Macaque study finally published
Reaction from experts to report
British scientists take another look at marmoset brains
Molecular genetics of prion diseases in France
Prion protein gene variation among primates
Amino acid and DNA sequence for rhesus macaque and marmoset Evolutionary link found between human and cow diseases
Commentary on molecular evolution result of Krakauer et. al

Study showing BSE causes CJD in monkeys published

1996 Nando.net ... Reuter Information Service
Jun 26, 1996


Macaque study finally published

LONDON -- A study showing monkeys can develop a new strain of the human version of mad cow disease after being injected with brains from infected cattle was published on Wednesday. A Swiss specialist called the study worrying but said many more questions needed to be answered.

The French scientists who conducted the study held a news conference earlier this month to publicise their findings, which they wrote up in a letter to the science journal Nature. Nature published their letter on Wednesday.

British scientists said in March they had identified a new strain of the brain-wasting Creutzfeldt-Jakob Disease (CJD), the human form of bovine spongiform encephalopathy (BSE or mad cow disease), and said it was likely the victims had caught it from eating infected beef.

Corinne Lasmezas, a neuro-virologist at the Atomic Energy Commission, and colleagues at the French army health service said they had injected brain material from BSE-infected cattle into the brains of two adult macaques and one newborn.

Nearly three years after inoculation "the two adults developed abnormal behavioural signs including depression for the one, edginess and voracious appetite for the other." They also started to show the difficulty in moving and tremors that typify the disease.

The baby monkey showed signs even sooner. All three were killed and their brains examined -- currently the only way to positively diagnose BSE or CJD. They had deposits of the abnormal prion protein, a mutated brain protein that most scientists think causes the disease.

The pattern was "strikingly" similar to that seen in the new CJD cases but different from the pattern seen in the brains of macaques that had been infected with normal CJD.

Although the monkeys were injected with CJD the researchers said there was probably no difference between having the infectious agent injected and eating it. Other tests have shown the abnormal prions survive very high temperatures and would likely make it through the digestive system unscathed.

Adriano Aguzzi, a neuropathologist at University Hospital of Zurich, said it was worrying that the small amounts of cow brain material used in the experiment infected the monkeys. "It is unsettling that these amounts are well within the range of brain tissue present in commerical food products for human consumption until a few years ago," he wrote in a commentary in Nature. "On the other hand, however spectacular (and worrying) these findings are...we can hope that the oral route of administration will be considerably less efficient."

Aguzzi said the study left many questions unanswered, such as whether the monkeys could get the new CJD variant from eating infected cattle brains and if so, what was the minimal infectious dose. He also said it could be doubtful whether macaques were a good model for human disease.


French scientists see mad cow-CJD link in monkeys

PARIS (Jun 13, 1996)

French government scientists said on Thursday they had found the first experimental evidence of a link between mad cow disease and its human equivalent, Creutzfeldt-Jakob disease, in work on monkeys.

Two neurologists working for the Atomic Energy Commission and the French army health service said their research showed striking similarities between brain lesions in monkeys injected with the crushed brains of cows infected with bovine spongiform encephalopathy (BSE), and a new form of CJD observed in humans.

Their study, to be published by the British scientific journal Nature, lends new weight to the theory that BSE can be passed to humans, although they stressed it did not prove that.

Scientists Corinne Lasmezas and Jean-Philippe Deslys said they had injected the brain concentrate into two adult macaque monkeys and a newly-born one in 1991.[[These are described in the NY Times as rhesus monkeys (Macaca mulata--webmaster]] Three years later, all three began to show behavioural disorders -- anxiety, nervousness and depression -- and had developed identical brain lesions and died. "This is the first experimental evidence supporting a link between BSE and the new form of Creuzfeld-Jakob disease in man," Lasmezas told a news conference.

The scientists, who had been studying BSE, said they had only been alerted to the possible link with CJD by the British announcement and had then compared their results with new cases of the human disease observed in Edinburgh, Scotland in mid-May in sufferers under the age of 40.

Deslys said the brain lesions were "very close, strikingly similar" and constituted "a first experimental argument for a causal link between the bovine illness and the new form of CJD in the British patients."

He said the lesions were flower-shaped patches surrounded by cavities. The researchers said they injected the concentrate directly into the monkeys' brains rather than giving it to them orally. They declined to say firmly whether BSE can be transmitted to humans but said their findings called for further research.

In March, British scientists said they had identified a new form of CJD that was probably caused by eating beef infected with BSE. The new strain shows a slightly different pattern from earlier forms, first identified early in the century.

British government experts say BSE was caused by feeding cows the remains of sheep that had been infected with scrapie, their own version of the disease.

Copyright © 1996 Nando.net
Copyright © 1996 Reuter Information Service


Nature press release:

The article will appear in the issue of 27th June in Nature's Scientific Correspondence section. Nature received the paper on 6 June 1996, and it was accepted on 12 June 1996. The fact that we are publishing these results as Scientific Correspondence, and not as a Letter to Nature, is a reflection of our judgement, based on referees' comments, that the full significance of these observations has yet to be established, though the observations themselves are of great interest --- Dr. Campbell, editor of Nature


Comment from Professor Richard Lacy of the National CJD Surveillance Unit :

"Serious, worrying confirmation of our worst fears ... The monkeys were injected 5 years ago which is exactly spot on in relation to the epidemic ... I am afraid it is certainly not good news"

Comment from Dr Ironsides (SEAC )

"It strengthens the hypothesis that BSE could be the cause of CJD, but it doesn't go as far as to prove it absolutely."

Scientists revive test in hunt for cow-to-human link

BY NIGEL HAWKES
The Times: Britain:June 15 1996

BRITISH scientists plan to look again at experiments in which "mad cow" disease was transmitted to marmosets [[New World monkeys of the famil Callithricidae --webmaster]] in the early 1990s. A French team has reported that brain lesions seen in macaque monkeys infected with BSE bear a close resemblance to those in young people who have developed Creutzfeldt-Jakob disease (CJD).

At the time the marmoset experiments were carried out, the new form of CJD had yet to be identified or described. Gerald Wells of the Central Veterinary Laboratory, one of the team responsible, said that at first sight they appeared to conflict with the French findings: "So far, I haven't seen a full version of the French data but, on the assumption that their material is very like the new variant of CJD, our results are not similar."

The team has already looked again at the distribution of prion protein in preserved material from the brains of the marmosets, and found that it is not the same as in the CJD cases. Now there is a proposal to use the material for further experiments to infect mice, with identical experiments using material from CJD patients. If both cause identical symptoms in mice, it would demonstrate a common origin.


Baker, H. F., R. M. Ridley, and G. A. H. Wells.
"Experimental Transmission of BSE and Scrapie to the Common Marmoset."
_Veterinary Record_ 17 April 1993: 403-406.

The species used was Callithrix jacchus.


Molecular genetics of prion diseases in France

Laplanche JL; Delasnerie-Laupretre N; Brandel JP; Chatelain J; Beaudry P; Alperovitch A; Launay JM
Service de Biochimie, Hopital Saint-Louis, Paris, France. Neurology 44: 2347-51 (1994)

Human prion diseases are characterized by the accumulation in the brain of an abnormal form of the prion protein. Prion protein polymorphisms seem to play a key role in the pathogenesis of these diseases, probably by enhancing the amyloidogenic properties of the protein. We performed prion protein gene (PRNP) coding sequence analysis in 57 French subjects with Creutzfeldt-Jakob disease (CJD) and found a mutation of the PRNP coding sequence in nine subjects (15.8%); the mutation corresponded with a known family history of CJD in only three of these subjects. In 41 definite and probable cases without known PRNP mutations, codon 129 genotyping revealed an excess of the homozygous 129Met/Met genotype corresponding to a 3.4-fold increased risk of developing CJD when compared with the two other genotypes. We also found that the 129Val/Val genotype, which mainly governs susceptibility to iatrogenic CJD, does not seem to predispose to sporadic CJD.


Prion protein gene variation among primates

Schatzl HM; Da Costa M; Taylor L; Cohen FE; Prusiner SB
J Mol Biol 245: 362-74 (1995)

Prion diseases are manifest as genetic, sporadic or infectious neurodegenerative disorders in humans and animals. The prolonged incubation times that accompany the transmission of prions between species are due, at least in part, to differences in prion protein (PrP) sequence. To examine the species barriers between non-human primates and humans, we sequenced the open reading frames (ORF) of 25 PrP genes from apes and monkeys. Comparison of the PrP genes of these animals with that of humans showed amino acid identities ranging from 92.9 to 99.6%. [[There are some 250 amino acids in the prion protein, so this translates to 1-18 amino acid differences -- webmaster]]

While phylograms of primate PrP sequences revealed a novel branching pattern for the apes, the genomic organization of all the primate PrP genes was similar, with the entire ORF contained within a single exon. Alignment of variant residues in primates, rodents and domestic animals showed no concordance with the mutations that segregate with human prion diseases or with polymorphisms that modulate disease in humans, mice and sheep. Most substitutions were conservative and, characteristically, clustered outside the four putative alpha-helical regions that are thought to form a four-helix bundle in the cellular isoform of PrP (PrPC).

Deletion of one of five Gly-Pro rich octarepeats from the N-terminus of PrP was seen in some species, while squirrel monkeys had an additional octarepeat; squirrel monkeys have been frequently used as experimental hosts for transmission of human prions. Alignment of primate and other mammalian PrP sequences suggests that codons between 90 and 130 have a profound influence on the transmissibility of prions from one species to another.


Rhesus macaque [Macaca mulatta] prion gene

Schatzl,H.M., Da Costa,M., Taylor,L., Cohen,F.E. and Prusiner,S.B.
Prion protein gene variation among primates
J. Mol. Biol. 245 (4), 362-374 (1995)

MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSP GGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHNQWHKP SKPKTSMKHMAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQ VYYRPVDQYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQY EKESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG

1 atggcgaacc ttggctgctg gatgctggtt ctctttgtgg ccacatggag tgacctgggc
61 ctctgcaaga agcgcccaaa gcctggagga tggaacactg gaggcagccg atacccgggg
121 cagggcagcc ctggaggcaa ccgctaccca ccccagggtg gtggtggctg ggggcagcct
181 catggtggtg gctgggggca accccatggt ggcggctggg gacagcctca tggtggcggc
241 tggggacagc ctcatggtgg tggctggggt caaggaggtg gcacccacaa tcagtggcac
301 aagcccagta agccaaaaac cagcatgaag cacatggctg gtgctgcagc agctggggca
361 gtggtggggg gccttggcgg ctacatgctg ggaagtgcca tgagcaggcc cctcatacat
421 tttggcaatg actatgagga ccgttactat cgtgaaaaca tgtaccgtta ccccaaccaa
481 gtgtactaca ggcctgtgga tcagtacagc aaccagaaca actttgtgca cgactgcgtc
541 aatatcacga ttaagcagca cacagtcacc accaccacca aaggggagaa cttcaccgag
601 accgacgtta agatgatgga gcgcgtggtt gagcagatgt gtatcaccca gtacgagaag
661 gaatcacagg cctattatca gagaggatcg agcatggtcc tcttctcctc cccgcctgtg
721 atcctcctga tttctttcct catcttcctg atagtgggat ga

Marmoset [Callithrix jacchus] prion gene

MANLGCWMLFLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSP GGNRYPPQGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPS KPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQV YYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYE KESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG

1 atggcgaacc ttggctgctg gatgctgttt ctctttgtgg ccacatggag tgacctgggc
61 ctctgcaaga agcgcccaaa acctggagga tggaatactg ggggcagccg atacccaggc
121 cagggcagtc ctggaggcaa ccgctaccca ccacagggtg gtggctgggg gcagcctcat
181 ggtggtggct gggggcaacc tcatggtggc ggctggggac agccccatgg tggcggctgg
241 ggacagcctc atggtggtgg ctggggtcaa ggaggtggca cccacagtca atggaacaag
301 cccagtaagc caaaaaccaa catgaagcac gtggctggtg ctgcagcagc tggggcagtg
361 gtggggggcc ttggtggcta catgctggga agtgccatga gcaggcccct catacatttt
421 ggcaatgact atgaggaccg ttactatcgt gaaaacatgt accgttaccc caaccaagta
481 tactacaggc ccgtggatca gtacaacaac cagaacaact ttgtgcacga ctgcgtcaat
541 atcacgatca agcagcacac ggtcaccacc accaccaaag gggagaactt cactgagacc
601 gatgttaaga tgatggagcg cgtggttgag cagatgtgta tcactcagta cgagaaggaa
661 tcccaggcct attaccagag aggatcgagc atggtcctct tctcctcccc acctgtgatc
721 ctcctgatct ctttcctcat cttcctgata gtgggatga


New link found between human and cow diseases

LONDON (Apr 24, 1996 4:12 p.m. EDT) -- Scientists working in Britain published evidence Wednesday that could show mad cow disease and its human equivalent, Creutzfeldt-Jakob Disease, are closely related. They said brain proteins linked with both diseases show very close genetic similarities.

Writing in the science journal Nature, David Krakauer and colleagues at Oxford University's zoology department said this could explain why people could develop CJD from eating meat from cows infected with Bovine Spongiform Encephalopathy (BSE or mad cow disease). It could also explain why people never got CJD from eating sheep infected with scrapie, the ovine version of the disease.

The British government said last month that people might possibly get CJD from eating beef after scientists identified a new strain of CJD, which is always fatal, in 10 victims.

No one knows for sure what causes either disease. But a naturally occurring protein called a prion is known to be involved. Recent studies have indicated that normal prions are important to the function of brain cells. In diseases like BSE and CJD the prions somehow become mutated and brain cells die off, creating the typical spongelike appearance of the brain.

Krakauer and colleagues decided to find out if there were any similarities between prions in cows and in humans. "Scrapie, which has been around for 200 years, has never been shown to transmit to human populations," Krakauer said in a telephone interview. "BSE may have. If that's the case, what we need to find is a similarity we share with cattle that we don't share with sheep. That's what we have found."

Krakauer, an evolutionary biologist, looked at the prion gene and closely examined the sequence, or order, of the amino acids on the gene. They compared cow prion genes and human prion genes to those found in other animals such as sheep, rodents and monkeys and found two striking similarities in the sequence for cows, humans, chimpanzees and gorillas. No other animal shared these similarities, they said.

Looking back at the evolution of the gene, they found that people, apes and cattle had independently evolved the change. "It sort of popped up at the terminal branches of this evolutionary bush," said Krakauer. "Therefore, we have to take it quite seriously...It is the first data that uniquely links us and cattle."

In addition, the difference was in a region of the gene that other studies show may be involved in the acquisition of BSE, CJD and other spongiform encephalopathies. The changes could be there for a number of reasons. But, they said: "As an incidental consequence...these changes might also have predisposed humans toward a strain of prion disease occurring in (cattle)."

Krakauer said more study was needed, especially into whether these amino acid sequences were indeed involved in development of the deadly brain diseases.


Copyright © 1996 Nando.net
Copyright © 1996 Reuter Information Service

Commentary on molecular evolution result of Krakauer et. al [Listserve 5.15.96 -- webmaster]:

Krakauer et. al have a fairly good case for an unfortunate happenstance of convergent evolution of prion protein secondary structure of cows and higher primates. There are 11 hot spots on the human prion gene for familial CJD that give rise to the rogue catalytic conformer. The critical areas are matched in the cow, even though the cow differs overall from human at 30 amino acids. So the argument is that the species barrier is less than what it otherwise might have been.

Tthe thesis could have been expanded to explain why zoo felids get the disease but not the canids. [But that still leaves us with the 3 spongiform German ostriches.]

Will et. al sequenced the entire open reading frame for prion protein from the young v-CJD victims and found them all (8/8) homozygous normal, ruling out familial CJD.

Basically, for proof of cow to human transmission, someone is going to have to demonstrate by immunofluorescence that traces of the rogue cow protein is present in human victims. Note that this need not be an abnormal sequence -- it could have been normal but converted in the cow from a rogue scrapie or rogue bovine sequence.

In this scenario, the cow has intermediated the passage of scrapie from sheep to humans [which apparently does not aoccur otherwise]. Thus some sheep somewhere had the scrapie mutation. The problem is passed on to a normal sheep [whether by hay mite or offal], then to a normal cow [whether by hay mite or offal], and then to a genetically normal person [through diet or cosmetics].

It is a dead certainty that "familial" BSE occurs naturally in cattle etc. all over the world. If, repeat if, the rate is similar to familial CJD in humans [say, 1 per 1,000,000 per year], then the US, for example, with 103,000,000 cattle, would have 103 cases each year. The FDA is careful and correct to say that these haven't been observed [yet]; APHIS makes a PR blunder by saying point-blank it doesn't occur. This is biological rubbish that will damage their credibility later. Just look at those Medline mutant bovine sequences.


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