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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 1996-1997

Summary of Research Grants

Cellular-based Assays to Detect Toxic Agents and Elucidate the Underlying Genetic and Biochemical Mechanisms of Toxicity
Michael Scott Dubow, PhD
McGill University, Montreal, Quebec
The basis of this research is the principle that cells will augment or repress the expression of specific genes, upon exposure to bioavailable doses of a toxic agent, in order to re-orient cell physiology to cope with the stress. To identify and quantitate the activity of these genes, the investigators have prepared a "library" of luciferase gene fusions in the bacterium E. coli and have identified a number of clones whose luminescence changes upon cellular exposure to a variety of compounds. These clones are being validated for use as luminescent "biosensors" to detect toxic agents. Dubow and colleagues will now focus on bacterial clones which are induced by the anti-oxidants selenium and DMSO, and the pesticides bromacil, chlordane, dinoseb, and the plastic stabilizer tributyltin. These and related studies will be used to develop molecular probes (and ultimately antibodies) for cellular-based assays for toxicity and safety evaluation.
Epidermal Cell Expression of Co-Stimulatory Activity in the Induction of Allergic Contact Dermatitis
Anthony Gaspari, MD
University of Rochester School of Medicine, Rochester, New York
Gaspari and colleagues propose to develop laboratory techniques to identify allergens based on genetically modified cultured human cells. The methodology is based upon the hypothesis that the regulation of epidermal antigen presentation is critical for the primary inductive phase of allergic contact dermatitis as well as the secondary (recall) phase of the allergic reaction. The investigators are building on previous observations which revealed that the expression of costimulatory molecules by epidermal keratinocytes is an important event in the process of allergic contact dermatitis. In the current studies, they plan to study the gene for the costimulator B7-1 by cloning the promoter elements into a reporter gene and testing them for cell-specific activity and response to cytokines that are known inducers of B7-1 expression. The B7 promoter test will serve as a model to study other costimulatory molecules and develop a molecular screen for allergens, based on multiple endpoints.
Fluorescent Probing of Oxidative Stress and Antioxidant Efficacy in a Cell Culture Model
Valerian E. Kagan, PhD
University of Pittsburgh, Pittsburgh, Pennsylvania
Extensive evidence implicates oxygen and lipid free radicals in a host of degenerative diseases and acute conditions. Lipid peroxidation is considered one of the major contributors to oxidative injury; inhibition of lipid peroxidation is extremely important in maintaining good health. The model being developed by the investigator will probe oxidative stress induced by chemicals and test antioxidants in biochemical models with in vivo-like metabolic pathways of activation and/or repair. Specific aims for the first year include optimization of the cell culture model for fluorescent probing of oxidative stress, validation of the model as a predictor of oxidative stress of chemicals, and validation of the model as a predictor of protective effects of antioxidant compounds.
R3-Organotypic (Raft) Epithelial Culture System for Contact Dermatitis Testing
Craig Meyers, PhD
Pennsylvania State University, Hershey, Pennsylvania
New information from in vivo and in vitro studies provides evidence that keratinocytes can play a pivotal role in allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD). Myers and colleagues have developed an in vitro keratinocyte culture system to assess the ability of commercial and therapeutic products to induce ACD and ICD. They propose to evaluate the human epithelial raft culture system for its efficacy by defining the expression of immunoregulatory molecules in human epithelial raft culture tissues following treatment with a contact dermatitis-inducing chemical early in its development. Gene expression will be measured and a dose-response determined. Lack of a fully developed cornified layer will allow the toxicant more direct access to the underlying epithelial cell layers, providing a more sensitive measurement of the compound's contact dermatitis-inducing capacity.
In Vitro Embryonic Gene Expression as Markers for Developmental Toxicity
Ellen K. Silbergeld, PhD
University of Maryland at Baltimore, Baltimore, Maryland
Methods to detect effects of chemical exposure on early development remain relatively limited. Silbergeld and colleagues therefore propose to investigate measurement of early embryonic gene expression as a system to detect effects of toxicants on development, using the murine two cell embryo. The two cell embryo represents the earliest stage at the gene expression unique to the new organism can be detected. Hybridization and immunologic techniques are used to identify specific proteins. The project will compare effects of several model toxicants under in vitro and in vivo conditions, in order to validate the two cell embryo model. The model toxicants include vinblastine, lead, and diethylstilbestrol, ethylene glycol and isotretanoin.
In Vitro Assay for Hapten-Specific Priming of Human T Lymphocytes
Wayne J. Streilein, MD
Schepens Eye Research Institute, Boston, Massachusetts
Streilein and colleagues have previously determined that it is possible to study in vitro the earliest inductive events in contact hypersensitivity using either murine skin or blood as a cellular source. Recent work has revealed that murine Langerhans cells that have been cultured for three days and then hapten-derivatized, display the capacity to activate unprimed hapten-specific cells. The T cells activated in this manner cause contact hypersensitivity when injected in vivo into hapten-derivatized skin. Dendritic cells of the spleen and the blood also acquire the capacity to activate hapten-specific T cells in vitro. The investigators have demonstrated that peripheral blood mononuclear cells can reveal the "fresh" and "cultured" phenotypes, and that primed T cells are preferentially activated by DNCB-derivatized cells. These results, together with evidence suggesting that naive T cells exposed in vitro to DNCB-derivatized peripheral blood mononuclear cells can be activated, lead them to conclude that human epidermal and blood cells can similarly be manipulated in vitro to activate naive T cells that mediate contact hypersensitivity.