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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 1999-2000

Summary of Research Grants

  • Francesco Botré, PhD
    Development of Enzymatic Inhibition Bioelectrodes for the Direct Determination of Phycotoxins
  • Franco Celada, MD, PhD
    Experiments Using a Computer Model of the Immune System
  • James Jester, PhD
    In Vitro Irritancy Test Using Telomerase Transfected Human Corneal Cells
  • Jeffrey M. Macdonald, PhD
    A Bioartificial Liver Lobule for "Toxicity Testing"
  • Philip S. Magee
    A New Approach to Modeling Allergic Contact Dermatitis
  • German Torres, PhD
    Behavioral and Molecular Effects of Cocaine in Drosophila Melanogaster
  • Donna Volpe, PhD
    Comparison of Semi-Solid and Suspension Assays for the Evaluation of Thrombocytopenia-Inducing Agents
  • Steven E. Wilson
    Development of Reversibly Transformed Human Corneal Epithelial Cells as an Optimal In Vitro Model
  • Fu-shin X. Yu, PhD
    Bovine Corneal Organ Culture: An Ex Vivo Model for Chemical Toxicity Tests

    Development of Enzymatic Inhibition Bioelectrodes for the Direct Determination of Phycotoxins
    Francesco Botré, PhD
    La Sapienza, Rome, Italy
    The goal of this research program is to develop biosensor-based analytical methods for the analysis of phycotoxins (toxins produced by algae which are responsible, when ingested, of various diseases in man) in mussels. These new methods should at least integrate, but hopefully substitute, the commonly applied "global toxicity tests", i.e. highly invasive bioassays on mammalians (in which the mussel extract is either injected or mixed with the food), which are, at present, the only internationally recognized methods for the screening of these toxins in seafood.

    Enzymatic inhibition biosensors (EIBs), being a particular form of response-based analytical devices, would reveal a good compromise between purely "biological" and "physico-chemical" techniques. An EIB represents in fact a sort of "artificial bioindicator", partially combining the flexibility of a biological effect-based test and the selectivity of a physico-chemical assay. In other words, the screening analysis to detect the possible contamination of seafood by phycotoxins would be carried out by "feeding the biosensor" and monitoring the alteration of a specific biochemical parameter, rather than "feeding the mouse" and following the development of generic toxic effects.

    The first part of the project is aimed to develop a lab-scale prototype for the direct determination of algal toxins belonging to the DSP ("diarrhetic shellfish poisoning") group; the same analytical approach will then be extended to the assay of other phycotoxins. A further development would be the realization of disposable biosensors, produced by the technique of screen printing, to be used for timely planned monitoring of risky areas and possibly also for shipboard operations.
    Experiments Using a Computer Model of the Immune System
    Franco Celada, PhD
    Hospital for Joint Diseases, New York City, New York
    Milestones for the Year 1999

    1. Modelling of vaccination strategies. We will evaluate the effectiveness of vaccination over a wide range of immunogenicity gaps between vaccine and wild-type antigen, pertaining to epitope and MHC-peptide differences.
    2. We will study this problem in a new version of the IMMSIM model featuring both the humoral and the cellular immune responses. We will also study vaccines capable of shifting the balance between the two responses.
    3. Performing vaccination when the wild-type antigen is mutable. We will evaluate changes of affinity maturation of the immune response versus epitope and T-peptide mutations in the foreign invader.

    Description of the Project in Lay Language

    Experimentation is the key feature of modern sciences; it consists of reproducing natural phenomena under controlled conditions and measuring its effects against those expected by the scientists' projections, which will thus be confirmed or refuted. In biology, experiments must be done in organisms similar to those under study. In human biology, since ethics forbids experiments in man, in mammals -- from primates to mice -- are the choice organisms that support the advancement of knowledge. It is part of a scientist's mission to try to reduce experimentation in living animals to a minimum. The result, of course, should not be jeopardized; in certain cases it may be substantially enhanced. A giant step in this direction has been the introduction of the culture of tissues and cells, allowing tests and experiments to be conducted in vitro instead of in vivo. In basic immunology, experiments unthinkable in the animal can be realized in the test tube, where cell hybridization has yielded monoclonal antibodies, and such phenomena as presentation of antigen and T-B cell cooperation have been elucidated. These cell cultures are not substitutes for boring/necessary safety tests; they are the new avenues of fundamental research, the kind that twenty years ago required hundreds of mice and rats for each experiment.

    Our activity, a tight collaboration between the P.I. and Dr. Philip Seiden, a physicist and Research Staff Member Emeritus with IBM's T.J. Watson Research Center, has been focused toward a further extrapolation, the experimentation in machina, that is, constructing a model immune system in the computer and conducting experiments with it. We believe that by showing that our model can be applied to real research problems we will foster its use by more and more immunologists (and, eventually, biologists), thereby catalyzing a cultural transition among bench scientists.
    In Vitro Irritancy Test Using Telomerase Transfected Human Corneal Cells
    James V. Jester, PhD
    The University of Texas Southwestern, Dallas, Texas
    Eye irritation testing is recognized as important in determining the safety of consumer products where manufacture or use may lead to accidental exposure and damage to the eye. Irritancy testing as currently performed, however, requires the use of live animals for which there are no recognized alternative replacement tests. The long-range goal of our work is to first develop and then validate an alternative replacement test using a human tissue culture model that reconstructs the anterior, exposed portion of the human eye. A critical first step will be the generation of extended life-span human cells from the anterior part of the eye or cornea that show structural and functional characteristics similar if not identical to normal cells. To establish these cells we will insert into corneal cells a gene encoding the enzyme telomerase that controls the number of times a cell divides, greatly extending if not indefinitely the life the cell while maintaining the normal cellular characteristics unlike cells that are immortalized using various cancer genes. During
    Year One we will clone human corneal epithelial, keratocyte and endothelial extended life-span cell lines following insertion of telomerase and establish the cellular characteristics of each clone. In
    Year Two we will establish and characterize various 3-dimensional corneal tissue constructs. In
    Year Three, we will evaluate the response of 3-dimensional corneal tissue constructs to irritating materials be measuring the area and depth of injury following exposure to eye irritants producing a range of irritancy from slight to severe.
    A Bioartificial Liver Lobule for "Toxicity Testing"
    Jeffrey M. Macdonald, PhD
    University of North Carolina, Chapel Hill, North Carolina
    We are tissue engineering liver using the body's natural tissue-replacing cells, progenitors, for toxicity testing. Tissue engineering is a new field combining cell biology and engineering. The liver is the organ primarily responsible for metabolizing toxicants and is the main port of entry for toxicants and especially drugs. We combine concepts in stem cell biology and cell signal pathways (hormones, paracrine, and autocrine factors), with a bioengineering analytical tool [magnetic resonance spectroscopy (MRS)] and a bioengineered device (a hollow-fiber bioreactor). Just x-rays used in computer tomography (CT), MRI and MRS are non-ionizing and do not cause DNA mutations.

    We isolate progenitors and culture them in a coaxial hollow-fiber bioreactor perfused with media and extracellular matrix containing the molecules that elicit signal pathways common to progenitors in vivo. A coaxial hollow-fiber bioreactor is simply two synthetic semi-permeable hollow tubes, a smaller tube inserted into a larger tube. The cells grow between the two fibers and media flows in compartments that 'sandwich' the cells. The coaxial fibers mimic the very small physiological unit of the liver, the liver lobule. Therefore, we call this a bioartificial liver lobule. We feed the liver glucose and glycine labeled with stable 13C rather than radioactive 14C. These stable-labeled nutrients and their metabolites are monitored, and the effect of a toxicant on primary metabolism is determined by taking biochemical pictures using MRS at continuous timepoints during toxicant exposure.

    The goal of this project is to establish the bioartificial liver lobule for toxicity testing by determining the effect of an environmental toxicant, dibromoethane (a carcinogenic fumigant), on the biological system (i.e., toxicodynamics). The in vivo toxicodynamics will be correlated to secreted stable-labeled nutrient metabolites so that a rapid, high-throughput toxicodynamic MRS toxicity testing assay can be developed.
    A New Approach to Modeling Allergic Contact Dermatitis
    Philip S. Magee, PhD
    BIOSAR Research Project, Vallejo, California
    During the past two years, a broad spectrum of electrophilic haptens that cause sensitization and subsequent contact dermatitis have been modeled for reactivity with protein end-groups. This key reaction with the cell surface identity molecules of several allergen presenting cells, especially the epidermal Langerhans cells is the initiating step in sensitization. The computational database now in hand allows the estimation of reactivity for many new haptens and the method permits the direct evaluation of specific new haptens where this is not possible.

    At the end of the second grant year, a data set of 25 haptens evaluated by the guinea pig maximization test (GPMT) at 4 levels (1 = non or very weak; 2 = weak; 3 = moderate; 4 = strong or severe) was compared with computed values of the same haptens. The agreement was excellent and fully consistent with the GPMT. Although more work needs to be done to expand this comparison between live animal and computational results, it is clear that the method has commercial value in providing companies with rapid and low-cost information to enable the setting of priorities.

    During our third year study, many more haptens will be studied will be studied in direct comparison with lab animal results to fully validate the method. In addition, the large amount of computational data we now have will be used to construct a convenient scale of reactivities that will allow the rapid classification of new haptens and provide descriptors for future structure-activity studies.
    Behavioral and Molecular Effects of Cocaine in Drosophila Melanogaster
    German Torres, PhD
    State University of New York, Buffalo, New York
    Final Report
    Understanding the behavioral, molecular and genetic mechanisms involved in the actions of cocaine remains a challenging issue in neuroscience. Many experimental approaches have been used to elucidate which biological processes contribute to the development of a drug-dependent state. Rodent and non-human primate models have been most thoroughly and successfully applied in the investigation of drug-induced behaviors. Yet, questions fundamental to understanding cocaine addiction remain. For instance, what are the molecular and genetic mechanisms specifying cocaine-related behaviors? As with other issues in neuroscience, an experimentally tractable animal is important to answer such questions. Although studies with rodents are beginning to shed some light on the above issue, these animals with their complex and intricate nervous systems, are not always suitable for studying basic questions related to genes and behavior. In addition, even when genetic manipulations are possible, they are not easy. Therefore, one approach to understand the effects of cocaine on behavior is to study "simpler" systems such as Drosophila. This organism, which has extensive biochemical information, is amenable to studies of genetic processes that are common to all animals. Also, genetic manipulations in Drosophila are relatively easy, and therefore information about highly conserved cellular processes derived from fly studies can be readily extrapolated to vertebrates, including humans. We propose to take advantage of these outstanding experimental features of Drosophila to determine the use of this non-mammalian organism as a model to study the behavioral and molecular effects of cocaine.
    Comparison of Semi-Solid and Suspension Assays for the Evaluation of Thrombocytopenia-Inducing Agents
    Donna A. Volpe, PhD
    Food and Drug Administration, Laurel, Maryland
    In the treatment of cancer with drugs, a frequent harmful effect is the decrease in the number of blood cells. One cell type that can be reduced is the platelet, which is involved in blood clot formation. A severe decrease in the number of circulating platelets, called thrombocytopenia, can result in life-threatening hemorrhage, or uncontrolled bleeding. As strategies to alleviate thrombocytopenia improve, it would be beneficial to understand a new drug's potential to reduce platelet counts in patients. Megakaryocytes in the marrow give rise to functional platelets through the processes of proliferation and maturation. Hematopoietic clonal assays are utilized during drug development as a method of predicting bone marrow toxicity. Megakaryocyte progenitors are routinely cultured in semi-solid assays that are tedious, time consuming and subjective. A more effective alternative is a liquid suspension assay that evaluates drug effects by measuring the megakaryocyte population by flow cytometry. The specific aims of the study are to:

    • evaluate the effects of four drugs on megakaryocyte colony formation in a clonal assay
    • develop a liquid suspension assay to assess toxic drug effects on megakaryocyte maturation
    • compare results of the two assays to preclinical and clinical endpoints to determine if they are qualitatively and/or quantitatively predictive of thrombocytopenia.

    These in vitro methods use human cells to predict a frequent clinical dose-limiting toxicity during cancer therapy. Such a refinement can lead to the reduction of animal studies in preclinical drug development and a better predictive model eliminating the need for interspecies extrapolations.
    Development of Reversibly Transformed Human Corneal Epithelial Cells as an Optimal In Vitro Model
    Steven E. Wilson, M.S., MD
    University of Washington, Seattle, Washington
    Appropriate in vitro models could markedly reduce animal usage in testing for irritation or injury during safety evaluation of consumer products. A promising new in vitro test has recently been developed, the transepithelial permeability to fluorescein (TEP) test. In this model, the barrier function to fluorescein of a multi-layered culture of transformed human corneal epithelial cells is analyzed to model corneal epithelial injury in the living eye. The available transformed human corneal epithelial cells, however, continuously express the transforming genes that extend the life span of the cells so that sufficient cells are produced for study. The genes used (such as SV40 large T antigen) extend the life span of cells by blocking important regulators of cell proliferation, apoptosis, and differentiation. It is unlikely that these cell lines could provide an optimal model for a critical differentiated function of the corneal epithelium such as barrier function since differentiation is affected. The aim of this study is to develop methods to genetically engineer human corneal epithelial with DNA sequences that allow the transforming genes such as SV40 large T antigen to be turned on to grow the cells and off to allow the cells to normally differentiate for testing. Optimal cells will be studied by evaluating normal functions of differentiated corneal epithelial cells such as differentiation markers and normal responses to growth factors that stimulate proliferation. Finally, we will test the utility of the engineered cell lines in the TEP test to ascertain whether they provide a good model for pharmacological testing.
    Corneal Organ Culture: An Ex Vivo Model for Chemical Toxicity Tests
    Fu-Shin Yu, PhD
    The Schepens Eye Research Institute, Boston, Massachusetts
    For five decades, the Draize test has remained the accepted method for evaluating the potential of test material to cause eye irritation or injury. Criticisms of this method center around the inhumane treatment of animals and the irreproducibility of the subjective scoring procedure. There is a great demand for a mechanistic based in vitro testing system that will minimize the use of animals in chemical toxicity tests. Recently, we have adapted a simple, long-term organ culture method as an ex vivo model for chemical toxicity tests. Here, toxicity can be assessed by applying test chemicals to the surface of the cultured corneas. Since this system more closely resembles an in vivo testing system than cell culture, it should serve as an appropriate model for chemical safety tests. The corneas we use are prepared from the bovine (or porcine) eyes, economical and resourceful by-products of meat industry, no live animals are euthanized for testing. Using this system, we will examine the corneal response to chemicals in several paradigms.

    1. The activation of AP-1 and NF-kB, two well-known stress-responsive proteins controlling gene expression, in response to chemical stimuli will be used as endpoints for ocular irritancy.
    2. Disruption of corneal barrier function by test chemicals will assess by opacity, epithelial fluorescein retention, and leakage of metabolic enzymes.
    3. Recovery of epithelial function after exposure of the corneal organ culture to tested chemicals will be monitored and used for assigning final ratings of chemicals.

    These ex vivo evaluations can be used for accurate prediction of irritation potential in vivo and offer reliable alternative to the use of live animals.