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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 2007-2008

Summary Of Research Grants

Development Of A High Throughput Method To Screen For Gene Expression Changes For The Prediction Of The Skin Sensitization Potential Of Chemicals
G. Frank Gerberick, PhD
Procter & Gamble
Allergic contact dermatitis is a frequent occupational health problem and is the most common type of chemical allergy in humans. Therefore, there is a need to identify those chemicals that can cause skin sensitization. For this purpose guinea pig, and more recently mouse test methods have been used. However, there is a need to eliminate completely the use of animals in skin sensitization safety assessments. In recent years, our understanding of the cellular mechanisms involved in allergic contact dermatitis has increased substantially. It is known that dendritic cells (DC) residing in the skin play a key role in the development of allergic contact dermatitis. Given the importance of these cells in the initiation of skin sensitization, it seems appropriate to explore whether there are opportunities to develop alternative approaches to hazard identification based upon chemical-induced changes in the gene expression profile of these cells. This project will explore the use of Luminex® xMAP® bead-based technologies for the development of a high throughput screening method to rapidly measure gene expression changes in allergen-treated DC-like cell lines. The observed effects on gene expression in these cell lines will be compared to data derived from exposing human peripheral blood derived-DC to the same chemical allergens. The goal of this project is to provide a quick, easy and more cost-effective in vitro method to evaluate a large number of chemicals in order to identify genes that when measured in vitro will aid in the prediction of the skin sensitization potential of chemicals in vivo.
Neural Stem Cells: A Powerful Alternative Model To Cultured Animal Neurons For Developmental Neurotoxicological Studies
RF Halliwell, PhD
University of the Pacific
Neural Stem Cells may revolutionize the treatment of human disease and the discovery of new drugs. They may also prove powerful in vitro models for the investigation of the toxicological effects of drugs and environmental agents on the nervous system. Stem cells might ultimately replace animal–derived tissues for scientific experiments.

Stem cells are self-renewing with the capability of forming any cell type, tissue or organ in the body. Stem cell-derived neurons (SCNs) can now be grown in vitroallowing their properties to be studied but currently there is little data establishing their functional properties. Indeed there is evidence that SCNs express some nerve cell protein markers but may not display their electrical properties. The goals of this project are to characterize the physiological and pharmacological properties of neurons derived from the embryonal carcinoma-derived stem cells, and to establish their validity for the investigation of neurotoxins. This study will utilize electrophysiological and some molecular biological methods. The main aims of this project are to determine (1) the properties and electrical activity of SCNs maintained in cell culture; (2) the identity of the receptors (through which drugs and toxins can act) that are expressed in these neurons and (3) the sensitivity of SCNs to selected neurotoxins. These experiments will provide new data on the functional properties of neurons derived from stem cells and their value as simple in vitromodels for the investigation of the neurotoxicological actions of xenobiotics and the biological mechanisms that underlie their actions.
A Closed-Head Recording Device For Neurologic Studies In Laboratory Animals
Lisa Krugner-Higby, DVM, PhD
University of Wisconsin-Madison
Devices that can be surgically fixed to the heads of animals such as rats, cats or monkeys have been important tools to study the function of the nervous system. These experiments have contributed significantly to our knowledge of how the brain processes information from our eyes and optic nerves.  Similar experiments are also important in the study of diseases of the nervous system associated with aging, such as Alzheimer’s disease and Parkinson’s syndrome.  However, these devices pose very significant issues for the welfare of the animals.  ‘Head caps,’ as these devices are commonly called, can serve as a focus for infection because they leave portions of the nervous system open to the external environment.  Our laboratory has developed an electrode array for recording microEEG that can be used to form the basis of a fully-implantable system for studying the central nervous system.  The electrode array has been tested in animals in preliminary experiments.  The array will eventually be connected to an implantable telemetry device.  The telemetry device will receive electrical signals from the electrode array in contact with the membranous covering of the brain and transmit them as digitized information to a receiver linked to a computer and appropriate soft ware.  A fully-implantable system will not leave portions of nervous system open to the external environment and therefore vulnerable to infection.  This will provide a significant improvement in the welfare of animals used in neurological research.
Zebrafish As An In Vivo Model System For Identifying Developmental Neurotoxicants
Pamela Lein, PhD
Oregon Health & Science University
There is evidence that exposure of the developing human nervous system to toxic chemicals can cause changes in the behavioral, emotional and intellectual function of children.  There is, therefore, much interest in screening chemicals to identify those with potential to cause developmental neurotoxicity.  Tests currently approved for this purpose primarily use rodents and are complex and expensive in terms of scientific resources, time and animal use. We propose to evaluate the zebrafish as an alternative model for screening chemicals for possible adverse effects on the developing brain.  We will use a specific transgenic line of zebrafish (NBT-GFP) in which motor neurons express green fluorescent protein (GFP) to measure axon outgrowth and quantify motor behavior at varying times following exposure to a panel of toxicants generally regarded as neurotoxic.  The reasons for choosing the zebrafish include:  (1) The cell and molecular mechanisms that control the normal development of the zebrafish nervous system are remarkably similar to those in humans, increasing the validity of using this model to assess potential risks to the developing human nervous system.  (2) The expression of green fluorescent protein (GFP) in specific populations of zebrafish neurons coupled with the fact that zebrafish are transparent, allows non-invasive imaging of these GFP-labeled neurons in the same animal over time, thereby significantly reducing the number of animals required for developmental neurotoxicity testing.  (3) The small size, rapid nervous system development and short life cycle of zebrafish are favorable for adapting this model system for testing large numbers of chemicals.
Opioid Analgesics And Their Role In Post-Surgical Gut Stasis And Anorexia In The Rabbit
Diana Scorpio, DVM, MPH
Johns Hopkins University
Gastrointestinal side effects of opioid (narcotic) drugs are common in many mammals, including humans, and are characterized by lack of appetite, nausea, and constipation.  Usually, these effects are temporary and of little consequence in humans and most mammals, although in rabbits, decreased intestinal motility can be devastating; it can contribute to longer recovery after surgery and can cause subsequent illness, which can be problematic when performing scientific studies.  Rabbits have complex intestinal functions that are sensitive to change caused by illness, stress, or surgery.  A temporary interruption in normal intestinal movements can result in decreased appetite and reduced stool production.  In New Zealand White rabbits that undergo abdominal surgery, we have clinically observed that certain opioids like buprenorphine (same family as morphine), when used for extended periods after surgery, relieves pain but may contribute to reduced appetite, diminished stool production, and weight loss.  These effects seem to occur in the absence of pain from surgery.  Use of non-steroidal anti-inflammatory drugs such as meloxicam (same class as ibuprofen) for pain after surgery may help to reduce these intestinal complications while allowing for a smoother, shorter post-surgery recovery and rapid return to normal eating habits, stool production, and well-being.
Microfluidic Systems For Reducing Animal Use In High Throughput Toxicity Assays
Glenn M. Walker, PhD
North Carolina State University
The system developed in this project will address the growing need for alternative animal models in toxicological studies. To meet this need we propose to develop an in vitro microfluidic screening system, capable of simultaneously testing many compounds, that will minimize or eliminate animal use in irritant testing. A novel polymer will be used to construct the microfluidic devices so that a wide range of test compounds, including organics, can be used for irritant screening. Cultures of human epidermal keratinocytes (HEK) will be maintained within the devices and used as the model system, thus eliminating animal use for these studies. We will validate the irritant potential of benzene and toluene, components in a wide variety of consumer products, by exposing them to HEK and assaying for the secreted pro-inflammatory markers IL-8 and TNFa. The microfluidic screening system will use one one-hundredth the volume of a traditional 96 well titer plate, thus reducing the amount of cells and reagents needed for irritant screening. The proposed microfluidic system will have wide-ranging impact in all areas of toxicology since it can be used with any cell type and with any soluble chemical compound.
Operant Conditioning Of Sheep And Goats
Lara Weaver, DVM
Tufts University
In the first year of this project, we aim to devise and optimize a program of positive reinforcement training (PRT) in sheep that can be accomplished in 14 days or less by an individual with little or no previous animal training experience. Once an effective method of training has been devised, we will design and perform a study testing the hypothesis that sheep that have undergone PRT will exhibit lower indices of stress (behavioral and physiological) compared to sheep that are untrained, when both groups are subjected to identical procedures. During this first year, video clips and other relevant training materials will be collected for use in developing a training module designed to rapidly train laboratory animal personnel in the technique of PRT in sheep. It will likely take longer than 1 year to finish the training module and this would not be expected to be completed within the first year of this project.

Project summary: The goal of this study is to develop a relatively rapid and reproducible method of training sheep and/or goats to better acclimate them to procedures commonly performed in a research setting. Positive reinforcement will be used to train the sheep or goats to accept a halter and walk on a lead; stand in a sheep blocking stand; undergo physical examination including body condition scoring, rectal temperature collection, hoof examination and trimming; and clipping of the hair coat as needed; and undergo blood, urine and feces collection with minimal restraint. It is hoped that by acclimating sheep and/or goats to these commonly performed procedures, they will experience less stress in the research setting.  Ultimately the goal of this work is to develop a training method that could be incorporated at other research facilities thus benefiting sheep and goats in a variety of research settings.
Morphologic And Genomic Approaches To Evaluate The Zebrafish Embryo Test As An Alternative Screening Model System To Predict Developmental Toxicity
Hilda Witters , PhD
VITO - Flemish Institute for Technological Research - Belgium
Developmental toxicity is a major issue in children's health worldwide. The developing human system is susceptible to many toxicants, and chemical exposure during development may cause lasting metabolic deficits. Such damage can range from subtle to severe, and it may impose substantial burdens on affected individuals, their families, and society. Testing compounds for developmental toxicity endpoints is an important societal and scientific goal.

Zebrafish is an established animal model for many aspects of human development and diseases that will benefit from extended embryological knowledge and genomic research. The first specific aim of the project will be the determination of the developmental time course of early physical malformations and neurobehavioural dysfunctions caused by a selection of candidate compounds in a dose range matter. The second aim will be a detailed investigation of the cellular and molecular mechanisms (fingerprints of genes), contributing to these (d)effects as observed for the most potent compounds.

The overall purpose of this research project is to utilize zebrafish embryos, as a simple and fast high-throughput assay to predict for potential malformations and dysfunctional effects during early development, caused by exposure to different classes of chemicals. This short term zebrafish assay might in the first place replace and reduce the number of mammals currently used for testing of chemicals. And eventually as it does contribute to an improved and cost-efficient test strategy for numerous chemicals, it may lead to reduction or prevention of the majority of induced birth defects.
3-D Sertoli Cell/Genocyte Co-Culture System: In Vitro Model For Developmental Testicular Toxicity
Xiaozhong Yu, MD, PhD
University of Washington
The development of in vitro models that monitor and which can identify altered testis development would provide important alternatives to in vivo testing. Such methods would allow for the assessment of reproductive and developmental effects induced by environmental agents. These models would also provide information regarding mechanisms of toxicant action in the testis, leading to improvements in the interpretation of data obtained from in vivo systems and thus lead to significant refinement and reduction of in vivo animal use. In our previous research we demonstrated that our three-dimensional (3-D) Sertoli cell/gonocyte co-culture (SGC) system permitted the formation of a testicular-like multilayered architectural bio-structure that mimic in vivocharacteristics of seminiferous tubules. We feel strongly that this novel in vitro SGC system will provide investigators with a simple, efficient, and highly reproducible alternative for the assessment of reproductive toxicity and the screening of testicular developmental toxicants. Thus, the purpose of this study is to further refine and characterize our in vitro 3-D SGC culture as a simple, efficient tool for screening testicular developmental toxicant by using a range of known in vivomale reproductive and developmental toxicants. This will be a systems based analysis using genomics and GO-quant technologies. We will also apply this 3-D SGC system to develop appropriate cell-based assays and biomarkers of susceptibility for testicular toxicity through the use of real-time nanotechnology biosensor assay methods. The establishment of a simple, efficient in vitro 3-D SGC culture model has the potential to greatly reduce the number of animals used through its ability to increase and refine the type of information obtained.