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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 2002-2003

Summary of Research Grants

Biosensors (EIB) for the Determination of Phycotoxins
Francesco Botré, PhD
Dipartimento di Controllo e Gestione Delle Merci del Loro Impatto Sull'Ambiente, Universittá Degli Studi Di Roma, Rome, Italy
The overall 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 last part of the project is aimed to develop disposable, monoenzymatic biosensors produced by the technique of "screen printing" for the direct determination of algal toxins belonging to the DSP ("diarrhetic shellfish poisoning") group. These novel analytical devices should be used for timely planned monitoring of risky areas and possibly also for shipboard operations.
Development and Use of an Immortalized Human Goblet Cell Line for the Use in Ocular Toxicity Testing
Darlene Dartt, PhD
The Schepens Eye Research Institute, Harvard University, Boston, Massachusetts
The conjunctival epithelium along with the cornea make up the ocular surface. The conjunctiva has long been used as a component of the Draize test. Mucin secretion from conjunctival tissue has been one of the endpoints measured by this test. The Draize test has been the accepted method by which a variety of agents have been evaluated with respect to their ability to cause irritation and injury to the ocular surface. Unfortunately the inhumane treatment of animals during this test procedure has often been overlooked. To replace the Draize test, many laboratories have used a variety of cell lines and primary cultures of corneal epithelial cells and conjunctival squamous cells. No one, however, has been able to culture conjunctival goblet cells. The long term goal of our proposal is to develop a cell line of human conjunctival goblet cells with an extended life span which will be able to be used for ocular irritancy testing. We hope to accomplish this goal by fulfilling the following specific aims:

  1. Develop a cell line witn an extended life span by telomerase transfection of primary cultures of human goblet cells;
  2. Characterize the new cell line by using established morphological, histochernical, immunocytochemical and biochemical markers known to be associated with goblet cells; and
  3. Determine the effects of neurotransmitters and growth factors on mucin and protein secretion from this cell line and the effect of ocular irritants on these secretions.

The development of a human goblet cell line with an expanded lifespan that secrete mucin and other antibacterial proteins is a useful model that could be potentially use in place of the Draize test.
Development of an In Vitro System to Test the Effects and Toxicity of Anti-Inflammatory and Anti-Fibrotic Drugs in the Human Liver
Geny Groothuis, PhD
University of Groningen, Groningen, The Netherlands
Liver cirrhosis is a fatal liver disease and belongs to the top ten causes of death in the Western World. Cirrhosis is characterized by, among others, excessive deposition of connective tissue (fibrosis) leading to loss of liver function. To date, no adequate drug therapy is available due to the fact that the potential drugs show either lack of effect or serious side effects. The only effective treatment is liver transplantation.

In addition the pathogenesis of the disease process, being the onset and progression of fibrosis and its reversibility, are only partially understood. Research concerning the pathogenesis and the development of drugs is usually performed in animal experiments, which is accompanied by relatively high discomfort for the animals.

In this project an in vitro system using liver slices will be developed in order to be able to investigate the pathogenesis of the disease without treatment of the animals, thereby reducing the number and the discomfort of the animals used. Moreover, the system will allow us to test drugs, developed in our institute for efficacy and toxicity in vitro and in vivo, as is currently the case. In addition, such an in vitro system can be applied to human liver tissue, enabling us to study human specific features of the pathogenesis of liver fibrosis and cirrhosis. This will contribute to a safer first administration of newly developed drugs in man and to a reduction, replacement and refinement of animal experiments.
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. For the development of a human based tissue culture model we have generated extended-life, telomerase transfected human corneal cells that show growth regulation and differentiation that is similar if not identical to normal human corneal cells. We have also developed a Human Corneal Construct (HCC) using these cells that resembles, in part, normal human corneas. The goal of this research is to evaluate the effectiveness of an HCC Test to predict ocular irritation potential of a wide range of irritants from slight to severe. They will also identify a simple biochemical end point that can be used by other laboratories to determine ocular irritation potential on a standard, commercial basis.
Reducing Postoperative Pain and Distress in Mice
Alicia Z. Karas, DVM
Tufts University, Boston, Massachusetts
Postoperative pain in laboratory rodents is often not adequately addressed by investigator, for several reasons. One reason for lack of giving pain medications to rodents is the inability to detect overt signs of pain or distress. There is also a genuine lack of knowledge about the administration and effects of pain medications that may lead to their omission from protocols. Although doses of analgesics for rodents are listed in the most current laboratory animal texts, and a few articles have been published regarding suggested techniques, studies to show true efficacy are comparatively rare. There is thus a need to make published information available regarding the presence and extent and treatment of postoperative pain in the mouse.

The weight loss and behavioral data that we have completed thus far tell us that some factor about surgery leads to a serious physiologic/metabolic setback in mice. It may or may not be pain, and main may be one of many contributing factors. We are proposing to study a number of interventions that might improve the welfare of the mouse portoperatively. These interventions will consist of pain medications, fluid and nutrition support, thermal support, and combinations of all interventions. The objective of our work is to use a relatively small number of mice to study techniques and interventions that will reduce postoperative distress. If we are successful, we can make recommendations to investigators and ICES that will not only improve welfare of mice, but theoretically result in less morbidity, and remove a potentially significant source of confounding from studies.
Differential Effects of Environmental Enrichment for Mice
Hal Markowitz, PhD and Clifford Roberts, DVM
University of California, San Francisco, California
Perhaps the most important refinement that can be made in the use of living animals in biomedical research is to improve the ways in which they "model" the disorders for which we seek cures. In this research we evaluate the effect of a number of different daily living conditions and exercise regimen for mice on their behavior and disease progress. Mice that are models for familial Amyotrophic Lateral Sclerosis (ALS) will be assigned to four groups: traditional caging; cages with "mouse houses"; cages with exercise wheels; and a group with wheels in their home cages and individual exercise regimen involving training six days a weeks. Control animals of the same strain will be assigned to identical groups.

Using standardized tasks to evaluate the behavioral abilities of the experimental animals as their disease progresses we will assess the utility of these different living conditions in slowing or accelerating progress of the functional disorder. Examples of these tests, which closely mirror the tests made on human ALS patients, include ability to balance on a rotating rod, gait analysis, and monitoring of food consumption. The control animals will be used to compare these results with normal behavioral change associated with increased age. Differences as a function of caging conditions are not often properly considered and seldom used as a critical component of experimental design. If our results do show differential outcomes we anticipate that methods similar to these will be used increasingly by researchers to refine their use of experimental animals.
Genomic Approaches to Defining Pain and Distress in Mice
Norman C. Peterson, DVM, PhD
Johns Hopkins University, Baltimore, Maryland
This project will utilize micro-chip technology to simultaneously monitor the expression of thousands of genes in mice brains in response to inflammatory pain delivered to a hind paw. By using this technology to develop a "pain stimulus gene expression map", we will be able to better characterize pain perception in mice. Ultimately, this will provide us with a means to more objectively identify conditions that cause pain and distress in laboratory animals, and to develop specific methods/reagents that inhibit or alleviate it in animals and people.
Measures to Reduce Stress Caused by Experimental Procedures
Bert van Zutphen, PhD and Vera Baumans, DVM
University of Utrecht, Utrecht, The Netherlands
Animals that are used in biomedical research are often submitted to routine experimental procedures. These procedures may cause an imbalance of the animal's homeostasis ('stress'. The purpose of the present project is to measure the animal's stress response to such procedures and to investigate the options for refinement. Is it possible to reduce the stress response through environmental enrichment or through conditioning of the animals? In this study the focus is on injection techniques in mice. It is anticipated that the stress response not only depends on the experimental procedure per se but also on the animal's environmental circumstances (e.g. social housing vs. individual housing; physically enriched housing vs. standard housing) and on the degree of conditioning.

Thus, groups of animals housed under different conditions, will be submitted to routine experimental procedures and the (difference in) stress response will be measured.

In the first year the effects of different kinds of housing conditions will be studied in two mouse inbred strains (BALB/C and C57BL/C). At a later stage the effect of conditioning will be subject of study.

Parameters for measuring the stress response include heart rate, body temperature, activity, food intake, body weight and corticosteroids.

The application of radio-telemetry is needed for stress-free collection of data on heart rate and body temperature. For this purpose a transmitter must be implanted into the abdomen of the animal. Studying the consequences of this procedure is also part of the present project.

Milestones for the first year will be to find answers to the following questions:

  • What is the stress response of the animal to IP injections?
  • What is the influence of physical or social enrichment on the stress response?
  • What is the effect of the strain on the stress response?

Development of an in vitro Cell Model of the Human Blood Brain Barrier
Senén Vilaró, PhD
School of Biology, University of Barcelona, Barcelona, Spain
The main obstacle for efficient drug targeting to the brain lies on the blood-brain barrier (BBB). Separating brain from blood at the level of the endothelial cells from cerebral capillaries, this cell barrier strictly controls the entry of solutes into the central nervous system. To assess the toxicity or bioavailability of drugs in development, academic groups and pharmaceutical companies would benefit from an in vitro model of BBB, based on brain endothelial cells in culture. In response to the need of such in vitro models, the main objective of our proposal is to develop easy-to-use stable immortalized endothelial cell lines from human brain microvessels, which will retain their functional properties and allow to develop an in vitro model of the human BBB as a highly reliable and reproducible alternative method to animal experimentation for pharmacological and toxicological testing of CNS-directed compounds.

Using the isolation procedure developed during the first year of CAAT funding, primary cultures of microvascular endothelial cells will be obtained from total brain samples from post-mortem multidonor patients. As BBB properties are quickly lost in culture, immortalization will be performed on early primary cultures using viral constructs expressing the human telomerase, as immortalizing gene. Selection will be based on expressing the human telomerase, as immortalizing gene. Selection will be based on growth properties and expression of specific brain endothelial markers. Positive immortalized subpopulations will be characterized, in solo or in co-culture with astrocytes, for transendothelial electrical resistance and permeability to standard molecules with known BBB profiles. A correlation will be established between in vitro and in vivo permeability values. Once validated, this in vitro cell model of human BBB will constitute a valuable alternative to animal use for product screening and safety tests. Its application in large high through-put screening will accelerate the CNS-oriented drug discovery, and decrease the R&D costs for pharmaceutical industry.
Development of Reversibly Transformed Human Corneal Epithelial Cells as an Optimal In Vitro Model
Steven E. Wilson, MS, MD
University of Washington, Seattle, Washington
Many potential drug and cosmetic products must be tested for ocular toxicities prior to evaluation in humans. In many cases, living animal models are utilized for testing these substances. Since the corneal epithelium is one of the primary tissues that can be injured by drugs or cosmetics, it is our hope that an alternative in vitro testing strategy can be developed that will spare large numbers of experimental animals. Previous investigators have developed corneal epithelial cell lines from animal and humans. However, these previous cell lines have been continually (constuitively) transformed with specific genes such as SV 40 T antigen. Unfortunately, permanent continual expression of those proto-oncogenes results in loss of the normal differentiated state in these epithelial cells.

With the funding from CAAT, we have already developed methods for reversible transformation of human corneal epithelial cells. We have demonstrated that these cells can be induced to proliferate when provided with an inducer triggering expression by the vector, but that the cells assume a more differentiated cell type when the inducer is removed from the culture.

In the last year of the study, it is our intention to utilize a different transforming gene called te lomerase that is present, but closely regulated, in normal human cells and other animal cell types that undergo continuous proliferation. This normal gene is less likely to produce chromosomal abnormality in the epithelial cell lines. We will generate regulatable vectors that express this telomerase gene and insert these into cultured human corneal epithelial cells. We will study normal differentiated functions to select appropriate cell lines that will be of special importance for use in toxicity testing and other applications in which cultured human corneal epithelial cell lines are needed for research.
Evaluation of CYP3A Induction with Engineered Cell Lines
Bingfang Yan, DVM, PhD
University of Rhode Island, Kingston, Rhode Island
Drugs entering the human body undergo extensive chemical conversions through an array of diverse catalytic proteins referred to as drug-metabolizing enzymes. Among them, cytochrome P450 (CYP) enzymes rank first among the phase I biotransformation enzymes in terms of catalytic versatility and the number of drugs they metabolize. Approximately twelve CYP enzymes are involved in drug-metabolism with CYP3A enzymes being the most abundant and responsible for the metabolism of two thirds of drugs. CYP3A induction by many drugs is known as an important contributing factor to many failures of therapy or severe toxicity. Recent studies demonstrate that activation of the pregnane X receptor (PXR) is responsible for CYP3A induction by many drugs, providing a rationale for establishing an in vitro screening system. The objective of the proposed studies is to develop a PXR-based in vitro screening system for CYP3A induction. Stable transfectants have been prepared to express steady levels of the PXR and to harbor a PXR responsive reporter. In the final period of support, we will focus on the optimization of the screening conditions such as selection of the best responsive transfectants. Screening of CYP3A inducers has been exclusively dependent upon the use of human hepatocytes and animals. The availability of human livers and the use of a large number of animals are the primary limited factors in rapidly and accurately predicting the effects of drugs on CYP3A expression. The screening system to be established 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.