Michael Noel Hart, MD, Marquette University - US grants

The purpose of these proposed studies is to examine directly the responses of intraparenchymal cerebral arterioles under various pathological and physiological conditions, and to compare these responses to the responses of pial arterioles. Arteriolar diameter will be determined using two different methods: 1) the diameter of pial arterioles will be measured in vivo, and 2) wall-to-lumen ratio of parenchymal and pial arterioles will be determined in cerebral tissue rapidly frozen with supercooled isopentane. These studies will be performed primarily in rats. The first specific aim will be to determine the response of parenchymal arterioles to increases in blood pressure beyond the autoregulatory range of cerebral blood flow, and to determine whether changes in arteriolar caliber are related to disruption of the blood-brain barrier. Acute hypertension will be produced with occlusion of the thoracic aorta, and the degree of barrier disruption will be determined with ultrastructural quantitation of pinocytosis in arteriolar endothelium. The second specific aim will be to determine the effects of chronic hypertension on dilator and constrictor responses of cerebral arterioles, and to determine whether prevention of vascular hypertrophy normalizes arteriolar responses. In these studies, the responses of innervated and denervated cerebral arterioles in stroke-prone spontaneously hypertensive rats will be compared during hypercapnia and during increases in blood pressure. Sympathetic denervation prevents cerebral vascular hypertrophy in these rats. The third specific aim will be to compare the responses of parenchymal and pial arterioles during autoregulation. Blood pressure will be reduced with controlled hemorrhage and increased with aortic obstruction. The fourth specific aim will be to compare the responses of parenchymal and pial arterioles to altered cerebral metabolism. The response of cerebral arterioles to bicuculline-induced seizure will be compared with the response to hypercapnia. The fifth specific aim will be to determine the response of parenchymal arterioles to sympathetic stimulation. The cervical sympathetic trunk will be stimulated unilaterally in cats. The studies will provide valuable information relevant to hypertensive encephalopathy, the cerebral vascular complications of chronic hypertension, and to the regulation of cerebral vascular resistance. Although performed in animals, these studies will be applicable to human disease.

Human vasculitis is a common disease entity. Currently the only known cause of autoimmune vasculitis is immune complex deposition which has been demonstrated in some types of vasculitis but not in others. A lymphocyte-mediated pathogenesis has been suspected but not proven in some vasculitides, especially those occurring in conjunction with collagen-vascular diseases. Nor has a model for an autoimmune cellular vasculitis been developed to the best of our knowledge. We plan to address this gap through the further development of a murine model of autoimmune vasculitis. In this model, lymphocytes are co-cultured with syngeneic endothelium or smooth muscle. After activation, the lymphocytes are injected into syngeneic hosts with a resultant vasculitis seen in various organs. It is hypothesized that either the lympocytes are activated to surface antigens in co-culture and then cross-react with cells in vivo or that the lymphocytes escape normal suppressor regulation by being co-cultured in vitro, or both. The objectives of this project are to further develop this model of autoimmune vasculitis while at the same time addressing some of the pathogenic immune mechanisms which allow the lesions to develop. The specific aims of this project are to: 1) enhance the number and severity of vasculitis lesions by attenuating suppressor cell systems in culture and in the hosts through the use of cytoxan, irradiation and anti-IJ+ sera; 2) to determine the phenotypes of cells in the lesions as well as the role of immunoglobulin and complement; 3) to investigate the relative contributions to the lessions of injected lymphocytes versus host lymphocytes; 4) to assess the morphologic types of cells in he lesions as well as the vascular damage by electron microscopy; 5) attempt to clone activated, effector T-cells; and 6) to identify the stimulating antigen(s) in the co-culture system.

This is a competitive renewal grant request for continued studies of vasculitis, a disease of humans frequently associated with collagen-vascular type autoimmune diseases. The murine model to be exploited is one in which splenic cells are co-cultured in vitro with syngeneic microvascular smooth muscle (SM) followed by transfer of the activated (immunized) cells into syngeneic recipients resulting in a distinct vasculitis. Preliminary studies have shown strong MHC restriction (syngeneic response much greater than allogeneic) governing spleen cell activation to SM that can be blocked by anti-Ia in vitro. The hypothesis to be tested is that lymphocytes recognize SM and MHC antigens in vitro to become activated. After transfer into syngeneic hosts the lymphocytes recruit cells that also recognize SM and MHC antigens in attacking vascular SM. This proposal will address both the in vitro lymphocyte activation events as well as the in vivo effector events in the host mice. Specific aims related to in vitro events include determination of mRNA for class 11 antigen in SM with the use of cDNA probes and determination of whether SM can present antigen to antigen-specific T cell clones. Specific aims related to the effector (in vivo) events include determination of the possible role of SM specific antibodies in affected mice and determination of the effector cell(s) in the lesions by light and electron microscopy. Several SM activated lymphocyte clones (L3 T4+ and Lyt 2+) have been developed and will be used to determine their ability to elicit vasculitis as well as their specificity for SM.

This proposal is devoted to studies of the immune properties of large vessel endothelium and smooth muscle. Studies are designed that will determine the ability of large vessel smooth muscle and endothelium to activate, stimulate proliferation of, and present antigen to various subtypes of T lymphocytes; to determine whether aortic smooth muscle supports in vitro proliferation of granulocytes and produces message for colony stimulating factors; to characterize immunologically important cytokine-inducible targets on large vessel smooth muscle and endothelial cells; and to correlate the morphologic properties and growth characteristics of smooth muscle cells with immune functions. It has been increasingly appreciated that large vessel smooth muscle and endothelium interact with lymphocytes in many different ways. Numerous studies have been devoted to determination of the effects of lymphocyte-secreted cytokines on smooth muscle and endothelium. The thrust of this study will be the opposite, mainly to determine the effects of smooth muscle and endothelium on lymphocytes and granulocytes. The key questions and hypotheses underlying these proposed studies are derived in part from recent studies in which we have shown that microvascular smooth muscle is a good antigen presenting cell under our conditions and also supports the colony proliferation of granulocytes in vitro. Microvascular smooth muscle also differs considerably from microvascular endothelium in that each activates different subsets of lymphocytes and under different respective conditions. It will be important to determine whether large vessel smooth muscle, which is normally involved in the atherosclerotic process, possesses similar or different immunologic properties from that of microvascular smooth muscle.

The primary goal of the Wisconsin Alzheimer's Disease Center (ADRC) Neuropathology and Biomarkers Core (Core D) is to facilitate the investigation of Alzheimer's disease (AD) and other dementias by University of Wisconsin (UW) investigators. The Core will accomplish this goal by providing complete and state-of-the- art characterization of disease pathogenesis and progression through biomarker, genetic and neuroimaging analysis, definitive neuropathologic diagnosis at autopsy and the preparation, banking and provision of frozen and fixed, non-demented control and AD brain tissues. In addition, the Core will enhance access to specialized proteomic and genomic technology to empower research progress. In particular, the Core will: Specific Aim 1: Perform rapid autopsies, obtain and archive representative frozen and fixed tissue blocks from multiple brain regions on deceased individuals who are enrolled in the Wisconsin ADRC as well as normal elderly controls, and generate NACC compatible diagnostic information. Specific Aim 2: Provide biomarker and genetic analyses in support of Wisconsin ADRC investigators and the procurement and management of tissues (blood, saliva, CSF). Specific Aim 3: Provide high quality, cost effective, cell and molecular neuroscience equipment, technology, assays, expertise, and training to Wisconsin ADRC investigators. This includes access to fluorescence and confocal microscopy, as well as specialized cell and molecular technology including Real Time PCR, DMA microarray analysis, 2-Dimensional gel electrophoresis, mass spectrometry, DMA and peptide synthesis and sequencing and metabolomics analysis. Specific Aim 4: Provide a service and infrastructure for AD-related brain imaging research. 4a) Advise investigators on protocols and analytic approaches;4b) Link the expertise of the vast brain imaging community at UW to the needs of Wisconsin ADRC investigators;4c) Collect a minimal standardized set of brain images on all ADRC participants at baseline;4d) Provide a location for investigators to store their images and analyze their data. In aggregate, these integrated services will greatly enhance basic and translational AD and dementia research on the UW-Madison campus. RELEVANCE (See instructions): An integrated set of core neuropathology, biomarkers and imaging services in this core will greatly empower ADRC researchers to implement transdisciplinary translational studies of AD. The core is organized to meet the needs of the ADRC as a whole and is oriented toward early detection of AD.

NEUROPATHOLOGY CORE (CORE D) - PROJECT SUMMARY The Neuropathology Core of the Wisconsin ADRC (ADRC) will support cutting edge cellular and molecular neuroscience technologies to the ADRC patient population and their properly banked tissue in the service of enhancing research regarding early detection, treatment outcome, and basic mechanisms of AD. The core will facilitate clinical-pathologic and translational research on normal aging, mild cognitive impairment (MCI), AD and other dementias by providing to Wisconsin ADRC and outside investigators a comprehensive resource of biospecimens, biomarker data, clinical data, and state-of-the-art diagnoses for MCI and AD, vascular, Lewy body, TDP-43, and mixed pathologies, from subjects in the Clinical Core and the Wisconsin Registry for Alzheimer's Prevention (WRAP). The Neuropathology Core will generate and assemble critical diagnostic information by combining clinical data (Core B), innovative neuroimaging (Core G) and biomarker assays collected during life with brain autopsy and morphologic, immunohistochemical, genetic, cell and molecular post-mortem analyses. The Neuropathology Core will also provide access to cutting edge tools (including genomics and proteomics) to enhance AD research on banked, frozen tissue. The Core will accomplish these goals by 1) collecting and archiving ante-mortem CSF, blood and DNA and post-mortem frozen and fixed tissue blocks from multiple brain regions on deceased individuals who are enrolled in the Wisconsin ADRC as well as normal elderly controls; 2) providing state-of-the-art postmortem diagnoses on Clinical Core subjects, collect NACC neuropathology data and make the results available to the family, relevant clinicians, qualified researchers, & NACC; 3) distributing ante-mortem and post-mortem biospecimens (brain, CSF, blood products, and DNA) and neuropathologic, genetic, biomarker and other data to suit the requirements of qualified research projects, both within UW-Madison and for national and international multi-center collaborations; 4) performing genetic and biomarker analyses in support of ADRC and outside investigators; and 5) providing high quality, cost effective, cell and molecular neuroscience equipment, technology, assays, expertise, and training to Wisconsin ADRC investigators. A flow of services is therefore envisaged whereby Wisconsin ADRC investigators and collaborators are able to obtain unambiguous post-mortem diagnosis, neuropathological data and tissues from the Neuropathology Core together with comprehensive biomarker data (Biomarker Services) and neuroimaging data (Core G). Moreover, advanced molecular and cellular analyses can be performed on tissues by the Biomarker, and Cellular and Molecular Neuroscience Services.