Skip Navigation
Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 2000-2001

Summary of Research Grants

Sensory Irritant Screening Using a Stable Cell Line Expressing the Vanilloid Receptor
Thomas K. Baumann, PhD
Oregon Health Sciences
When animals (including humans) are exposed to irritant chemicals in the air, they change the pattern of breathing in order to protect the lungs. Decrease in breathing rate in mice has for many years been used as a behavioral test for measuring sensory irritancy. The test forms the basis for setting a safe limit for human exposure to airborne chemicals. While very simple, the inhalation test requires exposing live animals to what may be unpleasant or harmful chemicals. We wish to develop an alternative method for sensory irritant screening. Studies of sensory irritation in mice showed that sensory irritation is a process which is mediated by a receptor protein. Experimental evidence suggests that the receptor is very similar to a protein which is present in sensory nerve cells and is activated by capsaicin (the irritant chemical which makes red pepper taste hot). The DNA sequence which codes for the capsaicin receptor (called the vanilloid receptor) in neurons has recently been deciphered and cloned. The goal of this research proposal is to insert the DNA and express the vanilloid receptor protein in a non-neuronal cell line. In contrast to neurons, non-neuronal cells have the ability to multiply. Once stable expression of the vanilloid receptor is achieved, and the new sensory irritant assay validated, then the cells could be grown for many generations and used to test chemicals for sensory-irritant potential. This assay would avoid exposing animals to chemicals or sacrificing animals to make nerve cell cultures for sensory irritant screening.
Experiments Using a Computer Model of the Immune System
Franco Celada, MD, PhD
Hospital for Joint Diseases, New York City, New York

  1. Milestones for the Year 2000
    1. Modelling of vaccination strategies. We will consider and evaluate the effect on vaccine effectiveness of a wide range of immunogenicity gaps between vaccine and wild-type antigen, pertaining to epitope and MHC-peptide differences. We will also study the effect of vaccination on humoral versus cellular parameters.
    2. Performing vaccination when the wild-type antigen is mutable. Evaluation of parameters: 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 science; 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, 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 -- such as the present project, a systematic study of vaccination -- we will foster its use by more and more immunologists (and, eventually, biologists), thereby catalyzing a cultural transition among bench scientists.
Genetically Modified Neurons Derived from Embryonic Stem Cells: A Versatile Cell Culture System for Research and Testing
Brian G. Condie, PhD
IMMAG - Developmental Biology Program Medical College of Georgia, Augusta, Georgia
Mouse embryonic stem cells (ES cells) are cell lines that retain the characteristics of early embryonic cells and have not yet become committed to forming specific tissues or cell types (i.e. nerves, blood cells). Through simple manipulations of the cell culture conditions mouse ES cells can be made to develop into many mature cell types including nerve cells. The ability of these cells to undergo development in culture makes them an important alternative to the isolation of embryonic or mature cells from animals. The experiments in my lab that are supported by the CAAT grant have two objectives. One goal is to develop simple methods to transfer specific genes into young nerve cells that are developing from mouse ES cells in culture. These methods would pave the way for the use of this cell culture system in a wide range of experiments that depend on the genetic manipulation of neural cells. The second goal is to develop a simple and general strategy to isolate pure nerve cell cultures from developing mouse ES cells. This technology would ensure that ES derived neuron cultures are uniform and reproducible, reducing variability and improving the reliability of tests that use these cell cultures.
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. We are currently inserting 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 the next year we will continue to clone human corneal epithelial, keratinocyte and endothelial extended life-span cell lines. We will also begin to establish and characterize various 3-dimensional corneal tissue constructs using these cells and determine how closely these constructs resemble normal corneal tissue. In the last year of this project we intend to test the response of these constructs to known ocular irritants.
Screening and Identification of Genes for Drugs in Drosophila Melanogaster
German Torres, PhD
State University of New York, Buffalo, New York
Human disease gene identification is increasing at an exceptional rate. Many genes are now thought to contribute to a variety of biochemical and behavioral deficits. Thus, characterization of a locus that controls sensitivity, for instance, to clinically relevant concentrations of cocaine and ethanol should take us from DNA sequence to biological and behavioral function. This progression, which will only be obtainable in model organisms, should significantly enhance our ability to unravel the normal function of genes or the mechanisms by which disruptions in these (same) genes result in specific disease pathologies. Recent studies give strong indications that Drosophila will be a key player for studying gene mechanisms underlying human disorders. Briefly, Drosophila melanogaster contains approximately 12,000 genes with a genome size of 165 Mb. Of these, the majority of DNA sequences have some similarity or motif to mammalian sequences. Thus, many fly genes have significant human homologs. Among the best matches are the human genes that code for DA, 5-HT, GABA, cAMP and other protein molecules mediating the actions of cocaine and ethanol.

One of the classical advantages of using fruit flies has been the extreme ease of making and identifying mutants. Further, other genetic manipulations in flies (e.g., P-element mediated germ-line transformations) are easy and cheap and thus information about human diseases learned from studying their fly homologs comes at an excellent price. In addition, the entire Drosophila genome has been sequenced; this elevates Drosophila further as a major eukaryotic model organism for studying functions of genes identified in human neuropathologies. Against these outstanding experimental features, we propose to use Drosophila as a model system for:

  1. screening and identifying gene-encoding proteins whose expression in brain are induced by cocaine and ethanol; and
  2. studying heritable genetic variance that underlies individual differences in sensitivity to cocaine and ethanol.

Rhythmically Stretching Dynamic Cell Culture: An in vitro Model to Study Particle Cell Injury
Akira, Tsuda, PhD
Harvard School of Public Health, Boston, Massachusetts
As the lung represents an enormously large surface area exposed to the environment, it is not surprising that exposure to airborne particulates is strongly associated with lung injury. However, the molecular and cellular mechanisms responsible for the particle-induced pathogenesis are not fully understood. We hypothesize that the mechanical contact between particles and the epithelial cells plays an important role in lung injury, and that the physical stimuli exerted on cells by particles may be greatly enhanced by the rhythmical motion of the alveolar epithelial cells associated with breathing--a factor entirely ignored in current in vitro studies with static cell culture systems, and may trigger subsequent cell response. To test this idea, as an alternative approach to inhalation or instillation animal experimentation, we propose to employ an in vitro dynamic cell culture model to study particle-induced cell injury. We plan to rhythmically stretch monolayers of the human aveolar epithelial cell line A549 employing a cell stretcher device, with physiologically relevant tidal breathing conditions, and simultaneously expose the cells to various particulates, such as (fibrous and non-fibrous) asbestos, glass, polyestyrene, and latex particles. The cells' responses to particle exposure with or without rhythmic stretching will be compared by measuring important "readouts" relevant to pathogenesis, such as proinflammatory cytokine production (e.g., IL-8). The role of receptor-mediated interaction between protein-coated particles and rhythmically moving cell surface receptors will also be assessed. The results of this study would help us to understand how physical insults exerted on the expanding and contracting alveolar epithelial cell surface lead to lung injury.
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.
Evaluation of CYP3A Induction with Engineered Cell Lines
Bingfang Yan, PhD
University of Rhode Island, Kingston, Rhode Island
Nearly all drugs and other foreign compounds absorbed by the body undergo metabolism, in which extensive chemical modifications usually take place as a result of the involvement of phase I and phase II biotransformation. Cytochrome P450 (CYP) enzymes are a family of heme-containing proteins and rank first among the phase I biotransformation enzymes in terms of catalytic versatility and the number of foreign compounds they metabolize. Many foreign compounds are shown to increase the expression levels of CYP enzymes thus alter the overall drug-metabolizing capacity. Such an alteration may accelerate the inactivation of drugs thus diminish therapeutical effectiveness or increase the formation of toxic metabolites thus cause tissue damage. The Food and Drug Administration requests the new drugs be tested as CYP inducers, which is usually conducted with human hepatocytes and animals. The availability of human tissues and the use of a large number of animals, however, are presenting major obstacles in rapidly and accurately screening daily increasing number of dug candidates. The proposed project is designed to develop and optimize an in vitro screening system for CYP3A induction. The CYP3A enzymes are the most abundant of CYP enzymes and involve the metabolism of two thirds of drugs and other foreign compounds. In this system, cells will be engineered to express appropriate proteins to support CYP3A induction, and the inductive effects will be monitored by determining a reporter activity, thus increasing the detection sensitivity. This system will significantly reduce the number of animal use and ease the supply of human tissues.
Bovine 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 on the inhumaned 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 developed an ex vivo model for chemical toxicity tests using a simple, long-term corneal organ culture method and tested several chemicals and consumer products. Our results showed that this system that closely resembles an in vivo testing, is an appropriate model for chemical safety tests. The corneas we use are prepared from the bovine eyes, economical and resourceful by-products of meat industry; no live animals are euthanized for testing. To perfect his system, our aims are:

  • To further elucidate how epithelial cells in organ culture respond to diverse classes of chemicals by assessing alteration of AP-1 and NF-B DNA-binding activity and normalize the data against known tonicities of test chemicals.
  • To investigate the changes in corneal function including impermeability and transparency and to correlate these with in vivo toxicity of known eye irritants.
  • To evaluate if recovery of epithelial functions after exposure of the corneal organ culture to a test chemical correlates with its ocular irritancy.

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