The U.S. Army Medical Research Institute of Chemical Defense (USAMRICD) at Aberdeen Proving Ground, Maryland, is the lead laboratory for executing the Department of Defense's medical chemical defense research program. The USAMRICD employs 141 permanent civilians and 55 military officers, and supplements this staff with National Research Council fellows, interns from the Oak Ridge Institute for Science and Education (ORISE) postgraduate internship program, and contract personnel. There are approximately 42 principal investigators, who carry out a research budget of over $16 million.
Their mission is to develop medical countermeasures against exposure to chemical warfare agents as well as against agents of biological origin. While accomplishment of this research mission necessarily requires in vivo studies, in the last 11 years, scientists at the USAMRICD have reduced the number of animals used in their research protocols by 92 percent.
These efforts to reduce use of animals in research are part of a larger program initiated by the Department of Defense, and more specifically by the U.S. Army Medical Research and Matériel Command (USAMRMC), of which the USAMRICD is a subordinate unit. In 1992, the U.S. House of Representatives Committee on the Armed Services issued a report on use of animals in DoD military experiments. The resulting action was the 1993 National Defense Authorization Act, requiring DoD to submit a comprehensive annual report on animal cost and use as well as in depth profiles of animal research and initiatives to promote alternatives to reduce, refine, and replace (the 3Rs). In 1995, DoD issued directives and policies that updated and redefined its requirements for the care and use of animals in DoD-sponsored programs. In adopting the 3Rs, the DoD also added a fourth, responsibility.
In 1993, the USAMRMC developed a management-by-objective program based on science and technology objectives, or STOs, with specific tasks under each objective. Among the major objectives established by the USAMRMC was one to develop reduction, refinement, and replacement strategies for use of animals in research. The goal was to develop technologies that would incrementally reduce reliance on animal and human subject research and improve the experimental conditions using animals. Current objectives are to reduce reliance on animals in research by 25 percent, by fiscal year 1999, using data from fiscal year 1991 as a baseline, and to introduce a minimum of one improvement per year in experimental protocols using animals.
The USAMRICD AAALAC-accredited since 1984 and with an established animal care and use committee overseeing animal research since the late 1970s was already in compliance with much of the Department of Defense directive when it was issued in 1995 and well on its way to meeting the goals of its Command's research objectives. Principal investigators at the USAMRICD have takenadvantage of emerging technologies in design of molecular modeling software and in development and maintenance of cell culture models to meet the animal reduction goals of the USAMRMC. In addition, scientists, where possible, have adopted the use of less sentient animal species.
Computer modeling of the molecular structure of nerve agents, physiological enzymes, and neurotransmitters is used to predict and eventually determine how these chemical compounds interact at the molecular level. A test compound that inhibits acetylcholinesterase (AChE) aging has also been studied via computer modeling to determine the specific molecular events that occur at the peripheral anionic site of AChE that subsequently prevent a potent AChE inhibitor, such as a nerve agent, from irreversibly binding to the acetylcholinesterase molecule. With this knowledge, the most likely candidates for chemical intervention in the prevention or treatment of nerve agent exposure can be determined.
|Figure 1. Cultured normal HeLa cells. This cell line is derived from a human cervical epithelial tumor cell.||Figure 1a. Cultured HeLa cells 24 hours after exposure to sulfur mustard. Note the loss of microvilli and multiple perforations and invaginations of the plasma membrane.|
Currently, 17 cell lines or cell cultures are used in research at the USAMRICD. While they are used in research studies across the Institute's program, they have particularly led to a reduction of animal usage in the research programs on blistering chemical warfare agents. In efforts to evaluate the effects of blister agents, or vesicants, particularly sulfur mustard, and to develop medical countermeasures against such agents, alternatives to animals have proven particularly useful. Several cell line or culture systems, such as peripheral blood lymphocytes, human epidermal keratinocytes (figs. 1 and 1a), and the HeLa, a human epithelial tumor line (figs. 2 and 2a), have been adapted and developed by scientists at the USAMRICD into valuable models for studying vesicant injury. In addition, studies have used a commercial human skin equivalent model and skin biopsies from the Cooperative Human Tissue Network. The USAMRICD has also developed the technology to process and generate a human epidermal model, which possesses typical structural components of human epidermis in vivo to include hemidesmosomes, anchoring filaments, and elements of a true basement membrane. The use of these alternative models has led especially to a decrease in the number of rodents required for these studies.
|Figure 2. Cultured normal human epidermal keratinocytes.||Figure 2a. Cultured normal epidermal keratinocytes 6 hours after exposure to sulfur mustard. Note the loss of microvilli, focal plasma membrane blebbing, and cellular detachment /loss subsequent to cell d eath.|
The USAMRICD also promotes the use of less sentient or less regulated animal species. Researchers at the USAMRICD use the Aplysia californica, a large naked marine mollusk or gastropod with anterior sensory tentacles, commonly referred to as the sea hare or sea slug. The Aplysia californica possesses rather large and discrete neural ganglia, unique neural anatomy that make these invertebrate animals an excellent and widely used model for neural transmission and neurotoxin research. The USAMRICD acknowledges that in certain sulfur mustard experimental protocols, SKH-1 hairless mice are an appropriate substitute for the hairless guinea pig. The main histological feature of skin lesions produced in a SKH-1 hairless mouse following exposure to sulfur mustard is the formation of microblisters at the dermal-epidermal junction. Similar microblisters also occur in sulfur mustard lesions of hairless guinea pigs. Whereas laboratory mice are not subject to the Animal Welfare Act, provisions for their care and use arecontained in the Guide for the Care and Use of Laboratory Animals.
Through these alternatives to animal research initiatives, the USAMRICD is 3 years ahead of schedule and has already exceeded the 25 percent reduction in use of animals required by the USAMRMC's research objective. From fiscal year 1991 to fiscal year 1996, the Institute's reliance on animals in its research dropped by 68 percent. The most dramatic drop was in use of rodents in research; 90 percent fewer rodents were used in research protocols in 1996 than were used in 1985. The number of publications resulting from research at the USAMRICD, however, has remained high and even increased slightly over the last several years. Approximately 34 percent of the Institute's 1996 publications were a result of research in which no animals or less sentient animal species were used.
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