Vasopressin (AVP), the antidiuretic hormone is a peptide hormone actively involved in the regulation of body fluid osmolality, blood volume, blood pressure, and cell proliferation via the stimulation of specific membrane-bound receptors classified into V1a-vascular, V2- renal, and V3-pituitary subtypes having distinct pharmacological profiles and intracellular second messengers. The secretion of AVP is an absolute requirement for the maintenance of fluid homeostasis as shown in human and experimental models of diabetes insipidus. The usefulness of natriuretic agents has been demonstrated in conditions associated with water and salt retention. However, there are diseases (hyponatremia of congestive heart failure, liver cirrhosis, nephrotic syndrome, and the syndrome of inappropriate secretion of antidiuretic hormone, SIADH) due to the inappropriate release of vasopressin and characterized by an excess retention of water. An agent that increases urine output by producing a free water diuresis would be of significant advantage in these conditions. This study is designed to evaluate the efficacy, safety, and tolerance of a single oral dose of VPA-985 in patients with hyponatremia due to SIADH. In addition, preliminary pharmacokinetic and pharmacodynamic parameters will be assessed. This study has just begun patient recruitment. Four to six patients with diagnosed SIADH between the ages of 18 and 75 years will be enrolled at this site.


This application addresses the broad Challenge Area (01) Behavior, Behavioral Change, and Prevention and specific Challenge Topic, 01-AA-102*: Functional Roles of Neuroimmune Factors in Mediating Behavior. The title of this proposal is "Alcohol-chemokine interactions and neurotransmission". Emerging research implicates a role for the innate immune system of the brain in the effects of alcohol on behavior. Astrocytes and microglia, the primary cell types that comprise innate immune system of the brain, are normal components of the brain and serve essential roles in normal brain development and function. Microglia and astrocytes also play critical defensive and repair roles during adverse conditions. Basic to their roles during both normal and adverse conditions is the production of neuroimmune factors such as cytokines and chemokines, which are signaling molecules that initiate or coordinate cellular actions appropriate to the need, be it cellular development, normal cell function or a defense/repair response to adverse conditions. Recent studies provide strong support for a role of neuroimmune factors, and in particular the chemokine CCL2, in alcohol use and abuse. Importantly, acute alcohol has been shown to increase levels of CCL2 in the hippocampus, a brain structure that is essential for cognitive functions such as short-term memory. CCR2, the receptor for CCL2, has been shown to be expressed in abundance in the hippocampus. Moreover, research has identified the hippocampus as one of several brain regions that play a central role in the cognitive deficits produce by alcohol abuse. An important target of alcohol action in the hippocampus is synaptic transmission and plasticity at the Schaffer collateral to CA1 synapse. Our studies show that CCL2 also alters the functional properties of this synaptic pathway. Thus, hippocampal synaptic function is a target of both CCL2 and alcohol action and a likely site for interactions between alcohol and CCL2 that could be a critical factor in the behavioral effect of alcohol. Alcohol actions in the hippocampus have been well documented but Information on the effects of CCL2 on hippocampus function is limited and interactions between alcohol and CCL2 at the level of neuronal function and synaptic transmission have not been studied. This information is critical to an understanding of the role of CCL2 in alcohol use and abuse and is the topic of the proposed studies. The studies will test the hypothesis that activation of the CCL2 signaling pathway in the hippocampus alters the actions of alcohol on hippocampal synaptic function and that these interactions between CCL2 and alcohol are also manifested at the behavioral level. To test this hypothesis, we will use variety of experimental approaches including electrophysiological recording of synaptic function, biochemical assessment of signal transduction pathway activation and behavioral testing. The studies will be carried out in transgenic mice that express elevated levels of CCL2 in the brain and their non-transgenic littermates as controls. Taken together, these studies will provide important new information that will significantly advance our understanding of the role of neuroimmune factors in alcoholism.


During the past year we have done extensive analyses of the data from both the child and adult components of the NHANES surveys. Two papers have been published or are in press and four others have been submitted. Below we describe the substantive issues addressed in this work. [unreadable] 1) We have completed the analyses of the prevalence estimates of GAD, panic, eating disorder, major depressive disorder/dysthymic disorder, ADHD and conduct disorders for the overall sample, by informant (child or parent), gender, race/ethnicity, and age group. In order to assess the severity of these conditions, we have also calculated prevalence by four alternative impairment algorithms. These data will provide the first population prevalence data on these seven major mental disorders in children ages 8 through 19 in a national probability sample of the US. [unreadable] 2) A separate project set out to describe the prevalence, demographic correlates, comorbidities, and service patterns for nocturnal enuresis. There are no published nationally representative prevalence estimates of nocturnal enuresis among children in the United States using standardized diagnostic criteria. The overall 12-month prevalence of nocturnal enuresis was 4.45%. The prevalence in boys was significantly greater than that in girls. Nocturnal enuresis was more common at younger ages and among black youth. ADHD was strongly associated with nocturnal enuresis. Only 36% of the enuretic children had received health service for nocturnal enuresis. Nocturnal enuresis is a common condition among children in the US. Few families seek treatment for nocturnal enuresis despite the potential for adverse effects on emotional health. Child healthcare professionals should routinely screen for nocturnal enuresis and its effects on the emotional health of the child and the family. Assessment of ADHD should routinely include evaluation for nocturnal enuresis and vice versa. Research on the explanations for the association between nocturnal enuresis and ADHD is indicated.[unreadable] 3) We investigated the prevalence of mental and physical comorbidity and health service utilization among 3,042 children aged 8 to 15 from the 2001 2004 NHANES. There was a strong association between mental and physical disorders. After adjusting for social and demographic characteristics, comorbidity was associated with a 2.5 times increase in healthcare utilization compared to those without comorbidity. Our findings confirm prior local population studies of comorbidity among youth and suggest that physical and mental comorbidity has a strong impact on healthcare utilization. Integrated treatment of mental and physical disorders is clearly indicated. Future studies should examine the explanations for systematic patterns of comorbidity in youth.[unreadable] 4) To determine the prevalence, sociodemographic correlates and comorbid medical conditions of recurrent headache in U.S. children, we investigated 10198 children aged 4 to 18 years old who participated in the 1999 to 2004 National Health and Nutrition Examination Surveys. We found that frequent or severe headaches including migraine in the past 12 months were reported in 17.1% of children, with greater rates among girls and older adolescents. Children with headache had more school absences and health care utilization. Asthma, hay fever, and frequent ear infections were more common in children with headache, with at least one of these occurring in 41.6% of children with headache vs. 25.0% of children free of headache (p<.0001). Children with headache were 13.6 times more likely to have all three of these conditions. We conclude that recurrent headache in childhood is extremely common and is associated with a variety of other medical conditions, particularly those with immunologic and inflammatory bases. The high frequency of recurrent headaches and their association with school absences suggest that headaches in U.S. youth have major public health significance. These results are a significant first step towards research to understand biologic mechanisms, identify more homogeneous subgroups in clinical and genetic studies, and develop better clinical management.[unreadable] 5) Our analytic team investigated patterns of mental and physical comorbidity among adults with severe headaches and migraine using merged data from the 1999-2004 survey years. The analyses include a comparison of the sociodemographic characteristics of individuals with versus without headaches as well as a description of the rates of comorbidity of both mental and physical disorders within both headache groups. In order to assess the relative impact of these comorbid conditions, we compared the responses to a series of questions on health care utilization and health perception across five groups (no headache, headache only, headache plus any physical condition, headache plus any mental condition, headache plus a physical and mental condition). [unreadable] 6) To understand the association between severe/recurrent headache and a range of cardiovascular and immunologic measures in the adult U.S. population, we used NHANES 1999-2004 surveys data including 14,503 adults and found that the major laboratory correlates of cardiovascular disease assessed in the NHANES including C-reactive protein, folate, homocysteine and HDL cholesterol differed among those with severe headaches/migraine compared to their non-headache counterparts. Moreover, people with severe headaches or migraine were more likely to have high BMI and diastolic blood pressure. These findings suggest that inflammatory mechanisms may play an important role in the pathophysiology of migraine as a risk factor for stroke and cardiovascular disease.[unreadable] 7) A parallel study to investigate the association of childhood headache with biomarkers for cardiovascular and cerebrovascular disease was also conducted among 11,770 children aged 4 19 years old in NHANES 1999 2004 data. We found mean values for homocysteine, C reactive protein, and body mass index were higher in children with than without headaches, and more children with headaches were in the highest quintile for these factors. Serum and red cell folate were lower in children with headache. More children with headache (32% in children < 12 years) thanMore children with headache (32% in children < 12 years) than without headache (18%) were in the highest quintile of risk for 3 or more of these factors. The findings indicate that these important risk factors for long-term vascular morbidity cluster with severe or recurrent headache in childhood or adolescence. Recognition of the subgroup among children with headache in whom these risk factors concentrate may permit improved preventive management. [unreadable] [unreadable] 8) These data provide new information on the significant role of comorbid disorders, specifically comorbid mood and anxiety disorders, on the impact of severe headaches or migraine, on health care utilization and health perception. Future analyses will then examine the risk factors and biological correlates of these conditions in the general population. We will prioritize our analyses to address the key study questions that are the focus of our own research.


The long range goal of this project is to develop methods of using supercritical carbon dioxide (scCO2) to achieve terminal sterilization that meets or exceeds the USP standard of 10-6 microbial survival probability and that produces a dry drug product in a single process. The objective of this application is to identify at least one set of conditions in which the act of drying drugs by the scCO2 process known as lyophobic precipitation (LP) can also sterilize the drug product in its dispensing container as well as the chamber in which the process is conducted. The rationale behind this project is that recent observations that scCO2 can be used to effect terminal sterilization of food and medical products without damaging the product will be adaptable to the LP drying process. Many drugs, including many anticancer agents, must be administered by non-oral routes (intravenous or intraperitoneal) and therefore must be sterile for injection. For stability reasons such drugs often must be dry as well as sterile. Current technology requires a two-step process of filter-sterilization followed by lyophilization under aseptic conditions to achieve safe, stable dosage forms. To accomplish the objectives of this application two specific aims will be pursued: (1) determine the level of sterilization of drug substance and container under normal LP conditions and using tested and untested added sterilizing agents, and (2) determine the level of sterilization of the chamber under normal LP conditions and using tested and untested added sterilizing agents. The strategy to accomplish the specific aims is to measure the level of killing of standard sterility-testing biological indicators added to containers of model drug under conditions that mimic the conditions that will eventually be used commercially. Similarly, challenge doses of biological indicators will be placed in the LP chamber to determine effectiveness of sterilization of the process with and without added sterilizing agents. The expectation is that at the conclusion of this Phase I SBIR period of support, precise conditions necessary to achieve a sterilization activity level of 10-6 for drug substance contained in a dispensing container and the chamber in which the lyophobic precipitation occurs will be identified. This information will guide a Phase II application to determine the effect of treatment conditions determined in this study on the chemical stability of existing oncology drugs that possess various reactive groups (e.g., alcohols, amines, thiols, carboxylic acids, etc.) that may prove susceptible to damage. PUBLIC HEALTH RELEVANCE: This project proposes a new technique to safely dry and terminally sterilize drugs in their dispensing containers. If successful, this approach will lead to simpler processing of injectable drugs that would increase safety and lower production costs.


This is a resubmission of the previously submitted proposal investigating the regulation of luminal breast cancer stem cells by HER2. We have addressed the previous critiques by adding an investigator with expertise in bone biology, Evan Keller, and a biostatistician, Kelley Kadwell. In addition, we have added new preliminary data and refined the research plan. The development of HER2 targeting agents represents one of the greatest advances in the therapy of breast cancer. Although the use of HER2 targeting agents such as trastuzumab has been limited to breast cancers with HER2 gene amplification, recent retrospective analyses of adjuvant trastuzumab studies suggests that a much wider group of breast cancer patients may benefit from this therapy. We propose that the biological basis for these surprising clinical findings relates to the cancer stem cell (CSC) model. We hypothesize that HER2 is selectively expressed in CSC populations in luminal breast cancers where it plays a major role in the regulation of CSC self-renewal. Furthermore, we hypothesize that the bone microenvironment induces HER2 expression expanding the CSC population in luminal breast cancer bone metastasis. This is mediated by RANKL-RANK-NF?B signaling in a process independent of HER2 gene amplification. We propose that the clinical efficacy of adjuvant trastuzumab in women whose breast cancers do not display HER2 gene amplification (HER2-negative) is due to the effective targeting of CSCs in micrometastasis in these patients. We propose to utilize established breast cancer cell lines, primary human xenografts and primary and metastatic breast cancer tissues from patients to directly test these hypothesies. These studies directly challenge the current clinical paradigm that only the 20% of women with HER2 amplified breast cancers benefit from adjuvant HER2 blockade which, if confirmed would have significant clinical implicaitons. These studies provide a strong biological rationale for the current intergroup trial NSABP-B47, a phase III study designed to determine whether adjuvant trastuzumab benefits women with HER2-negative tumors. Furthermore, the proposed studies have broader implications for the development of adjuvant cancer therapies. If CSCs mediate tumor recurrence, then effective adjuvant therapies will need to successfully target this cell population.


The primary objective of this project is to support the advanced development of candidate products for use in post-event settings following the intentional release of or in response to naturally occurring outbreaks of infectious diseases caused by NIAID Category A, B, and C Priority Pathogens. This contract may support formulation and manufacture of the individual vaccine components, as well as stability testing, nonclinical immunogenicity and efficacy testing in nonhuman primates, IND enabling GLP repeated dose toxicology, submission of an IND and eventual clinical evaluation.


Ca2+ entry through voltage-dependent Ca2+ channels is important in cardiac and vascular muscle excitation-contraction coupling. The Ca2+ regulated multifunctional protein kinase, Ca2+/calmodulin-dependent protein kinase II (CaMKII), has a very important role in signal transduction in nervous system, such as long-term potentiation (LTP) and memory. However, little i known as to whether this protein kinase also modulates the function of cardiac cells. Our studies demonstrate both spatially resolved and temporally distinct novel effects of CaMKII on L-type Ca2+ channel current (ICa) in cardiac cells. Either depolarization alone or calcium influx can increase the amplitude and slow the inactivation of ICa. The distinct volt e- and Ca2+-dependent effects persist with time constants of about 1.7 s and 9 s, respectively. Both effects are completely abolished by a specific peptide inhibitor of CaMKII. This CaMKII inhibitor also suppresses the prolongation of ICa induced by depolarizing holding potentials. An antibod specific for the autophosphorylated (activated) CaMKII, PY-66, is localized close to sarcolemmal membranes and the profile of CaMKII activation is qualitatively correlated with the changes in ICa under various conditions. Thus, the action of CaMKII on ICa is dually regulated by membrane depolarization and by calcium influx: the latter directly activates CaMKII while the former likely promotes the interaction between constitutive CaMKI and the membrane channel proteins. In contrast to the active CaMKII distribution, the intracellular distribution of the total CaMKII enzyme (visualized by using an antibody which specifically reacts with CaMKII k isoform) but does not sense its activation state is uniform with a higher nuclear distribution. This suggests that CaMKII is translocated to the cel sarcolemma following activation in cardiac myocytes. These findings provid new insights toward understanding the physiological function of the ubiquitous protein kinase, CaMKII in cardiac muscle cells as possibly in other type of cells as well.


The objective of the proposed project is the characterization of the properties of the different types of serotonin (5-hydroxytryptamine, 5-HT) receptors in the mammalian central nervous system (CNS). One part of this involves the use of in vitro binding assays utilizing ligands such as [H-3]5-HT, [H-3]PAT, and [H-3] ketsanserin, which are thought to label specific populations of serotonin receptors. A major emphasis of this work will be the pharmacological characterization of these receptors to determine the structural requirements of compounds for discrimination between different types of 5-HT receptors. In conjunction with the ligand-binding assays a program for the synthesis of new compounds will be carried out. Thus, information form the binding assays will be used in the design of new compounds for testing at 5-HT receptors. Another part of the project will be the characterization of functional 5-HT receptors, both in vitro and in vivo. This will include examination of 5-HT receptors that mediate contraction of the cerebral vasculature and 5-HT autoreceptors that regulate the release of 5-HT, as well as examination of central 5-HT receptors involved in the control of respiration, blood pressure, body temperature, and release of certain pituitary hormones such as prolactin and thyrotropin. The study of functional receptors will provide information as to whether the compounds are agonists or antagonists of 5-HT and whether the properties of the functional receptors correlate with those of the putative receptors measured by ligand-binding. As with the ligand-binding studies, a major emphasis of this portion of the project will be the attempt to determine the structural requirements of compounds for discrimination between different types of 5-HT receptors. Once these groups of receptors are classified and characterized, it is hoped that more specific drugs can be designed that will facilitate the study of the roles and actions of 5-HT in the CNS. It is hoped that information from such studies can be used for the design of new, more effective therapeutic agents for the treatment of behavioral or mental disorders that are thought to be linked to abnormal serotonergic function in the central nervous system.


In response to increase in ambient temperature or other noxious stimuli, all organisms generally respond by a rapid synthesis of heat shock RNA's and proteins. The regulation of heat shock gene transcription occurs by heat shock factor (HSF) which binds to heat shock elements (HSEs) in the gene. Even though HSEs are highly conserved among the species, there appears to be marked difference in HSFs. Two human forms of HSFs have been recently cloned from human B cell lymphoma and HeLa cells. In this study we have used both reverse - transcriptase polymerase chain reaction (RT - PCR) and standard cloning techniques to clone an HSF gene from human retinal cDNA library. Northern blot analysis of poly A+ RNA isolated from human retina indicated the presence of RNA of 2.4 kb size hybridizing with human [32P] HSF probe. The same probe was used to identify a number of cDNA clones of HSF in the human retinal library. These clones are presently being subcloned for sequence analysis. RT - PCR technique has also indicated that a form of human HSF is expressed in human retina. The existence and expression of a form of HSF gene in human retina underscores the possibility of induction of heat shock factor(s) by change in ambient temperature or by noxious stimuli as a means of protection of the retina from injury such as high temperature or oxidative damage.


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PROJECT SUMMARY (Description) Microglia are intimately involved in brain homeostasis and synaptic maturation. Recent studies suggest that developmental brain disorder may induce pathological microglia to impair the neural network. In this project, we will test whether these perinatal injury-induced pathological microglia have a unique lineage origin. The current prevailing view holds that microglia are derived exclusively from early CNS settlers of a yolk sac, and do not receive contributions by peripheral monocytes and derivtatives. Yet, our pilot studies suggested a far more complex situation, which bears great implications for perinatal brain injury/infection-related cognitive derangements. This project will test the hypothesis monocytes readily contribute to the brain microglial pool prenatally, but cease doing so shortly after birth. However, perinatal brain injury or infection widens the window of monocyte-to-microglia conversion to produce chronic pro-inflammatory microglia in adult brains. Aim 1: Generate CCR2-CreER mice using BAC recombineering to identify monocyte derivatives. We will produce tamoxifen-inducible CCR2-CreER mice using bacterial artificial chromosome recombineering transgenesis to detect monocyte-derivatives in the R26R-GFP reporter line. We will donate CCR2-CreER mice to the Jackson Laboratory to make this powerful transgenic tool readily available the research community. Aim 2: Compare monocyte-derived microglia in normal development or maternal immune activation (MIA) with and without mild hypoxia-ischemia (HI) in the neonatal period. We hypothesize that Monocytes transform to pro-inflammatory microglia to damage nearby synaptic structures after maternal immune activation (MIA) or endotoxin-sensitized hypoxia/ischemia (LPS/HI) in neonates. Aim 2a compares the number and morphology of monocyte-derived microglia in normal development and after MIA with and without mild HI injury. Aim 2b uses fluorescence-activated cell sorting (FACS) followed by RT- PCR analysis to compare gene expression by monocyte-derived microglia. Aim 2c employs confocal imaging analysis to compare the relationship of monocyte-derived microglia with adjacent synapses.


The transport of all classical neurotransmitters into synaptic vesicles depends on a H+ electrochemical driving force (??H+) generated by the vacuolar-type H+-ATPase. However, different transmitters depend to varying extents on the two components of ??H+, the chemical component (?pH) and the electrical component (??). Most of the vesicular transporters depend on ?pH, and mediate exchange of cytosolic transmitter for lumenal H+. However, the vesicular glutamate transporters (VGLUTs) depend primarily on ??, with an unclear role for H+. They also exhibit allosteric activation by Cl- and recent work has conflicted about their expression of a Cl- conductance. Adapting the use of electrophysiology to record currents associated with the VGLUTs at the plasma membrane, we have found that the VGLUTs exhibit a Cl- conductance allosterically activated from the lumenal side by H+ as well as Cl-. The Cl- condutance exhibits many of the properties previously described for vesicular glutamate transport and the two anions compete, suggesting they use a related permeation pathway. The long-term objective of this program is to determine how these mechanisms influence synaptic transmission, and the strategy is to characterize the mechanisms in heterologous expression systems, then test the role of these mechanisms in excitatory neurotransmission. We propose the following aims: 1) Identify residues responsible for allosteric activation of the VGLUTs by Cl- and H+. We will determine the effect of point mutations on the Cl- conductance associated with VGLUT expression in Xenopus oocytes, then extend the analysis to glutamate transport by functional reconstitution of purified protein. 2) Determine the role of allosteric regulation by Cl- and H+ in excitatory neurotransmission. Using VGLUT1/2 double knockout mice, we will rescue excitatory transmission with mutant VGLUTs defect in allosteric activation by Cl- and H+. We will also determine the effect of these and other properties on synaptic vesicle acidification. 3) Characterize the properties of currents associated with vesicular glutamate transport by whole endosome recording. We have recently detected currents associated with the expression of wild type VGLUTs on endosomal membranes. Importantly, these currents reflect the flux of glutamate as well as Cl-, and their characterization will provide basic new information about glutamate transport as well as the associated Clconductance. 4) Identify residues responsible for the differences in ionic coupling by different VGLUT family members\ (continuation of prior Aim 3). Bacterial members of the SLC17 family mediate the cotransport of organic anions with H+, raising questions about their relationship to the ??-drlven activity of the VGLUTs. In this aim, we will test the relationship between these closely related protein by introducing mutations that convert their coupling to that of the other.


We recently documented a case of naturally-acquired leprosy, near the LL end of the spectrum, in a sooty mangabey monkey (Cerocebus torquatus atys). The etiologic agent, identified as Mycobacterium leprae, and M. leprae isolated from a human leprosy case were each used to inoculate two normal manabeys. All four inoculated animals developed clinical and histopathologic signs of disseminated leprosy. This indicates that the mangabey may represent the first known nonhuman primated species that is susceptible to the experimental transmission of leprosy. The primary goal of the proposed studies is to establish the mangabey as a model for the study of experimental leprosy. Since the mangabey is rare in capativity and unvailable from importers, and additional goal is to establish a breeding colony to provide mangabeys for the proposed studies and to allow for future leprosyt research. Susceptibility will be studied by varying the dose and route of M. leprae inoculation. Natural transmission modes will be investigated by exposure of mangabeys to aerosols and to mangabeys with untreated, advanced leprosy. Each animal will be completely characterized prior to and at intervals after inoculation or exposure to M. leprae. The characterizations will include: serum chemistry values and hemograms; physical, clinical and neurological characteristics; and bacteriologic and histopathologic examinations of blood cells and biopsy materials. The following immunologic parameters will be studied: in vitro mitogen and antigen (e.g., lepromin) responses of lymphocytes (L); in vitro L immunoglobulin (Ig) production; B- and T-L numbers and subpopulations; suppressor/helper T-L activity; natural killer 9nk0 and antibodyt-dependent cellular cytotoxicity (ADCC); blood mononuclear cell cyclic-AMP levels; skin test responses; cross-reacting antigens between mangabey M. leprae and other mycobacteria; and serum levels of Ig's, C3 and C4, immune complexes, prostaglandins, sialic acid, rhematoid factor, and anti-M leprae antibody. The proposed studies will relate disease progress to changes in clinical, bacteriologic, histologic, and immunologic parameters. These relationships should define the extent to which the mangabey can serave as a useful model for the experimental study of human leprosy.


Cardiovascular disease is the leading cause of death worldwide. Current therapies for cardiovascular disease are associated with partial success, restricted access, delays, possible neurotoxicity and other important limitations. Our goal is to develop a novel therapeutic agent that is safer and more effective at dissolving the blood clots (thrombi) that cause heart attacks and strokes. Studies of humans and mice with lifelong deficiency of a2-antiplasmin (a2AP) have shown that it is the major regulator of blood clot dissolution. We have produced high affinity monoclonal antibodies that induce functional a2AP deficiency. We have shown that these monoclonal antibodies cause venous thrombi and pulmonary emboli to dissolve in vivo. They also accelerate the dissolution of cerebral arterial thrombi-thereby reducing stroke size without increasing bleeding. In this Phase I application, we will modify these promising antibodies by molecular engineering techniques to convert them into potential therapeutics suitable for human trials. In Aim 1 we will engineer and express a chimerized antibody and antibody fragment (Fab). In Aim 2 we will evaluate the relative abilities of the antibody and antibody fragment to bind and inhibit a2AP and enhance blood clot dissolution. With successful completion of these aims we will pursue a Phase II application to optimize the production of these molecules in order to examine their safety and efficacy in suitable pre-clinical models. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is the leading cause of death worldwide. Each year ~ 1.6 million Americans suffer a heart attack or stroke. The resulting death and disability costs the U.S. a staggering $316 billion a year. Current therapies are associated with partial success, restricted access, delays, possible neurotoxicity and other important limitations. This project seeks to develop a novel therapy for heart attacks and strokes that could markedly reduce death, disability and costs.


The University of Louisville proposes to establish a Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases (CPMBEID). Our long-term objective is to support the development of gene-based countermeasures and diagnostic approaches based on system changes that occur during host-pathogen interactions. Focusing on host-based bio-threat and emerging infectious disease research should allow the development of broad-spectrum interventions to infectious agents rather than the current paradigm of "one bug/one vaccine" approach so prevalent today. Our specific aims are to 1) meet the expanding needs of current researchers who work with Category A, B, or C priority pathogens at the University of Louisville; 2) expand facilities so that we can add an additional six faculty investigators who work with these agents; 3) assemble a unique mix of technologies in a free standing BSL3 facility so that the instrumentation and expertise is available on site to manipulate experimental materials and avoid the need for shipping material off site; and 4) to serve as a resource for investigators from other institutions in our region who can benefit from the unique mix of technologies we will assemble in the facility as well as be prepared to bring those technologies to bear in the event of a bioterrorism emergency. The University created a superb team of investigators and administrators to shepherd this project from application to operation. We are committed to working with NIAID and believe our missions can be mutually reinforced by building a RBL in Louisville.


The goal of the proposed research is to identify the functional groups at the active site of the mitochondrial F1-ATPase that participate in substrate binding and in the bond-making and bond-breaking steps that occur during catalysis. Analogues of ATP that are potential affinity labels for the enzyme will be synthesized for these studies. These analogues will also be used to determine if a nucleotide binding site exists in the enzyme in addition to the one that we have identified in the beta subunit with p-F-sulfonyl(14C)benzoyl-5'-adenosine. In addition studies on the topography of the ATP synthetase complex will be carried out. These studies will involve the modification of soluble coupling factors with soluble mercaptoalkylimidoesters followed by modification with Ellman's reagent to produce zwitterionic mixed disulfides that reduce the positive charge on the lysine residues to which they are attached by one. The zwitterionic derivatives will be fractionated by ion exchange chromatography and the resolved species will be converted to mixed disulfides which contain mitroaryl azides by reaction with 4-azido-2-nitrobenzenemercaptan. The modified coupling factors will be photolyzed in the presence of the F1-ATPase and depleted membrane preparations. Specific cross-links will be identified by two dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate in which the cross-links are cleaved after the first dimension. Since it contains the catalytic site for ATP synthesis and hydrolysis, the complete amino acid sequence of the beta subunit is being determined.


This competing renewal application is to provide for the continued maintenance of and activities associated with the SWAN Repositories of serum, plasma, urine, DNA and transformed cells generated from a 10-year study of a population of 3302 women from 5 ethnic groups who have been evaluated annually prior to, during and following the menopausal transition. These Repositories, an arm of the Study of Women's Health Across the Nation (SWAN), is meant to support, facilitate and extend the Core SWAN; additionally, the Repositories provide a mechanism for opening the resources of SWAN to the greater scientific community. Implementing activities associated with three proposed specific aims of this competing renewal will 1) provide for the continued management of the current 1.7 million Repository specimens and the additional specimens that will accrue as a result of fielding SWAN IV in 2009 to 2014; 2) expand the DNA Repository, the most frequently requested specimen type that is uniquely renewable because of our investment in cell immortalization; 3) promote effective information interchange about the SWAN Study, its data and the Repository resources through development of a 2-level web-based data warehouse; 4) provide for continued administration of the application review process for specimen utilization and administrative management of specimen distribution including Material Transfer Agreements; 5) engage in strategies to promote utilization of specimens; and, 6) expand the scope of the genetics studies associated with the SWAN study and its Repository.


Sequencing of the human genome and those of other organisms has revealed that a large percentage of pre-mRNAs are spliced in multiple patterns to produce mRNAs encoding distinct proteins. Although alternate splicing is widespread and must regulate the expression of thousands of gene products, the fundamental mechanisms that control it are not yet well understood. The RNA binding protein Tra2 has been shown to affect the regulated alternative splicing of several pre-mRNAs. In humans these include splice site choices in disease-associated genes such as SMN, tau and CD44. In Drosophila, Tra2 is required for the alternate processing of multiple mRNAs that are necessary for the regulation and realization of sexual differentiation. These include those of the doublesex, fruitless and exuperantia genes as well as tra2 itself. While the splicing activation function of Tra2 in doublesex RNA processing has been well studied and has served as an important paradigm for developmental control of exonic splicing enhancers, little is known about how Tra2 affects splicing of other pre-mRNAs. In this project the mechanism by which Tra2 represses splicing of a specific intron in its own pre-mRNA will be investigated and compared with regulatory elements used in the activation of doublesex splicing. Using an in vitro splicing assay in which recombinant Tra2 specifically blocks splicing of the M1 intron by Drosophila nuclear extracts, the regulatory elements through which Tra2 represses splicing will be identified and the hypothesis that Tra2 blocks the activity of a required exonic splicing enhancer upstream of the intron will be tested. Regulatory complexes that form on the Tra2 pre-mRNA will be compared to those that it associates with in the dsx splicing enhancer to define key elements that determine activation or repression of splicing. The veracity of mechanistic models developed from in vitro splicing studies will be tested by generating transgenic fly strains in which pre-mRNA and regulators with altered sequences are expressed in the tissue and stage of development where Tra2-dependent repression normally occurs. In further studies genetic approaches will be used to define the requirements for SR proteins and other splicing factors that cooperate with Tra2 in the control splice site recognition. These studies will elucidate the fundamental mechanisms by which individual splicing regulators can alter splicing in different ways for different pre-mRNAs. [unreadable] [unreadable]


A significant part of our investigation is concerned with the structure elucidation of fifteen new substances that we have isolated from cancer urine. Eleven of the fifteen unknowns (E,F,G,H,J,K,L,M,N,O, and P) are from the urines of colon carcinoma patients, three (unknowns D,I and Y) are from the urine of chronic myelogenous leukemia, and one (unknown X) is isolated from the breast carcinoma urine. In addition to these fifteen, three (unknowns alpha, beta, and gamma) are isolated from the normal urines. Preliminary information through uv, nmr and chromatography methods indicates that these compounds can be classified into the purines, pyrimidines, imidazoles and nucleosides. We suggest that these substances are derived most probably from the anabolic and catabolic pathways of tumor nucleic acids in man. Another part of our investigation deals with the study on the source of origin and on biochemical and clinical significance of the four new substances that have recently been identified in the Principal Investigator's laboratory. The new urinary compounds are: N6-succinyladenosine (1), N'-beta-D-ribofuranosylpyridin-4-one-3-carbox-amide (2), 2'-O-methyluridine (3), and 6-amino-3-methyl-5-(N-formylmethylaino)uracil (4). The source of origin of the compounds 2 and 4 will be determined by administration of selected radioactive precursors to normal and tumor bearing rats, followed by urinary analysis. The new anabolic modified nucleosides (1 and 2, Chart II) will be estimated by radioimmunoassay in the urines of the patients with colon carcinoma, chronic myelogenous leukemia, Hodgkin and non-Hodgkin lymphoma and in normal subjects. For the estimation of the compounds 3 and 4, a gas-chromatography-mass-spectrometry method using single and multiple ion monitoring techniques will be developed. On the whole this study will provide structures of several new urinary purines, pyrimidines, nucleosides and related compounds. Some of them in conjunction with other substances may well serve as quantitative tumor markers or indicators of tumor activity in assessing the treatment of certain cancers.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Detailed understanding of the molecular mechanisms of muscle contraction and the many other motile mechanisms involving actin-myosin type interactions is now facilitated by the availability of high intensity synchrotron X-ray sources. Limiting factor in this research, however, is current X-ray detection systems which fail to provide required time and spatial resolution simultaneously. The most promising design for detectors use scintillating phosphors coupled to charge coupled device (CCD). As the current phosphor screens exhibit long persistence effects and lower light conversion efficiencies, the suitability of these systems for proposed demanding time-resolved studies is contingent on having a phosphor with the fast decay characteristics, high spatial resolution, and high conversion efficiency. To address these specific needs, we propose to develop a large area imaging detector based on a novel structured scintillator. The scintillating screen will be fabricated using a sputter deposition process. For detecting X-ray energies typically used in macromolecular crystallography, this sensor will provide high resolution, high detection efficiency, and a fast time response, allowing the full potential of the modern synchrotron sources to be realized. Testing at BioCAT involves pairing these new scintilators with commercial EMCCD based cameras which have the promise to be useful, very cost-effective, X-ray detectors for specialized time-resolved applications


This is an open label Phase I study. At least 10 HIV-infected pregnant women between 14 and 28 weeks gestation will be enrolled. The women will receive triple combination therapy with indinavir, 3TC, and ZDV during the antepartum period, discontinue indinavir at the start of the intrapartum period while continuing 3TC, and ZDV, and restart indinavir, 3TC and ZDV during the post partum period thru the first 12 postpartum weeks. The mother and her primary care physicians will decide if the mother will continue the same regimen after 12 weeks post partum. The infant will receive 3TC and ZDV at birth and for six weeks following birth. Pharmacokinetic evaluations on cervical secretions and plasma will be performed at the antepartum, intrapartum, and postpartum visits in the women and postnatally in the infants. Baseline and study safety evaluations will include the monitoring of adverse experiences, clinical laboratory tests, physical examinations and vital signs. Temporarialy closed to enrollment awaiting FDA approval for modification.


7. PROJECT SUMMARY/ABSTRACT High-reliability organizations (HROs) such as aircraft carrier flight decks and nuclear power plants are organizations that operate hazardous technologies in a nearly error-free manner under trying conditions rife with complexity, interdependence, and time pressure. Case studies of HROs as well as in a few healthcare organizations that follow the principles of HROs suggest that a robust safety culture enables more reliable work processes, and thus safer performance. More tangibly, safety culture can be seen ?coming to life? in HROs through specific behavioral processes observed in front-line employees collectively termed collective mindfulness (CM). These five interrelated behavioral processes (also called safety organizing behaviors) are: (1) preoccupation with failure; (2) reluctance to simplify interpretations; (3) sensitivity to operations; (4) commitment to resilience, and (5) deference to expertise. Healthcare is increasingly examining and adopting the principles of CM as a means to improve quality and safety of care delivery. The critical need for achieving HRO status in healthcare is no more apparent than in neonatal perioperative care, which is among the highest risk services hospitals may provide. Neonates are highly vulnerable to iatrogenic events due to their small size, fragility, and exceptional sensitivity to environmental stressors. The objective of this pilot study is to measure the prevalence of CM in neonatal intensive care unit (NICU) and operating room (OR) teams and its impact on non-routine events (NREs) ? defined as any event that is perceived by care providers or skilled observers as a deviation from optimal care based on the clinical situation - during neonatal perioperative care. The project will leverage an active large prospective observational study (IRB-approved NICHD R01) that is defining the epidemiology of risk for neonates in the perioperative environment through an innovative analysis of NREs and contributory factors. We propose a two-year pilot study to characterize CM behaviors in NICU and OR teams and to measure their impact on patient safety as measured by the incidence and severity of NREs during NICU-to-OR handovers and subsequent care. Our Specific Aims are to: 1) Conduct a prospective observational pilot study of NICU and OR teams to: a) estimate the prevalence of perceived CM (i.e., self-reported using the SOS) during the perioperative period and b) delineate the relationship(s) between team attributes, case attributes, and perceived CM scores; 2) Determine the impact of perceived CM on the incidence and severity of NREs occurring during and across phases of neonatal perioperative care; and 3) Conduct a preliminary validation of a provisional behavioral marker system, by assessing the concordance of observed (expert ratings of A-V recordings) and perceived CM (self-reported SOS scores) in the same perioperative teams. We anticipate that knowledge gained from this study will lay the groundwork for a multi-center study on the impact of team-based HRO interventions on neonatal safety in the perioperative environment.


A method is outlined for obtaining information about the effectiveness of drug therapies in reducing the coagulability of blood in contact with biomaterials. This information will be gathered such that the relative effectiveness of current clinical drug therapies can be compared quantitatively in their ability to reduce the coagulatility of the blood with specific biomaterials as encountered in surgical procedures. The biomaterial will be exposed to the circulating blood of a canine via an ex-vivo (extracorporeal) test chamber and arterio-venous shunt. The ex-vivo technique has several advantages for evaluating the effect of pharmacologic agents on thrombus formation and coagulation dynamics: a) Ease in controlling blood flow, b) Continual exposure of the test surface to "fresh" blood so that cofactors are not depleted, c) Multiple samples can be run simultaneously, d) The material surface can be easily removed and the resulting thrombus quantitatively analyzed for its structure and composition, including platelet, fibrin, and erythrocyte content.


Identification of persons truly allergic to penicillin and cephalosporin antibiotics is still a major unresolved and expensive public health problem. Existing licensed skin testing material detects less than half of persons susceptible to anaphylactic reactions to penicillin, is of no value in diagnosing hypersensitivity in non-urticarial drug associated rashes, and when positive in urticarial rashes has the effect of over-estimating the extent of cross-hypersensitivity with other penicillins and cephalosporins. A major improvement in clinical skin testing materials for diagnosis of non-penicilloyl reaginic hypersensitivity will be sought by characterizing antigenically active fractions obtained by chromatography and isoelectric focusing from minor determinant solutions. Polylysines bearing sidechain-linked penicillin with nucleus modified to mimic minor determinants will be evaluated for their relevance in detecting allergy to minor determinants and compared with isolated purified MDM antigens. Evidence for a cell mediated immunopathogenesis will be sought in patients experiencing non-urticarial drug rash responses or allergic interstitial nephritis reactions to beta-lactam antibiotics. A guinea pig model of cell mediated immunity (CMI) to beta-lactam antibiotics will be used to validate in vitro assays for CMI (antigen-dependent DNA synthesis; agarose leukocyte migration inhibition) and to study the mechanisms and treatment of allergic interstitial nephritis. The CMI response of normal and hypersensitive humans will be compared using in vitro assays, and evidence will be sought for tolerogenic and suppressor mechanisms. Diagnostic testing systems that are practical and clinically useful as well as new information on mechanisms of reactions and potential modes of desensitization will result from these studies on penicillin allergy.


Melanona represents an important cause of cancer-related mortality in the Western world, and its incidence has increased steadily over the past 3 decades. Advanced melanoma remains largely refractory to most therapeutic approaches;in part, this may reflect the considerable genetic complexity underlying melanoma and most other solid tumors. To this end, a main goal of cancer genomics is to characterize genetic alterations at a large scale within human tumors, in hopes of deriving molecular classification schema that might aid targeted therapeutic interdiction. High-density SNP microarrays, which simultaneously interrogate more than 100,000 SNP alleles, have recently been adapted for high-resolution cancer genome analysis. Prior work by the Sellers/Meyerson group and others showed that SNP arrays enabled detailed characterization of chromosomal gains, losses, and loss of heterozygosity (LOH) regions, even in the absence of matched normal samples. In addition, preliminary studies on the NCI60 panel of cancer cell lines demonstrated that combined analysis of SNP array and gene expression data, followed by functional validation, represented a powerful means of tumor classification and cancer gene identification. We are therefore applying an integrated, functional genomic approach to the study of metastatic melanoma. Specifically, high-density SNP array and comprehensive gene expression data will be collected on a large set of patient-derived, short-term melanoma cultures. Computational algorithms will then be employed to define and order the meaningful deletions, amplifications, and LOH regions across the sample set. Next, gene expression data will be combined with chromosomal information to identify candidate cancer genes within relevant genomic lesions. Finally, selected candidate oncogenes will be validated functionally by a lentiviral shRNA approach. These approaches should provide robust grounds for melanoma classification and gene discovery, thereby offering new options for future therapeutic interdiction.


Dental caries is the single most prevalent chronic disease of childhood. Early childhood caries (ECC), a particularly severe form of caries, affects infants, toddlers and preschool children younger than 6 years. Among the general U.S. population, the prevalence of ECC is 25% among children 2-5 years (NHANES III). More recently, NHANES data from 1999-2002 found that the prevalence of caries in young children had increased from the 1988-94 time period, from 24% to 28% in 2- to 5-year-old children (Beltran-Aguilar, et al, 2005). As in the previous surveys, the 1999-2002 NHANES data revealed demonstrated profound disparities in Dental caries prevalence and severity based on race/ethnicity and poverty status. Among American Indian and Alaska Native (AI/AN) children, however, the disease burden is much higher, with 79% of 2-5 year old AI/AN children having ECC (IHS, 2001). The reason for this disparity is not understood, partially because a comprehensive study on the etiology of ECC in this high-risk population has not been undertaken. Until the etiology of the disease is fully understood, there is little hope of developing effective preventive strategies. The overall goal of this study is to identify risk factors for ECC among AI/AN infants and toddlers and to determine if Streptococcus mutans (SM) alone, or in combination with environmental and behavioral factors, increases risk of caries in Native American children. The Specific Aims are: 1) To determine the temporality and fidelity of transmission of Streptococcus mutans in Native American children, 2) To characterize expression of virulence determinants of specific clones of Streptococcus mutans, 3) To determine the composition of total cultivable flora, total Streptococcus mutans and total acid flora in plaque samples of Native American subjects over time, 4) To determine the incidence of ECC in Native American children through age 36 months, and 5) To identify dietary and nutritional risk factors for acquisition of virulent SM clones and ECC in Native American infants and toddlers. Working closely with the Health Board of the Aberdeen Area Tribal Chairman, we will work with the Oglala Sioux tribal community at Pine Ridge, South Dakota from which we will recruit 200 Native mother/child dyads within 1 month of delivery. Information on potential risk factors and caries status will be collected every 4 months until the child is 36 months. As part of our effort to train AI/AN researchers, we will hire a local program coordinator and Dental hygienists. We believe that this assessment of vertical transmission of specific SM clones using DNA fingerprinting approaches will provide valuable information on the acquisition of these cariogenic bacteria in these Native American children. [unreadable] [unreadable] [unreadable]


The major focus of research in my laboratory is transcriptional control of the pro-opiomelanocortin (POMC) gene. In particular we are interested in transcriptional factors that mediate the effects of stress and neuroimmune modulation on the POMC gene. We have previously described a transcriptional activator of POMC, designated PO-B, that interacts with the POMC promoter. PO-B represents the first defined transcriptional activator of POMC whose function has been verified by mutational analysis. Mutation of the PO-B binding site decreases basal transcription by 70% indicating a major role for this protein in POMC regulatory mechanisms. However, we do not know the physiological or pharmacological agents that regulate signal transduction pathways involving PO-B. Towards this goal we have recently purified PO-B to more than 90% homogeneity from HeLa cells. Interestingly deophosphorylation of the purified protein leads to a more 30-fold increase in DNA binding affinity. In this proposal we wish to rapidly extend these early observations by obtaining a full length cDNA clone of the PO-B gene. This is a defined project of limited duration that will enhance our knowledge of POMC transcriptional regulation and be a useful resource for other work ongoing in the laboratory. Since this laboratory was the first to identify and purify PO-B we would also like to obtain the full length clone in a timely fashion. The specific aims are: 1) To obtain reliable N-terminal and internal amino acid sequence data from purified PO-B. "Best-guess' oligonucleotide probes will be designed from these sequences. Specific PO-B cDNA sequences will be amplified with PCR from a human cDNA library using pairs of these oligonucleotides representing N-terminal and internal sequence. Amplified fragments will be recovered and sequenced to determine if they code for PO-B amino acid sequence. If this approach is unsuccessful, or only N-terminal sequence is obtained, we will use conventional screening of a HeLa lambda gt10 cDNA library identifying positive PO-B cDNA clones by hybridization with the 'best- guess' oligonucleotide probes. 2) PCR and conventional screening methods are not always successful approaches to identifying correct cDNA clones. Therefore, in parallel with the above experiments, we will also use expression screening. This method does not rely on obtaining any amino acid sequence from PO-B. For these studies we will screen a human fibroblast plasmid cDNA library that can be transiently expressed in mammalian cells. The identity of the full length clone will be confirmed by specific binding of the in vitro translated product to the PO-B cognate DNA binding element. The in vivo activity of the full length clone will be confirmed by the ability of transfected PO-B expression vectors to induce transcription from promoters harboring the PO-B binding site. If progress on PO-B cloning is rapid, we will examine its precise role in POMC transcription. We will perform mutational analysis of the protein to determine which domains are required for DNA binding, phosphorylation status and transcriptional activation. It is expected that these experiments will yield valuable information on the signal transduction pathways in which PO-B is involved.


Organisms capable of dramatic regeneration maintain adult cells that have the flexibility, or plasticity, to change their fate. This feature enables thm build replacement parts, like organs or limbs, to recover from damage or disease. Previous work on the project and other studies have revealed a broad propensity for specialized cells to display some properties of stem cells; they give rise to new cell types in response to injury. The dynamic changes that cells undergo during the transition from one fate to another could reveal the mechanisms that cells employ to enable them to switch their fates. Such insights will reveal basic features of cellular plasticity that could improve regeneration therapy in human cells. However, due to limitations in analyzing the global status of regenerating cells in vivo, little is known about how cells make the traversal from one state to another. The experimental plan proposes to provide a comprehensive view of cells as they transition to new fates during regeneration. The study utilizes plants because the model system provides a rare opportunity to model cellular plasticity. In addition, the mechanisms that regulate plasticity are well conserved across plants and animals and the plant's adept ability to regenerate will illustrate the full potential of cells to change their identity. The proposal takes advantage of a powerful model system that permits continual imaging of regenerating tissue and the analysis of the transcriptional contents of single cells as they traverse fates. This system includes an inventory of active genes for each cell fate, permitting quantitative analyses to track the complex identity of cells as they regenerate. Thus, the complete analysis will generate a model of the trajectory of regenerating cells in order to address basic questions about regeneration: What is the origin of the highly plastic cells that participate in regeneration? Do regenerating cells show proliferative behavior that resembles stem cells? Do fate transitions require losing all identity and reaching a ground state or younger developmental stage (dedifferentiation)? Or, do cells traverse directly to new fates? The results will point to processes that can be targeted to make regeneration more efficient. The innovation of profiling the complete transcriptional dynamics of cells and projecting them onto images of regenerating tissue is widely applicable to many developmental model systems. Thus, overall, the proposed work has broad impact on the field of regeneration.


To construct lotteries with abstract individual choice structures identical to critical organizational collective-outcome experiments involving voluntary participation. Two essential characteristics to be built into the lotteries are these: 1) the collective-outcomes (momentary rewards in these experiments) will be available to the subject whether he is present at a drawing or not, but the probabilities of the various outcomes will differ according to whether or not he is present; and 2) attendence at a drawing entails opportunity costs (e.g. the effort and time required to attend a drawing.) In the organizational research (by McMahon and Camilleri), the ratio of the utility $0.75 to $2.00 varied from nearly zero to close to unity in organizations differing by authority and consensus structures. The primary objective of the present research is to determine whether variations in the utility of monetary pay-offs observed in the organizational experiments can be attributed strictly to the number of possible outcomes in choice structure, or whether these variations are due to aspects inherent in the social aspects of the organizational experiments. S's will be offered 10 opportunities over five weeks to participate in a pure lottery. Three experimental conditions will be developed and executed, differing according to the number of outcomes that can occur, each outcome entailing a specific monetary reward ($2.00, $0.75, $0.00.) The lotteries will be performed by a remote terminal connected to the central CDC 6500 computer. The probabilities of participation (attendence) and the utility ratios at issue will be estimated by use of a choice model developed by Camilleri and Berger.


The objective of this proposal is the continued synthesis and development of anti-convulsant drugs related to 1) those dopamine agonists which can be considered as congeners of apomorphine, 9,8-dihydro-6H-dibenzo[c,g]azonines, and amonotetralin, and 2) aminoisoquinolines, and their evaluation as anticonvulsant drugs in the Anticonvulsant Screening Project (ASP) carried out by the NINCDS. In this research program we shall also investigate 1) the relationship of dopamine agonist response on the basis of the activity on DA sensitive adenylate cyclase, the competition of binding to various 3H-ligands compared with their anticonvulsant activity both from the ASP screen and from audio- or photic-induced seizures in rodents and baboons; 2) the continued study of the chemistry of aminoisoquinoline to make structural changes in this heterocyclic system and to assess the SAR in a systematic approach to structural modification; 3) the study of the acid catalyzed rearrangement of opium alkaloids which would lead to novel aporphines and related structures of interest for biological evaluation; 4) the continued development of sensitive methods for the quantification of N-n-prophylnorapomorphine (NPA), 2,10,11-trihydroxyaporphine (TNPA) and certain pro-drug derivatives of these agents in biological fluids using electron capture gas chromatography in combination with mass spectrometry. In order to facilitate absorption and pharmacokinetic properties, pro-drug derivatives of the most active anticonvulsant compounds will be investigated.


Bacterial identification using DNA hybridization probes has become increasingly sophisticated and accurate as probe technology and methods have improved. Up to now, however, instrumentation for detecting fluorescently- labeled probes has not been able to fully exploit the capabilities of such probes to identify high complex mixtures of genotypes in situ. Positive identification of single variant organisms in large populations of similar cells is also problematic. Fluorescent labeling methods are ultimately limited by the number of different spectral 'fingerprints' that can be distinguished by the imaging system that is used to measure and sort them. We propose to use a set of compatible fluorophores and recently developed KAIROS instrumentation and spectral deconvolution and sorting algorithms to increase the number of fluorescent tags that can be simultaneously distinguished, thus enabling accurate 'fingerprinting' of bacteria. In Phase 1 we will develop a set of these probes and demonstrate their efficacy on microscopic test targets. In Phase 2 we will apply the library of probes and the spectral deconvolution software to correctly identify highly complex mixtures of cells in situ by spectral sorting. This new technology for multispectral bacterial identification (Mbid) will benefit clinical and environmental microbiology as well as biotechnology. PROPOSED COMMERCIAL APPLICATION: Rapid and accurate fingerprinting of bacteria by multispectral fluorescent probes and fluorescence imaging microspectrophotometry is an enabling technology for both clinical and environmental aspects of microbial ID.


The only solution to the HIV epidemic in the developing world is a vaccine that either prevents infection or reduces transmission. Recent data from our laboratory suggest that CD4+ T cell responses along with CD8+ T cell responses against subdominant epitopes can lead to control of replication of SIVmac239. The goal of this proposal, therefore, is twofold. First, we would like to investigate the importance of CD4 help in an HIV vaccine. Second, we want to use innovative new technologies to vaccinate for specific peptides rather than whole proteins. This would eliminate the use of large, commonly seen vectors, thereby circumventing immunodominant responses against vector-derived epitopes. Our new vaccination regimen should induce both CD8+ and CD4+ T cells that efficiently control viral replication. Using newly developed, novel vaccine regimens, we hypothesize that vaccinating macaques with CD4 and subdominant CD8 epitopes will generate immune responses that should control SIV replication. In Specific Aim I of the R21, we will induce CTL specific for subdominant Mamu A*01-restricted SIV epitopes. In Specific Aim II we will engender multiple CD4+ T cell responses against epitopes that are commonly seen in SIV-infected elite controller rhesus macaques. In the R33, we will use the most effective vaccine regimen(s) from the R21 phase to engender CD4+ T cell responses and subdominant CD8+ T cell responses. Animals will be vaccinated with subdominant CD8+ T cell epitopes, CD4+ T cell epitopes, or a mixture of the subdominant CD8+ T cell and CD4+ T cell epitopes. These experiments will allow us to elucidate the role of CD8+ T cell responses against subdominant CD8+ T cell epitopes and CD4 help in an effective HIV vaccine. Project Narrative: Recent vaccine trials have failed to protect individuals from HIV infection or reduce transmission of the virus. Using newly developed vaccine modalities, we now have the tools to circumvent problems characteristically seen in common vaccine vectors as well as address the contribution of individual immune responses against HIV.


The Center for the Neural Basis of Cognition offers an interdisciplinary Ph. D. level educational program intended to create a cohort of excellent researchers who bring the skills, insights, and perspectives from a wide range of existing disciplines into the emerging new discipline of Cognitive Neuroscience. This program has now been in operation for almost 10 years. The program takes students from seven different departments spread over Carnegie Mellon and the University of Pittsburgh. Students are required to pursue a course of study including four core courses (Basic Neuroscience, Systems Neuroscience, Cognitive Neuroscience, and Computational Neuroscience) that often overlap very little with the core requirements of their home departments. Students also participate in a series of student and faculty research presentations, a student-run colloquium series and an annual retreat in which current issues are considered through a combination of research presentations and discussion groups focusing on special issues within the broad range of scientific activities encompassed by the Center for the Neural basis of Cognition, and students are encouraged to attend national and international meetings to gain exposure to contemporary research. There are currently 51 participants in the program, and the program's 17 graduates are all pursuing research careers, many of them either as assistant professors or as post-doctoral fellows in outstanding laboratories. We are adding a new research ethics series to the program and are joining in a broad recruitment effort to increase diversity among the participants. The program has been supported since its inception by an NSF research training grant that has now run its course and is not renewable. The current proposal seeks funding to allow the continuation of this program.


Studies on the regulation of cell proliferation and growth are important to our understanding of developmental biology and tumorigenesis. The long-term goal of the proposed research is to investigate the molecular mechanisms that regulate cell proliferation and growth. In order to address this question, the investigators will analyze the Tsc1 and Tsc2 tumor suppressor genes as well as genes interacting with them in the developing imaginal tissues of Drosophila melanogaster. Mutations in either TSC1 or TSC2 result in a hereditary disease - tuberous sclerosis. The disease occurs in 1/6000 births and is characterized by benign neoplasms known as hamartomas, in multiple organs. Tsc1 and Tsc2 are functionally conserved from flies to humans. The investigators have previously shown that Tsc1 and Tsc2 function together in the Insulin/PI3K/Akt signaling pathway, downstream of Akt and upstream of S6K. Tsc1, Tsc2, and other upstream components of the pathway affect both cell proliferation and growth, however S6K only affects cell growth. The mechanism by which Tsc1 and Tsc2 regulate cell proliferation is not yet known. The investigators propose to investigate the mechanism by which cell proliferation is regulated by Tsc1 and Tsc2. Specifically, the investigators propose to identify Tsc1/Tsc2 interacting genes by performing genetic modifier screens and by using microarray to identify genes regulated by Tsc1/Tsc2. The investigators will then select several Tsc1/Tsc2 interacting genes and further characterize the interaction between these identified candidate genes and Tsc1/Tsc2 as well as their role in the regulation of cell proliferation. This application for the mentored K08 award is submitted so as to enable the candidate to gain necessary didactic and scientific training in developmental and molecular genetics. The eventual goal is to independently investigate molecular mechanisms underlying human disease and develop targeted therapeutic strategy utilizing model organisms.


The focus of this U19 project, "Immunobiology of Acute Environmental Asthma", is to conduct mechanistic studies of the role of innate immune processes in mediation of acute environmental asthma. Epidemiological studies have clearly shown that asthma exacerbation linked to air pollutants is a major cause of asthma exacerbation. Endotoxin is a commonly encountered environmental pollutant found in ambient particulate matter and in occupational and domestic settings as well. Endotoxin can induce neutrophilic inflammation at high levels, and at low levels enhance response of asthmatics to airway allergen challenge. We have shown that endotoxin causes changes in airway monocytes and macrophages (increased CD14, CD80, Fc?RI and HLA-DR) which are associated with enhanced response to either innate or acquired immune stimuli. Our team has recently identified novel regulatory molecules of inflammation in the CATERPILLER family, specifically cryopyrin (which upregulates neutrophilic inflammation and monocytic function) and monarch-1, (which downregulates response to innate activation). Cryopyrin is of particular interest as it acts through formation of a Caspase-1 based inflammasome to cleave pro-IL-l? to active IL-1? (with downstream production of IL-6 and other mediators) and IL-18. Furthermore, cryopyrin is activated after ligation of either pathogen associated molecular pattern receptors (including CD14 facilitated binding of endotoxin by TLR4) or the P2X7 receptor by ATP (which is increasingly recognized as an endogenous danger signal released by host cells following non-specific cell injury). These dual activation pathways likely account for the similar actions of a wide variety of inhaled environmental contaminants, and represent novel targets for treatment of acute asthma. We will conduct mechanistic studies of the role of the CATERPILLER family members cryopyrin and monarch-1 (Project 1-J. Ting PI), and the role of NALP-1 and the purinergic receptor P2X7 (Project 2-B. Koller, PI) in rodent models of environmental asthma, in conjunction with a translational project (Project 3-D. Peden, PI) designed to determine the effect of interaction of endotoxin- and allergen-induced inflammation on the airway biology of allergic asthmatics. Human studies will be focused on the biology of airway monocytic cells and the expression of innate immune and the CATERPILLAR family of immune regulators and P2X7 receptors in the airway. In addition to assessment of the role of innate immune processes in regulating airway inflammation, we will examine the relationship of this inflammation on airway physiology, specifically mucociliary clearance. Decreased mucociliary clearance is an understudied process which mediates asthma exacerbation and is a feature of increased asthma severity. [unreadable] [unreadable] [unreadable] PROJECT 1: Novel and innate immune genes in asthma (TING, J) [unreadable] [unreadable] PROJECT 1 DESCRIPTION (provided by applicant): We recently discovered the CATERPILLER family which share structural similarities with the NB-LRR (nucleotide-binding, leucine-rich repeat) super-family of disease resistance (R) proteins that constitutes the plant immune system. In the animal kingdom, this family is also known as NOD or NLR. The clinical importance of this family is underscored by the genetic linkage of family members to a number of immunologic disorders. Among the human gene family members, several of these appear to mediate negative regulatory function in controlling an overzealous inflammatory response. Most notable is the Monarch-1 protein which blocks the function of NF-?B inducing kinase (NIK). Inhibition of NIK reduces the expression of an array of chemokines with relevance in asthma. Gene profiling of induced sputum from mildly asthmatic individuals suggests that the Monarch-1 gene is reduced in these individuals relative to controls, supporting the inhibitory role of this gene during inflammation. Another group of family members regulates IL-1 production. Most notable among these is cryopyrin which mediates formation of the inflammasome complex upon stimulation with a number of inducers. The inflammasome complex is required for procaspase 1 processing to mature caspase 1. In turn, caspase-1 is required for the processing of pro-IL-1 and pro-IL-18 to their mature forms. IL-1 and IL-18 are respectively important in inflammation and TH2 skewing. Cryopyrin is also important in mediating macrophage necrosis which exacerbates inflammation. Thus there are compelling reasons to believe that Monarch-1 and cryopyrin have crucial roles in asthma, however there is no in vivo data to indicate that this is the case. Furthermore we have shown that both of these proteins are ATP-binding proteins, and they exhibit ATPase activity, thus providing ways to modulate their function, which might be important leads to drug discovery. The goals of this proposal are: (1) To study the relevance of Monarch-1 in three animal models of asthma (OVA-induced, endotoxin, and house dust mite and delineate the biochemical effects of Monarch-1 in vivo and ex vivo. (2) To study the relevance of cryopyrin and a cryopyrin-adaptor (ASC) in asthma. (3) To study and identify factors which modulate the nucleotide-binding properties and ATPase function of Monarch-1 and cryopyrin. [unreadable] [unreadable] [unreadable]


The purpose of the proposed project is to provide minority (Black) undergraduate students in the Department of Biology, a carefully designed biomedical research that focuses on the use of video microscopy and digitized image analysis to monitor morphological changes in normal and malignantly transformed cells and to assess the impact of morphological modulations on tumorigenesis. The initial investigation will attempt to initiate morphologically variant cell types from transformed hepatocytes and a tumor cell line derived from mouse adenocarcinoma. Clonal derivatives from these cells will be examined, through the use of video counting and microdensitometry, for differences in their uptake and retention of fluorescent dyes (cell density), total cell area, shape factor (fraction for estimating the amount by which a cell varies from a circle), parameter, and nuclear/cytoplasmic ratio of normal and malignantly transformed cells. The clonal isolates will be used to determine the histological types of tumors arising from implantation of specific phenotypically variant cell lines. Karyomorphology of phenotypically variant cell lines will be determined to monitor possible chromosomal changes that may attend in vitro cytodifferentiation. It is felt that morphological and karyomorphological analysis of cells from a single tumor but variant phenotypes will provide valuable information on the series of cytological changes associated with cellular differentiation.


Continuation of the animal resource diagnostic and investigator laboratory in the Unit for Laboratory Animal Medicine is requested. The program consists of clinical, laboratory, and research elements which are guided by the academic standards of excellence prevailing at the University. The objectives are enhancement of experimental medicine and advancement of laboratory animal medicine. The specific aims are to diagnose and control naturally occuring diseases in animal colonies at the University, to call attention to problems which could disrupt research; to conduct research in laboratory animal medicine, including research on spontaneous or induced animal diseases, particularly diseases which could serve as models of human disorders; and to promote research training and education in laboratory animal medicine. Methods to be used will continue to include those of clinical medicine, clinical and anatomic pathology, immunology, and other relevant disciplines. Current research includes characterization of pathologic changes associated with aging in rats, immunobiology of Pasteurella multocida in rabbits, neutrophil function in rabbit pasteurellosis, animal model of thyroid dysfunction, latency and transformation of herpes viruses, and others.


Ultimate long term success of an auditory prosthesis requires an electrode design which is flexible enough for easy insertion, can bond sufficiently with metals to withstand bending and eliminate wicking of physiological fluids initiated at electrode contact points. Three fundamental objectives related to a flexible scala tympani electrode will be addressed: (1) Adhesion of metallic conductors to polymer (insulator) substrate, (2) etching and processing of metallic conductor to obtain optimum geometry and (3) evaluate physical properties of metal/polymer composite. A porous polysulfone will be evaluated for metal adhesion under hydrated conditions and with appropriate current stimulation. Delamination is a severe problem with present electrode configurations. Preliminary work has demonstrated that porous polymer surfaces enhance metal adhesion by mechanical interlocking. Preliminary studies with metal coating on porous polysulfone have demonstrated excellent adhesion even under prolonged hydrated conditions. Control of polymer surface energetics and surface porosity appears to enhance metallic coating adhesion sufficiently to warrant further investigation. A porous polysulfone will be evaluated for long term (greater than 2 months) adhesion, using Titanium undercoatings followed with platinum and rhodium coatings. Adhesion will be evaluated under hydrated conditions with appropriate levels of current stimulation. Flexural properties of electrode composites will be evaluated and compared to existing discrete 6-wire electrode systems currently in use for cochlear stimulation. Physical properties of the polymer-metal composite will be evaluated for processing via photolithography techniques. Both chemical and reactive ion etching (RIE) processing will be studied for generation of suitable electrode geometries compatible with current microcircuit fabrication technology.


We propose to develop Gene Therapy vectors that are targeted to particular cell membrane receptors, cell types and/or tissues. Development of these precise delivery systems will help overcome potential adverse reactions and high dosing requirements imposed by the current, non-targeted, traditional Gene Therapy vectors. The goal in Phase I is to construct a combinatorial library of chimeric Adeno-associated virus (AAV) vectors to screen for tissue-specific targeting during Phase II. Because of technical limitations, AAV-based combinatorial libraries were not previously available. We plan to use a unique new technology for building and subsequent selection of AAV vector libraries. Specifically, we will exploit molecular breeding of viruses by DNA shuffling to direct evolution in vitro of AAV capsid genes through recombination of multiple homologous parental sequences of known AAV serotypes. To construct the library, we will use a new AAV production technology based on insect cells and genome pseudotyping. Researchers at AGTC and UF Powell Gene Therapy Center, with proven track records in the AAV field, will join efforts to complete the project. During the feasibility study, we will demonstrate whether it is possible to build an AAV-based library of viable viral clones of at least 106 complexity using recombination of capsid genes of AAV1 and AAV2 serotypes. The successful completion of this step will allow us to proceed to the next stage of selection of vectors with a particular cell/tissue tropism completed during Phase II of the project. We anticipate to market AAV combinatorial libraries to users within the gene therapy community, as a product to select for delivery vectors targeted to a particular tissue or tailored to a particular application, e.g. targeted delivery of pro- apoptotic or suicidal genes to tumors. This project will represent a significant advance in the development of therapeutic gene delivery systems with precise targeting to improve patient outcomes.


This project has concerned the development of animal models of neurological disease produced by exposure to synthetic and naturally occurring neurotoxins in the environment. The interaction of various toxins and neurotransmitters in the environment. The interaction of various toxins and neurotransmitters and hormones in the CNS have provided the focus for combined behavioral and neurochemical studies emphasizing basic mechanisms of action of proposed neurotoxins. Major topics studied this past year were: (1) effects of heavy metals on neurotransmitter and neuroendocrine function; (2) neurotixic mechanisms of chemical and hereditary porphyrinopathies; (3) effects of artificial food colors on neuronal membranes and neurotransmission; (4) role of vestibular function in posture and motor function.


Production of candidate chikungunya vaccines for human clinical trials will be done in compliance with current Good Manufacturing Practices (cGMP) and released for use in human clinical trials. This will necessitate developing production methods based upon novel cell substrates and If these trials demonstrate safety and immunogenicity of this vaccine in human trials, further evaluation may take place in larger (Phase 2) trials which will necessitate production of the clinical trial materials (CTM) at large scale.


Accumulated clinical experience contains information relevant to clinical management and the clinical decision process. Utilization of prior clinical experience has been impeded by the frailties of the memory, the statistical biases of retrospectively recorded data, and most importantly, by difficult access to recorded clinical information. These problems are particularly acute in the major rheumatic diseases, where long patient courses, frequent fluctuations, interactions of problems in many organs, and uncertain natural history combine to create complicated clinical data. This project establishes a pilot Rheumatic Disease computer data bank network (ARAMIS), building upon previous national projects in standardization of clinical description. The network enables pooling of clinical data between institutions, inter-institutional studies, rapid access to large quantities of clinical data, and provides computer consultation and clinical decision procedures based upon this data. It demonstrates the feasibility of a chronic disease network, including successful function, fruitful collaboration, and documentation of appropriate levels of utilization. Evaluation of the contribution of the system to patient care, teaching, and research is being carried out by an evaluation team of biostatisticians, health economists, and physicians. The data bank is an essential tool to relate medical process to patient outcome. It can pinpoint areas of non-productive effort, leading to more efficient practice. By increasng the efficiency of clinical management studies it provides new information to the clinician. And by recognizing the unique characteristics of each patient it can assist in the development of individualized approaches to patient management. ARAMIS provides access to detailed, uniform clinical data for larger numbers of patients than previously amassed. Proceeding in concert with the National Arthritis plan, it seeks to provide the physician and the investigator with better information with which to manage the patient.


?DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is the third most common cause of cancer death in the USA. Germline mutations in the apc tumor suppressor gene, one of the key players in the development of CRC and an important component in the Wnt/beta-catenin signaling pathway, are responsible for familial adenomatous polyposis (FAP). Mutations that result in constitutive activation of the Wnt/-catenin signaling pathway can lead to colon cancer. Wnt(s) have diverse roles in regulating cell fate, proliferation, migration, and death. beta-Catenin is highly expressed in many cancer cell types and promotes growth and tumor formation. Elevated beta-catenin activity in carcinogenesis model systems and neoplastic tissues suggests that this enzyme is a valid target for chemoprevention. By using computational biology with BlueGene/L and GPU supercomputers, we have identified/synthesized several small molecule inhibitors of beta-catenin that are highly effective or have good potential to suppress colon carcinogenesis. In this application, we propose to use state of the art technologies to continue to identify, characterize, test and validate novel, nontoxic small molecule inhibitors of beta-catenin. Our approaches include determination of binding, binding affinities, identification of the specific binding sites, and examining the resulting structural changes by computational simulation using our supercomputer systems. We will validate the effectiveness of these inhibitors by performing protein binding assays, cell transformation assays and in vivo animal experiments, including xenograft models and the AOM-induced colon cancer and APCMin mouse models. Through these studies, we will develop more effective agents targeting beta-catenin with fewer side effects for the chemoprevention of colon cancer.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project studies avian heart development. This portion of the project concerns the processing of 4D (time-lapse + multiple focus plane) image sets to deconvolve image features between focus planes, to improve particle (cell tracking) accuracy and apply PSC-VB and other tools to view and analyze image sets.


The long-term goal of this proposed work is to characterize protein-protein interactions involving the enamel extracellular matrix proteins, as well as proteins integral to the plasma membrane of ameloblast cells, and characterize how these interactions regulate enamel formation. To achieve this the molecular biochemistry of biglycan, Cd63, Anxa2 and Lampl, and their relationship to the major enamel matrix proteins, will be studied. The hypothesis of this grant application is "protein-protein interactions involving secreted enamel matrix proteins and the secretory surface of ameloblast cells (Tomes' processes) play a key role in the formation of the enamelprismatic structure." To test this hypothesis we propose three specific aims. These are to: 1) define, using in situ hybridization and mouse-specific antibodies, the spatiotemporal expression patterns for Cd63, Anxa2 and Lampl within the murine developing incisor tooth, and relate these expression profiles to those of their respective ligand (enamel matrix protein partner), and the stages of enamel biomineralization; 2) demonstrate that the protein-protein interactions previously identified for the enamel matrix proteins and biglycan, Cd63, Anxa2 and Lampl occur in vivo and have physiological significance, and to define the minimal binding domains of Cd63 and Lampl that enable their interaction with amelogenin; 3) define and disrupt the receptor-mediated molecular mechanism that permits the enamel matrix expression levels of biglycan to influence or control amelogenin expression levels, and the subsequent events of enamel biomineralization. Knowledge gained from this and related studies may lead to better dental and non-dental materials, or biomimetics. This data will be critical to others in their pursuits to regenerate an entire tooth. For tooth regeneration to become a reality, the protein- protein interactions involving the key dental proteins must be known and understood.


This is the second competing renewal of our Program Project Grant, "Drugs of Abuse: Role of Protein phosphorylation" (DA 10044), which was initially funded in 1996 and again in 2001. The grant has been highly successful based on research productivity and initiation of new research projects. The overall objective of the Program Project Grant remains the same as the original, namely, to elucidate the molecular basis of the actions of drugs of abuse, particularly psychostimulants, in the dorsal striatum and nucleus accumbens. The addictive properties of drugs of abuse such as psychostimulants depend on their ability to augment dopamine neurotransmission in the basal ganglia. The Program Project Grant is organized in four Projects, a Scientific Core and an Administrative Core. The Administrative Core staff will support the integration of the researchers and institutions involved in the Program Project Grant. The main goal of the Scientific Core is to provide a research and technical support facility for all of the Program projects. Responsibilities of the Scientific Core will include the creation, characterization, and breeding of genetically altered animals;the design and execution of yeast two-hybrid studies;the production and maintenance of key reagents stocks and new reagents;and the performance of certain routine tasks required to accomplish the studies described in Projects I-IV. Project I, "Psychostimulants and dendritic spines," will focus on the role five regulators of actin dynamics play as targets of psychostimulants in the dendritic spines. Project II, "The Role of DARPP-32, CK1, and CK2 in Mediating the Molecular and Behavioral Effects of Drugs of Abuse," will extend previous studies of four key phosphorylation sites of DARPP-32 upon in vivo administration of drugs of abuse and chronic exposure to drugs of abuse. Project III, "Striatal Phosphoproteins and the Actions of Psychostimulants," which will be a subcontract carried out at Yale University School of Medicine, study the role(s) of RCS and ARPP-16 in mediating or modulating the actions of psychostimulants as well as study the roles of the three isoforms of PP1 (PPla, P and y) in the actions of psychostimulants. Project IV, "The Role of GPCR-interacting Proteins in the Actions of Pscyhostimulants," will utilize yeast two-hybrid screening to further investigate the multiple intracellular signaling pathways of GPCR and its protein-interacting subunits in mediating actions of cocaine, D-amphetamine, ecstasy and caffeine.


The focus of the proposal is in the area of breast cancer imaging and tumor characterization using oxygen sensitive phosphorescent labels and hybrid imaging modality. We propose to use simultaneous diffuse optical tomography (DOT) and magnetic resonance imaging (MRI) to obtain high spatial-resolution and quantitative functional images of breast tumors. Imaging in pre-clinical models with injected contrast agents and imaging the oxygen concentration within tissue are important for a number of clinical and scientific reasons. Low oxygen content in tumors is a marker of radiation-resistant tumor regions, and is also correlated with a poor treatment outcome. For basic research applications, it is also very important to understand how hypoxia develops in tumors and how it is correlated with exogenous and endogenous markers of low oxygen. During Phase I, in conjunction with standard diffuse optical tomography (DOT), we obtained high contrast images of hypoxic regions in breast tissue simulating phantoms using long-lived oxygen sensitive contrast agents and a simple lifetime imaging system with cooled avalanche photodiodes. In Phase II, a prototype of the CW and time domain lifetime imaging module will be developed which will have parallel acquisition across a cooled 16-element avalanche photodiode (APD) array for high resolution and high throughput imaging. The low-cost, compact lifetime imager module will be designed to be compatible with high magnetic and RF fields associated with MR units in hybrid imaging systems. Using this APD module in a dual-modality imaging setup at our collaborator's facility at Dartmouth College, NH (Prof. Brian Pogue, Thayer School of Engineering), phantom and animal model imaging will be performed to obtain functional optical images with high resolution and high contrast. The optical image reconstruction will be aided by the spatial and anatomical information obtained from simultaneously obtained high-resolution MR images in order to improve the accuracy of these images. The compact APD based detector prototype will have a small footprint and will be mounted inside the magnet of the MRI unit, dramatically decreasing the complexity of the current PMT based detection systems. PUBLIC HEALTH RELEVANCE: Hybrid imaging with simultaneous diffuse luminescence imaging and magnetic resonance imaging can be used to obtain high resolution functional maps of tumors based on their oxygen concentration. Hypoxic breast cancer detection is the primary target application, with the goal of improving diagnostic accuracy and treatment prognosis using exogenous contrast agents. Other applications could include brain imaging to monitor cerebral function or cerebral hemorrhage, and monitoring tissue and muscle oxygenation, histotoxicity, etc. [unreadable] [unreadable] [unreadable]