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In 18th century Britain, the most important commercial product was wool, involving in one way or another nearly one fourth of the population, and as the century wore on, the industrial revolution and a growing dominance of mercantilism put fine wool at a premium. In this setting, a discussion took place in the British Parliament in 1755 about the economic effects of a fatal and spreading disease in sheep, and the need for government to do something about it. Thus begins the recorded history of scrapie.
Scholars of the disease describe its unpredictable waxing and waning in different countries (and sometimes in different regions of the same country) over the next two centuries in England, France, Germany, and central Europe. It is not clear where or when the disease actually first appeared, although there is a suggestion that it was already present in Northern Europe and Austro-Hungary before the beginning of the 18th century. In general, the 18th and early 19th centuries saw a rapid extension of scrapie as a result of the practice of inbreeding to improve the quality of wool, and then as the practice abated, scrapie declined during the later 19th century. Nowhere, however, did it entirely disappear, and in Scotland scrapie was actually first recorded during this period.
Around the middle of the 19th century, veterinarians in England, France, and Germany initiated the scientific study of scrapie, including systematic neuropathologic examinations, and efforts to identify an infectious pathogen. French veterinarians finally succeeded in 1936 in transmitting scrapie to healthy sheep. In a grand historical irony, this experimental study was accidentally confirmed at the same time in England as a result of an outbreak of scrapie in several hundred sheep that had been immunized with a vaccine prepared from the brain tissue of sheep, some of which were later discovered to have had scrapie. The transmissible nature of scrapie was thus established beyond any doubt, although debate about the interplay between environmental and genetic factors continues to the present day.
Throughout the 1940’s and 1950’s, the accelerating pace of veterinary research yielded many new discoveries about the behavior of the causative agent, including the remarkable observation that infectivity survived a dose of ionizing radiation that was incompatible with the biologic integrity of nucleic acid, an observation that later led to the idea that the agent might consist only of protein.
In 1959, this endemic disease of sheep, unknown or ignored by medical science, was proposed by an American veterinarian to be analogous to a newly described disease of humans, called kuru, an epidemic neurological disorder found only in the Eastern Highlands of Papua New Guinea. In 1963, experiments to detect an infectious agent in kuru succeeded in transmitting disease to chimpanzees after incubation periods of 18-20 months. In the meantime, a neuropathological study of kuru had suggested a resemblance to Creutzfeldt-Jakob disease (CJD), first described in the early 1920’s by two German neurologists whose names comprise its eponym. CJD was therefore also inoculated into chimpanzees, and transmitted disease within 12-14 months.
The years following these discoveries were consumed by studies of the physical and chemical properties of the infectious agent, its distribution and titer in tissues of infected animals, and its host range. Eventually, in the early 1980’s, it was discovered that brain tissue could be purified to the point that only a single protein (called PrP, or “prion protein”) remained associated with infectivity. To the surprise of everyone, this protein was encoded by a normal host gene, and not by a foreign invader. All that we have since learned from molecular biology has added to the presumption of a self-replicating protein as the core or even sole constituent of the infectious agent. In the past decade, many further studies have been undertaken in different countries and laboratories in an effort to determine the precise basis of infectivity in transmissible spongiform encephalopathy (TSE), and at the same time find some means to protect both humans and animals from becoming infected.
We have learned that PrP is not distinguished from the universe of proteins by any unique structural features, and that its primary structure is identical both in healthy and diseased individuals. However, in diseased individuals its three-dimensional structure is altered, changing from a “floppy” soluble protein to a “stiff” insoluble amyloid, rather like turning a chiffon curtain into a Venetian blind (Figure). We have also learned that although visible pathological changes occur only within the nervous system, the infectious agent is also invisibly present in many visceral organs. The major pathway after oral infections first involves the tonsils, intestinal lymphatic tissues, and spleen, from which it spreads along nerves to the spinal cord and brain.
After identifying the gene that encodes PrP, more than two dozen different mutations were identified as responsible for the familial form of CJD, Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). All occur in a Mendelian dominant pattern of inheritance (like brown eyes and blue eyes), and all are experimentally transmissible to laboratory animals. Evidently, these mutations increase to near certainty the likelihood that during a single lifetime, the protein will transform itself into an abnormal configuration, and once transformed, the altered protein sets in motion the cascade of molecular events leading to the generation of amyloid with the property of self-replication. This, at least, is the theory. Molecular manipulation has shown that mice with no PrP remain healthy, and are totally resistant to experimental infection and disease, thus opening the door to the possibility that genetic engineering techniques might be able to eliminate the gene in family mutation carriers, and so prevent the genetic forms of human TSE.
While these basic research studies were going on, three outbreaks of CJD tested our ability to foresee potential problems, and found it wanting. Beginning around the mid-1960’s, a procedure to extract growth hormone from pituitary glands had been sufficiently refined to permit large-scale production and distribution to hormone-deficient patients. Glands were obtained from cadavers at autopsy, and were pooled in batches of up to 10,000 for each production run. In 1985, CJD was reported in three US patients, leading to the immediate replacement of native hormone by a recombinant product. Despite this action, CJD has now been responsible for 140hormone-related deaths, chiefly in France, Great Britain, and the U.S., after longer and longer incubation periods (up to 30 years) dating from the period when native hormone was used. It is clear that even when the potential risk was appreciated (nearly ten years before the first case of CJD), most of the damage had already been done because of the decades long “lead time” between peripheral route infections and disease; moreover, screening criteria were not always effective in preventing the inclusion of pituitaries from unsuspected cases of CJD.
Almost coincident with the growth hormone-CJD outbreak, contaminated dura mater grafts were also discovered to have caused iatrogenic disease: since 1988, 110 neurosurgical cases have died from CJD, the contamination again resulting from inadequate donor screening criteria, and batch-pooling before or during processing of cadaveric tissue. The lessons learned from these tragedies have prompted much more stringent regulations governing the collection and use of human-sourced biologicals, particularly those originating from central nervous system tissues.
Scrapie, meanwhile, had been quietly biding its time, waiting for the moment when, through human carelessness or lack of foresight, it would again attain the front ranks of medical attention. That moment came in 1996, with the recognition in British young people of a “new variant” of CJD (nvCJD) that has since with near certainty been traced to the consumption of tissue from cattle infected with spongiform encephalopathy (BSE), they having in turn consumed meat and bone meal nutritional supplements contaminated with scrapie-infected rendered sheep carcasses. It appears most likely that changes in the animal rendering process that occurred around 1980 allowed the scrapie agent to survive and infect cattle, the carcasses of which were then recycled through the rendering plants, leading to ever greater levels of cattle-adapted infectivity in meat and bone meal, and eventually producing a full-scale BSE epidemic.
Recognition of this source of infection led to the imposition in 1988 of a ruminant feed ban that by 1992 had turned the epidemic around, but the loss of some 180,000 cattle to date has brought the British livestock industry to its knees. BSE has also echoed through the tallow, gelatin, and pharmaceutical industries, all of which make use of bovine-derived products for human use, and even the blood-bank community has been seriously affected by virtue of the uncertainty about infectivity in blood donations from patients incubating vCJD. There are presently just over 90 verified cases of vCJD in the UK (plus 3 in France and 1 in the Republic of Ireland), and the number continues to grow at the rate of about 10-20 new cases per year: whether they represent a small group of susceptible individuals, or the leading edge of a major epidemic is still moot.
Despite these battle scars from engagements in applied science, we can look back with some satisfaction upon the accomplishments in basic science during the century now drawing to a close, and expect that during the early years of the 21st century, most of the remaining uncertainties will be resolved. These can be grouped into four broad categories: precise characterization of the infectious agent, elucidation of the mechanism of agent replication; prevention or treatment of disease; and continued exploration for other candidate diseases.
Although PrP is beyond doubt a necessary component of the infectious agent, formal proof that it is by itself infectious is still lacking. Such proof may come from continuing attempts to demonstrate a parallelism between infectivity and test-tube conversion of normal to abnormal protein, or the creation of a synthetic PrP-like protein that by itself is capable of transmitting disease to experimental animals.
Precise characterization of the PrP amyloid will not solve the question of its “replication”. What is it about PrP amyloid (as distinct from other types of amyloid) that gives it the ability to replicate and transmit disease to new hosts? We know that the amyloid of Alzheimer’s disease also comes from a normal host protein that in diseased individuals accumulates in the brain, but it does not have the ability to transmit disease to a healthy individual. Why this difference?
Neither of these two unknowns need inhibit research into disease prevention and therapy, which may come from a more general understanding of the process of amyloid formation. Chemical manipulation of the cellular pathways involved in PrP metabolism, or interference with the protein transformation to amyloid could become viable therapeutic approaches, and efforts to arrest and even reverse amyloid accumulation in experimental models are already beginning to show promise. Similarly, manipulation of the PrP gene (or its expression) in familial forms of disease will become feasible when genetic engineers overcome the technical problems that have generally prevented the successful results obtained in mice to be duplicated in humans.
Finally, we must continue to keep alert to the possibility that other diseases without known cause may share the apparently unique pathogenic mechanism of TSE, and so be susceptible to the same therapeutic approaches. We should also be prepared to admit that however interesting as a biological phenomenon, “replicating proteins” may not be found to cause other more numerically important disorders, but may forever remain confined to the small group of presently recognized “prion diseases” that pose (for the moment) a comparatively minor burden to public health.