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From: TSS ()
Subject: Re: Diagnosis of prions in patients should utilize novel strategy, team says
Date: February 22, 2005 at 9:31 am PST

-------- Original Message --------
Subject: Re: Diagnosis of prions in patients should utilize novel strategy, team says
Date: Mon, 21 Feb 2005 21:20:47 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE
References: <421A9F0B.6000407@wt.net>


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

Greetings list members,

my apologize, i failed to ask my question.
in this study, they state;

> Based on the smallest amount of prions that could detected by the two
> techniques, they found that the CDI was from 1000- to 100,000-fold
> more sensitive than Western blot analysis performed in six different
> research laboratories (Minor et al. Standards for the assay of
> Creutzfeldt-Jakob disease specimens. J. Gen. Virol. 85: 1777-1784, 2004).
>
> Explanation as to why IHC for prions is less sensitive than the CDI:
> IHC is routinely performed on formalin-fixed, paraffin-embedded
> samples of brain. Formalin fixation markedly decreases the ability of
> antibodies to bind to proteins in general, which greatly weakens the
> IHC signal for prions (PrPSc). In contrast, homogenates for the CDI
> are not treated with reagents that decrease prion antigenicity.
> Moreover, to concentrate the PrPSc for measurement by the CDI, the
> homogenates are exposed to phosphotungstic acid, which selectively
> precipitates both protease-sensitive and protease-resistant PrPSc that
> comprise prions, but not the normal prion protein conformer found in
> uninfected animals, PrPC. This step results in a higher concentration
> of PrPSc for detection by the CDI. Because the PrPSc was not exposed
> to proteases, the CDI measures all forms of abnormally folded PrPSc
> molecules. Protease-sensitive PrPSc can account for 50 percent of the
> total PrPSc. For Western analysis, homogenates of brain are treated
> with protease to eliminate PrPC; however, this step also eliminates
> protease-sensitive PrPSc leaving only protease-resistant PrPSc for
> Western blot detection and decreasing the PrPSc signal at least in half.
>

does this mean that the test they used to come to the conclusion that
the last inconclusive that was postive that later was found to be
negative, could be in fact postive? the fact that western blot was not
use even brings more concern. could someone from the USDA et al
please comment on this? could they please retest these last inconclusives?
why are we not using this test?

thank you,
kind regards,

terry


Terry S. Singeltary Sr. wrote:

> ##################### Bovine Spongiform Encephalopathy
> #####################
>
> Public release date: 21-Feb-2005
> Contact: Jennifer O'Brien
> jobrien@pubaff.ucsf.edu
> 415-476-2557
> University of California - San Francisco
>
>
> Diagnosis of prions in patients should utilize novel strategy, team says
>
> A technique for detecting prions in tissue, developed in recent years
> by UCSF scientists, is significantly more sensitive than the
> diagnostic procedures currently used to detect the lethal particles in
> samples of brain tissue from patients, according to a study performed
> by a UCSF team.
>
> The finding indicates that the diagnostic technique, known as the
> conformation-dependent immunoassay (CDI), should be established as the
> standard approach for brain biopsies of patients suspected of having
> the disease, they say. The team is exploring whether the CDI might be
> adapted to detect prions in blood and muscle.
>
> The finding suggests that reliance on the current methods for
> detecting prions in human brain tissue -- microscopic examination of
> tissue for the telltale vacuoles that form in brain cells and
> immunohistochemistry (IHC), which involves detecting prions in brain
> sections using prion protein-specific antibodies -- may have led to an
> under diagnosis of the disease in patients in recent years, they say.
> (A definitive diagnosis of the disease in humans is made only on
> autopsy, when a neuropathologist can analyze multiple brain regions
> for vacuoles and evidence of prions by IHC, and it is estimated that
> only 50 percent of human cases are autopsied, in part because many
> pathologists do not want to risk infection during the autopsy.)
>
> In the study, the team compared the ability of the CDI and the two
> traditional diagnostic techniques to detect prions in various brain
> samples from 28 patients diagnosed on autopsy as having one of several
> human forms of the disease -- sporadic, familial or iatrogenic
> Creutzfeldt-Jakob disease (CJD). While the CDI detected the
> biochemical signal for prions in 100 percent of the samples studied,
> the traditional tests failed to detect the prion in a high proportion
> of cases. For example, in an experiment that focused on 18 brain
> regions from eight patients with sporadic CJD, the CDI detected prions
> in 100 percent of the samples, while IHC detected them in 22 percent
> and routine tissue examination in 17 percent.
>
> "In about 80 percent of the different brain regions examined, prions
> were not consistently detected by either IHC or routine histology that
> measure vacuolation. In contrast, the CDI was always positive in all
> regions of the brain," says the lead author of the study, Jiri Safar,
> MD, associate adjunct professor of neurology and a member of the UCSF
> Institute for Neurodegenerative Diseases, which is directed by senior
> author Stanley B. Prusiner, MD, UCSF professor of neurology and
> biochemistry.
>
> "These findings indicate that histology and immunohistochemistry
> should no longer be used to rule out prion disease in single-site
> biopsy samples," says Safar. "The superior performance of the CDI in
> diagnosing prion disease suggests that the CDI be used in future
> diagnostic evaluations of prion disease, particularly for single-site
> brain biopsies during life"
>
> "If the traditional techniques are used at autopsy, they must be
> applied to many cortical and subcortical samples," says co-author
> Stephen J. DeArmond, MD, PhD, UCSF professor of neuropathology.
>
> Moreover, while the study examined the efficacy of the CDI in
> comparison to the two techniques routinely used by neuropathologists
> to detect prions in human brain tissue, previous studies at UCSF
> indicate that the CDI is also significantly more sensitive than
> Western blot analysis, the technology used with IHC to detect prions
> in brain tissue from cattle suspected of having bovine spongiform
> encephalopathy (BSE). That IHC and Western blot analysis are
> relatively insensitive methods, the researchers say, supports their
> ongoing assertion that the CDI should also be used to evaluate the
> brain tissue of cattle.
>
> "The studies reported here are likely to change profoundly the
> approach to the diagnosis of prion disease in both humans and
> livestock," says Safar.
>
> More broadly, the scientists say, the high sensitivity of the CDI
> suggests that CDI-like tests could also prove useful for diagnosing
> other neurodegenerative diseases, such as Alzheimer's disease,
> Parkinsons's disease and fronto-temporal dementias, all of which, like
> prion diseases, involve various forms of protein misprocessing. These
> diseases currently are diagnosed by neuropathological analysis and
> immunohistochemistry.
>
> "Whether immunohistochemistry underestimates the incidence of one or
> more of these common neurodegenerative diseases is unknown, but the
> CDI could shed light on these diseases," says co-author Bruce Miller,
> MD, UCSF A.W. and Mary Margaret Clausen Distinguished Professor of
> Neurology and director of the UCSF Memory and Aging Center.
>
> The finding will be printed on-line and in print on March 1, 2005 in
> Proceedings of the National Academy of Sciences.
>
> The study brings into high relief the different detection strategies
> of immunohistochemistry and the CDI, both of which involve revealing
> the presence of prions, known as PrPsc, by applying antibodies to
> brain tissue.
>
> Standard immunohistochemistry, developed in the DeArmond lab 20 years
> ago, involves using an enzyme known as a protease, or a combination of
> harsh acid and high temperature treatment, to destroy normal prion
> protein (PrPC), which is ubiquitous in brain tissue. Once this occurs,
> scientists apply fluorescently lit antibodies that react with residues
> of the relatively resistant abnormal prion protein (PrPSc), thereby
> highlighting it.
>
> The limitation of this technique is that scientists have since learned
> that there is a large part of the abnormal prion protein that is
> protease sensitive, and that portion escapes detection by the standard
> technique. Thus, this traditional method underestimates the level of
> PrPSc in tissue.
>
> The CDI addresses this limitation by revealing the region of PrPSc
> that is exposed in the normal PrPC but is buried in infectious PrPSc,
> using high affinity, newly generated antibodies that identify PrPSc
> through the distinct shape of the molecule, independent of proteolytic
> treatments. This makes it possible to detect potentially large
> concentrations of protease sensitive PrPSc molecules.
>
> Detractors would say that it is not necessary to detect the minute
> level of infectious agent that the CDI is capable of revealing, as it
> would be unlikely to be lethal, says Safar. But Prusiner and his
> colleagues maintain that any risk is too great when it comes to having
> prions in the food supply. In addition, because even low levels of
> prions are extremely resistant to inactivation, they may contaminate
> the environment for many years.
>
> Prusiner won the 1997 Nobel Prize in Physiology or Medicine for
> discovering that a class of neurodegenerative diseases known as
> spongiform encephalopathies was caused by prions. Prion diseases
> develop in humans, cattle, sheep, deer, elk and mink.
>
> The CDI was developed by members of the Prusiner lab. The CDI
> methodology has been licensed to InPro Biotechnology, Inc.
>
> Prusiner, Safar, DeArmond and other members of the Institute for
> Neurodegenerative Diseases are scientific advisors to, or own stock
> in, InPro.
>
> Other co-authors of the study were Michael D. Geschwind, Camille
> Deering, Svetlana Didorenko, Mamta Sattavat, Henry Sanchesz, Ana
> Serban, Kurt Giles, of UCSF, and Martin Vey, of Behring, Marburg,
> Germany, and Henry Baron, of Behring, Paris.
>
> ###
>
> The study was funded by the National Institutes of Health, the John
> Douglas French Foundation for Alzheimer's research, the McBean
> Foundation, the State of California, Alzheimer's Disease Research
> Center of California and the RR00079 General Clinical Research Center.
>
> The UCSF Institute for Neurodegenerative Diseases:
> http://ind.medschool.ucsf.edu/.
>
> FURTHER COMPARISON OF THE CDI TO THE STANDARD DIAGNOSTIC PROCEDURES,
> PROVIDED BY STEPHEN J. DEARMOND, MD, PHD, UCSF PROFESSORS OF
> NEUROPATHOLOGY:
>
> Explanation as to why the CDI is more sensitive than Western blot
> analysis: Studies at UCSF during development of the CDI showed that
> CDI could detect prions in brain homogenates at levels that fail to
> produce disease in animals (bioassay for prions). Therefore, the CDI
> is more sensitive than the bioassay method, which was considered to be
> the most sensitive technique for detecting prions. In contrast,
> Western blot analysis for prions is significantly less sensitive than
> the bioassay and is, therefore, significantly less sensitive than the
> CDI. Currently, the USDA uses a combination of Western blot analysis
> of brainstem homogenates and immunohistochemistry of the medulla to
> test cattle suspected of having bovine spongiform encephalopathy ("mad
> cow disease"). The relative insensitivity of IHC and Western blot
> analysis, says DeArmond, supports the UCSF scientists' ongoing
> assertion that the CDI should also be used to evaluate the brain
> tissue of cattle.
>
> DeArmond cites additional evidence about Western blot analysis from a
> World Health Organization (WHO) study group, which compared the CDI
> method with Western blots for detection of prions in sporadic and
> variant CJD brains. Based on the smallest amount of prions that could
> detected by the two techniques, they found that the CDI was from 1000-
> to 100,000-fold more sensitive than Western blot analysis performed in
> six different research laboratories (Minor et al. Standards for the
> assay of Creutzfeldt-Jakob disease specimens. J. Gen. Virol. 85:
> 1777-1784, 2004).
>
> Explanation as to why IHC for prions is less sensitive than the CDI:
> IHC is routinely performed on formalin-fixed, paraffin-embedded
> samples of brain. Formalin fixation markedly decreases the ability of
> antibodies to bind to proteins in general, which greatly weakens the
> IHC signal for prions (PrPSc). In contrast, homogenates for the CDI
> are not treated with reagents that decrease prion antigenicity.
> Moreover, to concentrate the PrPSc for measurement by the CDI, the
> homogenates are exposed to phosphotungstic acid, which selectively
> precipitates both protease-sensitive and protease-resistant PrPSc that
> comprise prions, but not the normal prion protein conformer found in
> uninfected animals, PrPC. This step results in a higher concentration
> of PrPSc for detection by the CDI. Because the PrPSc was not exposed
> to proteases, the CDI measures all forms of abnormally folded PrPSc
> molecules. Protease-sensitive PrPSc can account for 50 percent of the
> total PrPSc. For Western analysis, homogenates of brain are treated
> with protease to eliminate PrPC; however, this step also eliminates
> protease-sensitive PrPSc leaving only protease-resistant PrPSc for
> Western blot detection and decreasing the PrPSc signal at least in half.
>
>
> http://www.eurekalert.org/pub_releases/2005-02/uoc--dop021105.php
>
> TSS
>
> ######### https://listserv.kaliv.uni-karlsruhe.de/warc/bse-l.html
> ##########
>
>

######### https://listserv.kaliv.uni-karlsruhe.de/warc/bse-l.html ##########


-------- Original Message --------
Subject: Re: Diagnosis of prions in patients should utilize novel strategy, team says IHC GOLD STANDARD MY @SS RE-TEXAS MAD COWS???
Date: Tue, 15 Feb 2005 08:53:26 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE
References: <42116880.9090403@wt.net>


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

Greetings,

IHC gold standard my @ss. IHC is nothing more than a tool used by
the USDA to cover-up any inconclusives, such as mad cows in TEXAS
they insisted on covering-up. With these findings (however we have known
of this test by prusiner et al that is 1,000 times more sensitive, for some
time) we just had to wait for patent while all these supposedly inconclusive
go undetected in cattle. BUT what about these inconclusive by
Gambetti et al in humans. I think these findings warrant Gambetti
et al to go back and look over some of his 'NEGATIVES' in humans
with this test. i think he might be very surprised as to what he may
find, where he did not find anything the first time around.

NOW, what about them MAD COWS IN TEXAS ?
WE CANNOT let them cover these mad cows up.

WHAT ever became of this meeting ?

WAS suppose to happen just a few days ago.
I was sworn to secrecy, but that time has come and gone.
I want my cow! THE whole damn world knows they covered up
these TEXAS mad cows. USA BSE GBR should be raised
immediately to BSE GBR IV, until the brains of these inconclusives
are coughed up and retested. OR, are they toast by now???
IF they cannot produce these brains of these inconclusive cows,
then our questions have been answered...


January 14, 2005

Dr. John R. Clifford, Deputy Administrator, Chief Veterinary Officer
Animal Plant Health Inspection Service
1400 Independence Ave. SW, Room 317-E
Jamie L. Whitten Federal Building
Washington, DC 20250

Dear Deputy Administrator Clifford,

We have a number of questions about your November 23, 2004 announcement
that a cow, which had tested “not negative” in two runs of the Bio-Rad
ELISA quick test for mad cow disease, was “indeed negative for BSE.” We
are concerned because New Scientist reported last June that the false
positive rate after such repeated testing is “around one in 100,000 for
Bio-Rad.” We would appreciate a chance to meet with you this month to
discuss the questions below.

1. You have indicated that the Biorad screening test was run twice and
got a positive result both times. Were the two runs conducted by the
same or different technicians? Did they use the same or different brain
samples?

2. When the immunohistochemistry (IHC) test was conducted, what was the
condition of the brain when it arrived at the National Veterinary
Services Laboratory in Ames, Iowa? Did scientists note any deterioration?

3. How many slides were made and examined for the IHC test? Were they
from only the obex, or other areas of the brain? Atypical strains of BSE
have been found in Italy and Japan where the level of PrPres in the obex
was low or non-detectable, unlike in traditional BSE. What portion(s) of
the brain were examined?

4. Experienced technicians can sometimes disagree on the interpretation
of IHC slides. Does one technician review the slides or more than one
technician? If the latter, how many? Did they all agree on the conclusion?

5. The World Organization of Animal Health, known as O.I.E., recognizes
use of immunoblotting (also know as Western Blot) as a further
confirmation of the IHC test, and it is used in Japan and most European
countries. USDA used the Western Blot test in December 2003 along with
IHC to confirm the first case of mad cow disease in the United States .
In Japan and Belgium cows that tested positive on two quick tests (in
both cases using a Bio-Rad test), negative on IHC, yet positive on
Western Blot and are considered to be confirmed BSE cases. Was Western
blot or any other technique besides IHC used to confirm or rule out a
positive result on this November 2004 cow? If so, what was the result?
If not used, why not?

6. Were all the procedures referred to in your March 15, 2004
announcement of protocols to confirm any suspect positives utilized,
particularly the reference to use of “full battery of tests” that
includes, but is not limited to IHC? If not, what protocol was used and
what is your rationale for the differences?

7. Does USDA still have brain material from the cow in question? If so,
could it be sent to the World BSE reference laboratory in Weybridge,
United Kingdom for IHC and Western Blot analysis to confirm the USDA
finding? If not, why not?

8. Canadian press has reported that “Canadian authorities have been told
that the cow, from Texas, didn’t have the metal ID tags that cows born
here are given.” Is this correct? What was the age of the cow and where
had it lived?

We would like to request a meeting with you between now and the end of
January about these questions, which are very important to consumer
confidence in the safety of the food supply.


Sincerely,


Jean Halloran, Director Michael Hansen, Ph.D.
Senior Research Associate

From http://www.ars.usda.gov/is/AR/archive/dec04/tse1204.htm
Yamakawa, Y. et al. 2003. op cit.
De Bosschere, H., Roels, S. and E. Vanopdenbosch. 2004. Atypical case of
bovine spongiform encephalopathy in an East-Flemish Cow in Belgium. The
International Journal of Applied Research, 2(4). Accessed at
http://www.jarvm.com/articles/Vol2Iss1/DEBOSSCHERE.htm
In a technical briefing on the new BSE sureveillance plan, Dr. Ron
DeHaven clearly stated that USDA would use multiple tests that included
IHC: “The Department at NVSL will continue to use the
immunohistochemistry, or IHC, for quality control testing, and in
addition if any of the rapid screen test comes back with a suspect
positive then NVSL will use the IHC as well as other tests necessary to
confirm the results. . . Let me say up front that we expect that there
will be positive results on these screening tests, and that's just the
nature of the beast. That's because screening tests by design are
intended to be very sensitive and not to miss any positive animals. But
with that high degree of sensitivity also comes the possibility for
false positive test results. And again that's to be expected. any
suspect test results will be sent to NVSL for confirmatory testing with
the full battery of tests. That would include the IHC. From:
http://www.usda.gov/Newsroom/0106.04.html
From
http://edmonton.cbc.ca/regional/servlet/View?filename=ed-mad-cow20041122

Greetings again,

I was told that they were to meet on Feb. 9, 2005 about these inconclusives.
I have heard nothing of the outcome. Maybe we will hear soon.
BUT of course we will never know the results of that first TEXAS mad
cow they covered up, the stumbling and staggering one they refused to
test, and decided to render, head and all...

still disgusted in Bacliff, Texas USA

Terry S. Singeltary Sr.





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