Histopathology 1992, 20, 1-1 1

INVITED REVIEW

From slow virus to prion: a review of transmissible spongiform encephalopathies P.L.LANTOS Department of Neuropathology, Institute of Psychiatry, London, UK Accepted for publication 26 August 1991

LANTOS P.1,.

( 1 992) Histopathology 20, 1-1 1

From slow virus to prion: a review of transmissible spongiform encephalopathies Spongiform encephalopathies include seven neurodegenerative diseases: three in man (Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease and kuru) and four in animals (scrapie, mink encephalopathy, bovine spongiform encephalopathy and chronic wasting disease in deer and elks). They are all transmissible to a variety of species, and man-to-man propagation of the diseases in the form of iatrogenic transmission has been welldocumented. The infectious agent is highly unusual and the pathogenesis of infection remains controversial. The term prion was introduced to describe the proteinaceous infectious agent. Purification of this agent yielded a unique sialoglycoprotein, associated with the neuronal cell membrane, which is all or part of the infectious agent. Molecular genetics revealed variations in the prion protein; these are linked to or associated with the inherited forms of spongiform encephalopathies: familial Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker disease. The histological triad of spongiform change, neuronal loss and astrocytosis dominate the histological picture of spongiform encephalopathies. A recent case which did not develop any of the histological hallmarks of disease, but did have genetic abnormalities typical of the disease, indicates that the true incidence of Creutzfeldt-Jakob disease may be considerably higher than previously accepted, and a combination of molecular screening and immunohistochemistry for prion protein should complement traditional neuropathology to establish the diagnosis. The descriptive term of spongiform encephalopathy may now have to be abandoned in favour of prion disease. Keywords: transmissible spongiform encephalopathy, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, kuru, scrapie, bovine spongiform encephalopathy, iatrogenic transmission, prion proteins

Introduction It is one of the most puzzling phenomena of contemporary medicine that a rare and previously obscure group of neurological diseases has become the subject not only of scientific controversy, but also of intense political debate. The name of these disorders has been frequently changed during the last decade or so, reflecting our increasing knowledge, from slow virus diseases through subacute spongiform encephalopathies to prion diseases, the term which is now gaining widespread, if not universal, acceptance. The history of these diseases is Address for correspondence: Professor P.L.Lantos. Department of Neuropathology. Institute of Psychiatry, London SE5 8AF, tJK.

one of the most fascinating chapters of biomedical research’. In 1920 Creutzfeldt* described progressive dementia in a 22-year-old woman. The next year Jakob3reported three middle-aged patients who died demented a few months to 2 years after the onset of the disease. Whilst Creutzfeldt’s case does not fulfil the histological criteria, it was Jakob who defined the pathology of this dementing condition which has later become known as CreutzfeldtJakob disease (CJD).In 1957, following their visit to the Highlands of Papua New Guinea, Gajdusek & Zigas4 described a new degenerative neurological illness, known to the natives as kuru, which affected some of the local tribes. The similarities between kuru and scrapie, 1

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a n infectious disease of sheep, particularly on histopathological grounds, were noted by Hadlow in 1959’. Kuru was soon also linked to CJD‘ and the infectious nature of these two degenerative diseases was first raised. This view was confirmed after Gajdusek et al.’ reported the experimental transmission of kuru to chimpanzees and Beck et aL8 described the histopathology of the brain of kuru-infected animals. A cerebral biopsy from a patient with CJD was transplanted into a chimpanzee which became ill and developed the same histological abnormalities in the brain9. It was then realized that CJD, kuru and scrapie shared a common neuropathology and all three were transmissible. The infectious agent was thought to be, at this stage, a virus with long latency, and the term slow virus disease, a concept originally introduced by Sigurdsson in 1954“’, was adopted to describe these conditions. The viruses have, however, proved extremely elusive and most unconventional. Despite thorough research, viral particles were not identified. apart from the occasional early false sightings in the electronmicroscope, nor were viral antibodies recovered. Equally puzzling for a virus, it did not cause any inflammation in the brain. The inability to find the causative virus has led to the change of nomenclature to spongiform encephalopathies, a somewhat cumbersome descriptive term which, at least, adequately describes the most prominent histological hallmark of the disease: the spongiform degeneration of the cerebral grey matter. Currently, seven spongiform encephalopathies are distinguished: three in man (CJD, Gerstmann-Straussler-Scheinker’s disease and kuru) and four in animals (scrapie, mink encephalopathy, bovine spongiform encephalopathy and chronic wasting disease of deer and elk). All seven diseases affect the central nervous system, have long incubation periods and are transmissible. They are all invariably fatal. The aims of this review are to give a brief account of the neuropathology of transmissible spongiform encephalopathies with particular emphasis on CJD, the only spongiform encephalopathy relevant to histopathological practice, and to examine areas of current interest: iatrogenic infections, the epidemic of bovine spongiform encephalopathy. developments in molecular genetics in relation to the causative agent and current safety measures.

Human transmissible spongiform encephalopathies CREUTZFELDT- J A K O B D I S E A S E

Creutzfeldt-Jakob disease (CJD)is a relentlessly progressive dementia of fatal outcome. It usually starts in the presenium and runs a subacute or chronic course; the

duration can vary between 3 weeks and 8 years, but most patients die within 6 months”. Both sexes are thought to be equally affected, although the most comprehensive survey in England and Wales has reported a female preponderance of 1.68 to 1”. The same study found the annual incidence rate 0 . 3 per million, and a large survey of 329 cases between 1968 and 1982 in France reported an annual mortality rate of 0.5-0.6 per million, increasing to 1.1-1.2 per million in Paris”. These figures are in accordance with the worldwide mortality of the disease apart from wellknown clusters of increased incidence amongst Libyan Jews and in Slovakia, Italy and Chile”. About 1 5 % of the cases are familial, with figures ranging from 6% in England and Wales, and JapanI5to 4 5% in ChileIh.Although these epidemiological surveys indicate that CJD is a rare illness, in the light of recent developments the worldwide incidence has to be reassessed. Clinical features Clinically, three subtypes have been distinguished”: subacute, intermediate and amyotrophic. The commonest is the subacute variety, in which the prodromal symptoms of malaise, altered personality, emotional lability and sleep disturbance are followed by deepening dementia, ataxia, myoclonus and cortical blindness. The patients die, profoundly demented and usually emaciated, of terminal respiratory infection. The amyotrophic variety is rare, and the patients develop lower motor neuron signs and neurogenic muscle atrophy, together with dementia. Originally this type was thought nontran~missible’~ and different from the subacute form: however, recent evidence18supports transmissibility. In the intermediate type the terminal stage of the disease is preceded by a long period of focal or diffuse neurological signs’ l . The CSF is usually normal. CT-scan and magnetic resonance imaging may show cerebral atrophy, but they are more helpful in excluding other disease. Electroencephalography (EEG) is of considerable diagnostic value, showing characteristic alterations in two-thirds of the patients: repetitive sharp waves or slow spikes are later followed by synchronous triphasic sharp waves against the background of progressively suppressed cortical activity”. For the practising histo- or neuropathologist who is asked to perform a necropsy on a patient with the clinical diagnosis of CJD, the most relevant data are: profound dementia of relatively short duration in a middle-aged patient, myoclonus, and EEG abnormalities.

Gross pathology, histology arid ultrastructure Postmortem examination usually reveals terminal bron-

Transmissible spongiform encepkalopnthies

chopneumonia and otherwise normal internal organs in a wasted body. The brain may be normal, but more often shows focal or diffuse atrophy: in severe cases weighing 1000 g or less. The neurodegenerative process may affect both the cortical ribbon and the deep grey matter of the central nervous system, and, according to the topographical distribution, cortical, corticospinal, corticostriatal, corticostriatospinal and corticostriatocerebellar forms are distinguished”). The histological features afflicting the grey matter are spongiform change, neuronal loss and astrocytosis (Figure 1).The pathology is not even, and relatively normal cortical areas may alternate with severely affected stretches in which the cortical architecture has been completely destroyed. The spongiform degeneration is characterized by fine vacuolation of the grey matter. the vacuoles then enlarge and coalesce to form microcysts. This microcystic cavitation of the cortex may lead to status spongiosus, and in the most severe cases the cystic cortex collapses to form irregular clefts. Status spongiosus, however, is not unique to CJD: it may develop in elderly individuals, or those with Alzheimer’s disease or other conditions in which there is extensive neuronal fall-out. Neuronal loss is variable: in advanced cases it is severe and is accompanied by astrocytosis. In the corticostriatal form the caudate nucleus and the putamen show changes, in the spinal cord the motor pathways are affected, whilst in the cerebellar variant small amyloid plaques also occur. It is important to note that pathology is not restricted to the grey matter: the white matter is also involved and may show spongiosis, loss of myelin and astrocytic proliferation. Electronmicroscopy reveals vacuoles in the cell body and processes of neurons and astrocytes. Interestingly, a recent ultrastructural study of experimental CJD in mice emphasized the importance of neuronal, particularly dendritic, vacuolation, whilst astrocytic change could result from less than optimal fixation of human material”. Spongiform changes in experimental animals are thought to be derived from the cisternae of neuronal smooth endoplasmic reticulum2’. Immunocytochemistry using antibodies to prion protein gives a strong positive reaction (Figure 2 ) and is a useful diagnostic tool, particularly in cases without the typical histological changes.

lrrtrogeriic irlfections The transmissibility of CJD and kuru has been established beyond doubt and a variety of laboratory animals, including guinea pigs, hamsters, mice, rats, cats, goats, ferrets and primates have been infected. Man-to-man infection as iatrogenic transmission via corneal transplants and stereotactic electrodes has been well-documented. A 55-year-old woman developed CJD 1 8

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months after she had received a corneal transplant from a donor who had died of the disease2j. A young man of 17 and a woman of 2 3 developed CJD after they had been treated by stereotactic surgery for intractable epilepsy. The electrodes used in the operations had been previously implanted into the brain of a 69-year-old woman who had suffered from CJD. The young man died demented, aged 1 9 , of CJD, whilst in the case of the woman the clinical diagnosis was supported by EEG24. Several further cases raised suspicion, rather than being conclusively proven, of accidental transmission from person to person (for review see Corsellis’ and WelterZi), including a 2 8-year-old woman who developed CJD following a neurosurgical operation which involved the grafting of commercially available dura mater”. In 1985, reports of patients who developed and died from CJD following the administration of human growth hormone caused considerable concern in both the US and UK. The two American cases, both in men in their 20s, precipitated the ban on the use of cadaver-derived pituitary growth hormone. One patient was a 20-yearold who had received human growth hormone for 1 3 years17.The other patient, aged 23, had had injections of growth hormone for 8 years. Neither case had intracranial surgery and the diagnosis of CJD was histologically confirmed2x.Two further cases were reported in the US: both were men, one 32 and the other 3 7 years of age, and both had received human growth hormone. In these two cases the disease developed a somewhat unusual clinical course, in that cerebellar signs dominated with little evidence of dementia. Histological examination, however, confirmed CJD29.30. A woman who died of intercurrent respiratory infection had preclinical CJD and, although she did not have signs of progressive neurologic disease, the corpus striatum showed histological changes typical of CJD3*.The first British case was a woman of 2 3 who had undergone neurosurgery for the removal of a craniopharyngioma at the age of 2 years. Subsequently she was treated with growth hormone for 4 years and died from histologically verified CJD32.33. Six cases have now been reported in the UKJ4: two in Southampton (an additional case to the first reported British case there); two in London, and two in Cambridge (Anderson 199 1, personal communication: Duchen 1991, personal communication; Weller 1 9 9 1, personal communication). Overall there are 1 9 cases worldwide, 1 7 caused by growth hormone and two by gonadotrophin for the treatment of infer ti lit^^^". The patients develop the disease at a much earlier age than noniatrogenic cases: the six British patients had an average age of 23 years, ranging from 2 0 to 34. The incubation period is 14 years on average, with a range of 4-19 years. The average duration of illness is 6-12 months.

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P.L.Lantos

Transmissible spongiforin encephalopathies 5

Figure 2. Creutzfeldt-Jakob disease: positive reaction (arrows) for prion protein in the cerebellum. (Antibody to prion protein was kindly provided by Dr J.Hope of the Institute of Animal Health, Edinburgh.) ABC.

The clinical onset is dominated by kuru-like cerebellar syndromes. There are histological differences from the classical CJD: the cerebellum tends to be severely involved and plaque formation is usually prominent. There is convincing evidence which incriminates CJDcontaminated growth hormone as the source of infection. CJD is extremely rare in individuals under 30 years of age; only nine cases were found amongst more than 3000 cases worldwide. Moreover, the chance of three cases occurring in the US in one year has been ~ a l c u l a t e dto ~ ~be 1 in Although an epidemic caused by growth hormone treatment has not materialized, the actual number of young individuals who will eventually contract CJD as a result of treatment is not known. Large-scale epidemiological follow-up studies are essential to estabIish the full extent of iatrogenic transmission.

GERSTMANN-STRAUSSLER-SCHEINKERD I S E A S E

The clinical picture of Gerstmann-Straussler-Scheinker disease (GSS) is dominated by cerebellar ataxia, pyramidal signs and dementia. In some cases ataxia and other cerebellar signs predominate, whilst in others dementia is the presenting and most pronounced feature. The average age of onset is 40 years and the average duration is 5 years (with a range from 1 to 11 years)much longer than that of CJD. The precise incidence of GSS disease is not known, but it is estimated to be between 1 and 10 per hundred millionj6. Its neuropathology. originally described by Scheinker, is characterized by extensive amyloid plaque formation and spongiform change in the grey matter. The morphology of plaques is intermediate between that of kuru and Alzheimer’s disease. In the cerebellum, which is most severely

Figure 1 , Creutzfeldt-Jakob disease. a Spongiform degeneration and neuronal loss in the frontal cortex. b Striking astrocytosis in the putamen is demonstrated by glial fibrillary acid protein irnmunocytochemistry (GFAP. Ilako, IJK). Spongiforrn degeneration is also noted. a H & E. b ABC.

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affected, they may have single or multiple cores, but the neuritic components are absent or sparse. In the cerebral cortex and hippocampus they may have the radiating crown of abnormal neurites, similar to those of Alzheimer's disease. The amyloid of GSS disease is clearly different from that of Alzheimer's disease: it does not stain with antibodies against PA4 protein, but is immunoreactive to antisera against prion protein 2 7-3rJj7. Spongiform change is usually neither as prominent, nor as extensive as in CJD. There is widespread neuronal loss which is most severe in the cerebellum. Astrocytosis is particularly noticeable in relation to neuronal fall-out and plaque formation. However, both the clinical and pathological pictures may vary considerably. Most reported cases are familial, and the disease appears to be inherited as an autosomal dominant condition, although transmission to animals suggests that a n infectious agent is involved3'. Thus, the major interest of GSS, despite its rarity, lies in its dual nature: it is a disease which is both inherited and infectious.

KUBC

This is now a disease ofhistorical interest. Kuru occurred mainly in the Fore tribe of Papua New Guinea and was transmitted by cannibalism. The cerebral hemispheres show similar changes to CJD. More importantly the cerebellum is atrophied, particularly the phylogenetically older parts of the vermis and flocculonodular lobe. Histologically, there is loss of Purkinje and granule cells, astrocytosis and fibrillary gliosis throughout the cerebellar cortex and microglial response in the molecular layer. Amyloid plaques are abundant. The neuropathology of kuru, together with other spongiform encephalopathies, has been comprehensively reviewed*".

Animal transmissible spongiform encephalopathies SCRAPIE

Scrapie, known for over 200 years, has a worldwide distribution. It affects the central nervous system of most breeds of sheep and goats. The disease afflicts both sexes and most often develops in breeding ewes between the ages of 2 and 4 years. Only the central nervous system is involved, and the most prominent histopathological feature is the vacuolation of neurons in the brainstem, including the medulla oblongata. Spongiform degeneration of the neuropil and astrocytosis also occur. Neuronal loss may be detected, whilst demyelination is either

absent or slight. In common with human transmissible spongiform encephalopathies, there is no inflammatory response. Scrapie-associated filaments derived from brain extracts can be demonstrated by negative staining in the electronmicroscope. Extensive research has established four hallmarks of the infectious agent. First, scrapie is transmissible by inoculation, with a n incubation period ranging from 1 to > 5 years, although in genetically susceptible lines of sheep the latency can be reduced to 5 months. Secondly, the infectious agent replicates in vivo and can be maintained indefinitely by successive passages in sheep. Thirdly, the agent can be recovered in a 410 nm membrane filtrate, but not after filtration through a finer 2 7 nm membrane. Finally, the usual methods of disinfection do not inactivate the agent (for review see Kimberlin3'). Scrapie can be transmitted from both goat4" and sheep", as well as hamster". Studies on experimental scrapie have revealed that many biologically different strains of scrapie exist and it is thought that some of these may undergo mutation3'. Although scrapie is undoubtedly a n infectious disease, genetic factors also play an important role in its pathogenesis. For example, the incubation perind cf experimental scrapie is controlled by a single gene sip with two alleles (sA and PA).Most sheep carrying the sA allele develop experimental scrapie, indicating that sA is dominant for susceptibility, with the incubation period being longer in heterozygotes than in homozygotes. Sheep homozygous for the pA allele are less likely to develop the disease.

TRANSMISSIBLE M I N K ENCEPHALOPATHY

Transmissible mink encephalopathy was first observed in a mink herd in Wisconsin in 194745 . It is a rare disease which is likely to be scrapie in mink. Not surprisingly, the histopathology is similar to that of scrapie. with the exception of neuronal vacuolation being less severe. Transmissible mink encephalopathy can be experimentally transmitted to mink and to other species, including Chinese hamsters, golden hamsters and squirrel monkeys, but apparently not to mice (for review see Kimberlin3').

BOVINE SPONGIFORM ENCEPHALOPATHY

The first cases of bovine spongiform encephalopathy (BSE) were reported by Wells and his colleagues44 in 1987 following the aftermath of the epidemic a year earlier. The brain of the affected animals showed fine vacuolation of neuronal processes, corresponding to the spongiform change of the neuropil of the grey matter,

Transmissible spongiforrn encephalopathies

whilst the neuronal cytoplasm contained clear, single or multiple spaces distending the perikaryon. Each of the two forms of vacuolar pathology had a distinct distribution pattern, but the brainstem was usually most severely affected. Quantitative assessment revealed that the mean vacuolar densities were greatest in the medulla, midbrain and thalamus, whilst vacuolar changes in the cerebellum. hippocampus, cerebral cortex and basal nuclei were often minimal4'. Other forms of neuronal degeneration included occasional necrotic cells and spheroids. Neuronal loss had not been reported at this stage, but it might become obvious only on quantitative assessment. Astrocytic hypertrophy, with occasional gemistocytic forms, was demonstrated in the affected grey matter. In a few cases, small. sparse deposits of amyloid were found. Electronmicroscopy revealed spheroids or dystrophic neurites, containing predominantly neurofilaments with dense bodies and mitochondria. and tubulovesicular structures. An interesting feature of BSE is the uniformity of pathology, which contrasts with the variability of cerebral changes in scrapie. There is now compelling evidence to suggest that cattle in the UK were infected orally by the transmission of the scrapie agent from processed sheep protein, and thus BSE represents scrapie in cattle. Fibrils isolated from the brains of cows with BSE contain scrapie-associated protein, confirming that BSE is a scrapie-like disease4'. Epidemiological observations indicate that it is a new disease which has not been introduced by imported animals or animal products and that the only common factor is foodstuff containing meat and bone meal of infected animals. The disease is widespread geographically, with a higher incidence in southern England, with the risk increasing with herd size. There is no association with breed or sex and it is not a genetic disease, although there appears to be a genetic predisposition. The incubation period ranges from 2.5 years to at least 8 years". The effective exposure of cattle to infectious agents started suddenly in 1981-1982 following substantial changes in the processing of animal foodstuff. Lower sterilization temperatures and the omission of hydrocarbon solvent for the extraction of lipids during meat and bone meal production both could have facilitated transmission. BSE has been transmitted to mice, by inoculation of brain h o m o g e n a t e ~and ~ ~ more recently by dietary routes4Y.Other species appear to be also susceptible (for review see Wells et al.50). The facts that the infectious agent can cross the species barrier and BSE can be transmitted by the oral route raise the serious problem of risk to man by consuming beef or milk". Against the potential hazard it is argued that the oral route of challenge is relatively

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inefficient and the infectious agent is concentrated in the brain, spinal cord, lymphoreticular system and intestines, rather than in the muscles or milk. Moreover. there is no difference in the incidence of CJD between countries where scrapie has never been found and those like Britain and France where scrapie has existed for many years. Solution to this problem may lie in the longterm monitoring of the incidence of CJD to identify any increase over the baseline established for the years 1980-1984 in England and Wales51. In the end, however, the safety of beef has not yet been tested and may prove ~ n t e s t a b l e ~ ~ . OTHER A N I M A L TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

Chronic wasting disease of deer was first observed in Colorado between 1967 and 1979 and also in Wyoming54. Scrapie-like pathology developed in the grey matter of the central nervous system particularly in the brainstem and the hypothalamus. In addition, there were unusual focal lesions in the olfactory region of the frontal cortex. The disease can be transmitted by intracerebral injections of diseased brain into deer and ferrets. A similar disease has been reported in elkss5.

Prion proteins The intense research to identify the highly unconventional infectious agent causing transmissible spongiform encephalopathies has initially focused on scrapie and led to the discovery of prion proteins (PrP). The term prion was introduced to describe a proteinaceous infectious particle'h-60. Experimental evidence had already suggested that the scrapie agent was fundamentally different from traditional viruses. Improved protocol for the partial purification of the scrapie agent revealed a protein molecule, PrP 2 7-30, but surprisingly no nucleic acid, suggesting that protein alone may cause infection. PrP 27-30 is a neuronal cell surface sialoglycoprotein which is encoded by a single chromosomal gene. The human PrP gene is located on the short arm of chromosome 20. PrP 2 7-30 was found to polymerize into rods with the ultrastructural and histochemical features of amyloid. These prion rods, however. are different in their morphology and physicochemical properties from the scrapie-associated fibrils which had been previously demonstrated in a negatively stained electronmicroscope preparation following their isolation from scrapie-infected brains. PrP 2 7-30 was subsequently isolated not only from scrapie-infected brains. but also from animals with BSE, and from human and animal tissues infected with kuru and CJD",5y.

8 P.L.Lantos

Immunohistochemistry of formalin-fixed, paraffinembedded tissues indicates that prion proteins are produced mainly in the neurons of both normal and terminally ill animals". Moreover, in situ hybridization has shown that most PrP mRNA is localized in neurHowever, the possibility that prion protein may be produced in other sites cannot be excluded. Indeed, recently, prion protein was seen to accumulate in astrocytes during scrapie infection, raising the possibility that these glial cells may be actively involved in the replication of scrapie agent". The demonstration of abnormal prion protein from infected brains by immunoassay provides an additional diagnostic tool of high sensitivity and specificityh'. By using antisera raised against PrP 27-30. further prion proteins were detected in crude extracts of infected and normal brains, and these isoforms were subsequently designated as PrPS' (or PrP 33-35") and PrPC (or PrP 3 3-3 5'). respectively. The prion protein isolated from scrapie brains, PrPSC,is different from the normal cellular isoform PrP': PrP' is digested by proteinase K, whilst PrPS' is converted to PrP 27-30. PrP' is the biochemical precursor of PrPS', which is thought to be part or all of the infectious agent. Prusiners8 has accumulated an impressive range of data which indicates that PrP 27-30 is responsible for infectivity. The idea that infection can be propagated by protein alone is still controversial, since it is unprecedented. Currently there are two hypotheses to explain the multiplication of the infectious agent. According to the 'nucleoprotein' or 'virion' hypothesis, the agent is composed of a small nucleic acid which replicates conventionally and the PrP coat is then provided by the host cell. The 'protein only' hypothesis envisages PrP' being converted into PrPsc either by inoculation of exogenous PrPS' or spontaneously, as in GSS65.Experimental studies with transgenic mice are likely to shed further light on the method of transmission, latency and species differences. Transgenic mice inoculated with hamster prions developed scrapie after a shorter latency than control mice and produced PrP plaques normally found in hamsters but not in mice with ~ c r a p i e ~ ~ .

Molecular genetics of inherited human spongiform encephalopathies Molecular genetic studies have revealed six mutations associated with inherited human spongiform encephalop a t b ~ i e s ~ ~The . ' ~ .first was a 144 base-pair insert at PrP codon 53 in a British family suffering from CJD67.A similar insertion was found in an American family of British origin". These insertional mutations appear

to be associated with atypical manifestations of disease, A substitution of leucine for proline at PrP codon 102 was reported in both Caucasian and Asian families with the ataxic form of GSS d i s e a ~ e ' ~ This - ~ ~ . mutation, however, was not found in familial and sporadic CJD or in kuru, indicating that it may be responsible for amyloidogenesis and transmissibility of GSS disease but is not the sole cause of human spongiform encephalopathies7". At PrP codon 117 a substitution of valine for alanine was reported in a n American patient of German origin with the dementing form of GSS disease who also had pyramidal signs and parkinsonian features71. A further point-mutation, a substitution of lysine for glutamate. was found at codon 200 in two American families with CJD. Later it was revealed that the known clusters of CJD in Slovakia and amongst Lybian-born Israeli Jews are associated with the same mutation^^^,^^,^^. A mutation in codon 178, a substitution of asparagine for aspartic acid, first discovered in a large Finnish pedigree, results in spongiform change, neuronal loss and astrocytes without amyloid plaque^'^. Finally, a point mutation at codon 198 is associated, neuropathologically, with neurofibrillary tangles in addition to PrP immunoreactive plaques and mild spongiform change74a.A mutant human gene from GSS disease has reproduced spongiform encephalopathy in transgenic mice, indicating that a neurodegenerative process similar to human disease can be genetically modelled in experimental animals7'. Moreover, this experimental system supports the view that these mutations are responsible for the disease. Data from molecular genetics indicate that the primary structure of PrP appears to determine the following features of inherited spongiform encephalopathies: plaque formation and morphology: distribution of lesions leading to distinct clinical manifestations: and host range. Molecular genetic studies have contributed not only to the understanding of the pathogenesis of transmissible spongiform encephalopathies, but now also offer a practical, complementary diagnostic tool. A recent report described the finding, in a man of 36 years of age who did not have any of the histological hallmarks of spongiform encephalopathy. of a 144 base-pair insertion in the open reading frame of the PrP gene77.similar to that previously described in a family with neuropathologically confirmed familial CJD77.This report raises issues of fundamental importance. First, the incidence of CJD may be considerably higher, since a n unknown number of cases may go undiagnosed if the established histological diagnostic criteria are missing. Secondly, in order to establish the true incidence of these diseases a combination of genetic screening and the immunohistochemical

Transmissible sporigiform encephaloputhies 9

demonstration of the abnormal isoform of PrP should complement traditional neurohistology. Thirdly, the genetic basis of transmissible spongiform encephalopathies is likely to be more extensive than hitherto realised. Finally, the convenient descriptive term of spongiform encephalopathy may have to be abandoned in favour of prion disease.

Safety precautions for handling Creutzfeldt-Jakob material Since CJD is caused by a highly unconventional and elusive infectious agent, a great deal of uncertainty and confusion has been created concerning safety of performing postmortem examinations and handling pathological specimens. The iatrogenic transmission of a few cases and the reported higher incidence in health professionals, which is more apparent than real, have further added to the apprehension. Clear guidelines for clinical, surgical and pathological practices have now been e~tablished’~,~’. The brain and other parts of the nervous system are most infectious, whilst liver, lung and kidney are less likely to transmit the disease. The causative agent resists standard methods of disinfection and sterilization by heat, formaldehyde, 70% alcohol, UV-light and ionizing radiation. Formalin-fixed, paraffin-embedded material remains infectious and should be equally treated with utmost caution. However, formic acid has been recently found to eliminate the infectious agent completely without compromising tissue preservation. The inclusion of formic acid in the routine fixation by formaldehyde will thus remove the risk of infection and will provide histological sections of good quality8”. Autoclaving at 120°C for 1 h and the use of 1% dilution of hypochlorite containing 10 000 ppm chlorine are satisfactory for disinfecting instruments and exposed surfaces. Contaminated skin should be disinfected by 1~ sodium hydroxide for 5-10 min followed by copious washing with water”. Neuropathology departments have detailed codes of practice, and histopathologists who are requested to perform postmortem examinations may wish to seek advice from their neuropathologist colleagues.

Acknowledgements I would like to thank Dr J.Anderson (Addenbrooke’s Hospital, Cambridge), J. Collinge (St Mary’s Hospital Medical School, London), Professor L.W.Duchen (Institute of Neurology, London), Dr H.Fraser (Institute for Animal Health, Edinburgh), Professor M.A.Preece (Insti-

tute of Child Health, London), Professor R.O.Weller (University of Southampton) and Dr G.A.H.Wells (Central Veterinary Laboratory, Weighbridge) for the information they provided: Dr J.Hope (Institute for Animal Health, Edinburgh) for antibody to prion protein: Mr L.J.Doey for technical assistance: and Mrs LHayton for typing the manuscript.

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