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The 1985 outbreak of bovine spongiform encephalopathy, or mad cow disease, in the United Kingdom generated global awareness of a previously obscure set of neurodegenerative diseases called transmis- sible spongiform encephalopathies (TSEs). TSEs are caused by infectious agents. Yet, unlike all other known infectious diseases, TSE infectivity ap- pears to be associated with an abnormally folded protein known as a prion (Prusiner, 19821. There is no cure, prophylaxis, or fail-safe antemortem diagnostic test for TSEs, often called prion diseases. Infected hosts incubate a TSE for months to decades, and their health declines rapidly after the onset of clinical symptoms; death invariably follows within a period of months. Bovine spongiform encephalopathy (BSE) became an epidemic that af- fected hundreds of thousands of animals in the United Kingdom and in- flicted economic harm on the country's cattle and beef industries. Cases of BSE have also been reported in continental Europe, Israel, Japan, Canada, and elsewhere.) Exposure to BSE-infected beef products has given rise to a fatal human neurodegenerative disease called variant Creutzfel~t-Takob dis- ease (vCJD), first identified in 1996 (Will et al., 19961. As of September 2, 2003,140 definite or probable vCTD cases had been identified in the United Kingdom (National Creutzfel~t-Takob Disease Surveillance Unit, 2003), and a handful of cases had been identified in other countries. Estimates of the total number of people who will contract vCJD as a result of the BSE epi- iEDITORS' NOTE: After this report was completed, the first U.S. case of BSE was identi- fied in Washington State and was announced to the public on December 23, 2003.
6 ADVANCING PRION SCIENCE demic vary depending on assumptions regarding the incubation period, in- dividual susceptibility, and the level of exposure. The incubation period for another human prion disease, kuru, is 4 to 40 years (Huillard d'Aignaux et al., 20021. The origin of vCJD in prior-infected cattle raises the concern that chronic wasting disease (CWD), a prion disease spreading among North American deer and elk (Williams and Miller, 2002), could cause disease in people who consume venison from the affected regions. The European Commission has spent millions of euros on research to develop better diagnostics for TSEs, especially BSE, with modest success. Commercial diagnostic tests have been developed for rapid postmortem BSE detection and are used throughout the United Kingdom and Europe. These tests cannot detect prions present at low levels, however. The lack of highly sensitive, accurate, and rapid tests has led to such controls as cat- egorical importation bans and massive culling of herds to ensure the safety of beef products. To date the U.S. Food and Drug Administration (FDA) has received no request to approve a rapid test for the detection of human TSEs (personal communication, D.M. Asher, FDA, July 1, 20031. However, the U.S. De- partment of Agriculture's (USDA) Center for Veterinary Biologics has ap- proved the use of three rapid tests for the detection of CWD in cervids (personal communication, R. Hill, USDA APHIS Center for Veterinary Biologics, November 25, 20031. ORIGINS OF THIS STUDY The economic and health consequences of BSE and vCJD in Europe and the risk that U.S. military forces stationed abroad and their dependents could contract a TSE through infected beef or contaminated blood products led the U.S. Congress to pass a law establishing the National Prion Re- search Project (NPRP) in 2002 (U.S. Senate Committee on Appropriations, 20011. NPRP funds research on TSEs, with special emphasis on developing . . an antemortem ~ Agnostic test. Congress mandated that the U.S. Department of Defense (DOD) ad- minister the new project, which was delegated to the Army's Medical Re- search and Materiel Command (MRMC). MRMC administers grants through a two-tiered process of external scientific peer review, followed by programmatic review by a multidisciplinary group of DOD and civilian experts called an integration panel. MRMC requested that the Institute of Medicine (IOM) produce two reports assessing present scientific knowI- edge about TSEs and recommending the highest-priority research for fund- ing (Department of Army Contract DAMD17-02-C-0094, May 20021.
SUMMAR Y 7 In Tune 2002, IOM formed the Committee on Transmissible Spongiform Encephalopathies: Assessment of Relevant Science to conduct this study. The committee was charged with evaluating the state of prion science, especially as regards research needs in the area of diagnostics. The members of the committee were asked to examine novel technologies that might advance diagnostics; evaluate the reagents and assays used in prion research and recommend improvements; evaluate the adequacy of the TSE research infrastructure in the United States with respect to the number of investigators, physical facilities, and training needs; suggest opportunities for collaboration with foreign investigators; evaluate the threat of TSEs to U.S. military forces with respect to their food supply, their blood supply, and any other factors; provide advice on public health policies or surveil- lance programs that require new research or that might affect the military; and recommend additional research on ways to reduce or prevent TSEs. The committee's first report, released in January 2003, advised MRMC's integration pane! on the most promising avenues of research for developing antemortem TSE diagnostic tests. It also recommended ways to remedy key shortcomings in the U.S. infrastructure for TSE research and evaluated the degree of risk posed by TSEs to U.S. military forces and their dependents stationed abroad. This, the committee's second report, recom- mends the highest-priority research in TSE surveillance, prevention, and therapy. It includes an updated version of the material from the first report, with an additional chapter on the unique challenges of detecting TSE infec- tivity in blood. This summary presents the committee's conclusions and recommendations, which are also listed in Table S-1. Clearly, we believe all the recommendations in this report are impor- tant. Given that NPRP has a limited amount of resources, however, it can act only on a limited number of recommendations each fiscal year. There- fore, we prioritized our recommendations by placing them in one of three ranks, 1 being the highest (see Table S-11. We assigned approximately a third of the recommendations to each rank. The rankings are based on the following criteria: · Impact on public health · Impact on protecting animal health (mainly cattle) · Impact on protecting the U.S. economy (BSE/CWD) · Impact on advancing scientific understanding of prions (potential for breakthroughs) · The need to accomplish first rather than second (stepwise progres- sion of the science) · Return on investment · Likelihood of success (How feasible is it?)
8 ADVANCING PRION SCIENCE In addition to prioritizing all of our recommendations, we indicate in the body of the report the most urgent and critical areas of research related to recommendations 3.1 and 6.4. PRIONS AND PrPSC: DEFINITIONS AND USAGE This report uses the terms prion and PrPSc interchangeably in reference to the pro/ease-resistant protein associated with prion disease.2 The com- mittee is sensitive to the fact that such usage is controversial, however, so we want to explain our choice of words in the context of this controversy. The proteins PrPSc and prpC are both encoded by the PRNP gene on chromosome 20 in humans. Like all proteins, prpC has a characteristic con- formation. Under certain conditions, however, it folds into an abnormal shape that is associated with fatal neurodegeneration after a long incuba- tion period. This misfolded protein is called PrPSc. The committee believes that the preponderance of scientific evidence favors the hypothesis that priors, consisting of PrPSc, are associated with infection in TSEs. If prion aggregates are the infectious agent that causes TSEs and if a prion is the misfolded protein known as PrPSc, then by parallel reasoning, aggregates of PrPSc are the infectious agent of TSEs. However, some reputable TSE experts believe that PrPSc alone may not be sufficient to cause a TSE infection, although the protein may be associ- ated with and even necessary for such infection. A number of these investi- gators hypothesize that the infectious particle the prion may contain hid- den nucleic acid, additional proteins, or some other additional, essential material. This camp uses the term prion to refer to TSE infectivity, but does not equate prion with PrPSc. To respect and recognize this point of view, this report often uses the phrase "the infectious agent of ELSE under discus- sion]" instead of the term prion or PrPSc. The purpose of this report is not to resolve the controversy over the definition of a prion or to proffer the committee's opinion regarding the hypothesis that prions consist exclusively of the protein PrPSc. Rather, the purpose of this report is to call for additional research especially studies of the fundamental molecular structures and mechanisms related to TSEs- so that disparate views may converge and advance prion science. 2At times an additional term, PrPreS, is used synonymously with PrPSc. PrPreS is an abnor- mally folded prion protein that is highly resistant to digestion by the enzyme proteinase K (PK) and is strongly associated with prion disease. Unlike PrPreS, however, PrPSc demonstrates a gradient of resistance to PK. PrPSc is associated with infectious potential and with prion disease even in circumstances where it may be sensitive to PK digestion.
SUMMAR Y 9 BASIC BIOMEDICAL RESEARCH The committee determined that the main obstacle to developing sensi- tive, specific antemortem diagnostics for TSEs is the lack of knowledge about prions and their normal cellular isoform, PrPC. Recommendation 3.1:3 Fund basic research to elucidate (1) the structural features of priors, (2) the molecular mechanisms of prion replication, (3) the mechanisms of pathogenesis of transmissible spongiform encephalopathies, and (4) the physiological function of prpc. The committee believes basic research in these four areas will supply the knowledge required to advance TSE diagnostics more quickly than ap- plied research alone. The report text describes specific gaps in knowledge that need to be filled in each of the four areas. For instance, present models of prion conformation and tertiary struc- ture are neither complete nor conclusive. Defining the structural differences between PrP isoforms could enable scientists to synthesize a PrPSc-specific antibody probe or aptamer. Defining the structures of prpC and PrPSc at the sites where they interact during binding and conformational change could support the development of molecules that would block those interactions. Most experts believe the conversion of prpC to PrPSc and the accumula- tion of prions require the assistance of one or more molecules (Caughey, 2001) that may be easier to detect than prions themselves. These unidenti- fied ancillary or chaperoning factors could serve as surrogate markers for prion detection and as drug targets for TSE therapeutics and prophylaxes. Current gaps in knowledge of the pathogenesis of prion diseases pre- vent better characterization of diagnostic targets and strategies. The routes of transmission, the factors that influence host susceptibility, the lack of an immune response, the mechanisms of neuroinvasion, and the cause or causes of cellular toxicity all lack satisfactory explanations; filling these gaps would result in tests with greater sensitivity and specificity. In addition, isolating the multiprotein complexes that contain prions might make it possible to identify new cofactors important to the formation and stabilization of PrPSc and infectivity. Understanding the normal role of prpC might also reveal associated molecules and pathways that would be appropriate detection targets for TSE diagnostics. Investigators should clarify whether the basis for nerve cell dysfunction and death in prion disease is related to the toxicity of PrPSc, to 3For ease of reference, the committee's recommendations are numbered according to the chapter of the report in which they appear. For instance, recommendation 3.1 is the first recommendation in Chapter 3.
10 ADVaNCING PRION SCIENCE the loss of function of prpC as a result of its conversion to PrPSC and its aggregation during a prion infection, or to other factors. Finally, the committee concluded that too great a focus on applied rather than basic research would slow progress in TSE diagnostics. TSE DL\GNOSllCS Obstacles to Developing Antemortem Diagnostics for TSEs Conventional methods used to diagnose most infectious diseases, such as malaria, tuberculosis, hepatitis, and AIDS, fail to detect prion diseases for numerous reasons. First, a prion is a host protein with an altered con- formation such that the immune system does not recognize it as foreign and does not produce antibodies against it. Second, since a prion lacks DNA and RNA, it cannot be identified by molecular methods such as polymerase chain reaction and other nucleic acid-based tests, nor can it be identified by such customary methods as direct visualization under a light microscope, cultivation in the laboratory, or detection of specific antibodies or antigens by standard immunological methods. Moreover, prions are largely in- soluble, distributed unevenly in body tissues, and found in a limited set of tissues by currently available tests. PrPSc is neurotropic, so it ultimately affects cells of nervous system tissues. Yet where and how PrPSc progresses through the body before its final assault on the nervous system are largely unclear, complicating the ability to locate and detect it. In addition, the similarities between prions and the normal host cellular protein prpC pose a fundamental problem. Since it is normal to find prpC in healthy individuals, detection tests must differentiate between the normal and abnormal prion protein molecules. The most common strategy thus far has been to mix the test material with the proteinase K enzyme (PK), which digests normal prion protein but only a portion of the abnormally folded protein. Various techniques are then used to detect the residual PrPSc after digestion. This process incidentally reduces the small amount of original PrPSc captured, however, making the process less sensitive than the newer experimental methods that do not rely on PK digestion. The fact that only small amounts of abnormal prion protein may be available for detection in accessible living tissues such as blood, urine, and cerebrospinal fluid challenges diagnosticians to develop a sufficiently sensi- tive test. Such a test must differentiate not only between normal and abnor- mal prion proteins, but also, for some purposes, between one or more strains of PrPSc a challenge resulting from basic deficiencies in the understanding of prion strain diversity and the nature of strain variation. The ultimate objective of a prion detection test is to detect a single infectious unit while avoiding a false-positive test result.
SUMMAR Y 11 Presently Available TSE Diagnostics The diagnostic assays available today are generally used only after the death of an animal or person. These assays test brain tissue, where the greatest concentrations of prions are found during the terminal stage of disease. Standard histopathological and immunohistochemical techniques are used to view the tissue microscopically and identify characteristic vacu- oles, plaques, or other abnormal features and staining associated with prion diseases. The standard confirmatory test is the Western blot. Attempts made to date to develop accurate, rapid, and highly sensitive antemortem tests, especially for detecting prions early in the course of infec- tion, have largely failed. Also, most tests still involve PK digestion, and the specificity and sensitivity of tests that do not use PK require further demon- stration. Newer tests appear to have improved the limits of detection. As of this writing, however, most of the newer detection methods are experimen- tal and have not been independently verified and reported. Moreover, the sensitivity of these tests must still be improved by several orders of magni- tude if they are to reliably detect an infectious unit of priors. Recommendation 4.1: Fund research to clevelop new assays most likely to achieve quantum leaps in the quality of prion detection tools, rather than incremental improvements to existing tests. Any efforts to improve existing tests shouic! aim to increase their sensi- tivities by several orclers of magnitude (at least 103~. The optimal test shouic! detect less than 1 infectious unit (IU) of PrPSc per unit of ultimate product used (e.g., 1 liter of blooc! or 100 grams of beefl. Recommendation 4.2: Fund research to improve in vitro tech- niques that amplify small amounts of PrPSc to enhance the sensitivi- · ~ · ~ ties ot c Agnostic tests. The Need for Novel Approaches to Developing TSE Diagnostics Major improvements in TSE diagnostics must await the availability of novel testing techniques or of reagents designed to specifically target PrPSc. Recommendation 4.3: Fund research to clevelop novel methods and reagents that detect or bind to priors, inclucling new antibodies, pepticles, nucleic acids, synthetic derivatives, and chimeric mol- ecules. This research couic! leac! not only to better diagnostics, but also to better therapeutic and prophylactic strategies. Researchers have attempted a variety of novel ways to improve the sensitivities of tests for TSEs. The most promising of these techniques are summarized below.
12 Surrogate Markers ADVANCING PRION SCIENCE A strategy other than the direct detection of PrPSc is to detect surrogate markers of prion infection. Cells that have been injured by prion invasion may produce other unique proteins or protein mixes that can be detected. The committee determined that the rapidly expanding field of proteomics may offer new tools for the development of highly sensitive prion detection tests employing such surrogate markers. This strategy is being used success- fully for the detection of certain cancers (Petricoin et al., 2002a,b), and the committee recommends that it be applied to TSEs. Recommendation 4.4: Fund research to identify surrogate markers or signatures for the detection of prions or prion diseases. Prion Amplification As indicated in recommendation 4.2, the committee also sees promise in strategies for amplification of PrPSc material before further testing (Saborio et al., 20011. Analogous to the polymerase chain reaction tech- nique for amplifying small amounts of DNA, these strategies could signifi- cantly boost the power of prion diagnostics. Cell Culture Assays In vitro culture systems have been used for prion detection with moder- ate success. Yet the committee believes these assays would hold great prom- ise if a stable and robust cell culture assay were developed. Their speed and biological simplicity would make them highly effective in testing for TSEs. Recommendation 4.5: Fund research to improve techniques for propagating prions in cultured cells and develop new in vitro cell systems as a means to assay and study priors. Clinical Diagnostics Although clinical criteria for the characterization of prion diseases have been established, they are adjunctive at present. Neuroimaging offers prom- ise as a future clinical diagnostic too! for prion diseases. The committee concluded that newer magnetic resonance imaging techniques, positron emission tomography scanning applications, and multiphoton microscopy should be developed for antemortem detection of TSEs. Recommendation 4.6: Fund research to develop functional imag- ing for the presence of PrPSc in brain tissue, leading to an early
SUMMAR Y diagnostic test similar to the imaging diagnostics being developed for Alzbeimer's disease. TESTING BLOOD FOR EVIDENCE OF TSEs 13 The ability to detect prions in blood would enable diagnosis and treat- ment of a TSE at an early stage of the disease, ideally before neuroinvasion. It would also allow a TSE-infected donor to be identified and deferred be- fore his or her blood is taken and administered to others as whole blood, a blood product, or a blood derivative. In addition, a test for TSE infectivity in blood could allow donors currently deferred because of potential expo- sure to a TSE agent to reenter the donor pool. In animals, such a test would permit early recognition of infection and timely control procedures. The committee believes the development of a screening test for detecting prions in blood is desirable for all these reasons, but will be extremely challenging to accomplish. The findings of TSE transmission studies in animals give reason to be- lieve that prions can be found in blood (Brown, 20011. Most of those stud- ies used the most sensitive form of in viva assay, an injection of blood directly into the brain, to see whether the animals would become infected; some did. In addition, a limited number of studies showed that blood from infected animals could infect by the intravenous route. Those studies more closely simulate exposure by blood transfusion. Recent compelling studies in sheep demonstrated that both the BSE and scrapie agents could be trans- mitted to other sheep by blood transfusion (Hunter et al., 2002), support- ing the hypothesis that blood can serve as reservoir for infectious priors. Multiple transmission and case-control studies have failed to demon- strate that blood from patients with sporadic Creutzfel~t-Takob disease (sCTD) is infectious (Brown et al., 19941. By contrast, investigations into the transmissibility of the vCTD agent through blood transfusion are just beginning to gain momentum. Because a significant amount of prions ap- pears in the lymphoreticular system in vCTD cases but not in other varieties of human TSEs, the blood of vCTD-infected individuals may contain priors. The amount of vCTD prions that might be contained in blood and the amount that constitutes an IU are important public health questions. The committee concluded that there is a small but unknown level of risk of acquiring vCTD from blood products; therefore, more research to clarify the nature of that risk should be conducted. Recommendation 5.1: Fund research (1 ) to determine the amount of sporadic Creutzfel~t-lakob disease (sCID) prions and variant Creutzfel~t-Jakob disease (vCJD) prions in human blood and (2) to
4 ADVANCING PRION SCIENCE estimate the amount of PrPSc corresponding to one infectious unit of sCID and vCID prions in human blood. The technical feat of developing a prototype test to detect low, and possibly changing, levels of prions in blood is an enormous challenge. But that is only one of the steps necessary to field a new commercial blood- screening test. To avoid false-positive test results, multiple testing schemes should be developed so that results can be confirmed or refuted. Stable, standard, and reliable testing reagents should be developed. Biotechnology companies must be properly structured to successfully mass-produce a novel test. Users will need to develop ethically sound counseling and notification policies for those tested, especially to deal with positive tests. Developers will need to demonstrate and document the test's performance rigorously enough to achieve FDA approval. Finally, a market for the product must exist, or be created, to attract commercial investment and manufacturing. These tasks are achievable with great resolve. SURVEILLANCE FOR TSEs IN THE UNITED STATES Surveillance for TSEs in humans and animals can permit the detection of potential outbreaks of known or new TSEs and the monitoring of those TSEs known to occur in the United States: CWD and scrapie in animals, and sCJD, iatrogenic CJD (iCJD), familial CJD, familial fatal insomnia, sporadic fatal insomnia, and Gerstmann-Straussler-Scheinker disease in humans. Surveillance for TSEs in Humans The Centers for Disease Control and Prevention (CDC) monitors the U.S. population for human TSEs. It also conducts surveillance specifically for vC}D (Belay et al., 2003), even though there has never been a reported case of BSE in the United States. Human TSEs are reportable in 12 states. Identification and Autopsy of TSE Cases The diagnosis of TSE in humans almost always requires a neuropatho- logical examination. These examinations are conducted for the U.S. popu- lation by the National Prion Disease Pathology Surveillance Center (NPDPSC). Yet at least half of the estimated total number of deaths caused by TSE in the United States are not autopsied and confirmed by laboratory examination (Gambetti, 2002; Holman et al., 1996~. The committee con- cluded that the large percentage of undiagnosed TSE cases represents a major obstacle to thorough U.S. surveillance for human TSEs. Physicians
SUMMAR Y 15 and public health officials need to identify more suspected TSE cases, and more of those cases should be autopsied and sent to NPDPSC for neuro- pathological examination. As noted earlier, the knowledge that BSE can cross the species barrier into humans has generated concern that CWD, which is epidemic in U.S. deer and elk, could potentially infect humans as well. If so, the human form of CWD could manifest itself clinically in a form quite unlike that of the known human TSEs. Therefore, the committee believes it would be prudent for CDC to conduct TSE surveillance on all atypical cases of neurodegenerative disease. Recommendation 6.1: Provide funds to promote an increase in the proportion of cases of human neurodegenerative disease, especially suspected cases of transmissible spongiform encephalopathy, that are recognized and autopsied. Epidemiological Research on Human TSEs Improving U.S. surveillance for human TSEs will also depend on infor- mation gleaned from epidemiological studies that help define the target population and hone survey instruments. Recommendation 6.2: Provide funds to increase the number and diversity of epidemiological studies on human transmissible spongiform encephalopathies (TSEs) in the United States. In par- ticular, support research to identify potential cases of variant Creutzfel~t-lakob disease and new human TSEs possibly caused by the agent of chronic wasting disease. Surveillance for TSEs in Animals Chronic Wasting Disease Although CWD has existed in deer and elk in Colorado and Wyoming since at least the 1960s (Miller et al., 2000; Williams and Young, 1980), the disease has apparently4 spread to captive and free-ranging herds in 10 other states and two Canadian provinces since 1996 (Fischer and Nettles, 2003; Williams and Miller, 20021. Nationwide surveillance for CWD is improv- ing thanks to increased awareness of the disease and the availability of more resources, including a network of USDA laboratories that conduct neuropathological examinations on possible and probable CWD cases. 4It is also possible that increased awareness of CWD has brought preexisting cases to light for the first time.
16 ADVANCING PRION SCIENCE In addition, a multijurisdictional joint working group formed in re- sponse to a congressional mandate published a plan in 2002 for controlling the spread of CWD in the United States (CWD Task Force, 20021. The committee urges NPRP to support research proposals that complement the nationwide surveillance initiatives implemented by the joint working group. Recommendation 6.3: Provide funds to support the development of a nationwide surveillance system for chronic wasting disease in the United States. Bovine Spongiform Encephalopathy and Scrapie USDA runs surveillance programs for the other two animal TSEs of concern in this country: BSE and scrapie. There is no evidence that humans can contract TSE from eating scrapie-infected sheep tissue or from contact with sheep. As discussed above, humans can contract vCTD from BSE-in- fected beef products, but no U.S. case of BSE has been reported to date. USDA has continued to improve its BSE surveillance program, espe- cially in response to concerns about the economic and health consequences of an outbreak of BSE in U.S. cattle. The program targets the subpopula- tions of adult cattle most likely to have BSE. USDA tested 19,990 cattle brains for the disease in 2002 and 11,152 brains during the first 4 months of 2003 dramatic increases over the 5,272 brains examined in 2001 (Ani- mal and Plant Health Inspection Service, 20031. Research to Improve U.S. Capabilities to Conduct TSE Surveillance Many unanswered questions regarding the epidemiology, natural his- tory, and prevalence of animal and human TSEs in the United States limit the nation's ability to conduct the best possible surveillance for these dis- eases. First and foremost, NPRP should fund laboratory and epidemiologi- cal research to determine the likelihood that the infectious agent of CWD (PrPCWD) could infect humans. The transmissibility of PrPCwD to nonhu- man animal species, such as cattle, should also be studied further. Second, scientists must determine the characteristics of a host, an infectious agent, and the environment that affect an animal's susceptibility to a TSE. Third, the mechanisms of exposure to the infectious agent in nature, the routes of transmission, and the way the agent invades the host's central nervous sys- tem must be defined. Fourth, scientists must develop surveillance systems that are sensitive enough to identify new TSEs and new TSE phenotypes. Recommendation 6.4: Provide funds to expand research on the natural history, prevalence, distribution, exposure and transmis- sion characteristics, host susceptibility, and host range of transmis-
SUMMAR Y sible spongiform encephalopathies, especially chronic wasting dis- ease. ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 17 The only reliable strategy for protecting animals and humans from the infectious agents of TSEs is avoidance. To date, a few techniques applicable in specific circumstances have successfully removed or inactivated TSE agents from the principal avenues of transmission: food, animal feed, trans- planted tissue, and blood products. No successful therapy has yet been de- veloped. Therefore, USDA and FDA have enacted many regulations, some enforced more effectively than others, to prevent human and animal expo- sure to TSE agents. Preventing TSEs from Entering the Food Chain The U.S. formula for preventing exposure to TSEs through human food or animal feed has three elements (PL 107-9 Federal Inter-agency Working Group, 20031: 1. Do not import live animals or animal products possibly infected with a TSE agent. 2. In case a TSE agent penetrates U.S. borders, have measures in place that will prevent its spread. 3. Protect U.S. residents from possible exposure to TSE agents in food and other products that come partially or completely from potential carri- ers of TSE agents. Policies to Prevent the Entry and Spread of BSE The United States applies the above formula primarily to the infectious agent of BSE through four policies: the import ban, the feed ban, slaughter surveillance, and the ban on central nervous system (CNS) tissue and CNS- associated tissue in beef and beef products. Most of these policies have been challenging to enforce. A 2002 report of the U.S. General Accounting Office highlights several policies, especially the feed ban, that lacked adequate enforcement at the time the report was researched (GAO, 20021; full compliance with some of these policies has yet to be achieved. By contrast, a BSE risk assessment published in 2001 concluded that U.S. policies make this country highly resistant to the intro- duction and spread of the BSE agent (Harvard Center for Risk Analysis and Tuskegee University Center for Computational Epidemiology, 20011.
18 ADVANCING PRION SCIENCE Policy recommendations are beyond the scope of this committee's charge. However, further research could help beef processors detect rumi- nant CNS tissue in their products. USDA's Food Safety and Inspection Ser- vice (FSIS) tested end-product samples from 34 U.S. beef processors in 2002 and found that 35 percent of the samples contained CNS and CNS-associ- ated tissues (FSIS,2003~. Consequently, FSIS issued a more stringent en- forcement directive for the CNS tissue ban (FSIS,2002~. The most effective rapid CNS test available for beef processors cannot distinguish between CNS tissue from ruminants and that from other animals, such as pigs, which do not contract TSE (personal communication, D. Cliver, University of California, Davis, 2003~. Recommendation 7.1: Fund research to improve rapid, accurate, and affordable screening assays for central nervous system (CNS) tissue such that the assays can specifically identify CNS material from cattle in processed meat products. Economic Impact of ~ Single BSE Case The consequences of a single case of BSE discovered in a Canadian cow in May 2003 offered a glimpse of what the United States might experience if a case of BSE were to occur here. Separately, a federal interagency work- ing group concluded that a single case of BSE in the United States could have a greater economic impact on this country than the BSE epidemic has had cumulatively on the United Kingdom (PL 107-9 Federal Inter-agency Working Group, 2003~. Multiple Opportunities for Human Exposure to the CWD Agent While the United States has conducted three BSE risk assessments since 1991, it has conducted none to determine the risk of human exposure to the agent of CWD. It is possible, though unproven, that human consumption of deer or elk meat infected with the CWD agent could cause TSE. The pro- cessing of cervid meat is monitored only superficially, and processors gener- ally make independent decisions about what precautions to take (Means, 2003~. Moreover, some meat processors handle both game and beef, creat- ing a theoretical opportunity for cross-contamination. The poorly under- stood risks of the CWD agent should be characterized scientifically. Recommendation 7.2: Fund risk assessments that characterize the exposure of hunters, cervid processing establishments, and consum- ers to the infectious agent of chronic wasting disease.
SUMMARY 19 Preventing TSE Transmission Through Blood Products and Transplants Neither prions nor TSE infectivity has been detected in human blooct, blood products, or blood derivatives. Yet these substances remain theoreti- cal vehicles for the transmission of human TSE agents, especially the agent of vCTD, because scientists have transmitted natural scrapie and BSE to healthy sheep by blood transfusions (Hunter et al., 2002~. The FDA's Center for Biologics Evaluation and Research (CBER) man- ages blood safety issues for the U.S. population. As a result of lessons learned from the AIDS epidemic, CBER has tightened the entire framework for evaluating and ensuring blood safety (Hoots et al., 2001) and has instituted many policies to protect the blood supply from TSE infectivity. In the absence of a test to screen blooci donors for TSEs, the next-best approach is to inactivate or remove any prions in donated blood. A promis- ing too! for removing prions from plasma products and their derivatives appears to be a filter with pores 15 nanometers in diameter, which experi- mentally removed enough prions in blood to eliminate infectivity (Tateishi et al., 2001~. This filter should be validated further, however. Certain steps in the commercial processing of blood into plasma and its derivatives appear to reduce but not eliminate TSE infectivity in blood (Brown et al., 1999; Trejo et al., 2003~. The experimental use of gamma radiation to inactivate prions in blood also has reduced but not eliminated infectivity (Miekka et al., 2003~. Recommendation 7.3: Funs} research to develop novel methods for removing protons Prom or inactivating prions in blood products and tissues In vitro, using physical, chemical, or immune mechanisms alone or in combination. In contrast to blood, blood products, and blood derivatives, certain human tissues and hormones have been documental to transmit TSE agents from an infected human donor to a healthy human recipient. Present FDA regulations and guidance to industry have reduced the rate of this form of TSE transmission in the United States. The medical literature re- ports 267 human cases of iatrogenic transmission of TSE by tissues and hormones (Brown et al., 2000a)- all tragic cases, but small in number by global standards. Inactivating Prions The committee believes avoiding TSE infectivity is a preferred strategy, but is at times impractical. People working in research and reference labo- ratories, hospital operating rooms, and veterinary clinics may have little choice about their proximity to the TSE agent. Thus, it is important to
20 ADVANCING PRION SCIENCE develop methods to inactivate the agent on surfaces. Researchers in this area have focused on inactivating or destroying priors, primarily PrPSc. Standard biosafety methods that eliminate bacterial, viral, and fungal pathogens do not work with priors. Despite their relatively small size, prions are one of the most durable organic substances known. Even turning prion- containing animal tissue to ashes by heating it in a crucible at 600°C does not eliminate infectivity (Brown et al., 2000b). Investigators have tried to inactivate prions using many physical, chemical, and radiological treatments, with just a few successes (Taylor, 2000~. Combinations of steam heat and chemicals appear to work best. Some substances can reduce the prion load, but few can eliminate prions reliably and consistently. The inactivation of prions on glass or steel sur- faces appears to be particularly difficult and is a major infection-control concern in hospitals, where surgeons could use certain instruments to op- erate on prior-infected tissue in patients with preclinical TSE. In the ab- sence of reliable methods to clean instruments, disposal is the only depend- able way to prevent contamination, and that is a very expensive option. Hospitals need less costly techniques for preventing the inadvertent spread of TSEs among patients. Recommendation 7.4: Fed research to develop standard assays for the detection of PrPSC or TSE infectivity on the surfaces of reus- able medical instruments and materials, as well as research to de- velop better methods to disinfect such instruments and materials. The inactivation of prions contaminating a relatively small contained space, such as a laboratory work surface, operating room table, or surgical instrument, poses challenges. Far greater challenges are posed, however, by the inactivation of prions in a large contained space, such as an abattoir, or a large open space, such as a pasture. Knowledge of the natural degradation of prions in the environment is extremely limited. Standard methods for evaluating the presence and decay of TSEs in the natural environment are not available, and this has hampered our understanding of the threat posed by long-term environmental contamination by priors. Recommendation 7.5: Fund research to develop standard test methods for detecting prion contamination in environmental samples. The longevity of TSE infectivity specifically in soil has tremendous im- plications for the proper disposal of animals infected with a TSE agent and for these animals offal and rendered material. For years, the burial of dead farm animals has been a common, acceptable practice. However, large- scale burial of animal material potentially contaminated by TSE agents poses a significant risk (Scientific Steering Committee, 20031. Present research on
SUMMAR Y 21 this subject has not progressed far enough to provide a sufficient scientific foundation for policy making. Therefore, the United States should intensify research into disposal methods for prior-infected animals and animal tis- sues. Given that the United Kingdom and Europe need such methods ur- gently, this area of research appears ripe for international collaboration. Recommendation 7.6: Fund research to identify safe, cost-effective disposal mechanisms for animals and rendered waste infected with agents of transmissible spongiform encephalopathies. This research would best be conducted with a multidisciplinary approach involv- ing experts in such fields as prion biology, biochemistry, environ- mental engineering, and commercial disposal technology. Since the destruction of prions is so difficult, an alternative approach to prevention is to change host resistance to prion infection. This is the con- cept of immunization, which has been so successful in combating other infectious diseases (CDC, 19991. As discussed above, prions are made from normal host protein that is misfolded, so the host does not recognize them as foreign or mobilize a typical immune response as it does with pathogens containing DNA or RNA. This stymies the use of vaccines to prevent TSEs because vaccines work by exploiting the body's natural immune response. Nevertheless, studies in mice show some marginal positive effects in pro- longing life following prion challenge (Sigurdsson et al., 20021. Treatment for Human TSEs Many therapeutic agents used to treat human TSEs have produced dis- appointing results (Brown, 20021. Treatment barriers include the inability to diagnose TSEs in early preclinical stages, the appearance of clinical signs only after advanced CNS infection, the failure of many agents to cross the blood-brain barrier, intrinsic toxicity associated with the therapeutic agents, and difficulties in translating promising therapeutic results from the research laboratory to the clinic. Studies in cell-free systems, in cell cultures, and even in mice have shown reduction or even clearance of prions following treatment, yet these results do not guarantee a cure for humans. To date, no drug or other agent has demonstrated consistent or prolonged success in treating human TSEs. Some sophisticated, cunning experiments in animals and animal cells have demonstrated that certain modifications to the Prep gene can delay or prevent the development of TSE infectivity in a host challenged with a TSE agent (Holscher et al., 1998; Meier et al., 2003; Perrier et al., 20021. Although the array of effects of altered genes on molecular functions is not completely understood and can lead to unintended consequences, these studies help uncover important linkages between genomics and
22 ADVANCING PRION SCIENCE proteomics, building a stronger foundation for future endeavors to de- velop TSE therapeutics. The committee believes the most promising candidates for near-term therapeutics are synthetic products such as peptides, antibodies, or anti- body fragments (scFvs) that bind to specific epitopes on prpC or PrPSc and disrupt further PrP conversion (Enari et al., 2001; Heppner et al., 2001; Peretz et al., 2001; Sigurdsson et al., 2003; White et al., 20031. To realize the promise of these synthetic products, researchers will need to uncover and target specific structural sites on PrPC, PrPSc, or intermediate molecules involved in PrP conversion. Recommendation 7.7: Fund research to develop new therapeutic agents, including antibodies, that either block the conversion of prpc to PrPSc or disrupt the molecular mechanisms of pathogenesis of transmissible spongiform encephalopathies after this conversion has taken place. The most promising approach appears to be ra- tional drug design, which begins with knowledge of the tertiary structure of the protein or molecule that the therapeutic agent will target. The clues that lead to success in diagnostics will help uncover new thera- peutic agents. Likewise, having preclinical diagnostic tests for TSEs will make early treatment and a better prognosis possible. RESEARCH INFRASTRUCTURE The committee determined that prion science would advance more rap- idly in the United States if more investigators worked in this small research community and if more funds were consistently available. The U.S. infra- structure for TSE research, though of high quality, is small compared with its European counterpart. At present fewer than 20 principal investigators conduct prion research funded by the National Institutes of Health (NIH), the largest sponsor of TSE research in the United States. In fiscal year 2002, NIH spent $27.2 million of its total budget of $23.2 billion on TSE research (personal communication, R. Zalusky, National Institute of Neurological Disorders and Stroke, May 27, 20031. Furthermore, 75 percent of the NIH funds awarded for TSE research go to only two laboratories (personal communication, R. T. Johnson, special consultant to NIH on TSEs, 20021. Investigators and Facilities It has been difficult to attract new investigators to prion research for several reasons. First is the lack of available laboratory space and the high start-up costs associated with setting up a TSE research laboratory. Such
SUMMAR Y 23 laboratories have special containment requirements and rely on costly labo- ratory animals and dedicated equipment that cannot be shared with other researchers because of concerns about cross-contamination. Second, confu- sion exists within the TSE research community about biosafety-level (BSL) requirements. Formal standards should replace the presently informal and inconsistent guidance in this area. Third, it often takes years to reach ex- perimental end points because prion diseases have relatively long incuba- tion periods. This makes it difficult to attract doctoral and postdoctoral fellows, whose academic programs last for a relatively short time. Recommendation 8.1: Provide funds to attract and train more in- vestigators in prion disease research. In addition, for investigators conducting prion bioassay research, provide grants for 5- to 7-year periods. Recommendation 8.2: Provide funds to boost the capacity of the U.S. infrastructure for research on transmissible spongiform en- cephalopathies by expanding or upgrading existing laboratories, animal facilities, and containment laboratories (biological safety levels 2 and 3), and by building new ones. Recommendation 8.3: Provide funds to develop scientifically based biological safety level standards for laboratories conducting re- search that involves infectious agents known to cause transmissible spongiform encephalopathies. If embraced by the community of organizations that fund TSE research, nationwide BSL standards could serve as a common, consistent framework for regulations governing laboratory biosafety for the full spectrum of TSE research. Reagents Because prion biology is a relatively new science, many of the reagents and other materials used by investigators are not commercially available, so each laboratory has needed to produce its own. Consequently, standardiza- tion of these reagents and materials across different laboratories or even within the same laboratory is lacking. As a result, the experimental conclu- sions reported by investigators can be difficult to replicate and easy to chal- lenge. The committee views this as an area that would benefit from atten- tion and funding. Recommendation 8.4: Provide funds to support new or established transmissible spongiform encephalopathy (TSE) repositories that
24 ADVANCING PRION SCIENCE contain a collection of reference materials and genetically engi- neered animals (including transgenic mice), as well as reagents use- ful for developing TSE diagnostics and for other TSE research. All registered investigators involved in prion research should have ac- cess to these collections. Recommendation 8.5: Provide funds to support the U.S. Food and Drug Administration's development of panels of reference reagents needed to evaluate the performance characteristics of tests designed to detect the prion protein and TSE infectivity. These panels would be used to confirm the performance characteristics of test kits be- fore they are approved for public use, as well as to perform quality control on test kit lots before their release to the market. International Collaboration An additional strategy to improve TSE research capacity is to find more opportunities for U.S. researchers to work with investigators in Europe and elsewhere. The committee believes that exploiting opportunities for U.S. investigators to conduct TSE research on site in a European laboratory or to work in a collaborative fashion with a European investigator on a joint research project is not only feasible but also highly desirable. Recommendation 8.6: Provide funds to enable U.S.-based investi- gators of transmissible spongiform encephalopathies (TSEs) to col- laborate or train with TSE investigators internationally and to use TSE research facilities abroad. Exploiting such opportunities will expand the range of TSE research that U.S. scientists can conduct. THE RISK OF TSEs TO THE U.S. MILITARY The committee deliberated upon the risk of prion infection to members of the U.S. military and their families who are or have been deployed to Europe. Discussions focused mainly on the possibilities for consumption of beef products or transfusion of blood products contaminated by priors. The food and blood supplies of deployed U.S. troops are closely controlled and generally originate at U.S. sources. However, some beef products sold in U.S. military commissaries and post exchanges (commonly known as PXs) in Europe were procured from suppliers in the United Kingdom and continental Europe that later reported cases of BSE among their livestock. In exceptional circumstances, some therapeutic blood is also obtained from a host nation's medical facilities.
SUMMAR Y 25 The committee determined that the U.S. military is at an increased risk for acquiring vCTD as a result of its deployment to Europe. However, that risk was judged to be small and certainly less than that of the local popula- tion in the United Kingdom and other European countries reporting BSE. Recommendation 9.1: Use existing passive surveillance systems to monitor the incidence of Creutzfel~t-lakob disease and variant Creutzfel~t-lakob disease among individuals receiving medical care from the health systems of the U.S. Department of Defense and the Department of Veterans Affairs. CONCLUSION This final report of the IOM Committee on Transmissible Spongiform Encephalopathies: Assessment of Relevant Science provides an unprec- edented overview of the ways in which TSEs impact human and animal health from donated blood to deer hunting. The committee's recommen- dations are intended to provide guidance to the NPRP on the most pressing TSE research needs. This report should also serve as a useful reference for those interested in TSEs worldwide. Educated laypeople, physicians, scien- tists, and TSE experts should all find herein relevant and readable informa- tion about deadly diseases that appear to be caused by a new and mysteri- ous biological mechanism.
26 ADVANCING PRION SCIENCE TABLE S-1 Committee Recommendations by Functional Area and Priority for the National Prion Research Program Recommendation Priority (1 = highest) Basic Research 3.1 Fund basic research to elucidate: (1) the structural features of prions (2) the molecular mechanisms of prion replication (3) the mechanisms of pathogenesis of transmissible spongiform encephalopathies (4) the physiological function of prpC Improving Diagnostics Fund research to: 1 4.1 Develop new assays most likely to achieve quantum leaps in the quality of prion detection tools, rather than incremental improve- ments to existing tests. Any efforts to improve existing tests should aim to increase their sensitivities by several orders of magnitude (at least 103). The optimal test should detect less than 1 infectious unit (IU) of PrPSc per unit of ultimate product used (e.g., 1 liter of blood or 100 grams of beef). 1 4.2 Improve in vitro techniques that amplify small amounts of PrPSc to enhance the sensitivities of diagnostic tests. 4.3 Develop novel methods and reagents that detect or bind to priors, 1 including new antibodies, peptides, nucleic acids, synthetic derivatives, and chimeric molecules. This research could lead not only to better diagnostics, but also to better therapeutic and prophylactic strategies. 4.4 Identify surrogate markers or signatures for the detection of prions or 3 . prlon c .lseases. 4.5 Improve techniques for propagating prions in cultured cells and develop new in vitro cell systems as a means to assay and study priors. 4.6 Develop functional imaging for the presence of PrPSc in brain tissue, leading to an early diagnostic test similar to the imaging diagnostics being developed for Alzheimer's disease. Testing Blood for Evidence of TSEs 3 5.1 Fund research (1) to determine the amount of sporadic Creutzleldt- 1 Jakob disease (sCJD) prions and variant Creutzleldt-Jakob disease (vCJD) prions in human blood and (2) to estimate the amount of PrPSc corresponding to one infectious unit of sCJD and vCJD prions in human blood.
SUMMAR Y TABLE S-1 Continued 27 Recommendation Priority (1 = highest) U.S. Surveillance for TSEs Provide funds to: 6.1 Promote an increase in the proportion of cases of human neurodegenerative disease, especially suspected cases of transmissible spongiform encephalopathy, that are recognized and autopsied. 6.2 Increase the number and diversity of epidemiological studies on human transmissible spongiform encephalopathies (TSEs) in the United States. In particular, support research to identify potential cases of variant Creutzieldt-Jakob disease and new human TSEs possibly caused by the agent of chronic wasting disease. 2 2 6.3 Support the development of a nationwide surveillance system for 2 chronic wasting disease in the United States. 6.4 Expand research on the natural history, prevalence, distribution, exposure and transmission characteristics, host susceptibility, and host range of transmissible spongiform encephalopathies, especially chronic wasting disease. Assessment of Strategies to Prevent and Treat TSEs 7.1 Fund research to improve rapid, accurate, and affordable screening assays for central nervous system (CNS) tissue such that the assays can specifically identify CNS material from cattle in processed meat products. 1 7.2 Fund risk assessments that characterize the exposure of hunters, 3 cervid processing establishments, and consumers to the infectious agent of chronic wasting disease. Fund research to: 7.3 Develop novel methods for removing prions from or inactivating prions in blood products and tissues in vitro, using physical, chemical, or immune mechanisms alone or in combination. 7.4 Develop standard assays for the detection of PrPSc or TSE infectivity on the surfaces of reusable medical instruments and materials, as well as research to develop better methods to disinfect such instruments and materials. 2 2 7.5 Develop standard test methods for detecting prion contamination in 3 environmental samples. Continued
28 TABLE S-1 Continued ADVANCING PRION SCIENCE Recommendation Priority (1 = highest) 7.6 Identify safe, cost-effective disposal mechanisms for animals and rendered waste infected with agents of transmissible spongiform encephalopathies. This research would best be conducted with a multidisciplinary approach involving experts in such fields as prion biology, biochemistry, environmental engineering, and commercial disposal technology. 7.7 Develop new therapeutic agents, including antibodies, that either block the conversion of prpC to PrPSc or disrupt the molecular mechanisms of pathogenesis of transmissible spongiform encephalopa- thies after this conversion has taken place. The most promising approach appears to be rational drug design, which begins with knowledge of the tertiary structure of the protein or molecule that the therapeutic agent will target. Prion Research Infrastructure Provide funds to: 8.1 Attract and train more investigators in prion disease research. In addition, for investigators conducting prion bioassay research, provide grants for 5- to 7-year periods. 1 8.2 Boost the capacity of the U.S. infrastructure for research on transmis- sible spongiform encephalopathies by expanding or upgrading existing laboratories, animal facilities, and containment laboratories (biologi- cal safety levels 2 and 3), and by building new ones. Provide funds to: 8.3 Develop scientifically based biological safety level standards for laboratories conducting research that involves infectious agents known to cause transmissible spongiform encephalopathies. 8.4 Support new or established transmissible spongiform encephalopathy (TSE) repositories that contain a collection of reference materials and genetically engineered animals (including transgenic mice), as well as reagents useful for developing TSE diagnostics and for other TSE research. All registered investigators involved in prion research should have access to these collections. 8.5 Support the U.S. Food and Drug Administration's development of panels of reference reagents needed to evaluate the performance characteristics of tests designed to detect the prion protein and TSE 2 3
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