Project Summary/ Abstract: Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is the most common pediatric neurodegenerative disease caused by a mutation in the CLN3 gene. Children with JNCL experience progressive visual, cognitive, and motor deterioration with a decreased life expectancy (late teens-early 20s). There is currently no cure for JNCL, making research to identify novel drug targets crucial to extend the survival and quality of life of afflicted patients. Previous studies have shown that astrocyte activation precedes and predicts regions of neuronal loss in JNCL, suggesting a defect in supportive glial functions. Glutamate levels are elevated in the JNCL brain and neuronal loss is thought to occur, in part, via glutamate excitotoxicity. Currently, little is known about aberrant glutamate-glutamine cycling in astrocytes and no reports investigating neuron-astrocyte cross-talk or regulation in JNCL exist. Our prior studies revealed a significant decrease in glutamine synthetase coupled with altered expression of the glutamate transporters in CLN3?ex7/8 astrocytes both in vitro and in vivo. This proposal will utilize mice harboring a 1.02 kb mutation spanning exons 7 and 8 of CLN3 (CLN3?ex7/8) to study glutamate regulatory pathways both in vivo and in vitro. My preliminary data indicates that primary CLN3?ex7/8 astrocytes display decreased glutamate transporter expression and activity when exposed to stimuli present in the JNCL brain. By extension, this disruption in glutamate homeostasis in the context of CLN3 mutation threatens to disrupt neuron-astrocyte signaling pathways, which if chronically perturbed can induce neuron excitotoxicty. Indeed, my preliminary studies have demonstrated reduced Ca2+ signaling in CLN3?ex7/8 astrocytes concomitant with heightened Ca2+ transients in CLN3?ex7/8 neurons, suggesting that disruptions in glutamate cycling interrupt critical homeostatic cell signaling networks. Concurrent with impaired Ca2+ signaling, preliminary electrophysiology conducted on hippocampus slice cultures from 30-day-old mice show that CLN3?ex7/8 mice have higher population spike amplitude and field excitatory post-synaptic potentials. This data indicates that the CLN3 mutation causes an increase in neuron activation that would potentate elevated glutamate release. Collectively, these findings formulated the hypothesis that the CLN3 mutation perturbs glutamate cycling pathways, which potentially creates a toxic cellular milieu, culminating in neuron death. The Specific Aims of this proposal are: 1) To identify the mechanisms responsible for astrocyte glutamate dysregulation and altered astrocyte-neuronal signaling networks in the context of CLN3 mutation; and 2) To evaluate whether the S100B synthesis inhibitor ONO-2506 can limit neurotoxicity in CLN3?ex7/8 mice by enhancing glutamate transporter expression and glutamate uptake. Establishing key regulatory mechanisms of glutamate and astrocyte-neuron cross-talk in JNCL may unveil novel therapeutic targets to extend the quality-of-life for children suffering from this devastating neurodegenerative disease.


With a few important exceptions, all the neurons in the mammalian nervous system fall into two major classes: excitatory neurons, using glutamate as a neurotransmitter, and inhibitory neurons, using GABA (and in some regions also glycine) as a neurotransmitter. GABAergic neurons play the critically important roles of counterbalancing excitation and of channeling the flow of sensory and motor information in time and in space; loss of cortical GABAergic neurons or impairment of their function is a major factor underlying epilepsy and degenerative motor diseases. Their critical roles notwithstanding, GABAergic neurons remain poorly understood; in most regions they are interspersed among the more numerous excitatory neurons, and studying them has been seriously hampered to date by the lack of a means to identify them reliably in living tissue. The need addressed by the proposed study is for a novel method to identify and visualize GABAergic neurons in living preparations. We have recently made the first step towards addressing this need, by generating several lines of transgenic mice in which expression of green fluorescent protein is driven in a cell-specific manner in putative GABAergic neurons. These lines are potentially an extremely powerful research tool for studying GABAergic neurons in living preparations; however before they can be used for such studies, the molecular genetics of the transgene needs to be characterized, the fidelity of GFP expression in GABA neurons needs to be validated, and normal GABAergic function should be verified. Once these aims are accomplished, the mice will be made available to the scientific community through the NIH-sponsored Mutant Mouse Regional Resource Centers. The Exploratory/Developmental R21 Grant mechanism of support is appropriate, because the proposed project will demonstrate the validity and feasibility of a new tool, the GAD67-GFP transgenic mouse, which could have a major impact on the field of neuroscience by allowing investigators to address crucially important questions regarding GABAergic neurons, questions which were so far difficult or impossible to address.


AS-101 (Ossirene) induces the production of several hematopoietic growth factors in vitro, and protects mice from hematologic toxicity of external beam radiation or chemotherapy in vivo. The BRMP previously demonstrated similar effects of IL-1 before chemo/radiotherapy in animals, while a BRMP clinical trial demonstrated acceleration of platelet recovery from high-dose carboplatin when IL-1 followed chemotherapy. This protocol tests the ability of AS-101, by two doses and several schedules, to produce similar effects with high-dose carboplatin in pts. The objectives of this study are: 1) determine the toxicity of Ossirene and high dose carboplatin, 2) determine whether the sequence and/or schedule of drug is important, 3) determine which dose of Ossirene is superior in limiting bone marrow suppression from carboplatin. Pts. received carboplatin 800 mg/m2 and Ossirene, 3 or 6 mg/m2 pre or post carboplatin. Nine pts. were entered on this study. There were 2 minor responses. Pts. experienced significant hematologic toxicity, however toxicity did not prevent treatment.


A variety of endogenous and environmental DNA damaging agents pose a constant threat to DNA. Ultraviolet (UV) light from the sun is a common source of DNA damage in organisms, including humans. Exposure to UV light causes lesions in DNA that can impede DNA polymerases resulting in stalled replication forks. Failure to re-start stalled replication forks can have serious effects on cells, including interruptions in the cell cycle and chromosome breakage. Thus, UV-induced DNA lesions can destabilize the genome, increasing the chance of mutations. Such genome instability can lead to cancer. One way cells minimize the deleterious effects of damage that blocks DNA replication is through use of a specialized class of DNA polymerases that can replicate across the lesions in a process called translesion synthesis. DNA polymerase 7 (C. elegans POLH-1) is especially important for translesion synthesis past UV-induced lesions. Polymerase 7 and other translesion polymerases have high error rates when replicating undamaged template. Thus, their access to DNA replication forks must be tightly regulated to prevent mutagenesis. How translesion polymerases are removed from replication forks after TLS, to minimize their engagement with undamaged DNA, is not currently known. Recent work from our laboratory has elucidated a possible mechanism for how this might occur. According to our model, when POLH1-1 is recruited to replication forks, it is SUMOylated by GEI-17 E3 SUMO ligase, and stabilized long enough to perform translesion synthesis. The mechanism by which GEI-17 senses DNA damage to SUMOylate POLH-1 is not known. After translesion synthesis, POLH-1 is ubiquitinated by CDT-2 ubiquitin ligase, targeting it for degradation. Thus, we hypothesize that CDT-2-mediated degradation of POLH-1 limits its access to undamaged template, controlling POLH-1's mutagenic capacity. The experiments described in this proposal will address two outstanding questions related to this hypothesis. In Aim 1, I will use genetic and biochemical approaches to clarify the mechanism for how GEI-17 is recruited to sites of DNA damage to SUMOylate POLH-1. In Aim 2, I will use high throughput DNA deep sequencing to examine whether CDT-2 controls mutagenesis. PUBLIC HEALTH RELEVANCE: The variant form of the genetic disorder Xeroderma pigmentosum (XP-V) is caused by loss of polymerase 7 function. XP-V patients are hypersensitive to sunlight and have an increased predisposition for skin cancer. This pathology suggests that human DNA polymerase 7 limits the carcinogenic potential of UV light. A more complete understanding of polymerase 7 function, coming from my studies of POLH-1, could contribute to the eventual development of advanced cancer treatment therapies. PUBLIC HEALTH RELEVANCE: The experiments in this proposal will explore the regulation of DNA polymerase POLH-1 in Caenorhabditis elegans. Homologs of POLH-1 (DNA polymerase 7 in humans) minimizes mutations caused by UV light, thus limiting the development of cancer. Patients with a defect in the function of polymerase 7 are highly sensitive to UV light and have a predisposition for cancer. Thus, a detailed understanding of the molecular activities of polymerase 7 and its regulation may contribute to the creation of new cancer treatments.


Graft versus host disease (GVHD) is a debilitating and often fatal immunological condition induced by lymphoid cells grafted either experimentally or naturally into an immunoincompetent foreign host. Since children receiving bone marrow transplants or blood transfusions during the period of brain development are at risk of therapeutically-induced GVHD, we have studied the effects of GVHD on neonatal cerebellum, a well-defined model of developing human brain. The disease causes a decrease in cell accumulation (DNA synthesis and content and the numbers of cells in specific populations) and function (RNA content, synthesis and translational capacity; the number of cells migrating; and in the growth of Purkinje cell dendrites). The lack of inflammatory response or lymphocytic infiltration into the cerebellum suggests that a blood-borne factor is responsible. Amazingly, we found that many of the deleterious effects of GVHD on brain development could be reversed after the disease was stopped with alloantiserum immunotherapy. Here, we propose to study further GVHD-induced brain growth retardation and its amelioration with regard to: the timing of cell cycling and migration (autoradiography); cerebellar coordination of motor function (swimming behavior); the prenatal induction of GVHD (effects on prenatally formed cells and severity of postnatal events); the amount and distribution of the various forms of mRNA and the activity of each (cell-free protein synthesizing systems and 2-d gel electrophoresis; the significance of newly synthesized proteins during the disease). To investigate possible mechanisms involved in GVHD-induced brain growth retardation, we will address these questions; 1) Does cerebellar damage occur nonspecifically, i.e., regardless of histocompatibility antigen specificities? 2) Does GVHD serum inhibit growth in dividing cells in organ culture? 3) Is the blood brain barrier intact in GVHD? 4) How does GVHD effect the adrenal glands and their production of corticosterone? The general questions we expect to answer are: 1) In what manner does an immune process cause readily reversible alterations in neuronal cell functions which may be correlated with motor behavior? 2) Can immune processes regulate histogenesis and protein synthesis in non-neuronal rapidly dividing cell populations.


The purpose is to determine the effectiveness and tolerability of ucb L059 given in a twice daily regimen to existing AED treatment and to also determine the pharmacokinetic parameters (the measuring of the drug in the blood and urine) of both ucb L059 and its breakdown product ucb L057 in the age group of 6 to 12 years.


The menisci are C-shaped fibrocartilage disks that occupy the periphery of the knee joint and serve a number of significant mechanical functions including load distribution across articular cartilage, and joint stabilization. In the face of such mechanical demands, it is not surprising that the meniscus can become damaged. Unfortunately, surgical options for the treatment of damaged menisci are limited, primarily due to the poor vascularity and healing capacity of the central two thirds of the meniscus. Allograft implantation is complicated by the threat of transmission of infectious diseases, the difficulty of matching meniscal shape and material properties, and problems with maintaining cell viability. While meniscectomy (the complete or partial removal of the affected tissue) which is the most common orthopedic procedure performed in the United States can lead to the early onset of osteoarthritis. Providing a meniscal substitute engineered to function much in the way of the native tissue could delay or even avoid the onset of osteoarthritis. Meniscal substitutes, whether cell-seeded degradable scaffolds or synthetic implants, should ideally help to distribute loads across adjacent articular cartilage much in the way of the native tissue, thereby protecting the underlying articular cartilage. However, the properties of a substitute that are required to ensure that this functional capability is met are unclear. This is caused in part by the absence of a robust, comprehensive and physiologically relevant model within which the effect of substitute design variables can be parametrically studied. This lack of such a test, or series of tests, makes the design and evaluation of candidate scaffolds impossible, retards the regulatory pathway to commercialization, and leads to a situation where scaffolds are implanted in humans with little information about their ability to perform mechanically in the highly-loaded environment of the knee joint. Our goal is to define the relationship between meniscal substitute material and structural properties and joint contact mechanics under multiple physiological activities across a range of patient populations. The main element of the framework required to achieve this goal is a statistically-based, rapidly computable interpolator that will be built using results from experimentally-validated knee specific computational models. We will gather information about factors that most markedly affect the functional performance of the native meniscus and use this information as a template to identify the design space into which substitutes should fall if they are to function appropriately. Our goal is not to design a meniscal substitute; rather, we will demonstrate that our approach can establish a workable design space for use by those developing solutions for meniscal repair. Our efforts will culminate in the development of a new paradigm for the development and screening of any complicated tissue substitute, whether a non-degradable implant or a cell-seeded degradable scaffold prior to initiating more time consuming and costly animal and clinical trials. PUBLIC HEALTH RELEVANCE: Our goal is to define the relationship between meniscal material and structural properties and joint contact mechanics under multiple physiological activities across a range of patient populations. The main element of the framework required to achieve this goal is a statistically-based, rapidly computable interpolator that will be built using results from experimentally-validated computational models of the intact and meniscal substituted knee. Our approach will serve as a tool for rapid evaluation and design of functional substitutes and provide a new paradigm for the development and screening of any complicated tissue substitute, whether a non- degradable implant or a cell-seeded degradable scaffold.


PROJECT SUMMARY Caspase-2 (Casp2) has been described in the literature as an excellent target for Alzheimer's disease (AD) and has been implicated as therapeutically relevant in Huntington's disease (HD). Several non-apoptotic roles of Casp2 that may be relevant to neurotherapeutic discovery include its regulation of autophagy, oxidative stress, endoplasmic reticulum stress, and its effect on dendritic spines. Our own collaborative work provides evidence that ?tau314 (a non-fibillar, N-terminal tau fragment) levels are correlated with AD in humans. In rTg4510 mice, which express the hP301L tau associated with frontotemporal dementia (FTD), we have shown that Casp2 cleaves tau to form ?tau314 (H2N?YKPVD314), a heretofore-unreported tau fragment, and have linked this soluble tau fragment to reversible cognitive dysfunction. Given these exciting and promising results, we have initiated a comprehensive program to identify potent and selective Casp2 inhibitors. This platform includes standard structure-based design of covalent inhibitors based on the YKPVD314 cleavage site in tau and high-throughput screening (HTS). Herein we propose to employ fragment-based screening (FBS) to expand and complement the scope of this program, increasing the likelihood we will discover small-molecule inhibitors of Casp2. The single specific aim of this exploratory project is to identify and characterize fragments (low molecular weight and soluble compounds) that bind to Casp2. A commercial in-house fragment library will be screened against Casp2 using differential scanning fluorimetry (DSF). Active ligands will be characterized by surface plasmon resonance (SPR) and X-ray crystallography. Confirmed actives will be validated using SAR (structure-activity relationship) by commerce. Our working hypothesis is that the collection of well- characterized scaffolds we discover will constitute excellent starting points for future work focusing on the optimization and development of potent and selective small-molecule inhibitors of Casp2. The potential impact of this project on human health is considerable. There is an unmet medical need for therapeutic agents that can halt or reverse the cognitive decline associated with AD. This work will have a positive impact on the field of therapeutics to treat the cognitive decline observed in several diseases of the central nervous system, particularly AD, HD, and FTD. The eventual development of a selective inhibitor of Casp2 would address this unmet medical need and would represent a significant advancement in the field of AD.


The goal of this project is to increase the ability of virus vectors to deliver genes to the postnatal nervous system and to use these vectors to alter cell fate in the nervous system. Two strategies will be undertaken to expand the range and longevity of gene delivery to neural cells in the mouse nervous system. In the first scheme, retrovirus vectors will be generated which can confer stable gene expression on astrocytic cells grafted into the brain. The phosphoglycerate kinase promoter will be used to regulate expression of brain-derived neurotrophic factor (BDNF) for degenerating dopaminergic neurons in newborn weaver mice. These vectors will also be used to genetically modify astrocytic lines capable of migrating in the brain. In addition, one of the astrocytic lines will be converted to a packaging cell line which can be used to deliver retrovirus vectors to endogenous neural cells in vivo. Retrovirus vectors will also be generated to aid in functional analysis of the NF2 gene product in Schwann cells and tumor cells. Further we will try to develop an animal model for neurofibroma formation by retrovirus-mediated delivery of antisense RNA to repress expression of neurofibromin or by homologous recombination to knock-out the normal NF1 allele in Schwann cells from heterozygous NF1 knock-out mice. The behavior of genetically altered Schwann cells will be evaluated in a peripheral nerve injury-repair model. In the second gene delivery scheme we will try to improve herpes vectors for delivery of genes to neurons. A "piggy back" system of herpes simplex virus vectors will be developed using a combination of amplicon and recombinant virus vectors to combine complementary features of both vector types and to make them mutually dependent on each other. This system will be designed to allow limited replication of the vectors in glia-derived cells using glia-specific promoters to regulate expression of critical genes. Amplicon vectors will be used to deliver NGF and Oct2 to neurons at the time of infection in order to encourage entrance of the virus into latency, at the same time making preparation of vector stocks feasible by using a tet operon system to control expression of these genes in culture. The recombinant virus vector will be deleted for genes contributing to neurovirulence including ICP4 (or ICP27), ribonucleotide reductase, gamma 34.5 and UL41. The herpes vector system will be evaluated for gene delivery, stability of transgene expression and pathogenicity to neural cells in the brain. These new gene delivery systems will provide a basis for therapeutic intervention in animal models of Parkinson's disease and neurofibromatosis.


Studies to define the host immune response to human tumor has concentrated in part on characterizing the T cells within the tumor bed. The picture that has immerged is complex with respect to the functional responsiveness of these cells. Tumor infiltrating lymphocytes (TIL) contain responsive T cells that display normal effector functions. It is also clear that a significant portion of TIL from a variety of tumors are defective in their proliferative response. We have demonstrated that TIL derived from human RCC and B cell lymphomas have a reduced proliferative response that is reflected by a decrease in the IL2Ralpha surface expression and mRNA levels. While TIL can produce comparable levels of IFNgamma when compared to PBL they also display a diminished capacity to produce IL2. Our hypothesis is that while a segment of TIL are responsive the majority display a state of unresponsiveness that is rather limited in scope. Other functional parameters are thought to be intact even though most TIL have a diminished ability to proliferate and produce IL2. Aim 1 will determine which subsets of TIL are most affected. Cell sorting experiments "will determine if the proliferative defect is unequally distributed among T cell subsets (CD4+ and CD8+ cells) and if the defect relates to the state of activation of T cells in the tumor bed. Aim 2 will define the extent of the unresponsiveness by determining if other functional parameters are intact in the affected populations. Purified TIL subsets will be used to document whether TIL are defective in their ability to produce IL2. Whether the defect is limited to IL2 will he determined by testing TIL for the production of other cytokines such as IL4, TNFalpha and GM-CSF. Experiments will also determine if TIL are as competent as PBL to produce mRNA for perforin and serine esterases. Additional work will define the role various stimulation routes play in the unresponsiveness. An analysis of multiple functions induced by IL2R and TCR stimulation will help define the extent to which each of these receptors are affected. Experiments will also determine if triggering TIL via CD28 is normal since this pathway appears to be distinct from that induced by TCR. Aim 3 will determine if the reduced IL2Ralpha surface expression and IL2 production of TIL is related to alterations in transcription. RT/PCR will be used to quantitate mRNA levels of TIL and PBL. Included will be studies that determine if the reduction in mRNA levels is due to changes in the kinetics of mRNA accumulation, rate of transcription or mRNA stability. The goal of the proposed work is to begin to understand how broad the defect is, and to identify the subsets affected as well as which receptor pathways are altered in the unresponsive TIL. It is important to characterize quantitatively and qualitatively the T cell unresponsiveness within the tumor. An understanding of this anergy may provide ways to reverse or bypass this defect allowing for T cell activation and the development of a potent host immune response to tumors. Understanding T cell anergy in disease states such as cancer will be important for the development of new treatment strategies.


The objective of this study is to evaluate the effectiveness of therapeutic stockings to reduce the incidence of foot ulcers in high-risk diabetics, and to evaluate self-reported functional status among treatment groups. We expect that subjects treated with therapeutic stockings will have fewer foot ulcers and that their self-reported functional status will be higher than patients receiving standard care. Persons with diabetes with previous foot ulceration have a yearly recurrence rate of more than 30%, even at centers with specialized foot clinics. Therapeutic stockings are an inexpensive and practical mechanism to reduce pressure and shear forces on the foot and thereby reduce the likelihood of ulceration. However, there is very little clinical evidence to support the use of "therapeutic stockings". We will randomize 400 persons with diabetes and a history of foot ulceration to receive standard therapy (ST) consisting of education, regular foot care and protective shoes and insoles;or standard therapy with the addition of therapeutic stockings. The stockings used in this study are designed to reduce pressure and friction at the stocking-foot interface. The NeuroQol instruments will be administered to evaluate self-reported functional status. We will patients at centers in Temple, Texas, Georgetown, Texas, and two centers in Manchester, UK. Patients will be evaluated at least every three months for regular foot care after screening and provision of protective shoes and insoles. Patients will record daily use of therapeutic stockings, insoles and shoes in a log book. Patients will use a step counter to record daily activity. Therapeutic stockings are an inexpensive and practical tool to protect the feet of high risk patients from pressure and shear forces. This simple measure may significantly reduce the risk of foot ulcers in high risk patients.


A continuation of the CCOP grant will enable Hematology-Oncology Associates of Central New York, P.C. (HOACNY) to continue its active participation in a wide variety of ongoing clinical cancer research studies, including treatment and prevention trials. The goal of the program participation is to continue translating experience in cooperative group clinical research into expert cancer prevention, cancer control, diagnosis, and treatment (curative, palliative, supportive) for the greater community of central New York (Syracuse and surrounding rural counties). The Syracuse Hematology-Oncology CCOP aims to continue pursuing multi-disciplinary and multi-specialty participation in and awareness of clinical treatment and prevention protocols. The CCOP physicians and staff will continue in efforts to reach the professional and lay publics with current practice in the prevention and management of cancer and to encourage participation in research which has long term benefits for the entire community. The CCOP plans to increase recruitment of minorities to research trials and to maintain its high level of female participation in clinical trials. More specifically, the goal of the Syracuse CCOP physicians and staff is to maintain current levels of participation in clinical treatment trials while increasing the number of registrants on prevention and control trials. In the process of recruitment, the CCOP staff will be able to continue providing health education and screening related to cancer prevention and control.


We have continued during the past year to study those metabolic properties of the red cells of different animals which are associated with their viability in vivo and in vitro and which play a part in the transport of oxygen. We have also continued investigations on metabolic changes which take place during the maturation of the red cell. Our previous studies on the red cells of the alpaca, which presented interesting problems because of the unique adaptations of this animal to high altitude, have been extended to another cameloid, the dromedary. A program was begun this year to see what role the red cell of the diving animal might play in the special requirements of these animals for transport and conservation of oxygen. Metabolic intermediate analyses and glycolytic rate determinations have been performed on red cells from the bottlenose dolphin, the California sea lion and the gray seal. Work which has been in progress in this Laboratory for several years to try to explain the peculiar metabolic properties of pig red cells has continued with in-vivo experiments on C14-glucose utilization by the circulating red cells of the domestic pig. A project has been started this year to learn about the energy metabolism of fish red cells and analyses have been made of the glycolytic intermediates and soluble nucleotides of red cells from mackerel, dogfish and salmon. Work is also continuing on a comparison of the metabolic properties of mature circulating erythrocytes with reticulocytes obtained from rats and rabbits after treatment of the animals with phenylhydrazine and after severe bleeding.


This 2nd revision of an application for a competing renewal of an Independent Scientist Award seeks funding to support development of the candidate's research on the treatment of late-life bipolar disorders and his mentorship of young investigators. This research program addresses the need for evidence that can help individualize the management of these underserved patients by examining proposed clinical and neurobiological predictors of inter-individual differences in benefit from first line pharmacotherapies. Having initiated the multi-center GERI-BD study of acute treatment of manic bipolar elders, and its Coordinating Center, the candidate aims to use this platform to explore the utility of selected measures of frontostriato-limbic impairment as predictors of resistance to anti-manic treatment. Three Pilot Studies will focus on affect-laden executive tasks, neuroimaging of white matter integrity, and neurotransmitter-related genetic variation. Conducting these Pilot Studies will enable the candidate to expand his knowledge base in cognitive and affective neuroscience, neuroimaging and pharmacogenetics, and related analytic issues, through interaction with expert advisor/collaborators. This career enhancement is intended to foster development of his research to address the needs of elderly patients who do poorly with optimal standard treatments, and to study the management of other phases of bipolar illness in late life. It is also intended to support continuation and expansion of his mentorship activity. As the population ages, greater numbers of elders present with bipolar disorder. They suffer from severe symptoms, disability, and high mortality. These studies can provide findings that use relatively non- invasive and cost effective methods, derived from neuroscience and neurobiology, to develop safe and effective treatment approaches.


Chronic kidney disease-mineral and bone disorder (CKD-MBD) results in complex skeletal and metabolic phenotypes. Because of this, patients with advanced kidney disease have an increased risk of fractures. In patients with age-related bone loss, bone mineral density (BMD) estimates are a helpful predictor of fracture development. In CKD, however, the picture is unclear. While it may be of use, the presence of other metabolic derangements indicate that bone quality likely plays a greater role in the pathogenesis of CKD-related fractures than in those associated with age-related bone loss. Current treatment in patients with CKD-MBD is focused on suppressing elevations in parathyroid hormone. This is accomplished by calcitriol supplementation. While the effect of calcitriol in osteoporotic patients has been studied, its impact on bone quality and fracture risk n CKD patients is currently unknown. Also, recent analyses suggest that raloxifene, a selective estrogen receptor modulator, might be a useful intervention for patients with late stage kidney disease. This is supported by beneficial renal outcomes in patients on raloxifene as well as evidence demonstrating an additional non-cellular mechanism by which to improve bone quality. So, we hypothesized that CKD leads to alterations in bone quality that can be corrected by raloxifene and its combination with calcitriol. This will be tested through the use of a slowly progressive model of CKD-MBD, the Cy/+ rat. This study will compare the quality of skeletal tissue (independently of mass) in normal and Cy/+ rats. This will be accomplished by examining bones from 30-week-old rats for changes in bone quality. Specifically, outcomes will include tissue-level mechanical properties, mineralization, collagen composition (cross-linking, morphology, and mechanics), and bone matrix hydration (MRI). We expect rats with CKD to display lower bone strength, lower mineralization, lower collagen stiffness, lower matrix-bound water, and higher non-enzymatic cross-linking of collagen. The second major goal is to examine the effects of raloxifene on bone abnormalities present in Cy/+ rats. 25-week-old animals will be treated for 5 weeks (equivalent to one bone remodeling cycle). Primary outcomes will be determined by skeletal analyses (histology, microCT, bone density, mechanical testing, and bone quality measures), though the biochemical, renal, and vascular components of CKD-MBD will be assessed as well. Gene expression analyses will be performed to begin to identify signaling pathways involved in changes in biomechanical bone quality. We predict that raloxifene will improve the mechanical properties of bone by improving bone quality, while calcitriol will improve mechanical properties by increasing bone mass. Combination therapy should exceed all other treatments by positively impacting both quality and mass. An understanding of the detrimental impact of CKD on bone quality is a crucial step in preventing fractures in these patients. This study provides an important step in achieving this goal by examining these changes and their potential corrections in a rat model with the spontaneous and progressive development of chronic kidney disease.


The broad objective is to facilitate the immunologic control of cancer by providing information on tumor associated antigens. Specific aims are: 1. To elucidate the structures and antigenic roles of the carbohydrate and protein portions of CEA. 2. To isolate CEA-like materials from the tissues of both malignant and nonmalignant diseases in which elevated CEA levels have been reported in the serum. 3. To examine the CEA-like materials isolated to see if they are chemically identical to the material isolated from colonic adenocarcinoma. If they are different, attempts will be made to identify the chemical nature of the differences. 4. To assist other research groups in research on CEA and other tumor antigens through provisions of reagents and in training personnel in radioimmmune assay techniques. 5. To search for new tumor antigens which may be susceptible to isolation and purification for structural studies. 6. To develop and apply techniques other than radioimmune assay which may be helpful in establishing the role of the antigen in the evolution of the malignancy and may point the way to its application in the control of cancer. 7. To explore and develop techniques, such as mass spectrometry, which will enable structural information to be obtained on very small amounts of complicated materials. 8. To apply these and other techniques to structural studies of new tumor antigens as they become available.


The broad long-term objective of this research is to find and describe the genes responsible for 2 highly incapacitating hereditary disorders, namely, Diastrophic Dysplasia (DD) and progressive Myoclonus Epilepsy (PME). Nothing is presently known about the pathogenesis of these disorders. When these genes have been described the pathogenesis will become clarified and the way paved for treatment and prevention. It is particularly appropriate for these disorders to be studied in Finland, because they are rare elsewhere but common in Finland. Approximately 150 patients with DD (100 living) and 160 with PME (110 living) have been diagnosed in finland. The distribution of the genes is uneven considerable enrichment in small sub-isolates. This feature makes it possible to use homozygosity mapping in addition to conventional linkage. The first specific aim of this project is to generally map the genes causing DD and PME, with no available data about the nature of the genes of their products, the first will be to search for linkage with random RFLPs. Over 1500 RFLPs that span over 95% of the human genome are available; over 300 polymorphic probes are already in use in the laboratory of the PI. by choosing probes located less than 20 cM apart and distributed as evenly as possible along the chromes, region after region of the human genome will be excluded until positive linkage is found. Blood samples have already been collected and permanent lymphoblastoid cell lines established from 32 DD patients and 75 of their family members representing 14 pedigrees. These samples are sufficient to exclude or establish linkage. For PME the corresponding figures are 20 patients, 30 family members, representing 12 pedigrees. The collection of further samples for PME is underway and will be completed in 1989. Homozygosity mapping will be done using patient DNA from unrelated affected individuals. The second specific aim is to find markers as close as possible to the genes. This will be done by saturating the region with probes. Once closely linked, preferably flanking, markers have been identified, a method based on segregation analysis for carrier detection, and presymptomatic and prenatal diagnosis will be devised. The results of this research should increase the understanding of the biology of DD and PME and provide methods to diagnose and prevent these disorders.


Human vision seems to be a continuous process, uninterrupted by the eye blinks and saccades that usually occur every few seconds. We propose to explore this phenomenon in the following experiments: (1) Measure the time course of blinks that occur (a) spontaneously, without voluntary control or attention, (b) reflexly, in response to a puff of air or looming visual stimulus, and (c) voluntarily, on signal from the experimenter. (2) Evaluate the time course of any neural suppression that is found to accompany each blink. (3) Measure the extent and time course of the optical attenuation produced by lid closure. (4) Simulate the same attenuation in the open eye by external changes in the illumination of the visual field, and compare the visual effects of attenuation by lid closure with those of the equivalent reduction of external illumination. (5) Test the statement by Davson (1972) that, --"A movement of the eyes is generally accompanied by a blink and it is thought that this aids the eyes in changing their fixation point." (6) Test the extent to which the eyes rotate during blinks and the related question of whether blinks result in an apparent displacement of the visual field.


Pediatric cardiac arrest is a common and devastating condition which remains poorly understood. Mortality rates are extremely high and brain injury is the most common cause of death. The majority of research regarding cardiac arrest over the past 50 years has focused on improving rates of return of spontaneous circulation (ROSC), with significant progress leading to increased survival rates. However, without interventions to minimize organ injury, there is an increase in long-term health issues associated with our improved resuscitation practices. This has been termed the post-cardiac arrest syndrome, consisting predominantly of long-term neurological deficits. Indeed, several interventions that have been useful in improving ROSC, have not shown benefit in improving long term outcome. There have been numerous pre- clinical translational studies of cardiac arrest in adult animals demonstrating various pharmacological interventions to improve neuronal survival following global cerebral ischemia. However, to date, there are very few studies in pediatric cardiac arrest, as models are scarce. In the current proposal, we describe the first pediatric cardiac arrest model utilizing mice to study the effects of cardiac arrest on neuronal survival and test new therapies. Epidemiologic studies in adults have suggested that females have better outcomes after CA when compared to males. Numerous experimental studies in adult animal models have recapitulated this clinical data, showing that female animals exhibit significantly less brain injury following cerebral ischemia than males. The sex difference observed in experimental adult animals can be nearly completely explained by the high levels of estrogen in female animals, as removal of endogenous sex steroids (ovariectomy) increases female brain injury to male levels and estrogen replacement returns female injury to intact animal levels. Not surprisingly, we did not observe a gender difference in ischemic injury in pediatric mice, consistent with pre- pubertal state of low estrogen in both male and female animals. Interestingly, when estrogen is exogenously administered we observe a remarkable sex-difference in response to estrogen neuroprotection. We observed that a single intravenous estrogen dose administered at a clinically relevant time point following CA/CPR (30 min) provides protection to the female brain, while having no effect in the male brain. Therefore, the aims of the current proposal are designed to further characterize our novel pediatric cardiac arrest model and begin to elucidate the molecular mechanisms of sexually dimorphic estrogen neuroprotection observed at this developmental stage. Therefore, we will 1) establish the role of estrogen in determining neuronal injury following pediatric cardiac arrest and 2) determine the relative contribution of estrogen receptors alpha and beta (ER? and ER?) in estrogen neuroprotection. 3) Finally, determine the molecular mechanism of estrogen neuroprotection in pediatric cardiac arrest.


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. Mm-cpn is an Archaeal type II chaperonin of ~1 MDa. Its structural organization is simpler than TRIC, the mammalian type II chaperonin, because, while also a 16mer, it possesses only a single subunit isoform. We propose to determine its structures in different chemical states at near atomic resolution.


Cancer research relies on technologies that can interrogate the entire genome, detecting deletions, copy number variants and point mutations responsible for disease. It is becoming increasingly clear that epigenetic lesions in DNA and histone modification are at least as important, and probably more important, than genetic lesions as cells progress towards malignancy, and acquire various attributes, such as drug-resistance. Cancer research at Cold Spring Harbor Laboratory has been at the forefront of this "cancer genomics" approach, as well as research in epigenetic mechanisms that contribute to these and other diseases. Examples of research topics are summarized in the application, ranging from the role of methylation in breast cancer and chemoresistant AML to replication-induced senescence. Further, we plan to examine epigenomic profiles in model animals and plants, which undergo developmentally programmed changes in epigenetic profiles, and which use RNA interference to guide DNA methylation and histone modification patterns. To do this, we are requesting funds to purchase a Genome Sequencer FLX instrument from Roche Applied Science, which can reliably determine the sequence of the epigenome, even when cytosines are converted to uracil, because longer read lengths permit unambiguous mapping back to the reference genome. PUBLIC HEALTH RELEVANCE: As we learn more about how information is stored in our genome and controls biological processes it has become apparent that in addition to our basic DNA structure, modifications to it, such as the addition of methyl groups in specific places is of crucial importance. Changes in methylated DNA regions can change gene expression in normal development as well as lead to cancer if and possibly other diseases if not properly controlled. We are requesting equipment that will allow us to examine, in great detail and at very large scale, the changes to methylation in the genome so that we can better understand how this process occurs in normal biological processes as well as which genes it regulates in cancer.


In the developing nervous system, axons are guided to their targets by extracellular guidance cues. While much progress has been made in the characterization of these cues, the downstream signaling pathways are poorly understood. The goal of this project is to determine the role of the cytoplasmic proteins encoded by mig-10, a gene that is required for pathfinding of the SMDD axon. Preliminary evidence suggests that mig-10 might act to modulate signaling downstream of slit, a repulsive guidance cue. The expression pattern and locus of function will be determined for each of two MIG-10 isoforms. The hypothesis that mig-10 may modulate slit signaling will be tested by ectopically expressing both isoforms in the AVM, a neuron that is known to be sensitive to axonal repulsion by slit. Genes that interact with mig-10 will be identified through a candidate gene approach and a genetic screen. The products of the mig-10 gene are similar to a family of mammalian proteins that includes GRB-7, GRB-10, and GRB-14. These proteins are overexpressed in several types of cancers, but their function is poorly understood. Information from this project will be beneficial in understanding disorders of the nervous system as well as the molecular basis of cancer.


Retinoids are known to be important in a variety of processes during development. Previous studies have shown that both retinoid and FGF signaling are required for anteroposterior patterning. The specific aims are: 1) to determine if ets domain transcription factors interact with RAR and FGF signaling pathways, 2) to show the necessity for RAR regulation of Xcad expression in early anteroposterior development and 3) to show RAR and Wnt signaling act synergistically to pattern the trunk and posterior. We will employ MO-mediated gene knockdown and microinjection of specific reagents to determine the function of RAR isoforms, Wnt signaling as well as interactions with ets domain transcription factors. Whole mount in situ hybridization, real time PCR, microinjections, and embryological manipulations will be employed to discern the effects of loss-of-function in certain genes and their affects on the function of RAR, Wnt and FGF signaling. The long-term goal of the proposed research is to understand the role of retinoid signaling during vertebrate axial patterning. The results should be widely applicable to the study of other vertebrate model systems.


The long-term goals of the Duke/North Carolina Central University (NCCU) "Building Interdisciplinary Research Careers in Women's Health" (BIRCHW) program are (1) to contribute to improvement in women's health by providing training and experience in research methodology to new investigators interested in addressing important questions, (2) to foster innovative approaches to these questions by encouraging investigators to utilize the wide range of intellectual capital available through interdisciplinary training and collaboration, (3) to build the capacity of Duke and NCCU to provide leadership in women's health research, and (4) to serve as a model for broad-based interdisciplinary research training and collaboration for both universities. The program will reach these objectives by achieving the following specific aims: (1) providing didactic and practical training in the conduct of research in women's health for junior faculty across a broad spectrum of disciplines, focusing on 4 broad areas: Clinical Trials and Outcomes, Decision Making, Health Disparities, and Basic and Translational Research; (2) building upon existing interdisciplinary faculty relationships to foster productive and innovative collaborations; (3) creating new interdisciplinary research relationships between Duke and NCCU faculty; (4) providing additional mechanisms for the ongoing exchange of ideas, methods, and insights among the community of researchers at Duke interested in women's health; (5) providing a mechanism for recruitment and career development of junior faculty committed to the long-term goals of the program; and (6) enhancing overall research productivity of faculty by encouraging and facilitating interaction between clinician and non-clinician researchers. Trainees in the program will have a minimum of 2 years of mentored research training, including weekly seminars on research methods and career development topics, experience in peer review and grant writing, and completion of at least one project working in conjunction with a senior faculty research mentor. Trainees will be expected to complete and submit at least one application for independent research training by the end of their time in the program.


Mutations in LMNA, the gene for lamin A/C, have been shown to cause several forms of muscular dystrophy, a cardiomyopathy, familial partial lipodystrophy, one form of Charcot Marie Tooth neuropathy, mandibuloacral dysplasia, and Hutchinson Gilford progeria syndrome (HGPS). HGPS patients have many aging-like phenotypes, including a wizened appearance, alopecia, osteoporosis, and coronary heart disease. HGPS is a merciless disease; death occurs on average at age 13, nearly always from atherosclerosis in the coronary and cerebral arteries. A recent NIH workshop on HGPS concluded that a thorough understanding of vascular disease in HGPS syndrome represents a key research priority. In most cases, HGPS is caused by a de novo single nucleotide substitution in exon 11. This mutation generates a cryptic splice site, resulting in an in-frame deletion of 50 amino acids near the carboxyl terminus of the lamin A. Interestingly, the deletion prevents the endoproteolytic processing of prelamin A. The objective of this grant application is to create mouse models of HGPS for the purpose of exploring mechanisms underlying the strikingly increased susceptibility to atherosclerosis in HGPS. The Principal Investigator (PI), Dr. Stephen G. Young, is well-positioned to pursue this objective. For the past 10 years, the PI has worked both on the posttranslational processing enzymes required for the biogenesis of lamin A and on the genetic underpinnings of atherosclerosis. For both projects, the PI has relied heavily on studies with gene targeted mice. Recently, the PI showed that the targeted inactivation of Zmpste24 prevents the proper endoproteolytic processing of lamin A, leading to the accumulation of prelamin A. Of note, Zmpste24-/- mice have a host of phenotypes similar to those in HGPS. The PI has also created mice lacking other enzymes involved in the posttranslational processing of the lamins (protein farnesyltransferase, Rcel, and Icmt). The first aim of this application is to create, with gene-targeting approaches, authentic mouse models of HGPS. The second specific aim is to compare, at both cellular and whole-animal levels, phenotypes in HGPS mouse models and the Zmpste24-deficient mice. The third specific aim is to understand mechanisms underlying the strikingly high susceptibility to atherosclerosis in HGPS. The PI's laboratory has extensive experience in analyzing atherogenesis in mice and is thrilled with the prospect of exploring mechanisms of atherosclerosis in HGPS.


The primary objective of this research project is to study the modulating effects of green tea polyphenols on reducing hepatocarcinogen-induced oxidative damages in high-risk human populations. Oxidative damage induced by reactive oxygen species in vivo plays important roles in human hepatocarcinogenesis primarily caused by chronic infection of hepatitis B/C viruses and exposure to dietary aflatoxins. The level of 8-hydroxy-2'- deoxyguanosine, a biomarker for oxidative DNA damage, increases in hepatitis B virus surface antigen positive and aflatoxin-exposed humans and in aflatoxin- treated animals. Dietary antioxidants are important components of cancer modulating agents, which have been proven to effectively target carcinogen biomarkers, including oxidative damages, in high-risk human populations. Among various identified dietary associated antioxidants, green tea and its polyphenols have been shown to be safe and highly effective in inhibition of a variety of carcinogen-induced oxidative damages, mutagenesis, and tumorigenesis in in vitro bioassays and in vivo animal models. The general hypothesis underlying this proposal is that green tea polyphenols have a protective effect against oxidative stress or damage induced by aflatoxin and hepatitis B/C viruses through the mechanisms of modulating aflatoxin metabolism and oxidated DNA damage. The specific aims include: (1) to determine antioxidative role of green tea polyphenols in inhibition of the level of 8-hydroxy-2'-deoxyguanosine in urine samples collected from an intervention study of 120 participants who are double positive for hepatitis B virus surface antigen and aflatoxin-albumin adducts, and (2) to determine the modulating effect of green tea polyphenols on excretion of carcinogen detoxifying product, aflatoxin Bl-mercapturic acid in urine samples collected from the study participants. The results of this proposed study will help to understand the mechanisms of antioxidative role of tea polyphenols in modulating human hepatocarcinogenesis caused by hepatitis B/C viruses and aflatoxins.


Skeletal muscle insulin resistance, particularly that associated with central obesity, is a serious public health problem that is largely due to exercise deficiency. Exercise/muscle contraction, like insulin, stimulates muscle glucose transport by translocation of GLUT4 glucose transporters from intracellular sites to the cell surface. As this acute effect of exercise wears off, it is replaced by an increase in insulin sensitivity. Exercise also induces an adaptive increase in GLUT4 that results in increased insulin responsiveness. These effects are among the most important health benefits of exercise. While there has been much progress in the elucidation of insulin action, little is known regarding how contractions stimulate glucose transport. The initial signals appear to be the decrease in high-energy phosphates and the increases in cytosolic Ca2+ during contractile activity. These signals activate AMPK and CAMKII, both of which appear to mediate the increase in glucose transport. Thus the initial and final steps in the stimulation of glucose transport by contractions have been identified. However, the steps connecting activation of CAMKII and AMPK to GLUT4 translocation are unknown. Our goals are to elucidate the steps in the pathway by which contractions stimulate glucose transport and to investigate the mechanisms responsible for the increase in insulin sensitivity after exercise. Relative to the first goal, one of our aims is to determine if the contraction-mediated and the second, i.e. not wortmannin-inhibitable, insulin signaling pathway overlap or merge. Relative to this aim we will address the questions: Are small G-proteins of the Rho family activated by contractions? Does stimulation of glucose transport involve activation of tyrosine kinases? and, Is phosphatidic acid involved in the stimulation of glucose transport by muscle contractions? The second aim relating to the first goal is to determine whether activation of Rab4 is involved in the stimulation of glucose transport by contractions. It is our working hypothesis that this is the step at which the contraction-stimulated pathway and wortmannin-inhibitable insulin-signaling pathway merge. Relative to this aim we will address the following questions: Is phosphorylation of AS160 a step in the pathway by which contractions stimulate glucose transport? Is Rab4 activated in response to muscle contractions? Does inhibition of Rab4 activation prevents stimulation of glucose transport by contractions? and, Is activation of atypical protein kinases C-zeta/lambda involved in the stimulation of glucose transport? Relative to our second goal, one of our aims is to evaluate the hypothesis that during recovery from stimulation GLUT4 undergoing endocytosis cycle into a storage pool that is accessible to a weak insulin stimulus. Another is to determine whether the p38 MAPK is involved in mediating the exercise-induced increase in insulin sensitivity. A third is to answer the question, is activation of PKC-zeta/lambda involved in the exercise-induced increase in insulin sensitivity?


ABSTRACT Despite the availability of the human papillomavirus (HPV) vaccine that can prevent over 34,800 HPV-related cancers in the US every year, only 51% of girls and boys were up-to-date by 2018. Rural populations are the most impacted by HPV-related cancers. Best practices like the Announcement Approach training and systems communication have proven effective in increasing HPV vaccination, but rural providers struggle to access and implement such best practices. These data prompt the question: ?How can academic centers support HPV vaccination in rural primary care practices?? Although never tested for HPV vaccination, the ECHO (Extension for Community Healthcare Outcomes) Model is a promising implementation strategy (practice facilitation) that allows ?experts? at academic centers to connect with primary care providers to discuss best practices in care and complex cases managed within local practices. The objective of this R01 is to test two ECHO-delivered HPV vaccination communication interventions in rural primary care clinics. The first will provide Announcement Approach training (HPV ECHO); the second will provide this approach plus systems strategies to communicate with parents who initially decline vaccination (HPV ECHO+). The rationale for the project is that ECHO is a robust, highly-accessible platform to deliver best practices to rural providers and address the context-specific communication needs of parents. Our long-term goal is to improve HPV vaccination rates in rural clinics and reduce the health inequity rural populations experience in cancer outcomes. Aim 1 is to evaluate the impact of HPV ECHO and HPV ECHO+ on HPV vaccination among adolescents. We will conduct a 3-arm cluster randomized trial with 36 primary care clinics in rural Pennsylvania. Clinics will be randomized to: HPV ECHO, HPV ECHO+, or control. Our primary outcome will be change in HPV vaccine initiation (?1 doses) among adolescents, ages 11-14, at 12-month follow-up. Aim 2 is to evaluate the impact of HPV ECHO and HPV ECHO+ on implementation outcomes. Guided by implementation science frameworks, we will conduct a mixed-methods evaluation to compare interventions on acceptability, adoption, cost, penetration, and sustainability. Aim 3 is to evaluate the impact of interventions? vaccine information on secondary acceptance of HPV vaccination at the clinic level. We will also follow a subset of 200 vaccine-declining parents for up to 12 months to assess exposure to and impact of vaccine information from study arms versus naturally-occurring sources (e.g., social media) on secondary acceptance. Our expected outcome is to demonstrate the effectiveness of a highly efficient and scalable implementation strategy, ECHO, to support HPV vaccination in rural clinics. This study is innovative in leveraging existing infrastructure at academic centers to deliver best practices for HPV vaccination where they are needed most and in developing a greater understanding of the influences on decision making among vaccine-declining parents. We expect the project to have a significant impact on HPV vaccine uptake as we address the communication needs of both rural providers and parents.


[unreadable] Cystic Fibrosis is a fatal lung disease caused by misfolding and premature proteasomal degradation of the mutant Cl- channel CFTRAF508. CFTRAF508 is synthesized in the endoplasmic reticulum (ER), but its folding arrests at an unknown intermediate step and it is selected for degradation prior to passage to the plasma membrane (PM). Loss of CFTR function leads to defects in the hydration of mucosal layers that line glands and airways and chronic infections that death occurs due to lung failure. Hope for the treatment of CF comes from observations that folding defects in CFTRAF508 are rescued by compounds that either alter the cellular folding environment. This competitive renewal application for 2RO1 GM56981 seeks to aid in the development of therapeutics to treat Cystic Fibrosis by elucidating defective steps in the CFTRAF508 folding pathway and identifying ER quality control (ERQC) factors that select CFTRAF508 for degradation. The three major objectives of the proposal are as follows 1. We seek to define the mechanism by which mutations in CFTR cause it to misfold and be degraded the ubiquitin proteasome system. 2. CFTR is a polytopic protein that exposes surfaces in the ER lumen, ER membrane, and cytosol. 'Thus, we will identify the components of the ERQC machinery that sense the folded state of different sub-domains in CFTR. Then, we will determine how the action of the ER localized and cytosolic QC factors is coordinated. 3. We propose to apply the knowledge obtained in our basic studies to develop approaches to block the selection of CFTRAF508 for premature degradation and promote its proper folding. Overall, our studies will provide basic information on protein QC that will aid in the development of therapeutics to treat CF. [unreadable] [unreadable] [unreadable]


Malignant gliomas are associated with high morbidity and mortality. Standard therapies such as surgery, radiation therapy and chemotherapy do not offer effective options and local recurrence is the norm. However, being locally malignant nonmetastatic tumors, they may be amenable to control using effective tumor-targeted local therapies. TNF- related apoptosis inducing ligand (TRAIL/Apo2L) induces apoptosis in gliomas in vitro and prolongs survival in xenograft rodent models. It is nontoxic to normal organs in animal models including primates. However, the efficacy of local intracranial delivery of TRAIL, its toxicity to brain-adjacent-to-tumor and selectivity towards gliomas in humans remain to be fully explored. In this study, we will test the efficacy and toxicity of TRAIL in a living human glioma & brain slice model and in xenograft models. Our preliminary data suggests that soluble TRAIL is active against gliomas in vitro and in vivo and that its activity is modulated by the Akt pathway. We hypothesize that: a) soluble untagged TRAIL will be effective against human glioma and nontoxic to normal brain 2) Inhibition of Akt can sensitize human gliomas to TRAIL and increase survival in a glioma model 3) resistance to TRAIL is mediated by differing mechanisms in normal and tumor cells permitting selective targeting of resistance mechanisms in tumors. To test these hypotheses, we propose the following specific aims 1) determine the efficacy and toxicity of soluble untagged TRAIL expressed by an adenovirus in a mouse intracranial xenograft and an ex-vivo living human glioma slice model. 2) determine the in vivo effect and relevance of Akt inhibition to TRAIL-induced apoptosis in gliomas. 3) determine the mechanisms of TRAIL resistance in normal brain and resistant glioma cells in human living tumor/ brain slice model and the effects of modulating these mechanisms on TRAIL sensitivity. The combined use of human living brain slices and intracranial xenograft models are aimed at providing direct and highly relevant data on the efficacy of TRAIL against human gliomas and its toxicity against the brain. The results of this study could also potentially provide a basis for clinical trials using TRAIL or analogues against gliomas and allow rational selection of agents to modulate the tumor resistance to TRAIL. [unreadable] [unreadable]


This application attempts to clarify the mechanistic basis for the association between excessive adiposity and risk factors for prostate cancer-specific morbidity and mortality in men with clinically localized prostate cancer. The overall hypothesis is that excess adiposity alters prostatic exposure to nutritional, hormonal, and immunologic factors influenced by weight status and implicated in prostate cancer progression, and that inter- individual variation in gland-level exposure to these factors mediates the effects of adiposity on the pathologic presentation and course of the disease. To test this hypothesis, we will: Aim 1) conduct a prospective 2-year cohort study of incident cases of clinically localized prostate cancer to measure the association of adiposity with pathologic tumor features at diagnosis and 2-year risk of biochemical (PSA) failure after radical prostatectomy;Aim 2) measure fatty acids, insulin-like growth factor (IGF) axis activity, modulators of inflammation, and sex steroid hormone profiles in prostate tissue and periprostatic fat collected at surgery, and use mediation model analysis to determine which physiologic variables account for the effects adiposity on tumor characteristics and risk of biochemical failure;Aim 3) and evaluate the effects of post-treatment changes in adiposity on 2-year risk of biochemical failure. The cohort will consist of 540 men enrolled in the urology clinics of four Chicago-area medical centers who are awaiting prostatectomy for clinically localized prostate cancer. Adiposity will be quantified by anthropometry and dual energy x-ray absorptiometry at diagnosis and one year after surgery. Pathologic tumor characteristics will be determined by a single pathologist, and biochemical outcomes will be ascertained using a standard clinical protocol. Potential mediators to be studied include fatty acids, the IGF-1 receptor signaling pathway, adipose tissue-derived cytokines, eicosanoids products of arachidonic acid, and metabolites of estrogen and testosterone. Lifestyle factors that associate with more rapid progression of clinically early-stage prostate cancer are of increasing concern to health care providers and public health professionals. This research, to be completed over 5 years, will identify some of the biological reasons why obesity associates with more "aggressive" prostate cancer at diagnosis and decreases a man's chances for cure with surgery. It will also shed light on ways to prevent prostate cancer recurrence that may involve new or existing medicines or lifestyle changes. PUBLIC HEALTH RELEVANCE: Lifestyle factors that associate with more rapid progression of clinically early-stage prostate cancer are of increasing concern to health care providers and public health professionals. This research, to be completed over 5 years, will identify some of the biological reasons why obesity associates with more "aggressive" prostate cancer at diagnosis and decreases a man's chances for cure with surgery. It will also shed light on ways to prevent prostate cancer recurrence that may involve new or existing medicines or lifestyle changes.


Our long term goals are to understand the principles which underlie the information processing capabilities of neurons in the central nervous system. The goal of this proposal is to examine mechanisms of information processing in one region of the auditory brainstem, the dorsal cochlear nucleus (DCN). In the most superficial layer of this nucleus, a system of fine, unmyelinated parallel fibers (axons of granule cells) make numerous synapses on the principal projection neurons of the nucleus (pyramidal cells) and on small interneurons (cartwheel and stellate cells). The parallel fibers provide the substrate for unique and extensive interactions occurring orthogonal to the tonotopic axis of the DCN. Four specific hypotheses about the mechanisms of information processing by neurons in the mammalian DCN will be investigated. First, we will investigate the hypothesis that the activation of a specific subset of excitatory amino acid receptors, the N-methyl-D-aspartate (NMDA) receptors, can contribute to nonlinear facilitation of parallel fiber and auditory nerve inputs to pyramidal cells. Second, we will investigate the hypotheses that postsynaptic calcium entry associated with parallel fiber synaptic activity, either through NMDA receptors or voltage-sensitive calcium channels, can result in the long-term modification of synaptic or voltage-dependent conductances in postsynaptic cells. Third, we will test the hypothesis that the voltage- dependent discharge patterns of dorsal cochlear nucleus pyramidal cells are in part produced by a transient potassium conductance. Fourth, we will test the hypothesis that cartwheel cells are inhibitory to pyramidal cells. The experiments to test these hypotheses use intracellular, whole-cell tight-seal, field potential recordings, and optical recordings of calcium activity from an in vitro brain slice preparation, and whole-cell tight-seal voltage-clamp recordings from acutely isolated neurons. The results of these studies will provide important information about the function of the parallel fiber system in the cochlear nucleus, and will help to generate new theories about the role that this system plays in processing incoming acoustic information. These experiments will also set the stage for subsequent investigations of changes in central physiology that may occur as a function of peripheral acoustic trauma and aging.


Many theories have been proposed for the statistical mechanics of protein folding, typically derived from theories for less structured systems, such as random heteropolymers, diffusion-nucleation, random energies, or spin glasses. While these have certainly captured some important features of the physical chemistry of real proteins, such as cooperativity of folding and rapid folding from the random coil state, their derivation requires making some broad assumptions about the average behavior of polypeptides. In fact, the proteins of biological relevance consists of apparently very rare, moderately long amino acid sequences that permit the chain to fold rapidly- to a unique complicated native conformation, which depends greatly on the sequence. This suggests that theories focussing on average properties of long-chain heteropolymers may be over-generalizing and neglecting the important features of rare sequences folding to rare conformations. On the other hand, neither nature nor computer has sufficient time to exhaustively explore all conformations and all sequences for even small proteins. The idea here is to simply an otherwise realistic representation of polypeptides by reducing chain length, number of conformation states per residue, and choices of amino acid types until all sequences and all conformations can be exhaustively enumerated. By varying these parameters in the computationally feasible range, general conclusions can be detected and extrapolated to parameter values corresponding to real proteins. Since this model is so different from most theories, it is able to test their assumptions and conclusions about protein folding, such as the nature of the energy landscape and order parameters to describe the progress toward the native state. Questions to be addressed include: is there a general way to describe the folding of all proteins, or do some proceed by a recognizable pathway while others have innumerable routes? Can this model reproduce and explain the currently available experimental results on folding mechanisms and intermediates for certain particular proteins.