a historical primer of transmissible spongiform encephalopathy

[Guest guest]

Dear All,

A very worthwhile read from this week's BMJ, Cheers etc., Lynette.

_____________________________________

BMJ 1998;317:1688-1692 ( 19 December )

0000,0000,ffffhttp://www.bmj.com/cgi/content/fu\

ll/317/7174/1688

Clinical review

1755 and all that: a historical primer of transmissible spongiform

encephalopathy

Brown, senior research scientist, a

Bradley, private BSE consultant. b

a Laboratory of CNS Studies, National Institute of Neurological Disorders

and Stroke, Building 36, Room 5B20, National Institutes of Health,

Bethesda, MD 20892, USA, b Central Veterinary Laboratory, New Haw,

Addlestone, Surrey KT15 3NB

Correspondence and reprint requests to: Dr Brown pwb@...

In the political and economic turmoil of 18th century Europe, England

gradually established itself as the dominant trading

power. By far its most important commercial product was woollens,

involving in one way or another nearly a quarter of the

British population, which at 10 million people approximated the number of

British sheep. Sir Walpole, the embodiment

of pragmatic mercantile economics, was prime minister of an increasingly

powerful and commercially inclined Whig government,

and, with the agricultural revolution in full swing, the invention of the

flying shuttle and spinning jenny and the imminent

introduction of steam power would soon convert weaving from a handicraft

to a mechanical process. Wool, already in short

supply, was at a premium. In this setting, it is not surprising that in

1755 a discussion took place in the British parliament about

the economic effects of a fatal and spreading disease in sheep, and the

need for government to do something about it.1 Thus

began the recorded history of scrapie.

Summary points

Scrapie was first described in the 18th century, but it was not

proved to be a transmissible disease until 1936

Its position as the prototype of a small group of animal and

human spongiform encephalopathies, including

Creutzfeldt-Jakob disease, was not appreciated until a quarter

of a century later, in the early 1960s

The infectious agent, originally thought to be a " slow virus, "

has now come to be considered a conformationally

altered, self replicating form of a normal body protein, or

prion

Numerous mutations in the gene that encodes this protein have

been linked to familial forms of disease

Recent outbreaks of disease (bovine spongiform encephalopathy

and iatrogenic and " new variant "

Creutzfeldt-Jakob disease) have highlighted a disconcerting

disparity between advances in fundamental science

and practical common sense

Anecdotes and antidotes

Where or when the disease actually first appeared is unclear, although

there is a suggestion that it

was already present in northern Europe and Austro-Hungary before the

beginning of the 18th

century. It repeatedly occurred (or at least was exacerbated) in tandem

with exportations of

escorial and electoral merino sheep from Spain, either through

ecclesiastical, royal, or in some

cases private entrepreneurial activity. 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. As the

practice abated, scrapie declined during the later 19th century but did

not entirely disappear. In

Scotland scrapie was first recorded during this period. Most mentions of

the disease appeared in veterinary medicine manuals,

dictionaries, and articles devoted to surveys of livestock diseases.

One interesting example appeared in the German literature in 1759, from

which the following paragraph is quoted in its entirety:

" Some sheep also suffer from scrapie, which can be identified by the fact

that affected animals lie down, bite at their feet and

legs, rub their backs against posts, fail to thrive, stop feeding and

finally become lame. They drag themselves along, gradually

become emaciated and die. Scrapie is incurable. The best solution,

therefore, is for a shepherd who notices that one of his

animals is suffering from scrapie, to dispose of it quickly and slaughter

it away from the manorial lands, for consumption by the servants of the

nobleman. A shepherd must isolate such an animal from healthy stock

immediately because it is infectious and can cause serious harm to the

flock. " 2

In addition to the accurate clinical description of scrapie, two points

in this excerpt merit special emphasis: firstly, scrapie was

recognised as a contagious disease in sheep, and, secondly, scrapie was

not considered to be a human pathogen (at least, not

for the lower classes). Nothing we have learned in the past 250 years has

invalidated these observations.

Scientific beginnings

Around the middle of the 19th century, veterinarians in England, France,

and Germany initiated the

scientific study of scrapie, including systematic neuropathological

examinations, and made efforts

to identify an infectious pathogen.

In particular, Besnoit and his colleagues in the Toulouse school of

veterinary medicine recognised neuronal vacuolation as a characteristic

feature,3 and also attempted to transmit the disease to healthy sheep by

inoculation of brain and transfusion of blood from affected animals, and

by keeping symptomatic sheep with healthy sheep.4 The negative results

they reported after observation periods of up to several months were

certainly due to a failure to appreciate the extraordinarily long

symptomatic phase of infection, a failure that 70 years later would also

thwart the first experimental attempts to transmit a human spongiform

encephalopathy.

Undeterred by these results, the French veterinarian community continued

to explore the infectious nature of scrapie, and at

length Cuillé and Chelle, taking note of several epidemiological studies

that pointed to incubation periods of 18 months or

longer in naturally occurring disease, succeeded in 1936 in transmitting

scrapie to two healthy sheep by intraocular inoculation

of brain or spinal cord tissue from an affected animal.5 The incubation

period between experimental inoculation and onset of

disease varied from one to two years, shortest when inoculation was into

the brain, longest when a peripheral route was used.

In subsequent experiments, they also transmitted disease by using

intracerebral, epidural, and subcutaneous routes of infection,

and by passing brain tissue through a bacterial exclusion filter.

In a grand historical irony, this landmark series of experiments was

being confirmed at the same time in England as a result of an outbreak of

scrapie in several hundred sheep that had been immunised against louping

ill with a vaccine prepared from tissue

from the brain, spinal cord, and spleen of sheep that were belatedly

discovered to have been exposed to natural scrapie infection.6 The

transmissible nature of the scrapie agent was thus established beyond any

doubt.

Fig 1. The major players in the field

of transmissible spongiform

encephalopathy. ( " The Young Bull " by

us Potter (1625-1654) in the

Mauritshuis, The Hague, Netherlands)

Throughout the 1940s and 1950s, the accelerating pace of veterinary

research yielded many new discoveries about the

behaviour of the causative agent: its distribution through the body after

experimental and natural infection; its physical

association with cell membranes; its susceptibility to host genetic

factors; and its extraordinary resistance to standard methods

of inactivation. Especially notable were two seemingly contradictory

observations: Dickinson et al, using the methods of

classical genetics, identified a gene in both natural and experimental

infections that determined phenotypically different strains of

scrapie,7 and Alper et al showed that infectivity survived a dose of

ionising radiation that was incompatible with the biological

integrity of nucleic acid,8 an observation that led to several theories

about the agent being a membrane bound ligand, a

lipid-protein-polysaccharide complex, or even an unadorned protein.

Finally, and by no means least important, the successful

adaptation of the agent by Chandler to laboratory mice9 elicited a

collective sigh of relief from experimentalists, who had until

then been obliged to work exclusively with sheep and goats.

The human connection

Then, in 1959, this endemic disease of sheep, unknown to or ignored by

medical science, was proposed by the American veterinarian Hadlow to be

analogous to a newly described disease of humans, kuru, an epidemic

neurological disorder found in the eastern highlands of Papua New Guinea

that two years earlier had been introduced to Western medicine by

Gajdusek and Zigas. 10 11 The reverberations from these remarkable

insights are still being felt.

Pediatrician by training, virologist by experience, and genius by nature,

Gajdusek had just finished working in Sir MacFarland Burnet's laboratory

in Australia when an opportunity arose to revisit New Guinea. Once there,

he and Zigas, an expatriate of the Baltic states working as a medical

patrol officer, went into the highlands to have a firsthand look at kuru.

Just what it was about this disease, restricted to a remote and isolated

group of 15 000 people, that aroused Gajdusek's instincts about its

potential larger importance remains obscure, but during the next decade

he conducted a wide ranging campaign of scientific investigation, often

in a running battle with the Australian colonial bureaucracy, which

reacted rather peevishly to a perceived threat of " outside " medical

exploitation of its own territory.

Genetic, endocrine, nutritional, and toxic causes were explored and

particular attention was given to the possibility of an

infectious origin associated with the practice of ritual endocannibalism,

which, although not supported by either laboratory or

pathology data, was the theory favoured by everyone from missionaries to

bush pilots. All conceivable means of detecting an

infectious agent, including the inoculation of monkeys, were attempted,

with a uniform absence of success after observation

periods of two to three months. While these efforts were continuing

Hadlow's observation was published, leading to an

extension of experimental inoculations to chimpanzees kept under long

term surveillance. In 1965, three chimpanzees developed kuru 18-21 months

after having been inoculated intracerebrally with brain tissue from

different kuru patients.12

Well before this result was known, the neuropathologist Klatzo had

conducted an exhaustive study of the brains of 12 people

with kuru,13 commenting that they resembled only one other human disease

with which he was familiar Creutzfeldt-Jakob

disease (CJD), first described in the early 1920s by two German

neurologists. Brain tissue from a patient was inoculated into a

chimpanzee, and the disease developed 13 months later.14 Like scrapie,

kuru and Creutzfeldt-Jakob disease were subsequently

adapted to laboratory rodents.

An unexpected twist

In the years following these discoveries, the physical and chemical

properties of the infectious agent, its distribution and titre in tissues

of infected animals, and its host range were studied. Though knowledge of

the biology of the agent was advancing, knowledge of its molecular

biology remained scarce because of the technical difficulty of separating

the infectious agent from

contaminating host components. Alper's work had strongly suggested that

nucleic acid was not needed for replication, but without an alternative

molecule to work with, theories of replication directed by protein were

merely untestable intellectual gymnastics. This point was not lost on the

American neurologist Prusiner, who correctly saw that further advances

needed better purification methods. After several years of work, he

obtained a highly purified infectious preparation that yielded an

N-terminal peptide sequence sufficient for the corresponding cDNA to

" fish out " the encoding nucleic acid of a full length protein, known as

PrP.15 To the surprise of everyone, this coupling of biochemistry and

molecular biology gave birth to a protein that was encoded by a host gene

and not by a foreign invader.16 The concept of a conventional virus was

dealt a body blow.

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, and further support has

come from an unexpected sourcethe discipline of

epidemiology. As the diagnosis of sporadic Creutzfeldt-Jakob disease

became more precise, accurate surveys of disease

occurrence confirmed a combination of rarity and randomness that made the

idea of contagion difficult to entertain.17

Epidemiology also conspired with molecular genetics to show that the few

geographical clusters of the disease all resulted not

from environmental peculiarities but from familial disease due to a

genetic mutation in the gene on chromosome 20 that encodes PrP. Thus,

both sporadic and familial disease seemed not to be associated with any

outside influence: in the immortal words of Pogo, the American comic

strip character, " We have seen the enemy, and they are us. "

In the past decade, a remarkable amount of work has been done in

different countries and laboratories to determine the precise

basis of infectivity in transmissible spongiform encephalopathy, and to

find some means to protect both humans and animals

from becoming infected. On the first count, there have already been

spectacular results, whereas on the second count the

record is marred by three missed opportunities that instead led to

tragedies. Two of them (human growth hormone and dura

mater grafts) might have been foreseen; the third (bovine spongiform

encephalopathy) is best ascribed to plain bad luck.

Some successes . . .

We have learned that PrP is not distinguished from the universe of

proteins by any unique structural featuresit is in the lower middle range

in size (35 000 Daltons), has an octapeptide coding repeat region, two

asparagine-linked sugar moieties enclosed within a disulphide bridge, and

a glycolipid membrane anchor. Because its primary structure is identical

in both normal and

disease states, it has been concluded that the molecular basis of disease

results from a critical switch in the protein's mix of three dimensional

patterns from a predominantly -helix to -sheet configuration, changing

from a " floppy " soluble amyloid to a " stiff " insoluble amyloid, rather

like turning a chiffon curtain into a Venetian blind. This conversion has

also been accomplished in vitro, but a significant parallel change in

infectivity has so far been impossible to measure.

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Fig 2. Artist's conception of the

formation of abnormal PrP molecules, in

which a normal molecule with three

-helices (red coils) and one -sheet

(yellow plate) (left panel) is

configurationally altered by the proximity of an

abnormal molecule with one -helix and

four -sheets (right panel), leading to a

cascade of molecular aggregation into

visualisable deposits of amyloid in the

brain. Exactly how this happens remains

a mystery (Courtesy of

WGBH/NOVA, Boston, and BBC/HORIZON,

London)

In cells the protein moves along the usual pathways of endoplasmic

reticulum and Golgi apparatus in its post-translational

adolescence, attaches to the plasma membrane during its maturity, and

then in senescence re-enters the cytoplasm, where it is

catabolised. Its normal function is unknown, although it may be involved

in the process of synaptic repolarisations. In the

diseased host, the configurationally altered protein is either deposited

extracellularly as amyloid plaques or concentrated

intracellularly in the synaptosomal region. Either way, its catabolism is

impaired, and insoluble amyloid protein accumulates.

Although the only cells that seem to be morphologically and functionally

compromised lie within the nervous system, the

infectious agent is also present in many visceral organs, depending on

the host species, route of infection, and agent strain

(bovine spongiform encephalopathy, for example, has so far shown a very

restricted distribution in tissue). The most thoroughly studied forms of

transmissible spongiform encephalopathy are caused by oral infections,

particularly in experimental models of scrapie, where the major

pathogenic pathway, measured both by infectivity and presence of PrP,

starts with the tonsils, intestinal lymphatic tissues, and spleen and

goes via the splanchnic nerve into the spinal cord and on to the brain. A

minor alternative route shortcircuits the spleen and probably reaches the

brain via the vagus nerve. One scrapie model has recently found that B

cells and follicular dendritic cells in the spleen are critical for

neuroinvasion, but their exact role is not known.

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Fig 3. Known and speculative

interrelationships of transmissible spongiform

encephalopathy in animals and humans

More than two dozen different point and insert mutations identified in

the gene encoding PrP are responsible for the familial

form of Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker

syndrome, and fatal familial insomnia. All occur in a

Mendelian dominant pattern of inheritance, and all are experimentally

transmissible to laboratory animals. Evidently, these

mutations increase to near certainty the likelihood of the transformation

from -helix to -sheet protein, which in sporadic

disease occurs in only one per million people each year. Once transformed

(no matter what the triggering event), 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 genetic manipulation, although not yet feasible in humans, has

provided some extraordinarily interesting results in

experimental animals. Mice created with a PrP gene containing the

equivalent of one of the human mutations (P102L)

spontaneously develop a fatal spongiform encephalopathy.18 Conversely,

mice in which the PrP gene has been either made

dysfunctional or excised, and which thus do not produce PrP, are not

susceptible to experimental transmissible spongiform

encephalopathy, and perhaps even more surprising, live to old age in

perfect health (apart from an altered circadian rhythm).

This suggests that the PrP gene is redundant in mice, and thus perhaps

also in humans, opening the door to consideration of

gene ablation therapy in human transmissible spongiform encephalopathy.19

.... and some failures

To suppose that all of this basic knowledge might have translated into

practical solutions for the prevention of disease would be deceptive.

While these basic research studies were going on, three outbreaks of

Creutzfeldt-Jakob disease tested our ability to foresee problemsand found

it wanting. Beginning around the mid-1960s, a procedure to extract growth

hormone from pituitary

glands had been sufficiently refined to permit large scale production.

Glands were obtained from cadavers at necropsy and were pooled in batches

of up to 10 000 for each production run. In 1985, Creutzfeldt-Jakob

disease was reported in three patients in the United States, and native

hormone was immediately replaced by a recombinant product. Despite this

action, the disease has since been responsible for over 100 additional

deaths, chiefly in France, Great Britain, and the United States, 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 10 years before the first case of

the disease), most of the damage had already been done because of the

decades-long " lead time " between peripheral route infections and

verification of the disease; moreover, screening criteria were not always

effective in preventing the inclusion of pituitary glands from cadavers

in which Creutzfeldt-Jakob disease was unsuspected.

Almost coincident with this outbreak, contaminated dura mater grafts were

also discovered to have caused iatrogenic disease:

since 1988, more than 70 people who had received grafts during

neurosurgery have died from Creutzfeldt-Jakob disease, the

contamination again resulting from inadequate criteria for screening

donors and the pooling of batches of cadaveric tissue before or during

processing. These tragedies have prompted much more stringent regulations

governing the collection and use of biological products originating from

humans, particularly 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 young people in Britain

of a " new variant " of Creutzfeldt-Jakob disease (nvCJD, or the

Will-Ironside syndrome) that has since been traced with near certainty to

the consumption of tissue from cattle infected with spongiform

encephalopathy (BSE), they having in turn consumed meat and bone meal

contaminated with rendered sheep carcasses infected with scrapie.20 The

story contains elements that are still disputed, but it seems 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 infectivity in meat and bone meal, and eventually producing a

full scale epidemic of bovine spongiform encephalopathy. Recognition of

this source of infection led to the imposition in 1988 of a ban on

ruminants in cattle feed that by 1993 had turned the epidemic around, but

the loss of some 170 000 cattle to date has brought the British livestock

industry to its knees. The disease has also affected the tallow, gelatin,

and pharmaceutical industries, all of which use products derived from

cattle, and even blood banks have been seriously affected because of

uncertainty about infectivity in blood donations from patients incubating

nvCJD. There are currently just over 30 verified cases of nvCJD, and four

to five new cases a year: whether they represent a small group of

susceptible people or are the leading edge of a major epidemic is still

moot.

2000 and beyond

Despite these battle scars from engagements in applied science, we can

look back with some satisfaction on the accomplishments in basic science

during the century now drawing to a close We can 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

characterisation 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 (and a growing body of evidence points to the

likelihood that it is not only necessary in transmission but sufficient

to be considered as the infectious agent), formal proof is still lacking.

This may come from continuing attempts to show a parallel between the

amount of infectivity and the in vitro

conversion from normal to abnormal protein isoforms; or from the creation

of infectious synthetic PrP polypeptide sequences; or from the native

protein processed to crystalline purity, guaranteed free from any

contaminating molecular species, yet still able to transmit disease.

Precise characterisation of the infectious agent will not by itself solve

the question of PrP " 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 -A4 amyloid of Alzheimer's disease also derives from a

normal host protein that in diseased people accumulates

in the brain, but it does not have the ability to transmit disease to a

healthy person. Why this difference?

The answer to this fundamental biological question need not inhibit

research into disease prevention and treatment, 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 -helix to -sheet

configurational shift to amyloid (the central nervous

system version of a " beta-blocker " ) could become viable therapeutic

approaches, and efforts to arrest and even reverse

amyloid accumulation in experimental models are already beginning to show

promise. Similarly, it will be possible to manipulate the PrP gene (or

its expression) in familial forms of disease once genetic engineers

overcome the technical problems that have prevented the results obtained

in mice to be duplicated in humans.

Finally, we must remain alert to the possibility that other diseases

without known cause may share the pathogenic mechanism of transmissible

spongiform encephalopathy, and so be susceptible to the same therapeutic

approaches. We should also be

prepared to admit that " replicating proteins " may not cause other, more

numerically important, disorders but may forever remain confined to the

small group of " prion diseases " that are a comparatively minor burden to

human health.

Acknowledgments

We extend our apologies to the many contemporary investigators who,

because of constraints of space, could not be cited in

the text and references.

Competing interests: None declared.

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