Sunday, May 17, 2009

De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype

Sent: Saturday, May 16, 2009 9:49 PM

Subject: De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype

De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype

Marcelo A. Barria1,2, Abhisek Mukherjee1,2, Dennisse Gonzalez-Romero1,2, Rodrigo Morales1,2,3, Claudio Soto1,2*

1 George and Cynthia Mitchell Center for Neurodegenerative Diseases, University of Texas Medical Branch, Galveston, Texas, United States of America, 2 Department of Neurology, University of Texas Houston Medical School, Houston, Texas, United States of America, 3 Facultad de Ciencias, University of Chile, Santiago, Chile


Prions are the proteinaceous infectious agents responsible for Transmissible Spongiform Encephalopathies. Compelling evidence supports the hypothesis that prions are composed exclusively of a misfolded version of the prion protein (PrPSc) that replicates in the body in the absence of nucleic acids by inducing the misfolding of the cellular prion protein (PrPC). The most common form of human prion disease is sporadic, which appears to have its origin in a low frequency event of spontaneous misfolding to generate the first PrPSc particle that then propagates as in the infectious form of the disease. The main goal of this study was to mimic an early event in the etiology of sporadic disease by attempting de novo generation of infectious PrPSc in vitro. For this purpose we analyzed in detail the possibility of spontaneous generation of PrPSc by the protein misfolding cyclic amplification (PMCA) procedure. Under standard PMCA conditions, and taking precautions to avoid cross-contamination, de novo generation of PrPSc was never observed, supporting the use of the technology for diagnostic applications. However, we report that PMCA can be modified to generate PrPSc in the absence of pre-existing PrPSc in different animal species at a low and variable rate. De novo generated PrPSc was infectious when inoculated into wild type hamsters, producing a new disease phenotype with unique clinical, neuropathological and biochemical features. Our results represent additional evidence in support of the prion hypothesis and provide a simple model to study the mechanism of sporadic prion disease. The findings also suggest that prion diversity is not restricted to those currently known, and that likely new forms of infectious protein foldings may be produced, resulting in novel disease phenotypes.

Citation: Barria MA, Mukherjee A, Gonzalez-Romero D, Morales R, Soto C (2009) De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype. PLoS Pathog 5(5): e1000421. doi:10.1371/journal.ppat.1000421 Editor: David Westaway, University of Alberta, Canada Received April 7, 2008; Accepted April 9, 2009; Published May 15, 2009 Copyright:  2009 Barria et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institute of Neurological Disorders (R01 NS49173), the National Institute of Allergy and Infectious Diseases (P01 AI77774) and the National Institute of Aging (P01 AG14359). None of the sponsors or funders played any role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, or in the preparation, review, or approval of the manuscript. Competing Interests: Dr. Soto is an inventor on the PMCA technology and a Founder, Vice-President and Chief Scientific Officer of Amprion Inc, a biotech company focusing on the development of early and sensitive diagnosis for prion diseases and other disorders involving protein misfolding. * E-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000223/!


In this study we report spontaneous generation of PrPSc in vitro using a modified PMCA procedure with brain homogenate substrate. Misfolded protein was produced in two different animal species without the need to add seeds of in vivo generated PrPSc. At present it is unclear whether PMCA replicated a PrPSc intermediate already present in the brain homogenate or rather ‘‘spontaneous’’ misfolding was induced by the amplification cycles. In this context, a recent study reported the presence of small quantities of a PrPSc-like protein in healthy brains [20], but it is yet unknown whether this protein is infectious.

Although de novo prion formation was achieved before by PMCA using purified proteins in the presence of synthetic polyanions [4], the purpose of our study was to evaluate whether de novo generation of prions is possible under standard PMCA conditions. This is important to assess the validity of using PMCA for prion diagnosis. Another aim was to study whether different species of animals have a distinct propensity to form spontaneous prions under a given set of conditions. Finally, we aimed to characterize the newly generated infectivity and to show that the diversity of prions is perhaps larger than we currently think. Our results indicate that standard PMCA rounds consisting of 144 cycles (or less) did not show spontaneous formation of PrPSc, but longer rounds of 240 cycles were successful in replicating or generating misfolded protein. These findings suggest that de novo formation of PrPSc can be experimentally distinguished from replication of pre-formed PrPSc, indicating that the biochemical, conformational or stability properties of the PrP structures involved in both processes are probably different. Therefore, PMCA can be adapted to produce de novo generated PrPSc and it is likely that more drastic modifications of the procedure may lead to higher rates of spontaneous prion formation. Indeed, the higher rate of de novo PrPSc formation reported in the study by Deleault and colleagues, who used only 48 PMCA cycles per round [4], indicates that using polyanions and purified PrPC may favor spontaneous prion formation, suggesting that some factors in brain homogenate may prevent PrP misfolding.

Our data support the use of PMCA, (under conditions in which PrPSc de novo generation does not occur), for biochemical detection of prions and its potential application for TSE diagnosis. The usefulness of PMCA for highly sensitive and specific detection of prions in biological fluids, has been demonstrated by studies from us and other groups [21–25].

Strikingly, the rate and number of PMCA cycles required to produce de novo PrPSc was variable in distinct species. Considering that from the species studied humans are the only one in which sporadic disease is known to occur, we were surprised that de novo PrPSc was never obtained with human brain homogenates under the experimental conditions used. This contrast with the high levels of amplification of human PrPSc observed in samples seeded with vCJD (Fig. S1) or various sCJD brain homogenates (data not shown). The absence of spontaneous formation of PrPres in humans may be due to the different conditions for the preparation of the tissue, including several hours of post-mortem delay or the lack of perfusion before collection of the brain. However, this is unlikely, because similar results were obtained using brains of transgenic mice expressing human PrP subjected to the same conditions to prepare brain than the other rodent species. Our interpretation of these results is that human PrPC has a lower propensity to initiate misfolding than the rodent protein. The alternative explanation that factors present in the human brain may prevent conversion is unlikely considering the experiments with humanized transgenic mice. The appearance of sporadic disease in humans may simply reflect the longer life span that provides greater chances for stochastic processes of spontaneous misfolding. However, it is also possible that sporadic disease is actually more frequent in animals, because the rate of spontaneous illness in animals has not been systematically studied.

Several lines of evidence enable us to rule out the possibility that de novo PrPSc was the result of cross-contamination. First of all, conventional PMCA that allows us to amplify as little as a single particle of PrPSc did not show spontaneous formation of misfolded protein, even after extensive PMCA cycling (Fig. 1). Second, the rate of spontaneous generation of prions in different species does not correlate with the availability of infectious material in our lab. Moreover, the variable rate was surprisingly constant among different experiments, arguing that it is dependent on some intrinsic properties of the protein rather than a stochastic event as cross-contamination. Third, an experiment done using strictly prion-free equipment and reagents, performed in a new laboratory that was never exposed to prions and done by a person who has never been in contact with prion material showed very similar results as the one carried out in our standard facilities. Finally, the biochemical, structural and biological properties of de novo generated PrPSc in hamster were substantially different to those of various conventional hamster prion strains. Although all these evidences strongly indicate that the results are not due to crosscontamination, this possibility cannot entirely be ruled out.

Inoculation of wild type hamsters with de novo generated PrPSc produced disease in all animals. Strikingly, many of the disease characteristics were substantially different to those observed with several other hamster prion strains. In this study we directly compared PGP-h1 with 3 well-established strains (263K, HY and DY) and literature comparisons with other strains such as Sc237, SHa(Me7), MT-C5, SHa(RML), 139H and Me7-H, also show substantial differences [26,27]. It is important to note that PMCA amplification of PrPSc from various strains (of hamsters, mice, human or deer origin) faithfully propagates the strain characteristics [8–10,28,29]. Thus, the differences observed in the de novo generated material are not attributable to PMCA amplification, but to intrinsic differences of this new prion strain. In our direct comparisons the incubation time of PGP-h1 was significantly larger than 263K and HY strains, but much shorter than DY. Clinical signs were also clearly distinguishable from those observed in animals affected by other strains (see Video S1 and Video S2 in supporting online material). The animals inoculated with PGP-h1 were not hyperactive or aggressive, but not lethargic either. They exhibited a social withdrawal and lack of interest for the surroundings, which reflected in a much reduced horizontal and vertical activity and increased extent of inactive time in an open field test. The clinical differences on the PGP-h1 induced disease were likely the result of the severe brain damage produced, including extensive spongiform degeneration, PrPSc accumulation and brain inflammation. Remarkably, the brain areas targeted by vacuolation damage were significantly different from all other hamster strains studied and included a substantially greater extent of spongiosis in colliculus and a much reduced level of injury in cerebellum and cortex. The biochemical characteristics of PrPSc were also different in PGP-h1, mostly in terms of the higher quantity of PrPSc accumulated in the brain and the lower relative resistance to proteolytic degradation than the other hamster strains analyzed. Taken together this data demonstrate that de novo generated prions correspond to a novel strain of infectious material, able to generate a new disease in wild type animals. We are currently assessing the infectious properties of some of the other de novo generated PrPSc obtained in this study to examine whether or not new strains and new diseases are produced also in other species.

Our findings provide a model to study the possible origins of sporadic TSEs and a new avenue to investigate the mechanism and factors controlling spontaneous formation of infectious material. For example, using the modified PMCA reaction described in this study we could assess the sequence determinants of the variable propensity of PrPC in distinct species to undergo conversion into the pathological form. We could also study the contribution of other factors in brain to alter the rate of de novo generation of PrPSc. Finally, our data provides further support for the prion hypothesis, since misfolded and infectious protein was generated in vitro, without the need for addition of pre-existing PrPSc. The fact that the de novo generated PrPSc corresponds to a new strain of infectious material suggest that the diversity of alternative and transmissible foldings that PrPSc can adopt is much larger than usually thought. This is worrisome, because it raises the possibility that novel and perhaps more aggressive infectious prion foldings may spontaneously originate in diverse species, leading to the emergence of new and unpredictable forms of transmissible diseases.


Date: June 12, 2006 at 5:18 am PST

IF we all believe the BSe that the USDA is trying to put out now about atypical BSE in USA cattle just arising spontaneously, then we all should believe in the tooth fairy and santa claus as well.

IF USA scrapie transmitted to USA cattle long ago in experiments in a lab in Mission Texas did not produce UK BSE, but something very different, then why would USA TSE cattle produce the UK human version of mad cow i.e. nvCJD? IT wouldn't. USA sporadic cjd is increasing, the USA also has atypical human cases of unknown origin as well?

THERE are over 20 strains of scrapie, plus the atypical in sheep, and these strains are increasing in numbers.

SCRAPIE, CWD, AND TSE IN CATTLE i.e. ANIMAL TSE RAMPANT IN USA FOR DECADES, and amplified via rendering and feeding practices, where USDA triple firewalls against BSE were nothing more than a mere smoke screen.

NO test tube TSE by either Prusiner or Soto, to date, have ever produced a TSE identical to the sporadic CJD. IN fact, no test tube TSE has ever been produced that resembles _any_ natural field TSE.

IF you feed BSE tainted materials to cattle and primate, you have BSE and nvCJD. IF you feed USA sheep strain to USA cattle, you get USA TSE. IF you feed USA tainted cattle to humans, you get USA mad cow disease. IF you feed sporadic CJD to primate you get a CJD infected primate. NOTHING spontaneous about it at all.

USA is in a very unique situation. there are more documented TSE in different species than any other country, all of which have been rendered and fed back to animals for human and animal consumption, for decades. Millions exposed, and of these Millions, how many surgical and dental procedures have been done on these exposed, to pass on to others, via the 'friendly fire' mode of transmission?

IF, the spontaneous TSE was true, then this would be Prusiner and everyone else that is trying to cash in on this agent with there TSE rapid test, this would be there dream come true. IT would require mandatory BSE/TSE testing of all species, due to the fact you could not ever eradicate it through any intervention. BUT, then again, the spontaneous TSE is like believing in the tooth fairy or santa clause will be arriving at your house this year.

How long can this sharade continue $

How many more will become exposed and have to die $



EVEN the origin of sporadic CJD continues to be debated. A British researcher, Dr. Richard Kimberlin, points out that the age-specific incidence of sporadic CJD: is similar to the age-specific incidence of BSE.


Kimberlin argues that the bell curve of sporadic CJD indicates that this seemingly random human disease is probably also caused by infection: "The shape of the age-specific incidence curve... implies that infection with [a] common strain [of CJD] occurs in childhood or adolescence, and that the median incubation period is 40 to 50 years."

German researcher Dr. Heino Diringer similarly defends an infectious cause: "It seems more than likely that ... the sporadic cases of CJD always originate from direct or indirect transmission from animals to man."


1: EMBO J. 2002 Dec 2;21(23):6358-66. Links BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein.Asante EA, Linehan JM, Desbruslais M, Joiner S, Gowland I, Wood AL, Welch J, Hill AF, Lloyd SE, Wadsworth JD, Collinge J. MRC Prion Unit, Institute of Neurology, University College, Queen Square, London WC1N 3BG, UK.

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.


Monday, May 11, 2009

Rare BSE mutation raises concerns over risks to public health

Rare BSE mutation raises concerns over risks to public health

SIR - Atypical forms (known as H- and L-type) of bovine spongiform encephalopathy (BSE) have recently appeared in several European countries as well as in Japan, Canada and the United States. This raises the unwelcome possibility that variant Creutzfeldt-Jakob disease (vCJD) could increase in the human population. Of the atypical BSE cases tested so far, a mutation in the prion protein gene (PRNP) has been detected in just one, a cow in Alabama with BSE; her healthy calf also carried the mutation (J. A. Richt and S. M. Hall PLoS Pathog. 4, e1000156; 2008). This raises the possibility that the disease could occasionally be genetic in origin. Indeed, the report of the UK BSE Inquiry in 2000 suggested that the UK epidemic had most likely originated from such a mutation and argued against the scrapierelated assumption. Such rare potential pathogenic PRNP mutations could occur in countries at present considered to be free of BSE, such as Australia and New Zealand. So it is important to maintain strict surveillance for BSE in cattle, with rigorous enforcement of the ruminant feed ban (many countries still feed ruminant proteins to pigs). Removal of specified risk material, such as brain and spinal cord, from cattle at slaughter prevents infected material from entering the human food chain. Routine genetic screening of cattle for PRNP mutations, which is now available, could provide additional data on the risk to the public. Because the point mutation identified in the Alabama animals is identical to that responsible for the commonest type of familial (genetic) CJD in humans, it is possible that the resulting infective prion protein might cross the bovine-human species barrier more easily. Patients with vCJD continue to be identified. The fact that this is happening less often should not lead to relaxation of the controls necessary to prevent future outbreaks. Malcolm A. Ferguson-Smith Cambridge University Department of Veterinary Medicine, Madingley Road, Cambridge CB3 0ES, UK e-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000223/! Jürgen A. Richt College of Veterinary Medicine, Kansas State University, K224B Mosier Hall, Manhattan, Kansas 66506-5601, USA

NATUREVol 45726 February 2009

Sunday, May 10, 2009

Identification and characterization of bovine spongiform encephalopathy cases diagnosed and not diagnosed in the United States

Wednesday, February 11, 2009

Atypical BSE North America Update

February 2009

Both of the BSE cases ascertained in the US native-born cattle were atypical cases (H-type), which contributed to the initial ambiguity of the diagnosis. 174, 185 In Canada, there have been 2 atypical BSE cases in addition to the 14 cases of the classic UK strain of BSE2: one was the H-type, and the other was of the L-type.198 snip...end source : Enhanced Abstract Journal of the American Veterinary Medical Association January 1, 2009, Vol. 234, No. 1, Pages 59-72 Bovine spongiform encephalopathy Jane L. Harman, DVM, PhD; Christopher J. Silva, PhD

Atypical BSE North America Update February 2009

Thursday, April 30, 2009 FDA Issues Final Guidance for Renderers on Substances Prohibited From Use in Animal Food or Feed CVM Update Back April 30, 2009

Sunday, May 10, 2009

Meeting of the Transmissible Spongiform Encephalopathies Committee On June 12, 2009 (Singeltary submission)

April 20, 2009

National Prion Disease Pathology Surveillance Center Cases Examined1 (December 31, 2008)

National Prion Disease Pathology Surveillance Center Cases Examined1 (December 31, 2008)

Year Total Referrals2 Prion Disease Sporadic Familial Iatrogenic vCJD 1996 & earlier 42 32 28 4 0 0 1997 115 68 59 9 0 0 1998 93 53 45 7 1 0 1999 115 69 61 8 0 0 2000 151 103 89 14 0 0 2001 210 118 108 9 0 0 2002 258 147 123 22 2 0 2003 273 176 135 41 0 0 2004 335 184 162 21 0 13 2005 346 193 154 38 1 0 2006 380 192 159 32 0 14 2007 370 212 185 26 0 0 2008 383 228 182 23 0 0 TOTAL 30715 17756 1490 254 4 2 1 Listed based on the year of death or, if not available, on year of referral; 2 Cases with suspected prion disease for which brain tissue and/or blood (in familial cases) were submitted; 3 Disease acquired in the United Kingdom; 4 Disease acquired in Saudi Arabia; 5 Includes 20 cases in which the diagnosis is pending, and 17 inconclusive cases; 6 Includes 25 cases with type determination pending in which the diagnosis of vCJD has been excluded. Rev 2/13/09 National

*5 Includes 20 cases in which the diagnosis is pending, and 17 inconclusive cases; *6 Includes 25 cases with type determination pending in which the diagnosis of vCJD has been excluded.


it would be interesting to know what year these atypical cases occurred, as opposed to lumping them in with the totals only.

are they accumulating ?

did they occur in one year, two years, same state, same city ?

location would be very interesting ?

age group ?

sex ?

how was it determined that nvCJD was ruled out ?

from 1997, the year i started dealing with this nightmare, there were 28 cases (per this report), up until 2007 where the total was 185 cases (per this report), and to date 2008 is at 182. a staggering increase in my opinion, for something that just happens spontaneously as some would have us believe. i don't believe it, not in 85%+ of all sporadic CJD cases. actually, i do not believe yet that anyone has proven that any of the sporadic CJD cases have been proven to be a spontaneous misfolding of a protein. there are many potential routes and sources for the sporadic CJD's. ...TSS

Sunday, April 12, 2009

r-calf and the USA mad cow problem, don't look, don't find, and then blame Canada



Monday, April 20, 2009 National Prion Disease Pathology Surveillance Center Cases Examined1 (December 31, 2008)

Friday, November 30, 2007



i am reminded of a few things deep throat told me years ago;


The most frightening thing I have read all day is the report of Gambetti's finding of a new strain of sporadic cjd in young people......... Dear God, what in the name of all that is holy is that!!! If the US has different strains of scrapie..... why???? than the UK... then would the same mechanisms that make different strains of scrapie here make different strains of BSE... if the patterns are different in sheep and mice for scrapie..... could not the BSE be different in the cattle, in the mink, in the humans....... I really think the slides or tissues and everything from these young people with the new strain of sporadic cjd should be put up to be analyzed by many, many experts in cjd........ bse..... scrapie

Scrape the damn slide and put it into mice..... wait..... chop up the mouse brain and and spinal cord........ put into some more mice..... dammit amplify the thing and start the damned research..... This is NOT rocket science... we need to use what we know and get off our butts and move.... the whining about how long everything takes..... well it takes a whole lot longer if you whine for a year and then start the research!!!

Not sure where I read this but it was a recent press release or something like that: I thought I would fall out of my chair when I read about how there was no worry about infectivity from a histopath slide or tissues because they are preserved in formic acid, or formalin or formaldehyde..... for God's sake........ Ask any pathologist in the UK what the brain tissues in the formalin looks like after a year....... it is a big fat sponge... the agent continues to eat the brain ...... you can't make slides anymore because the agent has never stopped........ and the old slides that are stained with Hemolysin and Eosin...... they get holier and holier and degenerate and continue... what you looked at 6 months ago is not there........ Gambetti better be photographing every damned thing he is looking at.....

Okay, you need to know. You don't need to pass it on as nothing will come of it and there is not a damned thing anyone can do about it. Don't even hint at it as it will be denied and laughed at.......... USDA is gonna do as little as possible until there is actually a human case in the USA of the nvcjd........ if you want to move this thing along and shake the earth.... then we gotta get the victims families to make sure whoever is doing the autopsy is credible, trustworthy, and a saint with the courage of Joan of Arc........ I am not kidding!!!! so, unless we get a human death from EXACTLY the same form with EXACTLY the same histopath lesions as seen in the UK nvcjd........ forget any action........ it is ALL gonna be sporadic!!! And, if there is a case....... there is gonna be every effort to link it to international travel, international food, etc. etc. etc. etc. etc. They will go so far as to find out if a sex partner had ever traveled to the UK/europe, etc. etc. .... It is gonna be a long, lonely, dangerous twisted journey to the truth. They have all the cards, all the money, and are willing to threaten and carry out those threats.... and this may be their biggest downfall...

Thanks as always for your help. (Recently had a very startling revelation from a rather senior person in government here.......... knocked me out of my chair........ you must keep pushing. If I was a power person.... I would be demanding that there be at least a million bovine tested as soon as possible and agressively seeking this disease. The big players are coming out of the wood work as there is money to be made!!!

In short: "FIRE AT WILL"!!! for the very dumb.... who's "will"! "Will be the burden to bare if there is any coverup!"

again it was said years ago and it should be taken seriously.... BSE will NEVER be found in the US!

As for the BSE conference call... I think you did agreat service to freedom of information and making some people feign integrity... I find it scary to see that most of the "experts" are employed by the federal government or are supported on the "teat" of federal funds. A scary picture! I hope there is a confidential panel organized by the new government to really investigate this thing.

You need to watch your back........ but keep picking at them....... like a buzzard to the bone... you just may get to the truth!!! (You probably have more support than you know. Too many people are afraid to show you or let anyone else know. I have heard a few things myself... you ask the questions that everyone else is too afraid to ask.)


greetings again voice,

then i remind everyone to read this;

'As implied in the Inset 25 we must not assume that transmission of BSE to other species will invariably present pathology typical of a scrapie-like disease.'

Saturday, May 2, 2009


Sunday, May 17, 2009



Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA 77518

Labels: , , , , ,


Post a Comment

Subscribe to Post Comments [Atom]

<< Home