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From: TSS ()
Subject: Diagnosis of prions in patients should utilize novel strategy, team says
Date: February 14, 2005 at 7:12 pm PST

-------- Original Message --------
Subject: Diagnosis of prions in patients should utilize novel strategy, team says
Date: Mon, 14 Feb 2005 21:12:00 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy

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

Corinna Kaarlela, News Director
Source: Jennifer O'Brien

14 February 2005

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

"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

"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,

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:


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.



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