1221 Scrapie: Strategies, Stalemates, and Successes SCRAPIE is a fatal degenerative disease of the central nervous system (CNS) which betrays none of the usual immunological or pathological signs that an active infection is in progress. The relevance to man of research on this slow infection of sheep was firmly established after the suggestion by HADLOW in 1959 that kuru might be a human equivalent:1 this, in turn, led to identification of Creutzfeldt-Jakob disease as another of the group.2 The transmission of scrapie to laboratory rodents in the early 1960s, and the recog- nition that there are many strains of scrapie, enabled investigators to focus on two issues-the nature of the infectious agents, and the nature of the disease pro- cesses. The occurrence of different strains establishes that the agent has a "genome,"3,4 but whether this is a nucleic acid or some new coding molecule remains speculative. Several workers regard knowledge of the molecular structure of these agents as a prime objective which may even lead to an understanding of the disease process. So far, however, the success of this strategy has been more in terms of defining the formidable technical obstacles to the purification of scrapie agent than in identification of its constituents and structure. Much of the difficulty stems from the practical limitation of having to use animal infection assays as the only means of detecting the agent and for estimating the number of infectious units. Other workers have turned this limitation to advantage by studying the disease in detail; and, so far, this has been the more productive strategy for medical exploitation, having yielded experimental models for studying the development of some characteristic features of dementias of the Alzheimer type-for example, neuritic amyloid plaques, and the selective loss of hippocampal neurones.5 This approach does not rest on any assumption that these common dementias 1. Hadlow WJ. Scrapie and kuru. Lancet 1959; ii: 289-90 2. Gibbs CJ, Gajdusek DC, Asher DM, Alpers MP, Beck E, Daniel PM, Matthews WB. Creutzfeldt-Jakob disease transmission to the chimpanzee. Science 1968; 161: 388-89. 3. Dickinson AG, Fraser H. Scrapie: pathogenesis in inbred mice: an assessment of host control and response involving many strains of agent. In: ter Meulen V, Katz M, eds. Slow virus infections of the CNS. New York: Springer, 1977: 3-13. 4. Bruce ME, Dickinson AG. Biological stability of different classes of scrapie agent. In: Prusiner SB, Hadlow WJ, eds. Slow transmissible diseases of the nervous system, vol II. New York: Academic Press, 1979: 71-75. 5. Dickinson AG, Fraser H, Bruce ME. Animal models for the dementias. In: Glen AIM, Whalley LJ, eds. Alzheimer’s disease. Edinburgh Churchill Livingstone, 1979: 42-45. might also be unconventional infections (for which there is no substantial evidence6): the notion is simply that some common pathways are likely to be involved in specific types of CNS degeneration. There is agreement, from irradiation target studies, that scrapie agent is very small by viral standards.’ There is less accord on whether, despite its many peculiarities, it might be a very small, structurally conventional, virus8-that is, with a nucleic-acid core coding for proteins of one or more types, which provide a protective coat and often serve other functions (MEDAWAR’s "A virus is bad news wrapped in protein" is apt). Many workers prefer to rule out a virus, -and the reasons are several: the failure to recog- nise virions in scrapie-infected samples by electron microscopy; the apparent absence of neoantigens (though their production only at a low tolerogenic level might explain this); the very peculiar, slow and tightly controlled tempo of the disease, with the paradox of incubation exceeding normal lifespan in some extreme examples; and very high resistance of scrapie agent to physical and chemical inactivation, at least in crude homogenates. This point of how the scrapie genome may be protected is central to many of the research difficulties, but the failure to inactivate the infective agent with various nucleases, and its phenol sensitivity, dispose of one possibility-namely, that scrapie agent may be a viroid,9,IO a naked nucleic acid without a polypeptide product. Viroids rely on host components for replication and might cause disease by interfering with the host’s nucleic acid processing." I The simplest notion is that scrapie agent will eventually fit into the existing mainstream of biology, somewhere in the spectrum from viruses to viroids. Until complete purification has been achieved, the agent’s composition has to be deduced indirectly. There has been a recurrent difficulty in separating the agent from host cell components.9 It has some form of close association with cell membranes and, not surprisingly in view of this, aggregation of membrane components carrying the undislodged agent has been an obstacle. A common biochemical tactic, therefore, has been the empirical use of detergents, in the hope that one of them will separate remnant host molecules from what is perceived as "the agent"; but the use of too harsh a detergent reduces the infectivity. In these terms, the degree of purity of even the best preparation is still unknown. Some direct evidence supporting the general impression that proteins are required for the 6. Goudsmit J, Morrow CH, Asher DM, Yanagihara RT, Masters CJ, Gajdusek DC. Evidence for and against the transmissibility of Alzheimer’s disease. Neurology 1980; 30: 945-50. 7. Alper T, Cramp WA, Haig DA, Clarke MC Does the scrapie agent replicate without nucleic acid? Nature 1967; 214: 764-66. 8. Rohwer GR, Gajdusek DC. Scrapie-virus or viroid: the case for a virus. In: Boese A, ed. First International Symposium of Hertie Foundation. Weinheim: Chemie-Physik Verlag, 1979. 9. Millson GC, Hunter GD, Kimberlin RH. The physico-chemical nature of the scrapie agent. In- Kimberlin RH, ed. Slow virus diseases of animals and man. Amsterdam: North-Holland, 1976: 243-66. 10 Ward RL, Porter DD, Stevens JG. Nature of the scrapie agent: evidence against a viroid. J Virol 1974; 14: 1099-1103. 11. Dickson E. A model for the involvement of viroids in RNA splicing. Virology 1981; 115: 216-21.
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1221
Scrapie: Strategies, Stalemates, andSuccesses
SCRAPIE is a fatal degenerative disease of the centralnervous system (CNS) which betrays none of the usualimmunological or pathological signs that an activeinfection is in progress. The relevance to man ofresearch on this slow infection of sheep was firmlyestablished after the suggestion by HADLOW in 1959that kuru might be a human equivalent:1 this, in turn,led to identification of Creutzfeldt-Jakob disease asanother of the group.2 The transmission of scrapie tolaboratory rodents in the early 1960s, and the recog-nition that there are many strains of scrapie, enabledinvestigators to focus on two issues-the nature of theinfectious agents, and the nature of the disease pro-cesses.
The occurrence of different strains establishes thatthe agent has a "genome,"3,4 but whether this is anucleic acid or some new coding molecule remainsspeculative. Several workers regard knowledge of themolecular structure of these agents as a prime objectivewhich may even lead to an understanding of the diseaseprocess. So far, however, the success of this strategy hasbeen more in terms of defining the formidabletechnical obstacles to the purification of scrapie agentthan in identification of its constituents and structure.Much of the difficulty stems from the practicallimitation of having to use animal infection assays asthe only means of detecting the agent and for
estimating the number of infectious units. Otherworkers have turned this limitation to advantage bystudying the disease in detail; and, so far, this has beenthe more productive strategy for medical exploitation,having yielded experimental models for studying thedevelopment of some characteristic features ofdementias of the Alzheimer type-for example,neuritic amyloid plaques, and the selective loss of
hippocampal neurones.5 This approach does not reston any assumption that these common dementias
1. Hadlow WJ. Scrapie and kuru. Lancet 1959; ii: 289-902. Gibbs CJ, Gajdusek DC, Asher DM, Alpers MP, Beck E, Daniel PM, Matthews WB.
Creutzfeldt-Jakob disease transmission to the chimpanzee. Science 1968; 161:388-89.
3. Dickinson AG, Fraser H. Scrapie: pathogenesis in inbred mice: an assessment of hostcontrol and response involving many strains of agent. In: ter Meulen V, Katz M,eds. Slow virus infections of the CNS. New York: Springer, 1977: 3-13.
4. Bruce ME, Dickinson AG. Biological stability of different classes of scrapie agent. In:Prusiner SB, Hadlow WJ, eds. Slow transmissible diseases of the nervous system,vol II. New York: Academic Press, 1979: 71-75.
5. Dickinson AG, Fraser H, Bruce ME. Animal models for the dementias. In: Glen AIM,Whalley LJ, eds. Alzheimer’s disease. Edinburgh Churchill Livingstone, 1979:42-45.
might also be unconventional infections (for whichthere is no substantial evidence6): the notion is simplythat some common pathways are likely to be involvedin specific types of CNS degeneration.There is agreement, from irradiation target studies,
that scrapie agent is very small by viral standards.’There is less accord on whether, despite its manypeculiarities, it might be a very small, structurallyconventional, virus8-that is, with a nucleic-acid corecoding for proteins of one or more types, which providea protective coat and often serve other functions
(MEDAWAR’s "A virus is bad news wrapped in
protein" is apt). Many workers prefer to rule out avirus, -and the reasons are several: the failure to recog-nise virions in scrapie-infected samples by electronmicroscopy; the apparent absence of neoantigens(though their production only at a low tolerogenic levelmight explain this); the very peculiar, slow and tightlycontrolled tempo of the disease, with the paradox ofincubation exceeding normal lifespan in some extremeexamples; and very high resistance of scrapie agent tophysical and chemical inactivation, at least in crudehomogenates. This point of how the scrapie genomemay be protected is central to many of the researchdifficulties, but the failure to inactivate the infectiveagent with various nucleases, and its phenol sensitivity,dispose of one possibility-namely, that scrapie agentmay be a viroid,9,IO a naked nucleic acid without apolypeptide product. Viroids rely on host componentsfor replication and might cause disease by interferingwith the host’s nucleic acid processing." IThe simplest notion is that scrapie agent will
eventually fit into the existing mainstream of biology,somewhere in the spectrum from viruses to viroids.Until complete purification has been achieved, theagent’s composition has to be deduced indirectly.There has been a recurrent difficulty in separating theagent from host cell components.9 It has some form ofclose association with cell membranes and, not
surprisingly in view of this, aggregation of membranecomponents carrying the undislodged agent has beenan obstacle. A common biochemical tactic, therefore,has been the empirical use of detergents, in the hopethat one of them will separate remnant host moleculesfrom what is perceived as "the agent"; but the use oftoo harsh a detergent reduces the infectivity. In theseterms, the degree of purity of even the best preparationis still unknown. Some direct evidence supporting thegeneral impression that proteins are required for the
6. Goudsmit J, Morrow CH, Asher DM, Yanagihara RT, Masters CJ, Gajdusek DC.Evidence for and against the transmissibility of Alzheimer’s disease. Neurology1980; 30: 945-50.
7. Alper T, Cramp WA, Haig DA, Clarke MC Does the scrapie agent replicate withoutnucleic acid? Nature 1967; 214: 764-66.
8. Rohwer GR, Gajdusek DC. Scrapie-virus or viroid: the case for a virus. In: Boese A, ed.First International Symposium of Hertie Foundation. Weinheim: Chemie-PhysikVerlag, 1979.
9. Millson GC, Hunter GD, Kimberlin RH. The physico-chemical nature of the scrapieagent. In- Kimberlin RH, ed. Slow virus diseases of animals and man. Amsterdam:North-Holland, 1976: 243-66.
10 Ward RL, Porter DD, Stevens JG. Nature of the scrapie agent: evidence against aviroid. J Virol 1974; 14: 1099-1103.
11. Dickson E. A model for the involvement of viroids in RNA splicing. Virology 1981;115: 216-21.
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infectivity of scrapie agent has lately been offered byMCKINLEY and colleagues.12 Treatment of partlypurified brain homogenates with diethylpyrocarbonateremoved much of the infectivity, and subsequenttreatment with hydroxylamine restored, it.There is a logical barrier to the infectivity-assay
approach; but first a summary is needed of the moreincisive conclusions from irradiation studies. FromLATARJET’s analyses of the scrapie UV action-
spectrum,13 infectivity seems to be inactivatedvia two different types of molecule; the same is foundwith tobacco mosaic virus. This is what one would
expect if the infective units of scrapie consisted of asmall nucleic acid genome plus another molecule, suchas protein,. which was responsible for the agent’sinfective properties. One difficulty with the idea thatthe additional molecule could be a simple protein is thehigh resistance of scrapie agent to formalin: this wouldrule out some types of protein more firmly than others,and would indicate proteins or protein complexes withsmall numbers of exposed amino groups.An arrangement involving two components-a
genome plus some form of coating-thereforeconstitutes a close analogy with conventional viruses,where the coat, besides protection, often serves a
specific role in making viruses infective to particularhosts and cell types. But that is where the analogy mayend. One speculation is that the scrapie genome, beingvery small, needs only two functions-the ability to usehost processes for its replication, and the acceptance ofa coating of host molecules (which may well not beentirely specific) for its protection and infectivity; theterm virino has been proposed for this class. 14,15 If thiswere the case, some of the features of the disease and
problems with the research become self-evident. Therewould be no neoantigens; the "agent" would bedifficult to see; there would tend to be an initialbarrier to interspecific infection; and attempts to
separate the infective agent from all host moleculeswould be misconceived if the scrapie genome wasuninfective on its own. Disease could result either if thehost’s nucleic acid regulation or processing was dis-rupted, or if a vital function of the host was interferedwith during replication.In the 1930s it was generally thought that viruses
were proteinaceous, which is now easily seen as thetype of half-truth that puts the cart before the horse, byignoring the virus’s real genome. The rudimentarybiochemical techniques were part of the problem butthe intrinsic limitations of infectivity assays were
underemphasised: an example is STANLEY’s 1935
paper entitled "Isolation of a crystalline proteinpossessing the properties of tobacco mosaic virus".’612. McKinley MP, Masiarz FR, Prusiner SB. Reversible chemical modification of the
scrapie agent. Science 1981; 214: 1259-61.13. Latarjet R. Inactivation of the agents of scrapie, Creutzfeldt-Jacob disease and kuru by
radiations. In Prusiner SB, Hadlow WJ, eds. New York: Academic Press, 1979:387-407.
14. Dickinson AG, Outram GW. The scrapie replication-site hypothesis and its
implications for pathogenesis. In: Prusiner SB, Hadlow WJ, eds. Slow transmissibleviruses of the nervous system, vol. II. New York: Academic Press, 1979: 13-31.
The present logical stalemate with scrapiebiochemistry is, in essence, the same. There is noknown assay for "the scrapie agent", despite what isoften written. The only assay is for its infectivity: theUV irradiation data may well remain the more
informative, in showing that more than one molecularspecies is involved, until chemical assays are found forthe genome and any other necessary components of the
agent. A rigorous attempt to devise such an assay for theputative Creutzfeldt-Jakob agent genome, by means ofDNA nick-translation, has already been reported bythe MANUELIDIS’ team, but this was unproductive."
It is into this general context that the recent claim byPRUSINER must be set, that "Novel proteinaceousparticles cause scrapie". 18 He uses the neologism"prion" to describe the agent "because the dominantcharacteristics of the scrapie agent resemble those of aprotein". PRUSINER accepts that nucleic acids are notexcluded by existing data but, as others have done, goeson to propose various agent models excluding them.None of the latter can, however, account plausibly forthe various ramifications of the occurrence of differentstrains of scrapie. What PRUSINER’s group seems tohave demonstrated is that proteins of unknown
specificity, coded either by the host or in some form bythe scrapie agent, are necessary for infectivity, butwhether these are structurally integrated with thescrapie genome, or only in adventitious association
’ with it, is not known. The uncertainties at this stage aresuch that infectivity may be exclusively conferred onthe scrapie genome by relatively nonspecific host-coded molecules, in which case a biochemical
purification protocol will necessarily copurify one ormore of the infectivity determinants along with thegenome determinant. It will not be easy to distinguishthe possibility, with existing techniques, from anotherextreme model in which the genome is alwaysstructurally complexed with a single specific protein.In conventional virology it has, for example, beendifficult to prove whether host proteins are
incorporated into virions or merely adhere: vesicularstomatitis virus is one of the few cases of provenincorporation.The area of scrapie research which is now ripe for the
use of molecular biology is the host’s methods ofcontrolling the details of the disease. For this, a range ofscrapie strains are available-as tools, rather than asends in themselves. This is the wider framework inwhich scrapie should be seen-as a probe for analysingCNS degeneration generally, as in Alzheimer’s disease,irrespective of whether a similar slow infection is
thought to be at work. Indeed, the type of regulatoryprocessing and replication damage ascribed
speculatively to virinos could well be the types ofmolecular malfunctions which produce degenerative
16. Stanley WM Isolation of a crystalline protein possessing the properties of tobaccomosaic virus. Science 1935 81: 644-45.
17. Manuelidis L, Manuelidis EE. Search for specific DNA’s in Creutzfeldt-Jakobinfectious brain fractions using "nick translation". Virology 1981; 109: 435-43
18. Prusiner SB. Novel proteinaceous particles cause scrapie. Science 1982; 216: 136-44
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change in normal ageing. Other diseases caused byagents similar to that in scrapie are likely to be found,but there has been an unfortunate tendency to
speculate, without any rationale, that a greatassortment of diseases of unknown aetiology might alsobe unconventional slow infections. 18,19 Why are all theknown examples CNS diseases? According to oneinterpretation, different scrapie-like agents may targetonto any of the organ systems but this may only berecognised as disease in organs such as those of theCNS where the modest rate of damage is not
compensated for by cell turnover.
OBESITY: THE CANCER CONNECTION
IT is no news that obesity is bad news; the associations withcardiovascular, respiratory, and orthopaedic morbidity arewell known and readily explained. A positive correlationbetween body weight and cancer incidence is, however, lesswidely publicised and certainly cannot be regarded as self-explanatory. The link was established from actuarial recordsover forty years ago’ and has been upheld consistently eversince. Figures published in 1960 from the Metropolitan LifeInsurance Company of New York, for example, showed thatmen had a 16% and women a 13% excess mortality fromcancer if they were 20% or more above their ideal weight.2 2Several questions arise. Does the association hold for all
cancers? What is the mechanism? Is the cause excess bodyweight per se, or can we implicate a particular component ofthe diet or indeed some other feature of the lifestyle thataccompanies obesity? Is the effect reversible-i.e., will
slimming reduce the cancer risk? Only the first of these can beanswered with confidence. The overall age-specffic incidenceof malignant disease is about the same for populationscharacterised by subnutrition as for their counterparts in theU.S.A. or Western Europe where obesity threatens to
become the norm.3 Such international comparisons do, ofcourse, reveal great differences in occurrence rates for
particular tumours, and, where these differences have beenattributed to dietary factors, it is not always the over-fed whoare at a disadvantage. Among cancers that are much morecommon in third-world than in developed countries are thoseof oesophagus and liver, the former possibly associated withchronic deficiency of vitamin A,4 the latter with ingestion ofaflatoxin from badly stored cereals.5Nevertheless, surveys of mortality records from many
countries show a linear relation between the incidence ofcancers at several common sites (including colon, breast,endometrium, and prostate) and the average per caputconsumption of calories, fat, or meat protein. 6, These three
19. Gajdusek DC. Unconventional viruses and the origin and disappearance of kuru.Science 1977; 197: 943-60.
1. Tannenbaum A. Relationship of body weight to cancer incidence. Arch Pathol 1940;30: 509-17.
2. Metropolitan Life Insurance Company. Overweight, its significance and prevention.New York, 1960.
3. Doll R, Muir C, Waterhouse J, eds. Cancer incidence in five continents, vol II. Berlinand New York: International Union against Cancer, 1970.
4. Van Rensberg SJ. Epidemiological and dietary evidence for a specific nutritionalpredisposition to esophageal cancer. J Natl Cancer Inst 1981; 67: 243-51.
5. Linsell AC, Peers FG. Field studies on liver cell cancer. In: Hiatt HH, Watson JD,Winsten JA, eds. Origins of human cancer. Cold Spring Harbor Laboratories, 1977:549-56.
6. Lea AJ. Dietary factors associated with death rates from certain neoplasms in man.Lancet 1966; ii: 332-35.
7. Armstrong B, Doll R. Environmental factors and cancer incidence and mortality indifferent countries, with special reference to dietary practices. Int J Cancer 1975; 15:617-31.
factors are, almost invariably, closely correlated with eachother; but in careful studies of groups who (usually onreligious grounds) eat little meat or animal fat while
maintaining an adequate intake of calories and essentialnutrients,8,9 no specific association has been establishedbetween any major component of a "Western" diet and theincreased cancer risk.Much current work centres on the possibility that the total
amount of body fat influences the metabolism of potentialcarcinogens encountered as natural constituents of certainfoodstuffs or generated in the process of cooking or indeedintroduced as artificial food additives.l0 In the case of
hormonally influenced tumours (breast, endometrium,prostate) interest has centred at various times on the relationbetween obesity and circulating levels of oestrogens,prolactin, progestagens, or androgens. 10,11 Cancer of thecolon, on the other hand, has been attributed to lack of fibre,or bulk, in the modern diet which leads, in turn, to prolongedbowel transit time, an altered intestinal microbial flora, andenhanced production of carcinogens from bile acids. 12,13 Thefact that even this last, reasonably precise, hypothesis has yetto be formally validated speaks volumes for the logisticalproblems inherent in attempting to match biochemical
theory to epidemiological observation.On the issue of whether the influence of diet on cancer-
proneness is a continuous one or whether a pattern is set
during childhood, interesting, though conflicting, clues areprovided by studies on Japanese immigrants to the U.S.A. Astheir dietary habits changed towards those of their adoptivecountry, the incidence of colorectal cancer rose, even amongfirst-generation immigrants who were already adults whenthey left Japan. Breast-cancer rates, on the other hand, beganto rise only in second-generation immigrants who were rearedon a Western type diet. 14,15 5
A considerable body of data exists from animal experimentson the relation between body weight and malignant disease.Some caution must be applied in extrapolating the findings toHomo sapiens since most of the studies have been undertakenon laboratory strains of rats or mice which tend to be highlysusceptible to a wide range of spontaneous or induced
neoplasms, few of which correspond to the common tumoursof man. Nevertheless, the conclusion emerges withremarkable consistency that over-feeding increases both theincidence and the rate of progression of malignantdisease.16,17 Moreover, even when calorie restriction is
delayed until adult life, animals on such regimens tend to livelonger and to have fewer tumours than their overweight
8 Phillips RL, Garfinkel L, Kuzona JW, Beeson WL, Lotz T, Brin B. Mortality amongCalifornia Seventh-Day Adventists for selected cancer sites. J Natl Cancer Inst 1980;65: 1097-1107
9 Kinlen LJ. Meat and fat consumption and cancer mortality: a study of strict religiousorders in Britain. Lancet 1982; i: 946-49.
10. Armstrong BK The role of diet in human carcinogenesis with special reference toendometrial cancer In: Hiatt HH, Watson JD, Winsten JA, eds. Origins of humancancer. Cold Spring Harbor Laboratories, 1979: 557-65.
11. Reddy BS, Cohen LA, McCoy GD, Hill P, Weisburger JH, Wynder EL. Nutrition andits relationship to cancer. Adv Cancer Res 1980; 32: 237-45.
12. Burkitt DP, Walker AR, Painter NS. Effect of dietary fibre on stools and transit-timesand its role in the causation of disease Lancet 1972; ii: 1408-12.
13. Reddy BS, Hodges AR, Laakso K, Wynder EL. Metabolic epidemiology of large bowelcancer Cancer 1978; 42: 2832-38.
14. Haenszel W, Kurihara M. Studies of Japanese migrants. 1 Mortality from cancer andother diseases among Japanese in the United States. J Natl Cancer Inst 1968; 40:43-68
15 Buell P. Changing incidence of breast cancer in Japanese American women. J NatlCancer Inst 1973; 51: 1479-83.
16. Tannenbaun A, Silverstone H Nutrition and genesis of rumours In Raven RW, ed.Cancer; vol I London: Butterworth, 1957: 306-14.
17 Clayson DB Nutrition and experimental carcinogenesis a review. Cancer Res 1975;35: 3292-300
