Numerous human disorders arise from microdeletions and microduplications of relatively large genomic regions. These rearrangements can result in copy number alterations (CNAs) of one or more genes. Conditions arising from microdeletions and microduplications can manifest as multiple congenital anomalies (MCA) in patients. The most common phenotypic features observed in these patients include global developmental delay, mental retardation, cardiac defects and cranio-facial differences. Thus, diseases mediated by CNAs are referred to as 'genomic disorders' as large regions of the genome are altered leading to disorders in multiple organ systems. Many of the recurrent genomic disorders are mediated by aberrant homologous recombination between highly identical blocks of DNA sequences referred to as low copy repeats or segmental duplications (SDs) which may comprise up to 5% of the human genome. Despite this known correlation between SDs and genomic disorders, a significant proportion of SDs have not yet been associated with disease- causing genomic rearrangements. This observation suggests that other SD-mediated genomic disorders may exist but are currently undetectable mainly due to the low resolution of diagnostic techniques standardly used in molecular cytogenetics. We hypothesize that a significant proportion of children with MCA have a submicroscopic CNA that is not evident on standard genetic testing. We believe that a substantial number of these CNAs may be mediated by SDs or other unstable architecture within the human genome. We will test this hypothesis by analyzing patients with MCA using high resolution microarrays in order to detect disease-causing CNAs. Further, analysis of the genomic sequences at the rearrangement breakpoints will help determine what proportion of CNAs are mediated by predisposing genome architecture like SDs. The goal of this proposal is to identify previously undetectable, disease-causing CNAs in patients with MCA. The identification of genomic regions altered in MCA patients will allow a better understanding of the mechanisms underlying this group of disorders. We can then begin to assess the role of gene(s) in these regions that may be critical to early human developmental pathways. Relevance: Many children have genetic diseases that are undiagnosed due to limitations in current methods used for testing. These children may have very small deletions and duplications which can only be detected by techniques that allow a genomewide analysis. The identification of genomic regions that are deleted or duplicated in patients with birth defects will allow the identification of genes that are important in normal development.


The Lab of Immunoregulation is using molecular, biochemical and cell biological approaches to explore the viral envelope glycoprotein of HIV as a target of immunity. We know that envelope is a target of potent neutralizing antibodies made by people after infection. A mixture of just three human monoclonal antibodies to envelope is capable of protecting monkeys against challenge. Our goal is to develop vaccines that can elicit an immune response as strong as the response to infection. We have two approaches, comparable to the Salk and Sabin vaccines for polio. Our Salk-type vaccine is a particle with a lipid core and an array of HIV envelope glycoprotein on its surface, closely resembling a noninfectious version of HIV virus. These particles have the potential to enhance vaccine potency by up to 1,000-fold, as they have for other successful partulate vaccines. Our Sabin-type vaccine is a live rubella virus containing additional genes coding for the HIV envelope. Each cell infected by this attenuated virus looks to the immune system like a cell infected with HIV. However, the virulence of HIV is gone, only its antigens remain in the vaccine. If these vaccines can elicit a response as strong as to infection, they may be able to prevent or control a subsequent HIV infection. HIV-1 Neutralizing Antibodies from Live Attenuated Vaccines. Many of our most effective viral vaccines are live-attenuated viruses. However, for HIV vaccines, it seems that almost no degree of attenuation would be safe enough for human use. Instead, a number of live recombinant vaccines have been proposed. These have generally been defective viruses, capable of expressing HIV antigens in a single round of infection, but unable to replicate further. In this project, we have begun to make a live viral vector safe enough to express HIV antigens over multiple rounds of viral replication. Like HIV, rubella is an enveloped plus strand RNA virus which infects at mucosal surfaces. A single dose protects for life against mucosal and systemic infection with rubella virus. Live attenuated rubella vaccine is safe for human use. It has been given to millions of children around the world, including asymptomatic HIV+ children. It has no DNA intermediate, so there is no risk of integration and no reservoir of chronic infection. It is minimally pathogenic for adults and children, and its genetic stability is shown by the fact that it has not changed serotype in over 40 years. Ideally, a rubella/HIV hybrid could combine the growth and safety of rubella with the antigenicity of HIV gp120, but without the pathogenicity of HIV. With this vaccine, we may be able to induce a level and duration of immunity to HIV comparable to what has been achieved for rubella. We have used a full length, infectious cDNA clone of rubella, provided by Dr. Teryl Frey. We have identified an acceptor site in rubella where foreign sequences can be inserted without disrupting essential viral functions. For example, we inserted green fluorescent protein as a reporter gene at this site. When Vero cells were transformed with the DNA, they produced GFP in large amount. Significantly, the culture supernatants were infectious with virus that expressed GFP in the next round of infection. These viruses expressed all structural proteins, as measured by western blot, indicating that second strand RNA synthesis and the subgenomic promoter were both active. However, GFP expression was lost over two more passages of virus, although the rubella virus continued to grow without GFP expression. We are working on strategies to stabilize expression of the insert by preventing recombinants from outgrowing the GFP expressing strains. We will then express gp120 in the same manner. The resulting rubella/gp120 hybrid will be tested for growth and gp120 expression in vitro. It will then be tested for propagation in rhesus macaques and for the ability to elicit anti-gp120 antibodies (serum IgG and mucosal IgA), as well as safety and viral shedding and persistence. Macaques are the ideal host for demonstrating protection, since they can be infected with rubella and then challenged with an SHIV or SIV challenge strain expressing envelope glycoproteins of the same type or different from the vaccine strain. Novel Reagents for Making gp120 Conjugate and Particulate Vaccines. HIV envelope glycoprotein gp120 contains epitopes which are targeted by broadly cross-reactive neutralizing antibodies in humans and which depend on the native protein conformation. Monoclonal antibodies to these sites can neutralize a broad range of HIV-1 isolates and have protected monkeys against viral challenge by iv and oral routes. We have found a site on gp120, where foreign protein sequences can be inserted without disrupting the native folding of the neutralizing sites. By linking gp120 to a carrier protein capable of self-assembly, we could enhance the intrinsic vaccine potency of gp120 by assembling virus-like particles, while retaining important conformational sites needed to elicit these antibodies. When hepatitis B surface antigen (HBsAg)was inserted at this site, the resulting HBsAg-gp120 hybrids assembled particles efficiently. The particles sedimented at large size and banded at light density (1.22-1.25), consistent with a lipoprotein composition. On electron microscopy, they were 20 to 30 nm in diameter, similar to the spherical particles formed by native HBsAg. By analogy with native HBsAg particles, these contain about 100 to 200 copies of HBsAg-gp120, arrayed at a lipid/water interface. This recreates the natural milieu of gp120 on the surface of virions. Gp120 in the hybrids showed normal glycosylation, high affinity binding of the natural receptor CD4, and bound a panel of broadly reactive human neutralizing monoclonals, indicating that it was folded correctly in the native conformation. This is the first multimeric gp120 vaccine, and we anticipate that it may elicit a strong immune response in mice, rabbits and monkeys. We have now tested the immunogenicity of gp120 particles in mice and rabbits. Both species showed comparable antibody ELISA titers for particles as for monomeric gp120. However, the virus neutralizing activity of the particle-immune sera was much greater after two doses than for the monomer-immune sera. In some rabbits, the neutralizing titer was nearly as great as the ELISA titer, indicating efficient induction of neutralizing antibodies. The ratio of neutralization over ELISA titer for the rabbits was the same as for high-titered HIV-Ig from infected humans and was 30-fold greater than for animals immunized with gp120 monomers. If monkeys also produce high titered neutralizing anti-bodies, they will be challenged with titered stocks of SHIV virus of the same or different envelope type. We have expressed three envelope types: IIIB, 89.6, and SIV453 as particles. The first two correspond to SHIV challenge strains. The last one could be used to protect monkeys from a potent SIV challenge, which may more closely represent a natural HIV infection with a highly adapted virus. Through potent carrier and multimer effects, particulate forms of gp120 and other viral antigens may have a profound effect on HIV immunology. This project is funded by an intramural NIH grant. Transmembrane glycoprotein gp41 is another important target for HIV neutralizing antibodies. Human monoclonals such as 2F5 bind a site on gp41 close to the lipid bilayer. They can neutralize a broad range of HIV isolates, including fresh isolates. So far, vaccines have been unable to elicit similar antibodies. We hypothesize that, because they lack lipid, they cannot fold or display the 2F5 site correctly. We have expressed HBsAg-gp41 hybrids, which form gp41-rich particles. In these particles, gp41 is displayed at a lipid-water interface, and its transmembrane domain may span the lipid layer, just as it does on virus. We have produced sufficient quantities for immunological studies. These particles may be the first immunogen to recreate gp41 as it exists on the virus. Since the neutralizing sites on gp41 are conserved among diverse HIV isolates, antibodies elicited by these antigens have the potential to be broadly neutralizing. Antibodies to functional sites on viruses and prions. We have recently found that a variety of viral antigens, when expressed in tandem with HBsAg, will spontaneously assemble into 20-30 nm virus-like particles. The particles contain about 20% lipid, based on their density. The proteins are displayed on the surface of a lipid micelle. This milieu resembles the surface of an enveloped virus, as well as the local environment of a bacterial toxin bound to the surface of a cell. The HBsAg carrier is quite flexible in accepting other viral envelope proteins, while still forming particles. For example, the E2 and E1 envelope glycoproteins of VEE virus, as well as both E2E1 together, were expressed as HBsAg hybrids. All three forms assembled into particles. When expressed together, the E2E1 precursor was processed correctly into E2 and E1, and both proteins remained attached to the particles. This may be an ideal immunogen, since envelope proteins on the surface of a lipid particle resemble the same proteins on the viral surface. Immunogenicity will be tested in rabbits, and the resulting antibodies will be tested for the ability to protect cells in vitro from infection with VEE. Similarly, we have created hybrid particles containing protective antigen PA of anthrax. Antibodies to this protein are known to protect against anthrax. We anticipate that the particulate form of PA may greatly increase immunogenicity, allowing us to immunize with fewer doses and to maintain immunity with less frequent boosting than the current anthrax vaccine. In addition, PA was expressed in its active, cleaved form, which may elicit antibodies to sites involved in critical functions such as toxin subunit binding and translocation into the cell. PA particles will be tested for immunogenicity, and the resulting antibodies will be tested for the ability to block toxin-mediated killing of cell lines in culture. If successful, we will immunize mice and challenge them with anthrax spores, in collaboration with NIH scientists. This project incorporates FY2002 projects 1Z01BK009002-11, 1Z01BK009003-11, and 1Z01BK009008-04.


The general goal of this project is to study the cellular and molecular determinants of leukocyte interactions with components of the rheumatoid synovial microenvironment. In the past four years, we have shown that the transmembrane hyaluronate receptor (CD44) is a central, multifunctional proinflammatory molecule in RA, and is the same as the Hermes class of lymphocyte homing receptors that mediates mononuclear cell adhesion to synovial endothelial venules. Recent data have shown that CD44 represents a family of molecules with isoforms. In this project, we propose to define to define the proinflammatory functions of each of the isoforms of the CD44 with the goal of devising novel strategies of inhibition of CD44 proinflammatory function as potential new therapies for RA. Individual specific aims in this project are: 10 We will develop oligonucleotide probes that are specific for known isoforms of CD44. We will develop mabs that specifically react with the human CD44R1 (CD44E) form of CD44, and profile the expression of CD44 isoforms in normal and RA PB T cells, PMNs and monocytes, and in normal, OA and RA synovial T cells, macrophages and fibroblasts. 2) We will characterize the size of CD44 isoforms on purified purified synovial macrophages, T cells and fibroblasts, and compare with T cells, B cells, monocytes and PMNs and various cell lines. 3) We will We will study the origin of soluble CD44 in serum and synovial fluid. 4) recombinant (r) CD44-immunoglobulin (Ig) fusion molecules (rCD44Rg-2), 2), rCD44, rCD44R1 (CD44E), affinity purified transmembrane CD44, and purified soluble CD44S from synovial fluid and serum, we will determine the determine the immunoregulatory effects of various CD44 isoforms on in vitro monocyte function. 5) Using purified proteins, we will determine the the ability of regions of CD44 to bind to cytokines (IL3, GM-CSF, TGF-beta, FGF) and to bind to components of the extracellular matrix. Using cytoskeletal components, we will determine the ability of various forms of purified CD44 to associate with the cytoskeleton, and, using deletion mutants of CD44 cytoplasmic domain, define functional sequelae of CD44- cytoskeletal interactions. 6) To map functional domains of CD44, we will prepare a series of deletion mutants of CD44 extracellular or cytoplasmic regions. 7) Using a novel system of synovial tissue grown in vivo in severe combined immune deficient (SCID) mice, we will study the ability of autologous and allogeneic T cells to migrate to human synovial xenografts. Taken together, these studies should provide critical new knowledge necessary for the design of new therapies to interrupt multiple stages of inflammation in RA.


This loss of membrane-bound beta-adrenergic receptor recognition sites in frog erythrocytes during subsensitivity induced by exposure of cells to isoproterenol is due at least in part to the internalization of beta-receptor recognition sites. When the beta-adrenergic receptors are resensitized, the amounts of internalized receptor recognition sites are also returning to the normal. The internalized recognition sites of beta-adrenergic receptors have properties identical to those of membrane-bound receptors. Glycoprotein seems to play an important role in triggering this event. A series of tricyclic antidepressant drugs was found to inhibit the receptor internalization; these effects can be attributed to an inhibition of these drugs of the agonist binding to beta-receptor recognition sites in erythrocyte membranes. Inhibitors of transglutaminase diminish the extent of recognition site internalization with a concomitant prevention of the loss of membrane-bound receptors elicited by isoproterenol. There is an excellent correlation between the inhibition of recognition site internalization and the potency of these compounds to block the transglutaminase activity in vitro.


The Adult Clinical Core at the Beth Israel Hospital will serve to provide clinical specimen to the Dana-Farber Cancer Institute CFAR investigators, facilitate trials of therapies evolving from the basic research of CFAR members and encourage scientific exchange and collaborations between clinical and basic science investigators. The Beth Israel Hospital provides primary care to approximately 480 HIV-infected adults and has extensive NIH funded clinical research programs at the hospital, including a component of the Harvard/Boston City Hospital Aids Clinical Trials Unit (ACTU). The Adult Clinical Core will provide partial support for both a research nurse and a laboratory technician. The research nurse will provide CFAR investigators with clinical specimens from patients and carefully document the clinical histories of these patients for the investigators. The laboratory technician will establish and characterize HIV isolates from patients and maintain a bank of clinical specimens for the use of CFAR investigators. Both the research nurse and laboratory technician will also participate in the trials of therapies initiated through the Dana-Farber Cancer Institute-Beth Israel Hospital interactions. CFAR Cores will be made available to AIDS clinical investigators at the Beth Israel Hospital to strengthen the scientific base of their clinical studies. Finally, joint institutional conferences will be held to foster scientific collaborations and encourage the initiation of clinical trials of novel AIDS therapeutics.


A third of the world's population is infected with Mycobacterium tuberculosis (Mtb). Tubercle bacilli can remain inactive in lung lesions only to emerge decades later to seed new outbreaks of tuberculosis. In addition, tuberculosis is one of the most difficult bacterial infections to treat and continues to cause more deaths than any other bacterial infection. Bacilli exist in replicating and non-replicating states in a range of microenvironments that vary in oxygen concentration and nutrient availability. The bacilli that survive during latent infection likely exist in a non-replicating state and antimicrobials, effectie against actively growing bacteria, are often not effective against non-replicating bacteria. Population heterogeneity, the presence of more than one phenotypic variant in a clonal population, provides bacteria diverse mechanisms to endure environmental challenges. We have demonstrated that Mtb exists in two semi-stable states that appear to be epigenetically controlled. When mycobacteria were grown as a dense population, either by pellicle or settled growth, the vast majority of the population took on characteristics of a form termed pellicle. If bacilli were passaged starting with a solitary bacillus or just a few cells, however, the ensuing population shifted to a form termed solitary. One of the most striking contrasts between the solitary- and pellicle-prepared Mtb was the solitary form adapted to hypoxic and anaerobic conditions by maintaining high transcriptional activity, while the pellicle form failed to adjust t hypoxia and became truly dormant under anaerobic conditions. We hypothesize that populations of Mtb contain at least two phenotypic variants that allow for divergent responses to imbalances in proton homeostasis, particularly resulting from hypoxia. To test this hypothesis we will determine the differential abilities of the solitary and pellicle variants in adjusting to conditions that exert pressure on internal pH homeostasis. We will also identify regulatory elements that control the shift between the variants. Pellicle growth has historically been used to increase or maintain Mtb virulence during in vitro growth. Therefore, we will investigate if the pellicle-passaged variant is more virulent than the solitary form by using a mouse model of TB that reproduces hypoxic necrotic granulomas.


One in nine women suffer from pelvic floor dysfunction, including urinary incontinence and vaginal wall or uterine prolapse (VUP). Stress urinary incontinence (SUI) affects 38% of women over the age of 65 years and over 13 million women in the United States. Pelvic muscle strength is commonly assessed in these patients. However, current measurement techniques are either subjective or produce artifact, due to their non-isometric nature or contamination by intraabdominal pressure. During Phase I, we developed a second generation system that measures the isometric strength and contractile properties of female pelvic floor muscles. The system centerpiece is a novel intravaginal transducer that differentiates between intraabdominal pressure and levator ani force. During Phase II, system mechanics, electronics and software will be refined to improve system sensitivity, accuracy, and ease-of-use. Laptop- and Personal Data Assistant-based systems will be developed and validated. Clinical device performance will be confirmed by testing the null hypothesis in 120 women (40 healthy continent, 40 with VUP, 40 with SUI) that localized pelvic floor muscle defects visible on MR scans will correlate with pelvic muscle weakness. The system allows assessment of pelvic floor function and exercise intervention efficacy, and can provide biofeedback and adherence data during training.


DESCRIPTION: To date, no vaccine strategy has successfully induced potent broadly neutralizing antibodies (BnAb) against HIV-1. In vitro analysis, passive antibody transfer studies and analysis of antibody responses in the RV144 vaccine efficacy trial suggest non- neutralizing antibodies might contribute to protection against HIV-1 transmission. Three linear regions in the HIV-1 Env MPER, V2 and V3 have been implicated as potential targets of protective non-neutralizing antibody. We have constructed a novel recombinant Lactobacillus acidophilus vaccine platform that is orally delivered and induces antigen-specific mucosal IgA and systemic IgG against MPER peptides inserted into the bacterial surface layer protein. We have developed two different adjuvants for use with recombinant L. acidophilus, IL1 and flagellin (FliC). Specifi Aim 1 will determine the optimal adjuvant for mucosal immunization and whether responses are T-cell dependent or independent. Specific Aim 2 will test whether recombinant Lactobacillus acidophilus expressing candidate MPER, V2, and/or V3 epitopes can induce mucosal and systemic antibody responses that are protective against vaginal HIV-1 challenge in the HLA-DR transgenic (DRAG), humanized mouse model.


This project is designed to develop a means of replacing radioiodine, the radionuclide presently employed in the labeling of antibodies, with a radionuclide that reduces the absorbed radiation dose to the patient and has physical properties that can be more efficiently utilized by modern radiation detecting systems, specifically the gamma camera. During the past year, we have developed a procedure for labeling antibodies with indium 111. This procedure involves the conjugation of the IgG fraction of the antisera with iron-free transferrin using glutaraldehyde as the coupling compound. Indium 111 ions are then added to the solution containing the transferrin-IgG conjugate and are instantaneously chelated by transferrin. The projected goals for this year include the refinement of the conjugating procedures and the evaluation of the effectiveness of intravenously injected 111 In-transferrin-anti-CEA conjugate obtained from antisera raised in goats in detecting colonic tumors transplanted in animals. The results will be compared with those obtained with anti- CEA preparations labeled with I131 in the standard manner.


This research will focus on the management of diabetes and cardiac risk factors in elderly patients. Cardiovascular disease (CVD) and diabetes are two major public health problems, particularly in the elderly. By the year 2040, the elderly population in the United States will make up 20 to 24 percent of the population. It is projected that 19 percent of the elderly will be aged 85 or above and this population will grow to at least 12.8 million. An extensive literature review revealed that there has been research focusing on short and long-term outcomes in patients with either acute myocardial infarction (MI) or diabetes. Despite this, little is known about these combined conditions and their relationship to outcomes. In addition, management of diabetes and cardiac risk factors before and after MI have not been studied in the context of outcomes in these patients. The purpose of this preliminary clinical trial is to determine the relationship between management of diabetes and cardiac risk factors, and selected outcomes in elderly patients with diabetes following MI.


The incidence of infertility has increased 4% since the 1980s, with up to 20% cases having no known cause. One of the prevailing hypotheses is incompatibility between cognate egg and sperm proteins; however, very few pairs of interacting reproductive proteins have been identified in any organism. The best model for studying fertilization remains the marine gastropod abalone, where one of the first steps in fertilization involves the interaction between the sperm protein lysin and its egg coat receptor VERL. As a major component of the abalone egg coat, VERL is a giant, fibrous glycoprotein composed of ~22 ZP-N repeats that form intermolecular hydrogen bonds to create the highly stabilized and protective egg coat. Lysin creates a hole in the egg coat by competing for the hydrogen bonds, allowing sperm to pass and fuse with the oocyte. The VERL repeats are homologous to human egg coat proteins, and likely share a similar protein topology. However, the precise structural mechanisms that drive egg coat dissolution remains to be determined. To address this fundamental question in fertilization, two specific aims are proposed using state-of-the-art structural and proteomic approaches. In aim 1, multidimensional NMR will be used to characterize the structural basis of lysin-VERL interactions for three species of abalone. In aim 2, deep mutational scanning will be utilized to explore the specific adaptations acquired by lysin and VERL that permit species specific interactions, and simultaneously test theoretical models of sexual selection. The proposed research is innovative for its combined use of proteomic and structural techniques to characterize the evolutionary history of rapidly evolving reproductive proteins. The results are expected to shed insight into the core mechanisms that mediate egg-sperm interactions in abalone, and provide foundational information towards understanding the more complex mammalian system.


The formation of flagella and the synthesis of proacrosin will be investigated in primary cultures of mouse round spermatids. Initial experiments are designed to determine whether spermatids that do not make flagella in culture lack the centrioles necessary for axonemal nucleation. The requirements of protein, RNA, and tubulin biosynthesis for flagella formation will also be examined. The length of time for cell culture will be increased to ascertain the extent of tail maturation that can be obtained in vitro. Antibodies to the major fibrous sheath protein will be used to determine whether this component appears in the newly-formed flagella. The synthesis of proacrosin will also be examined to determine whether proacrosin contains N-linked oligosaccharides and whether these carbohydrate side chains are processed to the "complex" type. The carbohydrate side chains will be analyzed for mannose-6-phosphate groups. If these residues are found, it suggests that proacrosin is directed to the acrosome by the same phosphomannosyl receptor system that transports lysosomal hydrolases to the lysosome. The experiments of this proposal will provide the bases for long-term biochemical studies of mammalian spermiogenesis.


We plan to isolate and analyze zebra fish mutants with altered visual systems. Of particular interest initially will be mutants with altered organizations of retinal cell types, and specific enrichment procedures will be utilized to aid in the isolation of such mutants. The mutants will be analyzed by behavioral, anatomical, physiological and genetic methods. In order to increase the frequency of mutants in the populations subjected selection, we will expose fish to mutagens at appropriate stages in their life cycle. Since most mutations are recessive, we plan to generate homozygous fish from mutagenized eggs in order to enable us to recognize mutant phenotypes. Procedures for generating homozygous fish have already been developed in our laboratory.


Leukotriene B4 (LTB4) is a potent lipid chemo attractant that classically has been associated with myeloid cell chemotaxis. We isolated a novel murine seven transmembrane spanning G protein-coupled receptor, which we identified as BLTR1, an LTB4 receptor, and generated BLTR1-deficient (BLTR1-/-) mice by targeted gene disruption. Characterization of this mouse revealed that BLTR1 is responsible for LTB4- mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium. Further, despite functional redundancy with other chemo attractant-receptor pairs in vitro, loss of BLTR1 function significantly reduced eosinophil recruitment into the peritoneum in a model of peritonitis. Follow-up studies of BLTR1-/- mice have allowed us to make several novel observations about the roles of LTB4 and BLTR1 in leukocyte recruitment that will be the focus of this grant proposal. We have found that loss of BLTR1 function significantly and specifically impairs neutrophil transendothelial migration (TEM) in vitro and in vivo. Secondly, we have found that BLTR1 is highly expressed, functional on certain subsets of activated T cells, and important for their trafficking in vivo. Additionally, we have found that recruitment of BLTR1-/- T cells into the airways early in allergic pulmonary inflammation is profoundly impaired, despite preserved trafficking of these cells into the lung parenchyma. Based on these novel preliminary findings, the central hypothesis of this proposal is that LTB4 and BLTR1 play a critical role in facilitating the ability of leukocytes to traverse endothelial, interstitial and epithelial barriers. We hypothesize that interactions between leukocytes and barriers to migration they encounter, e.g. endothelial cells, tissue interstitium, and epithelial cells, induces the production of LTB4. We hypothesize that the LTB4 produced by these interactions in turn activates leukocyte BLTR1, which facilitates penetration of these barriers by the migrating leukocytes. We believe that the LTB4 biosynthetic pathway uniquely positions this chemo attractant-receptor pair for this role. LTB4 is produced by serial enzymatically catalyzed reactions, and hence can be produced much more rapidly than peptide chemokines, which require transcription and translation. The rapid kinetics of LTB4 biosynthesis enables leukocytes, endothelial cells, and epithelial cells to produce LTB4 contemporaneously with leukocyte migration across cellular and extracellular barriers. In this grant, we propose to investigate this hypothesis as it relates to neutrophil and lymphocyte recruitment. Specifically we propose to: (1) determine to cellular source of the LTB4 that facilitates neutrophil transendothelial migration; (2) define the roles of BLTR1 and CXCR2 in neutrophil recruitment into the airways; (3) determine the role of LTB4 and BLTR1 in antigen specific T cell trafficking; (4) determine the role of LTB4 and BLTR1 in T cell recruitment into the airways of the lung. [unreadable] [unreadable]


All forms of diabetes are due to a relative deficit of functional beta cells, yet adult human beta cells are resistant to attempts at replication, and serve as a poor experimental model for replicating human beta cells. Because of this paucity of models for adult human beta cell replication, we have developed a large Biorepository of rare human insulinomas, benign pancreatic adenomas that grow and over-secrete insulin, believing that they may provide information that will inform attempts at clues and drug targets and pathways for therapeutic human beta cell regeneration. Recently, we have provided the results of intensive genomic, transcriptomic and bioinformatic analysis of human insulinomas, comparing them to FACS-sorted pure human beta cells. Remarkably, we find that insulinomas display three cardinal features: 1) they almost universally contain recurring mutations, copy number alterations and gene expression abnormalities in members of the Trithorax Group of chromatin modifying enzymes, notably including KDM6A, MLL3 and/or MEN1; 2) they also almost universally display alterations in the Polycomb Repressive Complex of chromatin modifying enzymes and their targets, particularly YY1, EZH2 and H3F3A; 3) they almost universally display abnormalities in the chromosome 11, such as allelic loss of all or part of chromosome 11, and/or allele-specific expression and/or DNA methylation/imprinting abnormalities of the imprinted 11p15 region of chromosome 11, reminiscent of the other beta cell proliferative disorders, such as the Focal Variant of Hyperinsulinism and Beckwith-Wiedemann Syndrome. On the other hand, the mechanisms through with these events lead to beta cell proliferation and while maintaining the beta cell phenotype are unknown. More specifically, exactly how MEN1, KDM6A, MLL3, YY1, EZH2 and H3F3A modulate beta cell function and proliferation are largely unknown. The three Specific Aims of this application address this important knowledge gap. Aim 1. To Elucidate the Abnormal Pathobiology of Three Key Trithorax Members In Insulinoma vs. Beta Cells. Aim 2. To Define Abnormal Biology of Three Key Polycomb Repressive Complex Members in Insulinomas vs. Beta Cells. Aim 3. To Define 3-D Chromosome 11p15 Architecture in Insulinomas and Beta Cells.


This proposal is to study the local molecular interactions in cytochrome c oxidase from beef heart that control reduction of dioxygen to water and the conservation of chemical energy for sythesis of adenosine triphosphate. These studies will include freeze-trapping of intermediate species at temperatures between 10K and 280K as required. The chemical nature of stable or freeze-trapped cytochrome oxidase derivatives will be characterized by U.V. visible, and near infrared spectra, electron paramagnetic resonance spectra, and by vibrational spectra of ligand complexes with a3Fe and CuB. Fourier transform infrared spectroscopy has provided new information about the reduced oxidase in studies of the carbon monoxide complexes, a3FeCO in the dark, and CuBCO that results from photolysis at low temperature. This has opened new ways to measure structural interactions that control electron flow and oxygen reduction with minimum release of intermediates such as superoxide or peroxide. Pathological states that interfere with this control may cause altered forms that we observe by FTIR spectroscopy of the CO complexes of a3Fe or CuB. Conditions that affect the relative amount of these states will be explored. These studies apply areas of physics, spectroscopy, transition metal chemistry, and computer analysis of band shapes and reaction kinetics to problems of heart physiology and heart disease. The technology that is being developed in the conduct of these experiments will open yet new windows in our study of the heart.


PRODUCTION OF MUTANT CONGENIC MICE


Quantitative Imaging: Biostatistics and Medical Physics (SR-C). This Specialized Resource provides support for a wide range of imaging (MRI, nuclear and optical imaging) procedures and statistical functions for the clinical trials in ICMIC-3 as well as all the pre-clinical animal research for all Research Projects and Developmental Projects. The tasks and services to be performed by SR-C fall into six broad categories; 1) to make additional computational and centralized Image storage facilities available to lCMIC-3 investigators thereby facilitating data sharing between projects; 2) to make specialized image processing and display software available, allowing the sharing of the software licenses and centralizing support; 3) to provide software development capabilities for the performance of specialized processing and analyses including the automation of bulk processing tasks, format conversions and the development of new algorithms; 4) to consult on issues regarding imaging protocols, optimal acquisition settings, quantitation and on the modeling of tracer kinetics; 5) to provide biostatistical expertise in the design and analysis of research studies and to conduct statistical analyses and modeling of data generated by those studies; and 6) to conduct research leading to the development of new capabilities of common benefit to the iCMlC-3 projects.


Melanoma is a devastating form of skin cancer with limited therapeutic options. This project focuses on the signaling pathways in NRAS mutant melanomas, which accounts for approximately 20% of melanomas. Therapies that target RAS and MEK, a RAS effector, have not been clinical effective in mutant NRAS melanoma patients. In cell-based studies, we show that the response of NRAS mutant melanomas to the MEK inhibitor AZD6244 is heterogeneous, with a sensitive and an insensitive group. For the AZD6244 insensitive cells, there is a need to identify specific effectors that mediate mutant NRAS actions and additional pathways that are synergistic for NRAS signaling to form the basis of new therapeutic strategies. One protein that may prove to be important in this group is TANK-binding kinase 1 (TBK1), an atypical IkB kinase family member. In KRAS transformed cancer cells, TBK1 mediates oncogenesis through its phosphorylation and activation of the pro-survival kinase AKT. We show that NRAS expression increases TBK1 activation in melanoma cells. In mutant NRAS melanoma cells, we will determine if TBK1 regulates AKT activation, as measured by Western blot and kinase assays, through TBK1 depletion. To assess its potential as a therapeutic option, we targeted TBK1, via RNA interference or the pharmacological inhibitor BX795, in conjunction with MEK. We found that the combination leads to increased cell death in AZD6244 insensitive NRAS mutant melanoma cells. We have obtained a more specific inhibitor of TBK1, Compound II, and we will use it alone and in combination with AZD6244 in in vitro 3D collagen survival assays (which mimic the dermal environment) as well as in in vivo intradermal xenografts. In these experiments, we will be utilizing melanoma cell lines as well as cells dissociated from surgically resected NRAS mutant tumors. Additionally, we will analyze expression and activation of TBK1 in tumor microarrays of metastatic melanomas, associating our findings with NRAS genotype. In addition to TBK1, we predict that NRAS mutant melanoma cells also rely on genes that are not directly downstream of NRAS for survival. To identify signaling alterations in AZD6244 insensitive cell, we will use a short hairpin RNA (shRNA) library in a negative selection screen to find shRNA that are selectively toxic to AZD6244-treated AZD6244 insensitive cells. To validate the targets, we will knockdown the target using RNA interference to see if the lethal phenotype can be recapitulated. The validated targets will be examined in AZD6244 sensitive and insensitive cells and overexpressed in AZD6244 sensitive cells to see if they confer resistance to MEK inhibition. If targets or their involved pathways have inhibitors, then we will examine the effects, either alone or in combination with AZD6244, on cell death in 3D collagen survival assays. The data generated from this proposal may lead to the identification of TBK1, along with other proteins, as novel targets for NRAS mutant melanoma to be used in the clinical setting as part of a combination therapy regimen. Our long-term objective is to improve the treatment options for NRAS mutant melanomas.


Common use of condoms by young adults across all racial/ethic groups remains a formidable public health challenge, especially for African American males (Kennedy et al., 2007a,b,c; Kennedy et al., under review). As a call to action, a collaborative team of researchers proposes to conduct a four-year behavioral-driven research study to modify, implement and evaluate the efficacy of a promising brief condom promotion program in a two-arm randomized trial among African American males 18-24 years from 12 community centers on Chicago's South Side. Accordingly, we propose the following specific aims and related set of research questions: 1. Modify, implement, and assess the efficacy of a condom promotion program for African American males. 1.1. Will participants randomly assigned to the intervention report a statistically significant increase in the frequency of condom use and decrease in unprotected sexual intercourse (primary outcomes) relative to those assigned to the comparison group? 1.2. Will the intervention produce statistically significant positive effects on secondary outcomes (e.g., condom use self-efficacy, condom use decisional balance, etc.) relative to the comparison group? 1.3. Will the secondary outcomes mediate the relationship between intervention status and condom use (the primary outcome)? 2. Assess characteristics of program implementation and its relationship to changes in behavioral outcomes. 2.1. What was the level of implementation fidelity? 2.2. How is variation in implementation fidelity related to change in primary outcomes? For a nine-month formative phase, we will conduct a series of qualitative interviews with key informants and the target population to modify our existing program. Over the next 16 months, we will recruit 520 study participants from our recruitment sites to implement the intervention and comparison programs and assess the efficacy of the intervention program on a series of primary and secondary outcomes. Assuming 30% attrition rate (n=156), we expect to collect complete outcome data via Audio Computer Assisted Self Interview (ACASI) from about 364 participants over 12-months. In addition, we will conduct biological assessment of STDs to validate participants' self-reports on sexual behaviors at all assessment points. This proposed study is an extension of a recently completed NIH-funded R21 (PI: Kennedy) to develop, implement, and pilot a brief condom promotion program for African American males (Kennedy et al., 2007a,b,c; Kennedy et al., under review). We have assembled a collaborative team of experienced investigators and research partners to successfully implement this proposed project.


The focus of this project is development and refinement of statistical procedures for the design and analysis of screening, detection, and related studies in cancer. Statistical problems under investigation include sample size determination, comparison of analysis methods, and development of monitoring techniques and stopping rules. Each of these problem areas is common to screening and prevention trials in which the Division participates, but the methods for screening studies must address the special lead time and length biases inherent in screening programs. The research includes investigation of techniques to estimate and adjust for screening biases in the analysis of survival data, and the study of the relationship between long-term mortality and short-term outcome measures, such as stage shift. Probabilistic models of disease and screening are being developed to aid in the interpretation and evaluation of data from screening programs, and a breast cancer model relating doubling time to survival is under derivation in collaboration with the Biometry Branch, DCCP.


Each year, 6.2 million persons are newly infected with human papillomavirus (HPV) and 26,000 new HPV- related cervical, genital, and or pharyngeal cancers are diagnosed, resulting in >$4 billion in annual medical costs. Despite US guidelines for vaccinating all adolescents starting at age 11 with 3 doses of HPV vaccine, in 2012 only 53% of 13-17y females had >1 dose and 35% had 3 doses; 21% of teen males had a vaccination. The most effective strategy for improving vaccination rates is patient reminder/recall (R/R), in which providers send reminder messages by telephone, mail or other modalities to parents of children or teens who are eligible for vaccinations, or recall messages for vaccines that are overdue. Yet few practices use reminder/recall for any vaccinations because of costs and lack of personnel time; very few use reminder/recall for HPV vaccine. Statewide immunization information systems (IISs) now exist in 49 states to track childhood vaccinations, but they have not been used for reminder/recall for HPV vaccinations. We have united two leading immunization research groups (Rochester, NY and Denver, CO) for this dissemination/implementation project to determine whether IIS-based reminder/recall is more effective/cost-effective than practice- based reminder/recall in increasing HPV vaccination rates in adolescents. Our study has 4 aims. Aim 1 is to adapt R/R procedures used for children (protocols, clinical decision support, and message content) for state IISs to conduct reminder/recall for HPV vaccinations. Aim 2 is to assess the impact and cost-effectiveness of auto dialer (phone) IIS R/R in increasing HPV#1 and HPV#3 vaccination rates in adolescents 11-17 years. We will use a population-level, pragmatic comparative effectiveness trial, and randomizing counties to IIS R/R versus practice-based R/R training. We will apply the RE- AIM framework to evaluate the reach, effectiveness/cost-effectiveness, adoption and implementation of IIS R/R. Aim 3 is to assess the additional benefit and cost-effectiveness of adding mailed IIS R/R to auto dialer R/R for those with wrong or disconnected phone numbers or if no response to phone IIS R/R. This will involve a second pragmatic RCT, performed simultaneously with Aim 2, and also assessed using the RE-AIM framework. Aim 4 is to disseminate IIS R/R across New York and Colorado, develop a toolkit, and work with 4 other IISs and a technical advisory group to develop a sustainable IIS R/R system for the US. By the end of the study we will have a feasible, sustainable, cost-effective model for HPV vaccination reminders that can be used nationally to prevent cervical cancer and other HPV-related cancers.


Multiple myeloma (MM) is a B-cell malignancy that remains incurable in most patients. After treatment with high-dose chemotherapy and autologous stem-cell support, complete remission rates of 50% are achieved. However, relapses are inevitable in most patients. Additional measures are needed after transplantation to eliminate minimal residual disease, and immunotherapy is an appealing option for this purpose. We hypothesize that optimized dendritic cell (DC)-based immunotherapies are able to elicit a strong myeloma-specific cytotoxic immune response in immunocompetent patients that may effectively control or even eradicate residual tumor cells. Compared to idiotype protein (Id), myeloma cells contain a multitude of tumor antigens that can stimulate an increased repertoire of anti-tumor T cells. Indeed, our preliminary results demonstrate that myeloma lysate-specific T cells are promising effector cells for immunotherapy. In this project, we propose to examine the efficacy of intranodal vaccination of myeloma patients with tumor antigen-pulsed, CD40L-conditioned mature DCs. Newly diagnosed, untreated patients (most of who have nearly intact immune systems) will be targeted, and DC vaccines will be given to the patients prior to and after high-dose therapy in order to achieve a maximal tumor reduction without compromising the effects of immunotherapy. Thus, aim 1 of this project is to evaluate anti-tumor immune and clinical responses in patients receiving Id versus tumor lysate-pulsed DCs, to determine whether tumor lysate-pulsed DCs induce stronger responses. Antigens and keyhole limpet hemocyanin (KLH)-pulsed, CD40L-matured DCs will be used as the vaccine. CD40L-conditioned/matured DCs can activate CD8+ T cells directly and break T-cell tolerance. In Aim 2, we will evaluate whether re-infusion of blood T cells collected after initial DC vaccination helps to further expand specific T cells in vivo. Aim 3 is to evaluate whether long-term booster immunization could further augment anti-tumor immune and clinical responses in patients, and aim 4 to elucidate the biological mechanisms of the immunotherapy strategies by examining the induction, number and function of myeloma-specific T cells and subsets in blood and bone marrow of patients and whether immunotherapy may induce epitope spreading and loss of tumor antigens by myeloma cells. Anti-myeloma immunological and clinical responses are the primary and secondary end points of the studies. These innovative approaches will substantially contribute to the ability of immunotherapy to induce or improve long-term survival in patients with MM.


The research to be performed for this application will increase understanding of bacterial cell division. Although cell division is one of the most basic biological processes, there is relatively little known about what each component of the subcellular cell division machinery does and how the entire process is regulated. For this work the model organism Escherichia coli will be used. This organism is ideal for the study of cell division because (1) much about the basic genetics and cell biology of this organism is already known;(2) excellent genetic tools are available;and (3) the basic protein components of cell division in E. coli are highly conserved in other bacterial species. When a cell is about to divide, an essential protein called FtsZ polymerizes into a ring-like structure known as the Z ring at the center of the cell. Numerous other proteins, many of them also essential, are recruited to the Z ring. One of these proteins is FtsA. The ftsZ and ftsA genes are one of the most highly conserved bacterial gene pairs. Some of the activities of FtsA are known, but the molecular roles of FtsA remain elusive. The current line of thought is that FtsA regulates the formation of the Z ring by two mechanisms. First, FtsA tethers the ring to the inner membrane of the cell, connecting the Z ring to inner membrane proteins. Second, FtsA regulates the disassembly of the Z ring, so that it will constrict and divide the cell. The current data suggest that membrane binding, self-interaction and nucleotide binding are all critical for FtsA function and that each of these properties can be correlated to each other. Mutations that inhibit any one of these FtsA activities seem to affect FtsA-FtsZ interactions, which prevents normal cell division. The research proposed here will (1) determine how FtsA membrane binding and self-association are correlated and (2) define how each of these activities determines the ability of FtsA to affect the Z ring. Because they share some of the same properties, FtsA and MinC may affect FtsZ through analogous mechanisms. The study of bacterial cell division is important because it is a basic cellular process that needs to be understood and because it is a highly relevant target for potentially new antimicrobial drugs. PUBLIC HEALTH RELEVANCE: When bacterial cells cannot divide, they cannot multiply and spread. In this time of increasing bacterial resistance to antibiotic treatments it is critical that we decipher the mechanisms that allow these populations to proliferate. Understanding the mechanism of bacterial cell division will provide more targets for new antimicrobial therapeutics.


The long term goal of this work is to understand the underlying error and pathophysiology of cystic fibrosis, the most common lethal genetic disease of whites. In cystic fibrosis, there are inherited abnormalities in alpha-adrenergic, beta-adrenergic and cholinergic systems. This proposal will further elucidate the biochemical bases of these abnormalities and investigate whether they contribute to the development and progression of pulmonary disease. The beta-adrenergic defect is probably at the level of receptor-cyclase coupling, so this proposal will investigate Ns (a coupling protein), phospholipid methylation, receptor desensitization, and pharmacologic modulation of receptor-cyclase coupling in leukocytes from patients with cystic fibrosis, their parents, and appropriate controls. In vitro systems will be developed to investigate the cause of increased cholinergic responses in cytic fibrosis, such as cholinergic stimulation of lysosomal enzyme release from granulocytes, or of lymphoproliferation. Changes in intracellular calcium in response to cholinergic agents will be monitored with Quin 2, and the binding properties of muscarinic receptors on lymphocytes and granulocytes will be determined. To investigate the pathophysiologic significance of these autonomic abnormalities, three strategies will be used. First, using minimally invasive tests, like the cyclic AMP response to beta agents in leukocytes and power spectral analysis of heart rate variability, this project will test autonomic function in children with cystic fibrosis prior to the onset of significant disease. Second, autonomic function will be determined in tissues affected by the cyctic fibrosis disease process using the beta-adrenergic responses of nasal biopotentials in vivo and the cyclic AMP response to beta agents in tracheocytes obtained by bronchial brushing as test systems. Third, the relation of autonomic dysfunction to airway reactivity in parents of patients with cystic fibrosis and appropriate controls will be further defined. Thus, the study will determine if autonomic dysfunction is present early in the course of the disease and if it occurs in involved tissues, and will further elucidate the relation of autonomic dysfunction and airway reactivity. Such studies have implications for the development and progression of obstructive airways disease in general, and for cystic fibrosis in particular.


Many patients with hypertrophic cardiomyopathy have severe symptoms in spite of medical therapy with beta adrenergic blocking agents and/or calcium channel blocking agents. Recently we have been investigating the use of amiodarone, a benzofuran derivative with potent hemodynamic and antiarrhythmic properties in this same subgroup of patients and have noted an improvement in cardiac symptoms and an increase in exercise capacity. However, there remains a subgroup of patients who are intolerant of amiodarone or who do not improve on amiodarone and continue to have marked symptomatology. In response to a compelling clinical need in this subgroup of refractory patients, we felt it appropriate to explore other potential pharmacologic modalities. We have hypothesized that the functional and structural abnormalities in HCM are related to a primary membrane disorder leading to increased cytosolic calcium levels as a result of altered calcium fluxes involving both the myocardium and the vascular smooth muscle of the small intramural coronary arteries. Lidoflazine has been shown to be a potent calcium entry blocker, and has a cellular protective effect against calcium overload in vascular smooth muscle and cardiac muscle during ischemia, preventing ischemic contraction and myonecrosis. These properties of the drug afford an ideal mechanism for testing the above hypotheses, as well as offering a potentially important therapeutic alternative. The study will consist of 3 phases. The first phase to assess the clinical efficacy of the drug; the second to characterize the hemodynamic/metabolic correlates of the drug that may determine its efficacy; and the third to compare in a double blind fashion lidoflazine versus standard therapy. We have enrolled thus far 3 patients in phase I, two of whom have had symptomatic and exercise improvements.


Over half of the acute respiratory tract illness in infants and young children can presently be related to respiratory syncytial virus, a parainfluenza virus, an adenovirus, an influenza virus or Mycoplasmapneumoniae. The role of other agents apparently is much less important. The epidemiology of the more common agents is becoming increasingly understood, but often has shown surprising variations. Little is known regarding the pathogenesis of infection with these agents, particularly with respect to the factors of host resistance, and the role of local, specific cellular, or serum antibody in protection or in production of disease. In order more fully to understand these infections and their possible prevention we propose: (1) to determine (a) the relationship of non-bacterial agents to pediatric respiratory tract illness, (b) the clinical relationship of these agents. (2) to determine the pathogenesis of infection and development of resistance to respiratory tract viral infection. (3) to study the natural history of viral and mycoplasma agents.


It is currently understood that arterial baroreceptors control sympathetic nervous system tone, while vestibular end organs provide afferent input to a vestibulo-sympathetic reflex that normally augments the compensatory baroreflex. However, the chemical anatomy, connectivity and synaptology of the neural pathways underlying this integration remain largely unknown. The overall goal of the proposed project is to identify the neurotransmitters and modulators involved in mediating vestibulo-autonomic synaptic interactions. The four aims address the chemoanatomy and synaptology of these vestibular projections in adult rats and evaluate the overall hypothesis that different transmitter/modulator signatures and distinct synaptic architectural arrangements are utilized by vestibular projections to the caudal and rostral ventrolateral medulla (CVLM and RVLM, respectively). Aim 1 is to identify the locations of vestibulo-medullary and medullo- sympathetic projection neurons, determine the relative proportions of vestibular projections to CVLM and RVLM, identify the primary amino acid neurotransmitter(s) utilized by these pathways and determine whether these projections terminate on glutamatergic and/or GABAergic neurons in CVLM and RVLM. Aim 2 is to determine whether vestibular terminals in CVLM and RVLM co-express IAA-RP and/or L-citrulline. Aim 3 is to conduct a qualitative analysis of the main neurotransmitters and modulators of vestibulo-recipient cells in RVLM and CVLM, to test the hypothesis that glutamatergic vestibular afferents terminate on interneurons and on distal dendrites of GABAergic/IAA-RP-containing CVLM neurons, GABAergic and noradrenergic dendrites in RVLM, and directly on the somata of glutamatergic/IAA-RP-containing RVLM neurons with direct projections to the spinal cord. Aim 4 is to visualize and quantify parameters of vestibulo-sympathetic synaptology, and will test the hypothesis that the synaptology of vestibular terminals in RVLM involves complex interactions among multiple transmitter systems, whereas the synaptology of vestibulo-sympathetic terminals in CVLM reflects direct axo-dendritic contacts with CVLM output neurons. Throughout the project, the vestibulo-sympathetic pathways will be identified using anterograde and retrograde tract-tracing in tissue that will be further processed using additional immunofluorescent tags to identify transmitters and modulators of interest. Ultrastructural studies will employ immunogold labels to identify and quantify terminals and synaptic contacts. These studies will characterize the neurotransmitters, modulators and synaptic articulations utilized by vestibular projections to sympathetic pre-motor medullary neuron pools and will yield qualitative analyses and quantitative estimates of parameters of their synaptic architecture. This information does not currently exist in the literature, and is important for the development of evidence-based pharmacotherapeutics for the treatment of vestibulo-autonomic disorders such as orthostatic hypotension, as well as the development of more specific anti-hypertensive medications that do not elicit disabling vestibular side-effects such as dizziness and vertigo. PUBLIC HEALTH RELEVANCE: Orthostatic hypotension and vestibular side effects of drugs targeting the sympathetic nervous system (e.g. anti-hypertensive medications) impact large populations, especially the elderly. However, little is known about the neurochemical organization of vestibulo-sympathetic pathways. This project will provide fundamental information about the chemoanatomic and synaptic organization of vestibular projections to cardiovascular neurons in the ventrolateral medulla, may suggest new drug therapies to ameliorate orthostatic hypotension and intolerance, and may lead to the development of more specific anti-hypertensive medications that do not elicit disabling vestibular side effects such as dizziness and vertigo. [unreadable] [unreadable] [unreadable]


Neoplasms contain increased quantities of O-alkyl lipids. In order to detect surface membrane abnormalities which might exist in malignant versus non-malignant tumors, we have measured the O-alkyl lipid content of these structures. The surface membrane O-alkyl lipids of a series of transplantable metastasizing and non-metastasizing rat tumors was quantitated by photodensitometry. The results suggest that more rapidly growing and more malignant neoplasms contain greater quantities of O-alkyl phospholipids than less aggressive tumors. We are currently conducting a similar evaluation of a series of human mammary tumors obtained as surgical specimens and analyzed for estrogen and progesterone receptors. We are attempting to correlate the degree of malignancy of these tumors with their ether lipid content and with their concentrations of estrogen and progesterone receptors. An additional project in progress is an attempt to purify O-alkyl synthase, the enzyme responsible for the synthesis of O-alkyl dihydroxyacetone-phosphate. Although a method for purifying this enzyme has been recently published, we are taking a somewhat different approach involving novel forms of chromatography. Preliminary studies in this area involve isolation of this enzyme from Tetrahymena pyriformis. If successful, the procedure developed will be used on the enzyme found in tumors. In addition to the above, we are continuing work on the mechanism of action of O-alkyl synthase. In the past year, we have conducted experiments confirming the basic validity of our proposed mechanism and confirmed the work of others that the action may be, in part, reversible. We are also studying the stoichiometry involved in that aspect of the mechanism involving a hydrogen exchange and the release of fatty acid from acyl-dihydroxyacetonephosphate. Preliminary data suggest that the release of one proton is accompanied by the release of one fatty acid which retains both carboxyl oxygens. This unusual form of ester cleavage was recently demonstrated in this laboratory. The results have broader implications with regard to the mechanism of action of a related enzyme that may function via a similar mechanism, namely, the acylhydrolase which cleaves acyl-dihydroxyacetonephosphate. (A)


3D Microscopy with Ultraviolet Surface Excitation (3D-MUSE) Summary. We will create powerful techniques for performing 3D histology-quality imaging of small to large volume specimens (e.g., entire mouse or human organ) with extraordinary contrasts to address a very large number of preclinical, biotechnology, and clinical applications. We will combine Microscopy with Ultraviolet Sur- face Excitation (MUSE) (invented at Lawrence Livermore National Laboratory and extended at University of California at Davis), with 3D serial sectioning and block face imaging technologies (developed at Case West- ern Reserve University), to implement 3D-MUSE. Briefly, MUSE fluorescence imaging uses excitation at wave- lengths shorter than 300 nm, a range at which tissue penetration is limited to a few microns deep. This allows one to image?at sub-nuclear resolution?only the very surface of a piece of tissue, making it ideal for serial block-face imaging. Many common histological stains, autofluorescence, and fluorescent proteins excite at these sub-300-nm wavelengths and emit at their familiar wavelengths in the visible range. Because the excita- tion range is distant from emitted signals, images can be captured using a color camera, without additional fil- ters, providing a broad and informative color palette, beyond what is obtained using narrow-band-filter sys- tems. With color-sensitive volume rendering, and combined color and morphology-based segmentation, 3D microanatomy and pathology will be visualized and quantified. Image-guided histology will allow 2D/3D live- time monitoring to guide optional section acquisition for molecular studies using immunohistochemistry and manual or laser-capture micro-dissection. When fully realized, potential applications, out of many, include 3D microanatomy, mouse phenotyping, embryo cell lineage tracking, monitoring therapeutic nanoparticle delivery, as well as studies of cancer physiology, cancer immunotherapy, stem cells, toxicology, and biology. In all of these cases, we anticipate that there will be great value added by the third dimension. We will build on exper- tise in MUSE and block-face imaging systems to create test beds for evaluating 3D-MUSE. We will optimize tissue immobilization, staining, and imaging, and advance MUSE-specific software for 3D segmentation, visu- alization, and analysis. If our research is successful, we will transfer 3D-MUSE technology to commercial enti- ties that can create products for researchers across the world. It will likely be possible to create inexpensive, highly informative 3D-MUSE imaging systems that will empower many applications. Ultimately, the number of researchers using 3D-MUSE around the world will define project success.


Nearly 5 million people in the US alone are afflicted with Alzheimer's disease, and there is currently no means to prevent or treat the disease. Though the molecular basis of the disease is unclear, the deposition of amyloid plaques in the brain is a key hallmark of the disease. These plaques are widely believed to be pathogenic, and tremendous efforts have focused on the mechanism governing their formation. Amyloid plaques are composed of the 42-residue amyloid ? peptide (A?), which is generated through proteolytic cleavage of the amyloid precursor protein (APP) by ?-secretase. This enzyme is localized within cholesterol-rich lipid raft membrane domains, while the APP substrate exists in both the fluid-phase and lipid raft domains of cellular membranes. Therefore, the efficiency with which the A? peptide is generated may be governed by the distribution of APP between the raft and non-raft membrane domains. Recent work in the Sanders lab on the 99-residue C-terminal fragment of APP (C99) has revealed a cholesterol binding pocket within the TM domain (present in APP). These studies have confirmed that C99 binds cholesterol with high affinity in bilayers, which suggests that the localization of C99 (and APP) may depend on the distribution of cholesterol in biological membranes. We recently tested this hypothesis by characterizing the localization of C99 within phase-separated giant unilamellar vesicles (GUVs), which contain both fluid phase (L?) and liquid-ordered (Lo) domains. The results show that C99 is specifically localized within raft-like Lo domains. However, C99 variants carrying mutations that abolish cholesterol binding strongly prefer the non-raft L? phase. This confirms cholesterol binding directly affects the distribution of C99 within the membrane. The most intuitive explanation for this phenomenon is that C99 is driven into the Lo domain due to the high concentration of the cholesterol ligand, which leads to favorable binding energetics. However, thermodynamic evaluations of this partitioning suggest that binding energetics cannot account for the observed differences. Thus, the physical mechanism for this coupled binding and partitioning remains unclear. In the following, I propose a series of experiments aimed at dissecting the energetic contributions of both the membrane and the protein in the coupled binding and partitioning of C99. I will first use EPR spectroscopy to assess the binding energetics of cholesterol in Lo and L? like membranes. The results will suggest whether the cholesterol binding energetics are sensitive to changes in the bilayer. Next, I will use protein engineering and confocal fluorescence microscopy to determine how differences in the length and rigidity of the TM domain affect its partitioning. These studies will reveal the structural features of C99 that are critical for its sorting within te membrane. Finally, I will examine the structural dynamics of free and cholesterol-bound C99s using solution NMR in both Lo and L? like bicelles in order to determine how bilayers affect its binding mode. Together, the results will provide novel insights into the molecular basis of Alzheimer's disease and elucidate the molecular determinants of protein sorting within the membrane.


PROJECT SUMMARY DNA replication is undeniably important for life and as a consequence, cells have evolved mechanisms to monitor replication fidelity and to coordinate completion of replication with other cell cycle events. The goal of this project is to understand how cells choreograph the duplication of their chromosomes, and how defects in DNA replication may contribute to some recently discovered disorders in humans. Chromosome replication in eukaryotes is a process that involves the regulation of multiple initiation sites (origins) per chromosome. Origins show variation in timing-not all origins fire at the same time in S phase-and in efficiency-not all origins necessarily firing in every cell cycle. In addition, cells appea to maintain a backup set of origins (dormant origins) for use under special circumstances and these origins may differ in their basic control properties. Understanding how origin use is regulated is therefore critical for understanding how genome integrity is maintained. This notion is underscored by three recently described genetic disorders with links to replication defects: 1) Meier-Gorlin Syndrome with its point mutations in genes essential for origin licensing; 2) a point mutation in a gene encoding part of the replicative helicase that is associated with breast cancer in mice and growth abnormalities in humans; and 3) human segmental triplications with an inverted central copy that can be explained by a replication fork error. Yeast is an ideal model organism for studying DNA replication because of its small chromosomes, well defined origin sequences, ease of altering chromosome structure, and exceptional systems for genetic and genomic analysis. This project will apply a combination of molecular, genetic and genomics approaches to: uncover modulators of origin efficiency and timing in yeast, to explore/investigate how orderly replication contributes to genome stability use point mutations in the yeast orthologs of genes involved in human disorders to understand the downstream consequences of defects in replication as they relate to human health and disease, and to identify genetic and chemical suppressors of the defects determine whether DNA replication errors are responsible for a particular class of inverted amplicons that can contribute to cancer progression and congenital copy number variants. This project will address outstanding questions regarding the biology of chromosome replication control: why do origins initiate replication at different times, what distinguishes origins in different temporal categories, what i the molecular basis for inefficient origins, and what defects in replication fork progression predispose chromosomal loci to inverted amplification? In addition, this work will give insight int human disorders resulting from errors in chromosome replication and could lead to diagnostic or therapeutic advances.


Huntington[unreadable]s disease (HD) is a dominant neurodegenerative disease that is caused by the expansion of a stretch of CAG triplet repeats encoding polyglutamine (polyQ) within huntingtin (htt), the protein product of the HD gene. HD is considered to be the consequence of a deleterious gain-of-function caused by the expanded polyQ stretch that is unrelated to htt[unreadable]s normal function. Recent work suggests that although gain-offunction may play an important role in HD pathogenesis, a corresponding loss of normal htt function also contributes to the disease process. Our long-term objective is to use genetic and biochemical approaches to understand the role of htt[unreadable]s normal function in HD pathogenesis, and to discover new potential therapeutic strategies for the treatment of HD based on restoring normal htt function in HD. To accomplish this objective, we propose three specific aims that are designed to help us understand how the expanded polyQ stretch can affect normal htt function, and how a version of htt that lacks its normal short stretch of polyQ (?Q-htt) is able to rescue HD phenotypes in a mouse model for HD. In Aim 1, we will test the hypothesis that ?Q-htt is able to rescue HD mouse model phenotypes by enhancing autophagic clearance of mutant htt. We will focus on two potential mechanisms that may be responsible for ?Q-htt[unreadable]s effects. First, ?Q-htt may mediate the enhanced recognition of mutant htt aggregates by p62/SQSTM1, a polyubiquitin binding protein that can target such aggregates for autophagic degradation. Second, ?Q-htt may affect autophagic degradation of mutant htt aggregates indirectly by enhancing retrograde transport of autophagosomes to lysosomes. To test these mechanisms, we will characterize brains and primary neurons derived from mice expressing ?Q-htt with or without 140Q-htt expression, and conditional knockout mice lacking neuronal htt expression, for p62/SQSTM1 function. In addition, the efficiency of retrograde transport will be characterized in primary neuronal cultures by measuring organelle and dynein complex movement. Recently, we have also observed that increasing the length of the mouse htt polyQ stretch from 7Q to the normal human average length of 20Q can accelerate interactions of normal and mutant htt. To test the hypothesis that an interaction between normal and mutant htt can affect HD pathogenesis, we will compare in Aim 2, behavioral and neuropathological phenotypes in mice expressing 7Q/140Q htt, and in mice expressing 20Q/140Q htt. Finally, in Aim 3, we will test the hypothesis that mutant htt expression can influence the interaction of wild-type or ?Q-htt with their binding partners by using mice expressing epitope-tagged htt alleles to detect differences in the repertoire of normal and ?Q-htt interacting proteins in the presence and absence of mutant htt expression.


The North Central Cancer Treatment Group (NCCTG) is a regional organization made up of 10 community cancer centers with the Mayo Comprehensive Cancer Center serving as Operations Office and Statistical Center. It is designed to conduct cancer treatment research at the level of the community clinic where most cancer care is delivered. It is the intent of the NCCTG to produce the most complimentary clinical research interaction of community clinic, comprehensive cancer center, and national cooperative group. By bringing cancer patients into national research programs at an early stage of their disease, the NCCTG meets an expressed need of the National Cancer Program. Particular emphasis had been placed on multidisciplinary programs with strong participation of medical oncology, surgical oncology, radiation therapy, and pathology. We believe extraordinary high quality of research data has been achieved and we will continue our efforts to maintain and enhance quality control procedures. Significant and timely clinical research protocols are being conducted in colorectal carcinoma, lung cancer, breast cancer, pancreatic cancer, gastric cancer, gynecologic cancer, and brain tumors. Protocols for urologic cancer are under active planning. Whenever pertinent, protocols are randomized in design and concurrently controlled with untreated controls used when justified. A unique characteristic of clinical research within the NCCTG is unparalleled cost effectiveness. In spite of the severe handicap of grossly inadequate funding the NCCTG has proven itself to be a strong, viable, high quality, and productive clinical research organization.


Hepatitis C virus (HCV) is one of medically important RNA viruses and causes a wide spectrum of liver diseases, which are also a leading indication for liver transplantation in the United States. Given its high priority in public health, an in vito model for HCV life cycle is highly demanded for both basic and translational HCV research, however, current in vitro HCV models could only mimic some steps of viral life cycle. There is so far only one particular HCV genotype 2a strain, named JFH1, is able to be propagated in vitro. The JFH1-based cell culture has been established through the rescue of infectious viruses from cloned viral genomes, so called reverse genetics approach. A well-known constraint in such an approach is the unpredictability of viral mutations introduced internally or externally during the preparation of full-length viral clones. Some of these mutations may be lethal or detrimental for successful rescue of infectious viruses, in particular in the setting of HCV due to its coordinated genome network and remarkable genetic diversity. When the number of strains/clones tested is large enough to overcome the barrier of mutations from either heterogeneous viral population or experimental introduction, HCV cell culture could be established with strains other than JFH1. However, this goal is hard to be achieved by conventional reverse genetics that is notable for its tedious process. Based on our previous work, we thus propose to develop a rapid reverse genetics platform capable of screening clinical HCV isolates in a high-throughput manner. We hypothesize that the establishment of such a platform is feasible by the integration of our long RT-PCR technique (US patent No: 7,786,294 B2) and a seamless assembly method. The proposed platform is a plasmid-based reverse genetics in which long RT-PCR and 3'UTR amplicons of HCV will be fused into the target plasmid using the Gibson assembly method, followed by direct transfection into Huh-7 cells and subsequent measurement of potential infectious viruses. We will screen a total of 61 patient-derived HCV 1a strains that have already been characterized for viral mutations at the full-length genome level. The method developed with the proposed project has general applicability in RNA viruses other than HCV.


The proposed research focuses on the central unsolved problem of psychiatric epidemiology, the problem of how to measure psychiatric disorders independently of treatment status in the general population. Its major aim is to test the sensitivity and specificity of the newly developed Psychiatric Epidemiology Research Interview (PERI) as a first-stage instrument to screen functional psychiatric disorders among adults in a demographically complex urban population. To this end, two field operations would be conducted. In the first, we would investigate PERI's relationship to other potential screening instruments of a sample of 600 adults from the general population and calibrate PERI on a sample of 500 psychiatric patients with diagnoses of schizophrenia, affective psychosis, neurosis, anti-social personality, and other personality disorders. In the second field operation, we would examine PERI's relationship to two diagnostic instruments, the Present State Examination (PSE) and the Schedule for Affective Disorders and Schizophrenia (SADS). In this second study, interviews would be conducted with a full probability sample of 1400 adults from the general population. The research setting for these investigations would be Washington Heights, a section of Manhattan in New York City that surrounds the Columbia Presbyterian Medical Center. It is a demographically complex urban area containing large percentages of black and Puerto Ricans as well as members of more advantaged ethnic groups. The results should provide a textbook example of the development and testing of a two stage procedure for case identification and classification. The findings should also be important for needs assessment and to the planning and evaluation of mental health service in Washington Heights.


The aim of this research is to understand how the circulatory system is controlled, with the ultimate objective of determining which control factors are most directly involved in the production of essential hypertension. Over the past year we phased out the baboon research. In one aspect of the work we completed the lesions of the HACER (hypothalamic area controlling emotional responses), a region of the hypothalamus. Data analyses showed a dramatic correlation between the resting level of blood pressure and the decrease in that pressure level after hypothalamic lesions. Retrospective analysis of data obtained since 1986 showed that animals with spontaneous hypertension were returned to normotensive levels. In the second aspect, we found that dominant male baboons in social situations showed significantly elevated heart rate, blood pressure, and catecholamines during the presentation of food, whereas subordinate males did not show elevations in these variables.


ABSTRACT Stroke is one of the major medical concerns for United States military veterans. Stroke is devastating as currently no therapy is available to prevent stroke-induced neurological deficit. Prophylactic or therapeutic supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) has recently emerged as a highly promising neuroprotective strategy for stroke. Epidemiological studies have shown an inverse association between n-3 PUFAs intake and risk of ischemic stroke. Supplementation with n-3 PUFAs, especially DHA, effectively reduces the extent of brain damage and neurological deficits in the acute phase of ischemic injury in experimental animals. However, the long-term effect of n-3 PUFAs on brain damage and post- stroke neurological recovery is unknown. We have recently created transgenic (Tg) mice over-expressing the C elegans fat-1 gene encoding an enzyme that converts endogenous n-6 to n-3 PUFAs. Our preliminary studies with fat-1 Tg mice have suggested two-phase (acute and delayed) beneficial effects of n-3 PUFAs on ischemic stroke. The fat-1 Tg mice at either young (3-month-old) or old age (15-month-old) were remarkably resistant to focal ischemic injury compared to their age-matched wild-type (Wt) littermates, showing reduced infarct size and improved neurological functional performance up to 14 days after middle cerebral artery occlusion. We have found that angiogenesis and neurogenesis were robustly enhanced after ischemia in 3-month old fat-1 Tg mice compared to Wt littermates, suggesting that n-3 PUFAs promote post-stroke neurovascular regeneration. Strikingly, we found that post-stroke angiogenesis and neurogenesis were profoundly impaired in aged mice, but almost fully restored by transgenic expression of fat-1. This proposal attempts to further explore the long-term protective effect of n-3 PUFAs on focal cerebral ischemia. The goal is to develop n-3 PUFA supplementation as a novel, clinically feasible prophylactic and/or therapeutic strategy to promote long-term neurological recovery after stroke through stimulating the generation of functional blood vessels and new neurons. The central hypothesis to be tested in the current proposal is that prophylactic or therapeutic n-3 PUFA treatment improves long-term neurological outcomes after stroke by stimulating and enhancing post-ischemia brain repair, including augmented neurogenesis and angiogenesis. The following specific objectives are proposed: Aim 1. Test the hypothesis that n-3 PUFAs reduce long-term neurological deficits as well as brain tissue damage after focal cerebral ischemia in both young adult and aged mice. Two clinically applicable methods: 1) dietary delivery; and 2) augmented endogenous production of n-3 PUFAs via adeno-associated virus (AAV)-directed fat-1 gene expression, will be established to elevate brain levels of n-3 PUFAs. The effect of elevated n-3 PUFAs on ischemic brain injury induced by MCAO will be quantitatively evaluated. The endpoints of assessment include functional outcomes, infarct size, and white matter injury. Aim 2. Test the hypothesis that n-3 PUFA treatment enhances cerebral neurovascular regeneration, including augmented angiogenesis and neurogenesis after cerebral ischemia in both young adult and aged mice. The proposed studies will quantitatively determine the effect of n-3 PUFAs on post-stroke neovascularization and on neural stem cell proliferation, migration, differentiation and neuronal replacement following ischemic stroke. PUBLIC HEALTH RELEVANCE: NARRATIVE Stroke is one of the major medical concerns for United States military veterans. Stroke is devastating as currently no therapy is available to prevent stroke-induced neurological deficit. Prophylactic or therapeutic supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) has recently emerged as a highly promising neuroprotective strategy for stroke. Supplementation with n-3 PUFAs, especially DHA, effectively reduces the extent of brain damage and neurological deficits in the acute phase of ischemic injury in experimental animals. However, the long-term effect of n-3 PUFAs on brain damage and post-stroke neurological recovery is unknown. Our preliminary studies have suggested two-phase (acute and delayed) beneficial effects of n-3 PUFAs on ischemic stroke. We have found evidence that n-3 PUFAs promote post-stroke neurovascular regeneration. Thus, the objective of the current proposal is to develop n-3 PUFA supplementation as a novel, clinically feasible prophylactic and/or therapeutic strategy to promote long-term neurological recovery after stroke. First, we will test the hypothesis that n-3 PUFAs reduce long-term neurological deficits as well as brain tissue damage after focal cerebral ischemia in both young adult and aged mice. Second, we will test the hypothesis that n-3 PUFA treatment enhances cerebral neurovascular regeneration.


DESCRIPTION OF OVERALL CENTER (provided by applicant): The Kansas Interdisciplinary Center for PKD Research (Kansas PKD Center) was established to provide support for highly meritorious new research programs to explore the basic mechanisms causing polycystic kidney disease (PKD); to provide opportunities to bring new investigators into PKD research; and to foster opportunities for more interdisciplinary research in a dynamic, enriching, and interactive setting. The long term goal for the Kansas PKD Center is to find a treatment for PKD that will alleviate the suffering of patients and families affected by the disease. The central theme of the research projects in the Kansas PKD Center is "cell proliferation in polycystic kidney disease." This theme was chosen because Center investigators have been able to identify the cell proliferation defect in PKD as one that involves a cyclic AMP- and calcium-dependent mechanism. The focus will be on using the mouse as a model organism, and on human PKD cyst cells. The Kansas PKD Center is comprised of eight components. The Administrative Core will provide administrative support for Center investigators. The Biomaterials Research Core will establish and maintain a repository of human and animal biological materials for PKD research including primary cultured cells and tissues, and DMA and RNA from ADPKD, ARPKD, and normal human kidney tissues. The Core will also establish and maintain immortalized cell lines from mice carrying unique PKD gene mutations and will provide technical assistance and training in cell culture methodology for all Center investigators. There will be four Biomedical Research Projects: Project 1, "Calcium Regulation of cAMP-Dependent Proliferation," Project 2, "Polycystin-1 Mediated Calcium and cAMP Signaling," Project 3, "Role of Oxidant Stress in Progression of PKD," and Project 4, "Cux-1 and Cell Cycle Regulation in PKD."


Murine-based retroviruses continue to be developed as viral vectors, despite their association with oncogenic transformation and insertional mutagenesis. This is highlighted in the past month with the association of xenotropic murine leukemia virus-related virus (XMRV) with human diseases including chronic fatigue syndrome and prostate cancer. Although comparative retroviral/lentiviral analysis of integration sites has noted differences in the positioning within the host chromosome, the mechanism for tethering the murine-based viruses to the host chromosomes is not understood. Structural analysis of proteins can provide essential insights into function. This proposal develops and extends preliminary structural analysis of the N-terminal domain (NTD) of the Moloney Murine Leukemia Virus Integrase (M-MuLV IN) protein to define the function of this domain within the preintegrative complex. The structural analysis of this domain will be extended to XMRV, which maintains structural homology with MuLV IN. The first focus of the research is structural studies of the IN NTD. The MuLV NTD is distinct from that of HIV and avian retroviruses in that it encodes an additional 50 amino acids N-terminal to the HHCC zinc-binding domain. This adds complexity to the NMR structural analysis of the 105 aa dimer. Through development of a novel growth/labeling system in E. coli utilizing condensed cultures, a preliminary NMR structure of the MuLV IN NTD monomer has been obtained. Experiments modify this system utilizing amino acid auxotroph strains to determine the dimer structure by NMR. Structural comparison with the IN NTD X-ray structure will be made, for which diffraction quality crystals have been obtained. SAXS analysis of the full-length IN ( DNA) will be performed. The IN NTD structures will be critical in defining domain localizations within the synaptic complex. Based on the structural analysis, the predictive function of the MuLV IN NTD will be analyzed. Specifically, the role of the putative winged-helix domain to interact with chromatinized DNA or associated factors will be determined. Mutagenesis studies will identify the role of specific amino acids in the dimer interface as well as interacting with viral and host factors. The ability of known DNA viral tethering domains to functionally complement the mutant IN NTD domains will be examined. Insights into the tethering of IN to chromatin has broad applications into target-site selection, but also to the requirement for mitosis for MuLV-based vectors. The safety of retroviral-based vectors is dependent on the target-site selection during retroviral integration. Through this understanding, mechanism to alter or limit integration can then be rationally designed.


The literature on HIV risk behavior has shown that women who are at risk for HIV sexual disease transmission tend also to be at risk for a host of other issues, including increased substance use and past and current experiences with interpersonal violence. From increasing evidence in the relatively separate literatures on sexual risk behavior, substance use, and interpersonal violence (IPV), there is a pressing need for research to evaluate the intersection, or syndemic (Singer, 1994), of these risk issues with women's HIV sexual risk, so that interventions can attend to needs in domains that are interrelated and can theoretically effect behavioral changes of greater magnitude and greater duration. Bioecological Systems Theory (BST; Bronfenbrenner, 1993) may provide a framework for a greater understanding of the context of multiple risk behaviors in women. This prospective study will examine factors corresponding to the four levels of the BST model as they relate to the interaction of sexual risk behavior, substance use, and violence in women of lower socioeconomic status. Further, we will also examine changes in their reports over a six-month interval. The specific aims are: (1)To recruit 396 inner-city women living in urban housing developments between the ages of 18 and 45 in order to retain 375 participants; (2) To perform multiple assessments on factors pertaining to the levels of the BST model, as they relate to sexual risk, substance use, and trauma/victimization history; these levels and factors include but are not restricted to: a) Individual (Ontogenic) factors, b) Interpersonal (Microsystem) factors (the immediate context of the behavior and relationships with others); c) Community (Exosystem) factors (encompassing community structures, institutions, and the social networks surrounding the individual's life); and d) Socio-cultural (Macrosystem) factors (attitudes, practices, and convictions shared throughout society at large); (3) To perform similar follow-up assessments with these participants six-months and 12-months following the first set of assessments; and (4) To analyze the above variables in relation to occurrence of occurrence and severity of sexual risk behavior, substance use, and interpersonal violence (either perpetrated or as a victim of) using structural equation modeling to examine application of variables related to Bronfenbrenner's BST (1993) model. Findings from this research will provide important new information: (1) leading to greater insight of the dynamics and complexity of sexual risk behavior, substance use and trauma history in at-risk women; (2) needed to design interventions that attend to multiple risk issues in at-risk women; (3) needed to design effective strategies to prevent these issues from occurring; and (4) to evaluate the applicability and validity of BST to the context of women's sexual risk behaviors, substance use, and trauma history. PUBLIC HEALTH RELEVANCE: The primary goals of this project are to examine individual, interpersonal, community and sociocultural factors (Bioecological Systems Theory; Bronfenbrenner, 1995) as they relate to the interaction of sexual risk behavior, substance use, and trauma history/victimization in low income African American women living in urban housing developments in Milwaukee, as measured at three points over a 12-month interval. Findings from this research will provide important new information: (1) leading to greater insight of the dynamics and complexity of sexual risk behavior, substance use and trauma history in low income African American women; (2) needed to design interventions that attend to multiple risk issues in low income African American women; (3) needed to design effective strategies to prevent these issues from occurring; and (4) to evaluate the applicability and validity of Bioecological Systems Theory to the context of low income African American women's sexual risk behaviors, substance use, and trauma history.


Pro-inflammatory mediators, such as IL-1 beta and TNF-alpha, are known to inhibit smooth muscle contractility in part by acting directly on smooth muscle. Our hypothesis is that these mediators inhibit contractility by inducing or suppressing the expression of critical targets in the signaling pathways mediating contraction and relaxation. In preliminary studies on control and TNBS colitis muscle strips, and on freshly dispersed and cultured muscle cells, we have identified six novel cytokine (IL-1 beta)-mediated effects reflecting changes in the expression and/or activity of specific signaling targets. These include: (i) up-regulation of RGS4 and RGS12 expression, which leads to inhibition of initial Ca2+-dependent contraction by Gq and Gi-coupled receptor agonists, respectively; (ii) down-regulation of the endogenous MLC phosphatase inhibitor, CPI-17, which leads to inhibition of sustained RhoA-dependent contraction; (iii) up-regulation of SERCA2 and IP3R-I, which regulate Ca2+ uptake and Ca2+ release, respectively; (iv) up-regulation of PDE4D5 and PDE5A expression, which leads to greater degradation of cAMP and cGMP, respectively; (v) down-regulation of soluble guanylyl cyclase (sGC) expression via a PKG/JNK-dependent post-transcriptional mechanism, which leads to inhibition of cGMP synthesis; and (vi) inactivation of adenylyl cyclase (AC) via iNOS-dependent nitrosylation, which leads to inhibition of cAMP synthesis. Where examined so far, the effects elicited in IL-1 beta-treated colonic muscle were observed also in colonic muscle from TNBS colitis. The specific aims are to: (1) characterize the changes in the expression and activity of signaling targets (RGS4/RGS12, CPI-17, SERCA2 and IP3R-I) mediating initial Ca2+-dependent and sustained Ca2+-independent contraction; (2) characterize the changes in expression and activity of signaling targets (PDE3A, PDE4D5, PDE5A; sGC and AC) that regulate relaxation; and (3) characterize the transcriptional regulation of RGS4/RGS12 and CPI-17 by NF-kappa B and modulatory kinases (p38 MAP kinase, ERK1/2 and the PI 3-kinase/Akt/GSK3beta pathway), and the post-transcriptional regulation of sGC expression via a PKG/JNK/AP-1/HuR pathway. These studies will provide a comprehensive analysis of the mechanisms by which inflammatory cytokines inhibit contractility at cellular level, both in vitro and in an established model of colonic inflammation. [unreadable] [unreadable] [unreadable]