|
Items in cart [0] |
Questions? Call 888-624-8373 |
| Copyright © 2009. National Academy of Sciences. All rights reserved. Terms of Use and Privacy Statement |
Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 72
~ Diagnostics for Transmissible
~ soone`torm Encephalopathies
Diagnostic tests lie at the forward edge of all efforts to control hu-
man and animal diseases, for it is the diagnostic test that clarifies
the specific clinical problem. A reliable and speedy diagnosis leads
to appropriate clinical management and intervention. Yet very few diagnos-
tic tests are available to detect the infectious agents associated with trans-
missible spongiform encephalopathies (TSEs).
A number of tests have been developed for the rapid detection of PrP
specific to bovine spongiform encephalopathy (BSE) in the central nervous
system (CNS) tissue of cattle. At present, the European Community has
approved five such tests to screen slaughtered cattle for BSE. Similarly, two
rapid postmortem tests are available in the United States to diagnose chronic
wasting disease (CWD) in deer and elk. However, there is no test approved
by the U.S. Department of Agriculture (USDA) or the Food and Drug Ad-
ministration (FDA) to detect TSEs in live animals or people.
This chapter first addresses the unique challenges involved in develop-
ing antemortem clinical diagnostics for TSEs. This is followed by a discus-
sion of the diagnostic tools used at present and some of the newer tech-
niques being explored. The final section presents the committee's
recommendations for research that is likely to yield sensitive, reliable, and
cost-effective antemortem TSE diagnostics in the future.
In the case of animals, the ability to diagnose or detect an infection
drives food safety interventions, which can prevent the introduction of con-
taminated food into the food chain and offset economic damage to the food
production industry. In the case of people, the detection of infection can
avert the introduction of potentially infectious blood into the blood supply
72
OCR for page 73
DIA GNOSTICS FOR TSEs
73
system and be used to direct appropriate treatment. In addition, the use of
diagnostic tests for mass screening has epidemiological utility since it can
reveal the extent and distribution of a prior-related disease within a herd or
population, and can be used to monitor the effects of animal and public
health intervention strategies.
Most infectious diseases, such as malaria, tuberculosis, hepatitis, and
AIDS, can be diagnosed by established methods. This is not the case with
TSE. A prion cannot be identified by direct visualization under a micro-
scope, cultivation in a laboratory, detection of specific antibodies or anti-
gens by standard immunology methods, or detection of its nucleic acid by
molecular methods such as polymerase chain reaction (PCR). It consists
of host protein with an altered conformation such that the body does
not recognize it as foreign and does not produce antibodies against it. It
also lacks identifiable DNA or RNA, so it cannot be identified by PCR or
other nucleic acid-based tests. These factors make detecting the agent very
difficult.
TSE agents have other peculiarities that confound detection. They are
largely insoluble, distributed unevenly in body tissues, and found in a lim-
ited set of tissues by currently available tests. PrPSc (the pro/ease-resistant
protein associated with prion disease; see Chapter 2) is neurotropic, so it
ultimately affects cells of the nervous system tissues. Where and how PrPSc
progresses through the body before its final assault on the nervous system is
largely unclear, however, complicating the ability to locate and detect it.
The similarities between host prpC (the pro/ease-sensitive cellular pro-
tein) and PrPSc pose a fundamental problem. Since it is normal to find prpC
in healthy individuals, detection tests must differentiate between the two
proteins. The strategy thus far has been to mix the test material with the
proteinase K (PK) enzyme, which digests normal prion protein but only a
portion of the abnormal protein. Then various techniques, described below,
detect the residual PrPSc after digestion. Since this process incidentally re-
duces the small amount of original PrPSc captured, it is inherently less sensi-
tive than methods that do not rely on PK digestion.)
Thus two major factors challenge the designer of any useful antemor-
tem diagnostic test for a TSE agent. First, only small amounts of prions may
be available for detection in accessible living tissues, such as blood, urine,
and cerebrospinal fluid (CSF), necessitating an exquisitely sensitive test. Sec-
ond, since hosts incubating a TSE lack overt evidence of illness during most
of the period between infection and death, both infected and uninfected
animals and people would need to undergo antemortem diagnostic testing.
iThe degree to which PrPSc resists PK digestion depends on its strain. The limit of resistance
to PK digestion may relate to the conformation of each strain (Safer et al., 1998). Some loss of
PrPSc is also due to the test process itself.
OCR for page 74
74
ADVANCING PRION SCIENCE
Therefore, this test must be of sufficient specificity to differentiate accu-
rately between normal and misfolded PrP. For some purposes, the test would
also need to discriminate among one or more prion strains a challenge
heightened by basic deficiencies in our understanding of prion strain diver-
. , . . .
silty ant t" be nature ot strain variation.
The ultimate objective of a TSE diagnostic test is to detect a single
infectious unit (IU) while avoiding a false-positive result. In this report, we
define an IU as the smallest amount of infectious agent leading to an infec-
tion in a single person or animal exposed. Achieving the ideal TSE diagnos-
tic test will be complex because a single IU may not be equivalent to a single
infectious particle. In other words, a single IU of a TSE agent likely consists
of many aggregates of PrPSc, not just one aggregate. For further explana-
tion, please refer to the detailed definition of the term infectious unit in the
glossary at the beginning of this volume.
The quest for antemortem diagnostics will play a fundamental role in
controlling the spread of TSEs, yet current tests are largely unvalidated and
not readily available. Although the ultimate objective is far from being
achieved, scientists continue to invest much worthwhile effort into improv-
ing TSE detection methods.
CLINICAL DIAGNOSTICS
In general, diagnoses of prion diseases by clinical description or ancil-
lary clinical tests are not specific enough to confirm a specific prion disease.
In important circumstances, however, they give the clinician some clues
that may help support or question the diagnosis of a prion disease.
Differentiation of prion disease from other neurodegenerative diseases
and differentiation among different prion strains on clinical grounds are
problematic because affected individuals exhibit similar symptoms. Clinical
diagnostic criteria have nevertheless been established for sporadic
Creutzfel~t-Takob disease (sCTD) and variant Creutzfel~t-Takob disease
(vCJD) (Will et al., 20001. Some general clinical differences distinguish sCJD
from vCJD (see Table 4-11. For example, vCJD, unlike Creutzfel~t-Jakob
disease (CJD), occurs in patients generally younger than 40 years old; often
presents with early psychiatric and sensory neurological symptoms; and has
a longer duration of illness prior to death, usually more than a year (WHO,
2001a). Spencer and colleagues (2002) reviewed the early psychiatric mani-
festations of vCTD. They describe the clinical characteristics of the first 100
vCJD patients identified and conclude that "the combination of a psychiat-
ric disorder with affective or psychotic features and persistent pain, dysar-
thria, gait ataxia, or sensory symptoms should at least raise the suspicion of
vCTD, particularly if this is combined with any suggestion of cognitive im-
pairment" (Spencer et al., 2002:14821. Despite these differences between
vCTD and COD, they are not sufficient to establish a definitive diagnosis.
OCR for page 75
DIA GNOSTICS FOR TSEs
TABLE 4-1 Clinical Differentiation of sCTD and vCTD
75
Clinical Feature or
Supporting Clinical
Procedures
Classical sCJD
(M/M or M/V 1) vCJD
Average age at clinical onset
Length of survival from date
of clinical onset
Early psychiatric symptoms
El ectro enc ep hat ography
(EEG)
Magnetic resonance imaging
(MRI)
Cerebrospinal fluid
(CSF)
Histopathology of brain tissue
PrP immunohistochemical
. . .. . .
stalmng pattern or oram tissue
63 yr
4 mo
Unusual
Bi- or triphasic periodic
complexes
Increased signal in basal
ganglia, caudate nucleus,
and putamen
14-3-3 protein levels usually
elevated
No amyloid plaques
Punctate pattern
Immunohistochemical staining Negative
of tonsil or appendix tissue
PrPSc isotype by Western blot Type 1A
29 yr
14 mo
Common
Nonspecific, slow
Hyperintense signal in
pulvinar region of the
thalamus
14-3-3 protein levels not
usually elevated
100% florid plaques
Widespread plaque
. .
stalmng pattern
PrP present in tissue.
especially toward late stage
of disease
Type 2B
NOTE: M/M = methionine homozygous at codon 129; MIV 1 = heterozygous at codon 129,
subtype 1 (see Table 4-2).
Ancillary clinical testing typically supplements the medical work-up for
vCTD or CTD. The most helpful noninvasive tests have been electro-
encephalography (EEG); neuroimaging; examination of CSF; and, more re-
cently, tonsillar biopsy and prion strain identification by immunoblotting.
Evaluation of tissue obtained by brain biopsy establishes or excludes the
diagnosis of TSE in almost all cases, but brain biopsy is highly invasive and
is limited to cases in which a treatable condition must be excluded.
Electroencephalography
In typical cases of sCTD (see Table 4-2), the EEGs of more than 80
percent of patients show distinctive changes (Parch) et al., 1999~. The trac-
OCR for page 76
76
U.
. -
.=
U.
.~
.-=
~ no
be
u, be
O ~
Cal
Cal
Cal
Cal
._
o
._
Cal
so
o
o
o
._
Cal
._
Cal
Cal
Cal
¢
by
~ CO
~ o
.° ~
~ o
is
U)
~ ~ ~ ~ ~ ' E ~
to
U.
o o
be O
as, 1,, ~ \e, . ~ ~
o
o .-
=o
~ ~ ~ ~ ~ ~ ~ ·=
o ~
of ~ ~ ~ ~ ~ ~ ~
o ~
.~ .a ~1 .~^
{a X X ~ ~ C)
o 4~ Ct _ _ ~ ~ ~ ~ O
O O O ~ ~ ~ ~ ~ ~ it Ha
~ , () O
0
C~ C~ (S ~ ~
OCR for page 77
DIA GNOSTICS FOR TSEs
77
ing shows biphasic and triphasic periodic complexes in the clinical course
(Parch) et al., 1999), which are evident more than 90 percent of the time
with repeated tracings (Chiofalo et al., 19801. These periodic complexes are
observed less frequently in patients with the other subtypes of sC]D and in
familial C]D (Gambetti et al., 1999; Parchi et al., 1999) and have never
been found in patients with vC]D, although nonspecific siow-wave abnor-
malities can be seen.
Neuroimaging
Neuroimaging by computed tomography (CT) and magnetic resonance
imaging (MRI) can be useful, especially to rule out non-prior-related neu-
rological diseases. The CT result is usually normal, although the scan may
show atrophy in patients with a protracted clinical course (WHO, 19981.
This finding may be absent and is nonspecific. The MRI scan may also
show atrophic changes in patients with late-course disease. When patients
are evaluated by T2 MRI, proton- density-weighted MRI, or fluid- attenu-
ated-inversion-recovery MRI, there is an increased signal in the basal gan-
glia about 80 percent of the time (WHO, 1998), as well as gray matter
hyperintensities noted in diffusion-weighted imaging sequences in patients
with C]D (Mendez et al., 20031. MRI also can be used to help differentiate
vC]D from sC]D because the posterior puivinar region of the thalamus
shows a hyperintense signal in patients with vC]D. This puivinar sign is
present in 90 percent of patients with vC]D and is more than 95 percent
specific in selected cases, making it the best available in viva test for the
diagnosis of vC]D (~0, 2001b).
CSF Protein
A neuronal protein called 14-3-3 can be detected in CSF, particularly in
individuals who have diseases, such as sC]D, involving rapid neuronal de-
struction (Zerr et al., 20001. This protein may help differentiate TSE from
Alzheimer's disease and other dementias, where it is usually not detectable.
In necrotizing diseases such as stroke, viral encephalitis, and transverse
myelitis, the test for this protein may also be positive (Johnson and Gibbs,
1998; WHO, 19981. In more slowly progressing forms of C]D such as fa-
milial cases of human TSEs and vC]D, this CSF protein test is often nega-
tive. A WHO study (2001a) found the 14-3-3 test to be only 53 percent
sensitive when used to diagnose vC]D.
Tonsi! Biopsy
More recently, tonsil biopsy has been used for the presumptive identi-
fication of vC]D. Immunohistochemical testing for the prion protein in
OCR for page 78
78
ADVANCING PRION SCIENCE
these tissues has demonstrated that the protein is present in patients with
vC]D but not in those with sC]D (Hill et al., 1999; WHO, 2001a). The
postulated reasons for this difference include a strain effect, a species-bar-
rier effect, or the oral route of exposure in vC]D (Hill et al., 19991. There
have been too few case series to determine the sensitivity or specificity of
this ancillary test.
Although controversial, tests of tonsil and appendix lymphoid tissues
are being used to screen large, asymptomatic populations for TSEs. The
largest study to date was conducted in the United Kingdom (Hilton et al.,
20021. Between 1995 and 1999, Hilton and colleagues tested 8,318 tonsil
and appendix tissue samples from 10- to 50-year-old individuals and found
one appendix tissue sample that tested positive for PrPSc. From that result,
they estimated the prevalence of vC]D to be 120 per million in the United
Kingdom, with a 95 percent confidence limit of 0.5 to 900 cases (Hilton et
al., 20021.
It remains unknown how long before the onset of symptoms PrPSc ac-
cumulates in human tonsils or the human appendix and whether all positive
individuals will inevitably progress to the fatal CNS disease. Nevertheless,
studies of sheep naturally infected with the scrapie agent and of mice ex-
perimentally infected with that same agent have demonstrated that PrPSc is
detectable in lymphoid tissues long before clinical signs of neurological dis-
ease appear.
Brain Biopsy
In atypical cases of C]D, brain biopsy with histological examination for
spongiform changes, immunocytochemical staining, and Western blotting
for PrPSc, as well as analysis of the PrP gene, is diagnostic in virtually all
cases. However, this approach is seldom needed to diagnose patients with a
typical clinical course and consistent findings of classical sC]D by LEG,
MRI, and CSF analysis. Histological examination of brain tissue should be
performed for all patients with possible and probable cases of TSE, as well
as for all individuals with questionable neurodegenerative diseases at au-
topsy, to ensure that a new phenotype of prion disease is not missed.
In both sC]D and vC]D, histology typically reveals the spongiform ap-
pearance of the CNS tissues. However, amyloid plaque formations with the
characteristic morphology known as florid plaques are seen in all patients
with vCTD, whereas kuru plaques (without the characteristics of the florid
plaques) are observed only in patients with the sCJD subtype methionine/
valine 2 (M/V 2), which accounts for about 10 percent of all cases of sCJD
(Tohnson and Gibbs, 1998; Parchi et al., 19991.
OCR for page 79
DIA GNOSTICS FOR TSEs
79
Isotype by Western Blotting
Additional diagnostic precision has been made possible by the intro-
duction of PrPSc isotypes on the basis of the mobility of the PrPSc fragment,
which is PK-resistant, after gel electrophoresis and Western blotting
(Collinge et al., 1996; Monari et al., 1994; Parchi et al., 19971. According
to a widely used typing method, there are two major types (or strains) of
PrPSC in all forms of COD and fatal insomnia, including sCTD, iatrogenic
COD, vCTD, fatal familial insomnia (FFI), and sporadic fatal insomnia (sFI)
(Parch) et al., 19971.
PrPSc type 1 migrates to 21 kilodaltons (kDa) on gels after treatment
with PK and deglycosylation. PrPSc type 2 migrates to 19 kDa under the
same conditions. The different gel mobilities of the two PrPSc types are due
to the different sites of PrPSc cleavage by PK, resulting in PK-resistant frag-
ments of differing sizes. These two types codistribute with distinct disease
phenotypes and are conserved upon transmission to receptive animals.
Therefore, they fulfill the definition of prion strains, and this strongly indi-
cates that they have distinct conformations. Additional subtypes of PrPSc
have been distinguished on the basis of both the ratios of the three PrPSc
glycoforms (Parch) et al., 1997) and the profile generated by two-dimen-
sional gel electrophoresis (Pan et al., 20011.
In addition to the PrPSc type, the phenotype of human prion diseases is
influenced by the genotype at codon 129 of the PRNP, the site of a com-
mon M/V polymorphism. A classification of sporadic prion diseases has
been generated on the basis of the combination of the genotype at codon
129 and the PrPSc type (Parch) et al., 1996, 19991. This classification in-
cludes five subtypes of sCTD and sFI (see Table 4-21. Each of these subtypes
has distinct clinical and pathological features.
Despite these advances, clinical methods remain supportive rather than
diagnostic. As in virtually all other disease conditions, the diagnosis is most
reliable when obtained by combining information from the clinical exami-
nation, ancillary clinical tests, and laboratory tests. Yet even when all this
information is combined, present diagnostic tools lack sufficient sensitivity
and specificity. The development of tests to improve the early diagnosis of
human prion disease and to detect presymptomatic infections in humans
and animals more reliably is a major priority.
CURRENT LABORATORY DIAGNOSTICS
Histopathology and Immunohistochemistry
The first method used to confirm the diagnosis of a TSE is postmortem
neuropathogical examination of brain tissue from an animal or a human,
OCR for page 80
80
ADVANCING PRION SCIENCE
and this method remains the gold standard. WHO's position is that "a
definitive diagnosis of C}D including nvC}D New variant C}D] is estab-
lished only by neuropathological examination" (WHO, 1998:13~. Tissue is
collected; preserved in formalin; sectioned; stained; and then examined with
a light microscope, which is used to look for the characteristic pathological
abnormalities on histological examination. This procedure is generally aug-
mented with immunohistochemical staining of the tissue, which uses a PrP
antibody-tagged stain that affixes onto PrP. The stain will be abnormally
dark or dense in areas where an abnormal amount of PrP is present (see
Plate 4-1~. Electron microscopy can also be used to observe fibrils, called
scrapie-associated fibrils, in fresh postmortem tissue (Merz et al., 1983) as
well as in autolytic tissue (see Figure 4-1~.
Immunochemical Detection Methods
Five standardized commercial screening tests (two by Prionics AG and
one each by Enfer Scientific Ltd., Bio-Rad Laboratories Inc., and InPro
Biotechnology Inc.) have been approved by the European Commission (EC)
for use in the direct and rapid detection of PrPSc (Moynagh and Schimmel,
1999; EC, 2003~. The test by San Francisco-based InPro was developed in
the United States, while the other four tests were developed in Europe. Those
tests now on the market are used primarily in Europe.
In the United States, no rapid postmortem or antemortem diagnostic
test for human TSEs had been submitted to FDA for approval as of August
2003 (personal communication, D. Asher, FDA, 2003~. Furthermore, there
are no USDA-approved rapid postmortem or antemortem tests to diagnose
TSEs in sheep or cattle. USDA has approved three rapid postmortem diag-
nostic tests for CWD: the Bio-Rad enzyme-linked immunosorbent assay
(ELISA) by Bio-Rad of Hercules, California; the Dot Blot ELISA by Veteri-
nary Medical Research and Development (VMRD) Inc. of Puliman, Wash-
ington; and the IDEXX Her3Chek~ CWD Antigen Test Kit from IDEXX
Laboratories Inc. of Westbrook, Maine (IDEXX, 2003; personal communi-
cation, Rick Hill, USDA APHIS Center for Veterinary Biologics, November
25, 2003; USDA APHIS, 2002; VMRD Inc., 2003a). All three tests are
designed to detect the infectious agent of CWD in peripheral lymphoid tis-
sues of select cervids.2
2The Bio-Rad test is approved for use on tissue samples from mule deer, white-tailed deer,
and elk (personal communication, Rick Hill, USDA APHIS Center for Veterinary Biologics,
November 25, 2003; USDA APHIS, 2002). The VMRD test is approved for use on tissue
samples from white-tailed deer and mule deer (VMRD Inc., 2003a). The IDEXX test is ap-
proved for use on white-tailed deer tissue. All tests are approved for use exclusively in APHIS-
OCR for page 81
DIA GNOSTICS FOR TSEs
81
FIGURE 4-1 Electron micrograph of negatively stained fibrils composed of
PrP 27-30 from scrapie-infected Syrian hamster brains.
SOURCE: H. Wille, Institute for Neurodegenerative Diseases, University of California
San Francisco, September 9, 2003.
approved veterinary laboratories and for surveillance purposes only. The sensitivities and speci-
ficities are as follows:
Sensitivity ( % )
Specificity ( % )
No. Specimens
Bio-Rad 99.6 99.9 2,892
VMRD 91.5 100.0 298
IDEXX N/A N/A N/A
NOTE: N/A = not available.
SOURCE: Bio-Rad Laboratories Inc. (2003); VMRD Inc. (2003b).
OCR for page 82
82
ADVANCING PRION SCIENCE
TABLE 4-3 Estimated Detection Limits of the First Three EC-Approved
Postmortem Tests for BSE
Dilution of Homogenatea
Number of BSE-Infected Brain-Homogenate Samples Scoring
Positive
Prionics
Check Western
o
lo-l
.s
lo-2.o
lows
0-3.°
0-3.5
Test by
Enfer Scientific
6/6
15/20 (+2?)b
0/20
6/6
20/20
20/20
0/20
Test by Bio-Rad
6/6
20/20
20/20
20/20
18/20
1/20
0/20
NOTE: The data represent the number of samples testing positive/total number of
samples tested.
a ". . . positive brain homogenate of known infectivity titer was tested at dilutions in
negative brain" (Moynagh and Schimmel, 1999:105).
b Two samples rated inconclusive at this dilution.
SOURCE: Adapted from Moynagh and Schimmel (1999).
Tests Produced by Prionics, Enfer, and Bio-Rad
The rapid test most widely used to screen for BSE in Europe is the
Western blot test called Prionics Check Western, produced by Schlieren,
Switzeriand-based Prionics. Test kits are available for the diagnosis of
scrapie in sheep and BSE in cattle. The test uses gel electrophoresis with a
specific antibody against PrP after the prpC in homogenized brain tissue has
been digested by PK.
The Enfer and Bio-Rad tests use slightly different mechanisms for de-
tection. The test by Cashel, Ireland-based Enfer is an ELISA. After digestion
by PK, residual PrPSc binds to a capture antibody at the bottom of the
ELISA plate wells. After the wells have been washed, a detection antibody
complexed with an activating enzyme is added. This second antibody binds
to PrPSc and colors the substrate if PrPSc is present. The Bio-Rad test, cre-
ated in France, is also an ELISA, but in the initial step after digestion, it uses
two different antibodies to bind to different epitopes of PrPSc. This is the
most sensitive of these first three approved tests (see Table 4-3~.
Prionics-Check Luminescence Immunoassay (LIAJ and InPro Automated
Conformationally Dependent Immunoassay (aCDIJ
In April 2003, the EC approved two more rapid tests for the postmor-
tem detection of BSE Prionics-Check LIA and InPro aCDI (EC, 2003~.
OCR for page 97
DIA GNOSTICS FOR TSEs
97
oafs (such as those based on surface plasmon resonance), methods that de-
tect resonances in the microwave range, methods that detect changes in the
frequency of surface acoustic waves, methods that detect changes in the
frequency of piezoelectric cantilevers, microcalorimetric methods, methods
based on the field effect in transistors and capacitors, and methods that use
evanescent wave-dependent changes in Raman scattering on metallic
nanoparticles. With the exception of the latter, however, none of these meth-
ods is as sensitive as radioimmunoassays and ELISAs; nonetheless they of-
fer advantages, as they can be used with underivatized recognition and tar-
get molecules and can be coupled directly to optical or electrical readouts.
Evanescent wave devices, which are widely used, solve the issue of coupling
wet and dry elements by making the part of the apparatus that comes into
contact with the biological sample disposable. Modern Fourier transform
ion cyclotron resonance methods are capable of detecting about 1,000 mol-
ecules with a given mass:charge ratio and with unambiguous identification.
Wet Methods
More recently, wet methods have been developed that may have even
greater sensitivity for the detection of priors. One of these is the protein-
fragment complementation assay (Remy and Michnick, 19991. Another
couples recognition proteins with PCR-amplifiable DNA tails (protein
PCR). The resulting chimeric molecules can be used with existing real-time
PCR techniques and may allow extension of PCR to protein detection at a
level of 1 to 10 arbitrarily designated epitopes (I. Burbulis, R. Carison, and
R. Brent, unpublished results, 20021.
Use of any of these methods for the reliable detection of prions in clini-
cal and environmental samples requires that the prions be purified and con-
centrated. This requirement can be addressed by a variety of approaches.
Conclusions Regarding Reagents and Detection Methods
In broad terms, the present limitations to prion detection lie not in the
lack of methods but in the paucity of antibody and other recognition mol-
ecules specific for prion species, strain, and allelic variants and for the infec-
tious conformation. Efforts to select antibodies specific for the conforma-
tion of prions have been hampered by the lack of immunogenicity of the
revealed epitopes, the tolerance of the mammalian immune system to these
epitopes, the lack of an industrial-scale effort, and perhaps other factors as
well.
Whatever the reason for the failure of past efforts, the reasonable re-
sponse to the problem is to select more modern kinds of recognition mol-
ecules in vitro, bypassing the vertebrate immune system completely. Some
OCR for page 98
98
ADVANCING PRION SCIENCE
of these problems are common to many areas of application in the biologi-
cal sciences and have received high-level scientific and national attention
(Desai et al., 20021.
A daunting number of people and organizations own the rights to the
intellectual property needed to generate modern molecular reagents with
affinities for prion proteins and to use those molecules in detection schemes.
This situation may make the commercial application of such reagents diffi-
cult until patent-sharing schemes can be devised. Nevertheless, neither tech-
nical nor legal barriers block government or philanthropic groups from
funding the production of these reagents for use in detecting prion particles.
In summary, a wealth of natural and engineered molecules, along with
a variety of detection methods, have been developed for recognition of bio-
logical targets. Prion investigators must now apply these molecules and
methods to the development of selective, sensitive tools that can target PrPSc.
Once bound by specific reagents, prions become detectable and susceptible
to attack. That attack might employ catalytically active binding reagents,
such as ribozymes, that offer the potential for target inactivation.
Recommendation 4.3: Fund research to develop novel methods and
reagents that detect or bind to priors, including new antibodies,
peptides, nucleic acids, synthetic derivatives, and chimeric mol-
ecules. This research could lead not only to better diagnostics, but
also to better therapeutic and prophylactic strategies. [Priority 11
Surrogate Markers and Signatures of Prion Disease
Diagnostic approaches based on detection of indirect disease markers
have a long and inconsistent history. In general, these approaches have been
hindered by their lack of specificity (e.g., tests for the erythrocyte sedimen-
tation rate and C-reactive protein) or by their dependence on the generation
of a specific antibody that is delayed in all disease processes and absent,
altogether in some, including TSEs. Today, powerful methods for detection
of robust surrogate markers of disease create new opportunities for diagno-
sis and force reconsideration of these approaches. These methods are based
on genomic or proteomic techniques, focus on complex biological patterns,
and depend on pattern recognition algorithms.
All forms of mammalian pathophysiology and pathology are accompa-
nied by stereotyped and highly choreographed intra- and extracellular
changes in the diversity, abundance, and spatial distribution of
biomolecules. Mammalian biological systems are particularly sophisticated
and sensitive in their recognition of and response to perturbations. These
responses can be defined by complex changes in many classes of molecules,
OCR for page 99
DIA GNOSTICS FOR TSEs
99
including changes in DNA structure, RNA transcript abundance, protein
abundance and modification, and protein localization. Modern genomic
techniques have greatly facilitated comprehensive measurement of these
various changes in parallel. For example, changes in the abundance of RNA
transcripts for nearly all genes expressed in humans can be measured simul-
taneously and repeatedly over short periods of time by using DNA
microarrays. Similarly, changes in the abundance of oligosaccharides or
proteins among a massive number of species can be measured either by a
method that uses a solid-state format or by mass spectrometry.
Diagnostic and prognostic signatures can be identified by examining
complex patterns of biomolecules that occur in various disease conditions.
Pattern recognition methods fall into two categories: those that discover
possible signatures (class discovery methods) based on the association of
specific patterns with an outcome of interest, and those that test and vali-
date these signatures (class prediction methods). Both of these methods have
been used successfully to classify cancer subtypes, to predict survival, and
to evaluate response to therapy. Specific patterns of RNA transcript abun-
dance predict the outcomes for patients with various malignancies, such as
breast and lung cancer, lymphoma, and leukemia (Alizadeh et al., 20001.
Stereotyped, discriminant patterns of transcript abundance may also be
characteristic of the mammalian response to infection (Boldrick et al., 20021.
In a recent study using mass spectrometry, investigators were able to
identify a group of surrogate proteins in 50 of 50 patients with ovarian
cancer, including 18 patients with early-stage disease. This pattern was ab-
sent from 60 of 63 patients with a noncancer diagnosis (Petricoin et al.,
2002a). The same technique was used to diagnose prostate cancer in 36 of
38 patients whose diagnosis was blind to the investigators to identify cor-
rectly 177 of 228 patients without prostate cancer (Petricoin et al., 2002b).
In diagnosing prion infections, it appears reasonable to postulate that
there are patterns of altered transcript abundance or protein expression in,
for example, blood, lymph nodes, or cerebrospinal fluid that are character-
istic of infection. One approach is to conduct a blind search for such pro-
tein patterns by means of protein mass spectrometry (Petricoin et al., 2002b)
followed by isolation of recognition molecules directed against the proteins
that have been identified. The components that make up the diagnostic
pattern need not necessarily be directly involved in pathogenesis, nor must
they have a known function. This kind of approach, however, must rely on
rigorous evaluation with well-chosen control samples and on predictions
obtained from the results of tests with sets of test samples.
Recommendation 4.4: Fund research to identify surrogate markers
or signatures for the detection of prions or prion diseases. [Priority 31
OCR for page 100
100
ADVANCING PRION SCIENCE
Cell Culture Systems
Research gains leading to better diagnostics would be accelerated if
better cell culture systems were in place. These systems have significant
advantages over animal bioassay systems, the most important being that
they can greatly shorten the length of time required to complete the test.
At present, only a few lines of cultured cells can be infected with priors.
The efficiency of infection is low, the rate of PrPSc accumulation is slow,
and the yield of PrPSc is limited. In addition, the factors that determine
susceptibility to infection are poorly understood. Therefore, investigators
must find new cell cultures or mode! systems with bona fide CNS properties
that are susceptible to prions in vitro, as well as new ways to enhance the
efficiency of the initiation and propagation of infection (e.g., molecules that
enhance the conversion of prpC to PrPSc). This work would not only en-
hance the potential for the use of cultured cells to assay priors, it would
also shed light on the cellular mechanisms underlying prion replication and
nerve-cell death.
Recommendation 4.5: Fund research to improve techniques for
propagating prions in cultured cells and develop new in vitro cell
systems as a means to assay and study priors. [Priority 21
Clinical Neuroimaging
Recent improvements in clinical neuroimaging have shown increasing
utility in clinical diagnostics for TSEs. MRI is able to visualize the brain
lesions of patients with C]D and can even help in differentiating vC]D from
sC]D (as noted in Chapter 31. Newer scanning devices and tissue uptake
reagents will further increase the utility of this clinical tool.
MRI of vC]D patients has been helpful from a diagnostic point of view
because of the frequent and specific puivinar sign, an abnormality described
by Zeidler and colleagues (20001. Symmetric hyperintense signals have been
reported in the basal ganglia of patients with sC]D; however, this finding is
frequently absent and lacks specificity, making it less useful. For this rea-
son, investigators have examined new imaging methods that can enhance
the capabilities of present methods or provide very new technical ap-
proaches, such as multiphoton microscopy.
Multiphoton microscopy uses near-infrared light, which penetrates
more deeply than visible or ultraviolet light and permits imaging of micro-
scopic structures within the cortex of the living animal at an extraordinarily
high resolution with no apparent deleterious effects. To visualize p-amyloid
deposits in living transgenic mice with lesions like Alzheimer's disease, re-
searchers have used multiphoton microscopy with locally applied
fluorescently labeled antibody against p-amyloid or systemically adminis-
OCR for page 101
DIA GNOSTICS FOR TSEs
101
tered fluorescent derivatives of chemicals that bind to p-amyloid, such as
thioflavine A and Congo red (Bacskai et al., 2001; Christie et al., 2001;
Klunk et al., 20021. This in viva imaging approach has allowed character-
ization of the natural history of senile plaques and evaluation of antiplaque
therapy in mouse models of the disease. One could envision the application
of similar studies to transgenic mouse models of prion disease, especially
since thioflavine A and Congo red bind to PrPSc. The technique would en-
able characterization of the progression of PrPSc accumulation and localiza-
tion in animals or patients with disease by repeatedly imaging the same
diseased region of the brain over time.
Although multiphoton microscopy requires a portion of the skull to be
thinned or removed for the passage of light, modifications to this technique
may obviate this need. In addition, advances in detection sensitivity and
improved means of entry of p-amyloid-binding probes into the central ner-
vous system may allow the use of similar kinds of p-amyloid-imaging by
MRI and positron emission tomography for studies with humans (Bacskai
et al., 2002; Mathis et al., 2002; Shoghi-Jadid et al., 20021. These methods
may be valuable in the diagnosis of humans with prion disease, especially
individuals who are at risk for inherited or iatrogenic prion disease, and the
evaluation of TSE therapies.
Recommendation 4.6: Fund research to develop functional imag-
ing for the presence of PrPSc in brain tissue, leading to an early
diagnostic test similar to the imaging diagnostics being developed
for Alzbeimer's disease. [Priority 31
REFERENCES
Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H. Tran
T. Yu X, Powell JI, Yang L, Marti GE, Moore T. Hudson J Jr, Lu L, Lewis DB, Tibshirani
R. Sherlock G. Chan WC, Greiner TC, Weisenburger DD, Armitage JO, Warnke R.
Staudt LM, Levy R. Wilson W. Grever MR, Byrd JC, Botstein D, Brown PO, Staudt LM.
2000. Distinct types of diffuse large B-cell lymphoma identified by gene expression profil-
ing. Nature 403(6769):503-511.
Asante EA, Collinge J. 2001. Transgenic studies of the influence of the PrP structure on TSE
diseases. Advances in Protein CI7emistry 57:273-311.
Bacskai BJ, Kajdasz ST, Christie RH, Carter C, Games D, Seubert P. Schenk D, Hyman BT.
2001. Imaging of amyloid-beta deposits in brains of living mice permits direct observa-
tion of clearance of plaques with immunotherapy. Nature Medicine 7(3):369-372.
Bacskai BJ, Klunk WE, Mathis CA, Hyman BT. 2002. Imaging amyloid-beta deposits in vivo.
Journal of Cerebral Blood Flow and Metabolism 22(9):1035-1041.
Baron GS, Caughey B. 2003. Effect of glycosylphosphatidylinositol anchor-dependent and
-independent prion protein association with model raft membranes on conversion to the
pro/ease-resistant isoform. Journal of Biological CI7emistry 278(17):14883-14892.
OCR for page 102
102
ADVANCING PRION SCIENCE
Bessen RA, Kocisko DA, Raymond GJ, Nandan S. Lansbury PT, Caughey B. 1995. Non-
genetic propagation of strain-specific properties of scrapie prion protein. Nature
375(6533):698-700.
Bessen RA, Raymond GJ, Caughey B. 1997. In situ formation of pro/ease-resistant prion
protein in transmissible spongiform encephalopathy-infected brain slices. Journal of Bio-
logical Chemistry 272(24):15227-15231.
Bieschke J. Giese A, Schulz-Schaeffer W. Zerr I, Poser S. Eigen M, Kretzschmar H. 2000.
Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning
for intensely fluorescent targets. Proceedings of the National Academy of Sciences of the
United States of America 97(10) :5468-5473.
Biffiger K, Zwald D, Kaufmann L, Briner A, Nayki I, Purro M, Bottcher S. Struckmeyer T.
Schaller O. Meyer R. Fatzer R. Zurbriggen A, Stack M, Moser M, Oesch B. Kubler E.
2002. Validation of a luminescence immunoassay for the detection of PrP(Sc) in brain
homogenate. Journal of Virological Methods 101(1-2):79-84.
Bio-Rad Laboratories Inc. 2003. Chronic VDasting Disease. [Online]. Available: http://
www.bio-rad.com/ [accessed August 2003].
Birkett CR, Hennion RM, Bembridge DA, Clarke MC, Chree A, Bruce ME, Bostock CJ.2001.
Scrapie strains maintain biological phenotypes on propagation in a cell line in culture.
EMBO Journal 20(13):3351-3358.
Boldrick JC, Alizadeh AA, Diehn M, Dudoit S. Liu CL, Belcher CE, Botstein D, Staudt LM,
Brown PO, Relman DA. 2002. Stereotyped and specific gene expression programs in
human innate immune responses to bacteria. Proceedings of the National Academy of
Sciences of the United States of America 99(2):972-977.
Bossers A, de Vries R. Smits MA. 2000. Susceptibility of sheep for scrapie as assessed by in
vitro conversion of nine naturally occurring variants of PrP. Journal of Virology
74(3):1407-1414.
Callahan MA, Xiong L, Caughey B. 2001. Reversibility of scrapie-associated prion protein
aggregation. Journal of Biological Chemistry 276(30) :28022-28028.
Caughey B. Kocisko DA, Raymond GJ, Lansbury PT Jr. 1995. Aggregates of scrapie-associ-
ated prion protein induce the cell-free conversion of pro/ease-sensitive prion protein to
the pro/ease-resistant state. Chemistry and Biology 2(12):807-817.
Caughey B. Raymond GJ, Callahan MA, Wong C, Baron GS, Xiong LW. 2001. Interactions
and conversions of prion protein isoforms. Advances in Protein Chemistry 57:139-169.
Caughey WS, Raymond LD, Horiuchi M, Caughey B. 1998. Inhibition of pro/ease-resistant
prion protein formation by porphyrins and phthalocyanines. Proceedings of the National
Academy of Sciences of the United States of America 95(21):12117-12122.
Cervenakova L, Brown P. Soukharev S. Yakovleva O. Diringer H. Saenko EL, Drohan WN.
2003. Failure of immunocompetitive capillary electrophoresis assay to detect disease-
specific prion protein in buffy coat from humans and chimpanzees with Creutzieldt-
Jakob disease. Electrophoresis 24(5):853-859.
Chandler RL. 1961. Encephalopathy in mice produced by inoculation with scrapie brain ma-
terial. Lancet i:1378-1379.
Chiofalo N. Fuentes A, Galvez S. 1980. Serial EEG findings in 27 cases of Creutzieldt-Jakob
disease. Archives of Neurology 37(3):143-145.
Christie RH, Bacskai BJ, Zipfel WR, Williams RM, Kajdasz ST, Webb WW, Hyman BT.
2001. Growth arrest of individual senile plaques in a model of Alzheimer's disease ob-
served by in vivo multiphoton microscopy. Journal of Neuroscience 21(3):858-864.
Colas P. Cohen B. Jessen T. Grishina I, McCoy J. Brent R. 1996. Genetic selection of peptide
aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature 380(6574):548-
550.
OCR for page 103
DIA GNOSTICS FOR TSEs
103
Collinge J. Sidle KC, Meads J. Ironside J. Hill AF. 1996. Molecular analysis of prion strain
variation and the aetiology of "new variant" CJD. Nature 383(6602):685-690.
Cuille J. Chelle PL. 1939. Experimental transmission of trembling to the goat [translation
from French]. Comptes Rendus de l'Academie des Sciences 208:1058-1060.
DebBurman SK, Raymond GJ, Caughey B. Lindquist S. 1997. Chaperone-supervised conver-
sion of prion protein to its pro/ease-resistant form. Proceedings of the National Academy
of Sciences ofthe United States of America 94(25):13938-13943.
Desai M, Mishra B. Schwartz J. 2002. Designer Molecules for Biosensor Applications. Paper
presented at the National Science Foundation Banbury Workshop. Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory Press.
EC (European Commission). 2003. Opinion of the Scientific Steering Committee on the Field
Trial Evaluation of Two New Rapid BSE Post Mortem Tests. Brussels, Belgium: Health
and Consumer Protection Directorate-General, EC.
Ellington AD, Szostak JW.1990. In vitro selection of RNA molecules that bind specific ligands.
Nature 346(6287):818-822.
Engvall E, Perlman P. 1971. Enzyme-linked immunosorbent assay (ELISA): quantitative assay
of immunoglobulin G. Immunochemistry 8(9):871-874.
Fischer MB, Roeckl C, Parizek P. Schwarz HP, Aguzzi A. 2000. Binding of disease-associated
prion protein to plasminogen. Nature 408(6811):479-483.
Gambetti P. Petersen RB, Parchi P. Chen SG, Capellari S. Goldfarb L, Gabizon R. Montagna
P. Lugaresi E, Piccardo P. Bernardino G. 1999. Inherited prion diseases. In: Prusiner S.
ed. Prion Biology and Diseases. Cold Spring Harbor, NY: Cold Spring Harbor Labora-
tory Press. Pp. 509-583.
Harris D. 2002. Conventional Methods for PrPSc Detection. Presentation to the IOM Com-
mittee on Transmissible Spongiform Encephalopathies: Assessment of Relevant Science,
Meeting II. Washington, DC: National Academy Press.
Hill AF, Butterworth RJ, Joiner S. Jackson G. Rossor MN, Thomas DJ, Frosh A, Tolley N. Bell
JE, Spencer M, King A, Al-Sarraj S. Ironside JW, Lantos PL, Collinge J. 1999. Investiga-
tion of variant Creutzieldt-Jakob disease and other human prion diseases with tonsil
biopsy samples. Lancet 353(9148):183-189.
Hilton DA, Ghani AC, Conyers L, Edwards P. McCardle L, Penney M, Ritchie D, Ironside JW.
2002. Accumulation of prion protein in tonsil and appendix: review of tissue samples.
B ritish Medical Journal 325 (7365) :633 -634.
Horiuchi M, Caughey B. 1999. Specific binding of normal prion protein to the scrapie form
via a localized domain initiates its conversion to the pro/ease-resistant state. EMBO Jour-
nal 18(12):3193-3203.
IDEXX Laboratories Inc. 2003. IDEXX Laboratories Launches Diagnostic Kit for Chronic
VDasting Disease. [Online]. Available: http://www.idexx.com/aboutidexx/pressroom/re-
leases/20031117pr.cim [accessed November 25, 2003].
Ingrosso L, Vetrugno V, Cardone F. Pocchiari M. 2002. Molecular diagnostics of transmis-
sible spongiform encephalopathies. Trends in Molecular Medicine 8(6):273-280.
Johnson RT, Gibbs CJ Jr.1998. Creutzieldt-Jakob disease and related transmissible spongiform
encephalopathies. New England Journal of Medicine 339(27):1994-2004.
Kingsbury DT, Smeltzer D, Bockman J. 1984. Purification and properties of the K. Fu. isolate
of the agent of Creutzieldt-Jakob disease. In: Abstracts from the Proceedings of the 6th
International Congress of Virology, Sendai, Japan, VD47-6. P. 70.
Kirby L, Birkett CR, Rudyk H. Gilbert IH, Hope J. 2003. In vitro cell-free conversion of
bacterial recombinant PrP to PrP(res) as a model for conversion. Journal of General
Virology 84(Pt 4):1013-1020.
OCR for page 104
104
ADVANCING PRION SCIENCE
Klunk WE, Bacskai BJ, Mathis CA, Kajdasz ST, McLellan ME, Frosch MP, Debnath ML, Holt
DP, Wang Y. Hyman BT. 2002. Imaging Abeta plaques in living transgenic mice with
multiphoton microscopy and methoxy-X04, a systemically administered Congo red de-
rivative. Journal of Neuropathology and Experimental Neurology 61(9):797-805.
Kocisko DA, Come JH, Priola SA, Chesebro B. Raymond GJ, Lansbury PT, Caughey B. 1994.
Cell-free formation of pro/ease-resistant prion protein. Nature 370(6489):471-474.
Kohler G. Milstein C. 1975. Continuous cultures of fused cells secreting antibody of pre-
defined specificity. Nature 256(5517):495-497.
Korth C, Stierli B. Streit P. Moser M, Schaller O. Fischer R. Schulz-Schaeffer W. Kretzschmar
H. Raeber A, Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Rick R. Billeter
M, Wuthrich K, Oesch B. 1997. Prion (PrPSc)-specific epitope defined by a monoclonal
antibody. Nature 390(6655):74-77.
Marsh RF, Kimberlin RH. 1975. Comparison of scrapie and transmissible mink encephalopa-
thy in hamsters, II: Clinical signs, pathology, and pathogenesis. Journal of Infectious
Diseases 131(2):104-110.
Mathis CA, Bacskai BJ, Kajdasz ST, McLellan ME, Frosch MP, Hyman BT, Holt DP, Wang Y.
Huang GF, Debnath ML, Klunk WE. 2002. A lipophilic thioflavin-T derivative for
positron emission tomography (PET) imaging of amyloid in brain. Bioorganic and Me-
dicinal Chemistry Letters 12(3):295-298.
Mendez OK, Shang J. Jungreis CA, Kauier DI. 2003. Diffusion-weighted MRI in Creutzieldt-
Jakob disease: a better diagnostic marker than CSF protein 14-3-3? J Neuroimaging
13(2):147-151.
Merz PA, Somerville RA, Wisniewski HM, Manuelidis L, Manuelidis EE. 1983. Scrapie-asso-
ciated fibrils in Creutzieldt-Jakob disease. Nature 306(5942):474-476.
Monari L, Chen SG, Brown P. Parchi P. Petersen RB, Mikol J. Gray F. Cortelli P. Montagna P.
Ghetti B. Goldfarb LG, Gajdusek DC, Lugaresi E, Gambetti P. Autilio-Gambetti L. 1994.
Fatal familial insomnia and familial Creutzieldt-Jakob disease: different prion proteins
determined by a DNA polymorphism. Proceedings of the National Academy of Sciences
of the United States of America 91(7):2839-2842.
Moynagh J. Schimmel H. 1999. Tests for BSE evaluated: bovine spongiform encephalopathy.
Nature 400(6740):105.
Pan T. Colucci M, Wong BS, Li R. Liu T. Petersen RB, Chen S. Gambetti P. Sy MS. 2001.
Novel differences between two human prion strains revealed by two-dimensional gel
electrophoresis. Journal of Biological Chemistry 276(40) :37284-37288.
Paramithiotis E, Pinard M, Lawton T. LaBoissiere S. Leathers VL, Zou W. Estey LA,
Lamontagne J. Lehto MT, Kondejewski LH, Francoeur GP, Papadopoulos M, Haghighat
A, Spatz SJ, Head M, Will R. Ironside J. O'Rourke K, Tonelli Q. Ledebur HC,
Chakrabartty A, Cashman NR. 2003. A prion protein epitope selective for the pathologi-
cally misfolded conformation. Nature Medicine 9(7):893-899.
Parchi P. Capellari S. Chen SG, Petersen RB, Gambetti P. Kopp N. Brown P. Kitamoto T.
Tateishi J. Giese A, Kretzschmar H. 1997. Typing prion isoforms. Nature 386(6622):232-
234.
Parchi P. Castellani R. Capellari S. Ghetti B. Young K, Chen SG, Farlow M, Dickson DW,
Sima AA, Trojanowski JQ, Petersen RB, Gambetti P. 1996. Molecular basis of pheno-
typic variability in sporadic Creutzieldt-Jakob disease. Annals of Neurology 39(6):767-
768.
Parchi P. Giese A, Capellari S. Brown P. Schulz-Schaeffer W. Windl O. Zerr I, Budka H. Kopp
N. Piccardo P. Poser S. Rojiani A, Streichemberger N. Julien J. Vital C, Ghetti B. Gambetti
P. Kretzschmar H. 1999. Classification of sporadic Creutzieldt-Jakob disease based on
molecular and phenotypic analysis of 300 subjects. Annals of Neurology 46(2):224-233.
Pattison IH. 1966. The relative susceptibility of sheep, goats and mice to two types of the goat
scrapie agent. Research in Veterinary Science 7(2):207-212.
OCR for page 105
DIA GNOSTICS FOR TSEs
105
Pattison IH, Millson GC. 1961. Scrapie produced experimentally in goats with special refer-
ence to the clinical syndrome. Journal of Comparative Pathology 71:101-108.
Petricoin EF III, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM, Mills GB,
Simone C, Fishman DA, Kohn EC, Liotta LA.2002a. Use of proteomic patterns in serum
to identify ovarian cancer. Lancet 359(9306):572-577.
Petricoin EF III, Ornstein DK, Paweletz CP, Ardekani AM, Hackett PS, Hitt BA, Velassco A,
Trucco C, Wiegand L, Wood K, Simone CB, Levine PJ, Linehan WM, Emmert-Buck MR,
Steinberg SM, Kohn EC, Liotta LA. 2002b. Serum proteomic patterns for detection of
prostate cancer. Journal of the National Cancer Institute 94(20):1576-1578.
Prusiner SB, Bolton DC, Groth DF, Bowman KA, Cochran SP, McKinley MP. 1982. Further
purification and characterization of scrapie priors. Biochemistry 21(26):6942-6950.
Prusiner SB, Tremblay P. Safar J. Torchia M, DeArmond SJ. 1999. Bioassays of priors. In:
Prusiner SB, ed. Prion Biology and Diseases. Cold Spring Harbor, NY: Cold Spring Har-
bor Laboratory Press. Pp. 113-145.
Race RE, Fadness LH, Chesebro B. 1987. Characterization of scrapie infection in mouse neu-
roblastoma cells. Journal of General Virology 68(Pt 5):1391-1399.
Raymond GJ, Bossers A, Raymond LD, O'Rourke KI, McHolland LE, Bryant PK III, Miller
MW, Williams ES, Smits M, Caughey B. 2000. Evidence of a molecular barrier limiting
susceptibility of humans, cattle and sheep to chronic wasting disease. EMBO Journal
19(17):4425-4430.
Raymond GJ, Hope J. Kocisko DA, Priola SA, Raymond LD, Bossers A, Ironside J. Will RG,
Chen SG, Petersen RB, Gambetti P. Rubenstein R. Smits MA, Lansbury PT Jr, Caughey
B. 1997. Molecular assessment of the potential transmissibilities of BSE and scrapie to
humans. Nature 388(6639):285-288.
Remy I, Michnick SW. 1999. Clonal selection and in vivo quantitation of protein interactions
with protein-fragment complementation assays. Proceedings of the National Academy of
Sciences of the United States of America 96(10):5394-5399.
Rubenstein R. Carp RI, Callahan SM. 1984. In vitro replication of scrapie agent in a neuronal
model: infection of PC12 cells. Journal of General Virology 65(Pt 12):2191-2198.
Rubenstein R. Gray PC, Wehlburg CM, Wagner JS, Tisone GC. 1998. Detection and discrimi-
nation of PrPSc by multi-spectral ultraviolet fluorescence. Biochemical and Biophysical
Research Communications 246(1 ):100-106.
Saborio GP, Permanne B. Soto C. 2001. Sensitive detection of pathological prion protein by
cyclic amplification of protein misfolding. Nature 411(6839):810-813.
Sabuncu E, Petit S. Le Dur A, Lan Lai T. Vilotte JL, Laude H. Vilette D. 2003. PrP polymor-
phisms tightly control sheep prion replication in cultured cells. Journal of Virology
77(4):2696-2700.
Safar JG, Wille H. Itri V, Groth D, Serban H. Torchia M, Cohen FE, Prusiner SB. 1998. Eight
prion strains have PrP(Sc) molecules with different conformations. Nature Medicine
4(10):1157-1165.
Safar JG, Scott M, Monaghan J. Deering C, Didorenko S. Vergara J. Ball H. Legname G.
Leclerc E, Solforosi L, Serban H. Groth D, Burton DR, Prusiner SB, Williamson RA.
2002. Measuring prions causing bovine spongiform encephalopathy or chronic wasting
disease by immunoassays and transgenic mice. Nature Biotechnology 20(11):1147-1150.
Schatzl HM, Laszlo L, Holtzman DM, Tatzelt J. DeArmond SJ, Weiner RI, Mobley WC,
Prusiner SB. 1997. A hypothalamic neuronal cell line persistently infected with scrapie
prions exhibits apoptosis. Journal of Virology 71(11):8821-8831.
Schmerr MJ, Jenny AL. 1998. A diagnostic test for scrapie-infected sheep using a capillary
electrophoresis immunoassay with fluorescent-labeled peptides. Electrophoresis
19(3):409-414.
OCR for page 106
106
ADVANCING PRION SCIENCE
Schmerr MJ, Jenny AL, Cutlip RC. 1997. Use of capillary sodium dodecyl sulfate gel electro-
phoresis to detect the prion protein extracted from scrapie-infected sheep. Journal of
Chromatography B.: Biomedical Science Applications 697(1-2):223-229.
Schmerr MJ, Jenny AL, Bulgin MS, Miller JM, Hamir AN, Cutlip RC, Goodwin KR. 1999.
Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal
prion protein in the blood of animals that are infected with a transmissible spongiform
encephalopathy. Journal of Chromatography A 853(1-2):207-214.
Schmitt J. Beekes M, Brauer A, Udelhoven T. Lasch P. Naumann D. 2002. Identification of
scrapie infection from blood serum by Fourier transform infrared spectroscopy. Analyti-
cal Chemistry 74(15):3865-3868.
Shoghi-Jadid K, Small GW, Agdeppa ED, Kepe V, Ercoli LM, Siddarth P. Read S. Satyamurthy
N. Petric A, Huang SC, Barrio JR.2002. Localization of neurofibrillary tangles and beta-
amyloid plaques in the brains of living patients with Alzheimer's disease. American Jour-
nal of Geriatric Psychiatry 10(1):24-35.
Sigurdson CJ, Williams ES, Miller MW, Spraker TR, O'Rourke KI, Hoover EA. 1999. Oral
transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer
fawns (Odocoileus hemionus). Journal of General Virology 80 (Pt 10):2757-2764.
Soto C, Saborio GP, Anderes L. 2002. Cyclic amplification of protein misfolding: application
to prior-related disorders and beyond. Trends in Neurosciences 25(8):390-394.
Spencer MD, Knight RS, Will RG. 2002. First hundred cases of variant Creutzieldt-Jakob
disease: retrospective case note review of early psychiatric and neurological features. Brit-
ish Medical Journal 324(7352):1479-1482.
Tsuji K. 1994. Sodium dodecyl sulfate polyacrylamide gel- and replaceable polymer-filled cap-
illary electrophoresis for molecular mass determination of proteins of pharmaceutical
interest. Journal of Chromatography B.: Biomedical Science Applications 662(2):291-
299.
Tuerk C, Gold L. 1990. Systematic evolution of ligands by exponential enrichment: RNA
ligands to bacteriophage T4 DNA polymerase. Science 249(4968):505-510.
USDA (U.S. Department of Agriculture) APHIS (Animal and Plant Health Inspection Agency).
2002. USDA Issues Permit for Chronic VDasting Disease Antigen Test Kit. [Online]. Avail-
able: http://www.aphis.usda.gov/lpa/news/2002/11/cwdtest_vs.html [accessed June 3,
2003].
Vilette D, Andreoletti O. Archer F. Madelaine MF, Vilotte JL, Lehmann S. Laude H.2001. Ex
vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells express-
ing ovine prion protein. Proceedings of the National Academy of Sciences of the United
States of America 98(7):4055-4059.
VMRD Inc. 2003a. Chronic Wasting Disease Antigen Test Kit, dbELISA. [Online]. Available:
http://www.vmrd.com/products/testkits/detail.asp?CATNO=721 -960 [accessed June 29,
2003].
VMRD Inc. 2003b. Introducing New Diagnostics for CWD Dot Blot ELISA Test. [Online].
Available: http://www.vmrd.com/products/testkits/cwd/validation.htm [accessed August
2003].
Vorberg I, Groschup MH, Pfaff E, Priola SA. 2003. Multiple amino acid residues within the
rabbit prion protein inhibit formation of its abnormal isoform. Journal of Virology
77(3):2003-2009.
Wadsworth JD, Joiner S. Hill AF, Campbell TA, Desbruslais M, Luthert PJ, Collinge J. 2001.
Tissue distribution of protease resistant prion protein in variant Creutzieldt-Jakob dis-
ease using a highly sensitive immunoblotting assay. Lancet 358(9277):171-180.
Wells GA, Hawkins SA, Green RB, Austin AR, Dexter I, Spencer YI, Chaplin MJ, Stack MJ,
Dawson M. 1998. Preliminary observations on the pathogenesis of experimental bovine
spongiform encephalopathy (BSE): an update. Veterinary Record 142(5):103-106.
OCR for page 107
DIA GNOSTICS FOR TSEs
107
WHO (World Health Organization). 1998. Global Surveillance, Diagnosis and Therapy of
Human Transmissible Spongiform Encephalopathies: Report of a WHO Consultation.
Geneva: WHO.
WHO.2001a. The Revision of the Surveillance Case Definition for Variant Creutzfeldt-Jakob
Disease (VCJD): Report of a WHO Consultation. Edinburgh, United Kingdom: WHO.
WHO. 2001b. Working Group on International Reference Materials for Diagnosis and Study
of Transmissible Spongiform Encephalopathies (TSEs): Third Meeting. WHO Blood
Safety and Clinical Technology. Geneva: WHO.
Will RG, Zeidler M, Stewart GE, Macleod MA, Ironside JW, Cousens SN, Mackenzie J.
Estibeiro K, Green AJ, Knight RS. 2000. Diagnosis of new variant Creutzieldt-Jakob
disease. Annals of Neurology 47(5):575-582.
Wong C, Xiong LW, Horiuchi M, Raymond L, Wehrly K, Chesebro B. Caughey B. 2001.
Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free forma-
tion of pro/ease-resistant prion protein. EMBO Journal 20(3):377-386.
Yalow RS, Berson SA. 1959. Assay of plasma insulin in human subjects by immunological
methods. Nature 184:1648-1649.
Zeidler M, Sellar RJ, Collie DA, Knight R. Stewart G. Macleod MA, Ironside JW, Cousens S.
Colchester AC, Hadley DM, Will RG, Colchester AF. 2000. The pulvinar sign on mag-
netic resonance imaging in variant Creutzieldt-Jakob disease. Lancet 355(9213):1412-
1418.
Zerr I, Pocchiari M, Collins S. Brandel JP, de Pedro Cuesta J. Knight RS, Bernheimer H.
Cardone F. Delasnerie-Laupretre N. Cuadrado Corrales N. Ladogana A, Bodemer M,
Fletcher A, Awan T. Ruiz Bremon A, Budka H. Laplanche JL, Will RG, Poser S. 2000.
Analysis of EEG and CSF 14-3-3 proteins as aids to the diagnosis of Creutzieldt-Jakob
disease. Neurology 55 ( 6 ): 8 1 1 -8 1 5.
Representative terms from entire chapter:
dia gnostics
