Over 2 million adult cases of hepatitis E virus (HEV) are thought to occur annually in India alone, and HEV accounts for the majority of acute viral hepatitis hospitalizations worldwide, yet accurate population-based estimates of HEV burden remain scant. The hallmark and most concerning aspect of HEV is a high mortality in infected pregnant women. There have been few rigorous, population-based, prospective studies of HEV incidence, immunopathogenesis and natural history among pregnant women. Our current understanding of the risk factors and host characteristics that lead to severe consequences in pregnant women and neonates is largely based on hospital-based convenience samples. There is a need to carefully define the burden of HEV infection and disease in terms of maternal and neonatal consequences in a typical, endemic resource-poor population. To characterize the population- based epidemiology of HEV and describe maternal morbidity and mortality, pregnancy outcomes, and neonatal survival, we will 1) enroll and follow ~10,000 newly pregnant women through 3 months postpartum in a representative, rural Bangladesh population to identify the trimester-specific incidence of HEV infection, rates of illness, and risk factors for infection and hepatitis E disease and 2) recruit and follow pregnant women hospitalized with acute HEV infection and/or disease to characterize the immunologic, nutritional and other host factors associated with various treatment outcomes, including vertical transmission and neonatal survival. We will also isolate and sequence HEV from acutely ill women, for insight into the molecular epidemiology of HEV. We have over a decade of HEV research experience in this endemic population partnering with the ICDDR,B's Matlab Research Center, and we have estimated, in a smaller study, an incidence of HEV in pregnancy of ~40 per 1000 person-years. Diagnostic support will be provided by one of the most reputable HEV reference laboratories: Dr. Robert Purcell's Hepatitis Laboratory at the National Institutes of Health (Bethesda, Maryland). Co-investigators with expertise in immunology and access to the Johns Hopkins Becton Dickinson Immune Function Laboratory further strengthen this applications. The proposed project will be the first to quantify and characterize HEV infections in a large cohort of rigorously monitored pregnant women, and provide precise estimates of the burden and consequences of HEV in pregnancy and to the neonate. We will clarify the burden of this under recognized contributor to maternal mortality and morbidity in endemic areas, based on large population- based data, providing important information for future prevention and vaccine efforts.


The goal of this proposal is to increase the understanding of the mechanisms contributing the exacerbation of neurological complications in Human Immunodeficiency Virus (HIV) infected drug abusers. The use of dopaminergic drugs, such as cocaine and methamphetamine, increases both the incidence and severity of HIV-induced neurological disease in HIV infected individuals. These drugs act by increasing extracellular dopamine levels in the central nervous system (CMS). Studies show that increased extracellular dopamine exacerbates HIV induced CMS pathology, but the mechanisms that mediate this effect are not fully characterized. Macrophages, the major target for HIV in the CMS, also play a central role in the neuropathology of HIV infection. The preliminary data in this proposal show that dopamine treatment increases HIV replication in macrophages, and demonstrate the presence of dopamine receptors on the surface of the eels. These data indicate that dopamine may play a direct role in the development of HIV induced CMS pathology through dopamine mediated modulation of HIV infection of macropahges. To determine the mechanism mediating this effect, studies in this proposal will fully characterize the pharmacology and kinetics of dopamine-mediated increase in HIV replication in macrophages. HIV infection in the presence of dopamine receptor agonists and antagonists will determine which dopamine receptors mediate the increase in HIV replication in macrophages. To examine the mechanism mediating this effect, studies will examine crosstalk between the active dopamine receptors and the HIV receptor CD4, and coreceptors CXCR4 and CCR5 as well as dopamine-induced alterations in the viral entry, integration, and budding processes. Experiments will also use protein kinase inhibitors during HIV infection to examine the signaling pathways involved in dopamine enhanced HIV replication. Defining the mechanisms by which dopamine increases HIV replication in macrophages will contribute to the understanding and treatment of the heightened incidence and severity of neurological disease among HIV infected drug users. PUBLIC HEALTH RELEVANCE: HIV infection can result in a variety of neurological complications that are exacerbated by drugs of abuse. Defining the mechanisms by which drug abuse increases the incidence and severity of HIV induced neurological complications will increase the understanding of HIV infection in the brain and help to develop intervention strategies to limit the devastating consequences of neurologic impairment in HIV infected drug users.


Approximately 470,000 Americans have end-stage renal disease and most are treated with hemodialysis. A vascular access is required for performing the hemodialysis procedure and can be provided with a native arteriovenous (AV) fistula, a synthetic AV graft;or a central venous catheter. The AV fistula is the preferred type of vascular access because thrombosis rates, infection rates, access-related expenditures, and total healthcare expenditures all are lower for patients with fistulas than for those with either synthetic AV grafts or central venous catheters. However, the advantages of fistulas are counterbalanced by the substantially higher proportion of fistulas than grafts that are never able to be used for dialysis because of failure to mature adequately to support effective hemodialysis. Maturation failure is the major barrier to increasing fistula prevalence, and, for many patients, leads to multiple surgical procedures or prolonged use of central venous catheters, the least desirable type of vascularaccess. Recent studies suggest that 20-50% of new fistulas fail to mature. This application is a response to an NIDDK RFA to establish a consortium for designing and performing an observational cohort study of patients with new fistulas. We are applying to be one of the Participating Clinical Centers of the Consortium. The overall objective of this application is to identify determinants of fistula maturation outcomes in order to enable early identification of failing fistulas, elucidate mechanisms underlying fistula maturation, and identify potential targets for maturation-enhancing interventions. Our application has five specific aims. These include 1) determining the utility of ultrasound as a method for early identification of fistulas that are failing to mature, 2) evaluating the impact of pre-existing vascular function on fistula maturation outcomes, 3) identifying surgical factors that are associated with fistula maturation outcomes, 4) creating evidence-based criteria for fistula maturation, and 5) characterizing the clinical consequences of fistula maturation failure. These aims assume an enrollment of 75-100 subjects over two years at our site for an overall study cohort of 600 subjects. Several of our aims include sub-studies that will be performed in a subset of the subjects to provide additional mechanistic information about the fistula maturation process. Our established research focus and expertise in hemodialysis vascular access, our success and leadership as a Clinical Center for the NIDDK Dialysis Access Consortium (DAC), the multidisciplinary expertise of the collaborators for this application, and the resources available at Boston University make us ideally suited to serve as a Participating Clinical Center for this NIDDK initiative.


Hyperbilirubinemia is probably the most frequently diagnosed and treated condition in the human newborn. Treatment is aimed at preventing the entry of bilirubin into the brain because of the risk of permanent neurologic damage. The mode by which bilirubin enters the brain, its metabolic fate after entry, and the sites of toxic action are unknown. Using osmotic stress, the blood-brain barrier was opened in rats, permitting entry of bilirubin. However, in this acute model, the bilirubin was rapidly cleared, with a half-time of l.6 hours. To provide chronic exposure, especially in newborn animals, we are now studying two inbred strains of rats which lack glucuronyl transferase.


This research project aims to investigate a part of the neural control pathway for reach to grasp movements in cats. These movements are an essential part of many behaviors including feeding and defense. A reach to grasp movement was most likely utilized to pick up this document. When the movement fails, life changes drastically. To repair a deficit, an understanding of the basic neural control of the movement is necessary. The proposed research will contribute to this understanding. One of the fundamental aspects of the neural control of a movement is the organization of its control pathway in the nervous system. What areas of the brain control the movement? How do the control signals change as they progress through the brain? How is the control of the movement organized within the brain structures involved? Dr. Gibson's laboratory has concentrated on the control of the reach to grasp movement. His work has identified a pathway which appears dedicated to the control of the grasping aspect of the reach to grasp movement. This pathway includes the interpositus nucleus in the deep cerebellar nuclei and the magnocellular red nucleus. Neurons in these areas discharge sharply only to reach movements with a grasp. A grasp pathway suggests that there is a reach pathway. Recently, work in Dr. Gibson's laboratory has identified an area of the mesencephalic reticular formation where neurons discharge for reaching during the reach to grasp movement. Preliminary anatomical data from the lab suggests that the dentate nucleus projects to the reach related area of the mesencephalic reticular formation. The proposed research will first investigate the discharge from neurons in the dentate nucleus during reach to grasp movements. Next, the contribution these neural areas make to the reach to grasp will be studied through inactivation experiments of the dentate nucleus and the mesencephalic reticular formation. The deficits from the inactivation of each of the areas will be compared.


ABSTRACT Spinal cord diseases and disorders such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS) are debilitating, often resulting in loss of mobility and decreased quality of life for those affected. One promising treatment involves the transplantation of neural progenitor cells (NPC) into the spinal cord, which has been shown to have neuroprotective properties. However, most NPC-based therapies fail after reaching clinical trials, and without a method to monitor the injected cells and viability, locating injected NPCs is limited to postmortem histology. The ability to track and assess the viability of transplanted cells could be crucial in understanding whether the failure is technical or biological in nature, such as whether the injected cells were delivered to and remain at the correct location or survived transplantation. We propose the development of multi-modal contrast agents and a clinical ultrasound and photoacoustic (US/PA) imaging system for image guided delivery of trans- planted cells and longitudinal monitoring. Together with clinical magnetic resonance imaging (MRI), the use of US/PA imaging can allow for pre-, intra- and post-operative visualization of the cells. The MRI and US imaging modalities are well established in the clinic, and PA can be easily integrated with existing clinical US imaging systems. We hypothesize that visualizing the location and viability of injected neural progenitor cells obtained through the labeling of NPCs with contrast agents will allow for better understanding of transplanted cell behavior and improved translation of cell based therapies. Two contrast agents will be developed: photo-magnetic nano- particles, which will allow for photoacoustic and MRI tracking, and a dye-based apoptosis reporter, which will provide photoacoustic contrast upon apoptotic activity. These will be delivered to the cytosol of the neural pro- genitor cells and assessed for labeling efficiency and toxicity. Once optimized, labeled NPCs in different ratios of live to dead will be injected into the spinal cords of rats to assess the feasibility of distinguishing live and dead cells in vivo. After injection, the NPCs will be monitored longitudinally using trimodal US/PA/MR imaging. After validating the performance of the contrast agents in vivo, a clinical US/PA imaging system will be developed to demonstrate real-time image guided-delivery, US/PA/MRI longitudinal tracking, and PA viability assessment of the labeled cells. The ability to monitor the transplanted cells at every point during and after the procedure will allow for more accurate delivery of the cells and could elucidate common issues and behaviors of injected NPCs that could lead to therapeutic improvements. Furthermore, if successful, it will validate both the contrast agents and US/PA as a valuable tool for tracking and monitoring cell-based therapies to improve clinical translation.


This proposal seeks to understand in detail the role of alpha-lactalbumin in lactose biosynthesis ("lactose synthetase"). Alpha-lactalbumin, a common milk protein both plays an important regulatory role and is itself subject to a fine regulation at the levels of organ specificity, cellular structure and hormonal control of both its biosynthesis and secretion. In more detail this work is directed at examining structure, conformation and changes therein associated with galactosyl transferase function at the molecular level. This research utilizes the extremely powerful techniques of magnetic resonance to investigate the interactions of both substrates and the two proteins (galactosyl transferase and alpha-lactalbumin) involved in the biosynthesis of lactose. BIBLIOGRAPHIC REFERENCE: Lawrence J. Berliner and Shan S. Wong, "Mn(II) and Spin Labeled-UDP Binding to Bovine Galactosyl Transferase," Biochemistry 14, 4977-4982 (1975). Lawrence J. Berliner and J. Grunwald, "Immobilized Bovine Lactose Synthetase: A Method of Topographical Analysis of the Active Site." FASEB Abstracts 67th ASBC Mtg. 6-10 June 1976 San Francisco, CA.


Despite their prevalence in nature and as drug and therapeutic targets, many membrane proteins continue to evade structure determination by X-ray crystallography and NMR. The combination of site-directed spin labeling with electron paramagnetic resonance (SDSL-EPR) is becoming an increasingly popular method for the structural characterization of membrane proteins due to the relative ease with which they can be studied. However, SDSL-EPR does not yield high-resolution structures directly. The current proposal describes a new method, ROSETTAEPR, to overcome this obstacle. ROSETTAEPR will be a toolkit in which distance and accessibility restraints determined by EPR will be combined with Monte Carlo-based computational methods for the de novo structure prediction of proteins. After developing knowledge-based potentials derived from EPR experimental data, it will be benchmarked on proteins of known structure using both simulated and real EPR data. In addition, ROSETTAEPR and EPR experimental distance and accessibility data will be used to determine the LeuT apo, Na+, and Na+/leucine bound structural intermediates involved in leucine transport. LeuT is a bacterial homolog of the neurotransmitter sodium symporter (NSS) protein family, which includes the dopamine, serotonin, and norepinephrine transporters. While there are no high-resolution structures of the NSS transporters, extracellular-facing substrate-bound conformations of LeuT have been determined by X-ray crystallography. However, the current structures are static snapshots of the LeuT transport cycle; furthermore, they are believed to have been captured in a potentially inhibited form. Therefore, EPR spectroscopy has been employed to shed light on the dynamics of the protein. It was found that Na+ binding causes an increase in protein flexibility in the extracellular loops and hydration of the substrate permeation pathway, while subsequent binding of leucine causes the extracellular vestibule to close and become rigid. ROSETTAEPR will allow for the high-resolution structural elucidation of these intermediates based on low-resolution EPR data. PUBLIC HEALTH RELEVANCE: The dysfunction of neurotransmitter sodium symporter (NSS) proteins is a common characteristic of central nervous system (CNS) diseases, such as depression, anxiety, obsessive compulsive disorder (OCD), and epilepsy. Understanding how these proteins function on a structural level will aid in the development of new, more effective therapeutic agents that specifically target neural processes underlying mood, reward, and locomotion.


Ischemic preconditioning (IPC) refers to a phenomenon in which a tissue is rendered resistant to the deleterious effects of prolonged ischemia and reperfusion (I/R) by prior exposure to brief periods of vascular occlusion. Although first described over a decade ago, the mechanisms underlying the beneficial effects of IPC remain uncertain. However, preliminary data from our laboratory indicates that IPC prevents I/R-induced intestinal oxidant production, leukocyte/endothelial cell interactions, and venular protein leakage. Since the production of reactive oxygen metabolites from xanthine oxidase and other sources during reperfusion of ischemic intestine initiates the formation of chemotactic stimuli, induces the expression of endothelial cell adhesion molecules, and reduces intestinal levels of the potent antiadhesive agent nitric oxide, we hypothesize that IPC-induced reductions in leukocyte adhesion are mediated by the effect of preconditioning to reduce the postischemic oxidant production. To address this central postulate, four specific aims are proposed. One aim is to determine whether IPC prevents the increase in mesenteric xanthine oxidase activity or influence of IPC on the elaboration of proinflammatory stimuli (platelet activating factor (PAF), leukotriene B4(LTB4), complement activation) in postischemic small bowel. A third specific aim is to determine whether IPC attenuates the expression of P-selectin by the endothelium in postcapillary venules exposed to I/R. Since the beneficial effects of IPC on postischemic mesenteric microvascular dysfunction are reversed by topical application of adenosine deaminase during IPC or protein kinase C (PKC) inhibitors during the period of prolonged ischemia, fa fourth aim is directed at elucidating the mechanisms that may be involved in coupling adenosine production during IPC to the postischemic reductions in oxidant production, leukocyte adhesion, and venular protein leakage. To accomplish these aims, we will utilize intravital microscopic approaches to quantitate oxidant production, leukocyte/endothelial cell interactions, and venular protein leakage in rat mesentery. Mesenteric xanthine oxidase, nitric oxide synthase, phospholipase C, and PKC activities, nitrite/nitrate production, PAF and LTB4 levels, and the extent of complement activation will also be assessed. In vivo mesenteric P- selectin expression will be quantitated using a dual-radiolabeled monoclonal antibody approach. The proposed studies should not only substantially improve our understanding of the mechanisms whereby IPC reduces postischemic microvascular dysfunction in the intestine but should also provide a rationale for the development of novel therapeutic interventions in I/R that exploit the mediators of the preconditioning phenomenon.


The lab has previously shown that adipsin, a mouse adipocyte-derived serine protease, is secreted into the bloodstream and is markedly deficient in several models of genetic and acquired obesity, suggesting a possible functional role for this protein in the systemic or local regulations of energy balance. The overall goal of the proposed work is two-fold: to understand the role of adipsin in adipose physiology and obesity and to use the adipsin gene as a model to understand how obesity genes regulate the function of other gene promoters. Both of these goals are novel and will increase our understanding of the biochemical and genetic basis of obesity. Adipsin's direct role in the physiology of adipose cells will be approached in vitro by treating fat cells with adipsin and measuring changes in lipogenesis and lipolysis. Since adipsin activates the alternative pathway of complement via its intrinsic Factor D catalytic activity, the ability of various components of the alternative complement pathway to regulate adipose cell physiology will be studied, including complement-derived ligands such as C3a, C5a and Ba. In addition to an in vitro approach, adipsin will be administered acutely and chronically to obese and lean animals to ascertain the role of this protein and the alternative complement pathway in systemic energy balance in vivo. Multiple measures of systemic energy balance and hormonal regulation will be measured. Preliminary pharmacokinetic studies in lean and obese mice support the feasibility of altering adipsin levels in vivo by administration of the pure protein. New data in transgenic mice shows that a rodent obesity gene (db) suppresses adipsin expression through its action (direct or indirect) on the adipsin promoter. Complementary cellular and biochemical approaches will be used to study this pathway of signal transduction and identify the cis- and trans-acting factors involved in the "obesity-response element". Cultured adipocytes will be treated with serum from lean and obese mice and with various hormones to model the in vivo effects of obesity on the endogenous adipsin gene. By transfection of chimeric constructions and a series of deletions and mutations from the adipsin gene promoter into these cells, sequences involved in a putative "obesity-response element" will be mapped. A biochemical approach will involve mapping this response element in the adipsin promoter via DNA footprinting (and gelshift) analysis of the adipsin promoter DNA using nuclear extracts from lean and obese animals. The identity of putative "obesity-response elements" will be critically tested in vivo using point mutagenesis in the adipsin promoter and the construction of transgenic, obese mice. The trans-acting factor(s) binding to this response element and ultimately responsible for aberrant adipsin expression in obesity will be cloned and characterized by biochemical and new molecular genetic techniques.


Multiple lines of evidence have suggested that viral infection may play a pathogenetic role in biliary atresia (BA). However, searches for putative viral pathogens have been restricted to a small number of selected viruses and have not focused on virus-specific immune responses. The hypothesis underlying this proposal is that viral infection plays an etiopathologic role in biliary atresia. The purpose of the present proposal is three-fold: 1) Establish a Clinical Biliary Atresia Center. Five referral centers including the Johns Hopkins Pediatric Liver Center will recruit 10-20 new BA patients/year to add to the 73 BA patients in the present database. The database will gather demographic and clinical information as well as biological specimens pertinent to the viral infection hypothesis. 2) BA Peptide Project, (3-years) will employ a powerful new technique, bacteriophage peptide display, to search for novel antigenic epitopes specific to BA. BA sera will be utilized to "biopan" random peptide libraries to identify epitopes not recognized by control sera. One patient/control group from each of the Clinical Centers will be studied, for the greatest possible geographic, seasonal, and ethnic diversity. "BA peptides" will be sequenced and synthesized. Peptides will be used to immunoaffinity purify the corresponding monospecific antibodies from BA sera. Protein databases will be searched for homologous viral proteins. To determine if the BA peptides exhibit cross reactivity as HLA-A2+ restricted T cell epitopes, the peptides will be incubated with dendritic cells, antigen-presenting cells (APC) harvested from HLA-A2+ maternal peripheral blood lymphocytes (PBL). Maternal PBL will be incubated with the peptide-pulsed APC to identify peptide-specific CTL using target cell lysis assays. 3) The BA-Dimer Project, (1-year) will employ a sensitive new "dimer" technique to determine if HLA-A2+ biological mothers of BA (MBA) patients exhibit unique peptide-specific CTL. The dimer technique uses immunoglobulin as a molecular scaffold to produce a divalent peptide-HLA-A2-Ig complex. Dimers will be loaded with BA or homologous viral peptides and PBL from MBA and control mothers from each Clinical Center will be tested for dimer reactivity using FACS analysis. Novel BA-specific peptides, monospecific antibodies, and BA-dimers could be used for early diagnosis, identification of cellular targets and intrahepatic T cells.


We propose to continue a nationally visible and responsive center focused on the development of novel fluorescent biosensor and detection technologies for investigating pathways and networks in real time and high spatial resolution in living cells. The renewed center retains the combined experience and infrastructure of two existing centers in Pittsburgh: the Molecular Biosensors and Imaging Center at Carnegie Mellon University and the Center for Biologic Imaging at the University of Pittsburgh. The Technology Development (Core 1) component of the center will create a powerful toolbox of intracellular fluorescent biosensors and reporters that can be used to study many, if not all, of the pathway proteins and activities in living cells. This fluorescent biosensor development program integrates efforts across multiple disciplines, including dye chemistry, molecular biology, biochemistry, structural biology, modeling, cell biology, image acquisition and analysis, and high throughput screening. We have made substantial progress in this effort during the previous 3 years of funding. Four exciting Driving Biology Projects (Core 2) are essential to the technology development effort in Core 1. These DBPs are focused on important and currently un-addressable biological problems, and will serve both as test-beds for the technology and compelling demonstrations of the value of the biosensors and reporters developed in this program. The Infrastructure (Core 3) is provided through the Center for Biologic Imaging at the University of Pittsburgh. The role of the CBI is to act as the application and outreach ami of the project as a whole by testing the new biosensors with challenging biological problems. During the last cycle of this proposal the clear mission of the CBI evolved to become the catalyst in probe implementation, and to strengthen and broaden the impact of the new probes developed by MBIC. This is achieved by providing facilities and expertise to test and validate the probes in the context of the driving biological projects, and ultimately, the biomedical research community at large. The program contains a significant technology transfer component to disseminate concepts, knowledge, software, materials, and resources to users in both academic research labs and industry. This is achieved in our Infrastructure, Training, and Dissemination cores (Core 3,4, 5) and through the technology transfer activities in the Management core (Core 6).


Oxidants generated by activated white blood cells are critical to host defenses against microorganisms. However, overproduction of reactive species can damage host tissue. Indeed, white blood cells represent the cellular hallmark of inflammation, and oxidants have been implicated in tissue injury in inflammatory diseases ranging from atherosclerosis to neurodegenerative disorders to cancer.We have investigated four phagocyte-dependent pathways that oxidatively damage proteins in vitro. The pathways and their characteristic products are: myeloperoxidase and 3-chlorotyrosine; tyrosyl radical and o,o-dityrosine; hydroxyl radical and ortho-tyrosine; and reactive nitrogen species and 3-nitrotyrosine.Using two clinically relevant models of inflammation, we will study genetically engineered mice whose phagocytes are unable to produce specific oxidants. In the proposed research, we will ask three related questions. First, we will identify the pathways that generate chlorotyrosine, dityrosine, ortho-tyrosine, and nitrotyrosine in viva. The experiments will reveal whether a genetic deficiency of any of these oxidant-generating systems inhibits production of any of the chemical markers, thereby determining which pathway generates a particular marker in viva.Second, we will determine whether tissue, plasma, and urinary levels of the oxidized amino acids change in parallel. We will also investigate the absorption, metabolism, and urinary excretion of the oxidized amino acids. These experiments will determine whether plasma and urinary levels of these well-characterized products can be used as noninvasive markers of oxidative stress.Third, we plan to determine whether two proposed antioxidants-vitamin C or vitamin E- inhibit oxidative stress in our models of inflammation. Collectively, the proposed experiments will identify the oxidative pathways that cause phagocytes to damage tissues and will test the hypothesis that levels of oxidized amino acids in urine and plasma indicate levels of oxidative stress in vivo.


This integrated basic and clinical research program is directed at development of more effective therapy for malignant disease. The disciplines include pharmacology, both new drug and new drug usage principles, cell biology, transplantation biology, cellular kinetics and cell physiology. The specific research projects are: 1) Biometry Consultation Group; 2) Pharmacokinetically Designed Cancer Chemotherapy; 3) Synthesis and Biological Evaluation of 1-Aryl-3, 3-Dimethyltriazenes; 4) Prediction of Clinical Response to Arabinosyl-cytosine (Ara-C); 5) Study of Arabinosyladenine Toxicity In Vitro and In Vivo; 6) Detection of Proliferative Potential of Human Bone Marrow and the Prediction of Response and Relapse in Leukemia; 7) Cell Surface Antigens and Structure in Cancer Immunology; 8) Isolation and Characterization of Tumor-Associated Antigens Cross-reactive with BCG; 9) Clinical and Laboratory Studies on Mitogen-Induced Proliferation in Human Leukemic Cells; 10) Cellular Response to Antineoplastic Agents; 11) Cellular Chemotherapy and Kinetics.


This project is concerned with new understanding of the effects of molecular structure on fundamental parameters of hydrogen-bonding and the relationship of these parameters to acid-base phenomena generally. Both a physical picture of, and the energetics for, the specific solvation of organic solutes (both neutral and charged) with highly localized charges are being approached using models involving discrete equilibria for successive hydrogen bond attachments of individual solvent molecules. These equilibria are being determined in the gas phase using ion cyclotron resonance spectroscopic methods and in the condensed phase using nuclear magnetic resonance spectroscopic methods. Analysis of results by methods of linear free energy relationships and by ab initio molecular orbital calculations provides an effective complimentary approach.


Early nutrition can have far-reaching effects on behavior: emerging research has revealed the existence of sensitive periods during postnatal development in which the developing brain has heightened sensitivity to environmental influences, including flavors of foods. While children's innate taste likes and dislikes create obstacles to healthy eating, research in humans and other animals has revealed that early experience can affect these likes and dislikes. The challenge is to determine whether there are optimum times when experience promotes greater liking of the flavors of healthy foods. Our previous experiments have shown that, as with other mammals, what a nursing mother eats during the first months of lactation can influence her child's food preferences. We propose to conduct a detailed randomized within- and between-subject study of women and their infants during a 16-month window, varying the timing and duration of exposure to three vegetable flavors (carrot, beet, and cabbage) to determine effects on the acceptance of these flavors by children, their mothers' own liking of these foods, and the likelihood that mothers will feed these foods to their children after weaning. Based on solid, previous research in humans and other animals, the over-arching goals of the proposed research are to specify the timing (Specific Aim 1) and consequences (Specific Aim 2) of the sensitive period for flavor learning in infants via flavor variety in mother's milk, and to determine the effects of reproductive state (Specific Aim 3) and genetic variation (Specific Aim 4) on vegetable liking in newly parturient mothers and their children. The hypotheses behind this research, examining highly pervasive and under-investigated behavioral phenomena surrounding how to introduce vegetables to children's diets, focus on early life, unlike many other studies that attempt to modify food habits in older children, and focus on the maternal-infant dyad because mothers' food consumption and feeding choices during the sensitive period may have far-reaching consequences for children's food exposures and choices later in life. Knowledge regarding the most efficacious time mothers should eat foods to develop preferences can lead to intervention strategies for both infants and their mothers, and consequently their families. Furthermore, knowledge about the timing of a sensitive period for learning about flavors in breast milk will shed light on long-term effects of not being exposed to these flavors early in life. The knowledge gleaned from this basic behavioral work could be translated to effective behavioral interventions to promote vegetable acceptance that are effective and implemented widely. PUBLIC HEALTH RELEVANCE: Early experience modifies children's innate taste likes and dislikes, and promotes healthy eating behaviors. We propose a randomized trial to specify the timing and effects of the sensitive period for flavor learning in infants via flavor variety in mother's milk, and the effects of genetic variation and the mother's reproductive state on vegetable liking. This knowledge may lead to strategies for healthy eating interventions for infants, mothers, and families.


Project Summary Ion channels are membrane proteins that selectively regulate the movement of ions across cellular membranes. Ion channels play a role in many pathophysiological states, such as pain, epilepsy, metabolic disorders, obesity, diabetes, cardiac arrhythmias and renal disease. Studying the role of ion channels in disease is often slow because traditional patch clamp electrophysiology, although precise, is also a low throughput technique that can only be done by researchers who have specialized training and equipment. This bottleneck restricts the number of ion channel research problems that can be addressed by any lab or institution, compared to other drug targets, such as the G-protein-coupled receptors (GPCRs). Recent advances in ion channel technologies have demonstrated that studying ion channels can be done in high- throughput (HT). The first instrument that was specifically designed to study ion channels in HT was IonWorks HT. IonWorks HT was breakthrough technology because it enabled the study of ion channels in a 384-well ion channel instrument for the first time. The current market leader in HT electrophysiology (HT ephys) is Nanion. Nanion's SyncroPatch 384PE can record 384 cells in parallel, which can generate 20,000 data points per day. This gigaseal HT ephys instrument can measure the physiology and pharmacology of all classes of ion channels, including rapidly sensitizing ones such as alpha 7 nicotinic receptors. The Center for Chemical Genomics (CCG) is the well established home of HT biology and pharmacology at the University of Michigan (U-M), and has conducted over 200 high throughput screens over the last decade. The CCG's research team collectively has eight decades of pharmaceutical experience which provides them extensive knowledge of best practices in HTS assay development, validation, execution and informatics, and has the expertise to implement HT ion channel screening. However, there is currently no HT ephys instrument at U-M. Adding this highly scalable technology to the CCG, will allow the full exploration and exploitation of ion channel research and ion channel drug discovery at U-M and our collaborating partners.


This proposal seeks financial support for travel to Copenhagen. Denmark for the purpose of negotiating with the staff of the UCLA/University of Copenhagen Joint Center for Studies of Health Programs and the Danish National Committee for Health Information relative to the possible involvement of the Health Services Research Center of the University of North Carolina at Chapel Hill in the Danish National Health Education Project with regard to the evaluation of the Project's impact on the health promotive behaviors of the Danish people.


This project extends its previous work on excitation-contraction (E-C) coupling and relaxation (R) coupling in skeletal muscles of the frog and mammals to determine the mechanisms of these processess and their interactions that determine the flux of activator Ca2 ion regulating the contraction-relaxation cycle of the twitch and the tetanus. The studies of E-C coupling which depended previously on general electromechanical correlations in the twitch, now concentrate on the electromechanical "transforms" which specifically distinguish between the mechanical effects of the rising and falling phases of the spike, and which are used to determine possible mechanisms by which the mechanical threshold and the duration of the action potential modulate E-C coupling and thus vary the release of activator Ca2 ion and the associated active state. It is planned to determine the role in E-C coupling of Ca in T tubule action and to test further whether the latency relaxation is a sign of the release of Ca2 ion by the SR, this latter especially by D2O which, our work shows, greatly slows development of the LR. The role of the T tubules in generating the action potential, and especially their possible role in causing delayed rectification, will be investigated by studying action potentials and membrane current/voltage relations of detubulated fibers, which are treated with various agents that alter the rate of rise and fall of the spike. A new active state theory will be used for tracing the course of twitch and tetanus activity in relation to the flux of Ca2 ion as indicated by the E-C coupling electromechanical transforms, and by possible manifestations of R coupling and of regenerative Ca-release. The general procedures will be used to determine E-C and R coupling and active state course in normal and denervated slow and fast mammalian muscles. And certain special studies are planned to determine whether mouse dystrophy originates in a neurogenic or myogenic pathology.


Artemis has been shown recently to be involved in a human severe combined immunodeficiency syndrome (SCID) in which an absence of development of T and B cells is observed. This defect is due to a requirement for Artemis in the coding joint formation step of V(D)J recombination. In addition, patient cell lines were shown to be hypersensitive to ionizing radiation suggesting that Artemis is also involved in cellular responses to DNA damage. The goals of this application are to characterize the function of Artemis in regard to its role in mediating the cellular response to DNA damage. Specifically, we will determine the nature of proteins that Artemis interacts with, and examine its cellular localization before and exposure to genotoxic agents. Our findings show that Artemis is rapidly phosphorylated upon exposure of cells to DNA damage including that induced by both IR and UV. We have also shown that this phosphorylation is mediated by the PI3 kinases DNA-PK, ATM, and ATR. We will characterize this phosphorylation of Artemis and prepare phosphospecific antibodies that will be utilized for functional studies of Artemis. These functional studies will be conducted on cells defective in Artemis to determine its role in either DNA repair and/or cell cycle checkpoint pathways. It is expected that these studies will elucidate the function of Artemis in DNA damage response pathways, and thus provide an explanation for the radiosensitivity of the RS-SCID syndrome. Finally, we will prepare a knock-in mutant of Artemis in the mouse that is mutated at sites of phosphorylation mediated by ATM. This experiment is designed to determine if the modification of Artemis by ATM is involved in the immunodeficiency observed in AT patients.


Project Summary Transcriptional repressors represent the necessary counterweight to transcriptional activators in metazoan development. The mechanisms of transcriptional repression have been intensively investigated, but we lack crucial insights into how repressors act at a mechanistic level across many targets in the genome. In particular, the coordinated recruiting of transcriptional co-repressors can generate diverse effects on chromatin structure and modification, and we still lack insights on the functional significance of many changes that can be measured in `omics studies. To develop key insights into eukaryotic transcriptional regulatory mechanisms, we use the natural setting of the Drosophila embryo to identify basic biochemical processes in a developmental setting, where differential gene expression is used to drive the developmental fate of particular cells and tissues. In this proposal, 1) we will use genome-wide methods developed in our laboratory to identify direct biochemical, chromatin-based processes that are directed by a set of five endogenous transcriptional regulators that repress through short-range and long-range mechanisms. 2) We will study the in vivo activity of wild-type and mutant repression complexes to identify the contributions of distinct transcriptional co-repressors Groucho and CtBP, testing their contributions to quantitative and/or qualitative effects in chromatin modifications and gene regulation. 3) To identify the cis-regulatory context in which transcriptional repressors act on different enhancers, we will quantitatively measure and mathematically model the output of specific enhancers to uncover the fundamental quantitative properties of specific classes of repressors interacting with activators ? i.e. the common ?rules? by which these proteins interact on many target genes. The three interrelated aims will provide predictive tools for interpretation of genomic cis regulatory content of the metazoan genome, providing essential underpinnings for studies of evolution and disease in higher eukaryotes.


This proposal seeks STTR funding for the preliminary development of a Web-based screening tool to help elementary school health and mental health service providers quickly and accurately assess students' risk and protective factor status for adolescent substance abuse. Early identification and targeting of known predictors of adolescent substance abuse reduce its prevalence and its negative health, social, and academic consequences. The instrument will seek information from 3rd - 5th graders, and their parents and teachers. It will assess the social environment, individual characteristics, and functioning of children. Internet and computer technology will be used to increase there liability and validity of the child sell-report data, and facilitate the collection and scoring of data from children, parents, and teachers. Aims of Phase I are to: (a) create a preliminary version of the screening tool, (b)develop supplemental administrative and training materials, and (c) pre-test the child report section. Phase I tasks include developing the list of questionnaire items, computer formats, and supplemental materials. Expert consultation and cognitive testing with children will be used to evaluate the quality and feasibility of the tool. In Phase II, the preliminary instrument and administrative procedures will undergo pilot testing and large-scale psychometric testing. PROPOSED COMMERCIAL APPLICATION: The proposed screening tool has commercial applications among health and mental health practitioners working in elementary schools. Taking advantage of schools' existing investment in Internet technology, the tool will make comprehensive risk and protective factor screenings more feasible than existing options. The range of domains assessed, the child self-report component, computer scoring, and rapidly available individual and group results designed to guide intervention planning are unique and meet a market need.


Flavonoids constitute a group of polyphenolic compounds primarily known for their anti-oxidant properties. Studies on flavonoids show their promising use for the prevention and treatment of a variety of cancer, inflammatory and heart diseases. Quercetin is a naturally occurring bioactive flavonoid ubiquitously available in fruits, vegetables and other plants. Several studies have shown the bioactivity of Quercetin against a variety of tumor cells and angiogenesis. However, several other studies have also indicated the carcinogenic potential of Quercetin. Moreover, Quercetin has been described as one of the principal molecules of some toxic plants. Therefore, the conditions under which Quercetin is bioactive as an anti-tumor agent or as a carcinogen are not well known. Understanding how Quercetin and other flavonoids function at the molecular level is of paramount importance to public health because it will help us establish the conditions under which specific bioactive flavonoid compounds may or may not be beneficial for therapeutic purposes. [unreadable] [unreadable] In this study, we propose to determine the mechanisms of Quercetin bioactivity under conditions where specific protein regulators of cell signaling have been ablated. Our specific aims are to examine: (1) what genes are expressed or inhibited by Quercetin; (2) how Quercetin modulates cell survival or cell death through cell signaling regulatory proteins. To address these aims, firstly, we will study the global and differential gene expression profiles of cells, which are intact or null for the p53 protein, by treating them with Quercetin. We will also examine the effect of Quercetin on the expression and function of cell cycle and apoptosis proteins regulated by p53. Secondly, we will analyze the DNA-damage response pathway through examination of the ATM/ATR proteins and downstream events. Thirdly, we will examine the levels and activities of other key cell cycle and apoptosis regulatory proteins after treatment with Quercetin. To examine the role of key proteins identified through the initial treatment experiments, we will use cells knocked out for the proteins or we will knock down the proteins by short interfering RNAs. The results will help us understand if treatment with Quercetin influences cell cycle, apoptosis, and DNA-damage regulatory proteins in cells. In future studies, we will utilize knowledge from these aims to evaluate the effect that Quercetin exerts in tumor cells in vivo. [unreadable] [unreadable] [unreadable]


We propose to clone the entire I regions from BALB/c and AKR mice. We will identify, sequence and determine the organization of all class II genes with the I region. We will transfer class II genes into mouse L cells and normal bone marrow derived cells by DNA indicated gene transfer. We will study the DNA elements which control the expression of class II genes in the model mouse L cell system and normal hematopoietic cells. The class II genes will be altered by in vitro mutagenesis and their expression and functions studied after appropriate gene transfer experiments.


HPV plays an etiologic role in the development of a distinct subset of oral cancers that arise predominantly from the tonsils. HIV-infected individuals are at increased risk for tonsillar cancer. Oral HPV infection is therefore a newly appreciated public health problem. The objective of this proposal is to perform a comprehensive molecular epidemiologic study to determine the prevalence, risk factors, mechanisms of transmission, and natural history of oral human papillomavirus (HPV) infection in HIV-infected individuals and to identify precursor lesions for HPV-associated head and neck squamous cell carcinomas (HPV associated HNSCC). Tonsillar cancers comprise approximately 15-23% of all oral and oropharyngeal cancers. There was a 2-3% increase in tonsillar cancer incidence per year from 1973-1995 in men in the United States. Concurrent HPV and HIV infection may have contributed to this unexplained increase. Preliminary data suggest that the odds of oral and tonsillar infection by a high-risk HPV type are significantly elevated in HIV infected individuals, and that oral HPV infection is associated with sexual behaviors, severity of immunosuppression and the presence of potentially premalignant oral mucosal lesions. A prospective cohort study of 800 HIV-infected men and women with 36 months of semiannual follow-up is proposed to accomplish the following specific aims: (i) to determine the prevalence and type distribution of oral mucosal HPV infection and to characterize host, environmental (tobacco and alcohol) and biological factors (e.g. CD4 count, HIV viral load) associated with oral HPV infection; (ii) to identify specific sexual practices associated with presence of oral HPV infection after adjustment for confounders; (iii) to evaluate the relationship between high-risk HPVs (presence of HPV genomic DNA, viral load, integration) and tonsillar dysplasia and other oral mucosal abnormalities, and (iv) to determine if tobacco use, immunosuppression, and HPV type (low versus high-risk) affect the incidence and/or persistence of oral HPV infection. This proposal has the potential to yield information important for future efforts at primary prevention and early detection of HPV-HNSCC.


The recent studies indicated that the existing smoking prevention cessation strategies have failed to produce population reductions in teen smoking prevalence. This is despite significant progress in intervention development and increasing financial investments in anti-smoking activities. The investigators believe several possible explanations merit consideration for this paradox. First, effective smoking prevention programs have not been widely disseminated. Second, when adopted, teachers often fail to adhere to program guidelines. Third, teens who smoke are reluctant to identify themselves to seek cessation assistance. Fourth, smoking prevention and cessation programs often fail to account for salient individual differences among students. Finally, most existing smoking prevention and cessation programs do not utilize modern communication technologies and do not meet the demands of today's adolescents. The goal of the proposed project is to design, implement, and evaluate an interactive CD-ROM curriculum aimed at adolescent smoking prevention and cessation intervention. By interactive, we mean assessing each student individually and channeling him or her into the most appropriate intervention. The software will assess student smoking status, stages of change for smoking acquisition and cessation, level of addiction, and symptoms of depression. In this manner, recruitment of smokers is facilitated because both smoking and nonsmoking students will be asked to participate in the program and the intervention they receive will be customized to meet their individual needs. The intervention will also be tailored to gender and ethnicity. By utilizing CD-ROM the investigators hope to facilitate subjects' adherence to intervention protocols and subsequent program dissemination. The investigators hypothesize the intervention will result in: a) lower onset of smoking; b) greater smoking cessation outcomes; and c) impact on variables that mediate the process of change compared to an alternative treatment control group. The study design is an 18-month, group-randomized, controlled trial. Study participants will include students 14-16 years of age (10" graders at baseline) from 16 Houston area high schools.


During the past decade, researchers have emphasized the contribution of parent-teen relationships to adolescent adjustment and development. A general finding is that the quality of relationship with parents serves to support rather than obstruct the adolescent's growing independence and autonomy. While this finding points to the importance of parent-teen relationships, new questions have emerged. For instance, how is the quality of parent-adolescent relationships best conceptualized and assessed? What are the specific mechanisms that link the quality of the parent-teen relationship to the adolescent's adjustment and ability to function outside the home? How is the teenager's understanding of the parent-teen relationship influenced by his or her cognitive development and how does the teenager's growing cognitive understanding of the relationship influence his or her adjustment and well-being? The proposed project addresses these questions by viewing the parent-teen relationship from the perspective of attachment theory and emotion regulation. First, the study will attempt to further validate the Attachment Interview as an assessment of adolescent attachment security. More specifically, the validity of the Attachment Interview as a measure of individual differences in "styles of affect regulation" will be evaluated. Concurrent psychophysiological correlates of the different regulatory strategies identified in the interview will be examined as measures of inhibition and somatic activity. Second, measures of emotion regulation and attachment in the mother-teen relationship will be developed and the extent to which the ability to regulate negative affect in the Attachment interview generalizes to successful emotion modulation during parent-ten interaction will be tested. It is proposed that teenagers who are secure or who have the ability to modulate negative affect in the context of the Attachment interview will demonstrate more optimal patterns of interactions with parents in problem-solving and confiding tasks. Finally, the implications of attachment and emotion regulation for adolescents' ego-resiliency and well-being will be considered.


A group of current biomedical research projects which have a strong need for modern electron paramagnetic resonance equipment are outlined. These projects include studies of dipolar and exchange coupled binuclear cooper models for Type 3 biological copper, a detailed examination of the effects of ligand geometries and types on the EPR parameters of copper and manganese systems, investigations of superoxide dismutation and autoxidation mechanisms, vanadyl as a spectral probe of protein interactions and as an EPR-active phosphate analogue, and nitroxide spin-label probes of transketolase mechanism, lipid analogues, cooperative ligand binding in ferrihemoglobin, and details of cytochrome P450 action. A sophisticated EPR facility is described which will allow all of the projects presented herein to be carried out and will provide current generation EPR capability for the scientific community in northern Ohio and the surrounding area.


Neurodegenerative diseases are characterized by the progressive and relentless loss of neurons. One strategy to prevent or slow down neurodegeneration is to stimulate molecules that have neuroprotective activity. Using tissue culture systems we have demonstrated that FoxG1, a member of the Forkhead family of transcription factors, is necessary for the maintenance of neuronal survival. Furthermore, elevated expression of FoxG1 has strong neuroprotective effects. We propose to extend our studies in vivo and predict that elevated levels of FoxG1 will be also be protective in animal models of neurodegenerative disease. We propose to test this prediction by generating transgenic mice that express elevated levels of FoxG1 selectively in neurons. These mice will be crossed with two different mouse models of neurodegenerative disease and the effect on progression of neuropathology evaluated. The two specific aims of this proposal are - Aim 1: To generate transgenic mice overexpressing wild-type FoxG1, a constitutively-active form of FoxG1, and a dominant-negative form of FoxG1. We will use the mouse prion protein (mPrP) promoter to drive expression of the three forms of FoxG1 in mice. Aim 2: Analysis of the effect of FoxG1 overexpression on the brain and in mouse models of neurodegeneration. We will examine the effect of increasing FoxG1 activity, through expression of WT and CA FoxG1, on neuronal survival in the normal brain. In a second part of this aim we will examine whether elevated FoxG1 activity protects mice against neurodegeneration. We will cross FoxG1-overexpressing transgenic mice with R6/2 mice (a model of HD) and with p25/CDK5 inducible-transgenic mice (a model of AD). The project will result in the development of three new mouse lines expressing wild-type and mutant FoxG1 transgenic mice. FoxG1 transgenic mice do not currently exist. We feel that generating these mice and testing whether elevated FoxG1 can protect against neurodegenerative disease will shed insight into the function of FoxG1 in postmitotic neurons in vivo and could identify it as a target for the development of novel therapeutic strategies for neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: Previous work in our laboratory has demonstrated that elevated activity of the FoxG1 protein protects neurons against death. The goal of our project is to extend this observation in vivo by generating transgenic mice that express higher and lower activity of FoxG1. These mice will then be bred with existing models of neurodegenerative disease.


The proposed investigation focuses on the mechanism by which the strongly anticoagulant phospholipases from cobra venoms interfere with hemostasis. A hypothesis that strong anticoagulant phospholipases found in cobra venoms have a non-catalytic mechanism will be tested by incubation of the strongest anticoagulant phospholipase with tissue factor deprived of lipid and reconstituted with non-cleavable phospholipid. The basic anticoagulant phospholipase from Naja nigricollis venom will be chemically and proteolytically modified in an attempt to eliminate catalytic activity while retaining the anticoagulant effect. The enzyme will also be cleavedto obtain peptides which will be tested for anticoagulation. All three phospholipase isoenzymes from N. nigricollis will be sequenced to tru to identify cationic and external hydrophobic domains which can contribute to the anticoagulant effect. Reversibility of the anitcoagulant effect with antibodies specific to the anitcoagulant phospholipase will be examined. The classes of phospholipids cleaved and the kinetics of their cleavage from tissue factor and platelets by the three phopholipase isoenzymes will be examined. The lipid analyses will be extended further by the identificaion of fatty acids released during enzyme treatments. All lipid analyses will be correlated with functional studies of coagulation and/or platelet aggregation. These studies should prove insights into the mechanism of action of the strong anticoagulant phospholipases. They should also give new information about structural requirements of lipids critical to the coagulation process and platelet aggregation. Such anticoagulant enzymes might ultimately have clinical applications and should prove to be valuable research tools for future studies on hemostasis, thrombosis and their regulation.


The purpose of studies is to elucidate the role of female reproductive hormones relaxin, estrogen & progesterone in the etiopathogenesis of TMJ disease in women. We plan to determine association of relaxin, b-estradiol & progesterone with symptoms of TMJ disease in women using a case control study design. We also will determine if salivary levels of relaxin, b-estradiol & progesterone correlate with serum levels these hormones. We will assess if changes in relaxin, b-estradiol & progesterone over short period of time during pregnancy predispose to TMJ disease. We will evaluate, by a longitudinal prospective study design, whether aberrations in levels of relaxin, b-estradiol & progesterone during puberty and early adulthood are assoicated with the onset of TMJ disease.


The proposed research brings novel data to bear on the emergence of racial and socioeconomic disparities in health over the life course. We examine the intersection and mutual influence of three factors socioeconomic status (SES), cognitive ability, and health itself. Extant research has demonstrated amply that indicators of SES predict health outcomes, that health affects socioeconomic attainment, and that cognitive ability predicts both educational attainment and health. But how do such associations between SES, cognitive ability, and health arise over the life course? And what role does racial discrimination play in influencing each of these factors to create disparities by race? A theory of dynamic interplay of these factors across the life course is posited to explain the emergence of racial and socioeconomic disparities in general health, obesity, respiratory function, and depression. A California-based cohort, the Child Health and Development Study Adolescent Sample (CHDS-A) (n=2020), born 1959-1966, provides the novel data to evaluate this theory. Detailed information was collected at multiple time points from before birth until age 15-17 years for black and white offspring and their families. Sufficient numbers of black and white offspring from both high and low SES families were included in the sample. The data already collected include assessments during childhood and adolescence of SES, cognitive ability, and the selected domains of health. This project will add assessments at age 42-49 years. Analysis of the accumulated data will elucidate the relationships among fundamental sources of health disparities and inform the construction of policies and interventions designed to reduce such inequalities. Health disparities by socioeconomic status and race represent major public health issues in need of resolution. This study will provide novel data from a bi-racial birth cohort that has been intensively followed through adolescence and is now in middle-age adulthood. The analysis of key data collected in the proposed study will create an unusual opportunity to understand when and why health disparities emerge over the life course, and further guidance for shaping the content and timing of interventions to ameliorate observed disparities.


The adeno-associated virus 2(AAV)-based vector system has attracted considerable attention as an alternative to the more commonly used retroviral vectors for its potential use in gene therapy primarily because AAV is a non-pathogenic virus for humans, and the wild-type (wt) AAV genome has been shown to integrate into the human chromosomal DNA in a site-specific manner. However, a number of questions related to the basic molecular biology of the wt AAV interactions with normal human diploid cells in general, and that of the recombinant AAV in particular, remain largely unexplored. We have initiated systematic studies to pursue answers to these questions, and obtained preliminary evidence that the wt AAV interacts with normal human diploid fibroblasts in a manner that is distinct from that with human aneuploid cells. The hypotheses to be tested in this subproject are that the wt AAV integrates into normal human hematopoietic cells site-specifically at a site distinct from that characterized in human aneuploid cells, and that recombinant AAV genomes lacking the viral coding sequences (rep and/or cap) integrate in diploid hematopoietic cells at sites different from that for the wt AAV genome. The following Specific Aims will be pursued: 1. Comparison of patterns of integration of the wt AAV genome in human aneuploid and diploid hematopoietic cells, including purified populations of primitive progenitor cells from normal human bone marrow and umbilical cord blood: Using Southern blot and polymerase-chain reaction (PCR) analyses, the patterns of integration of the wt AAV genome will be compared in human aneuploid and diploid hematopoietic cells. 2. Evaluation of the role of AAV-encoded proteins in the site-specific integration of the wt and the recombinant AAV genomes: Recombinant viral vectors containing either the viral rep or the cap gene sequences will be used to evaluate whether AAV-encoded proteins play a role in the site- specific integration. 3. Molecular cloning and characterization of the AAV-integration sites in diploid cells, and comparison with that from aneuploid cells: In addition to direct cloning into bacteriophage lambda and plasmid vectors, PCR-based cloning strategies will be employed to obtain DNA sequences that contain the AAV-integration sites from human hematopoietic cells. The primary structure and transcriptional potential of these sequences will also be determined. 4. Evaluation of AAV-mediated transduction in vivo, and the potential for long-term expression of the transduced genes in a murine model system: Murine hematopoietic stem and progenitor cells will be transduced ex vivo, and following in vivo reconstitution of recipient mice, the patterns of integration of the wt and the recombinant AAV genomes as well as safety and efficacy of the AAV-based vector system will be examined. These studies will provide new insights into the basic molecular biology of the AAV-normal diploid cell interactions, and also evaluate the in vivo efficacy and safety of the AAV-based vector system prior to its potential use in human gene therapy. These studies also relate to our long-term interests in parvoviruses and human disease.


The 3' ends of eukaryotic messenger RNAs are formed by cleavage of the primary transcript, followed by addition of a poly(A) tail. Polyadenylation is required for RNA stability and the efficiency by which a gene's transcript is polyadenylated directly influences the level to which its mRNA accumulates and is translated. This is exemplified by a case of alpha-thalassemia in which a single point mutation was found in the poly(A) signal of the alpha-globin gene. One goal of the proposed research is to define the structural requirements of poly(A) signals. The experimental approach involves mutagenesis of portions of poly(A) signals followed by functional assays involving transfected COS cells or HeLa cell nuclear extracts. The mouse beta maj globin and SV40 early poly(A) signals will be studied in detail, as well as a variety of hybrid and synthetic constructs. The concept of poly(A) signals as interrupted palindromes is used as a guide for many of the experiments. The second goal of the proposed research is to elucidate the rules that govern poly(A) site selection. How poly(A) sites are selected when other poly(A) or splicing signals are located on the transcript will be studied using both in vivo and in vitro approaches. The relationship between poly(A) signals and introns will be explored in detail. These studies will give insight into the regulation of differential RNA processing.


Alcohol continues to be the drug of choice among American youth and teen drinking remains one of the nation's most serious public health problems. Yet in the Surgeon General's recent "Call to Action to Prevent and Reduce Underage Drinking" (USDHHS, 2007), he asserted that "underage drinking is not inevitable, and schools, parents, and other adults are not powerless to stop it." Accordingly, in line with scientific recommendations and NIAAA's (2006) research priorities, we propose to further develop and rigorously test two family interventions for adolescent alcohol problems, Multidimensional Family Therapy (MDFT) and a Family Motivational Interviewing Intervention (FMII). Family interventions have strong research and clinical traditions in the treatment of adult alcoholism and adolescent drug abuse, but little research has focused on family interventions for teen drinking. In this controlled trial, 250 youth ages 12 to 18 with alcohol-related crises in the ER or trauma center will be randomized to one of these two family interventions or to standard care. Both family interventions, MDFT and FMII, aim to potentiate the influence of the family to motivate and help youth to change, but they rely on different theoretical foundations and clinical techniques. Both family interventions provide 2 initial engagement sessions in the homes of participants within 72 hours of the ER visit. Youth in MDFT will then be engaged into a full course (3 months) of this family-centered treatment, and FMII youth will be enrolled into 3 months of behaviorally oriented 12-step group treatment. Standard care participants will be referred to the same group treatment as youth in FMII, but will receive no engagement or family sessions. We propose a randomized controlled trial with five aims: 1. To investigate the engagement potential and effectiveness of a family-centered intervention (MDFT) and Family Motivational Interviewing Intervention (FMII)/group for teens with alcohol-related crises;2. To explore differential treatment effects with comorbid adolescents;3. To examine the role of motivation and family factors as treatment mediators;4. To examine long-term abstinence, patterns and predictors of relapse up to 18 months follow-up;and 5. To compare the total and net monetary benefits to society of MDFT, FMII/group, and standard care. A multiple time point (intake, 3, 6, 9, 12, and 18 month follow-up), multiple domain and method assessment approach will be used to compare the interventions on their engagement and retention rates, clinical outcomes, and total and net monetary benefits to society. Latent growth curve modeling and other state-of-the-art statistical techniques will be used to test study hypotheses regarding adolescent and family change over time, differential effects of the interventions on comorbid youth, the mechanisms by which the treatments achieve their effects, and relapse patterns and predictors. The study has significant potential to advance adolescent alcohol treatment by providing new knowledge to providers and policy makers about effective and cost-beneficial family interventions for youth identified with alcohol problems in the ER and ultimately in other health care settings. PUBLIC HEALTH RELEVANCE: Adolescent alcohol abuse is one of the nation's most serious public health problems, and youth presenting for alcohol-related crises in the emergency room (ER) represent a particularly vulnerable group (Maio et al 2000). Findings from the proposed study have the potential to yield new knowledge for providers and policy makers to guide decisions about implementing the most effective and cost-beneficial alcohol interventions for youth in the ER and other public health care settings. Further, better understanding of the treatments'mechanisms of action and effects on comorbid problems, as well as youths'relapse patterns and predictors following these interventions, may guide further development of family-based interventions to halt the progression of alcohol problems among highly susceptible adolescents.


The first few divisions in C. elegans embryo has examples of both proliferative divisions in which both the daughters are equivalent, and differentiative divisions in which the daughters take on different fates. Studies are being undertaken of the dynamic properties of the cytoskeleton that are unique to differentiative divisions. In particular: factors are found to be segregated to one region of the cell prior to division; divisions are usually assymetrical due to an assymetry in the centrosomes of the mitotic apparatus; the mitotic apparatus is seen to align on a preformed axis of polarity. The purpose of this project is to use both in viva imaging and correlative electron microscopy to study these phenomenon so as to gain insights as to the mechanisms used in the generation of cell diversity The focus of this study is to elucidate the role of microtubules in positioning the spindle in early asymmetric cell divisions of Caenorhabditis elegans. We will use in viva fluorescence microscopy to characterize the organization and dynamics of microtubules during spindle alignment in wildtype and mutant embryos. We are especially interested in mutants that exhibit alterations in the dynamic organization of microtubules in order to identify genes involved in spindle alignment. In addition, we will examine the ultrastructure of cortical microtubule attachment sites. Results will provide insight into the basic mechanisms and molecular components underlying cleavage plane specification in determinative cell divisions.


Isolated rhesus monkey luteal cells offer a model system for the in vitro study of primate corpus luteum function and assessment of the direct influence of various factors on the corpus luteum during its lifespan. Initial topics of interest in this project include: 1) Morphological and functional correlations between the intact corpus luteum and subsequent isolated luteal cells, 2) Comparison of the in vitro steroidogenic potential of luteal cells obtained from corpora lutea at various stages of the menstrual cycle and pregnancy, and 3) evaluation of the influence of potential luteotropic and luteolytic agents on in vitro steroidogenesis of macaque luteal cells.


The past decade has seen the definition of large families and super-families of neural genes whose related but different sequences provide great opportunity if we can understand their functions and exploit their diversity. There can be no doubt that a major challenge facing modern neurobiology is the understanding and manipulation of gene function, both known and unknown. Institutions concerned with the critical issues of mental health and cognitive disability must look beyond gene identification and into the structure and function of the proteins encoded by these newly discovered sequences and the roles these proteins play in the development and behavior of the individual. While the techniques for gene discovery have become less expensive and more accessible, the most powerful techniques for the study of function have become more expensive and more technically demanding. It is increasingly difficult for any single investigator to be able to fully explore the structure of a gene and its regulation, the structure of the protein it encodes, the localization of the protein in the animal, the role of the protein in the normal animal, and the consequences of the absence or alteration of that protein in disease. However, successful exploitation of the gene discovery requires at least portions of each of these activities in part simply to set priorities for further studies. It is the purpose of the BCM-IDDRC cores to provide access to techniques and assays that will allow the investigator to make maximum progress, without undue duplication of effort. The Mouse Physiology Core is designed to provide BCM-IDDRC investigators with a battery of functional assays that will provide initial insight into the neurophysiologic consequences of a specific mutation. This core is considered a significant component of the BCM-IDDRC because it will help address the most common question following the creation of a new mouse mutant;"What is wrong with my mouse?" The BCM-IDDRC proposes to offer its investigators access to a battery of electrophysiologic assays that will help answer this question and direct the investigator's attention to experiments that might more directly address the role of a particular gene in generating a mental retardation or developmental disability phenotype. The BCM-IDDRC at Baylor College of Medicine is well established in studying synaptic transmission and synaptic plasticity in the central nervous system. For many years Dr. Rosenmund's laboratory has been investigating basic function and dysfunction of excitatory and inhibitory synapses as well as hippocampal electrophysiology and plasticity. Dr. Jeff Noebels has been a pioneer in the use of EEG techniques to understand the genetic and molecular basis of epilepsy, and specifically in the use of mouse models to understand epilepsy. The Mouse Physiology Core will be divided into two components. The Synaptic Physiology component of the Core will allow investigators to determine the basic attributes of synaptic function from cultured neurons as well as from acute slices from hippocampus. These preparations will allow for detailed examination of synaptic properties, circuitry function and synaptic plasticity. This information is particulariy germane to the mission of the BCM-IDDRC, given the well-documented role of the hippocampus in learning and memory, and the newly arising notion that autism and related diseases have their etiology (at least in part) at dysfunctional synapses. The procedures established will allow the assessment of several parameters related to normal synaptic physiology. For the presynaptic site, this includes determination of quantal content, readily releasable vesicle pool size, vesicular release probability, synaptic release probability, and several forms of short time facilitation and depression. For the postsynaptic site, this includes mlPSC and mEPSC amplitude and kinetics, GABAA, AMPA and NMDA receptor function, as well as the determination of synaptic and extrasynaptic receptor population. These measurements will be based on patch clamp whole cell recording techniques. Morphological analysis of dendritic structure, synapse formation and synapse activity are provided using quantitative light microscopy analysis. In slices, additional analysis of input-output relationships for various intensities of presynaptic stimulation as well as several short- and long-term forms of synaptic plasticity will be assessed, including: paired-pulse facilitation, post-tetanic potentiation, long-term potentiation (LTP), and longterm depression (LTD). Latter procedures will utilize extracellular recording in the hippocampal slice preparation, using ongoing standard protocols already used here. The Electroencephalography component of the Core will enable BCM-IDDRC investigators to evaluate the development of cortical excitability and brain function over prolonged periods in behaving animal models of mental retardation produced by genetic engineering techniques. Depressed excitability or abnormal brain rhythms are among the eariiest objective phenotypes of genetic human mental retardation syndromes. A high incidence of epilepsy is also associated with mental retardation, and the facility specializes in state of the art seizure detection techniques and assessment of seizure threshold. The ability to correlate spontaneous EEG activity with behavioral analysis by use of synchronized video/EEG monitoring is critical to the interpretation of the mutant nervous system phenotypes studied by the BCM-IDDRC.


Many elderly people complain of significant sleep disturbance. Complaints of multiple and prolonged awakenings and fragmented nocturnal sleep indicate that elderly individuals have difficulties in maintaining, rather than initiating sleep. Age-related changes in the circadian system underlie, at least in part, sleep disturbance. Timed exposure to bright light can alleviate such symptoms, although compliance to long-term treatments is poor. As such, alternatives and/or supplements to bright light are needed to relieve sleep disturbance. In humans, light phase shifts circadian rhythms according to a phase response curve. Nonphotic stimuli, such as social interaction, and sensory stimuli including auditory and olfactory cues, can also phase shift circadian rhythms in mammals and birds. Few studies have investigated rigorously whether nonphotic stimuli can affect human circadian rhythms. This study will determine whether an auditory stimulus, with an arousing component, can phase shift circadian rhythms in a sample of healthy male and female elderly (60+ yr old) subjects. An ancillary question will assess gender differences in response to auditory stimuli in the elderly. Subjects will spend two 4-day sessions in a sleep laboratory under constant dim light (less than 20 lux), receiving either a 2-h auditory stimulus or a control stimulus (absence of auditory cue) from 0100 h-0300 h on the second and third nights. Subjects will participate in both sessions, with presentation order counterbalanced among individuals. Polysomnographical sleep and core body temperature will be collected continuously and salivary melatonin samples will be obtained once each hour from 1800 h to 2400 h on Night 1 (baseline night) and Night 4 (post-stimulus night). To measure putative phase shifts, circadian phase of core body temperature minimum and the dim light melatonin onset (DLMO), before and after sensory stimulation, will be assessed in the control and experimental sessions. This study serves as the first essential step toward describing rigorously the effects of sensory stimuli on circadian rhythms in males and females. Completion of this project will allow for further exploration regarding the significance of nonphotic entrainers for the human circadian system, in particular as alternatives for relieving sleep disturbance in older subjects.


Cancer studies are limited by the availability of the types of data routinely collected by cancer registries. This project will collect data on comorbidity and hospital characteristics to evaluate survival effects in registry based studies. The objectives of this study are: 1) Link case records on rare cancer sites from central cancer registries with records from appropriate state health offices to obtain patient comorbidity information and hospital treatment information. Types of data from the state offices can include public datasets of inpatients data collected from state licensed hospitals. Examples would include datasets with a record for each inpatient discharged from a state-licensed hospital. Licensed hospitals could include general acute care, acute psychiatric, chemical dependency recovery, and psychiatric health facilities. 2) Covariate information will be merged into a database with case information to evaluate the rare cancer survival. 3) Linked data will permit the ability to construct follow-up of rare cancer patients from more detailed examination of treatment details and prognosis with facility information to uncover rare cancer outcomes and subsequent diagnosis and treatment of other health conditions, resulting in a more comprehensive understanding of rare cancer survival and treatment.


Although many studies have addressed the issue of the cellular components present in PHF none has defined the relationship of these components to PHF structure nor to each other. This lack of knowledge has resulted from a focus on components rather than their relationship. Further it has suffered from the necessity of a hypothesis with regard to likely components before a putative PHF component is examined. In the present proposal, an in vitro model system is developed utilizing the properties of polymers to define the relationship of identified PHF components as well as opening the possibility for identification of new PHF components. The utility of this approach is the possibility of assigning PHF components to a minimum of two classes, core or associated proteins, by non-immunochemical methods. PHF components detected by this protocol, but not identifiable by immunochemical approaches, will be defined by amino acid sequencing. These studies will provide an under standing of PHF as polymers current to that known for the most prevalent cellular linear polymer, the cytoskeleton.


Dystonia is a neurological disorder characterized by involuntary twisting movements or abnormal postures caused by excessive activation of specific muscle groups or abnormal co-activation of agonist and antagonist muscles. The neurobiological basis for this disorder is not well understood, and even the neuroanatomical substrates remain uncertain. While clinical studies of affected patients have traditionally associated dystonia with damage or dysfunction of the basal ganglia and its connections, laboratory studies of rodents have more consistently associated the disorder with dysfunction of the cerebellum and its connections. The overall goal of this proposal is to bridge our understanding of the gaps between the basal ganglia and the cerebellum in the expression of dystonia. Our hypothesis is that dystonia is not simply the reflection of dysfunction of a single motor control system, but may result from dysfunction in either the basal ganglia or cerebellum, or abnormal interactions between these two regions. In Aim 1 we will investigate the contributions of the basal ganglia in two well-characterized mouse models where dystonia is known to be triggered by dysfunction of the cerebellum. These studies will be valuable for demonstrating the involvement of the basal ganglia in these models as well as important interactions between these two motor control systems in the expression of rodent dystonia. In Aim 2 we will perform manipulations of the cerebellum in rhesus monkeys analogous to those that have been demonstrated to provoke dystonia in rodents. These studies will establish a role for abnormal cerebellar output in the genesis of dystonia in monkeys and have the potential to result in a novel monkey model for dystonia. Aim 3 is devoted to a careful reassessment of autopsy material from the cerebellum of dystonia patients. These studies will be valuable because prior autopsy studies, which traditionally focused almost entirely on the basal ganglia, have generally failed to disclose any consistent neuropathological changes in most forms of idiopathic dystonia. Overall, the studies described in this proposal have the potential to stimulate a major revision of current concepts concerning the neuroanatomical basis for dystonia. A more complete understanding of the neuroanatomical substrates for dystonia is of elemental importance for any future studies of dystonia addressing relevant pathogenetic changes at the molecular, cellular, and physiological levels. In addition, the studies will have direct relevance for modern neurosurgical approaches to the management of dystonia that involve focal brain stimulation or lesion techniques. [unreadable] [unreadable]


[unreadable] The HIV Evolution and Dynamics Meeting is a small annual HIV meeting with a 15 year history. This meeting has given people who perform data analysis, mathematician modeling, and HIV evolutionary analysis a specific opportunity to come together to discuss new research, both in terms of new studies and their implications, and new analysis tools and methods that can be applied to HIV. Mathematical and statistical aspects of the work can be discussed in greater depth than is possible in the larger HIV meetings, which primarily focus on experienial and clinical presentations. Historically this meeting has alternated venues between Europe and the US, to allow greater access to the meeting for researchers on both sides of the Atlantic. It can be one of the most important meetings of the year for HIV Scientists directly involved in the analysis of data. The meeting draws individuals who model in vivo dynamics and disease progression, global variation of HIV with considerations of the epidemic history, immunology and vaccine strategies, and individuals that study the emergence of drug resistance. The talks and posters range from important new studies that have complex aspects that may be presented more from a traditional view, but are discussed from an analysis perspective; mathematical oriented talks from scientists outside the HIV field who develop new analysis methods that might usefully be brought into the sphere of HIV research. On the order of 100 to 150 scientists attend, this number has depended on the venue in the past. There is ample time for discussion and developing contacts to facilitate and extend ongoing studies, and to initiate new collaborative efforts. The meeting has traditionally been supported by the OAR and the CDC with supplemental funding coming in through industry and the fund raising efforts of the primary organizer in any given year. We are requesting a continuation of the invaluable support received this grant for past 5 years which has permitted the attendance of junior investigators and participants from under developed countries. The UCSD Office of Continuing Medical Education has offered to support the financial and organizational infrastructure of the conference for the 3 year period for which we are requesting funding, and will work directly with the primary scientific organizer regardless of his or her home institution. The HIV Evolution and Dynamics Meeting is a small, intensive, annual HIV meeting with a 15 year history. This meeting has given people who perform data analysis, mathematical modeling, and HIV evolutionary analysis an opportunity to come together to discuss new research, both in terms of new studies and their implications, and new analysis tools and methods that can be applied to HIV. The information presented and generated by this meeting has potential important implications regarding HIV transmission, prevention, disease progression and treatment. [unreadable] [unreadable] [unreadable] [unreadable]


DESCRIPTION (Adapted from applicants abstract): An important part of the defense against parasites is the specific immune response of the host. The stimulus initiating the response is the binding of parasite-derived antigenic fragments to major histocompatibility (MHC) molecules and their presentation by these molecules to T lymphocytes. The MHC molecules vary from one individual to another and this variability is encoded in polymorphic Mhc genes. Some of the variant molecules are apparently incapable of presenting certain peptides to T lymphocytes and in such situations the capability of the host to respond to a parasitic infestation could be impaired. It is therefore important to understand the nature of the polymorphic variation in the Mhc genes. The main objective of the proposed research is to explain the origins, persistence, and significance of the Mhc polymorphism. The specific aims of this research are to determine what proportion of the allelic variation pre-and postdates the formation of a species; to define the mechanisms of Mhc diversification; to estimate the age of human allelic and haplotype polymorphisms; to identify the mechanisms possibly maintaining the Mhc polymorphism in natural populations; to provide evidence that parasites are indeed the main driving force behind the Mhc diversification; to establish the part played by population structure in the long-term persistence of Mhc polymorphism; and to formulate an all-encompassing theory of Mhc polymorphism. The research will be carried out on 3 model systems: the primates, the mouse, and the zebra fish. It will include both molecular analyses of the Mhc genes and the study of the Mhc in natural and model populations.


Ethanol is a teratogenic agent responsible for the fetal alcohol syndrom (FAS) that is characterized by mental retardation in children born to chronic alcoholic women. While the FAS invovles central nervous system (CNS) dysfunction, the cellular basis for understanding the defect related to central catecholaminergic (CA) neurons is not known. CA neurons in their CNS could be implicated in the FAS for the following reasons: (1) CA terminals provide a major input to the immature neocortex and neostriatum, and (ii) some biochemical evidence suggests that central CA neurons are susceptible to ethanol. CA terminals can be quantitatively assessed by fluorescence microscopic (FM) and electron microscopic (EM) morphometrics. The present proposal will use an FM/EM morphometric analysis to examine effects of ethanol on CA neurons in the neocortex and meostriatum of developing rats. By using glyoxylic acid-induced FM and 5-OH-DA EM techniques, parameters such as CA terminal density and CA synaptogenesis will be studied in neonates which have been exposed to ethanol through their meternal blood and breast milk from pregnant/lactating rats fed with 35% ethanol-containing liquid diets. This part of the study is to examine an early effect of ethanol on central CA neurons. These ethanol-exposed neonates will also be studied when they become adults, in order to examine long-term effects of ethanol on central CA neurons. Similarly, CA terminals will be evaluated by using glyoxylic acid FM method, and CA boutons in the adults will be studied by suing a potassium permanganate EM method. Since dendritic spine abnormalities of cortical neurons have been described in mentally retarded children, we will use the rapid Golgi method to evaluate effects of ethanol on dendritic spines of cortical neurons in adult rats exposed to ethanol during their prenatal and perinatal development. The goal of this proposal is to provide us a fundamental and ultrastructural basis for better understanding mechanisms by which ethanol affects CNS function.


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. Grant Number: R21-CA111988 Project Start: 22-FEB-2005 Project Original End Date: 31-JAN-2007 Project extended to January 31, 2008 Project Summary Surgical resection has been shown to be an effective treatment for secondary liver tumors, with five-year survival rates between 20-30%. Unfortunately, only select patients are candidates for resection, and it has a relatively high complication rate. About 35% of patients suffer minor complications, 15% have more serious complications, and the mortality rate is 2-5%. In addition, the average hospital stay is about two weeks, even without complications. Minimally invasive methods to thermally coagulate liver tumors have eliminated some of these disadvantages and have been shown to be an effective alternative treatment. The number of patients who receive such thermal ablation treatments is growing rapidly, and this technology is poised to enter widespread use. A way to substantially improve the safety and effectiveness of these treatments would be to have a method to easily and reliably quantify the tissue online that has reached a sufficient thermal exposure to induce thermal coagulation. Our hypothesis is that a novel magnetic resonance imaging (MRI) contrast agent that is activated at a threshold temperature level can be used to reliably successfully monitor thermal ablation of liver tumors. This agent consists of paramagnetic material encapsulated in a liposome shell. When encapsulated, the material has a minimal effect on the MR signal intensity. The liposome shell undergoes a phase transition at a temperature (Tc) that can be precisely chosen, and MR signal enhancement occurs which can be easily detected with standard imaging methods. With a Tc of 57[unreadable]C, which is the approximate threshold for thermal coagulation for heating of at least a few seconds, the signal intensity enhancement thus indicates the regions that are thermally ablated. Since perfusion is halted after thermal coagulation, any released paramagnetic agent will remain in the tissue for an extended period of time, clearly marking the ablation progression. Our preliminary tests of this agent in vivo have demonstrated its basic functionally. Here, we propose in vivo animal experiments to extensively characterize its performance and to test its effectiveness in monitoring thermal ablation of liver tumors. This research will lead to more effective and safer thermal ablation treatments in liver, and perhaps allow more difficult cases to be feasibly treated with thermal ablation methods, since it would provide straightforward and reliable online feedback of the treatment progression. Thesaurus Terms: contrast media, liver neoplasm, magnetic resonance imaging, neoplasm /cancer thermotherapy, technology /technique development image enhancement, liposome, noninvasive diagnosis bioimaging /biomedical imaging, histology, laboratory rabbit ICD: NATIONAL CANCER INSTITUTE IRG: RTB


The specific aims of this proposal are to a) purify melanoma-associated antigens of defined specificities, b) develop radio-immunoassay (RIA) and enzyme immunoassay (EIA), c) apply these sensitive and specific serologic assays to detect antibody and free circulating antigen levels in sera and other body fluids of patients with malignant melanoma, and correlate the results of RIA and EIA with clinical course of disease. It is expected that by developing the sensitive serologic assays and improving their specificities, these assays will become potentially useful tools for prognosis and diagnosis of human cancer. Cancer patients who are rendered disease-free by surgical removal of tumor can be monitored for the levels of free circulating tumor-associated antigens and their corresponding antibodies in the body fluids. Such information will be valuable to the physician for the management of human cancer. Besides being applicable as an immunodiagnostic aid, such information will expand the understanding and knowledge of tumor-host relationship. The isolation and purification of specific melanoma-associated antigens and their characterization will provide an insight into the immunobiology of human malignant melanoma.