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From: Terry S. Singeltary Sr. (
Subject: Bovine Spongiform Encephalopathies (BSE): A Review of Actions in North America
Date: December 31, 2004 at 2:14 pm PST

-------- Original Message --------
Subject: Bovine Spongiform Encephalopathies (BSE): A Review of Actions in North America
Date: Fri, 31 Dec 2004 16:04:12 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy

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

Bovine Spongiform Encephalopathies (BSE):
A Review of Actions in North America

> to date not in cattle muscle UK DEFRA
> mouse muscle (Bosque et al., 2002)

i think this might change soon ;
Last modified, 11/09/2004 13:42:49
BSE death cow's anomalous prion detected from peripheral nerve tissue,
suprarenal gland
First time from non-Specified Risk Material, or SRM
National Institute of Animal Health Animal announced on November 1 that
it had detected the anomalous prion protein that was the etiologic agent
of the mad cow disease, or BSE, or bovine spongiform encephaalopathy,
from the peripheral nerve tissue and the suprarenal gland of the cow of
the age in the mad cow disease for the dying infection 94 months on
March 9 this year.
Japan is obligating the removal of the Specified Risk Material, or SRM
such as the head, the spinal cord, the vertebral columns, and the small
intestines that accumulate the anomalous prion protein easily as a BSE
(bovine spongiform encephaalopathy) measures.
Because the mad cow disease etiologic agent was detected from a tissue
different from the Specified Risk Material, or SRM, the review of the
Specified Risk Material, or SRM might be urged on the Japanese Government.
International Symposium of PRION DISEASES for food and drug safety
national institute of animal health(only in Japanese)
The statement of the Ministry of Health, Labour and Welfare
(only in Japanese)
Yomiuri on line (only in Japanese)
Asahi on line(only in Japanese)
Mainichi on line(only in Japanese)

I acquired these abstract submissions over the weekend. maybe later we will
have access to this Symposium in full, but it will be several months... TSS


Variant Creutzfeldt-Jakob Disease

Ironside JW, Head MW, Knight R, Ward H
National CJD Surveillance Unit, University of Edinburgh, Edinburgh, UK

Variant Creutzfeldt-Jakob disease (vCJD) is a novel human prion disorder
which on the basis of experimental
strain typing appears to result from human exposure to the bovine
spongiform encephalopathy (BSE) agent,
probably by the consumption of BSE-contaminated meat products. This
disorder tends to present in young
adults (median age 28 y) as a psychiatric disorder often accompanied by
sensory abnormalities and later
followed by ataxia, myoclonus and other movement disorders. Death occurs
after a median duration of illness
of 13 months. MRI brain scans have shown that abnormal areas of high
signal occur in T2 and proton density
images in the posterior thalamus. All patients with vCJD have been
homozygous for methionine at codon 129
in the PrP gene. Pathologically, vCJD is characterised by florid plaques
in the cerebrum and cerebellum, and
by the presence of a PrP isotype (2B), which is distinct from other
human prion diseases. vCJD also differs
from other human prion disorders in that abnormal PrP is widely
distributed outside the nervous system in
lymphoid tissues. This has given rise to concerns that vCJD may be
transmitted by surgical instruments used
on these tissues, even if infectivity appears to be at lower levels than
in the brain. 2 cases of iatrogenic vCJD
infection have recently been reported following blood transfusion from
donors who subsequently developed
vCJD, one of whom was a heterozygote at codon 129 in the prion protein
gene and died before the onset of
clinical neurological disease. There are considerable uncertainties over
future numbers of vCJD cases in the
UK, since clinical cases appear to be declining in frequency, but
retrospective studies of PrP accumulation in
tonsils and appendix tissue have suggested that a few thousand
individuals may be incubating the disease in
the UK. Continuing surveillance is required in the UK and in other
countries where BSE has been identified.



Richard Knight
Clinical Neurologist, National CJD Surveillance Unit, UK

160 cases of variant CJD have been identified (149 in the UK). The
clinico-pathological profile of human prion
diseases is influenced by PRNP codon-129 genotype, prion ‘agent strain’
(or prion protein type) and mode of
acquisition. All tested cases of variant CJD have occurred in one
genotype (codon 129 MM), with one
presumed agent strain/one protein type and one probable cause: BSE
dietary contamination (but for one
instance of possible blood transmission). Therefore, it is perhaps
unsurprising that variant CJD has a relatively
uniform clinical picture, unlike the significant clinico-pathological
heterogeneity of sporadic CJD. Also unlike
sporadic CJD, it is predominantly affects the young with a relatively
long duration (median ages at onset:
vCJD 26 years, sCJD 66 years; median durations: vCJD14 months, sCJD 4
months in sporadic CJD).
Sporadic CJD typically presents in a clearly neurological way, most
often with a rapidly progressive dementia.
Variant CJD typically presents with a psychiatric or behavioural
disturbance; its essentially neurological nature
may not be apparent for some months.
Social withdrawal, loss of interest, dysphoria, anxiety, irritability
and insomnia are common early symptoms;
depression being a frequent initial diagnosis.
Sensory symptoms (typically unpleasant) are reported in around two
thirds of cases. Specifically neurological
signs appear at a median of around 6 months; memory/cognitive
dysfunction and cerebellar ataxia being the
commonest features.
Other neurological features include: pyramidal signs, and involuntary
movements (chorea, dystonia, tremor,
Definite diagnosis requires neuropathology. The clinical diagnosis of
variant CJD rests on: a clinical suspicion
of the condition, the exclusion of other possible diagnoses and the
appropriate use of supportive diagnostic
tests. The most useful non-invasive investigation is the cerebral MRI,
showing the so-called ‘Pulvinar sign’ in
most cases. Tonsil biopsy is useful in some.



Nikolai G Rainov 1,2
1 Department of Neurological Science, The University of Liverpool, and
2The Walton Centre for Neurology
and Neurosurgery NHS Trust, Liverpool, UK

Transmissible spongiform encephalopathies (TSE) are diseases believed to
be caused by accumulation of an abnormal
isoform of the prion protein (PrPsc) in the central nervous system.
There are sporadic, aquired and hereditary TSE.
Creutzfeld-Jacob disease (CJD) in its sporadic and variant form is the
most frequent and clinically important TSE. At
present there is no proven specific or effective treatment available for
any form of CJD, although drugs such as
quinacrine are being investigated in early clinical trials.
Pentosan polysulphate (PPS), a large polyglycoside molecule with weak
heparin-like activity, has been shown to prolong
the incubation period of PrPsc infection when administered to the
cerebral ventricles in a rodent scrapie model. PPS also
prevents the production of further PrPsc in cell culture models.
However, PPS penetrates poorly the blood-brain barrier
and only a minor fraction of orally administered drug may reach the CNS.
These properties of PPS prompted its cerebroventricular administration
in a total of 8 patients with vCJD and other TSE,
such as iatrogenic CJD and Gerstmann-Sträussler-Scheinker syndrome
(GSS). Long-term continuous infusion of PPS at
doses from 11 µg/kg/day to 110 µg/kg/d did not cause any drug-related
side effects. Follow-up CT and MRI imaging
demonstrated that brain atrophy may progress during PPS administration.
Proof of clinical efficacy has not been the aim
of these early studies, however one patient with vCJD survived for 37
months after initial symptoms and 30 months after
diagnosis, while the median duration of illness with vCJD is 13 months
(range 6-39).
Some lessons have been learned from the first cases. Surgery in a brain
affected by TSE may result in a higher rate of
surgical complications than might be expected in analogous cases without
TSE. Secondly, if clinically significant benefits
are to be expected, PPS administration should start as early as possible
in the course of the respective disease and
before irreversible loss of neurological function has occurred.
Further clinical, neuroradiological and laboratory investigations in the
setting of a prospective clinical study with
standardised follow-up protocol and data collection are essential in
order to assess the efficacy of PPS administration in
vCJD and in other TSE.


Peripheral Pathogenesis of Human Prion Diseases

Markus Glatzel1, Eugenio Abela1, Nicolas Genoud1, James Ironside2, and
Adriano Aguzzi
1Swiss National Reference Center for Prion Diseases, University Hospital
of Zürich, CH-8091 Zürich, Switzerland
2The National Creutzfeldt-Jakob Disease Surveillance Unit, Western
General Hospital, Edinburgh EH4 2XU, UK

Ante-mortem diagnosis of human prion diseases has been attempted by
brain biopsy, but is complicated by
insurmountable biosafety problems, is highly invasive, and is thus
generally considered obsolete in the
diagnosis of a human prion disease. As a consequence, there is currently
no minimally invasive ante-mortem
test available to confirm the clinical suspicion of a human prion
disease or to discriminate prion strains. By
studying the precise distribution of disease-associated prion protein
(PrPSc) in sporadic Creutzfeldt-Jakob
disease we have identified muscle as a putative target for the diagnosis
of Creutzfeldt-Jakob disease. These
investigations are supplemented by studies employing genetically
modified mice aimed unraveling the
molecular basis of muscular prion replication.


Further advances in the molecular and pathological diagnosis of
sporadic Creutzfeldt-Jakob disease subtypes.

Piero Parchi, Silvio Notari, Rosaria Strammiello, Sabina Capellari.
Laboratory of Neuropathology, Department of Neurological Sciences,
University of Bologna, Italy.

The characterization of at least six clinico-pathological phenotypes of
sporadic Creutzfeldt-Jakob disease
(sCJD) which largely correlate at the molecular level with the genotype
at codon 129 (MM, MV, VV) and either
one of two major types of PrPSc 27-30 with distinct physicochemical
properties (i.e. type 1 and type 2) has
provided the basis for a molecular classification of sCJD, and a
potentially powerful method for strain typing.
Despite the significant advances, however, additional work needs to be
to done to fully explore the whole
spectrum of sCJD variants and the issue of the molecular basis of
phenotypic variability in sCJD. The
discovery of subjects with the co-occurrence of PrPSc type 1 and type 2
constitutes a potential drawback for
the widespread application of this classification to CJD diagnostics and
epidemiology. Furthermore, there is
still some disparity among laboratories regarding the understanding and
nomenclature of PrPSc types and it is
still unknown whether a specific PrPSc type is associated with each sCJD
phenotypic variant. Lastly, the recent
identification of novel C-terminal fragments of PrP (PrP-CTF) has
provided a potential novel molecular marker,
but it is currently unclear to what extent PrP-CTF properties correlate
with the sCJD phenotype. We report on
our recent results on the above issues. I) Studying in detail a large
series of cases we found that the
co-occurrence of types 1 and 2 is not random and characterizes about 30%
of MM cases and 20-25% of the
sCJD population as a whole. Our data show that a correct molecular
classification can be reached in the
absolute majority of sCJD cases, but requires pathological and
biochemical analyses of 6-8 samples from the
cerebral cortex, thalamus and cerebellum. II) We further analyzed PrPSc
27-30 properties using a high
resolution gel electrophoresis system and varying experimental
conditions and found that pH varies among
CJD brain homogenates in standard buffers thereby influencing the
characteristics of PrPSc 27-30. We also
found that PrPSc 27-30 type 1 and type 2 are heterogeneous species,
which can be further distinguished into
molecular subtypes that fit the current histopathological classification
of sCJD variants. III) Finally, our latest
results indicate that PrP-CTF characterization is also useful for the
molecular diagnostics of some sCJD
subtypes. Supported by the Italian Ministry of Health (Ricerca
Finalizzata 1%/2001), the EU contract
QLK3-CT-2001-02345, and the G. Galletti Foundation.


Chronic Wasting Disease in Cervids in North America

Elizabeth S. Williams
University of Wyoming

Chronic wasting disease (CWD) is a transmissible spongiform
encephalopathy of free-ranging and farmed
cervids in North America that is distinct from scrapie of domestic
sheep, bovine spongiform encephalopathy,
and Creutzfeldt-Jacob disease of humans. The purpose of this paper is to
review the current status of CWD in
North America. The natural host range of CWD includes mule deer
(Odocoileus hemionus), white-tailed deer
(Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus
nelsoni). Experimentally, by intracerebral
or oral exposure, the host range is wider, but there appears to be a
significant barrier to infection of cattle and
humans. The exact mechanism of CWD transmission is not known but recent
studies indicate that direct
transmission, indirect transmission via environmental contamination, and
transmission associated with
carcasses are possible. Maternal transmission does not appear to play a
significant role in CWD. Although still
under investigation, polymorphisms in the prion protein influence CWD
pathogenesis in mule deer and elk.
Studies of CWD pathogenesis following oral exposure demonstrate early
widespread distribution of abnormal
prion protein in the lymphoid tissue prior to invasion of the central
nervous system. These data have lead to
techniques for CWD surveillance in deer based on testing retropharyngeal
lymph node. However, both brain
and lymph node must be tested in elk for highest sensitivity due to
differences in pathogenesis in this species
compared to deer. The unique nature of a transmissible spongiform
encephalopathy occurring in free-ranging
cervids is a serious challenge to wildlife managers and animal health
agencies in North America.


Bovine spongiform encephalopathy (BSE) in Japan

Takashi Yokoyama, Kumiko M. Kimura, Morikazu Shinagawa
Prion Disease Research Center, National Institute of Animal Health, Japan

Bovine spongiform encephalopathy (BSE) has become an important problem
not only for animal industry, but
also for public health. In Japan, BSE was first recognized in September
2001 by fallen stock surveillance.
Since October 2001, BSE examination for all cattle slaughtered at
abattoirs has started. In April 2004, all dead
cattle examination (over 24 months) has been conducted at livestock
hygiene service center. Samples positive
in enzyme linked immunosorbent assay (ELISA) are further subjected to
western blot (WB) and
immunohistochemistry (IHC). Thirteen BSE cases have been reported by
September 2004. Twelve cases
were classified as typical BSE, and the remained one was an atypical
BSE. Variant forms of BSE with atypical
histopathological and/or biochemical phenotype were reported in Italy
and France. Further study is required
for BSE prion characteristics.
To characterize BSE prion properties, brain homogenates of Japanese BSE
cases were intracerebrally
inoculated into wild-type mice. The first case (BSE/Chiba) was
successfully transmitted to rodents. The mean
incubation periods (409.0 days) in this experiment was preferably longer
than that of previously reported.
PrPSc distribution, prion titer, mice susceptibility and/or storage
condition of sample might be influenced the
result. Recently, we introduced transgenic mice that overexpress a
bovine PrP gene to overcome the species
barrier problem. These mice are expected to accelerate the transmission
experiment of BSE prion.
Transmission of atypical BSE case is undergoing by using these
transgenic mice.


How does host PrP control TSE disease?

Jean Manson1, R Barron1, N Tuzi1, H Baybutt1, Enrico Cancellotti1, P
Hart1, L Aitchison1, B.Bradford1, D King1 , R Moore2, D
Melton2, M Bishop3, J Ironside3, R Will3
1Institute for Animal Health, Neuropathogenesis Unit, Edinburgh, UK,
2University of Edinburgh, 3National CJD Surveillance
Unit, Edinburgh

PrP is central to the TSE disease process and has been hypothesised to
be the infectious agent.
Polymorphisms in the PrP gene of a number of species are associated with
different incubation times of
disease following exposure to an infectious agent and mutations in the
human PrP gene can apparently lead
to spontaneous genetic disease. Strains of TSE agent are proposed to be
generated and maintained through
differences in glycosylation or conformation of PrP and the barrier to
infection between species is thought to
be due to the differences in the sequence of PrP between different species.
In order to test these hypotheses, we have introduced specific
modifications into the endogenous mouse
Prnp gene by gene targeting. The mutated PrP gene is in the correct
location under the control of the
endogenous Prnp regulatory sequences and thus expressed in the same
tissues and amounts as the wild type
Prnp gene. This strategy therefore allows the effect of specific
mutations in the PrP gene to be assessed.
By altering the murine PrP coding region to that of another species we
have established that increasing
overall identity between host and donor PrP can lead to either an
increase or a decrease in incubation time of
disease in a strain dependent manner. We have introduced a point
mutation (101L) into the N-terminus of the
host PrP and shown that it dramatically changes the susceptibility of
the host to infection from different
species. We have in addition demonstrated that polymorphisms in the N
terminus (L108T) and C-terminus
(F189V) of host PrP both alter the incubation time of disease but each
operates by a different mechanism.
We have introduced mutations into the Prnp gene which prevent
glycosylation at each or both of the two
N-linked glycosylation sites of PrP. Inoculation of these mice with
infectivity has established that glycosylation
of host PrP can influence incubation time of disease, vacuolar pathology
and strain determination. We have
during these studies produced a model of TSE disease which contains high
levels of infectivity in the apparent
absence of PrPSc and we are using this model to define the nature of the
infectious agent.
We have thus established that the gene targeting approach can produce
models for TSE disease which
address fundamental questions associated with these diseases. We aim to
use these models to address
central issues including the origin of strains, the species barrier and
the nature of the infectious agent.


Prion interactions with the immune system
Neil A Mabbott
Institute for Animal Health, Edinburgh, UK

Many natural prion infections are likely to be acquired peripherally for
example, following ingestion of
prion-contaminated feed. Following peripheral exposure prions accumulate
in lymphoid tissues before
spreading to the brain. Using mice experimentally infected with scrapie
prions we have shown that mature
follicular dendritic cells (FDCs), expressing the host prion protein
(PrPc), are critical for replication of infection
in lymphoid tissues. Prion neuroinvasion is also dependent on FDCs as in
their absence disease
susceptibility is reduced. For example, temporary depletion of FDCs
before oral inoculation with prions
blocks the accumulation of disease-specific PrP in Peyer’s patches and
mesenteric lymph nodes, and
prevents neuroinvasion.
Studies in mice have shown that skin scarification is also an effective
means of prion transmission.
Following inoculation via the skin scrapie prions accumulate in the
draining lymph node in association with
FDCs. The accumulation of prions in association with FDCs is also
critical for the transmission of disease
from the skin to the brain, as disease susceptibility is reduced in
their absence. The mechanisms through
which prions are transported from the skin to lymphoid tissues are not
known. Langerhans cells (LCs) reside
in the epidermis and migrate to the draining lymph node after
encountering antigen. Our studies show that
LCs have the potential to acquire and degrade PrPSc following in vitro
exposure. To investigate the potential
role of LCs in prion transportation from the skin, we utilized mouse
models in which their migration was
blocked. We show that the early accumulation of prions in the draining
lymph node and subsequent
neuroinvasion was not impaired in mice with blocked LC migration. These
data therefore demonstrate that
although LCs have the potential to acquire prions they are not involved
in their transportation to draining
lymphoid tissues.
Thorough analysis of the early events in prion pathogenesis in lymphoid
tissues may identify potential
targets for therapeutic intervention.


Scrapie infection of SN56 cells: greater efficiency by
membrane-associated versus purified PrP-res
Gerald S. Baron1, Ana C. Magalhães2, Marco A.M. Prado2, and Byron Caughey1
1Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases,
NIAID, NIH, 903 S. 4th St., Hamilton, MT 59840

2Program of Molecular and Biochemical Pharmacology, Department of
Pharmacology, ICB, Universidade Federal de Minas
Gerais, Av. Antonio Carlos 6627, 31270-901, Brazil
The process by which transmissible spongiform encephalopathy (TSE)
agents, or prions, infect cells is
unknown. We employed a new highly susceptible cell line (SN56) and a
previously described cell line (N2a)
to gain insight into the mechanism of infection. The effect of
disease-associated PrP (PrP-res) association
with membranes on infection efficiency was examined by comparing
sustained PrP-res production in cells
treated with either scrapie brain microsomes or purified,
detergent-extracted PrP-res. When normalized for
quantity of input PrP-res, scrapie brain microsomes induced dramatically
enhanced persistent PrP-res
formation compared to purified PrP-res. Infected SN56 cells released low
levels of PrP-res into the culture
supernatant, which also efficiently initiated infection in recipient
cells. Interestingly, microsomes labeled with
a fluorescent marker were internalized by SN56 cells in small vesicles,
which were subsequently found in
neuritic processes. When bound to culture wells to reduce
internalization during the infection process,
scrapie microsomes induced less long-term PrP-res production than
suspended microsomes. Our
observations suggest that efficient infection of cells may involve a
transfer and/or internalization of membranes
containing PrP-res.


Slow dynamics of prion protein
Kazuo Kuwata
Division of Prion Research, Center for Emerging Infectious Deseases
(CEID), Gifu University

Although the conformational conversion mechanism from cellular (PrPC) to
scrapie (PrPSc) form of animal
prion proteins has not yet been elucidated, much evidence is
accumulating that potentially provides insights
into the conversion process at atomic resolution. We characterized the
critical aspects of the slow fluctuation
dynamics of the recombinant hamster prion protein, rPrP(90-231), based
on NMR relaxation analysis using
Carr-Purcell-Meiboom-Gill (CPMG) experiments, and compare them in detail
with the results from
high-pressure NMR. Residues exhibiting slow fluctuations on the time
scale of microseconds to milliseconds
are mainly localized on helices B and C (172-193 and 200-227), which
include locally disordered regions in an
intermediate conformer, PrP*, identified previously by high pressure NMR
(Kuwata et al., Biochemistry
12277-12283 (2002)). Moreover, chemical shift differences between two
putative exchanging conformers
obtained by the CPMG relaxation analysis and the linear component of the
pressure-induced chemical shift
changes are well correlated at individual residue sites. These
observations apparently support the notion that
both the CMPG relaxation and the pressure shifts represent slow
conformational fluctuations, and that these
slow motions in PrPC are fundamentally on the trajectories leading to
the transition to PrP*. Furthermore the
slow conformational exchange rates were not in proportion to the square
of the static magnetic field, indicating
that the slow dynamical trajectory of the prion protein is not simply
described by the semiclassical periodic
approximation, but includes bifurcation and/or singularities. Those
abnormal dynamical characterstics of prion
may be related to its pathogenicity.

Department of Prion Research
Tohoku University School of Medicine
2-1 Seiryo-cho Aoba-ku, Sendai 980-8575, JAPAN
Tel: +81-22-717-8233
Fax: +81-22-717-8148

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