All About Prions and Food

[Fat Guy]

do cows given doses of Acinetobacter calcoaceticus develop BSE?

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The last thing I read on the subject was a piece in the Wall Street Journal that said that Ebringer was having trouble securing funding to find that out:

While Dr. Ebringer's experimental work so far implies that indeed Acinetobacter is present in the diseased animals, what he has not shown is that he can cause the disease in animals by inoculating, or injecting, them with Acinetobacter. Therefore he has not satisfied the third postulate either.

Dr. Ebringer says he is eager to do that work, but has yet to secure funding for it.

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He says he has sought funding for the transmission study in the past, and is applying again

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Until then, his hypothesis remains an intriguing but inconclusive one.

[Fat Guy]

Can you show a single example of tranmission of spongiform encephalopathy via a pharmaceutical gel?

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Can you show a single example of tranmission of spongiform encephalopathy via eating beef?

[Fat Guy]

Not only is this simply incorrect, you have yet to explain how a virus or bacterium could survive treatment with nucleic-acid decomposing chemicals.

They shouldn't be able to. The issue with the studies I've seen that use those deomposing chemicals is that they then inject the end result into mice that are predisposed to get mouse-vCJD or what ever it's called. This is why John Collinge said his research group stopped using those mice. Granted, in the study we spoke of above, there was a control group given sugar water, but there wasn't a control group given any sort of toxic agent to see if general stress would trigger the TSE symptoms.

[Patrick S]

Not only is this simply incorrect, you have yet to explain how a virus or bacterium could survive treatment with nucleic-acid decomposing chemicals.

They shouldn't be able to. The issue with the studies I've seen that use those deomposing chemicals is that they then inject the end result into mice that are predisposed to get mouse-vCJD or what ever it's called.

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Huh? Maybe you could specify which studies are you looking at, Fat Guy, because the work I was referring to was done using scrapie prions, not BSE prions, on normal sheep, not mice genetically engineered to overexpress PrP. I don't know if these experiments were repeated with BSE prions (probably, I'll look), but the point still stands: there are infectious agents that cause spongiform encephalopathy that are not destroyed chemical and physical insults that scramble nucleic acids and destroy all known viruses and bacteria.

[Patrick S]

Can you show a single example of tranmission of spongiform encephalopathy via a pharmaceutical gel?

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Can you show a single example of tranmission of spongiform encephalopathy via eating beef?

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If by 'beef' you mean the muscle tissue of cows, then I'll repeat what I said above: I don't think beef per se is responsible for the transmission of BSE or vCJD. Rather it is the presence of small amounts of high-risk materials (e.g. nervous system tissue, intestines, lymph nodes, spleen) that sometimes works its way in to the beef during processing. If beef per se was infectious, we really would expect an enormous number of vCJD cases in the UK, rather than the 150-ish so far noted. Now, if the question is can I show case of spongiform encephalopathy transmission via those materials, the answer is absolutely, just check out the references I've already provided.

[Patrick S]

Not only is this simply incorrect, you have yet to explain how a virus or bacterium could survive treatment with nucleic-acid decomposing chemicals.

They shouldn't be able to. The issue with the studies I've seen that use those deomposing chemicals is that they then inject the end result into mice that are predisposed to get mouse-vCJD or what ever it's called.

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Huh? Maybe you could specify which studies are you looking at, Fat Guy, because the work I was referring to was done using scrapie prions, not BSE prions, on normal sheep, not mice genetically engineered to overexpress PrP. I don't know if these experiments were repeated with BSE prions (probably, I'll look), but the point still stands: there are infectious agents that cause spongiform encephalopathy that are not destroyed chemical and physical insults that scramble nucleic acids and destroy all known viruses and bacteria.

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As an example of this type of experiment, Safar et al (2005) subjected brain material from infected golden hamsters first to a purification process that, among other things, uses benzonase incubation. Benzonase is an enzyme that basically, chops up nucleic acid sequences into sequences smaller than ~20 bases. After purification, the material was subjected to strong UV radiation, at a wavelength (254nm) that breaks apart nucleic acids. The material was then innoculated in normal golden hamsters, and sure enough, still caused the disease. Towards the end of the article, Safar et al (2005) give some interesting commentary on the history of prions and the search for nucleic acids associated with them:

The search for a prion-specific nucleic began four decades ago. Transmission of scrapie to mice ushered in the application of biochemical experimentation aimed at revealing the composition of the infectious pathogen (14, 53). The startling results of ionizing and UV irradiation studies performed on mouse brain homogenates intensified the search for the nucleic acid genome of the putative "virus" causing scrapie (3, 4, 35). Those studies argued that the scrapie agent does not contain a nucleic acid, because it is extremely resistant to inactivation by irradiation at 254 nm and its target size, determined by ionizing radiation, is less than 150 kDa.

To search for a prion-specific nucleic acid, several different approaches have been taken. First, numerous procedures that hydrolyze or modify nucleic acids have been used to probe for the existence of a scrapie-specific nucleic acid (21, 53, 55). No procedure that hydrolyzes or modifies nucleic acids but does not alter proteins has been shown to reduce the titer of prions. [NOTE: "Titer" here means the quantity of material needed to cause the disease.] Second, molecular cloning and differential hybridization studies failed to identify a prion-specific polynucleotide (2, 48). Third, in biophysical studies searching for a small, prion-specific nucleic acid similar to a viroid, these nucleic acids were excluded (31, 46). Additionally, numerous unpublished studies have been performed in search of a prion-specific nucleic acid, but none has been found.

Naturally occurring prions. Unable to find evidence for a scrapie-specific nucleic acid in partially purified fractions prepared from Syrian hamster brains, the prion concept was introduced (55, 56). The possibility of a host-encoded, infectious protein along with several other scenarios was set forth to explain the apparent absence of a nucleic acid genome within an infectious pathogen. Opposition to the prion hypothesis was stout, but the opponents never identified the nucleic acid genome that they so vociferously championed (7, 11, 16, 39, 47, 63).

The resistance of purified prions to inactivation by procedures that hydrolyze or modify nucleic acids suggested that the putative genome of the prion was either quite small or did not exist. This resistance to inactivation raised the possibility that the prion genome was not only small but also well protected. Small, spherical viruses such the polio- and parvoviruses were known to be much more resistant to inactivation than larger viruses such as the one causing herpes (64, 68). But scrapie prions are much more resistant to inactivation by procedures that hydrolyze or modify nucleic acids than the sturdiest virus.

SNIP

The studies reported here as well as results from other recent investigations on synthetic prions and prion strains make the possibility of a prion-specific nucleic acid farfetched at best. There is no evidence for a prion-specific polynucleotide, and there is no unaccounted-for function that such a molecule would explain.

Safar et al, 2005. Search for a Prion-Specific Nucleic Acid. Journal of Virology 79, 10796-10806.

[Patrick S]

I also looked a little yesterday to see what evidence there might be for Ebringer's theory that the bacterium Acinetobacter calcoaceticus causes BSE/vCJD. The answer appears to be "none whatsoever." According to the first return from a Google search, Acinetobacter calcoaceticus is an opportunistic pathogen that is "ubiquitous in nature", and is "present as normal flora of the skin and throat of human beings." That's a problem for Ebringer: one of the most salient facts about BSE/vCJD is the very limited geographic distribution of these diseases (almost exclusively occuring in the UK). Why would a bacterium that is ubiquitous in nature cause disease in virtually exclusively in cows and humans in the UK? And why would the prevalence of these diseases appear to rise and fall in response to circumstances (like the ban on certain kinds of cattle feed) that have nothing to do with the bacteria?

[Patrick S]

I did a little more reading, and found a few more bits of interesting information.

BSE as an Autoimmune Disease

Regarding Ebringer's theory that BSE/vCJD is an autoimmune disease initiated by an immune response to a bacterium, I found some data that seems to be as definitive a refutation as one could ever hope for. In order to understand why the data is important, lets outline the theory. In Ebringer's theory, the body produces antibodies in response to a bacterial protein, but the antibodies also happen to match part of a protein on the nerve cells of the host. Thus, the immune system is induced to attack not only the bacterial cells in the body, as it should, but also the body's own nerve cells. It is the destruction of these nerve cells by the immune system that produces the characteristic symptoms of neurological disease. This is what happens in diseases like multiple sclerosis -- the body's immune system is attacking the myelin on nerve fibers.

Now, there is a elegantly simple way to test the hypothesis that BSE is an autoimmune disease analogous to MS: administer the agent to mice that lack the ability to generate antibody-mediated immune reactions. This has been done using so called SCID (severe combined immunodeficient) mice, which among other things do not have the functional B and T cells needed to generate an antibody-mediated immune reaction. Brown et al (1997) gave combined intraperitoneal and intracerebral injections of BSE cow brain to both SCID and normal immunocompetent mice. While the immunocompetent mice developed spongiform encephalopathy at a higher rate than did the SCID mice, the disease did in fact develop in both groups of mice (it is thought that the immune system facilitates replication of the prion in lymphatic tissue, hence the higher rate of disease in the immunocompetent mice).

The fact that SCID mice are at all susceptible to BSE would sure seem to rule out Ebringer's hypothesis that BSE is an autoimmune disease.

Brown et al, 1997. Severely Combined Immunodeficient (SCID) Mice Resist Infection with Bovine Spongiform Encephalopathy, Journal of General Virology 78, 2707-10

The Role of the PrP in Prion Disease

Ebringer is quoted as saying that ""Prions are not infectious particles. Instead they are the breakdown products of damaged nervous tissue" If I am interpreting Ebringer's statement correctly, he's implying that PrP is an "innocent bystander," and is not involved in causing the actual disease. If the prion theory is correct, however, the PrP protein is absolutely essential to the disease process -- according to that theory, the disease is caused when the misfolded PrP interacts with normal PrP, causing it to misfold as well, and the misfolded proteins polymerize together into plaques which disrupt the functioning of neurons. According to the prion hypothesis, if there is no PrP, the disease cannot develop.

These two views can be tested experimentally using mice that have been engineered to lack PrP (PrP knock-out mice). In experiments where mice lacking PrP (PrP0/0) were given scrapie prions, the disease did not develop, whereas the control mice (PrP+/+) with normal PrP expression developed disease on average 160 days later. Further, mice in which one copy of the PrP gene remains (PrP+/0), which express PrP at half the normal level, developed disease on average 270 days later, while mice engineered to express PrP at a higher rate than normal rate develop the disease even faster than normal mice.

So there is a consistent inverse relationship between the level of PrP expression and susceptibility to prion disease (see Weissmann and Flechsig, 2003 for a review). This makes perfect sense if the prion theory is correct, but would be hard to explain if PrP were just an innocent bystander.

Büeler et al, 1993. Mice devoid of PrP are resistant to scrapie. Cell 73:1339–47

Weissmann and Flechsig, 2003. PrP knock-out and PrP transgenic mice in prion research. British Medical Bulletin 66: 43–60

The Role of Pharmaceutical Gels in Transmitting the BSE/vCJD Agent

Steven stated earlier that he thought that transmission of the BSE agent by pharmaceutical gels was "in many ways more compelling" than tranmission by eating prions as contaminants in beef products. If what is meant here is the gelatin used in pharmaceutical gels, this can be pretty much ruled out, based on the fact that the gelatin manufacturing process is known to render the BSE agent inactive. Grobben et al (2004, 2005) tested this possibility experimentally using mice, and found that the process used to manufacture gelatin "reduced infectivity to undetectable levels". In contrast, the processes used to prepare meat and bone meal for cows, or to prepare meat for human consumption, are known NOT to reduce infectivity.

Grobben et al, 2004. Inactivation of the bovine-spongiform-encephalopathy (BSE) agent by the acid and alkaline processes used in the manufacture of bone gelatine. Biotechnology and Apllied Biochemistry 39(Pt 3):329-38.

Grobben et al, 2004. Inactivation of the BSE agent by the heat and pressure process for manufacturing gelatine. Veterinary Record 157(10):277-81.

Edited February 15, 2006 by Patrick S (log)

[Fat Guy]

Certainly, there are mountains of studies that support the prion theory and contradict competing theories. Each one I've ever seen has been called into question -- where I've understood the contrarian reasoning I've presented it above, though I'm not familiar with every single study so can't do so for each. But the larger question we as observers need to ask is how much credibility to assign to those studies. Are they untainted by agendas, are they being done in a context where competing theories are also being studied as aggressively, are the studies designed to test the theory or are they actually designed to prove it? Based on what appear to be the answers to those questions (no, no and no), I think we need to be skeptical -- actually, in this case, I'd say we should be downright suspicious given the particularly blatant politicization and agenda-mongering in the prion community.

[jsolomon]

FG, why are you so suspicious of scientists? Granted, there is a lot of high profile research going into BSE and the like, but there are also a lot of very dedicated, ethical, and moral scientists working on these questions.

I'm much more worried about the quality of the reporting on the research than anything else. If the reporting were of higher quality, then the issues regarding the finding of BSE would not have caused near so much alarm.

[Fat Guy]

As I commented above, in my opinion once you become so wedded to a hypothesis that you devote yourself to proving it rather than testing it you stop being a scientist. I'm not suspicious of scientists at all. I'm suspicious of people who call themselves scientists but aren't.

I'm equally suspicious of science writers, lobbyists, policy-makers and the various other groups that are collectively responsible for our often irrational responses to supposed scientific findings.

All this prion stuff may turn out to be right. Certainly the bulk of the findings, even the probably impartial ones, are pointing in that direction. It is far from a closed case, however, and the political aspects of so much of the research should cause us to take everything we hear with a grain of salt. What I've been trying to show here is that there are several reasonable positions on this issue, as opposed to the monolithic position that is presented as truth not only by the press but also by many in the scientific community.

[jsolomon]

and the political aspects of so much of the research should cause us to take everything we hear with a grain of salt.

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It's awfully rough to take the political environment out on the scientists. Just like any other group, they have to make their money according to the leading market indicators, and fortunately or not, most of the prion research money is doled out from federal coffers through the NIH or the NSF. We all know how political the budgeting process is in the US Congress. And, if Congress, Mike Johanns, Dr. Richard Carmona, or George Bush, decide that there is a need for research into prions, money will be opened up, researchers will hear about it, and apply for the money.

Does this make their research political? Opportunistic, to be certain, but all grant applications are.

For a historical perspective, when Dr.s Banting and Best suggested that diabetes was not an infectious disease, but a metabolic disease, they were openly laughed at. When insulin was shown to be a protein, and one with discrete structure, the skepticism was amazing. Both of those findings generated Nobel Prizes.

Does this all mean that prions are going to pan out? No. But, it does show that even the most educated minds of the time have their predilections and prejudices. However, earnest science is inherently self-correcting. But, it's not instantaneous.

Edit to correct Secretary of D^H^H^H^H^H^H^HAgriculture's name

Edited February 16, 2006 by jsolomon (log)

[Patrick S]

and the political aspects of so much of the research should cause us to take everything we hear with a grain of salt.

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It's awfully rough to take the political environment out on the scientists. Just like any other group, they have to make their money according to the leading market indicators, and fortunately or not, most of the prion research money is doled out from federal coffers through the NIH or the NSF. We all know how political the budgeting process is in the US Congress. And, if Congress, Mike Johanns, Dr. Richard Carmona, or George Bush, decide that there is a need for research into prions, money will be opened up, researchers will hear about it, and apply for the money.

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Of course its true that research funding can get politicized, and like you say, that generally can not be blamed on the researchers themselves. A great example of that, maybe the best example, is the way NIDA has historically refused to fund studies that might not support existing drug policy. So while no one should deny that funding can be politicized, there appears to be little or no evidence that this has happened for prion diseases. Its not that alternative theories for spongiform encephalopathies have all been dismissed without consideration, as has been implied. Rather, they have been considered and found wanting. In the case of BSE/vCJD as autoimmune diseases, for instance, the crucial experiments were done almost a decade ago, and the hypothesis failed the test spectacularly.

[Mallet]

The team of Telling et. al. referred to in the first page of this thread were just published in Science:

Prions in Skeletal Muscles of Deer with Chronic Wasting Disease

Rachel C. Angers,1* Shawn R. Browning,1* Tanya S. Seward,2 Christina J. Sigurdson,4Michael W. Miller,5 Edward A. Hoover,4 Glenn C. Telling1,2,3

The emergence of chronic wasting disease (CWD) in deer and elk in an increasingly wide geographic area, as well as the interspecies transmission of bovine spongiform encephalopathy to humans in the form of variant Creutzfeldt Jakob disease, have raised concerns about the zoonotic potential of CWD. Because meat consumption is the most likely means of exposure, it is important to determine whether skeletal muscle of diseased cervids contains prion infectivity. Here bioassays in transgenic mice expressing cervid prion protein revealed the presence of infectious prions in skeletal muscles of CWD-infected deer, demonstrating that humans consuming or handling meat from CWD-infected deer are at risk to prion exposure.

[Mallet]

And from today's Science issue:

Infectious Prions in the Saliva and Blood of Deer with Chronic Wasting Disease

Candace K. Mathiason et al.

A critical concern in the transmission of prion diseases, including chronic wasting disease (CWD) of cervids, is the potential presence of prions in body fluids. To address this issue directly, we exposed cohorts of CWD-naïve deer to saliva, blood, or urine and feces from CWD-positive deer. We found infectious prions capable of transmitting CWD in saliva (by the oral route) and in blood (by transfusion). The results help to explain the facile transmission of CWD among cervids and prompt caution concerning contact with body fluids in prion infections.

Edited October 6, 2006 by Mallet (log)