Paul Brown, PH.D - US grants

The proposed research will endeavor to determine: (1) afferent projections of hindlimb cutaneous nerves to cat lumbosacral dorsal horn (using both anatomical and physiological methods); (2) physiological connectivities among dorsal horn cells, using cross-correlation techniques; (3) properties of substantia gelatinosa neurons, using waveform recognition and source-sink mapping; and (4) responses of dorsal horn cells to a variety of quantitatively controlled inputs, such as single action potentials, action potential pairs, and larger inputs evoked by stimulating larger areas of skin.

We propose to test two hypotheses: (a) contralateral projections of primary afferents mediate crossed components of dorsal horn cell excitatory RFs; and (b) crossed dendrites of dorsal horn cells also mediate contralateral components of their RFs. In each experiment we shall inject horseradish peroxidase (HRP) into one low-threshold cutaneous axon on each side of the spinal cord, at least three segments apart. Then we shall inject HRP into cells in the dorsal horn, contralateral to the injected axons. We shall inject mostly cells whose RFs overlap those of the contralateral injected axons, and some cells whose RFs do not overlap those of the contralateral injected axons. The grey matter contralateral to each injected axon will also be mapped using extracellular recording to determine the somatotopy of the area. Data obtained in this manner will be used to determine whether (1) the contralateral components of dorsal horn cell receptive fields can be accounted for by crossed afferent axons and crossed dorsal horn cells dendrites; (2) crossed axons and dendrites can be accounted for as mediating the assembly of the contralateral RF components of dorsal horn cells; (3) all axons with RFs near the dorsal midline have crossed and uncrossed lateral dorsal horn projections; (4) all axons with RFs near the ventral midline have crossed and uncrossed medial dorsal horn projections; (5) all cells with RFs spanning the dorsal midline have dendrites reaching to the contralateral lateral dorsal horn; (6) all cells with RFs spanning the ventral midline have dendritic trees projecting to the contralateral medial dorsal horn; (7) no axons with RFs far from the dorsal and ventral midlines have crossed projections; (8) no cells with only ipsilateral components of their RFs have crossed dendrites; and (9) whether these generalizations apply to (a) the full rostrocaudal length of the spinal cord, (b) all receptor types (afferents), and (c) all convergence types (cells).

Our long-term objective is to determine the organization of cutaneous neurons of the dorsal horn, and their role in fine tactile discrimination. The somatotopic organization of the dorsal horn is essential to this role. We propose to study further the organization of cutaneous projections to the dorsal horn in order to test, refine or reject the presynaptic somatotopy hypothesis, which states that cutaneous afferents project only to those dorsal horn regions where their receptive fields lie within the receptive fields of dorsal horn cells. We have designed experiments to determine that aspects of the morphology of primary afferent projections to dorsal horn, and of dorsal horn cell dendritic trees, can be accounted for by the somatotopic organization of the region. These experiments will (a) test several specific predictions concerning single-axon projection patterns based on the presynaptic somatotopy hypothesis: and (b) correlate somatotopy and morphology of several identified types of primary afferents and dorsal horn cells. We will inject primary afferent axons in the white matter with HRP, followed by detailed mapping of the dorsal and ventral horns and Intermediate grey matter. The mapping will use extracellular multi-unit and single unit recordings, with reconstruction of all recording sites, as well as intracellular staining of as many spinal grey neurons as possible. Antidromic stimulation will be used to identify tract neurons. Computer-aided reconstruction will be used to allow us to model the formation of the monosynaptic components of dorsal horn cell receptive fields, and to predict the relative information-carrying ability of various ascending tracts in localization and two-point discrimination tasks. They will also be used in future studies of the development of dorsal horn somatotopy in neonatal animals, and reorganization following partial deafferentation.

This newly developed instrument permits imaging of dynamic microstructural and chemical changes of 'wet' sample materials in their natural state as they occur, without high vacuum. Housed in the MRL at Penn State it will be available to several groups for the study of chemically bonded ceramics, sol-gels, electronic ceramics, superconductors, diamond films, biological and composite materials.

The Mary Babb Randolph Cancer Center (MBR) at the West Virginia University Health Sciences Center proposes to develop, implement, host, and evaluate the North Central Appalachia Breast Cancer Education Summit (NCABCES) sponsored by the National Cancer Institute and the Susan G. Komen Foundation. The Summit will target Appalachian women age 50 and over in West Virginia and the 29 southeastern Ohio counties with an emphasis on rural, black and/or low income women with limited literacy skills. MBR's cosponsors will be Riverside Regional Cancer Institute in Columbus, Ohio, Charleston Area Medical Center in Charleston, West Virginia, the American Cancer Society, West Virginia and Ohio Divisions, the Ohio Health Department and the Ohio Bureau of Public Health. The Summit will cover information on breast cancer and early detection; program information on currently available screening and opportunities for joining regional and community cancer control coalitions interested in coordinating breast cancer education and screening activities; an example of a successful worksite education and screening program; and the latest information on how to reach rural Appalachian women. An RFP will be distributed at the Summit to encourage community coalitions to submit a proposal to hold a follow-up summit in their region or county. Summit analysis will include both process and outcome evaluations.

The objective of this propOsed program is to optimize the properties which can be developed in hydroxyapatite (HAp)-based dental restorative materials when they are formed near physiological temperature. HAp-based restorative materials potentially offer significant advantages in that HAp is compositionally close to tooth material and should exhibit very similar thermal conduction and thermal expansion. The opportunity exists to develop a "chemical" bond between an HAp-based restorative material and enamel or dentin, and physical properties can be tailored depending on the specific application. The basis for the approach is that basic and acidic calcium phosphates react in aqueous solution at low temperature to form HAp. A variety of studies carried out in our laboratory have demonstrated the viability of the approach. Depending on the specific conditions, HAp formation has been shown to reach a substantial degree of completion within 15 minutes by acid-base reaction. Based on the conditions in solution during reaction, HAp formation occurs under conditions compatible with both hard and soft tissue. In spite of these attributes, the reactions have not been well characterized with respect to kinetics, microstructural development, mechanical property development, and to the bond developed with tooth surfaces. Such development is related to both the characteristics of the reactants and to the conditions of reaction. The principal objective of this study is to establish these relationships. Once these have been established, property development can be optimized. Five task areas are envisaged. In the first task, factors which influence the kinetics of HAp formation will be investigated. These studies will determine of the rate-limiting factors in the reactions. The second task is to use high temperature processing of reactants to enhance both reactivity and property development. Compositions limited only to calcium phosphates will be investigated. A third task will study conditions in solution which influence the reactions forming HAp. These findings will be used to optimize property development in a fourth task. Included is an assessment of the effects of the inclusion of reactive and inert filler materials on property development. Materials will be tested mechanically and, based on failure analyses, microstructures will be controlled accordingly. In a final task, the quality of the bond developed with tooth surfaces will examined. Bonding will be examined depending on treatment of tooth surfaces in manner to identify opportunities likely to result in tooth- HAp restorative material interfacial regions of high strength and low porosity.

DESCRIPTION (Adapted from the Investigator's Abstract): This study will test the following hypotheses about the somatotopic organization of the hindlimb region of the cat dorsal horn: (1) the skin representation at each rostrocaudal (RC) level of the dorsal horn is determined but the miniature dermatomes of dorsal rootlets ("dermatomelets") at that level; (2) peripheral innervation density determines map scale (number of dorsal horn cells devoted to a unit area of skin); (3) the divergence of connections for a given afferent type. and (4) the convergence of afferent input upon a dorsal horn cell are constant throughout the map; (5) the distributions of afferent terminals and cell dendrites are determined by (1) - (4); (6) dorsal horn cell receptive fields (RFs) are determined by (1) - (5). RFs of axons in dorsal rootlets will be sampled using teased-fiber recording. RFs of dorsal horn cells at the same RC levels will be sampled using extracellular recording. Light touch innervation fields (IFs) of peripheral cutaneous nerves will be mapped electrophysiologically and the A fibers in the nerves will be counted. The probabilities of monosynaptic connections from afferents to dorsal horn cells will be determined with two methods: (a) single axons will be stimulated electrically throughout cells' RFs; and (b) single dorsal root ganglion cells will be stimulated intracellularly. Postsynaptic single unit response times minus arrival times of afferent action potentials will indicate synaptic delay. A model dorsal horn map with high resolution will be obtained from extracellular recording data. Primary afferent terminal distributions and dorsal horn cell dendrite distributions will be determined using intracellular injection of neurobiotin. These data will be used to construct a first-approximation model of the dorsal horn map of the skin, in three steps: (1) delineation of map gradients from dermatomelet data; (2) prediction of axonal arbors and dendritic tree geometries from the gradients; and (3) simulation of RFs of dorsal horn cells from calculations of probability of contact, based on axonal and dendritic distributions.

The Department of Mathematics, Statistics and Computer Science at the University of Illinois at Chicago will purchase computational equipment which will be dedicated to the support of research in the mathematical sciences. The equipment will be used for several research projects, including in particular research in Geometric Group Theory (Paul R. Brown); Topology of 3-Manifolds (Marc Culler); Algebraic Geometry (Anatoly Libgober); Theory of Finite Groups (Stephen Smith); and Number Theory (Jeremy Teitelbaum). Most of these projects are data-intensive and amenable to distributed computing techniques.

The glycolytic enzyme alpha-enolase (ENO1) is a tumor-associated antigen and therapeutic target, known to be overexpressed in a variety of cancers compared to normal tissues. Upregulation of ENO1 at the mRNA and/or protein levels have been demonstrated in cancers of pancreas, breast, colon, gastric, prostate, head and neck, kidney, lung, ovary, and brain, with its higher expression generally associated with more advanced diseases and a shorter disease free interval. ENO1 catalyzes the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway in the cytoplasm, while on the cell surface it functions as a plasminogen receptor. In cancer cells, ENO1 is believed to facilitate aerobic glycolysis (the Warburg effect) as well as plasminogen-dependent cell migration and invasion, thereby promoting cancer development and progression. In the clinical setting, patients with pancreatic and other cancers have been shown to harbor autoantibodies against ENO1 protein. In breast and lung cancer patients, anti-ENO1 autoantibody levels were decreased in the advanced diseases, while higher antibody levels against Ser419-phosphorylated ENO1 have been associated with longer disease free survival in pancreatic cancer patients. In another study, detection of autoantibodies to glycolytic enzymes, including ENO1 protein, was shown to precede the clinical diagnosis of breast cancer by several months. Autoantibody reactivity declined as sampling got closer to the time of diagnosis. ENO1 has also been demonstrated to elicit CD4+ and CD8+ mediated T cell responses, which correlated with the production of anti-ENO1 IgG, indicating the integrated T and B cell responses against the tumor antigen. Despite the presence of autoantibodies against ENO1, cancer patients do not exhibit signs of autoimmune diseases, suggesting a certain level of down-modulation of immune response to self-antigens in normal tissues. These findings support the notion that boosting host immunity against ENO1 tumor antigen may be safely achieved without increasing the risk of autoimmune diseases, and that it may prevent the development of cancers. To examine the feasibility and potential antitumor effects of ENO1 vaccination, Novelli et al. tested ENO1 DNA vaccine in two preclinical mouse models of pancreatic ductal adenocarcinoma (PDA) driven by constitutively activated mutant Kras oncogene, Pdx1-Cre;KrasLSL.G12D/+ mice (KC mice) and Pdx1-Cre;KrasLSL.G12D/+;p53R172H/+ mice (KPC mice). In both models, where mutant Kras is activated prenatally, animals develop a spectrum of pre-invasive ductal lesions, which mirror human pancreatic intraepithelial neoplasia (PanIN), shortly after birth. Human and mouse ENO1 are highly homologous with 95% amino acid sequence identity. Novelli and colleagues have shown that ENO1 DNA vaccine administered at 4 weeks of age followed by two booster shots given 3 weeks apart significantly delayed the development of PDA and improved the overall survival in the vaccinated animals as compared to control. ENO1 vaccination elicited anti-ENO1 antibodies that bound to the cell surface of syngeneic murine PDA cells and mediated complement dependent tumor cell killing. Higher levels of ENO1-specific Th1 and Th17 responses were demonstrated in the vaccinated mice, while the numbers of myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg) were decreased in circulation and in tumor tissues. Importantly, there were no overt signs of autoimmune diseases in any of the vaccinated animals. Safe and effective preventive measures are urgently needed to reduce cancer-associated morbidity and mortality, especially against highly aggressive cancers. Pancreatic cancer is one of the most lethal cancers in both men and women. Because pancreatic cancer is usually diagnosed at an advanced stage, the survival rate is extremely poor. Known risk factors for pancreatic cancer include cigarette smoking, obesity, chronic pancreatitis, diabetes, and family history of genetic syndromes associated with increased cancer risk, including BRCA2 gene mutation, Lynch syndrome, familial atypical multiple mole melanoma syndrome (caused by mutations in p16/CDKN2A), Peutz-Jeghers syndrome, and Von Hippel-Lindau syndrome. Other forms of aggressive cancer include triple negative breast cancer (TNBC), which accounts for 10-20% of invasive breast cancers. Risk factors for TNBC include race (African American), age (younger at diagnosis), and BRCA1 mutations. In summary, there are well-defined groups of individuals with an increased risk of pancreatic cancer or TNBC, who can be targeted for active screening and preventive intervention. The current project will examine the cancer preventive efficacy and immunogenicity of ENO1 DNA vaccine developed by Novelli et al. and determine immune correlates of protection, using genetically engineered mouse models of PDA and TNBC.