbor Veteran Administration Hospital, is commited to contributing protected time and resources to the applicant. The collaborators, Dr. Rossler and Dr. Johnson, as well as the core facilities at the University of Michigan will also provide expert help where it is needed. YUNG RL UNIVERSITY OF MICHIGAN, 1500 E. MEDICAL CENTER DRIVE, ANN ARBOR, MI 48109-0926 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES Crisp Data Base National Institutes Of Health hydroxyurea laboratory mouse T lymphocyte systemic lupus erythematosus drug adverse effect electroporation transfection gene expression cellular immunity cytokine interleukin 6 monoclonal antibody enzyme linked immunosorbent assay CD antigen CD4 molecule leukocyte adhesion molecule serology /serodiagnosis immunopathology DNA methylation high performance liquid chromatography ultraviolet radiation SDS polyacrylamide gel electrophoresis southern blotting autoimmunity gene targeting green fluorescent protein CRISP RPROJ MICHIGAN G CRISP/99/AR01977-02 CRISP/99/AR01977-02 199904 eng TARGETED DNA REPAIR OF PSORALEN ADDUCTS IN SKIN CELLS Research RPROJ This project focuses on DNA repair of sequence-specific damage caused by psoralens linked to antigene oligonucleotides (pso-AGOs) in human skin cells. The long-term goal is to elucidate the structural factors of DNA which govern its repair in general, and transcription-coupled repair in particular. The research will specifically aim l) To demonstrate specific gene suppression in skin cells caused by treatment with pso-AGOs and ultraviolet A light (UVA); 2) To characterize the targeted damage produced by these agents and determine how the subsequent cellular repair varies with specific sequences or sites in a gene; 3) To determine if DNA repair at one site influences repair at another; and 4) To use a system of differentiating keratinocytes to examine further how transcription controls repair. The research will be executed in the fully equipped laboratory of Professor Philip C. Hanawalt who co-discovered excision repair over 30 years ago and whose laboratory continues to do pioneering work in the field of DNA repair. The laboratory group, departmental and university environments consists of ample and outstanding intellectual and technical support. The research experience will allow the candidate to learn concepts and skills crucial to becoming an independent biomedical scientist. The award will thus serve as preparation to assume a junior faculty position in academic dermatology and to lead a competitive research effort in photobiology and photomedicine related to cutaneous diseases. OH DH STANFORD UNIVERSITY, STANFORD, CA 94305-5020 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES Crisp Data Base National Institutes Of Health psoralen cell differentiation adduct fibroblast electroporation transfection gene expression genetic transcription gene induction /repression radiation genetics human tissue western blotting DNA repair DNA footprinting nucleic acid structure nucleic acid sequence oligonucleotide ultraviolet radiation radionuclide keratinocyte tissue /cell culture DNA damage northern blotting gene targeting CRISP RPROJ CALIFORNIA G CRISP/99/AR02008-02 CRISP/99/AR02008-02 199904 eng LYSYL OXIDASES IN MATRIX DEVELOPMENT AND MAINTENANCE Research RPROJ The Mentored Clinical Scientist Development Award (K08) is intended to provide support for clinicians committed to research to facilitate their development into independent investigators. The K08 award is well suited for this candidate, who has completed clinical and research training, but requires more experience and publications before starting an independent research career. Dr. Hornstra will work in the laboratory of Dr. Steven Shapiro, under the guidance of Drs. Shapiro and Welgus. The sponsor, co- sponsor and collaborator at Washington University are well know for their expertise in the field of matrix biology. This unique environment will greatly enrich the candidate's potential to study the lysyl oxidases, important extracellular matrix cross-linking enzymes. Funding this candidates KO8 proposal will allow the generation of significant preliminary data and publications for future R29 or R01 research proposals. The objective of the research project is to investigate the role of lysyl oxidases in extracellular matrix development and maintenance. Lysyl oxidases are primordial enzymes which function to cross-link collagen and elastin in the presence of molecular oxygen, producing the strong and flexible ligaments and tendons of large animal bodies. In the human and now mouse, two lysyl oxidase genes have been identified: lysyl oxidase-1 and lysyl oxidase-2. In the mouse, only lysyl oxidase-1 (mLO-1) has been characterized, but my preliminary work has identified the murine lysyl oxidase-2 gene (mLO-2). These two genes exhibit differential expression in human and mouse cell lines and tissues. In many adult mouse tissues, LO-2 mRNA expression is readily detectable but LO-1 mRNA expression is undetectable via Northern Analysis. However, in fetal skin and lung fibroblast cell lines, LO-1 mRNA expression is slightly greater than LO-2, but LO-2 expression is detectable. These data suggest differential mechanisms of gene regulation and potentially cell-specific or tissue- specific expression during normal matrix development and maintenance. Lysyl oxidases are intimately involved with normal extracellular matrix development and turnover, but if abnormally regulated, may contribute to a variety of development and fibrotic disorders. The long term goal of this proposal is to delineate the role of the lysyl oxidase genes in the extracellular matrix of connective tissues during normal development, and in the future during pathological fibrosis. To accomplish this, we propose to generate lines of mice deficient in either mLO-2 and mLO-1 genes, examine the phenotypes of these deficient mice, and examine the differential expression and regulation of the LO genes. Our hypothesis is that either mLO-2 or mLO-1 deficient mice will have a specific phenotype due to the temporal and spatial differential expression of the LO genes. The phenotypes will result from he specific and independent functions of either LO, and this phenotype will be defined in the "knockout" models. These specific functions of either mLO-2 or mLO-1 could not be identified with the use of pharmacological inhibitors, which are non-specific. In the unlikely event that no significant phenotype is observed or can not be induced upon injury challenge, a double "knockout" with both LOs would be constructed in the future. HORNSTRA IK WASHINGTON UNIVERSITY MED CTR, 216 S KINGSHIGHWAY BLVD, ST LOUIS, MO 63110 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES 63110 Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse transgenic animal bleomycin hypertension extracellular matrix connective tissue development drug adverse effect site directed mutagenesis gene expression genetic promoter element genetic regulation gene deletion mutation immunocytochemistry wound healing nuclease in situ hybridization DNA footprinting skin disorder amine oxidoreductase enzyme activity enzyme deficiency lung injury gene targeting CRISP RPROJ MISSOURI G CRISP/99/AR02059-01 CRISP/99/AR02059-01 199904 eng ROLE OF CYTOTOXIC T CELLS IN MDX DYSTROPHY Research RPROJ The long term objectives of this proposal are to identify the role of cytotoxic T lymphocytes (CTLs), in the pathogenesis of Duchenne muscular dystrophy (DMD) using the mdx mouse as a model system. The anticipated findings will be significant in two regards: l) they will provide information on the functional importance of elevated CTL populations reported previously in DMD muscle, and 2) they will indicate the role of CTLs in currently encountered technical difficulties in myoblast transfer and adenoviral DNA delivery. The specific aims of the proposal are: 1) To determine if autologous CTLs lyse dystrophin-deficient myotubes at a higher frequency than dystrophin-containing myotubes, using conventional cytotoxicity assays; 2) To determine if factors released from mdx muscle enhance the ability of autologous CTLs to attack mdx myotubes, using cytotoxicity assays; 3) To identify if in vivo depletion of CD8+ CTLs in pre-necrotic mdx mice prevents muscle cell death, according to histological assays of necrosis; 4) To determine if muscle necrosis is reduced or absent in perforin- deficient mdx mice, using double mutant mice, deficient in dystrophin and perforin, that I will generate; 5) To determine if muscle necrosis is reduced or absent in fas- deficient mdx mice, using double mutant mice that I will generate. SPENCER MJ UNIVERSITY OF CALIFORNIA, 22-474 MDCC, LOS ANGELES, CA 90095-1752 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES Crisp Data Base National Institutes Of Health laboratory mouse transgenic animal cytotoxic T lymphocyte histopathology cellular immunity cell mediated lymphocytolysis test muscular dystrophy muscle pore forming protein cytotoxicity cytolysin dystrophin myotube CRISP RPROJ CALIFORNIA G CRISP/99/AR08439-03 CRISP/99/AR08439-03 199904 eng ERYTHROPOIETIC PROTOPORPHYRIA - MECHANISMS OF DISEASES Research RPROJ DESCRIPTION: (Adapted from investigator's abstract) Erythropoietic protoporphyria (EPP) arises from genetically determined partially deficient activity of the heme synthetic enzyme ferrochelatase (FC) that causes accumulation of protoporphyrin (PP) in rbc, plasma, liver and feces. PP causespainful cutaneous photosensitivity and may lead to fatal hepatotoxicity. Much remains unknown about the natural course of EPP, its pathogenetic mechanisms and inheritance patterns. Continuation of ongoing longitudinal investigations of an established EPP study population with a standardized protocol, and an existing data base built over 10 years in 24 subjects of diverse ethnic backgrounds, will further elucidate the natural course of the disease. Individual patients may benefit from early detection of adverse changes in hepatic function. Factors leading to development of fatal hepatotoxicity may be learned from retrospective analysis of the data base for patients who develop liver dysfunction when compared with the data of the population as a whole. Information about the variance in defective activity of FC in a diverse United States EPP population will be gained by measuring its levels in leukocytes of patients, family members and controls. This information will also aid in establishing the inheritance pattern(s) of the disease, which may be complex. Molecular genetic studies (restriction fragment length polymorphisms, detection and description of gene mutations) will be continued in genetic material isolated from blood of patients, family members and controls to further define the genetic heterogeneity of EPP, and will be correlated with clinical symptomatology, porphyrin burden and metabolic balance in blood and fecal distribution compartments, and FC activity in this diverse EPP population. Clinical and laboratory evaluations of unusual cases of several related forms of porphyria will be continued as a national resource function of the laboratory. Immunomapping of the microanatomical level of the epidermal-dermal separation and direct immunoelectron microscopic localization of immune reactants in blistering forms of porphyrias and "pseudoporphyrias" will be performed in skin biopsy specimens of patients with these disorders, to examine similarities or differences among them, and to target appropriate biomolecular components of the basement membrane zone for further study. The prevalence of the association of hepatitis B and C viral exposure and sporadic vs. familial porphyria cutanea tarda (PCT) in the United States will be determined by a multicenter study of blood specimens from PCT patients examining evidence of viral infection, levels of porphyrin accumulation and uroporphyrinogen decarboxylase activity. This information, and relevant historical, physical examination, and clinical laboratory data will be analyzed for statistically significant correlations. DELEO VA COLUMBIA-PRESBYTERIAN MEDICAL, 161 FT WASHINGTON AVENUE, NEW YORK, N Y 10032 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES 10032 Crisp Data Base National Institutes Of Health clinical chemistry biopsy pathologic process metalloenzyme molecular genetics gene mutation mutant restriction fragment length polymorphism immunoglobulin G patient /disease registry human subject liver toxic disorder protoporphyria congenital hepatic porphyria immunoelectron microscopy orphan disease /drug nonvisual photosensitivity longitudinal human study porphyrin metabolism human genetic material tag infectious hepatitis enzyme deficiency family genetics human data ferrochelatase CRISP RPROJ NEW YORK G CRISP/99/AR18549-23 CRISP/99/AR18549-23 199904 eng DEALING WITH RHEUMATOID ARTHRITIS--STRESS, COPING, AND OUTCOMES Research KATZ P VA MEDICAL CENTER, 4150 CLEMENT ST, SAN FRANCISCO, CA 94121 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES 94121 Crisp Data Base National Institutes Of Health chronic disease /disorder prognosis drug adverse effect fatigue self care patient care management health care service utilization human subject interview questionnaire statistics /biometry longitudinal human study coping health behavior social psychology pain sex difference antiarthritic agent rheumatoid arthritis physiologic stressor functional ability quality of life age difference stress management behavioral /social science research tag CRISP RPROJ CALIFORNIA G CRISP/99/AR20684-210043 CRISP/99/AR20684-210043 199904 eng ROLE OF GENETIC FACTORS IN DEVELOPMENT AND EXPRESSION OF RHEUMATOID ARTHRITIS Research CRISWELL LA VA MEDICAL CENTER, 4150 CLEMENT ST, SAN FRANCISCO, CA 94121 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES 94121 Crisp Data Base National Institutes Of Health polymerase chain reaction disease /disorder proneness /risk drug adverse effect allele gene expression gene frequency genetic polymorphism human subject serology /serodiagnosis statistics /biometry longitudinal human study radiography T cell receptor female rheumatoid arthritis rheumatoid factor caucasian American histocompatibility antigen functional ability human genetic material tag southern blotting women's health CRISP RPROJ CALIFORNIA G CRISP/99/AR20684-210044 CRISP/99/AR20684-210044 199904 eng CYTOTOXIC MECHANISMS IN CUTANEOUS DISEASE Research RPROJ This is a request for an additional five years of funding for "Cytotoxic Mechanisms in Cutaneous Disease" which has been funded to sixteen years to study the mechanisms of immunologic damage to keratinocytes and melanocytes, a central component in important skin diseases such as photosensitive lupus erythematosus, vitiligo, erythema multiforme, toxic epidermal necrolysis and lichen planus. We have found that the epidermis is intrinsically resistant to immunologic cytotoxicity, due in large part to resistance of basal keratinocytes and melanocytes to apoptosis induced by immunologic triggers. We hypothesize that this resistance to apoptosis in undifferentiated keratinocytes and melanocytes is maintained by "survival" signals provided by growth factor activation of receptors and by extracellular matrix activating cell surface integrins. We propose to test the effect of growth factor and integrin blockade on the susceptibility of melanocytes and keratinocytes to induction of apoptosis by ultraviolet radiation (UVR), ionophore, anti-Fas, and cytokines. Using combinations of blocking, rescue and transfection experiments, we will verify that survival signals in melanocytes and keratinocytes are transmitted through ras activation, and directly regulate expression of important proteins which control apoptosis, such as bc1-2, and perhaps bc1-x, Bax and Bad. We will also study regulation of these important proteins following nuclear translocation of p53, and important trigger of apoptosis induced by UVR. This proposal addresses the molecular and cellular biology of a fundamental characteristic of the basal layer of the epidermis: its intrinsic resistance to immunologic cytotoxicity. Although these anti- apoptotic defenses protect the skin from unwanted effects of inflammation, they may also allow favor survival melanoma and squamous cell carcinoma. NORRIS DA UNIVERSITY OF COLORADO, 4200 EAST 9TH AVE, BOX B153, DENVER, CO 80262 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES 80262 Crisp Data Base National Institutes Of Health biological signal transduction ionophore flow cytometry cellular pathology tumor suppressor gene gene expression oncogene neurotrophic factor human tissue cellular immunity enzyme linked immunosorbent assay western blotting immunopathology melanocyte integrin ultraviolet radiation radiation sensitivity receptor binding growth factor receptor cytokine receptor keratinocyte skin disorder cytotoxicity northern blotting programmed cell death CRISP RPROJ COLORADO G CRISP/99/AR26427-17 CRISP/99/AR26427-17 199904 eng LOCAL IMMUNOTHERAPY TO PREVENT SURGICAL INFECTION Research RPROJ Antibiotic resistant infection, catastrophic and costly, is an increasingly frequent complication of major surgery, trauma, and organ transplantation, and limits the use of biomaterial devices. Infection has been a principal barrier to the successful use of the total artificial heart, and for those in development and early use (e.g., ventricular assist devices and totally implantable artificial hearts), for xeno-transplants and engineered tissues. The emergence of methacillin and vancomycin resistant strains and of a large immuno- compromised and AIDS population, adds troublesome magnitude to the surgical infections disease problem. We report that pooled human immunoglobulin (IgG) preparations (natural polyclonal antibodies), delivered locally, to wounds and damaged tissues, or as biomaterial coatings, in the proximity of contaminating Staphylococcus aureus and Pseudomonas aeruginosa are highly effective in preventing infections in the presence or absence of biomaterials in four animal models in two species. Our hypothesis is that antibody preparations delivered locally to surgical and traumatic wounds, or on biomaterial surfaces (including engineered tissues) at the time of surgery, will prevent infection by enhancing opsonization, phagocytosis, and bacterial killing before antibiotic resistant and biofilm type infections are established. Contemporary pooled human IgG contains a repertoire of antibodies to current and evolving antibiotic resistant strains of bacteria. The studies we present of PLI are the first to demonstrate that the direct application of IgG may be utilized to prevent surgical wound and biomaterial infection. The pre-clinical studies we now propose are expected to further secure proof of concept in anticipation of human clinical trials by expanded investigation of (i) therapeutic dose range responses, for local application, (ii) time course studies, (iii) representative pathogens including antibiotic resistant strains, and (iv) biomaterial responses. (Aims 1 and 2). The mechanisms complement activation, blocking of adhesion, opsonization and phagocytosis) of IgG antimicrobial activity will be characterized in Aim 3. There are 25 million surgeries annually in the U.S. of which four million are at high risk for infection. PLI, based on sound immunologic principals and proof of concept studies, represents a novel, but simple, cost effective strategy and opportunity for effective broad spectrum prophylaxis and therapy, even in the presence of emerging antibiotic resistant pathogens, immunocompromised populations, and biomaterial centered infection. GRAINGER DW MEDICAL SCIENCES RSRCH INST, 520 HUNTMAR PARK DRIVE, HERNDON, VA 20170-5100 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES Crisp Data Base National Institutes Of Health laboratory rabbit laboratory mouse Staphylococcus aureus Staphylococcus epidermidis Pseudomonas aeruginosa Staphylococcus infection biomaterial evaluation biomaterial interface phenomena catheterization phagocytosis secondary infection postoperative complication immunoglobulin G bactericidal immunity passive immunization exotoxin complement pathway burn wound infection titanium adsorption surface property nonhuman therapy evaluation implant biomaterial compatibility CRISP RPROJ VIRGINIA G CRISP/99/AR26957-11A1 CRISP/99/AR26957-11A1 199904 eng BIOPHYSICAL STUDIES OF METABOLIC ACTIVITY AND CONTROL Research RPROJ For many years this laboratory has been developing and using radiation target analysis, a method for determining the mass of biologically active molecules. The laboratory has developed a variety of experimental techniques to properly utilize the inactivation of biochemically active material by ionizing radiation, specifically that from high-energy electrons. Radiation target theory has been successfully extended to complex biological systems. These techniques and theories have been utilized in studies of enzymes, receptors, transporters, and other structures of wide interest in the biomedical field. Often these radiation studies resulted in discovery of unexpected features or properties of these biologically active materials. The objectives of present studies are to determine the precise nature of damage in macromolecules caused directly by ionizing radiation and to utilize this knowledge in the application and extension of radiation target analysis. Several projects have been brought to fruition during the past year. The enzyme hyaluronan synthase is of great medical importance in human infections by streptococcus. Radiation studies revealed that each enzyme molecule requires fifteen molecules of a specific lipid in order to function. The enzyme ribonucleotide reductase is crucial in AIDS infection. It has a unique structure involving a stable free radical. In isolated subunits, this radical confers extraordinary radiation sensitivity which is not seen in the intact holoenzyme. Another enzyme, phenylalanine hydroxylase, is especially important in humans: a defect in this enzyme is the cause of phenylketonuria in newborns. Radiation studies of this enzyme have been continuing for several years. The enzyme has complex regulation mechanisms; in the purified state, radiation studies revealed the different structures involved in several of these mechanisms. Now the technique has been applied to the enzyme in the crude state, revealing unanticipated differences from those seen previously. It appears that purification has altered the structure of the enzyme complex. The method of radiation target analysis continues to reveal unique molecular properties of biologically active structures. Often these properties were unanticipated from studies using other techniques. This new knowledge has given a greater understanding of the mechanisms of these biological functions. In some cases these properties offer the possibility of external control or modification of these active structures. KEMPNER ES NIAMS, NIH 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES Crisp Data Base National Institutes Of Health biophysics enzyme structure transcription termination phenylalanine 4 monooxygenase protein structure ionizing radiation radiation dosage radiobiology radiation sensitivity glutathione transferase ribozyme enzyme activity structural biology hepatic lipase CRISP RPROJ A CRISP/99/AR27003-39 CRISP/99/AR27003-39 199904 eng SUPERANTIGEN INTERACTIONS WITH MHC CLASS II ANTIGENS Research JARDETSKY T NORTHWESTERN UNIVERSITY, 303 E CHICAGO AVE, CHICAGO, IL 60611 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES 60611 Crisp Data Base National Institutes Of Health Staphylococcus aureus Streptococcus pyogenes antigen presenting cell molecular site X ray crystallography antigen antibody reaction bacterial antigen staphylococcal enterotoxin analytical ultracentrifugation receptor binding antigen receptor T cell receptor arthritis MHC class II antigen musculoskeletal disorder SDS polyacrylamide gel electrophoresis autoimmunity superantigen CRISP RPROJ ILLINOIS G CRISP/99/AR30692-150032 CRISP/99/AR30692-150032 199904 eng GROWTH DIFFERENTIATION CONTROL IN PRIMARY KERATINOCYTES Research RPROJ Epithelial tumor development is the result of altered tissue homeostasis, due to deranged intracellular regulation and escape from the counteracting influences of a normal cellular environment. We plan to continue our work in this area, as it relates to the function of the three TGF-beta isoforms in the skin. Mice with specific knockout mutations of the TGF-beta1, TGF-beta2 and TGF-beta 3 genes have been recently developed. Homozygous TGF-beta 2 and TGF-beta 3 knockout mice die within a few hours after birth. To overcome this problem, we have developed a full-thickness grafting approach. When the skin of the newborn knockouts is grafted onto nude mice, it develops normally and can be maintained on the host for at least 1 year. We will use this approach to address the following questions: 1) TGF-beta control of keratinocyte growth and differentiation in vivo. We will test the hypothesis that the individual TGF-beta isoforms play distinct or only partially overlapping roles on control of keratinocyte growth/differentiation and apoptosis in the intact skin. We will analyze the epidermis of grafts from mice with each of the TGF- beta knockout mutations, as well as wild type controls, under basal conditions and under conditions of altered skin homeostasis, such as after TPA or retinoic acid exposure. 2) TGF-beta control of keratinocyte growth and differentiation in culture. We will complement the in vivo analysis with in vitro studies of cultured kerationocytes derived from both knockout and wild type mice, plus/minus TGF-beta exposure. In this manner, we will determine (1) which are the direct and specific effects of individual TGF-betas on keratinocyte growth, differentiation and apoptosis; (2) which of these effects are due to an autocrine versus paracrine loop. 3) TGF-beta control of skin tumor development. We will investigate the role that the individual TGF-betas play in skin carcinogenesis, with a special emphasis on TGF-beta3. For this purpose, TGF-beta knockout skins grafted onto nude mice will be tested for their rate of benign and malignant tumor formation, in response to a chemical carcinogenesis protocol or after crossing into a ras transgenic background. 4) TGF-beta control of keratinocyte tumorigenicity. The individual TGF-beta factors could play distinct autocrine and/or paracrine functions in control of keratinocyte tumor development. To address this question, we will evaluate (a) keratinocytes derived from TGF-beta knockout mice for their susceptibility to tumorigenic conversion after ras oncogene transformation, and (b) dermal fibroblasts derived from the same mice for their capability to suppress keratinocyte tumor formaiton. DOTTO G MASSACHUSETTS GENERAL HOSPITAL, BLDG 149 13TH ST, CHARLESTOWN, MA 02129 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES 02129 Crisp Data Base National Institutes Of Health athymic mouse transgenic animal cell differentiation cell growth regulation transforming growth factor skin neoplasm chemical carcinogenesis keratinocyte skin transplantation phorbol tissue /cell culture retinoate autocrine paracrine protein isoform CRISP RPROJ MASSACHUSETTS G CRISP/99/AR39190-11 CRISP/99/AR39190-11 199904 eng MODULATION OF LANGERHANS CELL FUNCTION BY ULTRAVIOLET B (UVB) RADIATION Research RPROJ Langerhans cells (LC) are skin-specific members of the dendritic cell (DC) family of antigen presenting cells. As members of this family, LC activate immunologically naive, antigen-specific T cells with high efficiency and, as residents of epidermis, are outermost sentinels for the immunologic recognition of environmental antigens, infectious microorganisms, and malignancies. Solar ultraviolet radiation between 295 and 320 nm (UVB) is responsible for sunburn and for the majority of skin cancers in humans. UVB radiation also prevents the immunological recognition of novel antigens, including contact sensitizers and tumor antigens in skin. Our working hypothesis is that UVB radiation exerts this effect (i.e., induction of immunological tolerance) by preventing the maturation of Lc into fully competent DC. Taking advantage of novel lines of Ia+ antigen presenting cells (XS lines) derived from newborn mouse epidermis, we propose four Specific Aims: 1) To confirm that XS lines are representative of epidermal l-C through precursor frequency analysis and cloning, phenotypic and functional comparisons with newly established short-term LC lines, and comparisons with other DC lines. 2) To study mechanisms of maturation in XS lines under the influence of different growth media, cytokines, reactive haptens, and a phorbol ester. Functional properties assessed at different states of maturation will include expression of surface molecules, production of cytokines, immunization with reactive haptens, and education of Th0 cells into Th1 or Th2 subsets. 3) To characterize mechanisms by which UVB radiation modulates the function of LC. We will study the impact of UVB on XS cell maturation and determine whether incomplete maturation accounts for distorted APC function (i.e., induction of clonal anergy in Th1 cells and/or preferential activation of Th2 cells). 4) To study the UVB- signalling pathways that lead to impaired antigen presenting function, by identifying UVB-inducible immediate early genes (IEG) in XS cells, characterizing chromophores and mediators responsible for IEG activation and functional changes. These studies of molecular mechanisms of UVB- induced damage to antigen-presenting cells in skin will provide new knowledge that ultimately may lead to the development of pharmacological agents to prevent the detrimental effects of solar radiation. BERGSTRESSER PR UNIV OF TEXAS SW MEDICAL CENTE, 5323 HARRY HINES BLVD, DALLAS, TX 75235-9069 1998 U.S. DEPT. OF HEALTH AND HUMAN SERVICES; PUBLIC HEALTH SERVICE; NATIONAL INST. OF HEALTH, NAT INST OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES Crisp Data Base National Institutes Of Health laboratory mouse leukopoiesis cell differentiation gene expression regulatory gene growth inhibitor immune tolerance /unresponsiveness antigen presentation skin neoplasm radiation carcinogenesis light adverse effect ultraviolet radiation radiation dosage skin Langerhans' cell tissue /cell culture CRISP RPROJ TEXAS G CRISP/99/AR40042-09 CRISP/99/AR40042-09