Recent journal papers
Books on BSE
Detecting BSE cattle with CSF test
Why is 14-3-3 protein found in new CJD test?
Japan researchers make mad cow breakthrough
CJD Therapy with anti-sense prion mRNA?
Prion infectious titre during incubation of Symptoms
The transgenic mouse experiment: what does it mean?
Rapid disease course in 8x prion gene over-expression
Risk of sporadic CJD with codon129 allele.
V-CJD In Ireland?
Strain typing of UK farmers with CJD
Collinge on risk to British population
Cytotoxicity of prion 106-126 unlike Alzheimer's A beta 25-35
Controversy over HSP binding to prion 180-2I studyBR> Book Review: Lethal Legacy by Stephen Dealler
Hideyo Inouye, Boston prion researcher
Herbert Budka and research in Austria
Maternal Transmission and milk production drop as a sign of BSE
Can US researchers legally possess BSE slides?
Kuru -- What really happened?
Chronic wasting disease shows TSEs loose in US

Japan researchers make mad cow breakthrough

Reuter Information Service Oct 17, 1996

TOKYO - A group of Japanese virologists has claimed success in culturing cranial nerve cells devoid of the protein believed to cause mad cow disease and similar infectious brain illnesses, Kyodo news agency reported on Thursday. A team at the University of Tokyo Agriculture Department would announce the results of an experiment to culture nerve cells free of prion at a meeting of the Society of Japanese Virologists on October 25, Kyodo quoted team members as saying.

Infection with abnormal prions has been associated with bovine spongiform encephalopathy, better known as mad cow disease, its human equivalent Creutzfeldt-Jakob Disease, and its sheep equivalent scrapie, the report said.

The researchers said by adding serum from cow embryos to nerve cells derived from mouse brains they succeeded in isolating nerve cells whose prion-producing functions had been lost.

Outbreaks of mad cow disease -- and suspected links to the human version -- earlier this year in Britain roiled world beef markets, costing millions of dollars in lost beef trade and triggering economic tension between Britain and its European Union trade partners.


V-CJD In Ireland?

An abstract has just appeared (October 1996) whose title suggests a new v-CJD vicitm has been found in Ireland. There were earlier reports, later questioned, of a few case in Germany and France.
Hawkins, S. A., Droogan, A. G., Esmonde, T. F., Allen, I. V., Will, R. G.:
The first Irish case of a newly recognized phenotype of CJD (abstr). 
Ann Neurol 1996; 40: 132.

Strain typing of UK farmers with CJD

Listserve 10.10.96

Strain typing of some UK farmers that have died of CJD is currently being done at the Neuropathogenesis Unit in Edinburgh. It is unknown if brain tissue from all four farmers that had BSE affected herds has been passaged in mice, or if brain tissue from the abattoir worker who died in February 1996 is being similarly studied. -----
The 3D pattern of TSE pathology in mice can possibly be used to distinguish different strains of CJD, but all mice and the method of inoculation have to be identical. According to the prion theory each passage to a non identical animal creates a new strain. The passage of BSE through humans with different prion proteins should tead to different pattern in mice after a second passage.


Cytotoxicity of prion 106-126 differs from Alzheimer's A beta 25-35

Hope J; Shearman MS; Baxter HC; Chong A; Kelly SM; Price NC 
BBSRC & MRC Neuropathogenesis Unit, Edinburgh. 
Neurodegeneration 5: 1-11 (1996) 
The abnormal form of the prion protein (PrPSc), a synthetic prion protein peptide fragment (PrP106-126) and fragments of the Alzheimer's protein precursor, APP, have been shown to be cytotoxic in vitro. We have used synchronous, clonal cell models originally developed to study the toxicity of the Alzheimer's disease amyloid peptide, A beta 25-35, to investigate the actions of PrP peptides. We found that the cytotoxicity of the PrP106-126 depends on its state of aggregation and the cellular expression of PrPc, and is independent of a loss of MTT reduction activity in the absence of cell death associated with the cellular effects of A beta 25-35. These factors may play a role in the lesion specificity of different pathological phenotypes of prion-protein related diseases.

Lethal Legacy

BSE - the search for the truth.
Stephen F Dealler Bloomsbury, Soho, UK ISBN 0 7475 2904 3

Dr Stephen Dealler's book "Lethal Legacy" is of hybrid genre-- imagine a cheerful and determined Franz Kafka had written "The Double Helix." In other words, a personal biological research odessey in an area controlled by a large and dark bureaucracy. The tone is mild, optimistic, and popular scientifice oriented. It covers the early days through to April, 1996 and includes a hilarious tour of US labs and personalities [all working in negative-pressure fume hoods, of course].

'Lethal Legacy ' contains, among other things, a to me fascinating and original unpublished proposal [Chapter 6, pages 112-132] on AGIs (alkaloidal glycosidase inhibitors from grazed forage) and scrapie, never funded for the usual MAFFka-esque reasons.

After considering anisotropic growth of unblocked prion multimers, Dealler addresses the molecular origin and biogeography of scrapie strains, proposing that forage AGIs affect prion asperagine glycosylation patterns. Dealler saw possibilities for treatment as well with these alkaloids. The author notes swainsonine causes a neurological disease of cattle and deoxynojirimycin from mulberries inhibits glycoprotein turnover. Australia and New Zeeland are [supposedly] scrapie free despite UK breed origins because the AGI-producing plants are missing, in Dealler's scenario. After getting the runaround and his brain picked by Searle-Monsanto's man at Oxford, Dealler gets a friendly reception from castanospermine expert Rob Nash (GC-mass spec man) at Kew Botanical Gardens and Winchester from RCH. With Eileen Lees given space in Bradford's lab, the search is on to identify AGIs in the British countryside. Some 250 plant species were collected, and most surprising to me, common plants like Bellis perennis [Asteraceae], which our scrapie-laden sheep in Oregon eat with great gusto, and bluebells were found to be quite strong AGIs in sheep brain assays. Nothing could be tested in sheep flocks because the Central Veterinary Lab In Weybridge wouldn't give permission.

It is discouraging to read about billions spent unproductively on the cull while not tuppence went to research. The US has a much better system with NIH and NSF: scientists evaluate grant proposals, not a secretive and unaccountable clique of agency bureaucrats. Little good science has come out of the UK, considering that it is their problem; they remain largely dependent on foreign researchers for answers, as many a committee has noted.


Web page for Hideyo Inouye, Ph.D.

 Hideyo Inouye, Ph.D.
Home page
Department of Biology
Boston College
Dr. Inouye works gon x-ray diffraction analyses of the Alzheimer's beta amyloid, prion and ttr fibers. Published results can be seen in abstract forms in his home page.

Milk production drop as a sign of BSE

Listserve 10.5.96

Most of the european dairy cattle are slaugthered only because of a reduction in their milk productivity. This happens when they have had only 1 or 2 calves in the Netherlands, after 3 or 4 calves in Germany or perhaps a bit later in the UK. When a cow becomes suffering from any disease, the milk productivity drops. In the case of BSE this will happen most probably slowly but pronounced and often with the consequence of the slaughtering of a infected cattle only a few weeks before other symptoms become detectable.

Therefor it is very important to know the quantitative correlation between the time point before they are killed because of BSE and the quantity of milk, they produced. It is very strange, that this correlation has never been published. If my hypothesis is correct and many older dairy cattle are killed because of an unrecognized BSE infection, then the britsh calculations of the mean incubation time of BSE, the frequence of maternal transmission and the duration of the BSE epidemy in the future are badly underestimated.

- The observed number of cases in UK annual birth cohorts
  peaked consistently (since 1986) at age 4-5 years.
 
- The within-herd incidence correlates with herd size.
 
- Unsatisfactory milk production, easy to observe,  has not been
  noted as typical 'first symptoms'.
 
- A very abrupt decrease in incidence in birth month cohorts appeared
  in the autumn 1988.
 
- A very large proportion of UK cases appeared in a few annual cohorts
  1986-88.
We know that calves from BSE-dams had higher BSE incidence than calves from non-BSE dams. The observed increased risk could be consistent with a 10-15 % (but not much higher) maternal transmission rate, and it _could_ be a result of genetic selection for susceptibility coupled with exposure to contaminated food. Were not the calves for the study selected from the 1988 as well as from the 1989 birth cohorts?

Maternal transmission would be consistent with _equal_ incidences in the 1988- and 1989 calves in the study. And genetic susceptibility coupled with food exposure would result in a difference: a lower incidence in the 1989-calves, as they were less exposed to contaminated food.


A reduction of milk productivity is a well known symptom >of BSE.. Colin Whitaker, the vet that discovered the first cases of BSE, wrote in his report regarding the first 10 cases at the same farm outside Ashford, Kent:

"Milk yield gradually drops and there is loss of condition in the later stages." (Presentation at a meeting of the British Cattle Veterinary Association in July 1987).  Later:

"In my paper I had a list of differential diagnosis, and the last one was 'a new scrapie-like syndrome'. The Ministry (MAFF) asked us not to use that (at the meeting in '87) - or told us not to use the word 'scrapie-like'. I think they were concerned that it would effect the sheep market." (Sept 23 1996) The word was blacked out at the meeting. Colin Whitaker's report was published one year later, in its original version.


As the cows of the study have not been subject to selective milk production related culling :-), the study should at least resolve whether the incidence peaks before age 7 year, or as you have suggested as a possibility, simply increases with age within that period. The Weybridge experiment and (mainly) the observation that the feed ban had an immediate dramatic influence on the incidence in birth month cohorts.

The Weybridge experiment was designed to be able to preclude the possibility of maternal transmission. In this it failed. The Weybridge experiment does _not_ preclude a maternal transmission rate at 30-45 % (although a much lower rate was observed under the conditions of the experiment) -- but at such a rate the decrease in UK incidence after the beef ban would have been less abrupt than observed.

With a 45 % maternal transmission rate (alert: back-of the-envelope-calculation!) we would be close to the point where each BSE case would reproduce itself in one of its female calves. The available data do _not_ indicate that this has happened!

A genetic selection for increased susceptibility to food exposure in the calves from BSE dams would still need food exposure to express itself as disease. This food exposure was less in 1989 than in 1988. Unfortunately, the number of 1989 calves in the experiment is (I think) quite small. If it is, it may be impossible to detect a difference.


Can US BSE researchers legally possess slides?

Listserve 10.11.96

Concerning slide and tissue importations for training veterinary pathologists in the US in the diagnosis of BSE, what are the legal implications involved? I am under the impression that, even in diagnostic labs, it is illegal to possess infectious materal for what is termed a "foreign animal disease." If this is true, then histology slides containing BSE would certainly fit into this category. We have some actual BSE training slides here at the lab, but I'm not positive what species they're from. (The brainstem appears sheep-sized.)

Response: All recipients of histological slides containing brain sections of BSE affected cattle received and are required to abide by an import permit that limits the types of procedures that can be done with the specimens. Specifically, work shall be limited to in vitro laboratory studies only and that the permit does not authorize direct or indirect exposure of domestic animals. In addition the specimens must be maintained at the original recipient instutution.


Detecting BSE cattle with CSF test

Abstract of a paper just published by V. Jones, T.C. Martin, P. Keyes & M. Dawson (CVL, New Haw, Addlestone, Surrey KT15 3 NB, UK [1996 Vet Rec Oct 12, 139, 360-363]:

Two-dimensional gel electrophoresis was used to analyse CSF from 75 suspect cases of BSE, 61 of which were confirmed by PM brain histopathology, and 38 normal cattle. CSF samples were also examined from cattle killed at periodic intervals through the incubation period following experimental challenge. Consistent changes were recorded in all CSF samples from the confirmed cases of natural BSE and also from cattle showing early signs of experimental disease.

The changes consisted of an increased intensity of staining of apolipoprotein E and the presence of two protein spots , as yet unidentified, of molecular weights 35 and 36 kDa, both with a pI of 5.5.

These changes were absent in the CSF samples from the normal cattle, from the clinically suspect cattle which were not confirmed as BSE and from the experimentally challenged cattle in the preclinical phase of infection.


Why does 14-3-3 protein work in new CJD test?

Does it interact or bind specifically to prion protein, either in situ on the cytoplasmic membrane, possibly modulating a function, or bind to rogue conformer? The other option is that 14-3-3 appears in CSF [only in CJD dementias] merely as a result of generalized neuronal breakdown and has no specific interaction with prion protein or plaque. [This fits 14-3-3 in recent stroke and viral encephalopathy CSF but not too well with other dementias.]

The 14-3-3 protein is highly unusual in structure with a alpha-helical large channel. It is ubiquitous [homologues in yeast, plants, etc] and has an activating role in other systems, nothing to do with prevention or repair of neuronal oxidative damage.

Some typical 14-3-3 Medline abstracts are organized by topic and posted locally::

1) Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides
on protein kinase C activation. Biochem J 315 ( Pt 1): 41-7 (1996)
 
2) Activation-modulated association of 14-3-3 proteins with Cbl in T cells.
J Biol Chem 271: 14591-5 (1996)
 
3) Association of a 14-3-3 protein with CMP-NeuAc:GM1 alpha
2,3-sialyltransferase. Biochem Biophys Res Commun 224: 103-7 (1996)
 
4) Isolation and characterization of a cDNA from Trichoderma harzianum P1
encoding 14-3-3 protein homolog. Gene 171: 123-7 (1996)
 
5) Function of 14-3-3 proteins [letter] Nature 382: 308 (1996)

Kuru -- What really happened?

Listserve 10.6.96

On the subject of cannibalism, which has become deeply entrenched in this topic and which has little basis in recorded observation. Collinge's new version is certainly one of the most detailed I have seen:

"Kuru was a disease mainly of young children and young women. But it was women and children who were involved in the cannibalistic feasts and their preparation. The men ate muscle rather than brains. "The pathogenicity of the agent is extremely high in this situation. Everybody who sits down at one of those feasts dies of kuru."

In reality, these feasts were not observed (by Gajdusek or any other visitor), but surmised. The nature of the feasts, lists of participants and other details were not collected. Despite this lack of any first hand evidence, these feasts are recorded in numerous scientific papers with a level of detail that increases with each repetition. They can not be relied on as accurate evidence of CJD risk factors.

What WAS observed was the ritual preparation of deceased tribal members. The skin, flesh and contents were removed from the skull with a range of stone and wooden (bamboo?) implements. By custom this was carried out by women and pre-pubescent boys. The gruesome black-and-white footage I have seen shows the process in some detail on what appears to be a disinterred corpse, not a fresh corpse, and in a manner that is not in the least reminiscent of food preparation - the object of attention was the skull, whilst the flesh and brain fell unheeded to the ground.

It is clear that women and children (both shown in the footage) were exposed to raw brain through lacerations to their hands, inhalation of spray and ingestion from their hands. It appeared to me that the flesh was re-interred and the bones taken elsewhere, as is common in Swat, Tibet, Venice or Greece (all of which I have also observed) and any number of other contemporary burial customs. Whilst it is possible that cannibilistic rituals took place, there is no hard evidence for it and none whatsoever of the "men ate the flesh and women at the brain" variety.

It is my belief that this is a misinterpetation of burial custom and nothing more. As this is not an area I have any particular expertise in, I will add that there is a growing body of literature that disputes the existence of any ritual human cannibalism, some of which details this particular example. Reports of cannibalism (viz. Ceausescu, Idi Amin, the Aztecs, Native Americans) tend to serve a political purpose.

The last sentence, "everybody who sits down at one of these feasts dies of kuru" is unsupportable. Rates of kuru were high, but it was not the leading cause of death. Precise epidemiological evidence linking kuru to particular forms of exposure is not available, but certainly indicates that the majority of those exposed to ritual preparation did NOT die from kuru.

Regarding the question of whether the Fore engaged in ritual cannibalism, I think the people who have objected to that statement have done so more because of the negative image this gives to indigenous New Guineans than because the evidence of cannibalism is lacking. Shirley Lindenbaum and Robert Glasse collected detailed anthropological information about the practice, based on interviews with the Fore.

Vincent Zigas, the physician who first observed Kuru before Gajdusek's arrival on the scene, tells a number of stories in his memoirs, titled "The Laughing Death," including an instance in which he claims to have personally witnessed an instance of cannibalism, and a conversation with a Fore native who matter-of-factly describes eating an enemy and complains that he didn't taste very good. He also describes an occasion when he was entertaining a foreign visitor and served a meal of sheep's brain. According to the story, Zigas's native cook ran out of sheep brain and took an autopsied human Kuru brain, sliced it up and was ready to start frying the pieces when Zigas happened to step into the kitchen and stopped what was happening!

When questions were raised regarding whether cannibalism occurred, Gajdusek reacted with anger and irritation, and pointed out that the people who raised the questions were people who were not on the scene to observe and had no basis for questioning what he considered to be incontrovertibly established fact. Furthermore, the fact that Kuru started to die out a decade after the New Guinean authorities began enforcing a ban against cannibalism provides strong evidence that the practice was occurring.

That said, however, Gajdusek has maintained for a long time that the actual ROUTE of exposure was probably not through oral ingestion but through handling of infected tissue, cuts in the hands, etc.


Collinge on risk to British population

London Observer Service (Oct 12, 1996) -- John Collinge's mice are doing nicely.... But... Collinge's message has a startingly gloomy content. [Survival of the mice] would simply mean that the species barrier between cow and human is sufficiently strong to prevent that transmission happening within the average lifetime of a mouse. It may still occur over a longer period. This is almost certainly the case, adds Collinge. "The new cases of CJD in humans have a startlingly uniform pathology: early age of onset, psychiatric disturbances and a relatively long period before death occurs, about 14 months. This indicates a single cause -- BSE -- though we cannot yet say whether it will lead to 20 or 100,000 deaths a year." Collinge says his research has revealed that about half the British population has a genetic mix of brain prions that should enable them to resist BSE. "Of course, that leaves out half the population who do not have the right brain prion mix, and they will be the ones who provide the pool of victims." ----- Listserve Commentary 10.8 96

I disagree with the conclusions of last paragraph, which I take as saying met/val or val/val or some such at codon 129 won't be in "the pool of victims." Collinge was just trying here to get a little of the bad news in the open while still offering some hope, so as not to throw the populace into a panic.

Instead, I think we are simply seeing progressive CJD (early age of onset, rapid decline) at the start of the epidemic in the most susceptible genetic background. Other populations would be looking at later onset, different symptoms, and slower progression of the disease. For example, they could look like cases of sporadic CJD rather than v-CJD.

As I read the middle-ground scenario in Table 30 in Dealler's "Lethal Legacy", 142,000,000 individual meals were eaten in which 10,000 or more lethal infectious units (IU)were present in that serving. As I read Table 29, again the middle-ground scenario, 18,000,000 people would have been exposed to a cumulative dose of 1,000,000 IUs or more. I also see in Table 5 significant numbers of under-2 calves showing BSE whereas Anderson et al quote an experiment with cows receiving 330 grams intra-cerebrally first showing symptoms at 40 months.

I would expect codon 129 to only modulate some parameters of the illness, not be all or none: For example, D178N M129 has a mean age of onset of 49 years, mean duration of 15 months, and presents in decreasing frequency dementia, ataxia, myoclonus, spongiform cerebral cortex, ... whereas D178N V129 has a mean age of onset of 45 years, mean duration of 22 months, and presents in decreasing frequency insomnia, dysautonomia, ataxia, myoclonus, and thalamic nuclei atrophy. Sporadic and iatrogenic CJD are similarly enhanced, but by no means exclusively so, I recall in homozygotes over heterozygotes.


CJD Therapy with anti-sense prion mRNA?

Listserve 10.9.96

This is a good therapeutic proposal that could easily be tested in mice. The experiment may be under way as we speak. Promising results have been published with other diseases. There is no technical problem is producing anti-sense oligonucleotides in quantity. A variation on this is to permanently knock out _one_ copy, more or less, of the prion gene (ie on one chromosome).

On the hopeful side, anti-prion mRNA, meaning mRNA complementary to the normally transcribed strand, would be expected to accomplish down-regulation of prion mRNA promoter and lower prion production itself and so abnormal prion. Gene dosage experiments, such as the 7x experiments of Prusiner, suggest that higher prion levels lead to exaggerated susceptibility and more rapid course of disease. Transfection under the control of a friendly promoter might produce a steady flow in situ of the desired oligonucleotides. Prion turnover is fairly rapid so buildup might be slowed. Even a total absence of prion takes some years to bring symptoms, so knocking back to half the steady-state level might be the best of both worlds.

On the less hopeful side, Collinge noted that prion mRNA is produced constitutively in adults; however, this is not true in neurodevelopment, as Prusiner discusses in the AnnRevMicrob 48 1994. In other words, cells have the potential to respond by some feedback mechanism to curtailment of prion production by trying harder (ie, more transcription starts, more translations per mRNA, etc), maybe resulting in the same end levels of production, especially over time. Then there is the matter of the blood/brain barrier, establishing the safety of any intervention, and early identification of people who might benefit.

Remember that while the prion gene is present in only one copy per chromosome, some other gene of unknown function is already producing mRNA rather complementary to the prion anti-sense strand. Your prion anti-sense oligonucleotides might interfer with this gene by binding to its anti-sense strand, with unkown effects. I have full text of all the relevent articles at www.mad-cow.org/Recent_Science.html and my previous posting is in the listserve Web archives


Prion Infectious Titre during incubation of Symptoms

Listserve comment 10.5.96

No cow data. From Dealler's 'Lethal Legacy', mouse and sheep spleen were at 100% final infectivity [800,000,000 and 10,000 IU, resp.] and steady (flat) 30% of the time into the incubation period for displaying overt symptoms.

Mouse brain: infectious titre increased steadily beginning at 40% of the incubation period but peaked and flattened at 75% at 10,000,000,000 lethal infectious units injected to the same species [IU] per gram.

Sheep brain: increased steadily with flatter slope with the earliest data point shown 65% of incubation period, so from 10,000 IU per gram to 120,000. Mink, goat, hamsters data is given as well.

In the UK, 142,000,000 individually lethal meals have been served (each over 10,000 IU) as per Table 30, page 299.

We don't eat mouse brain or spleen in the US but I hope they are disposing of these animals properly (incinerating under controlled conditions). One asymptomatic mouse has enough units to kill every mammal on the planet.


Controversy over HSP binding to prion 180-2I study

A recent paper (J. Virology July 1996) used the prion gene fused to glutathione transferase as bait in a yeast reporter system, recovering against a human clone library the heat-shock protein 60. A Swiss expert responds to questions on this paper.

Q1. The authors suggest that fusion to a highly soluble and over-expressed carrier protein may overcome problems of insolubility and quantity; one hopes the intact prion would hang off this as an independent and native domain. Do you see good prospects for this?

Q2. The authors then look at a set of deletion mutants of the prion protein, still fused, concluding that 180-210 was the binding recognition region for heat-shock protein 60. I doubted whether some of the deletion fragments would retain much in the way of native 3D structure. Do you think from your work that 180-210 alone would retain the relation between the alpha-helical structures?

Answer: The J. Virol. paper will cause a lot of confusion; the hsp60/PrP interaction is a pure artifact. Hsp60 is a typical cytoplasmic protein and does not occur in the endoplasmic reticulum, where PrP folds and is processed (The authors give a strange reference for the occurrence of Hsp60 in the ER, but I have talked to several experts in the field and everybody has confirmed that there is no hsp60 in the ER). Therefore, Hsp60 will never be in contact with PrP in the living cell.

Moreover, heat shock proteins such as hsp60 are often found as unspecific binding proteins during screening with the yeast two-hybrid system. I do not believe that the GST-fusion protein strategy will provide a useful tool to obtain large quantities of the whole PrP protein for its structure determination, since efficient cleavage and further purification of cleaved PrP will presumably be at least as difficult as trying to express PrP alone.

With regard to the native structure of the PrP segments in the GST fusions, it is not at all clear whether PrP's disulfide bond was formed in the constructs. However, the disulfide is absolutely essential for the native fold of the protein. Finally I do not believe that the segment 180-210 can fold to a native-like conformation, since 50 % of the last helix and the essential disulfide are lacking.

Coexpression of Hsp60 in conjunction with PrP in the cytoplasm will also never lead to native PrP, since disulfide bonds cannot form in the cytoplasm of any cell due to the reducing conditions in the cytosol.


The transgenic mouse experiment: what does it mean?

Survival of Collinge's transgenic mice would simply mean that the species barrier between cow >and human is sufficiently strong to prevent that transmission happening within >the average lifetime of a mouse. It may still occur over a longer period.

The toxic effect of the infectious agent is greatly modified by the amino acid sequence of the normal prion protein. This sequence of the normal prion protein clearly influences not only the species barrier, but also the pattern of damage. Therfore the incubation times of the transgenic Collinge mice depends not solely on the cattle-human species barrier, but also in the interaction of the normal but foreign human prion proteins with other components of the mouse.

Because the influence of the species barrier is only important at the beginning of the exponential process, it may be of minor importance for the incubation time. This was exactly, what John Collinge and many others overlooked with the interpretation of his results with BSE infected transgenic mice with prion proteins of human and mice origin.

It is very important to differentiate between the probabilities of heterologue or homologue conversion reactions. We will be unable to interpretate the running Collinge experiment because we do not know the influence of the second interaction, as it was impossible to interpretate the first Collinge experiment as he does, because not the species barrier but instead the homologue conversion reactions were the determining factors.

On the possible roles and effects of the glycosylation of the prion protein: it seems to not influence the conversion of the normal to the protease-resistent form Incorrect glycosylation may cause artificial results of the Collinge experiments with transgenic mice. Glycosylation influences the transport of the cellular prion protein to the surface. Key references:

Kocisko,D.A.; Priola,S.A.; Raymond,G.J.; Chesebro,B.; Lansbury,P.T.; Caughey,B.
Species-specificity in the cell-free conversion of prion protein to protease-resistant forms - a model for the scrapie species barrier
PNAS 1995 Apr 25; 92(9): 3923-7).

Lehmann,S.; Harris,D.A.
Effect of 2 pathogenic mutations on the cellular processing of the mouse prion protein
Journal of Cellular Biochemistry 1995; 1995(S21B): 104).



Very recent papers

*** Harrington published his 3rd paper in two weeks on the 14-3-3 test [formerly protein 190, 191] for CJD and BSE, using cerebralspinalfluid, in the Oct 3 [?} Lancet. First author was RG Will. They looked at last at CSF from v-CJD, getting satisfactory diagnostic results especially for CSF samples that had been stored properly. The test is likely to be a useful diagnostic adjunct but is probably not going to work too early on (nor be suitable for mass screenings).

Harrington has looked at CSF from cows with TSEs {test works quite well] but not from cows with specifically BSE. Oddly the test was first published in 1986 [no anti-14-3-3 then]. I don't know what is normally done in a slaughterhouse with CSF or whether it would be practical to take samples on the farm.

A week earlier, also in Lancet, a German group reported similar work on 14-3-3 and also used NSE, neuron specific enolase as a marker of CJD. Enolase is a boring cytoplasmic enzyme from glycolysis; evidently there is some isozyme specific to neural tissue. It wasn't quite as useful as 14-3-3. The great thing about NSE enolase is that there is a simple commercial test kit already out there, from Sandoz[?]

The presence of both proteins in CSF is attributed in all papers to brain cell death, not to speciific association with prion protein. Somehow, their presence is quite specific to CJD.

*** Prusiner's group has knocked apoE and GAFT in mice and found no impact on the course of TSE. This is progress. Apolipoprotein E has a big role in Alzheimer's and the e4 allele was also supposed to presuppose towards CJD. Glial fibrillary acidic protein has to do with astrocytic gliosis, also proposed in CJD pathogenicity. August 96 Neurolgy pg 449.

***Japanese group sequenced prion gene promoter in rat. Some exons outside the ORF. They say normal role of prion protein must be that of a "house-keeping" gene, from binding sites for Sp-1, AP-1, and AP-2 but no TATA, ie, the gene is consitutively expressed.

Measuring prion mRNA in varying tissues, brain and placenta came out on top. Hmmm.

*** Yet another Prusiner paper reports a 7x decrease in neuro-membrane fluidity as more fundamental than the long and growing list of _secondary_ physiological abnormalities in scrapie-infected =cell lines. (Sept 96 Neurology pg 771)

*** France is having a terrible problem with human growth hormone CJD, with 1.3% of all those treated already ill or dead. For some reason, much worse situation than in other countries.(Sept 96 Neurology pg 690)

*** Yet another octapeptide insertion family has been found in a Ukranian-US family. Here it is a penta-repeat R2 R2 R2 R2 R3 _without_ the silent wobbles found in Collinge's UK case. These mutations seem about as frequent as point mutations. Must have to do with secondary structure in the DNA and repeat slippage of the polymerase during replication. Long slow onset with many early psychological problems that were being treated with anti-depressants.

*** Codon 129 polymorphism data from Italy, vis-a-vis P102L GSS. A lot of phenotypic variability was found. (Sept 96 Neurology pg 734)

*** Lev Goldfarb and Paul Brown in the Ann. Rev. Med. 1995 pages 57-65. Being a book, it is not indexed by Medline [though 35 other articles on prions by these two oauthors are returned].

It has a nice review of iatrogenic CJD, including some incidence rate data by country for growth hormone CJD. 58 cases, incidence 0.1% US, 0.7% UK, 1.9% France. 4 cases from infertility treatment with gonadotrophin, all in Australia. There is also a nice table for genetic CJD, showing for each mutation, age of onset, duration, and clincal pathology [highly variable].

Most striking quotes:

"In Central Slovakia, CJD is the fifth most common cause of death." [Recall that while E200K M129 is the genetic basis there, it seems of recent origin and is in a high scrapie area.]

"Thus far, CJD transmission via blood or blood derivatives has not been proven, despite the fact that infectivity can be regularly shown in the blood of experimentally infected animals and occasionally in the blood of CJD patients."

Reference given for this is Paul Brown. 1993 "Infectious cerebral amyloidosis: clinical spectrum, risks and remedies." Dev Biol. Stand. 80:91-101 [puzzling, Dev Biol. Stand is indexed for 1993, but this abstract is not there.]


Interactions of prion proteins modulate neurodegeneration in transgenic mice.

Telling GC; Haga T; Torchia M; Tremblay P; DeArmond SJ; Prusiner SB Department of Neurology, University of California at San Francisco, 94143 USA. Genes Dev 10: 1736-50 (1996) Transgenic mice overexpressing approximately eightfold the mouse (Mo) prion protein (PrP) gene carrying the P102L mutation of GSS developed neurodegeneration between 150 and 300 days of age, while controls expressing the wild-type MoPrP-A transgene at the same level remained healthy. Mice overexpressing the wild-type MoPrP-A transgene were highly susceptible to inoculated mouse prions, exhibiting abbreviated scrapie incubation times of 45 days.

After crossing the mutant transgene onto a null (Prnp 0/0) background, the resulting Tg(MoPrP-P101L)Prnp 0/0 mice displayed a highly synchronous onset of illness at 145 days of age, which was shortened to 85 days upon breeding to homozygosity for the transgene array. Besides occasional PrP plaques and modest spongiform degeneration, Tg(MoPrP-P101L) mice suffered from a myopathy and a peripheral neuropathy. Disruption of the wild-type MoPrP gene increased the number of PrP plaques and the severity of spongiform degeneration.

Brain extracts prepared from spontaneously ill transgenic mice transmitted disease to Tg196/Prnp 0/0 mice, expressing low levels of the mutant transgene. Our results demonstrate that the presence of wild-type PrP genes, the level of PrP transgene expression, and the sequence of the transgene can profoundly modify experimental prion disease.


Dr. Herbert Budka
Klinisches Institut fuer Neurologie
Universitaet Wien
AKH 04J, PF 48
A-1097 Wien, Austria
Tel.+43-1-40400-5504, -5501, -5573
Fax +43-1-40400-5511, -5573

Research in Austria on Transmissible Spongiform Encephalopathies

As of Oct. 1996, Prof Budka does not have a web page. He has kindly rpovided a referenced review of TSE research in Austria, cmainly oncerning neuropathology in human.s.
Research in Austria on transmissible spongiform encephalopathies (TSEs, prion diseases) is centered at the Department of Neuropathology and Neurochemistry, Institute of Neurology, University of Vienna, in Professor Herbert Budka's group. According to the background of this group, their research concerns mainly the characterisation and pathogenesis of tissue lesions in human TSEs.

Prof. Budka leads an EU Biomed-1 Concerted Action (CA) on "The human prion diseases: from neuropathology to pathobiology and molecular genetics" 1,2 begun on Oct.1, 1994, and to expire on Dec.31, 1996.

It networks almost 100 European neuropathological and basic research laboratories dealing with neuropathologic diagnosis of, and tissue-based research in, human TSEs. Two CA meetings were held in Vienna in Oct. 19953 and in May/June 19964, respectively; the latter meeting addressed the highly topical issue of phenotypic variation in Creutzfeldt-Jakob disease (CJD) in Europe. A final CA meeting will be held Dec.6-7, 1996, in Vienna.

In two consensus reports, the CA has succeeded in defining criteria for neuropathological diagnosis5 of, and tissue handling6,7 in, human TSEs.

The first report is important for diagnosing human TSEs under generally accepted terms. The second has greatly contributed to alleviate fears about performing autopsies on patients suspected to suffer from TSEs; autopsies are most important for a definite diagnosis and are mandatory to identify the new variant of CJD recently recognized in the UK and France. A subsequent similarly designed CA proposal was submitted.

In addition, Prof. Budka participates in the EU BIOMED-2 Shared Cost Action entitled "The study of prion propagation, pathogenesis, and intermammalian species barriers in transgenic and other models" (Project Leader: Prof. J. Collinge) which is in the process of starting; its first meeting will be held in the second half of Oct. 1996.

Moreover, the Austrian Reference Center for Human Prion Diseases (÷sterreichisches Referenzzentrum zur Erfassung und Dokumentation menschlicher Prionen-Erkrankungen / ÷RPE; head: Prof. H. Budka) was recently established at the Institute of Neurology, University of Vienna 8. It will coordinate surveillance of human TSEs in Austria. As first success, we have identified the first Austrian case of CJD in a dura transplant recipient, and one CJD family with a yet undescribed PRNP molecular genetic constellation9.

Specific research topics investigated by Prof. Budka's group include the following:

1) CJD (human and experimental)

An epidemiological study of CJD in Austria10, based on the nationwide collection of neuropathologically diagnosed, i.e. definite CJD cases, showed a gradual increase of diagnosed CJD in recent years; in 1995, the mortality rate was 1.25 per million inhabitants which is the highest so far observed in Europe. Moreover, an update for the first half of 1996 gives an estimated rate for 1996 of 1.7 per million9. These data are important because Austria is a BSE-free country; the high CJD incidence is attributed to improved surveillance due to generally increased awareness of neurologists and the high autopsy rate in Austria11,12.

The previously described conjugal occurrence of JCD in an Austrian couple was not confirmed by modern immunocytochermical detection of the prion protein (PrP)13. This is an important argument against speculations on a possibility of environmental human-to-human transmission of CJD.

Several other studies have addressed, or are in progress to elucidate, the development of tissue pathology in TSEs: neuropathological overviews14-16; the presence of peculiar tubulovesicular structures which are specific to TSEs17; the type and pattern of PrP deposition in a large series18-20 which is an important point of reference when considering comparison with the newly recognised CJD variant in the UK; and the PrP deposition in the brain stem which might give some hints on spread from a possible infectious origin21. The distribution of parvalbumin positive neurons in brain correlates with hippocampal and temporal cortical pathology in CJD22.

Studies on experimental CJD showed that neuroaxonal dystrophy is an important part of the neuropathology of TSEs23. Another report on the sequential development of tissue pathology correlated with PrP deposition in experimental CJD is in preparation (Kordek et al).

2) Gerstmann Str”ussler-Scheinker disease (GSS)

The original Gerstmann family of Austria has been recently rediscovered and agreed to pedigree updating and clinical, neuropathological, and molecular genetic studies24. The classical P102L mutation was confirmed in the PrP gene (PRNP)25. A change in phenotype from the classic ataxic to a CJD-like dementing type was observed in a recent patient; comparative molecular analysis showed that the PRNP genotype was identical at codon 129 (M/M homozygosity) in both phenotypes24. This means that additional factors (other genes? environmental?) determine phenotypic expression in this inherited TSE.

Another study described the ultrastructural neuropathology of GSS26 which includes also tubulovesicular structures27. Microglia has been found as an important constituent of PrP plaques28. As in Alzheimer's disease, paired helical filaments dissociate from amyloid formation29. 3) Kuru

By cooperation with P. Brown's and D. C. Gajdusek's group at the NIH in Bethesda, MD, USA, one of the apparently last sets of archival Kuru brain specimens could be investigated with modern neuropathological methods including immunocytochemistry for PrP30,31. This is important because the new CJD variant in the UK and France has been considered to share clinicopathological characteristics with Kuru.


References

1. Budka H. Prion diseases - from molecular biology to neuropathology. J
Neuropathol Exp Neurol 1995;54 Suppl:75S-76S.

2. Budka H. The human prion diseases: from neuropathology to pathobiology
and molecular genetics. In: Baert AE, Baig SS, Bardoux C, et al., eds.
European Union Biomedical and Health Research. The Biomed-1 programme.
Amsterdam: IOS Press, 1995: 513-514.

3. Budka H, Hainfellner JA. The human prion diseases: Neuropathology,
pathobiology and molecular genetics. Vienna, Austria, 16 October 1995
(meeting report). Neuropathol Appl Neurobiol 1996;22:171-173.

4. Budka H, Hainfellner JA. Meeting report: Phenotypic variation in
Creutzfeldt-Jakob disease, May 31 and June 1, 1996, Vienna, Austria. Brain
Pathol 1996 (in press);6(4).

5. Budka H, Aguzzi A, Brown P, et al. Neuropathological diagnostic criteria
for Creutzfeldt-Jakob disease (CJD) and other human spongiform
encephalopathies (prion diseases). Brain Pathol 1995;5:459-466.

6. Budka H, Aguzzi A, Brown P, et al. Konsensusbericht: Gewebsbehandlung
bei Verdacht auf Creutzfeldt-Jakob-Krankheit und andere spongiforme
Enzephalopathien (Prionen-Krankheiten) des Menschen. Pathologe
1996;17:171-5.

7. Budka H, Aguzzi A, Brown P, et al. Tissue handling in suspected
Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies
(prion diseases). Brain Pathol 1995;5:319-322.

8. Budka H. Umfassend ¸berwacht. Referenzzentrum f¸r Prionen-Erkrankungen.
÷sterr źrzteztg 1996(19):34-36.

9. Hainfellner JA, Jellinger K, Diringer H, et al. Die
Creutzfeldt-Jakob-Krankheit in ÷sterreich. Wien Klin Wochenschr 1996 (in
Druck).

10. Hainfellner JA, Jellinger K, Diringer H, et al. Creutzfeldt-Jakob
disease in Austria. J Neurol Neurosurg Psychiat 1996;61:139-142.

11. Bignall J. CJD rates increase in Austria. Lancet 1996;348:602.

12. Boulton A. Rise in CJD is not a true increase. Brit Med J 1996;313:383.

13. Hainfellner JA, Jellinger K, Budka H. Testing for prion protein does
not confirm previously reported conjugal CJD. Lancet 1996;347:616-617.

14. Budka H. Neuropathological diagnostic criteria for Creutzfeldt-Jakob
disease (CJD) and other human spongiform encephalopathies (prion diseases):
submitted to WHO, 1996.

15. Budka H. Đbertragbare spongiforme Enzephalopathien
(Prionen-Krankheiten): ein einzigartiges biomedizinisches Paradigma, 1996
(in Druck).

16. Budka H. Transmissible spongiform encephalopathies (prion diseases).
In: Garcia JH, ed. Neuropathology: the diagnostic approach. Philadelphia,
PA: Mosby-Year Book, 1996 (in press).

17. Liberski PP, Budka H, Sluga E, Barcikowska M, Kwiecinski H.
Tubulovesicular structures in Creutzfeldt-Jakob disease. Acta neuropathol
1992;84:238-243.

18. Hainfellner JA, Budka H. Immunomorphology of human prion diseases. In:
Plotkin S, Fantini B, eds. Prion: Jenner, Pasteur and their successors.
Paris: Elsevier, 1996 (in press).

19. Budka H, Hainfellner JA, Guentchev M, et al. Neuropathologically
confirmed sporadic Creutzfeldt-Jakob disease: incidence in Austria, and
patterns and distribution of PrP deposits in brain (abstr). J Neuropathol
Exp Neurol 1996;55:661.

20. Hainfellner JA, Jellinger K, Gullotta F, et al. Immunomorphology of
cerebral and cerebellar prion protein (PrP) deposition in sporadic
Creutzfeldt-Jakob disease (abstr). Neuropathol Appl Neurobiol 1996;22 Suppl
1:7-8.

21. Pietrini V, Hainfellner JA, Trabattoni GR, Budka H. The brain stem in
Creutzfeldt-Jakob disease (abstr). Clin Neuropathol 1996;15:179.

22. Guentchev M, Hainfellner JA, Trabattoni GR, Budka H. Distribution of
parvalbumin positive neurons in brain correlates with hippocampal and
temporal cortical pathology in Creutzfeldt-Jakob disease (CJD). Clin
Neuropathol 1996;15:272.

23. Liberski PP, Budka H, Yanagihara R, Gajdusek DC. Neuroaxonal dystrophy
in experimental Creutzfeldt-Jakob disease. Electron microscopical and
immunohistochemical demonstration of neurofilament accumulations within
affected neurites. J Comp Pathol 1995;112:243-255.

24. Hainfellner JA, Brantner-Inthaler S, Cervenakova L, et al. The original
Gerstmann-Str”ussler-Scheinker family of Austria: divergent
clinicopathological phenotypes but constant PrP genotype. Brain Pathol
1995;5:201-211.

25. Kretzschmar HA, Honold G, Seitelberger F, et al. Prion protein mutation
in family first reported by Gerstmann, Str”ussler, and Scheinker. Lancet
1991;337:1160.

26. Liberski PP, Budka H. Ultrastructural pathology of
Gerstmann-Str”ussler-Scheinker disease. Ultrastruct Pathol 1995;19:23-38.

27. Liberski PP, Budka H. Tubulovesicular structures in
Gerstmann-Str”ussler-Scheinker disease. Acta neuropathol 1994;88:491-492.

28. Barcikowska M, Liberski PP, Boellard JW, Brown P, Gajdusek DC, Budka H.
Microglia is a component of the prion protein amyloid plaque in the
Gerstmann-Str”ussler-Scheinker syndrome. Acta Neuropathol 1993;85:623-627.

29. Liberski PP, Kloszewska I, Boellaard J, Papierz W, Omulecka A, Budka H.
Dystrophic neurites of Alzheimer's disease and
Gerstmann-Str”ussler-Scheinker disease dissociate from the formation of
paired helical filaments. Alzheimer's Res 1996;1:89-93.

30. Hainfellner JA, Liberski PP, Guiroy D, Brown P, Gajdusek DC, Budka H.
Neuropathology of a Kuru brain (abstr). Clin Neuropathol 1996;15:272-273.

31. Hainfellner JA, Liberski PP, Guiroy DC, et al. Pathology and
immunocytochemistry of a kuru brain. Brain Pathol 1997 (in press);7(1).

Other Books on BSE

Narang's book.  Not finished yet and mainly science, on the way.

Food 'scares' and the media. Glasgow University Media Group 1994. ISBN 09521669 4 1
 
Food and the media: Reporting health scares in S. Henson and S. Gregory
(eds) The Politics of food, 1994. Proceedings of an interdisciplinary
seminary held at the Univesity of Reading 7th July 1993. Reading:
Department of Agricultural Economics, Reading Univesity. ISBN 0 704905 38 X
 
Making an Issue of Food Safety: the media pressure groups and the public
sphere. In Donna Maurer and Jeffrey Sobal (eds) food Eating and
Nutrition as Social Problems: constructivist Perspectives, NY Aldine de
Gruyter 1995. ISN 0 202 30507 4.
 
Mad Cow Disease: The history of BSE in Britain. RW Lacey. Ipsela Press,
Jersey. 1995 ISBN 1 899516 00 X. Contains a lot of information showing
that much of what has come from the UK government has been misinformation.
 
Lethal Legacy. BSE - the search for the truth. SF Dealler. Bloomsbury
Press, Soho, UK. 1996 ISBN 0 7475 2940 X goes through BSE from the is
appearance to the day that cases are presumptively announced in humans.
Written as a story but with large amounts of information. Paperback.
 
Slow Virus Diseases. Zeman, Lennette, Brunson (Eds) The Williams and
Wilkins co, Baltimore, USA 1974. ISBN 0 683 09368 1
 
Spongiform encephalopathies. I.V. Allen ed. Published for the British
Council by Churchill Livingstone, Edinburgh ISBN 0 443 04928 9
 
Planned beef production and marketing. David Allen. BSP Professional Books,
Oxford, UK. ISBN0 632 02611 1 A useful book to work out what is
going on.
 
Rinderwahnsinn, BSE: die neue Gefahr aus dem Kochtopf Koester-Loesche,
Kari: Muenchen 1995, ISBN 3-431-03415-2. 
This book discusses the Mad Cow Disease, its origin and dissemination, it demonstrates how the hazards are played down and denied by authorities and politicians, it describes how infected beef can get into the shops in spite of all efforts, it explains why the BSE agents have to be kept out of the kitchen by all means. The author, Dr.med. vet. Kari Koester-Loesche, born in Luebeck, is a veterinarian and has published numerous scientific works, among other things, ""Disease with long-term effect. Documentation on Mad Cow Disease" (1994), "Fit against viruses and bacteria" (1995), and "The great epidemics, from Plague to Aids"(1995).)
BSE - The Facts Brian Ford. ISBN 0 552 14530 0, 208pp, 4.99, London: Corgi Books.
This is a sharp book aimed at explaining about TSEs and BSE. It is aimed at the general population and has been very popular because it has been well written and well published. The complexities of the disease and the politics that have gone with it are gone over and it is likely that this will be republished should further information appear.


Chronic wasting disease

Listserve comment 10.5.96

Cow and sheep herds in the US may already distributing a form of TSE, chronic wasting disease, into wild, never-captive deer and elk in northern Colorado and southern Wyoming, by local newspaper accounts at a hard-to-believe 1% level. It is absolutely imperative that hunters not use these brains for hand-tanning of hide or sausage, whatever the origin of the TSE. They have gotten really poor advice so far by Fish & Game and the hunting season is underway.

This should not be confused with the older situation where unsupplemented captive deer acquired a scrapie-like condition on fenced former sheep range in Colorado. Nor should it be confused with the captive elk with CWD from the Dakotas this year, which may have received bovine-based protein supplements. I have an additional concern about bison, being even closer to cattle genetically, but have not heard any reports.

I would personally speculate that CWD is cattle-to-deer/elk rather than sheep-to deer/elk because sheep would not have had supplement prior to turn-out. CWD is a newly reported condition; US scrapie has been around since the 1940's in many states. Whatever the origin, many questions are raised on horizontal and vertical transmission, vectors, and outdoor stability of agent.

This is a difficult situation for APHIS, whether or not to conduct (much less publish) lab animal _strain studies_ on CWD, as these have the potential for affixing the origin to public land livestock, whether sheep or cattle.

I also forgot to mention Norway, which has taken draconian measures to knock down the recent upsurge they have seen in scrapie (one supposes contaminated UK meal). There has been grave concern that this will get into reindeer from sheep aggressively grazed on common public lands, which has all sorts of racial overtones already with regards to native Lapp people.


Relative risk factors for sporadic CJD because of codon129.

Listserve comment 10.5.96

A doubling is seen for met-met frequency. There is not necessarily any direct relevence for v-CJD here. Note that "sporadic CJD" could actually lump, say, 3-4 separate causes [hypothetical example: spontaneous conversion, somatic mutation, pig-CJD, scrapie-CJD]. These could each have their own very distinct allele statistics -- we are only looking at the resultant sum.

The data were from 1990-93 so now there could be enough new sporadic cases for 1994-96 to perhaps look for any change to allele predisposition.

There is another common polymorphism in Japanese, at codon 220, 6% level. Not found at all in large series of Italians. It is difficult to say whether any polymorphism is truly non-disease causing. These are all genetic drift, not Darwinian-selected.

The codon 129 paper is Hum Genet 98: 259-64 (1996):

Abstract:
...We have determined the frequencies of predisposing mutations and allelic variants in PRNP and their relative contributions to disease. Systematic PRNP genotype analysis was performed on suspected CJD cases referred to the National Surveillance Unit in the UK over the period 1990-1993. Inspection of 120 candidate cases revealed 67 patients with definite and probable CJD, based on clinical and neuropathological criteria. No PRNP mutations were detected in any of the remaining 53 patients assessed as "non-CJD". A disease-associated mutation in the PRNP gene was identified in nine (13.4%) definite and probable cases of CJD, a reliable estimate of the incidence of PRNP-related inherited CJD based on a prospective epidemiological series.

Within the group of sporadic CJD patients (lacking PRNP mutations), we confirmed that the genotype distribution with respect to the common methionine/valine (Met/Val) polymorphism at codon 129 within PRNP was significantly different from the normal Caucasian population. The incidence of Met homozygosity at this site was more than doubled and correlated with increased susceptibility to the development of sporadic CJD. Unlike other recent studies, Val homozygosity was also confirmed to be a significant risk factor in sporadic CJD, with the relative risks for the three genotypes Met/Met: Val/Val:Met/Val being 11:4:1.