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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 2005-2006

Summary of Research Grants

Non-animal methodology to assess developmental immunotoxicology
Kathleen M. Brundage, PhD
West Virginia University
In the body, the immune system is important for maintaining the health of an individual by detecting and destroying damaged cells, bacteria, and viruses. An important player in this system is the white blood cell known as a B lymphocyte or B cell. B cells are responsible for making proteins known as antibodies that specifically attach themselves to bacteria or viruses aiding in their elimination from the body. Little is known about the consequences of chemical exposure on the developing human immune system (immunotoxicity). The majority of studies that have been performed have used animal data to assess human risk. In this study we will use human umbilical cord blood cells to assay the effect that chemicals have on B cell development. Ethanol (alcohol) will be used to test this assay system. Ethanol was chosen for several reasons including that fetal exposure to ethanol is common due to consumption of alcohol by pregnant women and mouse studies have demonstrated a decrease in the number of certain types of B cells in mice exposed in the womb to ethanol. A second part of this project will be to identify protein markers that may be useful in developing screening methods for immunotoxicity. The overall goal of this project is to establish a safe, reliable, non-animal method for evaluating immunotoxicity of B cells in the human immune system.
An in vitro flow adaptation chamber replaces animals in an ischemia/reperfusion model to study oxidant-induced signaling
Shampa Chatterjee, PhD
University of Pennsylvania Health System
Ischemia reperfusion (I/R), the temporary stop of blood supply to an organ followed by its reinstatement, is a cause of severe injury following surgery, organ transplantation or other kinds of obstruction. This injury is caused by an entity called the reactive oxygen species (ROS) generated by the lining of the blood vessels. In addition to injury, ROS also play a part in altering regular cellular processes and cause tissue and organ dysfunction. A better understanding of these processes would help in reduction of injury following I/R; however such studies also necessitate an extensive and indiscriminate use of animals. Here, we propose to drastically reduce the use of animals by an artificial capillary system (Fibercell) consisting of hollow fibers that can be coated with cells kept under flow using a pump so as to mimic the inner lining of blood vessels. I/R can be performed by stopping and/or restarting the flow in the system. The cells used to line the fibers are isolated from a few mice and rats by a procedure routinely carried out in our lab that allows for isolation fo endothelial (cells lining the vessels) cells and their expansion into larger cultures thereby preventing large scale sacrifice of animals. Earlier studies from our group have shown that ROS generation with I/R is triggered by the closure of a potassium channel on the cell surface. Therefore potassium channel openers will be used during I/R and ROS generation and the consequent cell damage will be examined to evaluate the potential therapeutic appliations of potassium channel openers.
Assessment of a pain scoring system for rhesus macaques
Kristine Coleman, PhD
Oregon National Primate Research Center
In recent years, there has been an increasing awareness of the need for improved pain asessment and management techniques for laboratory animals. Pain scales have been created for various species in an effort to reduce pain and sufering by allowing for timely intervention. However, many of the scoring systems that have been developed were based on one species and adapted to others. This practice may not be appropriate, given that expression of pain often differs across species. The overall goal of this study is to establish and validate useable guidelines for assessing pain in rhesus macaques (Macca mulatta). While nonhuman primates are common subjects in biomedical studies, there is a paucity of published pain scoring systems for these species. Having such a scoring system would allow primate veterinarians and researchers to refine current acceptable postoperative care practicies to reduce pain. In this study, we will monitor and assesss behavior and physiological variables such as heart rate, pupil dilation and activity in adult rhesus monkeys prior to and following a major surgery. We will then correlate these variables with variables that might predict pain levels, including the amount of analgesia (administered by the surgical staff), in an effort to get a better understanding of behavioral and/or physiological manifestations of pain. This information will help us develop and assess a species-specific pain scoring system for rhesus macaques.
In vivo imaging with biofluorescent virus
Diane E. Griffin, PhD
Johns Hopkins Bloomberg School of Public Health
Animals are essential for the study of how viruses cause paralysis and death due to encephalomyelitis and for determining how such infections can be treated. Traditionally, these pathogenesis and treatment studies involve examining the brains and spinal cords of mice at multiple times after infection to determine where the virus is, how much virus is present and the host immune response to the virus. We have shown that viruses engineered to express bioluminescent molecules such as firefly luciferase can be used to follow virus repliation and spread using only a small number of mice that can be followed individually over time. Emission of light for imaging requires injection of luciferin for each imaging session. In this project we will develop viruses that express a biofluorescent molecule that will emit light on its own without the need for luciferin. This will accomplish 2 goals - (1) quantitation of virus without the need for injection of luciferin and (2) potential expansion of the studies for imaging of both the virus and the host response. Imaging of the host response will use newly engineered mice that have a bioluminescent molecule (e.g. luciferase) that is a reporter for expression of genes that are turned on during inflammation of the brain. Much of the damage during virus infection of the brain is due to the inflammatory response that damages cells in the nervous system. These mice will allow this factor to be monitored without killing the mice and will allow us to determine the effects of treatmenton the inflammatory response.
Sea urchins as an alternative animal model for testing drugs and neurotoxicants as potential neuroteratogens
Jean M. Lauder, PhD
University of North Carolina School of Medicine
The purpose of these studies is to utilize sea urchin embryos, which use brain chemicals (neurotransmitters) to regulate their early growth and development, as biosensors to screen drugs or environmental neurotoxicants for potential deleterious effects on prenatal development. The experiments characterize malformations caused by particular test substances, identify which embryonic cell populations are affected, and investigate cellular and molecular mechanisms underlying these effects. Specific aim 1 determines the developmental time course of malformations caused by drugs (such as those used to treat depression, mood disorders, or seizures) and neurotoxins (pesticides, crop molds, retinoic acid), and determines which compounds (for example, folic acid), can prevent these malformations. In vitro fertilization of eggs is used to obtain embryos for testing. Embryos, developing in artificial sea water (ASW), are exposed to test compounds or control solutions and digitally photographed at times when normal embryos are at specific stages of development (e.g., mid-blastula, early gastrula). Knowledge of which compounds prevent malformations caused by specific drugs or neurotoxins will be used to design experiments in Specific Aim 2 to investigate cellular and molecular mechanisms contributing to malformations.
An in vitro model system to evaluate drug effects on B-lymphocyte survival and antibody production
Donna Muscarella, PhD
Cornell University College of Veterinary Medicine
The immune system is among the most sensitive of body systems to disruption by immunotoxic drugs and environmental pollutants. Testing chemicals in animals has identified some immunotoxic chemicals, but such tests are animal-use intensive, costly, and generally lack sensitivity. Furthermore, animal tests seldom reveal which cellular components and biochemical pathways are disrupted by immunotoxic agents. Consequently, there is the need to develop alternative in vitro systems that can provide the necessary mechanistic information that can enable investigators to better predict drug and chemical effects on immmune system function. One fundamental immune system activity that is a potentially important target of immunotoxic agents is the positive selection of specific clonal populations of B-lymphocytes (BLs) that ultimately generate the diverse repertoire of antibodies needed to recognize foreign antigens. This process takes place within the germinal centers of lymph nodes, where the interaction of BLs with specialized, follicular dendritic cells activates specific biochemical pathways that ultimately control BL survival and antibody production. However, these cellular interactions and biochemical pathways are susceptible to interference by exogenous drugs and chemicals. In this project a co-culture system of human BL-cell lines in contact with the HK-follicular dendritic cell line will be used to model the process of positive selection that occurs in germinal centers of lymph nodes. This in vitro germinal center model will be used to identify specific molecular targets and signaling pathways that are inhibited or activated by particular chemicals, ultimately causing disruption of productive interactions between BLs and HK cells. Candidate immunotoxic agents will be studied including environmental pollutants, anti-cancer drugs, and anti-inflammatory drugs. The specific pathway altered by these agents will be established and the information will be used to generate predictive models for potential immunotoxic effects of different classes of chemicals.
Development of a mathematical model to predict bacterial response to antimicrobial agents
Vincent Tam, PharmD
University of Houston College of Pharmacy
The emergence of bacterial resistance to antimicrobial agents is rising at an alarming rate. New agents must be developed rapidly and continuously to combat infections caused by these resistant pathogens. In pre-clinical development off new antimicrobial agents, a large number of laboratory animals are routinely used to explore the appropriate dosing regimens (dose and dosing frequency) to be investigated in clinical trials. However, the choice of these dosing regimens to be tested is often empiric. This trial-and-error approach is grossly inefficient and the use of laboratory animals is not always necessary in many of the conventional development phases. Our goal is to understand how mathematical modeling and simulation can be optimally incorporated in pre-clinical investigation of antimicrobial agents to streamline the development process. We propose to develop a methodology combining mathematical modeling and computer simulations to predict bacterial response to antimicrobial agents. The effect associated with an antimicrobial agent (given as different dosing regimens) could be evaluated efficiently using computer simulations. By guiding the choice of dosing regimens to be investigated, the proposed modeling system approach would enhance the efficiency of the antimicrobial development process and likely result in a significant reduction of the number of laboratory animals required for pre-clinical testing. Animals will unlikely be used at all in testing of agents predicted to have minimal antimicrobial activity, thus avoiding unnecessary use of the animals. For agents predicted to have antimicrobial activity, animal investigators could focus on only a few dosing regimens with high probabilities of success.
Identification of cell lines for use in in vitro assays for sensitization
Geert Verheyen, PhD
VITO - The Flemish Institute for Technological Research
Many chemicals can induce allergic contact dermatitis (ACD) in susceptible individuals. ACD represents an important occupational and consumer health problem and there is need for reliable methods that permit the prospective identification of chemical allergens. This is currently done by animal testing, but the availability of validated in vitro models is of high importance for the industry (cosmetics, pharmaceuticals, pesticides, ...). A promising in vitro alternative to animal testing is the use of human antigen presenting cells (APC). APC are essential in the initiation of allergic reactions. Unfortunately, APC that are currently used (e.g. CD34-derived dendritic cells (CD34-DC)) are obtained from peripheral blood or cord blood samples from donors, and have several drawbacks that limit their potential (e.g. interindividual variation, low cell numbers, limited shelf life, ...). Ideal would be to have an immortal APC-like cell line that can be used to develop high-throughput in vitro assays. Several candidate cell lines have been described in the literature, such as MUTZ-3, THP-1 and U-937 cells. Here we will use gene expression microarray analyses to compare these 3 cell lines after exposing them to model chemical allergens (e.g. DNCB, Nickel). The observed effects on gene expression will be compared to our already available data, derived from exposing CD34-DC to the same chemical allergens. The goal of this study is to identify the most relevant cell line (i.e. resembling CD34-DC) that can be used for the development of an in vitro assay for the identification of chemical allergens.
Use of ultrasonic vocalizations in pain assessment for laboratory rats
Daniel M. Weary, MSc, D. Phil
University of British Columbia
Laboratory rats are routinely subjected to surgical procedures, and it is important that they are provided with appropriate pain relief. However, in order to mitigate post-surgical pain in rats, we must first develop reliable methods of pain assessment. Promising pain assessment methods are currently under development for use with abdominal surgeries, but it may not be possible to generalize these methods to other types of surgeries. Rats are known to emit high-frequency vocalizations in response to painful stimuli, and these vocalizations can be reduced with the provision of analgesics. We will therefore investigate whether detailed vocalization analysis can be used as a general tool to assess pain in rats following routine surgical procedures. We will examine both undisturbed and handled animals, with and without analgesics, to determine whether post-operative pain changes the vocalizations of animals under these particular conditions. These studies will thus establish if rat ultrasonic vocalizations can be used as a reliable method of pain assessment in support of developing appropriate pain management regimes.