Frank M. Graziano - US grants

The guinea pig is an excellent model for studying pulmonary immediate hypersensitivity reactions. In this species, IgG1 is the major anaphylactic antibody, but IgE antibody can be produced under certain circumstances. For some time we have been studying the biologic activity of guinea pig IgE antibody as it relates to IgG1 reactivity and as a model of human IgE antibody activity in immediate hypersensitivity reactions. Therefore, the long range goal of our research has been and continues to be an understanding of the differences and relationships of IgG1 and IgE antibodies as they relate to pulmonary immediate hypersensitivity reactions in the guinea pig. The specific aims of our proposal are based upon three fundamentally important findings in our studies. Both IgG1 and IgE cause pulmonary smooth muscle contraction, but through separate receptors; differences in concentrations of mediators released when these receptors are activated; and evidence to suggest heterogenity of guinea pig lung mast cells. The questions we have formulated based upon these findings center around the issues of another mediator involved in the lung contractile responses, and whether different populations of mast cells (if they do exist) have IgG1 or IgE receptors on their surface and this is the basis for our tissue findings. Based upon techniques we have established in our laboratory, we will approach these questions in two ways. First, we will examine antibody sensitized pulmonary tissue utilizing the superfusion technique. In this procedure, both the contractile response and mediators released can be measured after antigen stimulation of sensitized tissue. In these experiments we will specifically be evaluating for other mediators (either released by mast cells or other pulmonary cells) involved in the contractile response. Second, we will isolate pulmonary mast cells from lung tissue, examine these cell suspensions for relationships and differences in mediator release mediated by IgG1 and IgE antibody, probe the isolated mast cell surface IgG1 and IgE antibody receptors, and examine these cells for functional and phenotypic heterogenity. Allergic disorders of the lung are important diseases occurring in many individuals. Our ability to examine the questions we have asked in both lung tissue and isolated lung cells gives us an important opportunity to investigate basic mechanisms of pulmonary immediate hypersensitivity reactions that could aid in our understanding of similar events in human lung.

The guinea pig is an excellent model in which to study mechanisms of immediate and delayed hypersensitivity. In our laboratory, we have preliminary evidence suggestive that we can make potentially important observations as to the role of homocytotropic antibody, mast cells, and basophils in guinea pig hypersensitivity states. Our data to date has demonstrated that both IgG1 and IgE can mediate polmonary contraction in the guinea pig and they seem to do so through separate tissue receptors. Furthermore, we have been able to isolate lung tissue mast cells and have the capability to study the biochemistry of these antibodies directly on this cell which has been shown to play an important role in immediate reactions. We also have the capability of isolating pure populations of blood basophilis (85-100% purity). Based upon these observations, we propose to study in detail the distinctive nature of the IgG1 and IgE pulmonary receptors. We plan to do this by suspending pulmonary tissue obtained from IgG1 or IgE sensitized animals in a superfusion chamber and simultaneously measure contraction and mediators released upon stimulation with specific antigen. We also would like to extend these tissue studies with homocytotropic antibody to the isolated pulmonary mast cell by studying this cell for IgG1 and IgE receptors and examining mediator release from these isolated cells when sensitized with IgG1 or IgE antibodies. A second separate but yet related part of our proposal is to examine the inflammatory provoking potential in guinea pig skin of isolated granules obtained from purified blood baseophils (90-100% purity). With this we hope to define some of the mechanisms of basophil involvement in delayed cutaneous reactions containing basophils. The similarity of the guinea pig to humans as far as primary mediators released by mast cells and basophils makes these studies of potential importance in understanding similar events in immediate and delayed hypersensitivity states in humans.

The primary aim of the Clinical Investigator Preparatory Program is to develop independent clinical investigators who are capable of leading clinical research programs that will attract extramural funding, advance the vision and mission of the University of Wisconsin-Madison, and foster their careers as academicians and scientists. A two-year curriculum that assembles existing mentors, didactic courses, seminars, workshops, research expertise, and institutional resources, has been designed with specific aims to provide graduate students, fellows and junior faculty knowledge and skills to: select and apply appropriate study design and statistics to one's own research; conduct clinical research according to professional and legal ethics; lead and manage a productive career in clinical research; acquire and maintain expertise in one's own research domain; teach and communicate scientific knowledge through verbal presentations; and write well-organized, logical journal publications, research proposals and grant applications. Several tracking mechanisms and evaluation methods will be used to determine the success of this program toward developing the careers of clinical investigators. They include: 1) a pretest-posttest questionnaire, 2) portfolio assessment, 3) progress reports and review by an advisory committee, 4) external program review, 5) post-program follow-up of multiple outcomes, and 6) participant peer review.

[unreadable] DESCRIPTION (Provided by the applicant): [unreadable] UW-Madison has the largest research commitment of any school or college on campus, receiving more than $130 million in research support in fiscal 2000. Approximately 580 faculty members, working in 25 departments and 19 centers and institutes, have active research programs covering virtually every aspect of basic or clinical biomedical research. The purpose of the Clinical Research Scholar (CRS) Program is to provide financial support for junior faculty who wish to participate in the UW-Madison Clinical Investigator Preparatory Program (K30 Program) and assure them protected time to conduct mentored clinical research. Specific objectives of the CRS program are to 1) provide an individualized didactic training program in biostatistics, study design, bioethics, scientific writing and presentation, leadership, teaching, responsible conduct of research and discipline-specific or interdisciplinary research; 2) provide salary support to protect at least 75% of a trainee's time for mentored research and training; 3) award trainees a Professional Certificate or Master of Science Degree to credential their preparation to conduct clinical research; 4) support and encourage the career development of women and minorities in clinical research; 5) maintain a comprehensive evaluation plan that assesses the trainees' accomplishments and the CRS Program's success. The applicants have highlighted the accomplishments of faculty mentors from a variety of disciplines including medicine, pediatrics, cardiology, oncology, women's health, geriatrics, psychiatry, psychology and surgery who can provide multidisciplinary research experiences that will foster the career development of clinical investigators. Furthermore, the UW-Madison CRS Program plan emphasizes recruitment of underrepresented minorities and includes an extensive evaluation plan that formatively and summatively assesses the trainees, mentors, instructors, curriculum and overall program plan. After completing the CRS Program, UW-Madison trainees will receive a Capstone or Graduate Certificate or a Master of Science degree that should have prepared them to be successful and independent clinical investigators. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our goal is to investigate expression of Toll-like receptors (TLR) on human conjunctival epithelium and determine the potential contributions of TLR activation by microbial ligands to ocular allergic inflammation. Studies examining conjunctival epithelial cells as immunomodulators have been focused on the effects of pro-inflammatory cytokines, while the direct effects of microbial products have not been examined. Preliminary studies suggest that conjunctival epithelial cells express a pattern recognition receptor, TLR2, and respond to Staphylococcus aureus peptidoglycan (SA-PGN) with mediator release and surface receptor upregulation. It is likely that simultaneous activation of conjunctival epithelial cells via TLRs and mast cell mediators is a frequent occurrence, which may contribute to the development of chronic ocular allergic disease. The first specific aim will focus on determining the repertoire of TLRs (focusing on TLR1,2,4,6,9 and accessory receptor, CD14) expressed by conjunctival epithelial cells and whether the TLRs expressed are inducible by cytokines and/or TLR ligands (SA-PGN and yeast zymosan for TLRs 1,2, and 6; Pseudomonas aeruginosa LPS for TLR4, CpG DNA for TLR9). This will include in vivo examination of cells obtained from the ocular surface of human subjects (normal vs various disease states) via impression cytology. The second specific aim will investigate activation of conjunctival epithelial cells with TLR ligands, specific binding of microbial products to their respective TLRs and signaling (accessory proteins MyD88, MD2, and TIRAP). Activation will be defined as upregulation of ICAM-1 and/or HLA-DR expression and/or enhanced release of TNFalpha, IL-6, IL-8, and/or RANTES. The third specific aim will examine the effect of simultaneous and sequential activation of conjunctival epithelial cells with TLR ligands and supernates from IgE-activated purified conjunctival mast cells. The results of combined activation will be compared to activation with either TLR ligands or IgE-activated mast cell supernates alone. Conjunctival epithelial and mast cells will be obtained from cadaveric conjunctival tissues. Techniques to evaluate protein expression will include flow cytometry, Western blot, Immuno-PCR (in impression cytology cells), and ELISA. Techniques to evaluate mRNA expression will include Northern blot and RT-PCR (in impression cytology cells). Techniques to examine binding specificity and signaling complexes will include blocking antibodies, co-immunoprecipitation and gel shift assays.