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From: TSS ()
Subject: Prion biology: the quest for the test
Date: September 17, 2007 at 7:49 am PST

Prion biology: the quest for the test


Adriano Aguzzi


The adaptation of the protein misfolding cyclic amplification
assay (PMCA) to use recombinant hamster prion protein (PrP) as a
substrate shows promise for both basic research applications and
clinical diagnostic assays.


Prions, the elusive infectious particles
responsible for transmissible spongiform
encephalopathies (Creutzfeldt-Jakob disease
in humans as well as various conditions
in wild and captive animals), have
been responsible for some of the most
tragic disasters in the history of medicine1.
For decades, biological materials derived
from subclinically infected organisms have
transferred the infection to others. Thus,
the development of sensitive, fast and reliable
assays represents an urgent and unmet
medical need. In this issue of Nature
Methods, Atarashi and colleagues from the
Caughey laboratory introduce a technique
that may be an interesting step toward that
goal and may also help to understand the
mechanisms of prion replication2.
For a long time, blood products were
not thought of as vectors of iatrogenic
prion transmission. The bovine spongiform
encephalopathy (BSE) epidemic has
changed that perception. BSE has infected
cows throughout the world, and as a result
of eating tainted beef, some 160 people
have contracted variant Creutzfeldt-Jakob
disease (vCJD). An undetermined number
of hitherto asymptomatic persons
may have also contracted infection. Some
of them have donated blood, resulting in
at least four cases of blood-borne transmission
of vCJD. Such transmission will
be likely witnessed time and again in the
coming decades3.
PCR-based nucleic acid testing technologies
have reached wondrous levels of
sensitivity and speed: it is now possible to
detect viruses in donated blood with sufficient
sensitivity to fully exclude transmission.
Yet prions do not contain nucleic
acids and consist primarily of proteins—
the sensitive detection of which lags well
behind that of nucleic acids. These hurdles
have become painfully evident when
addressing the prion detection question.
According to the protein-only hypothesis,
prions consist of PrPSc, a misfolded,
structured aggregate of a cellular constituent
called PrPC. A very large body of
evidence supports this hypothesis, and
PrP-deficient mice that lack PrPC are resistant
to prion infection4. Because PrPC is
prevalent in body fluids and shares most
epitopes with its aggregated counterpart,
termed PrPSc, prions have proved resilient
to immunodetection. In 1994, Caughey
and colleagues discovered that PrPSc can
catalyze the conversion of PrPC into more
PrPSc, but that process was inefficient and
detectable only with considerable efforts5.


Adriano Aguzzi is at the Institute of Neuropathology, CH-8091 Zürich,
Switzerland.
e-mail: adriano.aguzzi@usz.ch
© 2007 Nature Publishing Group http://www.nature.com/naturemethods
NATURE METHODS | VOL.4 NO.8 | AUGUST 2007 | 615
NEWS AND VIEWS


In 2001, another milestone was the invention
of the protein misfolding cyclic
amplification (PMCA) assay by Soto and
colleagues6, which can be loosely compared
to PCR, as it generates PrPSc from
monomeric substrates and a small amount
of template PrPSc. Prion amplification is
accomplished by repeated ultrasoundinduced
fragmentation of aggregates,
followed by accretion of more PrPC. Soto
and colleagues showed that PMCA can
propagate prions indefinitely7 and can be
used to diagnose prion infections in body
fluids8.
Four issues need to be overcome to
enable PMCA for clinically useful prion
detection. First, PMCA is prone to many
technical problems. For example, the
uneven energy distribution renders the
reaction highly sensitive to the positioning
of tubes in sonication chambers.
Second, PMCA is qualitative at best: the
amount of PrPSc amplicons does not correlate
well with the prion input. Third,
PMCA has not been done using chemically
defined components: the PMCA substrate
is whole-brain homogenate, which is not
easy to obtain—particularly in the case of
humans. Fourth, PMCA is extremely timeconsuming
and laborious, and requires
weeks to attain maximal sensitivity.
While the first two issues await resolution,
Atarashi and colleagues provide a
solution to the third problem and a relief
to the fourth one. By carefully titrating
the amplification ingredients, they succeed
in exploiting recombinant, bacterially
expressed prion protein as substrate
(Fig. 1) for the detection of as little as 50 ag
of hamster prion. This opens the way to
much-needed standardization assays. Also,
their technology amplifies prions within
48–72 h. The sensitivity is not quite as high
as that of the original PMCA but—assuming
that it is not restricted to hamster prions—
may still reach a range useful for
prion detection in body fluids. The technology
may also be helpful in determining
the parameters that affect prion replication
and may even find a place in the screening
of inhibitory compounds.
Will the technology described by
Atarashi and colleagues put an end to iatrogenic
prion transmissions? Unfortunately,
this is unlikely. For one thing, there is no
indication that their technique can sensitively
detect prions in blood. Atarashi and
colleagues successfully identified infected
hamster cerebrospinal fluid (CSF), but
CSF contains fewer inhibitory factors
than blood9. Second, the technique is still
a far cry from being high throughput. But
PMCA may become a second- or thirdtier
means of reprobing samples that may
falsely test positive in a high-throughput
assay.
The objects detected by PMCA are
unknown and may consist of ordered
aggregates of PrP. Such aggregates may
arise stochastically and may not always be
infective. Highly ordered polyionic scaffolds10
may promote formation of PrP
arrays that could spontaneously seed the
PMCA reaction. Indeed, prions have been
generated de novo from purified eukaryotic
PrP with mixtures of polyanions and
lipids using PMCA11. This discovery is
exciting but blows a kiss of death to diagnostic
PMCA. If PMCA generates or can
generate infectivity from noninfectious
material, it may be prone to generating
false positives. Finally, the possible generation
of prion infectivity from recombinant
material creates a biosafety quagmire for
large-scale deployment.
In summary, the medical need for a
blood test for prions is as acute as ever. The
study by Atarashi and colleagues marks a
good step in this direction. The methodology
is fascinating, and I am certainly
looking forward to trying it out in my
own laboratory. The clinical problem of
identifying potential sources of iatrogenic
infections, however, may require more
than biotechnological advances, and will
eventually necessitate a great deal of input
from other disciplines, including chemistry,
physics and engineering sciences.


Figure 1 | Prion replication and PMCA. Present thinking about the prion life
cycle is that the infectious moiety is a highly structured aggregate that
grows by
recruiting misfolded prion protein (left). The PMCA reaction as modified by
Atarashi (right) consists of a sonication step, which disrupts the PrPSc
aggregates,
followed by an ‘elongation’ step that entails incubation with recombinant
PrPC. Elongated aggregates are then subjected to additional cycles of
sonication
and elongation.

COMPETING INTERESTS STATEMENT
The author declares no competing financial
interests.


1. Aguzzi, A. & Polymenidou, M. Cell 116, 313–
327 (2004).
2. Atarashi, R. et al. Nat. Methods 4, 645–650
(2007).
3. Aguzzi, A. & Glatzel, M. Nat. Clin. Pract.
Neurol. 2, 321–329 (2006).
4. Büeler, H. et al. Cell 73, 1339–1347 (1993).
5. Kocisko, D.A. et al. Nature 370, 471–474
(1994).
6. Saborio, G.P., Permanne, B. & Soto, C. Nature
411, 810–813 (2001).
7. Castilla, J., Saa, P., Hetz, C. & Soto, C. Cell
121, 195–206 (2005).
8. Castilla, J., Saa, P. & Soto, C. Nat. Med. 11,
982–985 (2005).
9. Fischer, M.B., Roeckl, C., Parizek, P., Schwarz,
H.P. & Aguzzi, A. Nature 408, 479–483
(2000).
10. Deleault, N.R., Lucassen, R.W. & Supattapone,
S. Nature 425, 717–720 (2003).
11. Deleault, N.R., Harris, B.T., Rees, J.R. &
Supattapone, S. Proc. Natl. Acad. Sci. USA
104, 9741–9746 (2007).

© 2007 Nature Publishing Group http://www.nature.com/naturemethods
616 | VOL.4 NO.8 | AUGUST 2007 | NATURE METHODS
NEWS AND VIEWS

tss

Blood test for prion diseases reported
Aug 29, 2005 (CIDRAP News) – Scientists in Texas report they have found a way to detect abnormal prion protein in blood, an achievement that could lead to the first practical blood test for bovine spongiform encephalopathy (BSE) and similar diseases in living animals.

The test was used successfully to detect a prion disease in hamsters. If it proves effective in cattle and humans, it could help protect the blood supply from BSE, help determine the prevalence of the disease in US cattle, and assist researchers trying to assess how many people are unwittingly infected with variant Creutzfeldt-Jakob disease (vCJD), the human equivalent of BSE.

At present, BSE and related prion diseases, called transmissible spongiform encephalopathies (TSEs), can be definitively diagnosed only by examination of brain tissue after death. In the United Kingdom, BSE spread through cattle herds in the 1980s and early 1990s and led to more than 150 cases of vCJD in people, presumably as a result of eating beef from infected animals. The United States has had two BSE cases so far.

Because TSEs take years to produce symptoms, it is feared that many more Britons were infected unknowingly and will fall ill in the years ahead. The discovery in Britain of a few possible cases of transmission of vCJD through blood transfusions has fueled more concern.

The new blood test was developed by Claudio Soto and two colleagues at the University of Texas Medical Branch at Galveston (UTMB). Writing in Nature Medicne, they report that they devised a way to stimulate a tiny, undetectable amount of abnormal prion protein in a blood sample to multiply so that it reaches detectable levels.

"Our findings represent the first time that prions have been biochemically detected in blood," the authors state. Because the test appears to be highly accurate, it "offers promise for the design of a sensitive biochemical test for blood diagnosis of transmissible spongiform encephalopathies."

The method is called protein misfolding cyclic amplification (PMCA). It involves separating the "buffy coat" portion of a blood sample, adding a dose of normal prion protein from brain tissue to it, incubating the preparation at 37˚C, exposing it to sound waves, and repeating the process many times. If abnormal prion protein is present, it causes the normal prions to convert to the abnormal, misfolded form, forming small clumps, the report says. The sonic treatment breaks up the clumps into smaller bits, stimulating further conversion as the cycle is repeated. After a number of rounds of PMCA, the abnormal protein can be detected by an existing test, such as the Western blot.

The authors described the basic process in previous reports, and in the new article they write that they found a way to automate the process to speed it up and increase the number of cycles.

To evaluate the test, they used 12 healthy hamsters and 18 hamsters that had clinical signs of scrapie (the sheep form of TSE) after being inoculated with infected brain tissue. After six rounds of PMCA, abnormal prion protein was detected in blood samples from 16 of the 18 sick hamsters, but not in any of the samples from healthy hamsters. The findings signify 89% sensitivity and 100% specificity for the test.

In its current form, PMCA testing takes several days to yield a highly sensitive result, the report says. The authors say they expect to further increase the speed of the test.

The researchers predict that the development of a similar blood test for humans will have a "tremendous impact" on the beef industry, the safety of blood and blood products, and estimation of the number of vCJD cases. They suggest that the test could permit the diagnosis and treatment of vCJD early in its course, before the appearance of clinical signs and permanent brain damage. The disease is currently untreatable and always fatal.

In a UTMB news release, Soto commented, "The next step, which we're currently working on, will be detecting prions in the blood of animals before they develop clinical symptoms and applying the technology to human blood samples."

According to an Associated Press report, Soto also said he hopes to evaluate the test in animals that have been naturally infected with a TSE and in animals that have asymptomatic infections.

Castilla J, Saa P, Soto C. Detection of prions in blood. Nature Medicine 2005; early online release [Abstract]
http://www.nature.com/nm/journal/v11/n9/abs/nm1286.html

See also:

UTMB news release from EurekAlert
http://www.eurekalert.org/pub_releases/2005-08/uotm-cp082505.php


http://www.cidrap.umn.edu/cidrap/content/other/bse/news/aug2905bse.html

Published online before print May 29, 2007, 10.1073/pnas.0703910104
PNAS | June 5, 2007 | vol. 104 | no. 23 | 9551-9552


COMMENTARY
A simplified recipe for prions

Kil Sun Lee, and Byron Caughey*

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840


As long ago as the 1960s, it was proposed that the infectious agent of the transmissible spongiform encephalopathies (TSEs), or prion diseases, was composed of protein with no essential nucleic acid component. From the beginning, one basic idea was that the causative agent was a corrupted and pathological form of a host protein that could propagate itself by causing its normal homolog to convert to the pathological form (1). This concept, now commonly known as the "prion hypothesis" (2), has been extremely difficult to prove (3). Fungi were shown to have conceptually analogous protein-only prion pathogens (4), but TSE infectivity eluded clear biochemical characterization for decades. Complicating matters were the inherent difficulties in purifying TSE infectivity, which forms sticky, insoluble aggregates that are not amenable to rigorous purification techniques. Consistent with the prion hypothesis, the major component of infectious isolates was found to be an aberrant, partially protease-resistant form of the host's prion protein (PrPSc or PrP-res) (2). However, various other molecules, including nucleic acids, could often be found in even the purest preparations (e.g., refs. 5 and 6). Because there were typically 100,000 PrPSc molecules per infectious unit, it was difficult to exclude the possibility that other essential components of infectivity might be much less abundant. A standard approach to establishing the essential ingredients of any biological activity is the reconstitution of the activity from defined ingredients in vitro. However, despite the seductive simplicity and apparent plausibility of the prion hypothesis, many laboratories were frustrated for years in their attempts to assemble infectivity in defined cell-free reactions. Recently, striking progress has been made toward this goal, and, as reported by Supattapone and colleagues in this issue of PNAS (7), a defined recipe for producing robust . . . [Full Text of this Article]

*To whom correspondence should be addressed. E-mail: bcaughey@nih.gov


http://www.pnas.org/cgi/content/extract/104/23/9551

PDF]Freas, William TSS SUBMISSION

File Format: PDF/Adobe Acrobat -

Page 1. J Freas, William From: Sent: To: Subject: Terry S. Singeltary

Sr. [flounder@wt.net] Monday, January 08,200l 3:03 PM freas ...

snip...


I am beginning to think that the endless attempt to track
down and ban, potential victims from known BSE Countries
from giving blood will be futile. You would have to ban
everyone on the Globe eventually? AS well, I think we
MUST ACT SWIFTLY to find blood test for TSE's,
whether it be blood test, urine test, eyelid test,
anything at whatever cost, we need a test FAST.


snip...

http://www.fda.gov/ohrms/dockets/ac/01/slides/3681s2_09.pdf

Second, a single unit of vCJD-prion-infected

blood is sufficient to cause transmission of the

disease. This fact is particularly unsettling, as it

can only be taken to signify that the concentration

of ID50 units in blood is relatively high.

One ID50 unit is defined as the infectious

dose sufficient to establish infection in 50% of

recipients; animal experiments indicate that the

amount of prion infectivity needed to reach

one ID50 unit is much higher when prions are

administered intravenously than when they

are inoculated intracerebrally. ...


snip... full text ;


JUNE 2006 VOL 2 NO 6 AGUZZI AND GLATZEL NATURE CLINICAL PRACTICE NEUROLOGY 329

www.nature.com/clinicalpractice/neuro

go back to around recall date, and there is a multitude of mad cow nvCJD (only) blood recalls, and a great deal from Texas ;

http://www.fda.gov/bbs/topics/enforce/2006/ENF00955.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00956.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00957.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00958.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00959.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00961.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00962.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00963.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00964.html

http://www.fda.gov/bbs/topics/enforce/2006/ENF00965.html


[Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk
Materials for Human Food and Requirement for the Disposition of
Non-Ambulatory Disabled Cattle


THE infamous USA SPORADIC CJDs, something to ponder;

IF the USA TSE in cattle all does not look like UK BSE, why would all USA human TSE look like UK nvCJD???

over 20 strains of scrapie documented to date with new atypical strains now being documented in sheep and goat i.e. BSE.

atypical strains of BSE/TSE showing up in cattle in different countries?

ALL animals for human/animal consumption must be tested for TSE.

ALL human TSEs must be made reportable Nationally and Internationally, OF ALL AGES...

IN a time when FSIS/APHIS/USDA/FDA et al should be strengthening the TSE regulations, it seems corporate interest has won out again over sound science and consumer

protection from an agent that is 100% fatal for the ones that go clinical. With the many different atypical TSEs showing up in different parts of the world, and with GWs BSE

MRR policy (the legal policy of trading all strains of TSEs), the battle that has waged for the last 25 years to eradicate this agent from this planet will be set back decades, if not lost for good. ...

Terry S. Singeltary Sr.

P.O. Box 42

Bacliff, Texas USA 77518

9/13/2005

http://www.fsis.usda.gov/OPPDE/Comments/03-025IFA/03-025IFA-2.pdf


Subject: Fourth case of transfusion-associated vCJD infection in the United Kingdom
Date: January 18, 2007 at 8:32 am PST

http://lists.ifas.ufl.edu/cgi-bin/wa.exe?A2=ind0701&L=sanet-mg&T=0&F=&S=&P=9807


Subject: Re: Docket No. 2005N-0373 RIN number 0910-AF54 TSS SUBMISSION
Date: March 30, 2007 at 10:57 am PST

[Federal Register: March 30, 2007 (Volume 72, Number 61)]
[Proposed Rules]
[Page 15080-15081]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr30mr07-24]

=======================================================================
-----------------------------------------------------------------------

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Parts 211, 226, 300, 500, 530, 600, 895, and 1271

[Docket No. 2005N-0373]
RIN 0910-AF54


Use of Materials Derived From Cattle in Medical Products Intended
for Use in Humans and Drugs Intended for Use in Ruminants; Reopening of
the Comment Period

AGENCY: Food and Drug Administration, HHS.

ACTION: Proposed rule; reopening of the comment period.

-----------------------------------------------------------------------

SUMMARY: The Food and Drug Administration (FDA) is reopening until May
14, 2007, the comment period for the proposed rule published in the
Federal Register of January 12, 2007 (72 FR 1582). The proposed rule
would prohibit the use of certain cattle material in, or in the
manufacture (including processing) of, drugs, biologics, and medical
devices intended for use in humans and human cells, tissues, and
cellular and tissue-based products (HCT/Ps) (collectively, medical
products for humans), and in drugs intended for use in ruminant animals
(drugs for ruminants) and would also require new recordkeeping
provisions for medical products for humans and drugs for ruminants that
are manufactured from or otherwise contain material from cattle. The
agency is reopening the comment period in response to a request for
more time to enable industry to generate more information on products
that might be affected by the rule.

DATES: Submit written or electronic comments on the proposed rule by
May 14, 2007.


http://lists.ifas.ufl.edu/cgi-bin/wa.exe?A2=ind0703&L=sanet-mg&T=0&F=&S=&P=19128


http://lists.ifas.ufl.edu/cgi-bin/wa.exe?A3=ind0703&L=SANET-MG&E=quoted-printable&P=2791189&B=------%3D_NextPart_000_000C_01C772D3.8C9E50C0&T=text%2Fplain;%20charset=iso-8859-1


http://lists.ifas.ufl.edu/cgi-bin/wa.exe?A3=ind0703&L=SANET-MG&E=quoted-printable&P=2898631&B=------%3D_NextPart_000_000C_01C772D3.8C9E50C0&T=text%2Fhtml;%20charset=iso-8859-1&XSS=3

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




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