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More nvCJD found with tonsil test

Lancet Volume 353, Number 9148  16 January 1999
A F Hill, R J Butterworth, S Joiner, G Jackson, M N Rossor, D J Thomas, A Frosh, N Tolley, J E Bell, M
Spencer, A King, S Al-Sarraj, J W Ironside, P L Lantos, J Collinge 
Lancet has the fulltext freely available to non-subscribers who register. The article is too long [thirteen 19" screens] and complex to relay directly here. There is additionally a cautious commentary piece by Robert B Petersen, Case Western Reserve, that additionally calls attention to similar concerns of contamination of surgical tools used in appendectomies.

Methods:

Lymphoreticular tissues (68 tonsils, 64 spleens, and 40 lymph nodes) were obtained at necropsy from patients affected by prion disease and from neurological and normal controls. Tonsil biopsy sampling was done on 20 patients with suspected prion disease. Tissues were analysed by western blot to detect and type PrPSc, by PrP immunohistochemistry, or both.

Findings:

All lymphoreticular tissues obtained at necropsy from patients with neuropathologically confirmed nvCJD, but not from patients with other prion diseases or controls, were positive for PrPSc. In addition, PrPSc typing revealed a consistent pattern (designated type 4t) different from that seen in nvCJD brain (type 4) or in brain from other CJD subtypes (types 1-3). Tonsil biopsy tissue was positive in all eight patients with an adequate biopsy sample and whose subsequent course has confirmed, or is highly consistent with, a diagnosis of nvCJD and negative in all patients subsequently confirmed to have other diagnoses.

Introduction

"...Diagnosis of nvCJD in the living patient presents more difficulties than sporadic CJD. Cerebrospinal fluid 14-3-3 protein, usually positive in sporadic CJD, is presumably a marker of neuronal injury, and appears less useful in the more slowly progressive nvCJD.8 High T2-weighted signal in the posterior thalamus on magnetic resonance imaging (MRI), seen in several patients with nvCJD, has yet to be formally assessed as a diagnostic tool.8 Definite diagnosis of nvCJD has therefore remained neuropathological, at necropsy or brain biopsy.

"34 cases of nvCJD in the UK and a single case in France have been confirmed by neuropathology. While numbers have risen slowly to date, it remains entirely possible that a substantial epidemic of nvCJD will occur over the coming years. The extremely prolonged incubation periods of these diseases in human beings, allied with the effect of crossing a species barrier (which will further prolong mean incubation periods), means that it may be some years before the parameters of such an epidemic can be predicted with any degree of confidence. It is possible that significant numbers in the population are incubating this novel disease and that they might pass it on to others via blood transfusion, blood products, tissue and organ transplantation, and contaminated surgical instruments, and other iatrogenic routes. ...

"Prion replication occurs in the lymphoreticular system in sheep with scrapie and in rodent models of scrapie, and replication in the spleen and other lymphoreticular tissues may precede detectable neuroinvasion with prions by quite some time. Tonsil biopsy has been shown to be a useful diagnostic test in sheep scrapie--PrP immunohistochemistry is positive in about a third to a half the normal incubation period.

"PrPSc has been found in tonsil tissue in a necropsy sample from a neuropathologically confirmed case of nvCJD, suggesting that tonsil biopsy sampling may be useful for tissue diagnosis of human prion disease. We analysed a series of necropsy samples from patients with sporadic, inherited, and acquired human prion diseases and tonsil biopsy samples done for the investigation of patients with suspected prion disease.... "

Strain type 4t

"Human prion strains can be distinguished by both molecular mass and relative abundance of the three principal PrP glycoforms detected by western blotting of proteinase-K-digested brain homogenates. Fragment sizes of protease-treated PrP in tonsil tissue were indistinguishable from those seen in brain from patients with nvCJD. Figure 2A shows western blot comparison of a tonsil biopsy sample [lanes 5, 6] with (necropsy) tonsil and brain tissue from definite nvCJD [lanes 1, 2] and normal control tonsil [lanes 3, 4] with anti-PrP monoclonal antibody 6H4 (Prionics). After digestion with proteinase K an identical pattern of protease-resistant prion protein fragments was seen in the biopsy sample [lane 6] and in tonsil tissue from a patient with nvCJD confirmed at necropsy [lane 8]. While the glycoform ratios in nvCJD tonsil superficially resembled those seen in nvCJD brain, with a distinctive abundance of diglycosylated PrP, quantitation of these ratios revealed that they were different from those seen in brain (p less than 0.0001 with respect to the proportions at each of the three glycoforms [unpaired two-tailed t-tests]), but highly consistent between the nvCJD samples studied. The concentrations of PrPSc detectable in nvCJD tonsil varied and in some cases was about 10% of the concentration seen in nvCJD brain tissue."

[ The original grayscale gel is shown at Lancet.]

Discussion

The numbers of patients studied are still relatively small, but all definite or clinically highly probable cases of nvCJD studied had PrPSc detectable in tonsil tissue, or had abnormal PrP immunoreactivity, or both, and no case of other forms of prion disease were positive. These findings suggest that tonsil biopsy, at least in advanced disease, is both an extremely specific and sensitive diagnostic test. Our results, allied with previous studies that failed to detect PrPSc in lymphoreticular tissues of patients with sporadic CJD, show a clear distinction between the degree of lymphoreticular involvement in variant and sporadic CJD. This could be due to a prion-strain effect, with the BSE-like (bovine-spongiform-encephalopathy) strain that causes nvCJD being highly lymphotrophic in human beings, or could relate to a first passage or species-barrier effect.

The degree of involvement of the lymphoreticular system in prion pathogenesis is known to vary between prion strains in the same host and also between hosts infected with the same prion strain. It has also been suggested that prion replication is required in the lymphoreticular system on first passage of a prion strain from a different species to the inoculated animal. The route of exposure of the patients developing nvCJD may be crucial. We were unable to detect PrPSc in lymphoreticular tissues from patients with iatrogenic CJD. It is possible that the oral route of exposure, presumed to be the route of BSE infection of patients with nvCJD, results in a more pronounced lymphoreticular phase. In this regard, it will be of interest to study lymphoreticular tissues from patients with kuru.

The stage at which PrPSc is detectable in human tonsil in patients incubating nvCJD is unknown. In sheep scrapie and in animal models of prion disease, prion replication is typically detected first in the spleen and rises to a plateau before detectable neuroinvasion. Mice with severe combined immunodeficiency are highly resistant to prion infection if inoculated peripherally. The precise cell type or types involved in lympho-reticular prion replication and transport to the central nervous system remains controversial although several lines of evidence argue for a key role for follicular dendritic cells in peripheral replication.

Extrapolation from these studies would argue for an early pre-clinical involvement of the lymphoreticular system in nvCJD. This is supported by the finding of abnormal PrP immunoreactivity in an appendix removed 8 months before the onset of neurological symptoms in a patient subsequently confirmed, at necropsy, as having definite nvCJD. It is of interest that in cattle experimentally infected with BSE, infectivity (by mouse bioassay) is detectable only in the distal ileum at 6 months post-exposure, suggesting early involvement of Peyer's patches and other gut lymphoreticular tissue. These findings suggest that human tonsil biopsy samples may allow presymptomatic diagnosis of nvCJD. Indeed, it is possible, assuming sufficiently sensitive tests were available, that PrPSc will be detectable in human tonsil within months of exposure to BSE.

It is possible, although speculative, that all human beings incubating BSE could currently be detected from tonsil biopsy samples. It may therefore be possible to obtain useful prevalence information about preclinical nvCJD by anonymous screening of routine tonsillectomy samples, appendicectomy tissues and archival material. The methods described could, in principle, be used to screen cadaveric or live organs, or tissue donors, including bone-marrow donors, if the prevalence of preclinical nvCJD were of a sufficient level to justify this.

It is possible however, for the reasons outlined above, that secondary nvCJD cases, arising from iatrogenic exposure to nvCJD prions, may not have positive tonsil biopsy samples, and may be undetectable by these methods. It is also possible that the current patients affected by nvCJD will prove to be atypical in the degree of their lymphoreticular involvement. There is, at present, no epidemiological evidence to suggest an unusual dietary or occupational exposure to BSE amongst the patients currently recognised as having nvCJD, and therefore it is possible that they represent a particularly susceptible subgroup with unusual lymphoreticular sensitivity to BSE prions resulting in short incubation periods.

Experimental evidence to suggest infectivity of human blood in sporadic CJD has been questioned, and epidemiological studies provide no evidence that blood transfusion or blood products are a risk factor for the development of CJD.3 However, our finding of significant concentrations of PrPSc in lymphoreticular tissues of nvCJD patients, but not in classical CJD patients, together with evidence for a key role for mature B lymphocytes in prion pathogenesis, emphasised concerns that blood and blood products derived from patients incubating nvCJD may represent a greater risk than with material from classical CJD.

In this regard, it will be important to identify the cell type or types involved in propagating and carrying prion infectivity, or both, in peripheral tissues. Our studies are consistent with a role for follicular dendritic cells and it is possible that the involvement of B lymphocytes is simply to allow maturation of follicular dendritic cells. The intimate association of B cells and follicular dendritic cells in lymphoid follicles, with interdigitation of their cellular membranes, complicates dissection of their individual roles because glycosylphosphatidylinositol anchored cell-surface proteins such as PrP can transfer between cells [Some cites are provided below -- webmaster]. If prion propagation were to be restricted to non-circulating cell types such as follicular dendritic cells, this would not necessarily exclude transport of PrPSc by circulating cells such as B lymphocytes.

It is known that different tissues, and different cell types within tissues, glycosylate proteins differently. It is therefore unsurprising that the ratios of protease-resistant PrP glycoforms seen in the tonsil in nvCJD differs to some extent from those seen in brain tissue. However, diglycosylated PrP represents the predominant glycoform as in nvCJD brain, in clear distinction to the pattern seen in the brain in the various types of sporadic CJD, where monoglycosylated PrP predominates. Presumably, the glycoform ratios seen in nvCJD tonsil represent a superimposition of prion strain and tissue-specific effects. This tonsillar PrPSc type in nvCJD is designated type 4t to distinguish it from the type 4 pattern seen in the brains of all patients with nvCJD studied to date. Clearly, the PrP glycosylation patterns in tonsil of other human prion diseases cannot be compared because, if PrPSc is present in tonsil tissue in these patients, it is undetectable by current methods.

Comments (webmaster):

The graphics in the online Lancet article were of unacceptable quality. A reader has to blow up Figure 2B 8-fold in other software and do a pixel-by-pixel study to determine which strain type goes with which data point.

Strain types remain very poorly defined from a basic biochemistry standpoint. For example, monoglycosylated could be all at codon 181 in the polypeptide chain, all at codon 197, or any mix. Thus, strain type 1 could easily be a mix of very distinct strains. The gels are smears by the nature of this technique, intentional over-loading, the mix of molecular species (ie, ragged N and C termini, variable extent and nature of glycosylation), and varying affinities of antibody 6H4. No one has the slightest idea if the tonsil attaches the same or even similar carbohydrate to that of brain. These strain-types are desparately in need of MALDI-TOF to really define them as covalent structures, never mind for now differences in native conformations.

Figure 2A, the gels, were not offered as a high resolution image and its legend did not explain clearly what was in each of the 8 lanes. I fixed this and also recolorized the gel using an adaptive spectral palette to bringing out key features in the gel. This resulted in a very substantial improvement of gel visualization.

As I read this article, they found 5 new cases of nvCJD for a total of 40 UK+1 EU:

All 9 were met/met at codon 129. 5 are still alive. Ages at onset were 18, 21, 21, 22, 24, 28, 29, 34, 35 which average to 25.8. Three cases were in the DoH statistics page at the time the article was written [34 cases] and 1 has been added since. making for 5 new cases. 9 out of 20 patients with suspected prion disease had nvCJD; the 20 could potentially be 20 referals to the CJD Unit or alternately 20 referals that had passed further screening [but see below]. The 4 who died had clinical duration averaging 15 months, whereas the 5 further cases are averaging 14 months, suggesting that most of the 5 new cases would have appeared at DoH in the next few months using the previous surveillance method. {Further comments on nvCJD statistics below]

Two patients, with no family history of neurodegenerative disease on referral for biopsy sampling, were confirmed as E200K M129M and A117V M129V. These are two of the commonest CJD-causing mutations. The article does not state whether these are new kindreds .A M129V patient has been affected for over 33 months and is thought to have dementia with Lewy bodies.

One case diagnosed as sporadic CJD was confirmed on neuropathological examination; a second was diagnosed as sporadic CJD with a typical electro-encephalogram, but necropsy was not done. Of the seven living patients, five have made a partial or complete recovery, inconsistent with a diagnosis of prion disease.

Another patient, V129V, who presented with a most unusual clinical picture of very young onset progressive aphasia, has continued to deteriorate--no diagnosis having been reached. This case is potentially the first case of nvCJD in V129V.

They "were unable to detect PrPSc in lymphoreticular tissues from patients with iatrogenic CJD. It is possible that the oral route of exposure, presumed to be the route of BSE infection of patients with nvCJD, results in a more pronounced lymphoreticular phase. In this regard, it will be of interest to study lymphoreticular tissues from patients with kuru." It is not clear whether such tissue has been saved from older studies or whether autopsy material is needed from the weakly ongoing epidemic. Kuru is significant because it is the only other instance of sporadic CJD established to have an external route and because of large numbers of victims with common strain exposure. All other sporadic CJD is one of a kind (or better, has no basis for lumping at this time).

Although tonsil biopsy seems highly specific and a reliable enough leading indicator of nvCJD, at least for patients showing up at the two referral centres, the authors explicitly state more refined methods might detect material in the 3 tissues studied in other kinds of CJD.

Was it just fortuitous that the lymphoreticular system works so well for nvCJD or does it have to do with the dietary source of transmission? If the latter, and if dietary scrapie also transmits to humans (as an unrecognized class of sporadic CJD, then perhaps there too the lymphoreticular system will show an effect.

Expansion of the tonsil and appendectomy screen to a much broader segment of the UK population is warranted. Since an upper bound on the epidemic is the main goal, a few percent false positives would be irrelevent. The results here do not establish how early nvCJD can be detected in tonsils nor whether infected tonsils reliably predict future disease more than, say, a year prior to clinical symptoms.

Anticipated mis-quotations from this article

Most of the patients are still alive -- the tonsil biopsy was the only tissue studied. In others, lymphoreticular tissues (68 tonsils, 64 spleens, and 40 lymph nodes) were obtained at necropsy. No other body tissue was studied. The location of the lymph nodes tested is not stated. Some people will go beyond this data to say, "all lymphoreticular tissue must be infected, lymphoreticular tissue is everywhere, ergo the whole body is infectious." Actually, infectivity was not even studied in this article, only the presence of PK-resistant prion.

More broadly, there is a disturbing trend of people saying "all" sporadic CJD or "all" classical CJD is this or that. Here they looked at 8 lymph nodes from an unstated number of sporadic CJD cases. Sporadic CJD by definition is a catch-all term for anything and everything for which the etiology of the CJD is not yet known. nvCJD itself was initially classified as sporadic CJD. In one decade worldwide, there would be 50,000 cases of sporadic CJD. There could easily be a hundred distinct contributing component mechanisms. No one can sample this diversity adequately with 8 lymph nodes any more than anyone can sample 20 kinds of familial CJD with 1 lymph node (as here). The cumulative world total for sporadic CJD strain-typing probably does not 150 cases.

I see a distinct political agenda in deliberately mis-quoting good articles such as this: the use of "all" to make the blood sound safe because the donor was "only" a sporadic CJD victim and "all" of them were "shown" by so-and-so to be blah blah blah... Get back to the webmaster when you have looked at 10,000 cases with an exquisitely sensitive method.

Infectivity is just a concern vis-a-vis surgical passaging at this time. There are no proposed iatrogenic cases of nvCJD. No one can quantitatively compare the infectious titres likely to be passed with surgical tools to those already acquired ten years ago by eating hundreds of kilos of BSE beef. On the other hand, injection has been vastly more efficient than eating in animal models. Surgical transmission is a significant issue if the nvCJD epidemic is fairly small scale; if it is of biblical proportions, secondary transmission is more of an issue for other countries.

It is easy to misread the DoH statistics page. A line in the table gives the number of referrals, the number of each type of CJD identified, and the total. Of course, not every referral to the surveillance unit is a case of CJD, much less nvCJD, so the number of referrals is larger than the total CJD ["about half the cases referred in the past have turned out not to be CJD."]. A final disposition for each referral is implied, that is, each referral was some kind of CJD or it wasn't.

However it doesn't really say this anywhere in the table or legend. Indeed a case could drag on for months or years without disposition.

What is missing is a column tallying the referrals that are still in limbo [resp. those for which CJD is definitely ruled out]. Supposing that the Lancet series of patients represents 20 consecutive referrals consenting to tonsil biopsy, the article seems to be saying that of the current pool of referrals, 9/20 or 45% turned out to have nvCJD. There are 137 referrals - 47 total CJD = 90 referrals unaccounted for in 1998, a number to be lowered by the number of referrals already having definite not-CJD disposition.

Except this number should be higher to correct for a second missing column giving the number of 'outside' cases of CJD that came into the death tally without passing through referrals to the surveillance unit:

"Deaths: These columns show the number of deaths which have occurred in definite and probable cases of all types of CJD and GSS in the year shown. The figure includes both cases referred to the Unit for investigation while the patient was still alive and those where CJD was only discovered post mortem (including a few cases picked up by the Unit from death certificates). There is therefore no read across from these columns to the referrals column. The figures will be subject to retrospective adjustment as diagnoses are confirmed."

Assuming (with no real basis) that the two corrections balance each other and taking off the 20 cases of the Lancet article, 45% of 70 = 32 additional cases of nvCJD have shown up at the surveillance unit but not yet been sufficiently studied. For 1997, 82 referals remain unaccounted for, but presumbably a higher proportion received not-CJD disposition by now. For 1999, 200 new referrals eventually give 90 further cases. This line of reasoning leads to the conclusion that perhaps 73 cases of nvCJD have already occurred and that this coming year will see this total more than double.

Now the article itself does not say that the 20 patients came from the pool of in limbo referrals at the surveillance unit, though 3 of the authors are from there. In the acknowledgements, it says "We thank neurologists and neuropathologists across the UK for case referral to the Specialist Prion Disease Clinic at St Mary's Hospital and to the National CJD Surveillance Unit." This makes it unclear whether the cases referred to St Mary's are counted in the DoH statistics as referrals to the surveillance unit or counted as outside cases.

All in all, the DoH page could do a far better job in presenting surveillance data. We are left with no real idea how many unresolved referrals to all sources are out there and what percentage is currently going to nvCJD. Which brings us back to the "one-third" quote from R. Will. Tonsil biopsy has the effect of speeding case disposition; however, the article does not actually say that the test is being extended to everyone left in the referral pool. Effort might be (better) directed instead to the three cities where non-referral tonsils are being studied because this looks farther forward.

Examples of GPI transfer across cells from Medline:

Glycosyl phosphatidylinositol anchor.

Exp Nephrol 1998 Mar-Apr;6(2):148-51 
Kooyman DL, Byrne GW, Logan JS
Recently, we and others demonstrated the unique potential for glycosyl phosphatidylinositol (GPI) anchored proteins to transfer from one cell membrane to another in a process we termed 'painting'. The GPI-anchored proteins were shown to transfer intact and functional. The full significance of this phenomenon has yet to be fully realized, but implications exist in many areas including disease transmission (prions), cell protection (endothelial cells), and senescence (erythrocytes). It is of interest to note that cells exhibiting limited or no biosynthetic capacity (spermatozoa and erythrocytes) have been implicated thus far in cell-cell transfer of GPI-linked molecules. This observation demonstrates the potential for GPI-linked proteins to be 'painted' onto cells which otherwise may be incapable of expressing exogenous proteins. We show in this paper that GPI-linked CD59 and decay-accelerating factor will transfer intact from erythrocytes to endothelial cells in transgenic mice. We also demonstrate that the transfer process occurs under physiological conditions using several experimental models including organ and bone marrow transplantation. We detail the procedure to effect transfer of GPI-linked proteins from one cell type to another in either an in vivo or in vitro system.

Correction of the PNH defect by GPI-anchored protein transfer.

Blood 1998 Dec 1;92(11):4439-45
Sloand EM, Maciejewski JP, Dunn D, Moss J, Brewer B, Kirby M, Young NS
Hemolytic anemia is a major feature of paroxysmal nocturnal hemoglobinuria (PNH). Intravascular red blood cell (RBC) destruction is caused by increased sensitivity of the abnormal erythrocyte to complement-mediated lysis, due to the GPI absence of a membrane-bound glycosylphosphatidylinositol (GPI)-linked protein, which functions as an inhibitor of reactive lysis (CD59). Both in vivo and in vitro models have suggested the feasibility of cell-to-cell transfer of GPI proteins, and patients with hemolysis could potentially benefit from transfer of CD59 to their deficient erythrocytes.

Chicken Prion Tandem Repeats Form a Stable, Protease-Resistant Domain.

Biochemistry 1999 Jan 12;38(2):667-676 
Marcotte EM, Eisenberg D
"Prion-linked diseases, such as mad cow disease, scrapie, and the human genetic disorder Creutzfeldt-Jakob disease, are fatal neurodegenerative diseases correlated with changes in the secondary structure of neural prion protein. We expressed recombinant chicken prion protein [long allele] in Escherichia coli and purified the protein to homogeneity [98%].

Circular dichroism spectra of the 26 kDa recombinant protein closely resemble those of prion protein purified directly from healthy hamster brain. The chicken prion protein exists as a soluble, monodisperse monomer but can be forced to multimerize [dimer or trimer or tetramer not resolved, mechanism proposed: domain exchange at high concentration] following lyophilization and resuspension.

We analyzed the chicken prion protein domain structure by proteolysis and show that, unlike the mammalian homologues, the chicken prion protein N-terminal tandem amino acid repeats form a stable, protease-resistant [trypsin and PK] domain. This domain probably represents a physiologically functional unit.

As tested by both mass spectrometry and circular dichroism, the mature [reconstituted from E. coli, no glycosylation, no modified arginine] chicken prion protein does not bind copper, unlike synthetic peptides from the chicken prion N-terminus, suggesting that binding copper is not the physiological activity of the chicken prion. However, copper strongly destabilizes the prion protein and depresses the melting temperature by 30 degreesC, presumably by binding to the unfolded form of the prion protein.

The chicken prion N-terminus may have evolved to fold without a cofactor, unlike mammalian prion proteins, whose N-termini are disordered without cofactors such as copper present. Chicken prion offers an alternative to intractable mammalian prions for structural studies of the amino-terminal domain."

Comment (webmaster):

While this is a welcome study, thoroughly conducted and exposited in excellent detail, keep in mind that:

1. Copper proteins don't necessarily assemble spontaneously. There exist specific copper chaparones for superoxide dismutase and cytochrome oxidase etc. Copper is also treacherous in aqueous solution, Cu++ is not by any means the result of added cupric sulphate to water.

2. E coli does not provide the covalently modified arginines post-signal. It is important to determine whether bird prions contain this modification -- and what this modification is -- before structural studies. It would also be important to know whether all 3 potential glycosylation sites are utilized in bird. The effect of glycosylations is in effect to change the amino acid sequence from a small polar residues to new, very bulky very hydrophilic residues. While the target and product, one hopes, are on the protein surface and extend into the aqueous mileau without interacting too much with other residues, it still does not follow that there are no effects on 3D structure.

3. Both the Harris and Prusiner chicken alleles have an extra half repeat not found in any wild bird as well as extra full repeats. Chicken, like lab mice, goats, sheep, etc also have a couple of very improbable substitutions in the downstream domain. This is probably attributable to inbreeding, ie they are mutations, not PAMs. If they are harvested for broilers at 6 weeks, this is a very different than life as a jungle fowl in southeast Asia. Thus structural properties of chicken prion may not be applicable to 'real' birds. However, the principle conclusion of this paper, that the "random coil" model for the repeat region is wrong, is likely still valid.

5. Predicted 3D structures posted in May 1998 (A, B) for wildtype avian and mammalian prions do not need modification because of this paper. The repeat region is a very unusual situation where enough constrains existed to get at 3D coordinates from theory.

3,000 sheep survey completed (but not released)

MAFF web site, European Parliament documents, listserve correspondence 15 Jan 99
In July 1998, SEAC again called for work to investigate the possibility that BSE might be present in sheep but masked by scrapie. This need was stressed again in September by Prof Jeff Almond chairman of the SEAC sub-group on sheep.

The verbatim speeches in the conference on the lessons of the BSE crisis, organised by the European Parliament and Commission on 30 November- 1 December 1998 are now accessible from two links to two large Acrobat .pdf files. These speeches and discussions are in the native languages of the speakers with no translations.

In the discussion at the end of the first day (30 November), there was some information that I have not seen reported elsewhere, from Prof Roy Anderson who stated:

"A survey of brains from sheep randomly collected from abattoirs has just been completed in the United Kingdom. Very encouragingly, of the three thousand examined there was no evidence of BSE. That is a very encouraging result but a lot more needs to be done in Europe more broadly."

Question: what methods were used, which agency did the tests, and how many of them had sub- or pre-clinical BSE? And how many were scrapie-positive? If they had looked at 3000 scrapie-positives, how many would have been attributable BSE? The number of scrapie-positives would be of great interest in itself if the sample were really representative. That number is also a quality control on the sample and methods and a wonderful opportunity to quantitate what strains of scrapie are out there.

Response: According to what Anderson said, it was not by any diagnostic testing but by brain examination, which would find any clinical and some pre-clinical or sub-clinical cases; and none were found. The Central Veterinary Laboratory likely did the pathology testing(see below). Note that the SEAC public report of July 1998 contained the following section:

D. The survey of sheep scrapie strains since 1986.

To date nine UK isolates of scrapie contemporary with the outbreak of BSE have been strain typed. None has proved to be BSE-like and all seven that have been transmitted to mice have strain typing characteristics within the range expected of classical scrapie. In project SE 1919 at the Central Veterinary Laboratory, brains from sheep with scrapie born after January 1991 in flocks already known to have confirmed scrapie are being collected. Payment is offered to owners as an incentive to notify cases. Brains will be pooled according to sheep genotype at codons 136 and 171 to increase the opportunity of isolating a BSE-like strain. The project is still in the collection phase. A later collection phase is planned to include pools of lymphoreticular tissues as well as of brains.

A similar project (SE 1423) at NPU will attempt isolation from single sheep again selected according to genotype. The avoidance of pooling should increase the sensitivity of isolation but the extent of the project will not provide a nationwide screen. At a meeting on 1 February 1996, SEAC recommended that high priority should be given to a study to infect, by the oral route, scrapie-free sheep (preferably from New Zealand) of a PrP genotype known to be susceptible to BSE, and then investigate whether or not BSE was transmitted naturally to subsequent generations.

From the above date we conclude that results of a sufficiently representative survey of the strain characteristics of the infectious agent of scrapie in the national flock since the start of the BSE epidemic are not yet available. [end of SEAC July 1998 report extract]

John Wilesmith, of the Veterinary Laboratory Agency (formerly known as the Central Veterinary Laboratory) was asked about the study by J Ralph Blanchfield:

Yes, the work had been done at VLA, but Roy Anderson seems to have produced at the Brussels conference a "Chinese whispers" garbled version, wrong in the most important respect. The work to which he was referring was _not_ a search for BSE, but for scrapie strains. It was carried out on the brains of 3000 sheep, in two groups of 1500, one of sheep under 15 months, the other of sheep over 15 months. (They also have the tonsils of those sheep for further work). The work was carried out by "classical" histopathology, by electron microscopy and by immunocytochemistry, plus some immunoblotting. All of the sheep were negative for TSEs.

This is quite separate from the two projects SE1919 at VLA and SE1423 at NPU, referred to in the SEAC report of July 1998

Comments (webmaster):

This experiment is alluded to also on the MAFF site, sheep were taken from 130 abattoirs throughout Great Britain between August 1997 and August 1998 for this study. The MAFF age and geographical distribution of scrapie [pdf to friendlier gif] is recast below. Note that the data is not normalized to the number of sheep in each age class and goats are not broken out.

So this study is saying, roughly, that although there is anecdotally a fair amount of scrapie in England [2789 cases reported from 1980 to mid 1998] not very many of the clinical cases -- or pre/sub clinical but detectable as above -- are showing up at the slaughterhouse, with 0 in 3,000 being less than 0.03% (though under 15 months deflates the statistics). This includes but is not limited to the [experimentally defined] BSE strain of scrapie.

One just hopes they salted the sample with a few confirmed cases, including some experimental BSE scrapie, to verify that the detection system was working. My congratulations to the lab if they could find 1 salted case of BSE scrapie within the 3,000.

My impression of the tonsil test is that it is more of an early warning system, rather than a more sensitive test than brain IHC etc.; if so, it won't find any cases either unless scrapie is on an upswing. That must have also been the reason for including so many young sheep. I am guessing the most sensitive test is the capillary electrophoresis method of Schmeer or bioassay in high copy number mice.

Where then are the scrapie carcasses going that don't get turned in? Scrapie became notifiable on 1st of January 1993 in the UK. An additional program exists of buying scrapie sheep at good prices so as encourage farmers to turn these in for strain-typing. These programs would "interfere" with the abattoir program viewed as a scrapie incidence experiment. So this program really is addressing more the issue of clinically hidden scrapie.

The current number for the total sheep in the UK is not at hand but there were 42,885 farms with sheep in 1989. At 200 sheep each, the 3,000 sampled becomes 1 in 2800 tested. In 1997, 508 scrapie cases were reported to the government.

How many sheep slaughterhouses are still around? In the study mentioned above, sheep were taken from 130 abattoirs throughout Great Britain which sounds like a good assortment. On the other hand, just Cumbria, Oxfordshire, Devon, and Hereford/Worcester account for 1,627 of the cumulative 2,789 scrapie cases or 58%. The distribution of BSE-scrapie is of course unknown.

Do they dare do a similar bovine BSE survey with 3000 cows?

Surveillance:
Maff scrapie page

Compulsory slaughter with compensation was one of the 3 measures recommended by SEAC in May 1997 to improve the surveillance of scrapie in sheep and goats. The other two measures were: an abattoir survey of sheep tissues, and a postal survey of sheep farmers.

For the abattoir survey almost 3,000 sheep have been sampled from approximately 130 abattoirs throughout Great Britain between August 1997 and August 1998. Various diagnostic tests have been used to look for scrapie infectivity in the samples. The findings of the survey will be considered by SEAC and a report will be published in due course.

Although scrapie is a notifiable disease experts suspect that the true incidence is under-reported. It is not clear how many sheep are likely to be affected at any one time. Without such basic information it is very difficult to plan control of this disease. A better understanding of scrapie will lead to better initiatives for the future control of the disease and give farmers a better chance to protect themselves from the damage which scrapie can cause.

A postal survey to gain further information on the incidence of scrapie began on 12 November 1998. A questionnaire was sent to 11,500 randomly selected sheep farmers. The survey is being carried out independently of MAFF by the Agricultural Development and Advisory Service (ADAS), with the assistance of experts from the Central Veterinary Laboratory and the Institute for Animal Health. The survey is completely anonymous in order to encourage as high a response rate as possible. The survey team will not be able to identify the farms that participate. The survey also represents an opportunity for sheep farmers to express a view on the handling of scrapie. It has the support of industry representatives who have said that it is extremely important that farmers complete and return the questionnaires. The findings will be of interest to all those involved in the sheep sector and will be published in due course.

Compulsory Slaughter Scheme

Under legislation (listed below) which came into effect on 29 July 1998, it is now compulsory for sheep and goats displaying clinical signs of scrapie to be slaughtered. Compensation is paid to owners and MAFF disposes of the carcase.

The compensation payment per animal is equal to the average cull ewe market price in the calendar month two months prior to slaughter. For November 1998 this will be 15.53 for confirmed cases. A higher rate may be paid for suspect animals slaughtered where post-mortem laboratory diagnosis does not confirm scrapie. In such instances the owner will have to provide documentary evidence of the animal's value. This higher rate of payment is subject to a maximum of 400.

This legislation will improve the surveillance of scrapie by removing a disincentive to the farmer to report scrapie. It will improve controls on affected animals and provide additional information and materials for the study of the disease.

The compulsory slaughter scheme has been introduced through 3 Statutory Instruments:

-- The Sheep and Goats Spongiform Encephalopathy Order 1998 - provides for the compulsory slaughter of suspect animals.

-- The Sheep and Goats Spongiform Encephalopathy Regulations 1998 - provides for the conduct of veterinary investigations of premises where scrapie is suspected.

-- The Sheep and Goats Spongiform Encephalopathy (Compensation) Order 1998 - provides for compensation to be paid.

Antagonistic Interactions between Yeast Chaperones Hsp104 and Hsp70 in Prion Curing.

Mol Cell Biol 1999 Feb;19(2):1325-1333 
Newnam GP, Wegrzyn RD, Lindquist SL, Chernoff YO
The maintenance of [PSI], a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of [PSI] very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not.

These observations pointed to a mechanism for protecting the genetic information carried by the [PSI] element from vicissitudes of the environment. Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation. At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal level. In contrast, hsp104 deletion strains lose [PSI] even in the presence of overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of overproduced Hsp104.

Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of [PSI] under natural growth conditions.

US biologists propose launch of electronic preprint archive

Nature 14 Jan 99
An electronic challenge to traditional biomedical science journals is being planned by a group of US scientists. They propose to create an e-print archive similar to that established for physics at the Los Alamos National Laboratory in New Mexico by Paul Ginsparg in 1991.

The initiative is led by Patrick Brown, a researcher at the Howard Hughes Medical Institute at Stanford University School of Medicine, and David Lipman, director of the US National Council for Biotechnology Information (NCBI), which operates the PubMed and GenBank databases (and a distinguished genomics researcher).

Under preliminary proposals, the life science e-print archives would be established at a single website. Papers could be automatically posted, archived and distributed, with authors retaining copyright over their work. Articles would either be posted without refereeing, or peer reviewed by third-party professional societies or electronic journals, and labelled as such. Signed commentaries would also be posted along with articles.

E-print archives were first established in high-energy physics as an electronic alternative to distributing preprints. But by automating the process, Ginsparg created a large free electronic journal for papers that have not been peer reviewed . This year the archive will 'publish' some 25,000 papers in astrophysics, quantum physics, condensed matter theory and computer science.

The Los Alamos archives are the primary means of first publication in many areas of physics, although many papers are later published in journals. E-print archives for cognitive science have been established by Steven Harnad at the University of Southampton, and a computer research repository for the computer sciences was recently launched by Joseph Halpern and Carl Lagoze at Cornell University, New York state.

Lipman said last week that it was too early to comment on the plans in detail. But under one proposal being discussed, the archives would benefit from direct indexing and linking via NCBI's PubMed database.

Brown says this would guarantee wide exposure, as PubMed is one of the most widely used Internet services in the biomedical sciences. It offers access to 9 million bibliographic citations from 4,000 journals in Medline, and publishes abstract information provided by publishers before it has been indexed and made available on Medline.

PubMed has hyperlinks to full text in more than 300 journals, and links molecular biology citations to protein and nucleotide sequence databases, and from one Medline record to other related entries. The number of daily users has climbed from 35,000 to 70,000 over the past year, and PubMed now handles 1.5 million transactions daily.

Access to the proposed e-print archives would be free. The protagonists are said to be seeking funding for operating costs from the Howard Hughes Medical Institute and the US National Institutes of Health.

The initiative is likely to be welcomed in the research community. Fotis Kafatos, director of the European Molecular Biology Laboratory in Heidelberg, Germany, says: "I believe strongly that more open communication could benefit all the life sciences."

Kafatos says he would not like to see the established journals system dismantled. But he believes that an e-print archive would undermine the "scoop mentality" of some journals towards the acquisition of papers, and prompt a shift towards publishers providing greater editorial value.

The proliferation of specialized journals has compartmentalized knowledge in an artificial way, argues Kafatos. "Obviously the content of journals like Nature and Science appeals to a wide audience. But as a whole, biology is rapidly moving in the direction of dismantling barriers, and you need new ways to cut across disciplines." Electronics provides means for "more flexible organization of knowledge," he says.

Brown intends to invite leaders in the biomedical community to a workshop to discuss the proposals and encourage scientists to publish in the archive; biologists, unlike physicists, have no strong tradition of distributing e-prints.

The main obstacle facing the archive is the fear that publishing an e-print will preclude later publication in a peer-reviewed journal. Several journals (including Nature) have relatively liberal policies stating that publishing e-prints does not preclude publication, and others are expected to follow.

But some journals maintain that publishing on the Internet constitutes prior publication. Brown wrote to life-science journals this week asking them to publish "an explicit policy statement that distribution of a preprint, by means of a public electronic preprint server or Internet site, will not influence the decision of your journal to publish a paper". The letter was signed by 30 scientists including James Watson, Arthur Kornberg and Daniel Botstein.

Scientists Balk At Broad Data Release

Science Volume 283, Number 5400, Issue of 15 January 1999 Bruce Agnew
FREEDOM OF INFORMATION:

Prompted by concerns that the public may soon get access to all federally funded researchers' records, Congress, the Office of Management and Budget (OMB), federal research agencies, and the scientific community are on the verge of a great debate over the meaning of the word "all." By the end of this month--possibly within a few days--OMB is expected to take the first step toward implementing a new congressional mandate that "all data" produced with federal funding be given to anyone who seeks them under the Freedom of Information Act (FOIA) (Science, 6 November 1998, p. 1023).

But even before OMB formally launches the debate--it plans to issue a Notice of Proposed Rule Making and ask for public comment--science policy officials have begun building a case against too freewheeling an interpretation of the new law. Together with key congressional allies, they warn that federally funded research could be hobbled if scientists are forced to disclose experimental results before they are published in a scientific journal, before the end of an ongoing clinical trial, in a form in which human research subjects might be identifiable, or in a manner in which confidential data provided by collaborators might be revealed, among other issues.

Officials are urging scientists and scientific organizations to weigh in with their own worries. The Association of American Universities and the Council of Government Relations already have done so. In a 4 December 1998 letter to OMB, they not only cautioned against too-broad FOIA disclosure but also asked for clarification of who should bear the potentially "significant" costs involved.

National Institutes of Health (NIH) director Harold Varmus calls the provision "a potential burden--even a threat--to our investigators." A National Science Foundation (NSF) spokesperson says it could create "tremendous burdens" for both scientists and the agency. "Most of us believe there should be procedures in place and understanding of how you do share data," says Wendy Baldwin, NIH deputy director for extramural research. "The problem is, this is too blunt an instrument."

Not just blunt, but sudden. The FOIA provision, sponsored by Senator Richard Shelby (R-AL), was written into last October's massive omnibus appropriations bill at virtually the last moment, replacing language that originally ordered OMB simply to study the issue. The new 106th Congress could back away from Shelby's sweeping new FOIA requirement, and some members say it should. Representative George Brown (D-CA), ranking Democrat on the House Science Committee, last week introduced legislation to repeal the provision and start over--with hearings to determine what the data-access problem really is. "Documentation of this problem has been no more than anecdotal," said Brown in a statement. "We should not jeopardize this [government-sponsored research] enterprise by taking a hasty, ill-considered approach to remedy an alleged problem."

Even though FOIA provides exemptions for personal and proprietary data, Brown said individuals and companies might shy away. "Significant loss of voluntary participation in public health and biomedical research would be devastating," he said, adding that mandating release of all data "would undermine" intellectual property protections.

In addition, 22 other House members--six Republicans and 16 Democrats--joined Brown in a 7 December 1998 letter to OMB director Jack Lew warning of "a number of negative, unintended consequences." Co-signers include Representatives Vernon Ehlers (R-MI), an influential Science Committee member, and John Porter (R-IL) and James Walsh (R-NY), who chair the House Appropriations subcommittees that oversee the budgets for NIH and NSF. They might revisit the issue in this year's appropriations bills.

But congressional aides caution against betting that lawmakers will undo the FOIA requirement as nonchalantly as they enacted it. Some members want to let the process work and see if OMB can come up with a rule that the scientific community can live with, says one Science Committee staffer. That doesn't seem likely. No matter how imaginative OMB may be, it will be hard to soften the meaning of the words "all data."

Making (anti)sense of non-coding sequence conservation.

Nucleic Acids Res 1997 Sep 15;25(18):3580-3 
Lipman DJ
National Center for Biotechnology Information, National Library of Medicine, NIH, Building
A substantial fraction of vertebrate mRNAs contain long conserved blocks in their untranslated regions as well as long blocks without silent changes in their protein coding regions. These conserved blocks are largely comprised of unique sequence within the genome, leaving us with an important puzzle regarding their function. A large body of experimental data shows that these regions are associated with regulation of mRNA stability. Combining this information with the rapidly accumulating data on endogenous antisense transcripts, we propose that the conserved sequences form long perfect duplexes with antisense transcripts. The formation of such duplexes may be essential for recognition by post-transcriptional regulatory systems. The conservation may then be explained by selection against the dominant negative effect of allelic divergence.

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