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From: TSS ()
Subject: In Vitro Generation of Infectious Scrapie Prions
Date: April 21, 2005 at 11:57 am PST

-------- Original Message --------
Subject: In Vitro Generation of Infectious Scrapie Prions
Date: Thu, 21 Apr 2005 13:44:08 -0500
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy

##################### Bovine Spongiform Encephalopathy #####################


In Vitro Generation of Infectious Scrapie Prions

Joaquín Castilla1
Paula Saá1
Claudio Hetz1

and Claudio Soto1
Corresponding Author Contact Information
E-mail The Corresponding Author

1Department of Neurology, University of Texas Medical Branch, Galveston,
Texas 77555
2Centro de Biología Molecular, Universidad Autónoma de Madrid, 28049
Madrid, Spain
3University of Chile, Instituto de Ciencias Biomédicas, P.O. Box 625,
Santiago, Chile

Received 12 October 2004; revised 20 December 2004; accepted 11 February
2005. Published: April 21, 2005. Available online 21 April 2005.


Prions are unconventional infectious agents responsible for
transmissible spongiform encephalopathy (TSE) diseases. They are thought
to be composed exclusively of the protease-resistant prion protein
(PrPres) that replicates in the body by inducing the misfolding of the
cellular prion protein (PrPC). Although compelling evidence supports
this hypothesis, generation of infectious prion particles in vitro has
not been convincingly demonstrated. Here we show that PrPC ? PrPres
conversion can be mimicked in vitro by cyclic amplification of protein
misfolding, resulting in indefinite amplification of PrPres. The in
vitro-generated forms of PrPres share similar biochemical and structural
properties with PrPres derived from sick brains. Inoculation of
wild-type hamsters with in vitro-produced PrPres led to a scrapie
disease identical to the illness produced by brain infectious material.
These findings demonstrate that prions can be generated in vitro and
provide strong evidence in support of the protein-only hypothesis of
prion transmission.

Corresponding Author Contact Information
409-7470017; F.: 409-747-0020


From Microbes to Prions

The Final Proof of the Prion Hypothesis

Wen-Quan Zou and Pierluigi GambettiCorresponding Author Contact
E-mail The Corresponding Author

Division of Neuropathology, Department of Pathology, National Prion
Disease Pathology Surveillance Center, Case Western Reserve University,
Cleveland, Ohio 44106

Available online 21 April 2005.

Much like the microbe hypothesis put forth over 150 years ago, the
prion hypothesis can be definitely proven only if a prion disease is
engendered in a natural host from an infectious prion produced in vitro.
In this issue of Cell, Castilla et al. (2005)

come very close to accomplishing this goal by producing a prion disease
in a natural host from a prion entirely generated in vitro using a
PCR-like amplification system.

Corresponding Author Contact Information
216-368-0587; Fax: 216-368-2546


######### ##########

Study seeks to end prion controversy
Published on : 4/21/2005 1:14:00 PM
Category : World News
GALVESTON, Texas, April 21 (UPI) -- U.S. scientists said they have found
strong evidence that proves the controversial theory that proteins cause
mad cow disease and similar brain disorders.

Claudio Soto, of the University of Texas Medical Branch, said his
findings, which appear in the April 21 issue of the journal Cell, offer
"the best and final proof for the prion hypothesis."The prevailing
theory is mad cow and similar disorders are caused by an infectious
protein called a prion, but this has remained controversial because
prions have never been shown to be capable of causing disease.

In the study, Soto's team isolated prions from the brains of hamsters
experimentally infected with scrapie, a mad cow-like illness that occurs
in sheep.They amplified the prions and injected them back into healthy
hamsters, which subsequently came down with scrapie symptoms.

Some experts said, however, the study still leaves open the possibility
other pathogens may be the cause of these diseases.Pierluigi Gambetti of
the National Prion Disease Pathology Surveillance Center in Cleveland,
wrote in an editorial accompanying the Cell article that Soto's
experiment had not ruled out the possibility of other infectious agents
contaminating the original sample.

- -- Copyright 2005 by United Press International.


-------- Original Message --------
Subject: Study points to prions in brain disorders UPI
Date: Thu, 21 Apr 2005 14:04:58 -0500
From: "Terry S. Singeltary Sr."
To: Bovine Spongiform Encephalopathy

Study points to prions in brain disorders

By Steve Mitchell

Washington, DC, Apr. 21 (UPI) -- Scientists involved with a new study
released Thursday said it provides strong proof of the controversial
theory that infectious proteins called prions cause mad cow disease and
similar brain disorders in humans.

Some experts find the data unconvincing, however, and one researcher
recently presented findings he said robustly support a different
hypothesis: these diseases are caused by a bacteria.

"This is really the best and final proof for the prion hypothesis,"
Claudio Soto, a professor of neurology at the University of Texas
Medical Branch at Galveston and senior author of the new study, told
United Press International.

Stanley Prusiner, of the University of California, San Francisco, won a
Nobel prize in 1997 for his hypothesis that prions are the pathogen that
cause these fatal, brain-wasting diseases, collectively known as
transmissible spongiform encephalopathies or TSEs. These include mad cow
disease, Creutzfeldt Jakob disease in humans, chronic wasting disease in
deer and elk and scrapie in sheep.

The prion hypothesis has enjoyed widespread acceptance among TSE
specialists, but it also has remained controversial because prions
isolated from a TSE never have been shown to be infectious. Prions exist
in two forms in the body: a normal form whose function is uncertain and
a misfolded form that is associated with disease.

The new study, published in the April 21 issue of the journal Cell,
describes how Soto and colleagues isolated misfolded prions from the
brains of hamsters experimentally infected with scrapie. Using a method
they developed called protein misfolding cyclic amplification, or PMCA,
they significantly increased the number of prions and then placed them
in a test tube with normal prions.

The misfolded prions are thought to cause the normal prions to transform
into the misfolded, disease-causing variants.

The team diluted the samples over and over again, to ensure the only
remaining prions were those generated in the test tube and none of the
original prions from the hamster brain were left. Then they injected the
samples into the brains of hamsters -- which developed symptoms of TSE.

Laura Manuelidis, a TSE expert and section chief of surgery in the
neuropathology department at Yale University -- who has long doubted the
prion hypothesis -- reached the opposite conclusion from Soto.

"It's about the best proof that (prions) aren't infectious," Manuelidis
told UPI.

She noted the study shows prions obtained through PMCA had less
infectivity than the same amount of prions from the original hamster
brain sample.

"They got amplification but they didn't see any increase in
infectivity," she said.

Soto's team acknowledged this problem in their journal article, noting
"the reason for the lower level of infectivity ... is unknown and
currently under investigation."

Manuelidis, who thinks the bulk of the evidence points toward the TSE
pathogen being a virus, said she is not committed to that position.

"I'm firmly in the 'I don't know' camp," she said. If researchers could
prove prions are the infectious agent in TSEs, she said, she readily
would acknowledge it, but this study does not meet that criteria.

Neil Cashman, a TSE expert at the University of Toronto, said the study
offered solid support for the prion hypothesis.

"I find the article convincing," Cashman told UPI. He added, however, he
would like to see other groups repeat the methodology and obtain the
same results, because previous similar experiments have failed to find
increased infectivity.

Wen-Quan Zou and Pierluigi Gambetti, of the National Prion Disease
Pathology Surveillance Center in Cleveland, argued Soto had not ruled
out the possibility of other infectious agents as the causative pathogen
of TSEs.

"Although the study ... makes significant progress toward proving the
prion hypothesis, it still falls a bit short of achieving this goal,"
Gambetti and Zou wrote in an editorial accompanying the paper. "For
example, the possibility remains that RNA or other molecules present in
the original brain preparation might be amplified along with (prions)
and might play a critical role in conferring infectivity and specificity
to the new (prions)."

Frank Bastian, a pathologist at Tulane University in New Orleans, whose
hypothesis is TSEs are caused by a bacteria, doubted the study for
similar reasons.

Bastian said the processes used by Soto's team -- to ensure other
pathogens were destroyed in the original sample of hamster brain -- may
not have been entirely effective, particularly against Spiroplasma, the
strain of bacteria he thinks is the cause of these diseases.

"I suggest that if Spiroplasma are in the original preparation, that
they could survive" the disinfection measures, Bastian told UPI. He said
he has exposed cultures of Spiroplasma to some of the same procedures
and not seen a decrease in infectivity.

Bastian presented research at the American Academy of Neurology meeting
last week. He said his lab had succeeded in isolating the Spiroplasma
bacteria from the brains of deer infected with chronic wasting disease
and the brains of sheep infected with scrapie. He said the findings
offered further support of his hypothesis that Spiroplasma are the
pathogens behind TSEs.

Bastian's lab is currently injecting the isolated Spiroplasma into
animals to see if they contract symptoms of a TSE. "If we can fulfill
that, then we have proved that it's a cause," he said. If that happens,
then scientists will "have to seriously consider criteria of the
bacteria hypothesis," he said.

The problem Bastian faces is that his funding may run out before he can
complete the animal inoculation studies, which will take about a year.
His current grant ends in July and it can be difficult to get funding in
the TSEs field for studies not focused on prions.

"I'm going to go as far as I can go," Bastian said.


Steve Mitchell is UPI's Medical Correspondent. E-mail:


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