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This final installment in our feature series on prions and prion diseases focuses on current research efforts to understand mechansims of prion disease transmission and susceptibility. We also discuss efforts to develop technologies that will yield rapid and sensitive field diagnostic tests. Introduction |
In May 2003, a single sick cow in Alberta, Canada, sent meat futures and U.S. restaurant stocks plummeting. The eight-year-old cow had been slaughtered in January and its brain tissue sent to the UK where it was confirmed positive for Mad Cow Disease [BSE (Bovine Spongiform Encephalopathy), 1]. Although the cow never entered the food chain and all of the other cows in the herd were destroyed, the reaction to the news affected global markets and closed the United States border to Canadian beef imports.
In spite of the progress made in understanding prion biology, prion research assumes new importance as the economic and health havoc these diseases can wreak becomes more evident (2–4). Understanding the transmission of the prion diseases between species and developing rapid and sensitive diagnostic tests are key areas of current research.
The 1980s outbreak of BSE in Britain probably resulted from cows eating feed that contained material from Scrapie-infected sheep (5).
Since that outbreak, over 100 cases of variant Creutzfeldt-Jakob Disease (vCJD) have been diagnosed in
people who presumably ate prion-infected beef (6). Researchers are attempting to discover how prions cross the “species barrier” and
whether prion diseases of animals other than cattle can spread to humans.
The BSE prion spreads from cattle to humans at low frequencies, and some people are concerned that the prion that causes Chronic Wasting Disease (CWD) in deer and elk can do the same. Two studies investigated sporadic human prion disease in patients from Wisconsin and Colorado (USA) whose only risk factors were consuming venison. These studies found no link between chronic wasting disease and CJD (7,8). A third study indicated that the CWD-associated prion can convert normal prion protein from deer and elk to the infectious protein in vitro but that it was not
as efficient at converting the human or bovine proteins in this study. The
authors of this study suggest that a molecular mechanism of some kind limits the susceptibility of humans, cattle and sheep to CWD (9).
However, whether this mechanism is sufficient to prevent transmission of CWD to
humans at low frequencies is unclear. More work is needed to determine the nature of this
mechanism.
The human prion diseases CJD and vCJD present different patterns of pathology in the diseased tissues, and CJD is believed to arise spontaneously or from iatrogenic sources, while vCJD is believed to result from infection with the BSE prion. As of April 2003 all known cases of vCJD involve individuals in which both copies of the prion gene encode the amino acid methionine at position 129 (129MM) of PrPc (normal human cellular protein; PrPsc is the pathogenic, misfolded protein). Homozygosity at the same locus also seemed to predispose individuals to sporadic and iatrogenic cases of CJD. Collinge and colleagues at the Medical Research Council in Britain have produced a mouse that is a 129MM homozygote (10). These mice develop a disease similar to vCJD of humans when challenged with the BSE prion. Unexpectedly, they can also develop a phenotype similar to that of sporadic CJD as well.
The specific pathologies of each type of prion disease are attributed to distinct “strains” of prions, with each strain reflecting differences in protein conformation or modification, even though the primary amino acid sequences are identical. The vCJD and human CJD prions are thought to represent such “strains.” The transgenic mouse results challenge this line of thinking. Much work remains to determine the precise molecular differences that result in the strain “phenotypes” of prion diseases, and modeling the disease in mice with specific genotypes should lead to a better understanding of the factors that influence strain properties.
Population genetics research also provides clues to the relationship between host genotype and susceptibility to prion diseases. Kuru is a human spongiform encephalopathy described in tribes that practice cannibalistic rituals, such as the Fore tribe in Papua New Guinea. In a study of the Fore group, strong selective pressure for Methonine/Valine heterozygotes at position 129 is noted in women above age 50. These women have presumably participated in ritualistic cannibalistic feasts before such feasts were prohibited in the early 1950s. The genotypes of the unexposed population at this position are at equilibrium (11). Additional analyses suggest that the prion gene is under balancing selection pressure, perhaps in a manner similar to the sickle cell anemia gene, where there is a heterozygote advantage. If this is true, the results imply that prion diseases have been a major cause of death during human evolution, and that an individual’s genotype may predict risk for developing a prion disease.
In addition to understanding transmission of and susceptibility to prion diseases, diagnostics are another major thrust of prion research. The infamous Alberta cow was diagnosed by postmortem autopsy of the brain. Diagnostics for prion diseases rely on biopsy of neural tissue or infectivity experiments that require long incubation times. Prion detection using antibody-based assays is fairly rapid but still requires hard-to-get nervous tissue where prions are most highly concentrated. Increased sensitivity will be required to detect prion protein in easily accessible body tissues or body fluids such as blood or urine (12) to develop rapid and reliable field tests for infectious prion proteins.
Forensic scientists have benefited from PCR technology that is used to amplify DNA present in tiny quantities in a sample. Amplifying the number of prions from a sample would increase the sensitivity of diagnostic tests and allow analysis in much the same manner. Saborio et al. developed a cyclical, in vitro method for amplifying prions that is conceptually similar to PCR (13). Instead of relying on an enzyme to catalyze a reaction, this method (protein-misfolding cyclic amplification; PMCA) relies on any minute quantity of pathogenic prion (PrPsc) present in a sample to nucleate the conversion of excess normal protein (PrPc). The process involves multiple cycles of PrPsc incubation followed by sonication. In the Saborio study, five cycles resulted in the conversion of 97% of the PrPc to PrPsc. If this assay is developed further, it may be used as a “front end” amplification procedure to detect prions in a variety of peripheral tissue samples.
A second technology that could allow prion detection from blood samples is RNA aptamer technology (12). Aptamers are single-stranded oligonucleotides of either RNA or DNA that assume a specific conformational structure, and as a result, bind very specifically to proteins or other small molecules (14). Developing an antibody that can distinguish PrPc from PrPsc has proven difficult, and aptamers may be better suited for this purpose. SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been used to find aptamers that specifically bind proteins, peptides and small molecules (15). Weiss et al. have used SELEX to screen an RNA aptamer library for aptamers that specifically recognize PrPc (16). An aptamer that specifically recognizes could be used to identify PrPsc in a variety of samples including blood and might even be used to clear PrPsc from contaminated samples (16,17).
As a result of prion diseases, Canada banned the import of U.S. deer and elk. Illinois temporarily banned deer from Wisconsin, and the U.S. banned the import of cattle from Canada. Almost every developed country has banned beef imported from Britain for at least a period of time. These draconian measures are directed at a group of diseases that are invariably fatal and that are not well understood. Once the mechanism of transmission of prion diseases between individuals and between species is better understood, scientists and governments can implement effective containment procedures that take advantage of rapid and sensitive diagnostics. And hopefully, one sick cow will no longer be able to send the global economy reeling.
References
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