0 - COUVERTURE + DER2

Transcription

May 24th-28th
Content
Welcome
OIE’s Missions
Industrial Partners
Programme
Oral Presentations
Poster Session 1
Poster Session 2
Poster Session 3
NeuroPrion Members
Participant List
Index
3
7
11
21
29
79
115
153
187
191
215
Welcome
Welcome
O
n behalf of the NeuroPrion
Network of Excellence (NoE),
we are pleased to welcome you in
Paris to Prion2004, our first international
conference on Prion diseases.
The NoE NeuroPrion, endorsed by the
European Commission, aims at reinforcing
the protection of human and animal health
against prion diseases and avoiding new
BSE/prion crises.
The network is a self-structured organization
composed of 52 members from more than 20
countries corresponding to the main
European public research teams in the field
of transmissible spongiform encephalopathies
(TSEs) or prion diseases.
NeuroPrion will coordinate the research
efforts of its partners by identifying and
filling the gaps within the existing
programmes and using joint project actions.
The deliverables of these projects are
planned to impact on public health policies
and agricultural monitoring. A new
infrastructure dedicated to prion research
will facilitate the exchanges within
NeuroPrion and other research groups from
all parts of the world. Additionally, it will
enable the CEA to offer training
opportunities for young researchers.
NeuroPrion will also constitute a platform
for communication between the Scientific
Community and Society. The Society’s
expectations and concerns will be integrated
into the permanent global reflection held
within the Network to define strategies and
accurate priorities and develop suitable
cooperations with industrial partners. It
should lay the ground for a transparent
dialog allowing to prevent further TSE
related societal crises.
Prion2004 is the first major event organised
by NeuroPrion. This conference will offer
the opportunity to communicate the latest
scientific results, as well as endeavour to
trigger new interactions and collaborations
amongst major stakeholders in the Prion
field. Tuesday to Thursday will constitute the
scientific core dedicated to oral and poster
communications. The days of Monday and
Friday will be dedicated to establish
cooperations between Science and Society
and will be open to the media.
We would like to gratefully acknowledge the
funding of NeuroPrion from the European
Commission and address special thanks to
the Commissariat au Plan, the Conseil
Regional and the Senat Presidence for their
strong support in the organisation of this
event. Moreover we would like to stress the
fairness of our industrial partners (BeckmanCoulter, BioRad, Pourquier Institute, Merial
and Prionics), who joined the spirit of
NeuroPrion and contributed to making
Prion2004 a memorable event.
Last but not least we have to thank you all
for your participation, because
communication and cooperation within and
across the different stakeholders is the most
valuable outcome of a conference. In this
respect we wish you all a pleasurable and
inspiring time in Paris.
THE ORGANISING COMMITTEE
Jean-Philippe Deslys,
Corinne Ida Lasmézas,
Jens Schell,
Steve Simoneau
005
OIE’s
Missions
Industrial
Partners
EXEMPLARY COLLABORATION BETWEEN
INSTITUT POURQUIER
AND INRA FOR THE
DEVELOPMENT
OF TSEs TESTS
Philippe POURQUIER - Scientific Director - 326, rue de la Galéra 34097 Montpellier - France
Tel. 33 (0)4 99 23 24 25 - Mob. : 33 (0)6 60 74 63 98 - Fax : 33 (0)4 67 04 20 25
E.mail : philippe.pourquier@institut-pourquier.fr - Website : www.institut-pourquier.com
INTRODUCTION
Institut Pourquier is specialised in the development, production and distribution of
reagents for the diagnostic of large animal infectious diseases.
Institut Pourquier was specialised for decades in the production and distribution
of a lyophilised vaccine against smallpox. With the definitive suppression of small
pox vaccination in 1984, Institut Pourquier moved completely its activity towards
the production of veterinary diagnostic reagents intended to eradication program
settled by the EEC (Brucella, Bovine Leukosis Virus, Aujezsky disease …).
The following years, the company progressively extended its diagnostic lines to a
panel of livestock infectious pathologies of economic importance or epizootically
character. On another hand, it strongly developed its international implantation,
particularly in European community and in Oceania.
This line is widely based on ELISA techniques of antigen or specific antibody
detection in biological samples (serum, plasma, milk, faeces) using immunological
or molecular technologies.
Today, Institut Pourquier is one of the European leaders in the field of animal disease diagnostic kit production.
Institut Pourquier disposes of all technologies and experience allowing manufacturing of reagent kits (in a stable form and ready-to-use format). The methods are
developed to be easy to implement, robust and viable.
Institut Pourquier is progressively becoming a privileged partner for the valorisation of technologies developed by veterinary research centres of international
renown (I.N.R.A., Istituti zooprofilattici, A.F.S.S.A., V.L.A., etc…)
At the beginning of the BSE crisis, the Institut Pourquier was not involved at all
in TSE diagnostics. From the beginning of 2001, Institut Pourquier tried to apply
its immuno assay experience to the development of Scrapie and BSE tests.
This was made possible within the framework of a collaboration between Institut
Pourquier and I.N.R.A.
I.N.R.A./INSTITUT POURQUIER COLLABORATION
The collaboration between Institut Pourquier and I.N.R.A. was exemplary,
owing to an excellent competence synergy. The development team, composed at
the beginning by Philippe Pourquier and one engineer, was strenghtened by a
PhD student and supported by I.N.R.A. scientists.
It allowed the development of a very-well positioned test in two years and with
very limited human resources.
I.N.R.A. contribution
- Supplying of monoclonal antibody panels from different origins
- Knowledge in prion field brought by INRA teams of Jouy-en-Josas- France
(Molecular Virology Unit), Toulouse and Tours
- Supplying of Scrapie positive brains
Institut Pourquier contribution
- Expertise in development of immuno assays
- Various contacts for the supplying of positive and negative sample panels
- Experience in industrial production of diagnostic kits.
Preliminary results were presented in the Open call for the expression of interest
to participate in a programme for the evaluation of tests for the diagnosis of TSEs
in ruminants, in two different files : one for Scrapie (brainstems and lymphoid tissues), one for BSE.
AIMS OF OUR PARTICIPATION TO THE NOE NEUROPRION
The aims of our participation to the NoE NeuroPrion are multiple :
- To be involved in a scientific community for an experience and biological share,
allowing a better evaluation and improvement of the diagnostic tools already
developped by the Institut Pourquier.
- To acquire a better knowledge of the Prion biological mechanisms opening the
way to the development of an ante mortem test
Programme
Monday,May 24th 2004
13H 30 - 15H 45 SCIENCE AND SOCIETY
Chair: L. Court
13:30
13:40
14:20
15:00
Introduction to Prion2004
What is Society expecting from Science
The Principle of Precaution and Economic aspects
BSE and its Impact on Europe
Louis Court
Alain Etchegoyen
François Ewald
Jean-Philippe Deslys
15h 45- 16h 15 Coffee-Break
16H 15 - 18H 30 DIAGNOSTICS
Chair: P. Brown
16:15
17:00
17:45
Complexity and beauty of the intracellular protein traffic
Stuctural Biology and Protein Diseases
Kuru: The settings of Paradigms
Günter Blobel
Kurt Wüthrich
Carlton Gajdusek
023
Tuesday,May 25th 2004
8H 30 - 9H 35
RISK ASSESMENT
Chair: M. Ricketts / F. Guarnieri
08:30
08:40
08:55
09:15
Introduction
Role of the OIE
A General Framework for Prion Safety Risk Assessment
Quantitative Assessment of the Risk of Transmission of BSE
by Tallow-Based Calf Milk Replacer
Maura Ricketts
Bernard Vallat
Franck Guarnieri
Larry G. Paisley
9H 35 - 10H 10 ANIMAL TSE I
Chair: J. Langeveld / M. Caramelli
09:35
09:55
Epidemiological analysis of data for scrapie in Great Britain
Prevalence of infection in cull sheep from scrapie-affected flocks
in the UK
Thomas Hagenaars
Susane C. Tongue
10H 40 - 11H 15 ANIMAL TSE II
Chair: J. Langeveld / D. Matthews
10:40
11:00
Prevention of TSEs in animals through PrP genetic selection:
new PrP haplotypes and the principles of disease association.
Experimental evidence on maternal transmission of scrapie
and BSE in sheep
Wilfred Goldmann
Nora Hunter
11H 15 - 12H 10 HUMAN TSE I
Chair: B. Will / J-J. Hauw
11:15
11:35
11:55
Strain typing studies of human and animal TSEs
The European CJD Surveillance system: current concerns
Increased proportion of genetic prion disease in Hungary
Moira Bruce
Robert G. Will
Gabor G. Kovacs
12h 10- 15h 10 Lunch and Poster Session
15H 10 - 15H 50 HUMAN TSE II
Chair B. Will / J-J. Hauw
15:10
15:30
vCJD in the UK 1995-2003: a case-control study
of potential risk factors
Use of non human primates for CJD risk assessment
Hester J.T. Ward
Corinne I. Lasmézas
15H 50 - 17H 00 PRION STRAINS I
Chair: T. Baron / J-M. Torres
15:50
16:05
16:20
024
How does host PrP control TSE disease?
Investigating natural scrapie strain diversity in ovine
PrP transgenic mice
Analysis of experimental ovine BSE agent through
transmission studies to Tg(OvPrP4) mice expressing
the ovine prion protein
Jean Manson
Hubert Laude
Anna Bencsik
17H 30 - 18H 45 PRION STRAINS II
Chair: T. Baron / J. Manson
17:30
17:50
18:05
18:25
Initial findings on experimental transmission of atypical BSE to mice
Unusual Prion Protein Characteristics in Isolates from Ovine Brain
collected in the UK Scrapie Surveillance Programme.
Atypical scrapie cases in German and French sheep carrying
the scrapie susceptible and also the presumably resistant PrP alleles
Efficient propagation of Nor98 scrapie agent in transgenic mice
Fabrizio Tagliavini
Roy Jackman
Anne Buschmann
Sylvie L. Benestad
Wednesday,May 26th 2004
8h 30 - 10h 10 Diagnostics
Chair: H. Laude / R. Gabizon
08:30
08:50
09:10
09:30
09:50
Differential diagnosis of BSE in sheep through rapid in vitro tests.
Monoclonal antibodies diagnosing disease-associated conformations
of non-infectious prion protein
Detection of Transmissible Spongiform Encephalopathies in blood
A novel blood based TSE diagnostic test
Selective and efficient immunoprecipitation of PrPsc can be mediated
by non-specific interactions between monoclonal antibodies and SAFs
Emmanuel Comoy
Carsten Korth
Stuart Wilson
Bruno Oesch
Jacques Grassi
10h 40 - 11h 30 Diagnostics
Chair: M. Beekes / P. Parchi
10:40
11:00
11:15
Surrogate Makers: Host responses to TSE infection
Urine from Scrapie infected hamsters comprises
low levels of prion infectivity
Detection of peripheral PrPsc in non-human primates infected
with iatrogenic, sporadic and variant CJD agent
Laura Manuelidis
Ruth Gabizon
Christian Herzog
11h 30 - 12h 00 Plenary Session
Chair: H. Kretzschmar / S. Benestad
11:30
CWD, iatrogenic CJD: An Overview
Paul Brown
15h 00 - 16h 45 Plenary Session
Chair: H. Kretzschmar / J. Badiola
15:00
15:30
16:00
The Amyloid Theories of Alzheimer’s and Creutzfeld-Jakob Diseases :
Devising Specific Therapeutic and Diagnostic Strategies
Protein Mysfolding, Molecular Evolution and Human Disease
How the Mad Cows changed America.
Collin Masters
Chris Dobson
Stanley B. Prusiner
17h 15 - 18h 30 Decontamination
Chair: D. Riesner / M. Pocchiari
17:15
17:30
17:50
18:10
Iatrogenic CJD and Growth-Hormone:
Conclusion of INSERM Expert Committee
Acid inactivation of Prions Efficient at elevated temperature or high acid concentration
Mechanisms of Heat and Chemical Inactivation of TSE Models
New Perspectives in the Decontamination of Prions
Henry Baron
Detlev Riesner
Robert Somerville
Guillaume Fichet
025
Thursday,May 27th 2004
8h 30 - 9h 50 Therapy I
Chair: F. Tagliavini / A. Williams
08:30
08:50
09:10
09:30
Prion Protein Conversions, TSE Infections, and Therapeutics
Where does disease-associated prion protein deposit in the human brain?
A left-handed, parallel beta-helical architecture as a model for the structure
of the scrapie prion protein
Neurotoxic PrP conversion intermediates.
Byron Caughey
Matthias Preusser
Holger Wille
Steve Simoneau
10h 20 - 11h 25 Therapy II
Chair: G. Forloni / I. Ferrer
10:20
10:35
10:55
11:15
11:35
Neuronal cell death triggered by PrPC signaling in vivo.
A genetic assay for cellular prion proteins: N-terminal determinants
in PrPC facilitate neuroprotection against pro-apoptotic action of Doppel
BAX Deletion rescues neuronal loss but not neurological symptoms
in a transgenic model of inherited prion diseases
Role of Prion Proteins and their Receptor and Co-Receptor Molecules
in the Prion Life Cycle
Glycogenome expression and prion diseases :New targets for therapy?
Anthony Williamson
David Westaway
Roberto Chiesa
Stefan Weiss
Agnès Barret
15h 00 - 16h 30 Therapy III
Chair: H. Budka / S. Lehmann
15:00
15:20
15:40
15:55
16:10
Human prion disease therapeutics: from molecular strategy
to clinical evaluation
Prion infected cell cultures: from basic to applied research
New anti-prion drugs discovered by SIFT-based high-throughput
and high-content screening
A novel generation of heparan sulfate mimetics for the treatment
of prion diseases
Active immunisation of C57BL/6 mice with PrP peptides
associated with oligo-CpG delays prion progression .
John Collinge
Sylvain Lehmann
Uwe Bertsch
Karim T. Adjou
Martine Bruley Rosset
17h 00 - 18h 15 Therapy IV
Chair: H. Budka / T. Slaviadis
17:00
17:20
17:35
18:00
026
Novel targets for experimental therapy and prophylaxis against
prion infections
Re-routing intracellular trafficking of prion protein in neurones:
a novel therapeutic approach?
Poster Awards
Conclusions
Hermann M. Schätzl
Alun Williams
Friday,May 28th 2004
Commissariat à l’Energie atomique in Fontenay aux Roses
09:00 - 10:00
Press Conference
10:00 - 12:00
Official Inauguration of a New Neuroprion Research Platform
with special guest : Philippe Busquin
(Commissioner for Research of the EU)
restricted to representatives of NoE members
14:00 - 18:00
Open-Visit of the New Neuroprion Research Platform
15:00 - 17:00
Workshop : Science and Communication
Funding of Prion Research within Europe and America
027
Oral
Presentations
Oral-01
A GENERAL FRAMEWORK FOR PRION SAFETY
RISK ASSESSMENT
FRANCK GUARNIERI & DEIRDRE MURRAY
ARMINES Ecole des Mines de Parism Pôle Cindyniques
The purpose of this paper is to describe the prion risk assessment framework that is being promoted by the European
Network of Excellence NEUROPRION. It makes visible how NEUROPRION intends to conduct prion risk
assessments within the context of risk analysis. In the framework adopted, risk analysis is a process consisting of risk
assessment, risk management and risk communication. Each item is discussed in the paper, with a special emphasis on
risk assessment which provides the scientific basis for risk analysis.
Risk assessment methodologies have evolved generally somewhat separately among the different disciplines of food
safety, animal health and human health. An objective of NEUROPRION is to promote harmonisation of risk assessment
methodologies across several disciplines. Therefore, the following framework provides a basic model for prion risk
assessment, that merges the terminology and principles of traditional health risk assessment, with animal and human
health approaches.
The present paper will discuss several topics such as definition of risk analysis, steps in risk assessment and different
approaches to risk assessment. Some inputs will be also given on possible ways to contribute to prion risk management
and communication.
031
Oral-02
QUANTITATIVE ASSESSMENT OF THE RISK OF
TRANSMISSION OF BOVINE SPONGIFORM
ENCEPHALOPATHY (BSE) BY TALLOW-BASED
CALF MILK REPLACER
LARRY G. PAISLEY AND JULIE HOSTRUP-PEDERSEN
Danish Institute for Food and Veterinary Research, Department of Epidemiology and Risk Analysis
A Monte Carlo simulation model was constructed to assess the risk of BSE transmission to calves by calf milk-replacer
(CMR). We assumed that any BSE infectivity in the CMR would be associated with the allowable levels of impurities
in tallow used to manufacture the milk-replacer. Scenarios using three different levels of impurities, six different
distributions of the BSE infectivity titers of CNS tissues and with and without inclusion of specified risk
material(SRM)were modelled. Our results suggest that tallow-based CMR could have been responsible for some BSE
infections in nearly all simulations. The reduction in the allowable impurities in tallow and the exclusion of SRM have
greatly reduced -- but have not eliminated --the risk of BSE transmission by CMR. Sensitivity analysis showed that the
distribution of the BSE infectivity titer had the greatest influence on the risk. The results of the simulations are
associated with much uncertainty.
032
Oral-03
EPIDEMIOLOGICAL ANALYSIS OF DATA FOR
SCRAPIE IN GREAT BRITAIN
T.J. HAGENAARS (1,2), C.A. DONNELLY (1), AND N.M. FERGUSON (1)
1 Department of Infectious Disease Epidemiology, Imperial College London, 2 Quantitative Veterinary Epidemiology,
Division of Infectious Diseases, Wageningen University and Research Center, AB Lelystad
In recent years, the control or eradication of scrapie and any other transmissible spongiform encephalopathies (TSEs)
possibly circulating in the sheep population has become a priority in Britain and elsewhere in Europe. A better
understanding of the epidemiology of scrapie would greatly aid the development and evaluation of control and
eradication strategies. Here we bound the range of key epidemiological parameters using a combination of relatively
detailed pathogenesis and demography data, more limited data on susceptibility and incubation times, and recent survey
data on scrapie incidence in Great Britain. These data are simultaneously analysed using mathematical models describing
scrapie transmission between sheep and between flocks. Our analysis suggests that occurrence of scrapie in a flock
typically provokes changes in flock management that promote termination of the outbreak, such as the adoption of
selective breeding, and that a large fraction of cases (possibly over 80%) goes undetected. We show that the data
analysed are consistent with the reproduction number of scrapie lying in the range 1.5-6, consistent with previous
epidemiological studies.
033
Oral-04
PREVALENCE OF INFECTION IN CULL SHEEP
FROM SCRAPIE-AFFECTED FLOCKS IN GREAT
BRITAIN
S. C. TONGUE*, M. M. SIMMONS**, P. WEBB**, N. BUSK**, S. GUBBINS*
* Scrapie Epidemiology Group and ** Pathology Department, Veterinary Laboratories Agency - Weybridge,Woodham
Lane, New Haw, Addlestone, Surrey, KT15 3NB. UK.
Field studies of the epidemiology of scrapie have necessarily focused on clinical disease, although the ultimate aim of
disease control is to reduce and eliminate infection. Estimates for the prevalence of infection are available (e.g. from
abattoir and fallen stock surveys), but it is difficult to relate these to the occurrence of clinical disease. Consequently, it is
important to collect information on the prevalence of infection in scrapie-affected flocks with known incidence of clinical
disease.
The aim of this study was to investigate the prevalence of infection within scrapie-affected flocks that had known prion
protein (PrP) genotype profiles and recorded within-flock epidemics, including clinical incidence, case profiles and
control methods used. More than 400 cull sheep of known age, PrP genotype and origin were purchased from 14
scrapie-affected flocks. A range of tissues (obex, tonsils, submandibular and medial retro-pharyngeal lymph nodes) were
collected post mortem from each animal and examined using immunohistochemistry (IHC). 27 animals from seven flocks
were confirmed positive by IHC. Preliminary analysis of these results gives an overall estimate for the prevalence of
infection in the cull sheep of 6.6% (95% C.I. 4.4-9.5%), while the prevalence in individual flocks varied from 0% to
15.4%. The PrP genotypes of the positive sheep were ARQ/ARQ, ARR/VRQ, ARQ/VRQ, ARH/VRQ and
VRQ/VRQ. Considerable variation was seen between flocks, reflecting differences in the stage of within-flock epidemics
and in the age and genotype profiles of the flocks.
This work was funded by the Department for the Environment, Food and Rural Affairs, (Defra).
034
Oral-05
PREVENTION OF TSES IN ANIMALS THROUGH
PRP GENETIC SELECTION: NEW PRP
HAPLOTYPES AND THE PRINCIPLES OF
DISASSOCIATION.
WILFRED GOLDMANN 1, STEWART BURGESS 1, GERRY O’NEILL 1, FIONA HOUSTON 2, JIM FOSTER 1, MATTHEW
BAYLIS 2 AND NORA HUNTER 1
1 Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, 2 Compton Laboratory, Institute for Animal Health,
Compton, Berkshire,
The crucial genetic elements in the host response to prion diseases are the level of expression of the prion protein in
tissues relevant to the TSE pathogenesis and the primary sequence of the prion protein. Our approach to investigate the
PrP genetics in ruminants is therefore twofold: (1) to identify rules governing the PrP gene regulation and (2) to
understand the mechanisms that lead to the association of PrP polymorphisms with disease. Our aim is to define general
principles of PrP genetics which help to eradicate animal TSEs, such as scrapie or Chronic Wasting Disease by breeding
for resistance.
We have studied conserved sequence motifs in the PrP gene promoter. Using gel shift assays to analyse the binding of
nuclear proteins isolated from ovine cell cultures and tissue samples to radiolabeled DNA we could show for the first
time the specific binding of transcription factors YY1 and E4BP4 to the PrP promoter. The binding of YY1 was
dependent on the C substitution in the ovine promoter position 5382, whereas E4BP4 binding was enhanced for the
ovine promoter compared with the human and murine promoter. The T/C-5382 polymorphisms was linked to the
A136R154Q171 allele in UK Cheviot sheep. We propose a model of differential promoter regulation, which may have
relevance for the different susceptibility to TSEs observed for ARQ homozygous sheep.
We also analysed the ARQ allele of ruminants for additional amino acid substitutions and can add new variants to the
ever increasing number of PrP alleles (total sheep ARQ-Xnnn variants: 24, total goat ARQ-Xnnn variants:17). Our
analysis confirms that natural scrapie in ARQ/ARQ animals is not caused by pathogenic amino acid substitutions. More
importantly, we show that the ovine ARQ-L168 PrP is associated with survival after experimental BSE challenge and
may be a new target allele for genetic selection.
035
Oral-06
EXPERIMENTAL EVIDENCE ON MATERNAL
TRANSMISSION OF SCRAPIE AND BSE IN SHEEP
NORA HUNTER AND JIM FOSTER
Institute for Animal Health, Neuropathogenesis Unit, Edinburgh
It is the aim of many countries to eradicate TSEs in sheep by breeding and selection for resistant PrP genotypes.
However the rate at which clinical disease disappears is dependent on the natural routes of transmission of infection
between animals, something which is poorly understood. It is one of the old dogmas of natural scrapie that one route of
spread of infection is from ewe to lamb: maternal transmission. In a series of experimental studies of both BSE and
SSBP/1 scrapie lasting more than 8 years, we have evidence for a low level of maternal transmission of scrapie but this
does not seem to be the case with BSE. In our BSE studies none of the susceptible offspring of experimentally infected
sheep has itself developed BSE. The results of these studies will be presented and the implications discussed.
036
Oral-07
STRAIN TYPING STUDIES OF HUMAN AND
ANIMAL TSES
MOIRA BRUCE, AILEEN BOYLE, IRENE MCCONNELL, DIANE RITCHIE, MARK HEAD & JAMES IRONSIDE
Institute for Animal Health, Neuropathogenesis Unit, Edinburgh and National CJD Surveillance Unit, Western
General Hospital, Edinburgh
Strain typing methods, based incubation periods and neuropathology in non-transgenic mice, have been used for many
years to explore epidemiological links between TSEs in different species. BSE from cattle produces a characteristic
disease pattern on primary transmission to mice, which is also seen in transmissions from a range of experimentally and
naturally BSE-infected animal species. The disease patterns in mice challenged with sheep scrapie have been variable,
with no individual source resembling BSE. However, transmissions of vCJD from humans to mice have given closely
similar disease characteristics to those of BSE. In contrast, sCJD rarely produces clinical disease within the lifespan of
mice, although a high proportion of challenged mice in some sCJD transmissions develop TSE neuropathology in old
age. Type 1 PrPSc sCJD sources have transmitted most readily to mice. We have extended these studies by
characterising TSE strains isolated on serial mouse passage. For BSE from cattle, two distinct mouse-passaged strains
have been isolated from each source: 301C and 301V by passage in PrP-a and PrP-b mice respectively. The strains
isolated from vCJD by passage in PrP-a and PrP-b mice closely resemble 301C and 301V, providing further compelling
evidence of a link between vCJD and BSE. There has been no overlap in the mouse-passaged strains derived from BSE
and natural scrapie sources, collected before or after the start of the BSE epidemic. However, despite the variable
results seen on primary transmission of natural scrapie, only a limited number of serially mouse-passaged strains has
been isolated from these sources. This suggests that strain variation in field scrapie may be less extensive than previously
suspected. The mouse passaged strains isolated from sCJD sources are distinct from the strains isolated either from
vCJD or from any animal TSE.
037
Oral-08
THE EUROPEAN CJD SURVEILLANCE SYSTEM:
CURRENT CONCERNS
RG WILL
National CJD Surveillance Unit, Western General hospital, Edinburgh
A surveillance system for all forms of human TSE was started in a number of European countries in 1993 and has now
been extended to all member states. The main current aim of this study is to identify and characterise cases of variant
CJD, but the study has also allowed pooling of data to allow detailed analyses of the distribution and characteristics of
all forms of human TSE.
The numbers of cases of vCJD in the UK are higher than in any other country although cases of this condition have
been identified at low levels in a number of other countries. Epidemiological data is consistent with the hypothesis that
BSE is the cause of vCJD, but despite extensive human dietary exposure to the BSE agent, the epidemic of vCJD in
the UK appears to be in decline.
Concern that some cases of sporadic CJD might also be caused by a zoonosis has been raised by recent evidence
including the identification of a novel form of BSE in Italy. Data from the European CJD surveillance system do not
support this hypothesis, but continued sytematic study of human TSEs remains important.
038
Oral-09
INCREASED PROPORTION OF GENETIC PRION
DISEASE IN HUNGARY
KOVACS GG, MAJTENYI K
National Institute of Psychiatry and Neurology, H-1021 Budapest,
.
The proportion of cases defined as familial prion disease shows considerable variability in different epidemiological
surveys. Usually, familial Creutzfeldt-Jakob disease (CJD) is thought to comprise 10-15% of all CJD cases. Recent
observations point out that CJD may have a genetic etiology despite the lack of family history, also supporting the more
reliable term “genetic” instead of “familial”. To evaluate the proportion of genetic CJD cases we have analysed a 10-yearperiod between 1994-2003 of definitive CJD cases. At this stage 23.6% of the 106 examined cases is definitive genetic
CJD according to recent WHO criteria. However, genetic analysis of 4 cases is still in progress. The majority of genetic
cases carried the E200K mutation of the prion protein gene (PRNP) with predominance of methionine homozigosity at
codon 129 and no valine homozygote was found. Of the 25/106 genetic CJD case, only 15 had a family history of CJD,
while the remaining cases presented as sporadic disorders. We noted the increased ratio of relatively young patients in
our series since 31% was younger than 55 years. Neuropathological examination demonstrated that additional cases,
lacking full length PRNP analysis, show a peculiar stripe-like pattern of PrP immunoreactivity perpendicular to the
surface of the molecular layer in the cerebellum which is suggested to be related to the E200K mutation. Thus, the
proportion may be considered to be even higher than 23.6%. We conclude that lack of family history is not relevant to
exclude genetic CJD and that Hungary represents a higher risk for genetic CJD similarly to Slovakia.
This work was supported partly by the EU QoL project SEEC-CJD and by ETT69/2003 for GGK.
039
Oral-10
VARIANT CREUTZFELDT-JAKOB DISEASE IN THE
UK 1995-2003: A CASE-CONTROL STUDY OF
POTENTIAL RISK FACTORS
WARD HJT(1), EVERINGTON D(1), COUSENS S(2), SMITH-BATHGATE B(1), LEITCH M(1), HEATH C(1), COOPER S(1),
KNIGHT RSG(1), SMITH PG(2), WILL RG(1)
1 National Creutzfeldt-Jakob Disease Surveillance Unit, Edinburgh, UK, 2 London School of Hygiene & Tropical
Medicine, London, UK
Variant Creutzfeldt-Jakob disease (vCJD) was first reported in 1996 in the United Kingdom. There is now strong
evidence that vCJD is caused by exposure of humans to the same agent that causes bovine spongiform encephalopathy
(BSE). Oral ingestion of food contaminated by the BSE agent would appear the most likely method by which the
human population acquired the BSE agent, with mechanically recovered meat suggested as one means by which the
BSE agent may have been introduced into food. However, direct evidence for the dietary hypothesis is lacking and other
potential routes of primary or secondary transmission, including iatrogenic transmission through surgery, medicines or
related products, transmission through occupation, and transmission through other less obvious routes also need to be
considered.
World-wide a number of studies have investigated risk factors for sporadic CJD, however, this is the first study to
investigate risk factors for vCJD. Since 1996 people with vCJD in the UK have been identified prospectively by the
NCJDSU and definite and probable cases recruited into a case control study. Three types controls were recruited for
each case: from the same primary care practice, the same referring hospital and the community. A close relative of the
patient or control was interviewed using a standard semi-structured questionnaire.
Here we present the findings of a recent analysis of a wide-range of potential risk factors for vCJD, including dietary,
medical, occupational and exposure to animals and animal products. This study includes 143 cases of vCJD occurring in
the UK (to November 2003), where 94% of all cases worldwide have occurred to date.
040
Oral-11
USE OF NON HUMAN PRIMATES FOR CJD RISK
ASSESSMENT
LASMÉZAS C, HERZOG C, SALËS N, ETCHEGARAY N, RIVIERE J, DESLYS JP
Commissariat à l’Energie Atomique, Departement de la Recherche Medicale BP 6, Fontenay-aux-Roses , France
The occurrence of variant Creutzfeldt-Jakob Disease (vCJD) linked to human contamination with the BSE agent by
tainted food has posed a new challenge to medical authorities in charge of protecting human health against iatrogenic
disease transmission. The specific concerns raised by vCJD (large tissue distribution of the infectious agent) have been
compounded recently by the detection of even very low amounts of PrPres in muscle in different species and in
peripheral organs of sCJD patients.
The context of uncertainty regarding the number of vCJD patients including possible secondary cases due to
interhuman transmission, the infective dose for humans as well as the exact pathogenesis of the infection renders risk
assessments very difficult. Using the non-human primate species cynomolgus macaque which has been shown to
faithfully reproduce several of the characteristics of vCJD when inoculated with the BSE agent, we aim at laying the
foundations of an experimental model of the human disease to provide basic data useful to this endeavour.
We will report over the efficiency of the various routes of transmission of the BSE agent in primates and some aspects of
tissue distribution of PrPres after intravenous or oral infection. In depth analyses of tissue distribution of PrPres using a
highly sensitive detection method are the subject of the accompanying abstract by Herzog et al. Specificities of the
peripheral replication phase of the BSE agent in the lymphoid tissues of primates will be described. A picture will be
drawn of the advances made with regards to our understanding of the cattle to human interspecies transmission of the
BSE agent and disease pathogenesis.
041
Oral-12
HOW DOES HOST PRP CONTROL TSE DISEASE?
JEAN MANSON, R BARRON, N TUZI, H BAYBUTT, M BISHOP, ENRICO CANCELLOTTI, P HART, L JAMIESON, L
AITCHISON, E GALL, B BRADFORD, D KING
Institute for Animal Health, Neuropathogenesis Unit, Edinburgh., University of Edinburgh., 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.
We have introduced mutations into the Prnp gene which prevent glycosylation at each or both of the two N-linked
glycosylation sites of PrP and are using TSE infection of these mice to investigate the role of PrP glycosylation in strain
targeting and strain determination. We have investigated the role of the sequence of the host PrP gene in determining
susceptibility by inserting point mutations or replacing the murine PrP gene with that of human or bovine PrP. This has
produced a model of TSE disease which contains high levels of infectivity in the absence of PrPSc and we are using this
model to determine 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.
042
Oral-13
INVESTIGATING NATURAL SCRAPIE STRAIN
DIVERSITY IN OVINE PRP TRANSGENIC MICE
ANNICK LE DUR1, TANH LAN LAI1, NATHALIE BESNARD2, AUDE LAISNE1, GAÎLLE TILLY2, THIERRY BARON3,
DIDIER VILETTE1, VINCENT BERINGUE1, JEAN-LUC VILOTTE2, HUBERT LAUDE1
INRA Virologie Immunologie Moleculaires 1 and Genetique Biochimique et Cytogenetique 2, AFSSA, Virologie-ATNC
3, France
Serial transmission to a panel of congenic mouse lines, where characteristics of incubation time and brain vacuolation
areas give distinct patterns, is the current way of investigating strain biological diversity in natural sheep scrapie.
Following inoculation of natural scrapie isolates to mice, the disease commonly requires 1 or 2 years to develop, and a
proportion of isolates fail to be transmitted. As a consequence, a limited number of isolates have been studied yet, and
the extent of the natural scrapie strain variation remains largely unexplored.
In an effort to develop an improved model for transmission of sheep scrapie, we have expanded a series of transgenic
mice (tgOv) that express the VRQ (Val136-Arg154-Gln171) allele of ovine PrP on a null mouse PrP background. This
allele is associated with the highest susceptibility to the disease in many flocks. Mice of the tgOv line tg338, expressing
high PrP level (~ 10-fold that in sheep brain), were inoculated intracerebrally with more than 40 scrapie sources
including sheep of distinct PrP genotypes and of various geographical areas in Europe. In contrast with earlier studies
using non-transgenic mice, all isolates transmitted successfully, generally with a ~ 100% attack rate. However, the
incubation time to death differed widely among the isolates, ranging from 70 to 750 days. More than half of the isolates
transmitted in <= 200 days post-inoculation. No correlation was found between the donor PrP genotype and the
transmission efficiency. The isolates were found to distribute in distinct groups according to the molecular signature of
brain PrPsc and the survival time on secondary passage, and all differed from BSE agent experimentally transmitted to
sheep. Altogether these results argue that tgOv mice overexpressing the VRQ allele may provide a valuable alternative
to conventional mice for field scrapie strain characterisation.
043
Oral-14
ANALYSIS OF EXPERIMENTAL OVINE BSE AGENT
THROUGH TRANSMISSION STUDIES TO
TG(OVPRP4) MICE EXPRESSING THE OVINE
PRION PROTEIN
A. BENCSIK1; S. LEZMI1; S. PHILIPPE2; L. CHOUAF-LAKHDAR1; C. CROZET1; J. SAMARUT3; T.
BARON1
1.Unite ATNC, AFSSA, Lyon, 2.Unite Epidemiologie, AFSSA, Lyon, 3.ENS Lyon, Laboratoire de Biologie
Moleculaire et Cellulaire, Lyon,
To study experimental ovine BSE, we infected through peripheral routes (intraperitoneal or intrasplenic) 4 Lacaune
sheep (2 ARQ/ARQ versus 2 ARR/ARR genotype) using a French BSE brain sample. Only ARQ/ARQ animals
developed scrapie like symptoms, the first one died at 672 days post inoculation (d.p.i.), the second one at 1444 d.p.i..
Abnormal prion protein (PrP) was detected in each case in the brain and in some peripheral organs such as lymphoid
organs and enteric nervous tissues. To further analyse experimental ovine BSE compared to natural sheep scrapie, we
inoculated this reference material to the Tg(OvPrP4) transgenic mouse line expressing the ovine prion protein
(A136R154Q171) in neurons, as it is a sensitive model for transmission studies of ovine spongiform encephalopathies.
This model revealed unexpected potentialities in the discrimination of BSE agent among sheep flocks, since infection
with experimental ovine BSE from the first sheep (672 d.p.i.) of ARQ/ARQ genotype leads to the occurrence of
numerous typical florid plaques in Tg(OvPrP4) mice brain. By contrast, florid plaques were never detected in any other
mice (Tg or C57Bl/6) or in these Tg(OvPrP4) mice inoculated with different scrapie isolates or BSE sources. Thus, the
presence of florid plaques could be considered in our model as a criterion to discriminate BSE from natural scrapie in
sheep.
To further explore this property, Tg(OvPrP4) mice were also inoculated with the second experimental ovine BSE (1444
d.p.i.) of ARQ/ARQ genotype. We will present the first results of this transmission study analysing the incubation
periods, the PrP biochemical and histopathological signatures and the distribution of abnormal PrP through a
comparative study using a PET-blot analysis. We will also explore the existence of this property in this model, in
particular when instead of brain tissue, a peripheral tissue accumulating abnormal PrP is used as the inoculum.
044
Oral-15
INITIAL FINDINGS ON EXPERIMENTAL
TRANSMISSION OF ATYPICAL BSE TO MICE
F. TAGLIAVINI (1), R. CAPOBIANCO (1), C. CASALONE (2), M. MANGIERI (1), S. SUARDI (1), L. LIMIDO (1), M.G.
BRUZZONE (1), C. CORONA (2), P.L. ACUTIS (2), D. GELMETTI (3), L. CAPUCCI (3), G. ZANUSSO (4), S. MONACO (4),
M. CARAMELLI (2)
(1) Istituto Nazionale Neurologico Carlo Besta, Milano, (2) Istituto Zooprofilattico Sperimentale del Piemonte, Liguria
e Valle d’Aosta,
(3) Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna, (4) Clinica Neurologica dell’Universite
di Verona, Italy
A novel BSE phenotype has been recently identified in Italy, marked by the presence of PrP-amyloid plaques and a
PrPres type of lower molecular mass with predominance of the monoglycosylated species. These data suggest that
typical BSE and the amyloidotic form of BSE (termed BASE) may be related to different prion strains. To investigate
this issue we set up experiments of transmission to SJL, RIII, C57Bl and VM mice, challenged both i.c. and i.p. with
BSE, BASE and vCJD brain homogenates. At the time of writing, the experiments have been in progress for 325 days
and initial results are available for SJL mice. All SJL mice injected with BSE and vCJD have developed clinical signs
of disease with mean incubation periods of 274 ± 5.3 and 272 ± 4.0 days (± s.e.m.) respectively, while the mice
challenged with BASE are still free of neurological symptoms. This clinical difference between groups is paralleled by a
difference in the appearance of abnormalities at Magnetic Resonance Imaging. While BSE- and vCJD-infected mice
showed T2 signal hyperintensity in the diencephalon 304 days after infection, no changes were observed in mice
challenged with BASE. Neuropathologic examination of BSE- and vCJD-infected mice sacrificed at the terminal stage
of disease showed PrPres immunoreactivity in form of diffuse and focal deposits in the somatosensory cortex (middle
layers and subpial region), striatum, hypothalamus and medulla. Immunoblot analysis of brain homogenates showed a
PrPres profile with a diglycosylated dominant pattern similar to that of BSE. Conversely, no PrPres was detected by
immunohistochemistry or Western blot analysis in the brain of three SJL mice challenged with BASE, and sacrificed
304 and 318 days after infection for control. Noteworthy, PrPres was present in germinal centres of the spleen of both
BSE/vCJD- and BASE-infected animals. These data support the view that BSE and BASE are caused by prion strains
with different biological properties.
045
Oral-16
UNUSUAL PRION PROTEIN CHARACTERISTICS IN
ISOLATES FROM OVINE BRAIN COLLECTED IN
THE UK SCRAPIE SURVEILLANCE PROGRAMME.
R.JACKMAN, L. THORNE, S.EVEREST, D.BARNICLE, J. EDWARDS
Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK
During 2002-2003, the screening for abnormal prion protein by the BIO-RAD PrP ELISA (PlateliaTM) in caudal
medulla from cull sheep detected 50 positive cases in 32,200 animals tested. However, half of these survey cases were
apparently normal when examined by histopathology for vacuolation or by immunohistochemistry and/or confirmatory
Western blotting (WB) methods for the presence of disease-associated abnormal PrP (PrPsc). These results are
therefore unconfirmable and have been described as “Unclassified” or “Anomalous” Scrapie Survey results. Similar data
is arising from the current 2003/2004 Survey. These unconfirmed positive occurred in a variety of PrP genotypes but the
majority of animals were ARR/ARR, AHQ/AHQ homozygotes or ARR/AHQ heterozygotes, regarded as relatively
resistant to scrapie. It was therefore essential to determine whether the positive signals obtained by the PlateliaTM
screening process were non-specific (non-prion) in nature, or represented an unusual form of the prion protein. Our
initial studies on the “Unclassified” samples were targeted at the hypothesis that there is a form of PrP present which,
whilst more resistant to Proteinase K than is PrPc, is also less resistant to proteolysis than “classical” PrPsc - based on
the observation that the Platelia utilises less PK to eliminate PrPc than other, traditional, methods. Analysis of the
Platelia extracts by PAGE / Western immunoblotting has clearly demonstrated the presence of such a partially PKresistant prion protein. However, these results do not indicate any association between this unusual form of the prion
protein and TSE infectivity or disease.
046
Oral-17
ATYPICAL SCRAPIE CASES IN GERMAN AND
FRENCH SHEEP CARRYING THE SCRAPIE
SUSCEPTIBLE AND ALSO THE PRESUMABLY
RESISTANT PRP ALLELES
A. BUSCHMANN1, J.-N. ARSAC2, A. BENCSIK2, J.-Y. MADEC2, U. ZIEGLER1, G. ERHARDT3, G. L¸HKEN3, T. BARON2,
M.H. GROSCHUP1
1. Federal Research Centre for Virus Diseases of Animals, Isle of Riems, Germany, 2. AFSSA-Lyon, Unite VirologieATNC ª, Lyon France, 3. Department of Animal Breeding and Genetics, Justus-Liebig University of Giessen, Germany
Since the introduction of an intensified scrapie surveillance in 2002, the number of notified scrapie cases has increased
dramatically throughout the European Union. The testing is carried out using one of the four BSE rapid tests that have
passed the EU evaluation. These tests include an indirect ELISA, a colorimetric sandwich ELISA, a chemiluminescent
sandwich ELISA, and a Western blot. In retrospective examinations among the German and French scrapie cases, it
became obvious that a substantial amount of the samples (up to 40%) could not be detected using three of the four
available rapid tests. These nonuniform reaction patterns may be determined by differences in the antigenicity and
physicochemical characteristics of the pathological prion protein that is used as a diagnostic marker. We propose that this
indicates the existence of at least one atypical scrapie isolate / strain that has lately been observed in several European
countries in the frame of the TSE monitoring program. One of these atypical strains is the “Nor98” strain that has been
described in Norway. In further studies on such atypical ovine samples, we found a distribution pattern of protease
resistant PrP that varies distinctly in terms of localisation and the type of deposition from that of PrPSc in classical
scrapie cases. In classical scrapie, the major amount of PrPSc is detectable in the obex region as granular and plaque-like
depositions. Interestingly, this atypical reaction pattern was not directly linked to a specific allele of the ovine prion
protein. We even detected such atypical cases in two German and three French sheep carrying the PrPARR allele in
homozygous form that is supposed to mediate a relative resistance to scrapie. However, the pathological prion protein of
none of these phenotypically different sheep samples showed biochemical properties that may indicate a BSE infection.
Similar divergent results have never been observed during rapid testing of bovine samples for BSE.
047
Oral-18
EFFICIENT PROPAGATION OF NOR98 SCRAPIE
AGENT IN TRANSGENIC MICE
BENESTAD S.L.(1), LEDUR A.(2), ANDRÈOLETTI O.(3), LANTIER F.(4), BRATBERG B.(1), SARRADIN P.(4), LAUDE H.(2)
1.National Veterinary Institute, Oslo, Norway, 2.Unite de Virologie Immunologie Moleculaires, INRA Jouy-en-Josas,
France, 3.UMR959 INRA-ENVT, Toulouse,France, 4. INRA PII, Nouzilly, France
Since 1998, 35 atypical sheep scrapie cases, designated Nor98, have been identified in Norway. They had unique
epidemiological, clinical and histo-pathological features compared to the classical scrapie cases (1). In particular, neither
spongiform changes nor PrPSc, as demonstrated by IHC, could be observed in the DMVN at the level of the obex. The
unusual biochemical profile of PrPres was characterised by a fast migrating band around 12kD in western blot.
Moreover, 20 of the 35 Nor98 cases carried at least one AHQ allele of Prnp.
These observations raise important issues with regard to the surveillance and control of sheep scrapie. A crucial question
is whether any transmissible agent is associated with Nor98 cases. Indeed, a first series of experiments aiming to transmit
disease by inoculation to several conventional mouse lines were inconclusive.
In this study we have examined the susceptibility of the tg338 transgenic mouse line to intracerebrally inoculated Nor98
material. Tg338 mice overexpress the VRQ allele of ovine Prnp on a mouse PrPO/O background, and were previously
reported to enhance the transmission of natural sheep scrapie isolates to mouse (2). Two brain homogenates derived
from affected sheep of either AHQ/AHQ or ARQ/ARQ genotype were tested. On primary transmission all mice died
with scrapie signs with a mean survival time around 300 days. WB analysis revealed the accumulation in the brain of
PrPres with the atypical Nor98 profile, while no PrPres could be detected in the spleen, similar to that observed in
Nor98-affected sheep. Secondary passages in tg338 mice led to similar findings, consistent with the view that Nor98
agent retained its original strain phenotype upon transmission to VRQ mice. Altogether these data demonstrate that
Nor98 cases are caused by an authentic, infectious TSE agent with characteristics unprecedented in sheep.
1)Benestad et al., Vet.Rec. 2003, 153: 202-8; 2)Vilotte et al., J.Virol. 2001, 75: 5977-84
048
Oral-19
DIFFERENTIAL DIAGNOSIS OF BSE IN SHEEP
THROUGH RAPID IN VITRO TESTS.
EMMANUEL E. COMOY, FREDERIC AUVRE, DOMINIQUE MARCE, SOPHIE FREIRE, EVELYNE CORREIA AND JEANPHILIPPE DESLYS.
CEA/DSV/DRM, Groupe d’Innovation Diagnostique et Therapeutique sur les Infections - Prions, Fontenay-aux-Roses,
France.
The risk of BSE in sheep and goats with its major health and economic consequences has urged the need of rapid strain
typing methods able to warrant that animals found prion-positive are not infected with BSE. Moreover the discovery of
atypical prion strains in cattle reinforces the need of adapted rapid discriminative tests.
We previously developed a rapid and sensitive biochemical diagnostic test for BSE in cattle which sensitivity turned out
to be close to the mouse bioassay*. Since, we have developed a western blot as sensitive as the screening test based on
ELISA as a confirmation test. We now propose a strategy which uses first western blot and second immunohistology as
a two-steps method for strain typing. Long-term inoculation to animal laboratory could then be limited to unusual cases
for full characterisation.
To this purpose, we exploited the differences in resistance to proteolysis of the prion protein depending on prion strains.
The combination of controlled protease digestion of PrPres with the use of several antibodies has allowed us to
selectively distinguish BSE strain from scrapie strains in sheep. Moreover on the same basis, we were able to
differentiate scrapie strains. In order to increase the efficiency of strain typing developed for western blot, we have used
immunohistochemistry which provides additional information on the localisation and the shape of the PrPres aggregates.
This new protocol has been adapted to be used on frozen samples provided for biochemical analysis.
The combination of these methods, the robustness of which has now to undergo trial evaluations, clearly offers the
possibility of a very efficient prescreening of ruminant samples. Once validated such a strategy could constitute, after a
first high-throughput screening of TSE-contaminated animals, a system to warrant an additional level of protection for
the consumer and the industry.
*Deslys, Comoy et al, Nature, 2001
049
Oral-20
MONOCLONAL ANTIBODIES DIAGNOSING
DISEASE-ASSOCIATED CONFORMATIONS OF NONINFECTIOUS PRION PROTEIN
S. RUTGER LELIVELD1, RALF KLINGENSTEIN1, VISHWANATH R. LINGAPPA2, CARSTEN KORTH1
1Institute for Neuropathology, Heinrich Heine University of Duesseldorf, Germany, 2Departments of Physiology and
Medicine, University of California, San Francisco, USA
Simple models of prion replication hypothesize that the normal form of the prion protein, PrPC, is converted into PrPSc
through both a direct interaction between these conformational isoforms and contact to assisting molecules. In vitro
translation of PrP mRNA has established that PrPC can adopt three topological orientations corresponding to three
different conformations. Thus, during synthesis, the PrP polypeptide can be inserted N?C or C?N into the ER
membrane, yielding CtmPrP or NtmPrP, respectively, or fully translocated, yielding SecPrP. CtmPrP is associated with
neurodegeneration when mutants favoring the CTM topology are produced or prion infection occurs. Ligands
specifically detecting CtmPrP would thus indicate the presence of prion infection in wild-type animals or humans.
We have established a protocol of screening hybridoma supernatants of PrP0/0 mice immunized with recombinant PrP
or purified CTMPrP by immunoprecipitation against in vitro translated PrP conformers. Here, we report on two such
conformational mABs that can be used for improved, sensitive diagnostics of prion infection.
The N-terminal domain of PrP contains four consecutive octarepeat sequences that preferentially bind copper ions.
Copper binding causes a structural rearrangement that likely influences the yet undetermined biological function of PrP
isoforms. We have isolated a mAB, termed 7VC, that distinguishes between copper-free and -bound octarepeats, an
effect that was only observed for peptides containing more than two octarepeats, suggesting that the octarepeat domain
is a highly regulated conformation-dependent ligand.
Another mAB, 19B10, selectively binds NtmPrP in the in vitro translation system. In non-infected N2a cells, 19B10
induced cell death arguing that PrP conformers corresponding to NtmPrP in vitro also exist in vivo and and may be
involved in cell death signaling events.
Supported by the BMBF, Germany, and a grant from the NINDS of the NIH, USA.
050
Oral-21
DETECTION OF TRANSMISSIBLE SPONGIFORM
ENCEPHALOPATHIES IN BLOOD
AMIN LANE, JOSEPHINE OLIVER, EMMA QUARTERMAN, CHRISTOPHER STANLEY, STEVEN DEALLER, STUART WILSON
Microsens, London
Background. We have developed the PrPSc-specific ligand, Seprion, into a very simple microplate immunoassay which
removes the need for sample preparation, including proteinase K, in post-mortem brain assays. This Seprion Assay has
100% sensitivity and specificity compared to current gold standards (1) and has received USDA approval for use in
CWD and BSE. We present here our progress in developing the assay for detection of TSEs in blood.
Method.
Sheep. A crude cellular fraction was made from the blood of five scrapie infected sheep and from a number of control
animals. Briefly, 20 ml of water was added to 5 ml of citrated blood. The non-lysed non-RBC cell fraction was collected
by centrifugation and resuspended in Seprion Capture Buffer. PrPSc was assayed as described in (1). Briefly, PrPSc
from the lysed cells was captured to immobilised Seprion ligand on Seprion-coated magnetic beads and detected with an
anti-prion AP conjugate.
Human. Blood from an iatrogenic human CJD patient was tested in a similar manner to the sheep blood and tested
against a number of human blood controls.
Results. All five scrapie-infected animals gave an increased signal in the assay compared to uninfected controls. Similarly,
the iatrogenic CJD patient showed an increased signal compared to the human controls.
Discussion. PrPSc was detected in 5/5 scrapie-infected sheep and in none of the control animals. Similarly, PrPSc could
be detected in the blood of an iatrogenic CJD patient. We are currently expanding these studies to include more scrapieinfected animals and more uninfected controls of sheep and human blood, the results of which will be shown.
Reference 1. Lane, A; Stanley, C; Dealler, S; Wilson, S.M. 2003. Clin. Chem., 49, 1774-1775.
051
Oral-22
TSE DIAGNOSTICS IN THE FAST LANE
PRICE, P., NAYKI, I., ISWANTI-STANEK, D., MEIER, M., STADELMANN, M., BUCHMANN, A., K¸B-LER, E., MEISSNER, K.,
SIDLER, M. AND B. OESCH
Prionics AG, Switzerland
Current testing for BSE is performed by rapid diagnostic tests which are based on Western blot or ELISA technologies.
Some of the EU approved ELISA tests involve laborious sample preparation steps such as centrifugation or precipitation
and require sophisticated laboratory equipment. The PrionicsÆ-Check PrioSTRIP is a new rapid diagnostic test for
transmissible spongiform encephalopathies (TSEs) including BSE, scrapie and chronic wasting disease. In contrast to
currently used rapid post mortem tests, the PrioSTRIP test is a dip-stick based immunochromatographic assay, similar to
lateral flow devices. It is based on the antibody-mediated detection of the pathological form of the prion protein PrPSc.
The design of the PrioSTRIP forces the analyte and the antibodies into close proximity thereby accelerating the reaction
kinetics and thus minimizing incubation times. The lateral flow based format uniquely combines high testing speed with
simple handling, resulting in very low total operation costs, while maintaining high reliability. The diagnostic
performance of the PrioSTRIP was evaluated under field conditions for screening of BSE in cattle and compared to
officially validated and accepted reference methods. The total number of negative samples was 4668 and the total
number of positive samples was 241. The result of this study demonstrates that the PrioSTRIP performs as well as the
reference methods, i.e. it shows 100% sensitivity (no false negative results) and 100% specificity (no false positive
results). We conclude that the PrioSTRIP opens new avenues to cost-effective, simple and accurate prion diagnostics.
052
Oral-23
SELECTIVE AND EFFICIENT
IMMUNOPRECIPITATION OF PRPSC CAN BE
MEDIATED BY NON-SPECIFIC INTERACTIONS
BETWEEN MONOCLONAL ANTIBODIES AND SAFS
NATHALIE MOREL1, STEPHANIE SIMON1, YVELINE FROBERT1, HERVE VOLLAND1, CHANTAL MOURTON-GILLES2,
ALESSANDRO NEGRO3, CATIA SORGATO4, CHRISTOPHE CREMINON1 AND JACQUES GRASSI1
1CEA, Service de Pharmacologie et d’Immunologie, 2 CNRS UMR 5160, Faculte de Pharmacie, 3 CRIBI, University
of Padova, 4 University degli Studi di Padova, Dipartimento di Chimica Biologica,
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders affecting humans and animals and
are characterized by the accumulation in brain tissues of an abnormal isoform of the prion protein named PrPsc. PrPsc
is the only direct unambiguous marker known for TSEs and its detection is the basis of most of the diagnostic tests for
prion diseases.
Recent studies1-4 have described monoclonal antibodies (mAbs) that specifically immunoprecipitate PrPsc as found in
TSE-infected brain in the form of scrapie-associated fibrils (SAFs). By screening 53 mAbs, including many anti-PrP
antibodies, we made the observation that the same results can be obtained by using several mAbs of various specificities
independently of the properties of their binding site (paratope). These results strongly suggest that a significant
proportion of mAbs can interact non-specifically with SAFs through non-specific, paratope-independent, interactions
which specifically immunoprecipitate PrPsc when these mAbs are immobilized on a poly-disperse solid phase (micro
beads), thus favouring the binding of mAbs to highly polymerized PrPsc as found in SAFs. Our work raises serious
questions concerning the actual specificity of antibodies previously described as specifically recognizing PrPsc since the
hypothesis of non specific interaction between monoclonal antibodies and SAFs was not taken into account.
Whatever the nature of the interactions between mAbs and SAFs, some antibodies proved to allow a very efficient and
fully selective immunoprecipitation of PrPsc in absence of PK treatment and centrifugation step. This opens the door for
the development of a rapid and simple diagnosis test for TSE with improved sensitivity.
References
1.
Korth,C. et al. Nature 390, 74-77 (1997).
2.
Paramithiotis,E. et al. Nat. Med. 9, 893-899 (2003).
3.
Serbec,V.C. et al. J. Biol. Chem. 279, 3694-3698 (2004).
053
Oral-24
DETECTION OF PERIPHERAL PRPSC IN NON-
HUMAN PRIMATES INFECTED WITH IATROGENIC,
SPORADIC AND VARIANT CJD AGENT
HERZOG C, RIVIERE J, ETCHEGARAY N, SALES N, DESLYS JP, LASMÉZAS C
Commissariat à l’Energie Atomique, Departement de Recherche Medicale, BP.6 Fontenay-aux-Roses
Abnormal PrPsc accumulation can be detected in lymphoreticular organs such as spleen and tonsil of vCJD patients. A
recent study revealed low amounts of PrPsc in spleen and muscle of certain sporadic CJD patients. This clearly
challenges the hypothesis that peripheral PrPsc is a unique feature of vCJD. Consequently, different issues pertaining
to the possible iatrogenic transmission of human TSEs are being put into question and a systematic analysis of
extraneural PrPsc is urgently needed. We used non-human primates (Macaca fascicularis) infected with sporadic,
iatrogenic, and variant CJD agent, offering the advantage of the unique availability of many tissues and organs obtained
under precise experimental conditions. Since assay sensitivity is the critical point in detection of peripheral PrPsc in
human patients, we developed purification methods optimized for each tissue. They allow for easy and complete sample
homogenization and PrPsc extraction in a form amenable to analysis using the Biorad ELISA test. We can detect PrPsc
at a concentration of 1x10-5 lower than that in brain tissue, which is to our knowledge the highest sensitivity for human
tissues currently described. This method should solve the problem of variations in human sample testing by giving high
sensitive and reproducible results for all tissues. We confirmed low amounts of PrPsc in muscles of sporadic CJD
infected primates. To understand the variations observed in different human studies, we screened several muscle
samples. We found that muscle PrPsc distribution depends on the type of muscle within one individual and on the
human TSE strain. This could explain the difficulties in extraneural prion detection. We are currently extending our
study to other organs and results will be discussed with regards to their implications for risk of iatrogenic transmission.
Moreover, we propose this method as a diagnostic tool for PrPsc detection in human biopsy samples.
054
Oral-25
URINE FROM SCRAPIE INFECTED HAMSTERS
COMPRISES LOW LEVELS OF PRION INFECTIVITY
KARIV-INBAL Z*, BEN-HUR* T, GRIGORIADIS** N, GABIZON R*
Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah University Hospital, Kiryat
Hdassah, Jerusalem Israel
The question of whether prion diseases can be transmitted by body fluids has important epidemiological, environmental
and economical implications. Although infectivity has been demonstrated in blood from scrapie-infected sheep, no
infectivity was reported as for today in any prion disease urine. Following our findings, which suggest that a protease
resistant isoform of PrP may be present in prion urine, we set to investigate whether urine collected from scrapie,
infected hamsters can transmit infectivity to normal hamsters. Normal and scrapie urine samples were inoculated to
normal hamsters by several routs of infection. Following such inoculations, three animals succumbed to scrapie disease,
after prolonged incubation times ranging from 350-700 days. Several additional hamsters in the groups inoculated with
prion urine presented symptoms of subclinical prion disease, including low levels of PrPSc in their brains and extensive
gliosis. None of the animals inoculated with normal urine succumbed to disease or could be shown to be infected
subclinically. Further examination of the infectious urine fractions indicated they comprise an array of protease
resistant peptides, among them protease resistant light chain immunoglobulin. Our results constitute preliminary
evidence that urine secreted from prion infected animals may constitute a reservoir of the prion disease infectious agent
055
Oral-26
HOST RESPONSES TO TSE INFECTION
LAURA MANUELIDIS
FMB11, Section of Neuropathology, Yale Medical School, New Haven, USA
Because the infectious component of TSE agents has not been identified, we assumed specific host inflammatory
responses to a foreign agent might be recruited early in CJD infection. We used cDNA arrays to identify differentially
expressed transcripts in infected mouse brains. Using RT-PCR analyses we were able to discriminate a new set of host
response genes that together can be used to diagnose early and later stages of progressive disease. These genes were
upregulated >10 fold between 30-40 days, well before the PrP abnormalities that begin only after 80 days. Parallel mock
inoculation with normal brain showed no comparable increase in these transcripts. At later stages of symptomatic
neurodegenerative disease (100-110 days), selected transcripts rose by as much as 100 fold. Some of these genes are
different from those found in symptomatic Alzheimer’s disease, and thus may help differentiate different types of
dementing disease in people. Additionally, since specific agent strains can target different cell types, several of these
molecular markers may help identify particular agent strains. Because both myeloid microglia and peripheral myeloid
cells are infectious in CJD, the myeloid cell markers discovered here may also facilitate rapid early diagnosis of
CJD/TSEs from accessible peripheral tissues such as spleen and blood. The above responses to progressive infection
challenge the concept that the immune system does not recognize TSE infections because the agent is composed only of
the host’s own misfolded prion protein. The above host responses to infection, as well as many other findings, are most
simply explained by invasion and replication of an exogenous virus.
056
Oral-27
THE AMYLOID THEORIES OF ALZHEIMER’S
AND CREUTZFELDT-JACOB DISEASES : DEVISING
SPECIFIC THERAPEUTIC AND DIAGNOSTIC
STRATEGIES.
C.L. MASTERS1 AND K. BEYREUTHER2
1. Department of Pathology, The University of Melbourne, and the Mental Health Research Institute of Victoria,
Melbourne, Australia, 2. Center for Molecular Biology, The University of Heidelberg, Germany
As the molecular basis of Alzheimer’s disease becomes more clearly defined, then more therapeutic targets can be
validated and taken into the clinic. Currently there are several approaches to the Ab amyloidogenic pathway which have
now reached early phase clinical trials:
1) g-secretase inhibition. The search for effective modulators of the proteolytic processing of APP has yielded at least
seven classes of compounds. Several of these classes have now entered early phase clinical trials, one of which has been
abandoned because of adverse events.
2) Metal-Protein Attenuating Compounds (MPACs). Interactions of metal ions [Zn(II), Cu(II)] with Ab have multiple
effects including promoting aggregation, lipid penetration and engendering harmful redox activity. Compounds which
have the property of attenuating A1b-metal interactions are therefore of interest in promoting Ab clearance from the
brain and ameliorating Ab toxicity.
3) Immunization. Immunization with Ab promotes its clearance from the brain. This strategy, applied to humans, has
caused an anticipated adverse autoimmune response. Novel epitopes on Ab may yet provide a strategy to bypass this
serious adverse effect.
4) Cholesterol _metabolism. Lowering cerebral cholesterol may decrease Ab production, possibly through the bsecretase pathway. The use of statins are now being actively investigated in prospective studies.
5) Ab-binding proteins. Various classes of molecules have been shown to bind to Ab aggregates in vivo, and some of
these (glycosaminoglycans, for example) may inhibit fibrillization.
An increasing number of therapeutic targets within the central Ab amyloidogenic pathway have emerged. Each in their
own way may provide a definitive test of the Ab amyloid theory of Alzheimer’s disease. Analagous approaches to the PrP
theory of CJD are also now in development, and some strategies developed for AD may be directly transferable to
CJD.
057
Oral-28
PROTEIN MISFOLDING, MOLECULAR
EVOLUTION AND HUMAN DISEASE
CHRISTOPHER M. DOBSON
University of Cambridge, Department of Chemistry, Cambridge United Kingdom
Protein folding is perhaps the most fundamental process associated with the generation of functional structures in
biology. There has been considerable progress in the last few years in understanding the underlying principles that
govern this highly complex process. Recently, much research has also focused on the realisation that proteins can
misfold in vivo and that this phenomenon is linked with a wide range of diseases. We have been investigating in
particular the nature of the amyloidogenic conditions, that include Alzheimer's disease, type 2 diabetes and the
spongiform encephalopathies, e.g. BSE and CJD, in which protein misfolding leads to the aggregation of proteins, often
into thread-like amyloid structures. Our studies have led us to put forward new ideas concerning the origin of the
various diseases associated with their formation. We have also speculated more generally that the need to avoid
aggregation could be a significant driving force in the evolution of protein sequences and structures.
References:
M. Vendruscolo, E. Paci, C.M. Dobson and M. Karplus, “Three Key Residues Form a Critical Contact Network in a
Transition State for Protein Folding”, Nature 409, 641-646 (2001).
C.M. Dobson, “Getting Out of Shape - Protein Misfolding Diseases, Nature 418, 729-730 (2002).
M. Dumoulin, A.M. Last, A. Desmyter, K. Decanniere, D. Canet, A. Spencer, D.B. Archer, S. Muyldermans, L. Wyns,
A. Matagne, C. Redfield, C.V. Robinson and C.M. Dobson, “A Camelid Antibody Fragment Inhibits Amyloid Fibril
Formation by Human Lysozyme”, Nature 424, 783-788 (2003).
F. Chiti, M. Stefani, N. Taddei, G. Ramponi and C.M. Dobson, “Rationalisation of Mutational Effects on Protein
Aggregation Rates Using Simple Physical Principles”, Nature 424, 805-808 (2003).
C.M. Dobson, “Protein Folding and Misfolding”, Nature 426, 884-890 (2003).
058
Oral-29
CONCLUSIONS OF INSERM EXPERT
COMMITTEE ON IATROGENIC CREUTZFELDTJAKOB DISEASE RELATED TO THERAPY WITH
CADAVERIC HUMAN GROWTH HORMONE IN
FRANCE
HENRY BARON
ZLB Behring, Departement of External Affairs, France
France's National Institute of Health and Medical Research (INSERM) appointed an Expert Committee to examine
and elucidate the potential cause(s) of prion contamination in vials of cadaver-extracted human growth hormone (hGH)
produced in France under the national therapeutic hGH program coordinated and supervised by Association France
Hypophyse. The Committee was composed of French and international experts in various scientific domains (prion
diseases, endocrinology, peptide/protein biochemistry, orphan drugs in paediatric diseases, pre-clinical safety of biological
products, etc.). Its mission was to analyse the circumstances surrounding the iatrogenic Creutzfeldt-Jakob disease
(iCJD) tragedy related to cadaver-extracted hGH in France, where more than half the total number of cases worldwide
have been reported, and attempt to understand and determine the principal cause(s) of this tragedy, taking into account
the level of established scientific knowledge at the time the prion contamination presumably occurred, i.e., in the years
prior to 1985. This was a purely scientific exercise, construed neither to attribute blame nor to exonerate fault, but
rather to carry out an optimal scientific analysis of pertinent issues and factors which may have had some bearing on the
ultimate, unfortunate outcome. Multiple documents were provided to all Committee members for their consultation and
study. The Committee examined and discussed epidemiological data which allowed designation of a critical "high risk"
period for contracting hGH-related iCJD in France covering the interval between mid-1983 and mid-1985, as well as
issues related to the collection of pituitary glands (under the direct supervision of Association France Hypophyse),
extraction and purification of hGH (under the supervision of Institut Pasteur), and quality control testing,
pharmaceutical formulation and final batch release (under the supervision of Pharmacie Centrale). The outcome is
presented here.
059
Oral-30
ACID INACTIVATION OF PRIONS - EFFICIENT AT
ELEVATED TEMPERATURE OR HIGH ACID
CONCENTRATION
THOMAS R. APPEL (1,2,3), RALF LUCASSEN (2,3), MARTIN H. GROSCHUP (4) AND DETLEV RIESNER (2,3)
1 IMB, Leibniz Institut f¸r Molekulare Biotechnologie Jena e.V., 2 Institut f¸r Physikalische Biologie, Universität
Düsseldorf, 3 Biologisch-Medizinisches Forschungszentrum, Universität Düsseldorf, 4 Bundesforschungsanstalt für
Viruserkrankungen der Tiere, Insel Riems
New and unpublished data on acid inactivation of prions will be presented.
Infectious prion rods and non-infectious aggregates of the corresponding recombinant protein rPrP(90-231) were treated
with hydrochloric acid. The amount of prion protein (PrP) remaining undegraded at various incubation times, acid
concentrations and temperatures was quantified by Western blot. Prion rods and PrP aggregates showed a similar PrP
degradation kinetic. However, prion infectivity was inactivated much faster than PrP. Acid degradation - of PrP in prion
rods and of aggregated PrP - was proportionally dependent on HCl concentration up to 6N, and kinetically first order in
PrP. The infectivity time constant is 0.54 per hour (1N HCl, 25 °C). In comparison, 0.3N NaOH at 30 °C is orders of
magnitude more efficient (>10^3 per hour). Complete loss of infectivity and PrP reduction to < 2% was observed by 8N
HCl at 25 °C (1 h), or by 1N HCl at 85 °C (1h). The slow acid inactivation of prion rods at room temperature is
attributed to a structural effect, i.e. solvent inaccessibility of fibrillar protein aggregates. Temperatures above 45 °C yield
exponentially faster acid degradation, due to thermally induced disaggregation with an activation energy of 50 kJ/mol.
Structural homology between prion rods and non-infectious aggregated PrP is indicated by a similar degradation kinetic.
060
Oral-31
MECHANISMS OF HEAT AND CHEMICAL
INACTIVATION OF TSE MODELS
ROBERT A. SOMERVILLE, NICOLA GENTLES AND KAREN FERNIE
Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, UK.
The infectious agents causing TSE diseases are notoriously difficult to inactivate. The need to re-assess methods of TSE
decontamination has been well recognised. We are investigating mechanisms of inactivation using combinations of heat
and inactivating chemicals. Heating under hydrated conditions causes biphasic inactivation above temperatures which
vary according to TSE strain, but not PrP genotype. High pH (> pH 11) reduces the temperature at which inactivation
takes place. Heating and dehydration can lead to the creation of heat-resistant, stabilised forms of TSE agents which
present the greatest challenge to the development of effective inactivation protocols. Exposure to SDS has shown that
there is little inactivation at concentrations below 1%, even at high temperatures, with the exception of one TSE model,
301V in SV mice. Inactivation of 301V in VM mice is enhanced at pH 10 and proceeds monophasically. The chaotrope,
guanidine hydrochloride also inactivates monophasically with respect to concentration and temperature. Its effect is
independent of TSE strain.
A two component model of the TSE infectious agent has been developed based on TSE inactivation properties. It
proposes that if fully hydrated, the agent is dissociated by heat in a biphasic reaction into its two components.
Dehydration stabilises the structure making it refractory to dissociation. Denaturation with guanidine hydrochloride
shows a trend to monophasic kinetics with no difference between TSE models suggesting that chaotropic denaturation
primarily affects a single invariant component of the agent. SDS also inactivates monophasically, suggesting that one
component is the primary target for SDS denaturation. However TSE-model-specific differences in rates of
denaturation, suggests interactions with other components of the agent’s structure; and at least one of these components
differs in structure between TSE models.
061
Oral-32
NEW PERSPECTIVES IN THE DECONTAMINATION
OF PRIONS.
G. FICHET (1, 2), E. COMOY (1), C. DUVAL (1), C. DEHEN (1), A. CHARBONNIER (1), K. ANTLOGA (3),
C.I. LASMEZAS (1), G. MCDONNELL (3), J.P. DESLYS (1).
1. CEA/DSV/DRM, Groupe d’Innovation Diagnostique et Therapeutique sur les Infections ‡ Prions, 2. ANJOU
RECHERCHE / VEOLIA WATER, Laboratoire Central, 3. STERIS Ltd., Jays Close, Viables, Basingstoke, United
Kingdom.
The high resistance of prions to classical methods of decontamination and evidence that prion diseases can be
transmitted iatrogenically on medical devices pose a unique infection control challenge to healthcare facilities, and could
constitute a major public health concern with regard to the large tissue distribution of the vCJD agent in humans. There
is therefore an urgent need for new evaluation methods adapted to prion surface contaminations as well as efficient and
practicable innovative decontamination procedures.
An in vivo test method using stainless steel wires contaminated with prions has been adapted to the hamster 263K
model. Moreover, a new in vitro protocol of surface contamination compatible with subsequent biochemical detection of
PrPres from the treated surface has been developed to explore the mechanisms of action of methods under test. These
models were used to investigate the efficacy of innovative physical and chemical methods of inactivation.
We confirm in this model that the prion infectivity has a unique affinity to surfaces. Harsh chemical decontamination
methods (NaOH 1 N, NaOCl 2%) were verified as efficient, but the recommended 134°C autoclaving led to incomplete
decontamination.
We then tested different chemical formulations procedures which are more compatible for the treated surfaces. Our data
show that among the various treatments tested, two of them provided “full” surface decontamination i.e. more than 5 logs
reduction of infectivity :
- an alkaline cleaner
- a phenolic derivative.
A gas vaporisation device amenable to the treatment of dry surfaces including electronic components was as efficient as
the recommended 134°C autoclaving.
We will discuss the three types of mechanisms which our study suggests for these different treatments. Combinations of
procedures can be immediately proposed to secure medical and surgical instruments as well as various complex surfaces.
062
Oral-33
PRION PROTEIN CONVERSIONS, TSE
INFECTIONS, AND THERAPEUTICS
BYRON CAUGHEY, GERALD S. BARON, ANA CRISTINA MAGALH„ES, MARCO ANTONIO PRADO* AND DAVID KOCISKO
Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840 USA., *Department of Pharmacology, Federal
University of Minas Gerais, Belo Horizonte, MG, Brasil
The conformational conversion of the normal, protease-sensitive prion protein (PrPC) to the protease-resistant prion
protein (PrP-res or PrPSc) is critical in transmissible spongiform encephalopathies (TSE) or prion diseases. Our studies
of PrP conversion and the effects of cofactors have provided insight into the molecular bases for PrP-res formation, TSE
species barriers, and agent strains. Cell-free studies of the interactions of the PrP isoforms in membranes suggest that the
propagation of infection between cells requires transfer of PrP-res into the membranes of recipient cells. Also supporting
this conclusion is the observation that membrane-associated PrP-res preparations are more efficient at infecting cultured
cells than membrane-free preparations. In the course of these studies, we have developed murine SN56 (septal neuronal)
cells as a new experimental model for investigating acute TSE infection of cells, the subcellular trafficking of PrP-res,
and the effects of neuronal differentiation.
Chronically scrapie-infected cells have also been adapted for use in a high-throughput screen for inhibitors of PrP-res
formation. Many different classes of inhibitors have been identified which can serve both as potential therapeutic agents
063
Oral-34
WHERE DOES DISEASE-ASSOCIATED PRION
PROTEIN DEPOSIT IN THE HUMAN BRAIN?
PREUSSER M*, KOVACS GG*, STROHSCHNEIDER M, VOIGTLAENDER T, BUDKA H (*CONTIBUTED EQUALLY)
Medical University Vienna, Institute of Neurology, AKH 4J, Vienna, Austria
Disease-associated prion protein (PrPd) deposits in distinct immunostaining patterns in the nervous system, influenced
mainly by the molecular phenotype and codon 129 genotype. Previous observations suggest mainly a synaptic
localization, but there are other morphological types of deposits. On one hand, these may be extracellular, e.g. in plaques
or patchy/perivacuolar deposits. On the other hand, some deposits are associated with cells, like perineuronal and
synaptic type immunoreactivities. Studies defining the exact location and cellular association of various morphological
deposits in the human brain are lacking. Co-localisation studies have been restricted by technical difficulties including
the damaging effect for other antigens of the pretreatment protocol used to enhance immunostaining for PrPd. After
having found a convenient pretreatment protocol, we systematically evaluated the co-localisation patterns of
morphologically different PrPd immunodeposits by confocal laser microscopy. In addition to the most prominent colocalisation with synaptophysin, PrPd may also co-deposit with connexin 32, a gap-junction related protein. Both
dendrites and axons harbour small granular PrPd deposits. Furthermore, occasionally astrocytes and microglia may also
contain PrPd granules. Highly aggregated deposits are focally ubiquitinated. We conclude that 1) PrPd is not exclusively
associated with chemical but also with electric synapses; 2) axonal transport may be a relevant route of PrPd spread in
the brain; and 3) activated microglia and astrocytes may play a pathogenetic role in PrPd processing or degradation.
064
Oral-35
A LEFT-HANDED, PARALLEL BETA-HELICAL
ARCHITECTURE AS A MODEL FOR THE
STRUCTURE OF THE SCRAPIE PRION PROTEIN
HOLGER WILLE1,2, CÈDRIC GOVAERTS3, DAVID A. AGARD4,5, FRED E. COHEN1,3,4, AND STANLEY B. PRUSINER1,2,4
1Institute for Neurodegenerative Diseases, Departments of 2Neurology, 3Cellular and Molecular Pharmacology, and
4Biochemistry and Biophysics, and 5Howard Hughes Medical Institute, University of California, San Francisco, CA
94143, USA.
The insolubility of the scrapie prion protein (PrPSc) has frustrated attempts to solve its structure by X-ray
crystallography or NMR spectroscopy. Knowledge of the structure of PrPSc is a prerequisite to attempts at structurebased drug design studies. Previously, we reported the discovery of two-dimensional (2D) crystals of N-terminally
truncated PrPSc (PrP 27-30) and a redacted miniprion (PrPSc106) that can be studied by electron crystallography
(Wille et al., 2002). The limited quality of these 2D crystals required the use of a single-molecule image-processing
approach. The final averages allowed us to map the differences between PrP 27-30 and PrPSc106, thereby localizing the
N-linked sugars and the internal deletion of the miniprion. Optical spectroscopy indicated that most residues of the
internal deletion are in a beta-sheet conformation. These data were used to constrain structural models of PrPSc,
suggesting a parallel beta-helix as the key element of the structure. The limited resolution of the data did not allow us to
distinguish between left- or right-handed architectures or whether the unit cell contained three or six protein molecules.
Here we report results obtained with a FEI Tecnai electron microscope, which provided projection maps of significantly
improved quality. The higher-resolution data were used to constrain the structural models further. The new data clearly
argue for a trimeric arrangement within the unit cell. Improved difference maps between PrP 27-30 and PrPSc106
confirm the positions of the N-linked sugars as well as the internal deletion of the miniprion. These new data and
analysis of the molecular models strongly favor the left-handed beta-helix as the putative fold for PrPSc. Recent data
from several laboratories, obtained by different methods, indicate that the amyloid fibers that are present in Alzheimer’s
disease, Parkinson’s disease and several other diseases may also be based on left-handed, parallel beta-helical
architectures.
065
Oral-36
NEUROTOXIC PRP CONVERSION
INTERMEDIATES.
STEVE SIMONEAU(1), FRANZISKA WOPFNER(2), HUMAN REZAEI(3), JULIEN COMTE(1), MAXIME LEFEBVRE-ROQUE(1),
JEANNE GROSCLAUDE(3), HERMANN SCHÖTZL(2), CORINNE IDA LASMEZAS(1).
1. Department of Medical Research, Commissariat à l’Energie Atomique, Fontenay-aux-Roses, 2. Institute of Virology,
Technical University of Munich, Germany, 3. Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
The mechanisms of neurodegeneration linked to the accumulation of the abnormal prion protein (PrP) during TSEs are
not well understood. It is unlikely that large PrP aggregates are responsible for the observed neuronal death. Rather,
intermediate reactive species would be more likely to interfere with the normal function of PrP or directly induce
neuronal death. In order to shed some light on this still puzzling feature of prion diseases, we have performed an indepth study of neuronal toxicity induced by two full-length PrP conversion intermediates, namely alpha-helical dimeric
PrP and beta-sheeted PrP. We show that both PrP entities are highly toxic to neurons and that the mechanism of cell
death implicated is apoptosis. Several neuroprotection assays were carried out with a series of antibodies and molecules
in order to gain insight on the origin of the neurotoxic signal. Remarkably, the neurons were shielded from the toxicity
using an antibody specific for the 106-126 “transmembrane” region of PrP, demonstrating the requirement of this domain
for toxicity. We performed cell surface biotinylation, to investigate the mechanisms of neurotoxicity. Notably, we
observed differences in the processing of alpha/beta PrP conformers sharing identical primary sequences implicating the
role of the three-dimensional structure and oligomerization state of the proteins. Because heparan mimetics, which block
prion replication in vitro and in vivo also had a neuroprotective effect, we investigated the link between neurotoxicity
and infectivity. We were unable to detect infectivity of PrP dimers in the mice bioassay, even after two passages
demonstrating that it could not serve as a seed to propagate disease. Thus we have identified for the first time full-length
neurotoxic PrP species. Our data highlights several key points for PrP neurotoxicity which could serve as targets for
neuroprotective-driven therapeutic intervention.
066
Oral-37
NEURONAL CELL DEATH TRIGGERED BY PRPC
SIGNALING IN VIVO.
LAURA SOLFOROSI, JOSE CRIADO, MICHAEL OLDSTONE, BRUNO CONTI AND R. ANTHONY WILLIAMSON
The Scripps Research Institute, La Jolla, CA 92037, USA
In the absence of the cellular prion protein (PrPC), the disease-associated isoform, PrPSc, appears not to be intrinsically
neurotoxic, suggesting that PrPC itself may participate directly in the prion neurodegenerative cascade, possibly via a
specific signaling mechanism.
To investigate this possibility, we established an experimental model in which PrPC-specific recombinant monoclonal
antibodies (Mabs) were stereotaxically injected into mouse brains. Two PrP-specific MAbs, IgGs D13 and P, each
recognizing epitopes within the 95-105 region of PrP, caused extensive apoptotic neuronal loss throughout the treated
hippocampal and cerebellar regions of injected mice at 24 h, but not 12 h, after antibody injection. Equivalent
contralateral injections of control antibodies produced no neuronal damage. Similarly, no damage was detected when
monovalent Fab fragments of IgGs D13 or P were independently injected into mice in the same manner. Intriguingly
injection of a third PrP-reactive antibody, IgG D18, binding to neuronal cell-surface PrPC between residues 133-157,
did not manifest any neuronal injury.
We hypothesise that, upon complexation with IgGs D13 or P, PrPC was efficiently dimerized, thereby initiating an
apoptotic cascade in neurons, possibly through PrP docking with an unidentified secondary molecule. We reason that
the D18 antibody, although binding well to the neuronal cell-surface, was either inefficient at crosslinking neighboring
PrPC molecules, or, alternatively, sterically obscured a region of PrPC that interacts with its putative signaling partner,
thus preventing activation of apoptosis. In prion-infected brains, neuronal loss may occur when oligomeric forms of cellsurface PrPSc undertake the PrPC-crosslinking role performed in our experiments by these MAbs. Thus, PrPC may be
co-opted twice in prion disease, once as a substrate for conformational conversion into nascent PrPSc, and additionally
067
Oral-38
A GENETIC ASSAY FOR CELLULAR PRION
PROTEINS: N-TERMINAL DETERMINANTS IN
PRPC FACILITATE NEUROPROTECTION AGAINST
PRO-APOPTOTIC ACTION OF DOPPEL
BETTINA DRISALDI, JANAKY COOMARASWAMY, PETER MASTRANGELO, BOB STROME, JING YANG, M. AZHAR CHISHTI,
OTTO WINDL, ROSEMARY AHRENS, HANS KRETZSCHMAR, PAUL E. FRASER, HOWARD T.J. MOUNT AND DAVID
WESTAWAY
Centre for Research in Neurodegenerative Diseases, Department of Medical Biophysics, Department of Medicine,
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, and LudwigMaximillians University, Munich, Germany
PrPC, an enigmatic protein encoded by the Prnp gene, is the precursor to the infectivity-associated prion protein PrPSc.
When expressed in the CNS, the PrP-like Doppel (Dpl) protein encoded by the adjacent Prnd gene produces an ataxia
resulting from apoptotic death of cerebellar neurons. Building on the ability of wt PrPC to block this process we have
used transfection of primary cultures of cerebellar granule cell neurons (CGNs) to explore activities of these two
proteins. Wild type PrPc expressed from the endogenous Prnp gene or from plasmid vectors blocked Dpl-induced
apoptosis and this neuroprotective activity was nullified by deletions encompassing either the N-terminal charged region
(Prnp delta23-28) or all five octarepeat motifs (Prnp delta51-90). Protective activity was also absent from a Prnp allele
with glycine residues in place of histidine within the four PHGGG/SWGQ octarepeats responsible for copper-binding in
vitro. In the case of Dpl a series of overlapping deletions defined a major role for a single region - residues 101 to 125
encoding helices B and B’ - in promoting neuronal apoptosis. The granule cell neuron assay described here reveals close
concordance with analyses of Prnp alleles performed in transgenic mice and will provide insights into the attributes and
sub-cellular localizations required for the action of PrPC upon a model substrate
068
Oral-39
BAX DELETION RESCUES NEURONAL LOSS BUT
NOT NEUROLOGICAL SYMPTOMS IN A
TRANSGENIC MODEL OF INHERITED PRION
DISEASE
ROBERTO CHIESA1,2, PEDRO PICCARDO3,4, LISA NOWOSLAWSKI5, SARA DOSSENA1, KEVIN A. ROTH5, BERNARDINO
GHETTI3, AND DAVID A. HARRIS2
1Dulbecco Telethon Institute (DTI) and Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario
Negri, Milano, 2Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis,
3Division of Neuropathology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA; 4Center for
Biologics Evaluation and Research, Food and Drug Administration, Rockville, 5Department of Pathology, University of
Alabama at Birmingham (UAB)
A nine-octapeptide insertion in the prion protein (PrP) gene is associated with an inherited form of prion disease.
Transgenic (Tg) mice that express the mouse homologue of this mutation (designated PG14) accumulate in their brains
an insoluble and weakly protease-resistant form of the mutant protein that resembles PrPSc. As this form accumulates in
the brain, Tg(PG14) mice develop a progressive neurological syndrome characterized clinically by ataxia, and
neuropathologically by astrogliosis, synaptic-type PrP deposition, and cerebellar atrophy with massive apoptotic
degeneration of granule neurons (Chiesa et al., PNAS 97:5574-5579, 2000).
Bax, a pro-apoptotic gene of the Bcl-2 family, plays a key role in regulating cell death in the nervous system. To analyze
the role of Bax in the Tg(PG14) phenotype, we crossed Tg(PG14) mice with Bax knockout (Bax-/-) mice. We found
that the age at which symptoms began and the duration of the clinical phase of the illness were not altered in
Tg(PG14)/Bax-/- mice. However, Bax deletion effectively rescued cerebellar granule neurons from apoptosis. The
granule cell layer was preserved in Tg(PG14)/Bax-/- mice, and activated caspase-3 staining, TUNEL labeling, GFAP
immunoreactivity and DNA laddering were all greatly reduced. In contrast, Bax inactivation failed to prevent shrinkage
of the molecular layer and loss of synaptophysin-positive synaptic endings. Moreover, Tg(PG14)/Bax-/-mice still
displayed synaptic-like PrP deposition in the molecular and granule cell layers. These data indicate that synaptic loss
induced by accumulation of mutant PrP, rather than neuronal death, is primarily responsible for neurological dysfunction
in Tg(PG14) mice.
069
Oral-40
ROLE OF PRION PROTEINS AND THEIR
RECEPTOR AND CO-RECEPTOR MOLECULES IN
THE PRION LIFE CYCLE
STEFAN WEISS
Laboratorium für Molekulare Biologie Genzentrum-Institut für Biochemie der LMU München, Germany
We identified the 37 kDa laminin receptor precursor (LRP) as an interactor for the cellular prion protein (PrPc) (1) and
proved that the 37 kDa/67 kDa laminin receptor (LRP/LR) (2) and HSPGs (3,4) act as the cell surface receptor and
co-receptors for PrPC, respectively.
LRP/LR is required for PrPSc propagation in neuronal cells (5). Knock-down of the 37 kDa/67 kDa LRP/LR in mouse
brain by ectopic transgenic expression of antisense LRP RNA resulted in a significant reduction of LRP/LR levels in the
cerebellum and the hippocampus (6), suggesting that these mice might be useful for further investigations of the role of
LRP/LR in prion pathogenesis.
Data from the Semliki-Forest-Virus (SFV) system suggest that 37 kDa/67 kDa LRP/LR and PrPc both act as receptors
for the infectious scrapie prion protein. Pentosan polysulfate (SP 45) and the heparan mimetic HM2602 significantly
reduced moPrP27-30 cell binding.
Live cell imaging technology with moLRP-DsRed and EGFP-moPrP allowed us to monitor the movement of these
molecules on the cell surface and intracellularly.
Yeast Two hybrid analyses showed that the prion-like protein Doppel (Dpl) fails to interact with itself, the 37 kDa/67
kDa LRP/LR and the prion protein proposing that Dpl and PrP are not or only marginally related with respect to their
ligand binding behaviour (7).
Single chain anti-LRP/LR antibodies (scFvs) and siRNAs directed against LRP mRNA might act as powerful
therapeutic tools in the treatment of TSEs.
(1) Rieger, R. et al. (1997) Nat Med, 3, 1383-8.; (2) Gauczynski, S. et al. (2001) EMBO J, 20, 5863-5875.; (3) Hundt,
C. et al. (2001) EMBO J, 20, 5876-5886.; (4) Warner, R.G., et al. (2002) J Biol Chem 277, 18421-18430; (5) Leucht,
C., et al. (2003) EMBO Rep 4, 290-295; (6) Leucht, C. et al. (2004) Transgenic Res 13, 81-85. ;(7) Hundt, C & Weiss,
S. (2004) Biochim Biophys Acta, in press.
070
Oral-41
GLYCOGENOME EXPRESSION AND PRION
DISEASES :NEW TARGETS FOR THERAPY?
A.BARRET1, P-F GALLET2, L. FORESTIER2, C.JAYAT-VIGNOLLES2, R.JULIEN2 AND J-P. DESLYS1
1Commissariat à l’Energie Atomique, GIDTIP/DSV/DRM, Fontenay-aux-Roses, 2UMR 1061, Unité de Génétique
Moléculaire Animale, INRA/Université de Limoges
The changes in glycogenome expression and function are still poorly understood in prion neurodegenerative disorders.
While the prion protein glycosylation does not seem to be directly involved in the disease, natural heparan sulfate
moieties have been reported to interact with the binding of PrP to the laminin receptor (LRP/LR) and treatments with
heparan sulfate mimetics can cure infected cells.
In the present study, we have investigated if altered glycosylation genes expression could be associated with prion
replication using the ScGT1 cellular model. The analysis by DNA micro-array including 178 specific glycosylation genes
resulted in the identification of several genes for which the expression was significantly modified in scrapie infected GT1
cells. The highest transcription differences were found for two genes : one is overexpressed and encodes for a GalNAc
transferase enzyme implicated in chondroitin sulfate synthesis and the second one is underexpressed and encodes for a
sulfotransferase. These results have been confirmed by real-time PCR in two cellular models of infection. The still
ongoing analyses at the protein level using specific antibodies have already shown alterations for the sulfotransferase
expression .This observed underexpression appears to be specific of a peculiar glycosylation metabolic chain and it is to
note that we observed no modification of the expression of 31 other unrelated sulfotransferases in the infected cells. This
phenotype is completely reversed when cells are cured by an heparan mimetics treatment. Ongoing studies include the
expression analysis of these two enzymes in vivo to understand more precisely their biological function in prion diseases.
We will discuss the potential mechanisms underlying the disregulations observed which induce modifications of specific
sugar moieties and of their negative charges. We will envisage the consequences of our findings for the definition of new
therapeutic targets.
071
Oral-42
HUMAN PRION DISEASE THERAPEUTICS: FROM
MOLECULAR STRATEGY TO CLINICAL
EVALUATION
John Collinge
MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, National Hospital for
Neurology and Neurosurgery, Queen Square, London WC1N 3BG
Prions appear to be composed principally or entirely of abnormal isoforms of a host-encoded glycoprotein, prion protein
(PrPC). The central molecular event in prion replication is thought to be the conversion of PrPC into a self-propagating
conformational isomer that accumulates as aggregated material (PrPSc). Mice devoid of PrP, are fully viable but do not
replicate prions or develop neurodegeneration following experimental inoculation. Conditional knockout of neuronal PrP
expression in a developed adult nervous system also does not cause neurodegeneration. Remarkably, such conditional
knockout during established CNS prion infection prevents onset of clinical disease and leads to reversal of early
pathology despite continued accumulation of PrPSc and prion titres to levels seen in end-stage clinical disease. PrPC,
rather than PrPSc, therefore appears the rational therapeutic target. Progress in the development of candidate drugs will
be discussed. In anticipation of such therapeutics, and at the request of the UK Department of Health, a protocol and
infrastructure for the clinical evaluation of therapeutics has been established: the MRC PRION-1 trial. The protocol has
been prepared in discussion with UK patient groups, modified in the light of pilot studies, and has recently obtained
multicenter ethics approval. The drug quinacrine is currently under evaluation and an up to date summary will be
presented of progress on this study.
072
Oral-43
PRION INFECTED CELL CULTURES: FROM BASIC
TO APPLIED RESEARCH
SYLVAIN LEHMANN
Institut de Genetique Humaine du CNRS, UPR 1142, MONTPELLIER, France
Cell culture models of Transmissible Spongiform Encephalopathies include chronically prion infected cell lines, as well as
cultures expressing variable amounts of wild-type, mutated or chimeric prion proteins. These cell lines have been widely
used to investigate the biology of both the normal and the pathological isoform of the prion protein. They have also
contributed to the comprehension of the pathogenic processes occurring in Transmissible Spongiform Encephalopathies
and in the development of new therapeutic approaches of these diseases. Here we will present recent findings using
these cell models including the detection of pathologic PrP in the nucleus of infected cells, which may play a role in prion
neurodegeneration (MangÈ et al., J Cell Sci, in press), and the study of phosphorous dendrimers and their therapeutic
effect both in vitro and in vivo (Solassol et al., J Gen Virol, in press).
073
Oral-44
NEW ANTI-PRION DRUGS DISCOVERED BY SIFTBASED HIGH-THROUGHPUT AND HIGH-CONTENT
SCREENING
UWE BERTSCH, KONSTANZE WINKLHOFER, JÖRG TATZELT, THOMAS HIRSCHBERGER, JAN BIESCHKE, PETRA WEBER,
PAUL TAVAN, HANS A. KRETZSCHMAR, AND ARMIN GIESE
Zentrum fuer Neuropathologie und Prionforschung, Ludwig-Maximilians-Universitaet, Muenchen, Max-Planck-Institut
fuer Biochemie, Martinsried; Theoretische Biophysik, Biomolekulare Optik, Ludwig-Maximilians-Universität,
Muenchen
A possible therapeutic approach to the currently untreatable prion diseases may be the blockade of interactions between
PrP-C and PrP-Sc resulting in a block of the conversion and PrP-Sc propagation. Based on the Scanning for Intensly
fluorescent Targets (SIFT-) technique, which relies on single molecule fluorescence correlation, we have developed a
largely automated assay system for the screening of substance libraries, which identifies new drugs that interfere
specifically with the association of alpha helical to purified authentic aggregates of the disease related scrapie-isoform
PrP-Sc. We validated the assay system with known antiprion compounds, of which in particular the cationic lipid
DOSPA proofed to be a specific inhibitor of this early step in the process of prion propagation and therefore was used
for positive control reactions. In a screening of 10 000 compounds with this assay 250 substances were identified as
primary hits, which displayed an inhibitory effect on the association of alpha-helical PrP with PrP-Sc in a single
measurement at a concentration of about 10 µM. Subsequent determination of the dose response curves for these
substances narrowed down the number of substances with a half maximal inhibitory effect at concentrations below 100
µM to 80. Of these eight substances displayed an inhibitory effect in vivo on prion propagation in scrapie-infected N2a
cells in a three-day treatment at a concentration of approximately 10 µM.
074
Oral-45
A NOVEL GENERATION OF HEPARAN SULFATE
MIMETICS FOR THE TREATMENT OF PRION
DISEASES
K.T. ADJOU1,2, S. SIMONEAU1, N. SALËS1, F. LAMOURY1, D. DORMONT1, D. PAPY-GARCIA4, D. BARRITAULT3, J.-P.
DESLYS1 AND C.I. LASMÉZAS1.
1) CEA, DSV/DRM, Fontenay aux Roses , 2) Ecole Nationale Veterinaire d’Alfort, 3)Laboratoire CRETT, CNRS
FRE2412, University Paris XII-Val de Marne, 4) OTR3 Sarl, Creteil, France.
The accumulation of PrPres, the protease-resistant abnormal form of the host-encoded protein PrP plays a central role in
transmissible spongiform encephalopathies (TSEs). Human contamination by bovine spongiform encephalopathy (BSE)
has propelled many scientific teams on a highway for anti-prion drug development. In this study we report that heparan
sulfate mimetics (HMs), originally developed for their effect on tissue regeneration, abolish prion propagation in scrapieinfected GT1 cells (ScGT1). PrPres does not reappear for up to 50 days post-treatment. When tested in vivo, one of
these compounds, HM2602, hampered PrPres accumulation in scrapie- and BSE-infected mice and prolonged
significantly the survival time of 263K scrapie-infected hamsters. Interestingly, HM2602 is an apparently less toxic and
more potent inhibitor of PrPres accumulation than Dextran Sulfate 500 (DS500), a molecule known to exhibit antiprion properties in vivo. Kinetics of PrPres disappearance in vitro and unaffected PrPc levels during treatment suggest
that HMs are able to block the conversion of PrPc into PrPres. We speculate that HMs act as competitors of
endogenous heparan sulfates known to act as co-receptors for the prion protein. Since these molecules are particularly
amenable to drug design, their anti-prion potential could be further developed and optimised for the treatment of prion
diseases.
075
Oral-46
ACTIVE IMMUNISATION OF C57BL/6 MICE WITH
PRP PEPTIDES ASSOCIATED WITH OLIGO-CPG
DELAYS PRION PROGRESSION .
MARTINE BRULEY ROSSET, ANNE SOPHIE BERGOT, CLARA BALLERINI, RAOUL TORERO-IBAD, AND PIERRE
AUCOUTURIER.
INSERM E 209, Hopital St Antoine, Bat Kourilsky, 75012 Paris, France.
The absence of an immune response during TSE is likely due to the fact that the normal prion protein isoform (PrPc) is
a self-antigen. We demonstrated that 30-mer PrP peptide immunisation of wild-type C57BL/6 mice could lead to a
specific immune response when CpG oligodeoxynucleotides, but not complete Freund s adjuvant, were used as
adjuvants. Vaccination with P158-187 generated IFN-É◊ and IL-4 secreting splenic specific T-cells and few antibodyproducing B-cells. In contrast, P98-127 did not increase the number of IFN-É◊ and IL-4 secreting T-cells but induced
anti-PrP antibodies. Thus, tolerance to PrP can be overcome when peptide immunisation is associated with CpG, and
the pattern of response depends on the peptide. We examined the efficiency of T-cell versus antibody responses on
disease progression in mice infected with the 139A scrapie strain. Peptide-specific T cell responses decreased rapidly in
P158-187 immunised mice. In contrast, all mice immunised with P98-127 developped an antibody response, the level of
which was maintained throughout the whole period of observation. Surprisingly, mice that displayed undetectable serum
antibody titers one month after immunisation with P158-187, began progressively to secrete antibodies. Analysis of
lymphoid populations indicated a progressive increase of CD19+ B cells with a concomittant decrease of CD3+ T cells in
lymph nodes of peptide-treated mice. P98-127 and P158-187 treatments weakly but significantly delayed the onset of
clinical signs of disease and prolonged survival as compared with mice treated with CpG only. The amount of splenic
PrPSc was lower in peptide-treated mice, particularly with P158-187. Transient skin lesions were observed only in mice
treated with P158-187. These results indicate that active immunisation of C57BL/6 mice can lead to anti-PrP specific T
and B immune responses and delays the progression of prion disease. Possible autoimmune reactions remain to be
documented.
076
Oral-47
NOVEL TARGETS FOR EXPERIMENTAL THERAPY
AND PROPHYLAXIS AGAINST PRION INFECTIONS
SABINE GILCH, ALEXA ERTMER, MAX NUNZIANTE, GUNNAR SCHULZ, FRANZISKA WOPFNER, MARIA OBOZNAYA, ELKE
MAAS, CLAUDIA KEHLER, CHRISTOPHER BRUNS, INA VORBERG, HERMANN M. SCHÄTZL
Institute of Virology, Technical University of Munich, Munich, Germany
No effective prophylactic or therapeutic regimens are available against prion diseases. We focused our initial anti-prion
efforts on strategies targeting the expression of PrPc on the plasma membrane. We have extended this by introducing
RNA aptamers and polyclonal auto-antibodies directed against surface located PrPc or accessible conversion
intermediates. In order to induce auto-antibodies we used a mouse PrP tandem construct expressed in E. coli. Using this
immunogen we were able to overcome auto-tolerance against murine PrP in wild-type mice to a certain extent without
inducing obvious side effects. Treatment of prion-infected mouse cells with anti-PrP auto-antibodies inhibited the
endogenous PrPSc synthesis, demonstrating their neutralizing capacity. Interesting insights in epitope reactivity
depending on nature of immunogen and self-/non-self situation were obtained. In addition, we could show T-cell
reactivities. Our data point to possibilities of developing means for an active immunoprophylaxis or immunotherapy
against prion infections. An alternative anti-prion approach came from recent work pointing to a role of PrP in signaling
events. In this context, we screened substances interfering in signaling pathways in prion-infected cells for effects on
PrPSc propagation. We identified one compound which was effective in inhibition of PrPSc accumulation. Prioninfected cells could be rapidly 'cured' in a time- and dose-dependent manner, without effects on biogenesis and cellular
trafficking of PrPc. The compound mainly affects pre-existing PrPSc by reducing its half-life time, probably induced by
functional up-regulation of lysosomal proteases. We demonstrated that these effects are most probably caused by
interference with the tyrosine-kinase c-abl. Our studies show that interference in specific intracellular signaling cascades
can have impact on prion clearance/degradation and might provide novel targets for prophylaxis and therapy of prion
diseases.
077
Oral-48
RE-ROUTING INTRACELLULAR TRAFFICKING OF
PRION PROTEIN IN NEURONES: A NOVEL
THERAPEUTIC APPROACH?
C. BATE (1), R. WILSON (2), J. BREWER (2), A. WILLIAMS(1,3)
1. Department of Veterinary Pathology, University of Glasgow Veterinary School, Glasgow, 2. Department of
Immunology and Bacteriology, Western Infirmary, University of Glasgow, 3. Department of Pathology and Infectious
Diseases, Royal Veterinary College,
Whilst direct interaction of various compounds with PrPSc may lead to disruption of prions and loss of infectivity, other
therapeutic approaches include inhibiting the mechanisms by which prions infect neurones and cause their degeneration.
In the present studies, the squalene synthase inhibitor squalestatin was used to reduce the cholesterol content of three
prion-infected cell lines and dispersed lipid rafts on their plasma membrane. This action prevented the accumulation of
PrPSc in all three cell lines. The effects of squalestatin were dose-dependent and evident at nanomolar concentrations.
Squalestatin also prevented the killing of ScN2a cells by microglia and non-infected neurones treated with squalestatin
became resistant to the otherwise toxic effect of prion preparations or PrP peptides. The protective effect of squalestatin
coincided with a reduction in the activation of the phospholipase A2 pathway and the prostaglandin production that is
associated with neuronal injury in prion disease. Furthermore, squalestatin treatment altered the intraneuronal
trafficking of prion peptide away from its normal recycling pathway and into degradative lysosomes. The
neuroprotective effects of squalestatin treatment were reversed by the addition of water-soluble cholesterol to neurones.
These studies indicate a pivotal role for cholesterol sensitive lipid rafts in controlling PrPSc formation, prion
neurotoxicity and in the activation of pathways leading to neuronal death, and provide a further avenue for developing
therapeutic strategies.
078
Poster
Session 1
Poster Session 1
A-01 DETECTION OF POLYMORPHISMS IN THE PRION
PROTEIN GENE IN TH BELGIAN SHEEP POPULATION :
SOME PRELIMINARY DATA
S. ROELS1, C. RENARD1, H. DE BOSSCHERE1, R. GEEROMS1, M. VAN POUCKE2, L. PEELMAN2 & E. VANOPDENBOSCH1
1 National Reference Laboratory for Veterinary TSE, Department of Biocontrol Veterinary and Agrochemical Research
Centre (CODA/CERVA) 2 Laboratory of Animal Genetics and Breeding, Faculty of Veterinary Medicine, Ghent
University,
In this study, 3721 Belgian sheep were genotyped for the PRNP polymorphisms at codons 136, 154 and 171, including
clinical healthy animals (n=3103), healthy animals at the slaughterhouse (n=102) and animals in 5 TSE/scrapie positive
farms (including a Nor98 farm; n=516). In most of the examined Belgian sheep breeds, the majority of the genotypes
found are considered to be TSE/scrapie resistant (R1-R2). These findings implement that for the majority of the Belgian
sheep population, a selection towards resistant genotypes will not give major problems. The genotypes of the 27
TSE/scrapie positive animals (all considered to have a status from moderate resistant to high sensitive; R3-R5) confirm
the correlation between genotype and positive TSE status.
Roels et al. (2004) Vet. Q. 26, 3-11.
A-02 NEUROPATHOLOGICALLY DISTINCT PRION STRAINS
GIVE RISE TO SIMILAR TEMPORAL PROFILES
OF BEHAVIOURAL DEFICITS
BOCHE D, CUNNINGHAM C, DEACON R.M.J, RAWLINS J.N.P. AND PERRY V.H.
CNS Inflammation group, Southampton Neurosciences group, School of Biological Sciences
Mouse-adapted prion disease strains have been characterised by their different neuropathological profiles and incubation
times but the behavioural consequences have not been well studied. The current study compares the behavioural profile
produced by the strains ME7, 79A, 22L and 22A in C57BL/6J mice. We show here that there are no clear differences in
the time of onset of the first behavioural impairments on burrowing, glucose consumption, nesting or open field activity
in the strains ME7, 79A and 22L. 22A-infected animals were not impaired on these behaviours until much later,
consistent with the long incubation time of this strain. Similarly, excepting 22A, the late stage motor impairments did not
appear at significantly different times among these strains. Despite behavioural similarities, these animals showed clear
neuropathological differences at the onset of overt clinical signs. All strains showed clear microglial activation in both
hippocampus and thalamus (ME7>79A>22L). 79A showed marked white matter pathology. Neurodegeneration in the
hippocampal CA1 layer was statistically significant in the ME7 and 79A strains, whereas only 22L and 22A showed
statistically significant neuronal loss in the cerebellar Purkinje cell layer. Hippocampal synaptic loss was more marked in
ME7 animals than other strains while 22L showed more severe cerebellar synaptic loss. All strains showed clear thalamic
neuronal loss. These behavioural similarities coupled with clear pathological differences may give insights into key
circuits whose early dysfunction underlie the effects of infection with different prion disease strains. Such behaviour
testing is also the ideal approach to study therapeutic intervention in prion disease.
081
Poster Session 1
A-03 COMPARTMENTALIZATION OF PRION ISOFORMS WITHIN
THE REPRODUCTIVE TRACT OF THE RAM
ECROYD H, THIMON V, SARRADIN P*, DACHEUX J-L, AND GATTI J-L.
Gamètes Males et Fertilité, UMR 6175 INRA-CNRS, Station de Physiologie de la Reproduction et des Comportements.
*Equipe Génétique et Immunité, Station de Pathologie Infectieuse et Immunologie, Institut National de la Recherche
Agronomique.
Cellular prion protein (PrpC) has been shown on sperm and in the reproductive fluids of the male. We show by RTPCR and Northern blotting, that prion mRNA is present in the testis and epididymal tissue but differences in the
isoforms present are due to post-transcriptional or post-secretory processing. Antibodies directed against the C-terminal
sequence near the GPI-anchor site, an N-terminal sequence and against the whole protein showed that the Prp isoforms
are compartmentalized within the reproductive tract of the ram. Immunoblotting with the three antibodies showed that
the complete protein and both N- and C-terminally truncated and glycosylated isoforms are present within male
reproductive fluid. Moreover, we demonstrate that in these fluids, the PrpC isoforms are both in a soluble state as well as
associated with small membranous vesicles (epididymosomes). In the soluble state, we found Prp to be part of a high
molecular weight complex in association with a number of other proteins. We also report that only one major
glycosylated 25kDa C-terminally truncated PrpC isoform is associated with sperm from the testis, epididymis and
semen, and this form is also present in the sperm cytoplasmic droplets that are released during maturation. Significantly,
this C-terminally truncated isoform was found to glycosylated indicating that C-terminal membrane anchorage is not
required for glycosylation of the protein. This C-terminal truncated form is also associated with membrane lipid rafts
present in the mature sperm suggesting a role for it in the terminal stages of sperm.
A-04 ALPHA-SYNUCLEIN ACCUMULATES IN THE BRAIN OF
SCRAPIE-AFFECTED SHEEP AND GOATS
K.T. ADJOU1,2, S. ALLIX1, S. BACKER1, H.C. NGUYEN1, C. COUQUET3, M.-J. CORNUEJOLS3, J.-P. DESLYS2, H.
BRUGÈRE1, J. BRUGÈRE-PICOUX1 AND K. EL HACHIMI4.
1) Ecole Nationale Vétérinaire d’Alfort 2) CEA, GID/TIP/DSV/DRM, 3) Laboratoire départemental vétérinaire 4)
EPHE/INSERM U106, Hôpital la Salpétrière.
082
Transmissible spongiform encephalopathies (TSEs) are characterized by the post-translational accumulation of the hostencoded prion protein (PrP) in an amyloid misfolded form (PrPsc). Several lines of evidence indicate that the conversion
process of PrP into PrPsc can be mediated by cofactors. a-synuclein, a presynaptic nerve terminal protein has been
immunohistochemically detected in lesions of Alzheimer’s and Parkinson’s diseases. It has also been detected in sporadic,
iatrogenic, and nvCJD.
The purpose of this study was first to investigate whether a-synuclein immunoreactive deposits are present in the brain
of small ruminants affected with scrapie, second to determine a possible co-localisation between PrPsc and a-synuclein,
third to investigate the astrogliosis reaction upon accumulation of a-synuclein.
Brains of ewes and goats infected with natural scrapie were analysed. PRNP genotyping was performed in sheep by
Labogena laboratories. Ewes (ARR/ARR genotype) and goats without neurological diseases were used as healthy
controls. a-synuclein and PrP accumulation were revealed by immunocytochemistry.
Our results showed i) many granular deposits of a-synuclein in the brain of affected animals, particularly in the
hippocampus and cerebellum, ii) many granular deposits were observed in the cornu ammonis and subiculum of the
hippocampus, compared to the diffuse and no granular staining seen in healthy controls, iii) granular deposits of asynuclein mainly in the granular cell layer of the cerebellum, iv) and no constant co-localisation of PrPsc and a-synuclein
was observed. Moreover we observed, in the hippocampus, a prounounced astrogliosis associated to the accumulation of
a-synuclein and independent of the presence of PrPsc. In conclusion, we showed that a-synuclein accumulates in the
brain of small ruminants affected with natural scrapie, indicative of a metabolism perturbation of a-synuclein as a
common mechanism involved in human and animal TSEs.
Poster Session 1
A-05 ESTIMATES OF SMALL RUMINANT TSE
PREVALENCE IN FRANCE IN 2002
E. MORIGNAT, G. CAZEAU, A.-G. BIACABE, J.-L. VINARD, A. BENCSIK, J.-Y. MADEC, C. DUCROT*, T.
BARON, D. CALAVAS
AFSSA Lyon, France. *INRA Theix, France.
An active surveillance program of transmissible spongiform encephalopathies (TSEs) was implemented in France in
2002 among four categories of small ruminants (sheep/goats in abattoirs/rendering plants) in order to estimate the
prevalence of TSEs. 78,283 animals meeting the inclusion criteria were sampled from April to December 2002 and
included in the analysis. 167 were found positive with the rapid tests, and confirmed positive with a reference test. The
prevalence was 6.94 ‰ for fallen sheep, 1.04 ‰ for fallen goats, 0.92 ‰ for healthy slaughtered sheep and 0.14 ‰ for
healthy slaughtered goats. The functional analysis of this program highlighted three biases: a potentially non-random
sampling scheme in both rendering plants and abattoirs, a heterogeneous geographical sampling ratio and the use of two
diagnostic tests of unequal performance. Simulations taking into account the different biases were run in order to
estimate the prevalence of TSEs . The comparison of the TSEs prevalence calculated from the raw data set with the
simulation results showed that the effect of non-random sampling was minor as compared to both the heterogeneous
geographical sampling ratio and the use of two different diagnostic tests.
The full simulation (weighted according to the size of the sheep and goat populations per county and standardised
according to the sensitivity of the diagnostic tests) ranged between 10 % lower and 80 % higher than the raw prevalence
in the same category of animal. This evidence clearly indicates that the combination of a heterogeneous geographical
sampling scheme and the type of rapid test used can dramatically affect the prevalence estimates.
This highlights the importance of the study design and data collection in estimating the prevalence of the disease. When
potential biases cannot be avoided, they should at least be taken into account carefully in order to get the best-least
biased estimates of the prevalence.
A-06 EVIDENCE FOR THE IN UTERO TRANSMISSION OF
SCRAPIE IN SHEEP
J.FOSTER, W.GOLDMANN, D.PARNHAM, D.DRUMMOND, S.EATON, C.MCKENZIE, K. FERNIE A.CHONG, M.J.A.
MYLNE & N.HUNTER
Institute for Animal Health, Neuropathogenesis Unit, Edinburgh, UK
This study has shown that scrapie can be transmitted from an experimentally infected, parturient ewe to her unborn
lamb. Embryo transfer enabled the PrP genotype of embryo donor and recipient sheep to be chosen according to
susceptibility to SSBP/1 experimental scrapie. SSBP/1 was recognised as the causal infection in at least three and
probably five lambs born to these experimentally infected surrogate dams. The distinctive pattern and intensity of PrPSc
immunostaining produced in the brain of infected sheep, compared to those with natural scrapie, confirmed an SSBP/1
infection. Two of these lambs were derived by laproscopy, which meant that the infection was transmitted to the
developing foetus from its infected recipient dam as an in utero infection.
083
Poster Session 1
A-07 WHAT IS THE CLINICAL STATUS OF BSE
TEST POSITIVE CATTLE AND WHY DID THEY ESCAPE CLINICAL
SURVEILLANCE ?
G. CAZEAU, C. DUCROT, E.COLLIN, G. DESJOUIS, D. CALAVAS
G. Cazeau, D. Calavas AFSSA Lyon, France. C. Ducrot INRA Theix, France E. Collin, G. Desjouis SNGTV, France
A Mandatory Reporting System (MRS) was set up in France in December 1990 in order to detect the animals showing
evocative symptoms of Bovine Spongiform Encephalopathy. Since June 2000, four active surveillance programs have
been implemented to reinforce the surveillance, based on the BSE screening of fallen stock and slaughtered cattle with
rapid tests. All the cases detected through these programs have to be retrospectively investigated in order to estimate the
clinical status of these cases at the time of death or slaughter, and to understand why the MRS failed to detect them.
Each inquiry involved the interview of the farmer and the veterinarian.
A first analysis of the 181 inquiries recorded at the French Agency of Food Safety (AFSSA) until September, 1, 2002,
provided helpful elements i) to understand the status of the BSE positive animals detected through the active
surveillance programs, ii) to consider them in epidemiological studies, and iii) to understand the relationships between
the different surveillance systems for BSE.
It showed that almost all the cases from fallen stock were animals showing clinical signs, two thirds of them showing
clinical features which should have led to a suspicion of BSE. No clinical sign had been noticed for two thirds of the
cases detected at the abattoir and eight per cent for those from fallen stock.
It pointed out the difficulties of clinical surveillance, particularly the fact that farmers do not routinely call their
veterinary practitioner for a sick animal, and the problem of insufficient neurological investigation.
Since this date, other inquiries have been received which brings to a total of 304 questionnaires. This will permit to
better analyse the relationships between MRS and active surveillance programs over time and space.
A-08 TREND OF THE BSE EPIDEMIC AND SOURCES OF
INFECTION FOLLOWING THE FEED BANS IN FRANCE
DUCROT CHRISTIAN*, CALAVAS DIDIER#, ABRIAL DAVID*, MORIGNAT ERIC#, LABONNARDIÈRE CLAUDE*, JARRIGE
NATHALIE#
* Unité d’Epidémiologie Animale, INRA Theix, France. # Unité Epidémiologie, AFSSA Lyon, Lyon, France
084
A feed ban of meat and bone meal (MBM) for cattle started in France in 1990 to control BSE, and a ëreinforced feed
ban’ in 1996 was based on the removal of cadavers and specified risk material from MBM. The data from the active
surveillance programs at the abattoir and fallen stock since 2000 were used to analyse the trend of the epidemic in the
different birth cohorts. Logistic models were run to compare the BSE prevalence on successive birth cohorts, using a
pair-wise method of controlling for age at testing. The results showed different trends between the Jul93-Jun94 and
Jul94-Jun95 cohorts, with a significant increase (OR=2.3) of the BSE prevalence at the abattoir on the whole French
territory but a plateau on fallen stock in western France. The results were homogenous over the next two birth cohorts,
with a significant decrease; the Jul95-Jun96 birth cohort was significantly less affected than the July 94-June 95 one OR=0.5 for France-abattoir (FA), 0.3 for western France-fallen stock (WF)-, and the birth cohort Jul96-Jun97 was
significantly less affected than the Jul95-Jun96 one (OR=0.2 for FA and WF). The decrease matches the reinforced feed
ban in 1996 if we consider an average infection at 1 year old.
The main hypotheses of the sources of infection for the cases born after the feed ban or reinforced feed ban are the crosscontamination with poultry or pig feed and the infection of by-products. To test them, a spatial study of BSE accounting
for the dairy versus beef cattle demographic was carried out, with a disease mapping of the risk (Poisson Process). It
showed that both the cases born after the feed ban and those born after the reinforced feed ban were not randomly
distributed, and highlighted the geographic areas with a higher risk, that were mostly the same for both types, which
suggests a common contamination source. The link between the disease mapping and the assumptions on the sources of
infection is been studied now.
Poster Session 1
A-09 CANADIAN BSE SURVEILLANCE
STEFANIE CZUB
National BSE Reference Laboratory/CFIA, Canada
BSE is a neurodegenerative disorder with extended incubation time, slowly-progressive disease course and fatal
outcome. Epidemiological and molecular data suggest that BSE is capable of infecting a number of mammalian species
including humans. To determine the prevalence of BSE within the cattle population and the effectiveness of mitigation
measures to prevent further spread of the disease, to meet health and international trade objectives, identification of
infected animals through surveillance of targeted populations is required. In 1990, Canada named BSE a reportable
disease and active surveillance was implemented in 1992. The surveillance system is based on testing mature cattle with
clinical signs compatible with BSE, delivered through the TSE Veterinary Diagnostic Laboratory Network which is a
collaboration of federal and provincial governments. In addition to passive surveillance, samples from mature cattle are
actively collected at provincial and federally inspected abattoirs, from rabies-negative submissions and from submissions
from private practitioners to provincial and academic veterinary diagnostic laboratories. In 2002, testing of samples
derived from the dead stock population was introduced. Since 1992, a total of 10.591 animals was tested for BSE, thus
exceeding international requirements established by the OIE. The detection of the Canadian BSE case in May 2003
resulted in an increased surveillance of risk populations. BSE surveillance samples are tested within the TSE network
laboratories by immunohistochemistry and, recently, by rapid tests. Confirmation of suspect cases is the responsibility of
the National BSE Reference laboratory. Its mandate includes also diagnostic services, quality control for the TSE
network, training, validation of and serial release testing for BSE rapid tests.
A-10 BSE INSPECTION IN JAPAN AND FINDING OF ATYPICAL
PK-RESISTANT PRION PROTEIN (PRPRES) IN AN
APPARENTLY HEALTHY 23-MONTH-OLD HOLSTEIN STEER
YOSHIO YAMAKAWA1, KENÍICHI HAGIWARA1, KYOKO NOHTOMI1, YUKO NAKAMURA1, YOSHIMI HIGUCHI2, YUKO
SATO2, TETSUTARO SATA2 AND THE EXPERT COMMITTEE FOR BSE DIAGNOSIS, MINISTRY OF HEALTH, LABOUR AND
WELFARE OF JAPAN3
Department of Biochemistry& Cell Biology1 and Department of Pathology2 of National Institute of Infectious Diseases,
Japan. 3Ministry of Health, Labour and Welfare, Tokyo, Japan.
An ELISA-positive specimen from a 23 month-old healthy Holstein steer slaughtered on September 29, 2003, in Ibaraki
Prefecture (Ibaraki case) was sent to National Institute of Health for confirmation. The histology showed no
spongiform changes and IHC revealed no signal of PrPSc accumulation. However, WB analysis of the homogenate
prepared for ELISA revealed a small amount of PrPSc with an electrophoretic profile different from that of typical
BSE-associated PrPSc. The characteristics were (i) low content of the di-glycosylated molecular form of PrPSc, (ii) a
faster migration of the non-glycosylated form of PrPSc on SDS-PAGE, and (iii) less resistance against PK digestion as
compared with an authentic PrPSc specimen. The DNA sequence of the PrP coding region on ibaraki case was the same
as that appearing in the data base (GenBank accession number: AJ198879).
Though the clinical onset of BSE is usually at around five years or later of age, a two year-old case with the clinical
signs was reported. We encountered another case that accumulation of typical BSE-specific PrPSc in young cattle (a 21month old Holstein steer). Variant forms of BSE similar to our cases, i.e., with atypical histopathological and/or
biochemical phenotype, have been recently reported in Italy and in France. Such variant BSE was not associated with
mutations in prion protein (PrP) coding region as in our case.
Ministry of Agriculture, Forestry and Fisheries of Japan (MAFF) announced a ban of feeding ruminants with meat
bone meal (MBM) on September 18, 2001, and a complete ban was made on October 15 of the same year. According to
the recent MAFF report, the previous seven cases of BSE in Japan were cattle born in 1995-1996 and possibly fed with
cross-contaminated feed. However, the two cattle in this report were born after the complete ban. Whether contaminated
meat bone meal was implicated in the present cases remains to be investigated.
085
Poster Session 1
A-11 PRP DETECTION AND IMMUNOPHENOTYPING OF
LYMPH NODES, LYMPH AND BLOOD CELLS FROM SHEEP
EXPERIMENTALLY CHALLENGED WITH SCRAPIE.
EATON S L.*, CHIANINI F.*, GONZALEZ L.** ROCCHI M.*, JEFFREY M. **& REID HW*.
*Moredun Research Institute,Pentlands Science Park, SCOTLAND ** Lasswade Veterinary Laboratory Pentlands
Science Park, SCOTLAND
The role of the lymphoreticular system and the immune cell involvement in scrapie pathogenesis is still relatively
unknown. In this study we aim to investigate the changes in the immune system when lambs are experimentally infected
with scrapie at a regional site. Fourteen six month old ARQ/ARQ Suffolk lambs were cannulated at the prefemoral
efferent duct in two phases (early and late). Each phase comprised of 7 lambs: 6 were challenged with a clarified scrapie
brain inoculum and 1 with a clarified scrapie free brain inoculum. In the early phase cannulation occurred 3 days before
challenge and in the late 164 days post challenge (d.p.c.). The challenge was delivered via the sub cutaneous route in the
drainage area of the prefemoral lymph node.
Lymphadenectomies of the challenged and contra lateral lymph nodes took place at 16 and 180 d.p.c. in the early and
late phase respectively. Tonsil biopsies were carried out at 365 d.p.c. to assess the development of infection. Flow
cytometry was performed on blood and lymph samples and on fresh lymph node cells using a panel of monoclonal
antibodies (Moab) recognising leukocyte surface antigens (CD4, CD8, CD14, CD21, gamma delta TCR and MHC
Class II). Moab FH11 was used to label PrP. Contra lateral and challenged lymph nodes and the tonsil biopsy samples
were formalin fixed and immunohistochemically labelled for the abnormal form of prion protein (PrPd) using R145
Moab. Flow cytometric analysis indicated that there were a larger number of positively labelled CD21 mononuclear
cells at 180 d.p.c. in the challenged lymph nodes in comparison to the contra lateral lymph nodes. PrPd accumulation
was detected in a challenged lymph node of one animal at 180 d.p.c. and in 9 out of 12 of the tonsil biopsy samples which
indicated a successful scrapie challenge. However, the absence of early phase changes suggests that the sub cutaneous
route may not be an effective method for delivering infectivity to the local node.
A-12 PATHOGENESIS OF SCRAPIE AND BSE IN TRANSGENIC
MOUSE MODELS
BERNADETTE LYNAM, DAVID O'CONNELL, LUCIANO BACILIERI, MARK ROGERS
Prion Research Unit, Department of Zoology, University College Dublin, Ireland.
086
Detection of the abnormally folded prion protein, PrPSc, in infected tissues is a fundamental step in the study of the
pathogenesis of TSEs including Scrapie and BSE. Traditional methods of detection of this protein include the protease
digestion of PrPC and PrPSc with subsequent detection of the protease resistant PrPSc by Western blot. More recently,
conformation dependent immunoassay (CDI) techniques have been developed that detect the abnormally folded protein
without the need for protease digestion. This may facilitate the detection of protease sensitive PrPSc species in certain
tissues and may provide a sensitive means of studying sub-clinical TSE disease.
We have developed a tissue repository of infected tissues from Tg20 murine PrPC over-expressing mice. These mice
have been infected by intracranial (IC), intraperitoneal (IP) and oral routes of infection with murine scrapie & murine
BSE strains. We have employed Western blot and developed a CDI to analyse these tissues. The time course of
infection, the route of infection and the strain of PrPSc have been analysed using these techniques. Our preliminary
findings will be presented.
Poster Session 1
A-13 TSE IN GERMAN CERVIDS ?- FIRST RESULTS OF THE
YEARS 2002-2004
STEINBACH F, SCHETTLER E, BLASCHE, T, KAPS I*, MEUSSDOERFFER F*, FRÖLICH K
Institute for Zoo and Wildlife Research, GERMANY. *Cenas AG, E.-C. GERMANY
TSE occur as chronic wasting disease (CWD) in north American cervids since more than 30 years. Until now, CWD
has been observed in mule deer (Odocoileus hemionus), white tailed deer (Odocoileus virginianus) and Rocky
Mountain wapiti (Cervus elaphus nelsoni). The incubation period ranges from several months to years and death is
inevitable. Until now, there is no information that TSE (BSE, CWD or scrapie) exists in cervids from Germany or any
other European country. Accordingly, the objectives of our study are to (1) determine the prevalence of TSE in deer in
Germany and (2) to compare various commercial rapid BSE tests regarding their specificity and sensitivity for TSE in
different cervid species.
Within a period of three years approx. 10,000 brain (obex region) and lymph node samples will be examined for TSE by
rapid BSE post mortem tests (Bio-Rad ELISA). The samples originate from roe deer (Capreolus capreolus), red deer
(Cervus elaphus elaphus), and fallow deer (Cervus dama dama) >18 months. Special emphasis will be provided on
collecting samples from cervids living in regions where BSE or scrapie has been diagnosed and on individuals that show
clinical signs like cachexia or CNS disorders. Moreover, a considerable number of samples from captive cervids will be
tested. The specificity and sensitivity of various BSE tests will be analysed using CWD positive tissue from North
America and brain samples (without proteinase digestion) of cervids from Germany.
Until January 2004, more than 4,000 samples of cervids from Germany were tested negative by Bio-Rad ELISA. The
samples originated from allover Germany. First results on rapid tests show that the Prionics Western blot, Prionics LIA,
and BioRad BSE ELISA detect both proteinase K resistant prion protein (PrPres) of CWD positive animals and
cellular prion protein (PrPc) of German cervid species. There are differences regarding handling and sensitivity of the
tests. Further details will be presented.
A-14 A CLINICAL PROCEDURE TO IMPROVE THE
REPORTING RATE OF SUSPECTS OF BSE
CAREDDU M. E, D’ANGELO A*,MAURELLA C., ZANINI A.*, CARAMELLI M., RU G.
CEA - Centro Encefalopatie Animali - TSE National Reference Laboratory Istituto Zooprofilattico Sperimentale di
Piemonte Liguria e Valle d’Aosta;*Dept.of Animal Pathology, University of Turin,Italy
Since 2001 in the frame of the passive surveillance for BSE more than 160 suspects were carried out in Italy. The
distribution by year of suspects has been uneven: 10 in 2001, 96 in 2002 and 62 in 2003.Even if from a clinical point of
view changes in sensation are one of the most common features in BSE, they are often missed by veterinarians on field.
Because of paucity of number of suspects we have implemented a clinical procedure whose aim is to identify in the
bovine population a subset of animals with hyperresponsiveness to evoked stimuli. This subset may be used as an easily
accessible source of cattle to be submitted to a neurological examination in order to increase the rate of suspects of the
mandatory reporting system. A clinical procedure consisting in 4 types of standardised tests was set up: tactile stimulus
(on head and neck), flexible stick test (brushing the hough and provoking kicking), auditory test (metallic noise), flash
test. We have compared three different cattle populations: 1) 267 cattle coming from 52 herds from Valle d’Aosta
(whose 122 cattle visited twice); 2) 1,451 cattle coming from 14 BSE outbreaks of which 169 visited at slaughterhouse
and 1,282 visited on farm; 3) cattle on which clinical suspects were done.Individual bovine data were collected with a
standardised form, entered in a database and analysed with Stata 8. In all groups stimulus-specific prevalence rates of
hyperresponsiveness were calculated after an evaluation of the reproducibility of the clinical procedure. Our results show
that the clinical procedure proposed might be very effective in improving the reporting rate of suspects of
BSE.Moreover analysing the official suspect reports it is evident that behavioural changes are often missed and not used
in order to formulate a suspect of BSE.
087
Poster Session 1
A-15 AN ANALYSIS OF THE INFLUENCE OF THE S138N AND
R151C SHEEP POLYMORPHISMS ON THE PROCESSING OF A
PRPCARQ-EGFP CLONE.
BIRKIR THOR BRAGASON AND ASTRIDUR PALSDOTTIR
Institute for Experimental Pathology, Department of Virology and Molecular Biology, University of Iceland, Iceland.
Two rare PrPC polymorphisms, S138N and R151C, have been detected in Icelandic sheep. The incidence of S138N is
similar in scrapie and control sheep, suggesting that it is a neutral genotype. Several sheep with R151C (16
heterozygotes and 1 homozygote) were identified in a scrapie herd, in which the disease had probably been present for a
few years before notification. Only one of them, an ARQR151C/VRQ heterozygote, tested positive for scrapie. This
suggests that the R151C polymorphism can confer some resistance to scrapie infection.
The aim of this project is to evaluate if S138N and R151C affect the processing of PrPC, with emphasis on the
possibility of alternative disulfide bonding of R151C. Three PrPCSheep-EGFP clones were constructed, with the
genotypes ARQ, ARQS138N, and ARQR151C. The clones were processed into the secretory pathway, as determined
by co-localization with Golgin-97. They were glycosylated, as determined by a PNGase assay, and were transported to
the plasma membrane, as determined by biotinylation of surface proteins with sulfo-NHS-biotin. Non-reduced extracts
(whole cell) from transfected N2a cells were examined by Western blot with anti-PrPC. They show, in addition to the
PrPC-EGFP band, an identical band in the ARQ and ARQS138N samples, of a size that could correspond to a dimer.
This band disappears after reduction with ‚-mercaptoethanol. A larger reduction-sensitive band is present in the
ARQR151C sample, this band is approximately 18 kDa larger than the band in the other samples. These “dimer” bands
are biotinylated with sulfo-NHS-biotin suggesting that they reach the plasma membrane. Immunoprecipitations with
PrPC antibodies from whole cell extracts of transfected N2a cells show that, with some antibodies, less ARQR151C is
precipitated than ARQ and ARQS138N, even though identical PrPC-EGFP protein amounts are present. This could be
due to structural differences.
A-16 CELLULAR PRION PROTEIN OF THE WILD RODENT
SPECIES CLETHRIONOMYS GLAREOLUS CAN BE CONVERTED
INTO PRPRES USING IN VITRO CONVERSION REACTIONS
NIKLAS PIENING, UWE BERTSCH, UMBERTO AGRIMI, HANS A. KRETZSCHMAR
Niklas Piening, Uwe Bertsch, Hans A. Kretzschmar: Ludwig-Maximilians-Universität München, Zentrum für
Neuropathologie und Prionforschung, Germany. Umberto Agrimi:Instituto Superiore di Sanità,Italy.
088
Transmission studies with bank voles (Clethrionomys glareolus), a wild rodent species, have revealed that compared to
mouse these rodents are highly susceptible to natural scrapie but show apparent resistance to BSE infection. Comparison
of the prion protein primary sequence of voles, mouse, sheep and cattle has identified the four amino acid residues
M109, N155, N170 and E227 that are likely to be responsible for these different species barrier effects.
We are exploring the role of the primary amino acid sequence in the differential susceptibility of the bank vole to scrapie
and BSE using the cell-free conversion assay described by Kocisko et al. (1994).
Therefore the sequence of the bank vole PrP gene and mutagenised versions of it were cloned into suitable expression
vectors, allowing expression in mammalian cells. Cellular vole PrPC as well as the closely related hamster PrPC have
been purified from mammalian cells by immunoprecipiation and were converted into their protease resistant states by
PrPSc prepared from voles infected with the mouse-adapted scrapie strain 139A. We will further explore the differential
susceptibility of bank voles to scrapie and BSE in respect to the role of the primary amino acid sequence.
Poster Session 1
A-17 THE ANIMAL TISSUE BANK OF CATAUNYA: A SOURCE OF
ANIMAL PRION DIDEASE SPECIMENS
MÁRQUEZ M 1., VIDAL E 2., FERNÁNDEZ H 3., FERNÁNDEZ J 3., MATÉ C 3., PUMAROLA M 4.
1Animal Tissue Bank of Catalunya (BTAC), Institut de Neurociències, Universitat Autònoma de Barcelona (UAB),
SPAIN. 2Laboratori PRIOCAT, CReSA Foundation, UAB, SPAIN. 3Barcelona Zoological Park, SPAIN. 4Medicine
and Surgery Department, Veterinary Faculty, UAB, SPAIN.
The Animal Tissue Bank of Catalunya (Banc de Teixits Animals de Catalunya, BTAC) is placed in UAB. This bank is in
the service of research, diagnosis and education and is linked to several centres that provide material to the bank namely
the Barcelona Zoological Park, the Animal Pathology Service of the UAB Veterinary Faculty and of special interest the
PRIOCAT Lab (The Catalunya Reference Animal Prion Diseases Laboratory).
BTAC is a non profit entity which collects, processes, stores and distributes animal nervous tissue samples to the
scientific community, offering samples of a great variety of species and a wide range of ages. Non pathologic tissues and
pathologic specimens including TSEs are available. TSE material corresponds to field cases of BSE and Scrapie
diagnosed within the active TSE surveillance program in Catalunya.
BTAC has a well established protocol for sample processing which can be modified upon the applicant requirements;
routinely, a half of the sample is frozen at -80° C and the other formaline fixed and paraffin embedded. Apart from CNS
in some instances samples from other tissues are also collected.
All samples included in the bank are submitted to a quality control check in order to assure the optimal conditions of the
processed tissue for research. Furthermore, a team of specialized pathologists carries out a thorouhg
anatomopathological examination to characterise the samples. These and other data about the tissue, i.e. samples origin,
clinical information, processing details etc, are also included in our data base.
BTAC is coordinated with other animal and human tissue banks and actually develops, apart from its own research lines,
collaborations with other groups and, in fact, the bank is open and willing to establish new research collaborations.
The bank samples are made available upon request and after the approval of such request by the BTAC Scientific
Committee.
More information in the web page http://quiro.uab.es/btac.
A-18 ESTABLISHMENT OF RAPID PRPSC DETECTION
PROTOCOL IN LYMPHOID TISSUE AND APPLICATION TO
SCRAPIE SURVEILLANCE OF DOWNER LIVESTOCK IN JAPAN
KIMI SHIMADA, YOSHIFUMI IWAMARU, MORIKAZU IMAMURA, HIROKO HAYASHI, MARY JO SCHMERR, MORIKAZU
SHINAGAWA, TAKASHI YOKOYAMA
Prion Disease Research Center, National Institute of Animal Health; JAPAN
An abnormal isoform of prion protein (PrPSc)accumulates in both brain and lymphoid tissues of sheep and goats with
scrapie. Rapid western blot (WB) procedure for PrPSc detection in lymphoid tissues was established and has been
applied to the surveillance of downer livestock in Japan. In this program, brain and palatal tonsil tissues were examined
using WB. Out of 138 diseased or dead animals (sheep or goats) examined, one case of scrapie was detected. Upon
examination of other sheep in the same herd, we identified two more scrapie-infected sheep. All three sheep had the
scrapie-susceptible genotype (MARQ/MARQ at codons 112, 136, 154, and 171 of prion protein gene). However, while
the clinically scrapie-affected sheep harbored PrPSc in the brain and palatal tonsil, the two sheep in the pre-clinical stage
harbored PrPSc in brain, but not in palatal tonsil. This study shows that PrPSc accumulation in palatal tonsil is variable
in natural scrapie, even among genetically susceptible sheep. Established protocol could detect PrPSc rapidly and more
sensitively, and further surveillance might be required to solve PrPSc accumulation mechanisms in palatal tonsil.
089
Poster Session 1
A-19 PREDICTABLE PATTERNS OF BSE IN CATTLE
POPULATIONS
ALINE A. DE KOEIJER
Quantitative Veterinary Epidemiology (QVE), Department of Infectious Diseases, Animal Sciences Group,Wageningen
University and Research Center, The Netherlands:
Age structured modelling can help in analysing age distribution patterns in BSE cases, and deriving from those for
instance knowledge on the BSE prevalence in the past. Using such methods, one can also derive from a basic BSE risk
assessment, the most likely age groups where BSE is to be expected. Such methods can be very useful for optimizing
and targetting surveillance of BSE.
A-20 EXPRESSION PROFILING IN SCRAPIE-INFECTED MOUSE
BRAINS USING MICROARRAY TECHNOLOGY
WEI XIANG1, OTTO WINDL1, GERDA W¸NSCH1, MARTIN DUGAS2, ALEXANDER KOHLMANN3, INGO M. WESTNER1 AND
HANS A. KRETZSCHMAR1
1Institute of Neuropathology, Ludwig-Maximilians-University Munich, Germany: 2Department of Medical Informatics,
Biometrics, and Epidemiology, Ludwig-Maximilians-University, Germany. 3Laboratory for Leukemia Diagnostics,
Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University, Germany.
The pathogenesis of prion disease is still unclear. The aim of this study was to identify the differentially regulated genes
during prion disease as well as the abnormal intracellular or intercellular pathways that may be responsible for the
pathogenesis of prion disease. We applied Affymetrix ME430A microarrays containing more than 22,000 transcripts and
compared the global gene expression profiles from scrapie-infected mouse brains with those from brains of uninfected
and mock-infected mice. The mice were intracerebrally inoculated with two mouse adapted scrapie strains, ME7 or
RML. Microarray data were analysed by Significance Analysis of Microarrays, revealing 121 genes whose expression
increased at least twofold in scrapie-infected mouse brains with an estimated false discovery rate of <5%. These genes
encode proteins involved in proteolysis, protease inhibition, signal transduction, immune response, cell adhesion, cell
growth/maintenance, and molecular metabolism. The up-regulation of the majority of these genes coincided with the
accumulation of PrPSc and the activation of glia, the important pathological events which may contribute to neuronal
cell death. Our observations support a link between the selectively regulated genes reported in this study and
neurodegenerative changes associated with prion disease. These findings generated several novel insights into gene
expression profiles correlated with prion disease.
090
Poster Session 1
A-21 OBSERVATION OF A MATERNAL TRANSMISSION OF
SCRAPIE IN SHEEP
BRUGÈRE-PICOUX J.*, COUQUET C**. CORNUEJOLS M.-J.**, FRÉMONT A.**, EL HACHIMI K.*, ALLIX S.*, ADJOU
K.*, CHI NGUYEN T.H.*, COMOY E.***, DESLYS J.P.***, BRUGÈRE H.*
* Ecole Nationale Vétérinaire Alfort **Laboratoire Départemental d'Analyses et de Recherches avenue du Professeur
Joseph de Léobardy *** Commissariat à l'Energie Atomique,GIDTIP, France
Within framework of eradication of scrapie in France, we have received an ewe the 11 of january 2002 in the regional
laboratory of Limoges with suspicion of scrapie. The clinical signs were wasting, woll loss, and trembling. Ten days later,
the ewe lambs with difficulties and with assitance. These help permit us to make an isolation of the ewe lamb without
contact with the mother, especially without absorption of the colostrum. The lamb was isolated in an other place where
there was no possibility of horizontal transmission of scrapie and it received only lactoreplacers.
The deterioration of the condition of the ewe, with locomotor disturbances and finally decubitus, lead us to decide an
euthanasia the 6 of February, 2002. The confirmation of natural scrapie was obtained by Western Blot (Prionics test)
and histopathology.
In last august 2002, first subtle clinical signs of natural scrapie appear in the ewe lamb (trembling, licking). Some weeks
later, the signs were more obvious : pruritus, locomotor disturbances, wasting. With an evolution to the decubitus,
euthanasia was decided the 18 of december 2002. Confirmation of natural scrapie was obtained by Western Blot,
histopathology and immunohistochemistry. The ewe lamb was ARQ/VRQ.
It is the first confirmation of maternal transmission of scrapie in sheep with a short incubation time (7 months). As
placenta can be infectious in sheep, this transmission can be foetomaternal.
A-22 LATEST TRENDS IN SCRAPIE REPORTING IN GB
MOHAMMAD ALI, VICTOR J. DEL RIO VILAS
Scrapie Epidemiology Group, CERA, Veterinary Laboratories Agency-Weybridge
Passive surveillance of scrapie has recorded a significant number of events since the disease became notifiable in 1993 in
Great Britain (GB). Scrapie clinical cases, despite potential under-reporting, have provide the best possible picture of the
incidence of the disease in the past.
Reporting trends are regularly monitored by the Veterinary Laboratories Agency (VLA) in the UK. Since 1998, when
new regulations came into force and compensation payment was introduced, the number of flocks reporting clinical
scrapie sheep has declined. A clear, sudden drop was observed in some regions of GB (mainland Scotland, Shetlands)
very recently.
Trying to describe this fall, 1998-2003 quarterly reporting flocks counts were analysed and an estimate of the likelihood
of such drop occurring obtained. We show that the previous falling trend, keeping all things equal, would not explain the
sudden drop and zero counts found in some regions of GB during the last quarter of 2003.
We discuss the potential causes and their implications. In 2003 new European Regulations imposed more rigorous
control measures on farms if disease was confirmed. This may be behind the described fall by discouraging farmers to
report. If so, similar patterns may be observed in active surveillance of fallen stock although there is no clear evidence to
support this last claim.
Ultimately, a shortfall of cases arising from the statutory notification leaves the active surveillance of abattoir and fallen
stock as the only sources of data on naturally occurring scrapie. Biases resulting from design and operational problems
concerning both data sources may have an effect on the observed epidemiology of the disease.
091
Poster Session 1
A-23 SEARCHING FOR AN INDICATOR OF BSE
SURVEILLANCE EFFICACY: BOVINE MORTALITY
GAGNA C., INGRAVALLE F., RU G., MAURELLA C., CARAMELLI M.
Piemonte Liguria e Valle d’Aosta
In the frame of active surveillance for BSE, emergency slaughtered animals, animals with non-specific clinical signs at
ante mortem examination and fallen stock represent “risk categories for BSE”. It is assumed that bovines which test
positive for BSE are more likely to be find in those groups than in regularly slaughtered animals. Due to huge cost
surveillance is going to be targeted on these categories in future and regularly slaughtered animals will be randomly
tested. Beginning in February 2001 Italy tested all fallen stock for BSE, while EU legislation enforced testing of a
sample of this subpopulation till august 2002. Aim of this study is to assess if observed bovine mortality could be a
suitable indicator of efficacy of the Italian BSE surveillance system. Analysis of bovine mortality by region in Italy is
based on data from national BSE testing database and national bovine population database. Rate of mortality have been
directly standardized by age and breed (as a proxy of production type) in order to correct for these potential
confounders. Standardised mortality rates per year have been compared by region and with the national rate, anomalies
have been observed as excess or defect of mortality by region and expected fallen stock have been calculated for these
regions based on national mortality rate. Given the expected fallen stock number, expected BSE cases for each region
have been calculated applying the crude national rate for this category, in order to assess if some cases could have escape
the surveillance system. Results, at least for 2002, suggest that no cases have been lost in the fallen stock category, but
the large Confidence Interval for expected cases (CI 95%: 1 - 11 ) does not allow to make definitive conclusions. Work is
in progress to better define the analysis, including data from 2003 activity. Data on mortality by region should be used
routinary to evaluate efficacy of BSE surveillance focusing on areas where is underreported.
A-24 IATROGENIC SCRAPIE IN ITALY: FURTHER EVIDENCES
BARIZZONE F, BONA C, MAURELLA C, GAGNA C, CARAMELLI M, RU G
Piemonte Liguria e Valle d’Aosta
092
In Italy a vaccine was suggested as the best explanation for the increase of scrapie outbreaks observed in the second half
of the 90’s. Aim of this study is to test that iatrogenic hypothesis.
A list of flocks which bought that formol inactivated vaccine against contagious agalactia in the years 1994-1997 was
obtained. Data analysis were restricted to the 8 Regions where the vaccine had been distributed and using the 2002-2003
National Database of Scrapie Active Surveillance of in Italy. Prevalence Ratios (PR) [95% C.I.] between animals tested
from exposed flocks vs those from unexposed flocks were calculated along with Relative Risks (RR) [95% C. I.]
comparing incidence of new outbreaks among exposed flocks vs unexposed flocks in which at least 20 subjects per flock
were tested. The comparison were extended to the complete list of exposed flocks.
Since 1997 in the 8 Regions involved 109 outbreaks have been observed: 36 of them were exposed to the vaccine (33%).
The list included 175 flocks exposed, 15 of them experienced scrapie (8.6%). In the years 2002-2003 we tested 58,871
animals from unexposed flocks with 53 Scrapie cases (0.09%) and 382 animals from exposed flocks with 8 scrapie cases
(2.09%): PR was 23.3 [11.1 - 48.6]. Twelve outbreaks were identified among 489 unexposed flocks (2.45%) and 5
outbreaks among 36 exposed flocks (13.89%) resulting in a RR 5.7 [2.1 - 15.2]. The RR of all exposed vs unexposed
was 3.5 [1.7 - 7.3]. These findings strongly suggest that exposure to the vaccine was associated with at least part of the
Italian scrapie outbreaks. We acknowledge the “Istituto Zooprofilattico Sperimentale del Lazio e della Toscana” which
kindly provided data. This study was supported in part by a grant (HSP) from the Italian Ministry of Health.
Poster Session 1
A-25 A CLINICAL PROTOCOL TO IMPROVE THE SCRAPIE
SURVEILLANCE
BONA C., MAURELLA C., D’ANGELO A.*, CAREDDU M.E., BOTTERO P*., CARAMELLI M., RU G.
CEA- National Reference Centre of TSE, Istituto Zooprofilattico Sperimentale di Piemonte, Liguria & V.Aosta. Turin,
Italy; *Dept. of Animal Pathology, University of Turin, Italy.
Since a reliable ante mortem diagnostic test for the detection of TSE is missing, the clinical diagnosis of scrapie suspects
is based upon the neurological signs observed. These signs share with many other neurological diseases and often is very
difficult to recognise a TSE. Aims of this work are to identify neurological signs consistent with scrapie in the study
population, to standardise a clinical protocol easy to handle and useful to passive surveillance of scrapie in sheep and to
evaluate validity and reliability of the proposed clinical protocol.
We have visited 130 sheep applying a clinical visit protocol obtained after a pilot study and using a standardised form to
collect the clinical signs. Moreover 35 sheep have been visited by two different operators to evaluate the reliability of the
proposed method.
Sheep have been classified into three different groups. A database ad hoc was implemented and the collected data have
been analysed with EpiInfo 6.04. Odds Ratios were used to identify association between signs and scrapie; predictive
values were computed for selected clinical signs and K of Cohen was used to assess the degree of the interobserver
agreement.
Most recurrent signs are those reported in literature, readily identifiable and anamnesis and nibble show high negative
predictive values. Animals with anamnestic history of typical symptoms showed a larger proportion of clinical signs
when compared with those chosen by random criterium animals.With regard to the reliability some signs showed a good
K value, whereas others do not. Work is still in progress on this topics in order to increase the size of sample and to
refine the assessment.We suggest that the use of this standardised protocol would be useful in field to rapidly and
correctly evaluate animals for scrapie and/or neurological impairment and could improve passive surveillance of sheep
TSE.
This study was supported in part by a grant (HSP) from the Italian Ministry of Health.
A-26 PRION PROTEIN (PRPC) IMMUNOCYTOCHEMISTRY AND
EXPRESSION OF THE GREEN FLUORESCENT PROTEIN REPORTER
GENE UNDER THE CONTROL OF BOVINE PRP GENE PROMOTER I
ANNE-MARIE HAEBERLÉ1, FRANÁOISE BLANQUET-GROSSARD2, JEAN-YVES CESBRON2, CATHERINE LEMAIRE-VIEILLE2
AND YANNICK BAILLY1
1Neurotransmission & Sécrétion Neuroendocrine CNRS UPR2356, IFR37 des Neurosciences, Strasbourg, France,
2Transmission & Pathogenèse des Maladies à Prion, CNRS FRE2685, Université Joseph Fourier, Grenoble, France
Expression of the cellular prion protein PrPc by host cells is required for prion replication and neuroinvasion in
transmissible spongiform encephalopathies. As a consequence, the identification of the cell types expressing PrPc is
necessary to determine the target cells involved in the cerebral propagation of prion diseases. In order to identify the
cells expressing PrPc in the mouse brain, the immunocytochemical localization of PrPc was investigated at the cellular
and ultrastructural levels in several brain regions. In addition, we analyzed the expression pattern of a green fluorescent
protein reporter gene under the control of regulatory sequences of the bovine prion protein gene in the brain of
transgenic mice. Using a preembedding immunogold technique, neuronal PrPc was observed mainly bound to the cell
surface and presynaptic sites. Dictyosomes and recycling organelles in most of the major neuron types also exhibited
PrPc antigen. In the olfactory bulb, the neocortex, the putamen, the hippocampus, the thalamus and the cerebellum, the
distribution pattern of both the green fluorescent protein and PrPc immunoreactivity suggested that the transgenic
regulatory sequences of the bovine PrP gene were sufficient to promote the expression of the reporter gene in neurons
which express immunodetectable endogenous PrPc. Transgenic mice expressing PrP-gfp may thus provide attractive
murine models to analyze the transcriptional activity of the Prnp gene during prion infections as well as the anatomopathological kinetics of prion diseases.
093
Poster Session 1
A-27 NEW FUNCTIONAL, STRUCTURAL AND ULTRASTRUCTURAL
INSIGHTS ON THE GABAERGIC SYSTEM DURING SCRAPIE:
RELEVANCE FOR NEURODEGENERATIVE MECHANISMS
2,3 ESSIA BOUZAMONDO-BERNSTEIN*, 4 STEPHANIE D. HOPKINS, PATRICIA SPILMAN, 2 DIANE LATAWIEC, 3 JANE
UYEHARA-LOCK, JIRI SAFAR, 4 HENRY J. RALSTON III, 1,2,5 STANLEY B. PRUSINER AND 1,2,3 STEPHEN J.
DEARMOND
1 Department of Neurology,2 Institute for Neurodegenerative Diseases, 3 Department of Pathology, 4 Department of
Anatomy and W.M. Keck Foundation. Center for Integrative Neuroscience, 5 Department of Biophysics and
Biochemistry, UCSF, USA
The goal of this study was to test whether morphological and functional abnormalities of GABAergic synapses correlate with
accumulation of the abnormal, protease-resistant prion protein, rPrPSc, during the course of scrapie in Syrian hamsters (SHa).
We focused on the GABAergic system because of reports that early and selective loss of GABAergic neurons underlies many
of the clinical features of prion diseases. Three approaches were used: (a) quantitative morphology both at the cellular and
subcellular levels to determine whether GABAergic neurons and their processes were selectively lost; (b) measurements of
evoked [3H]-GABA release from synaptosomes to test for functional abnormalities and (c) correlation of synaptic
abnormalities with the kinetics of rPrPSc accumulation.
At the preterminal stages of scrapie, we found: (a) a significant increase in the density per unit volume of GABAimmunopositive neocortical cell bodies by stereology; (b) an abnormal aggregation of synaptic vesicles in presynaptic boutons;
(c) a decrease in GABA release from cortical and thalamic synaptosomes, suggesting that trafficking of g-aminobutyric acid
(GABA) was abnormal; and (d) a strong correlation of the morphological and functional abnormalities with the kinetics of
rPrPSc accumulation.
Our results support the growing body of evidence that synapse loss precedes cell body loss and ultimately leads to cell death.
Thus a precise knowledge of the timing of functional and morphological abnormalities is a prerequisite to determine whether
emerging pharmaceutical treatments of Creutzfeldt-Jacob disease (CJD) will be effective. Early therapeutic strategies clearing
PrPSc from the brain and stimulating reactive synaptogenesis may give a new optimistic perspective in CJD treatment.
A-28 FIRST BELGIAN NOR98 SCRAPIE CASE DIAGNOSED VIA
ACTIVE SURVEILLANCE
H. DE BOSSCHERE 1, S. ROELS 1, S. L. BENESTAD 2, E., VANOPDENBOSCH 1
1 National Reference Labaroratory for Veterinary TSE, Veterinary Agrochemical Research Centre (CODA/CERVA),
Belgium, 2 National Veterinary Institute, Department of Pathology, Norway,
094
Scrapie is a fatal transmissible spongiform encephalopathy caused by prions. Several (classical) scrapie strains have been
described based on lesion profiling in mice. In Belgium, since April 2002, all sheep older than 18 months are tested with a rapid
test (Bio-Rad) through the active TSE surveillance program. Five of the 6 outbreaks in 2002 showed a classical scrapie lesion
profile, but in one case the positive sheep showed special features. The ewe was apparently healthy and presented for slaughter.
According to the active epidemio-surveillance protocol only part of the medulla oblongata around the region of the obex is taken
out. The sample was repeatedly tested positive with the rapid test. PrPSc Western blot (WB) analysis was positive showing a
PrPsc glycoprofile with a strongly marked lower band at ~ 12 kDa, compared to a classical scrapie glycoprofile. The “special”
glycoprofile of the present case was confirmed by Benestad S. and coworkers in Norway. The sheep PrP genotype was
A136R154Q171 homozygous.The unusual characteristics of the present case are: 1/ only one (of the 55) animals of the flock was
affected; 2/ no lesions were present in the brainstem (obex) as compared to the described lesion profiles of classical scrapie cases;
3/ the absence of PrPsc immunolabelling in the area of the obex; 4/ the PrPsc glycoprofile of the present case differed clearly
from the glycoprofiles found in isolates of classical scrapie strains and the BSE strain, and is not distinguishable from the Nor98
glycoprofile. This type of scrapie, may question the scrapie active epidemio-surveillance protocol from a diagnostic point of view.
This may be of significance for future sampling in scrapie surveillance programs and confirmation tests.
Reference : H. De Bosschere, S. Roels, S. L. Benestad, E., Vanopdenbosch. A Nor98 scrapie case diagnosed in Belgium via
active surveillance. Veterinary Record (accepted)
Poster Session 1
A-29 FAST RETROGRADE SPREADING OF SCRAPIE IN
HAMSTER SCIATIC NERVE
FRANCO CARDONE, MEI LU, MAURIZIO POCCHIARI, LUIGI DI GIAMBERARDINO.*
Istituto Superiore di Sanità, Rome, Italy., *E.N.S., Paris, France.
The scrapie agent reaches the CNS via the peripheral nerves at a reported rate of 1-3 mm/day, hardly compatible with
retrograde axonal transport. Here we revisited this estimate by sequential sectioning the sciatic nerve after footpad
inoculation. Seven groups of Syrian Golden hamsters were injected into the footpad of the left hindlimb with 20 µl of a
10% brain suspension of a 263K scrapie-affected hamster. In 5 groups the sciatic nerve was sectioned 50 mm from the
footpad 1, 2, 3, 5 and 7 days after injection. In one group (the negative group) the nerve was cut 5 min after injection.
The seventh group (the positive group) was not cut. Incubation periods were assessed by monitoring the animals for
clinical signs of scrapie.All the animals of the positive group had short incubation periods, 81.4 ± 3.0 days (mean ± SD,
n=22, range 76-90) indicative of intranerval spreading. While all the animals of the negative group showed long
incubation period, 184 ± 25 days (mean ± SD, n=10, range 106-248), indicative of blockade of intranerval spreading and
of systemic spreading.In the 7 and 5 days cut groups the great majority of animals (8 out of 9 and 7 out of 9 respectively)
had short incubation periods (82±4, and 81±3), indicating intranerval spreading at a rate of no less than 10 mm/day
(50mm/5days). In the 3 days cut group 3 animals out of 8 had short incubation periods (82±1), pointing to a possible
intranerval spreading rate of 17 mm/day. In the 2 and 1 day cut groups only 1 animal out 9 and 1 out of 6 respectively
showed short incubation period, with intranerval spreading rates as high as 25 and 50 mm/day respectively.These results
strongly support the notion that the intrasciatic spreading rate of the scrapie agent of the strain 263K is most likely
higher than 17 mm/day.
A-X CHRONIC WASTING DISEASE AND CERVID POPULATIONS:
THE STANDPOINT IN EUROPE
DOLORES GAVIER-WIDEN AND ELVIRA SCHETTLER
Department of Wildlife Diseases, National Veterinary Institute (SVA), Sweden, Institute fo Zoo-and Wildlife Research,
Germany
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy of free-ranging and farmed cervids. Even
though there is no evidence that CWD may affect humans, it is recommended that consumption of meat of products
derived from infected animals be avoided. This is a precaution in view of the similarities between human and animal
transmissible spongiform encephalopathies and of the yet unknown aspects of CWD. CWD has been recognized only in
North America, except for a single case of an infected elk exported to Korea. CWD has not been reported in Europe.
CWD affects mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk
(Cervus elaphus nelsoni). Subspecies of these hosts, such as the red deer (Cervus elaphus elaphus), are probably also
susceptible. CWD is laterally transmitted by mechanisms not yet clear. Infection may also be contracted from the
environment, which becomes contaminated by the shedding of PrPCWD of infected animals, probably into faeces and
saliva.
A brief description of the farmed and wild cervid populations in Europe with emphasis on the potential CWD-targetspecies will be presented. The recommended samples for testing, the type of tests that are licensed and routinely used in
North America as well as other tests that can be used for CWD will be mentioned. Until the date, the level of testing
conducted specifically for TSEs in cervids in Europe has been very limited, mostly applied to passive surveillance, and
insufficient to exclude the possible occurrence of CWD. Some countries, for example Germany, are working on active
surveillance programs. A significant proportion of the meat consumed in Europe is derived from farmed deer, semicaptive reindeer and hunted free-living cervids. Some practical issues related to the application of TSE testing on wild
animals will be discussed.
095
Poster Session 1
A-X RECOGNITION OF NOR98 TYPE PRION DISEASE IN THE
SWEDISH SHEEP POPULATION
D. GAVIER-WIDEN, M. NOREMARK, S. BENESTAD, M. SIMMONS, L. RENSTROM, B. BRATBERG, U.
CARLSSON, M. ELVANDER, C. HARD AV SEGERSTAD
National Veterinary Institute (SVA), Sweden (Gavier-Widen, Noremark, Renstrom, Hard af Segerstad, Elvander). National
Veterinary Institute (NVI), Norway (Benestad, Bratberg). Community Reference Laboratory (CRL), VLA, UK (Simmons).
European Commission, Food and Veterinary Office FVO, Grange, Dunsany, Co Meath, Ireland (Elvander, new address)
Within the framework of the active surveillance for TSE in sheep in Sweden four cases of the atypical form of scrapie,
Nor98, were identified during 2003. This study describes the age, breed, genotype and clinical signs of the affected
sheep, as well as the results of the rapid test (ELISA, Bio-Rad) and of the confirmatory testing based on PrPSc
immunohistochemistry (IHC) and western blot analysis (WB). In the first case, no histological lesions or
immunohistochemical staining were observed in the brain stem. In the cerebellum, mild vacuolation and diffuse thingranular positive immunostaining were observed throughout the molecular layer. WB (Sheep and Goat, Bio-Rad)
performed at NVI in samples from mesencephalon, cerebellum and cerebral cortex showed a clear positive signal with
the characteristic Nor98 glycopattern. The IHC performed at SVA, NVI, and CRL gave similar results. The second case
showed similar results to the first one, with positive immunostaining in the cerebellum and WB with a Nor98 pattern.
The third and fourth cases were derived from rendering plants and the confirmation of Nor98 was based mostly on WB.
As for classical scrapie, whole flock slaughter, disinfection and a ban on repopulation for 7 years was applied. All culled
animals tested negative for TSE. The only known cases of classical scrapie in Sweden occurred in 1986. Classical scrapie
was ruled out based on confirmatory testing conducted in different laboratories. Nor98 differs from classical scrapie and
exhibits new features in its epidemiology, frequency of genotypes affected, clinical signs, histopathology, distribution of
PrPSc in the brain, and characteristics of the immunostaining and immunoblotting profiles. Our understanding of prion
disease in sheep is being challenged and extended by such newly described atypical cases, but much further work is
required before the implications of these observations on animal or human health can be fully defined.
DIS-01 MMUNOHISTOCHEMICAL CHARACTERISTICS OF PRPD
ARE NOT ALTERED BY HOST GENOTYPE OR ROUTE OF
INOCULATION FOLLOWING BSE INFECTION OF SHEEP.
S MARTIN, L GONZALEZ, A CHONG, F HOUSTON, N HUNTER, M JEFFREY.
1VLA-Lasswade, Pentlands Science Park, Midlothian 2 IAH Neuropathogenesis Unit, Edinburgh EH9 3JF, 3IAH, Compton,
096
In previous studies we have shown that immunohistochemical labelling patterns using N terminal PrP antibodies are
different when natural and experimental sheep scrapie sources and sheep experimentally dosed with BSE are compared.
The intracellular accumulations of disease specific PrP (PrPd) in ARQ/ARQ genotype sheep orally infected with BSE
are not immunolabelled by antibodies which recognised the upstream segments of the flexible tail of the PrP molecule. In
contrast, intracellular PrPd accumulations in different sheep scrapie sources are labelled with antibodies to the upstream
segments of the PrP molecule irrespective of the PrP genotype. In order to determine whether the immunohistochemical
properties of BSE infected sheep are influenced by the route of inoculation or genotype of the host, we have now
examined seven groups of clinically sick sheep, which had previously been intra-cerebrally or intravenously challenged
with BSE. The genotypes examined following intracerebral inoculation were VRQ/VRQ (n=5) ; VRQ/ARQ (n=2);
ARQ/ARQ (n=5) and ARR/ARR (n=5). ARQ/ARQ sheep showed the most widespread and abundant accumulations of
PrPd when compared with other genotypes. No peripheral PrPd was detected in the ARR/ARR genotypes. The
intensity of PrPd accumulation in visceral tissues was less than that found previously in orally dosed sheep. A further
two PrP genotypes , AHQ/AHQ (n=3), ARQ/AHQ (n=5) were examined following intravenous BSE challenge. These
two groups showed similar tissue distribution patterns and levels of labelling. Irrespective of the route of inoculation or
genotype of sheep , the affinity of N terminal PrP antibodies for intracellular PrPd in both brain and lymphoreticular
system were the same as described previously for orally dosed ARQ/ARQ sheep. These results suggest that truncation of
BSE PrPd is not influenced by genotype or by route of inoculation.
Poster Session 1
DIS-02 THE EFFECT OF OVINE PRP GENOTYPE ON THE PRPD
DEPOSITS IN THE MURINE BRAIN AFTER PRIMARY TRANSMISSION.
CLAIRE HORROCKS, STEVE RYDER, JOHN SPIROPOULOS
Veterinary Laboratories Agency, Weybridge, UK
The mouse bioassay is still the current ëgold standard for scrapie strain characterisation despite the increasing number of
promising molecular approaches. The first step of any scrapie strain typing project involves inoculation of ovine brain
material into panels of inbred mice (primary isolation). The VLA has a number of large projects involving scrapie strain
characterisation in mice. Over 200 brain homogenates from natural scrapie cases have so far undergone primary isolation.
In addition to the traditional methodology (recording of incubation period and lesion profile) we have used
immunohistochemistry (IHC) to further analyse the brains of mice derived from primary isolation. Here we report our
observations from 51 transmission experiments of natural scrapie cases inoculated into C57Bl mice. Murine brain sections
from 206 randomly selected mice were stained by IHC using the polyclonal antibody Rb486. An average of 4 mice (range
1-5) per inoculum were examined. For each mouse the patterns of disease-specific PrP (PrPd) deposits were recorded.
Granular deposits of PrPd in the neuropil of the grey matter were observed in all murine brains examined. Plaques were
observed in 57 out of 76 mice inoculated with brain homogenates derived from ARQ/ARQ sheep, but in only 2 out of
108 mice inoculated with brain material sourced from VRQ/VRQ mice. One mouse out of 22 inoculated with
homogenates generated from ARQ/VRQ sheep showed plaques.
Our results show that in scrapie transmission experiments, the sheep genotype influences the type of PrPd deposits
observed in the brains of mice. Plaques were mainly detected in mice inoculated with ARQ/ARQ derived inocula. This
contrasts with observations in the sheep brain, where plaques are almost exclusively associated with the VRQ/VRQ
genotype. Further research is required to fully understand the effect of PrP genotype on the variability of the pathogenic
mechanisms of the scrapie agent.
DIS-03 INFLUENCE OF THE ROUTE OF INFECTION ON THE
CLINICAL AND PATHOLOGICAL PHENOTYPE OF SCRAPIE IN
OVINE TRANSGENIC MICE
VINCENT BERINGUE (1), ANNICK LE DUR (1), OLIVIER ANDREOLETTI (2) AND HUBERT LAUDE (1)
(1) INRA, Virologie & Immunologie Moléculaires, (2) INRA-ENVT, Physiopathologie Infectieuse et Parasitaire des
Ruminants, France
Because of the species barrier, several passages are necessary to produce a 100% attack rate with a stabilized incubation
period in conventional mice inoculated with natural sheep scrapie isolates. Once cloned by limiting dilution, a pure
murine prion strain has been created. Such strains have been very useful in deciphering scrapie pathogenesis. Infectivity
or PrPSc detection in tissues of peripherally-infected mice have shown that prions may replicate in the lympho-reticular
system long before the central nervous system is involved and the disease becomes apparent. From the currently
available data, it would appear that the strain phenotype, as defined by the nature of the clinical signs and the
distribution of spongiform changes and of PrPSc in the brain, is similar whatever the route of infection suggesting the
existence of clinical target areas specific of each prion strain, irrespective of their entry road into the brain.
In this study, we used a new rodent TSE model consisting of the scrapie 127 strain propagated in tg338 transgenic mice
overexpressing ovine PrP. This strain derives from a natural sheep scrapie isolate that was serially transmitted to these
mice without any obvious transmission barrier, and biologically cloned by limiting dilution. We compared the pathology
induced by the peripheral or intracerebral inoculation of this strain. Surprisingly, we found that the clinical signs as well
as PrPSc levels and distribution in brain greatly differed among the routes of infection. Secondary intracerebral
transmission performed with brain material from mice inoculated either centrally or peripherally produced the same
pathology, consistent with no change of the strain phenotype. These results therefore indicate that the route of infection
may strongly influence the apparent phenotype of a scrapie strain. The implication of this finding with regards to the
natural disease and its diagnostic will be discussed.
097
Poster Session 1
DIS-04 NOVEL GENE TARGETED TRANSGENIC MICE AS
MODELS FOR CODON 129 DISEASE ASSOCIATION IN
CREUTZFELDT-JAKOB DISEASE.
MT BISHOP (1), RG WILL (1), L AITCHISON (2), H BAYBUTT (2), E GALL (2), P HART (2), N TUZI (2), JC MANSON
(2).
1 UK National CJD Surveillance Unit, Western General Hospital, Crewe Road, Edinburgh, UK. 2 IAH,
Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, UK.
With the aim of defining susceptibility in human TSE disease and the role of codon 129, a gene targeted mouse model
expressing the human prion protein gene was developed. This allows for expression of transgenic prion protein
mimicking that of the wildtype murine gene in both CNS and peripheral tissue. Level of expression can therefore be
ruled out as effecting transmission studies. We have produced three lines of mice expressing the three codon 129 variants
(Met/Met, Met/Val, and Val/Val). These lines were inbred to ensure any differences in transmission between the lines
was due to the differences in the PrP genes and not to additional genetic background effects. Mice have been inoculated
via intracerebral and intraperitoneal routes with vCJD(Genotype: MM, PrP isotype: 2B), sCJD(MM1),
sCJD(MV2A), sCJD(VV2A). The preliminary results presented will show that:
- Direct replacement of the murine gene has successfully lead to variation in incubation times, dependent on the
inoculum strain.
- Homology of PrP between host and source of inoculum is not a requirement for shorter incubation times.
- Both inoculum strain and host PrP codon 129 genotype determine TSE incubation time.
- Codon 129 genotype of the host has a significant effect on TSE incubation time.
DIS-05 HISTOPATHOLOGICAL AND IMMUNOHISTOCHEMICAL
PROFILES OF SPANISH SCRAPIE AFFECTED SHEEP.
ACIN, C.1, MONLEON, E. 1, HORTELLS, P. 1, MONZON, M. 1 AND BADIOLA, J.J. 1.
1 National Reference Centre for TSEs in Spain. University of Zaragoza. Veterinary Faculty. Spain.
098
Strain typing of transmissible spongiform encephalopathies (TSEs) has been carried out by the transmission of scrapieisolates in several mouse lines. Recent investigations asses the the vacuolar lesion profile, the glycoprofile and the
phenotype of disease-specific PrP deposition in the brain and other tissues of affected sheep We described the
histopathological and immunohistochemical profiles showed in 24 scrapie-affected sheep from a retrospective study.
Another study has also been performed in 9 animals on different stages of the disease that had the same genotype and
breed. All the animals of the study were genotyped by sequencing techniques.
The results of the retrospective analysis, following the method described by Wood et al. (1997), shown that there were
differences between animals of the same breed belonging to the same outbreak, probably due to different stages of the
disease. In animals of the same breed belonging to different flocks, probably these differences were due to different
strains that were present in each outbreak.
Concerning to the immunohistochemical study of the different stages, the animals of the preclinical stage shown
perivascular and perineuronal patterns. This could be due to the preliminary deposition of PrPsc in the first stages of the
disease, without invading the neuronal pericarion, and ascending through the astrocytic cells of the vessels. In the
animals of the clinical stage, they shown the same pattern described by Gonz lez et al. (2002) in the Welsh Mountain
PrPVRQ/PrPVRQ breed what could imply the presence of a new strain in the field.
In these studies, as described by other authors, there were several features that couldn't be controlled, as the age, stage
of the disease, dose, route and age at infection. It is possible that these aspects could modify the characteristics of the
lesional and immunohistochemical patterns.
Poster Session 1
DIS-06 GLYCOSYLATION OF PRP AND THE SPECIES BARRIER
IN TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES.
FRANCES WISEMAN, ENRICO CANCELLOTTI, NADIA TUZI, HERBERT BAYBUTT, LORRAINE AITCHISON, JAMES IRONSIDE*
AND JEAN MANSON
Institute for Animal Health-NPU, Ogston Buliding. *CJD Surveillance Unit, Western General Hospital, Edinburgh,
UK
Host encoded PrP is central to the transmissible spongiform encephalopathies. Transmission of TSE between
mammalian species generally results in prolonged incubation times and often does not lead to overt clinical disease.
However, sub-clinical TSE pathology and accumulation of disease associated, PrPSc in brain is frequently apparent. It
has been suggested that the principal component of this species barrier is PrP incompatibility. However, transmission
experiments to transgenic mice have shown that factors other than the amino acid sequence of PrP are important to the
species barrier. Inter species differences in the post-translation modification of PrP, such as glycosylation, may be one
such influence.
Murine PrP is glycosylated at two sites in vivo (N180 and N196). To address the role of PrP glycosylation in TSEs,
mice with amino acid substitutions that prevent N-glycan addition have been produced by gene targeting. We have
shown these mice have an altered pattern of PrP glycosylation, here we present full biochemical characterisation of this
altered protein. In constrast to cell culture work, we demonstrate that in vivo un- or partially glycosylated PrP does not
exhibit spontaneous PrPSc like properties. We are using these transgenice mice to investigate the importance of
glycosylation of PrP to strain determination and the cross species transmission of a number of TSE agents.
DIS-07 EFFECT OF PRP POLYMORPHISMS AND MUTATIONS
ON TSE TRANSMISSION
R.M. BARRON, V. THOMSON, H. BAYBUTT, N. TUZI, J. MCCORMACK, D. KING, R. MOORE, D.W. MELTON AND J.
MANSON
(1)Institute for Animal Health, Edinburgh, UK. (2)University of Edinburgh, UK
The host PrP gene is known to play a central role in the Transmissible Spongiform Encephalopathy (TSE) diseases.
The expression of PrP is required for transmission of disease, and some mutations/polymorphisms in PrP are associated
with the development of specific forms of disease. We have used gene targeting to introduce a number of
polymorphisms and mutations into the murine PrP gene. Any alteration in disease phenotype/strain characteristics when
compared to the control mice can therefore be attributed to the introduced mutation. These lines of mice have been
challenged with several isolates of infectivity, both from primary human sources, and well characterised mouse scrapie
strains. We have found that the 101L mutation in murine PrP can alter incubation time, targeting and species specificity
with some, but not all strains of agent.
Two natural PrP polymorphisms occur in laboratory strains of mice at amino acids 108 and 189, which determine the
Prnpa (108L/189T) and Prnpb (108F/189V) alleles. Gene targeting experiments have shown these polymorphisms are
the main factors involved in the control of incubation time in Prnpa and Prnpb lines of mice. These polymorphisms have
now been targeted separately into the murine Prnpa gene producing 108L/189V and 108F/189T lines of mice.
Inoculation of these lines with ME7 and 301V strains has shown that residues at codon 189 exert the major control over
incubation time. However the combination of residues at 108 (homozygous or heterozygous) is also critical in
determining the incubation time of disease, and may model the risk to humans of homozygosity/heterozygosity at codon
129.
The results of transmissions to transgenic mice show that alteration of a single amino acid can have a major effect on
both susceptibility to disease, and the incubation time of disease. This implies that even small changes in the host PrP
genotype can significantly alter disease phenotype caused by different strains of TSE agent.
099
Poster Session 1
DIS-08 SPORADIC CJD VERSUS FCJD R148H: ARE THE
PRION STRAINS THE SAME DESPITE THE DIFFERENT ETIOLOGY?
PASTORE M.,W. ZOU, RJ CASTELLANI, M. COLUCCI, S. CHEN, Z. HUA, K. BELL, S. CHIN, P. GAMBETTI
Pastore, Zou, Castellani, Colucci, Chen, Gambetti: Division of Neuropathology, Department of Pathology, Case Western
Reserve University, USA.Hua, Bell, Chin : Division of Neuropathology, Department of Pathology, College of Physician a
The molecular basis of prion diversity has been related to changes in protein conformations where the shape of misfolded proteins
determines the type of strain. The prion strain in turn is influenced by the etiology (familial, sporadic or transmitted by infection)
and by the PrP genotype, as determined by the presence of pathogenic mutations and the genotype at codon 129, the site of a
metionine/valine polymorphism. The question also has been raised of whether some phenotype is actually shared among prion
diseases with different etiology: fCJD E200K is believed to reproduce often the histopathological features of the sporadic CJD
subtype that is associated with metionine homozygosis at codon 129 and type 1 scrapie prion protein (sCJD MM1); fCJD
D178N is thought to resemble sCJD with valine homozygosis at codon 129 and type 1 scrapie prion protein (sCJD VV1). The
founding of a novel PRNP mutation associated with a new phenotype mimicking sCJD with heterozygosis and type 2 scrapie
prion protein (sCJD MV2) gave us the opportunity to compare the phenotype and PrPSc features. Conformational-dependent
immunoassay showed that the PrPSc associated with the mutation is more resistant to Gdn-HCl denaturation than that from
sCJD MV2. The two-dimensional immunoblots revealed differences in number and localization of PrPSc and PrPc isoforms in
the two diseases as detected using antibodies specific for different PrP regions. Furthermore the substitution of the amino acid at
codon 148 in the first alpha-helix of PrP affected the recognition of the protein by the 6H4 antibody and provided a tool to
discriminate between wild type and mutated protein and to determine the allelic origin of protease-resistant PrP in fCJD R148H.
In conclusion even if a detailed analysis of PrPSc suggests that the familial and sporadic forms are associated with distinct PrPSc
strains, probably due to the presence of the mutation, these variations might not be sufficient to cause divergent phenotypes.
DIS-09 SEARCHING FOR BASE: AN ONGOING STUDY
M.I. CRESCIO, P.L ACUTIS, C. MAURELLA, M. MAZZA, F. INGRAVALLE, G. RU, P. GAZZUOLA, L. CAPUCCI§, G.L.
ZANUSSO ‡, M. CARAMELLI, C. CASALONE.
CEA-Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D'Aosta, Italy § Istituto Zooprofilattico della
Lombardia e dell'Emilia-Romagna, Italy ‡ Department of Neurological and Visual Sciences, University of Verona, Italy
100
A new atypical BSE phenotype was recently found in Italy by the authors (1), in a study on the whole brain of eight
positive BSE cattle. This new BSE variant was characterised by a prion protein (PrPsc) molecular weight lower than
typical BSE and by a different glycoform ratio. Peculiar amyloidotic PrPsc deposition was found at
immunohistochemistry allowing the authors to name it Bovine Amyloidotic Spongiform Encephalopathy (BASE).
At the same time new BSE phenotypes, even different from BASE, were identified in France (2) and Japan (3). For this
reason it seems necessary to carefully examine all the BSE cases.
Purpose of this work is to present the data regarding features of PrPsc at Western Blot and immunohistochemistry of all
the 118 BSE cases so far detected in Italy. The study was carried out only at the level of the obex, owing to the lack of
the whole brain in most of the cases. Obtained results showed that only the obex of the two already known cases
presented the BASE features. All the other 116 cattle were different from BASE and likely from French and Japanese
BSE variants, but displayed the classical BSE phenotype.
On the basis of the paucity of the BASE cases and of other epidemiological available data, it is not possible at the
moment to perform any useful statistical analysis of these two atypical phenotypes, but only to describe their features.
They are both in the right tail of the age distribution and represent the 1,7% of all BSE cases within large Poisson's
confidence interval (95%C.I. 0,21%-6,12%).
This study was supported by Ministry of Health grant IZSPLV 001/01.
1. Casalone C., Zanusso G., Acutis P., Ferrari S., Capucci L, Tagliavini F., Monaco S. & Caramelli M (2004) Proc Natl
Acad Sci U S A, Mar 2; 101(9):3065-70
2. Biacabe A.G., Laplanche J.L., Ryder S.& Baron T.( 2004) EMBO Reports 5, 110-115
3. Yamakawa Y, Hagiwara K, Nohtomi K, Nakamura Y, Nishijima M, Higuchi Y, Sato Y, Sata T; (2003) Jpn J Infect
Dis. Oct-Dec; 56(5-6):221-2.
Poster Session 1
DIS-10 A SINGLE PRP GENE POLYMORPHISM DETERMINES
DIFFERENTIAL SUSCEPTIBILITY TO TSES: TRANSMISSION STUDIES
AND IDENTIFICATION OF PRPSC ALLOTYPES BY HPLC-MS
C. CARTONI°, R. NONNO°, M.E. SCHININÀ*, G. VACCARI°, M. DI BARI°, M. CONTE°, F. CARDONE^, U. AGRIMI°
°Istituto Superiore di Sanità, Department of Food Safety and Animal Health.*Department of Biochemical Sciences and
Centro di Eccellenza BEMM, University la Sapienza. ^Istituto Superiore di Sanità, Department of Cell Biology and
Neuroscience, Rome, Italy.
Interspecies transmission of TSE is highly dependent on PrP amino acid sequence and polymorphisms of the PrP gene are
important factors in conditioning the susceptibility of prion diseases in human and animals. The bank vole (Clethrionomys
glareolus), a wild rodent species, is more susceptible to natural scrapie and several others TSE sources compared to the mouse.
The PrP gene of bank vole is polymorphic at codon 109, codifying for Ile or Met. This codon is the homologous to the mouse
108 whose polymorphism Leu/Phe is linked to differences in TSE susceptibility. In this study we investigated the transmission
efficiency of several TSE sources in bank voles with 109Met/Met, 109Met/Ile or 109Ile/Ile PrP genotypes. Preliminary results
show that 5 different scrapie isolates, as well as a case of familiar CJD (Val210Ile), transmitted with shorter survival times in
109Met/Met bank voles. On the contrary, the transmission of the mouse-adapted scrapie strain 139A was equally efficient in
the three bank vole genotypes. In order to investigate if these differences were related to the species barrier or derived from the
different TSE strain used, we studied the efficiency of intra-specific transmission of different TSE in the secondary passages
from 109Met/Met to the three bank vole genotypes. Preliminary results suggest that Met or Ile at codon 109 are differentially
targeted depending on the TSE strain.
Identification of PrPSc allotypes, which accumulate in the brain of heterozygous affected animals, is valuable to investigate
whether this polymorphism is critical for the pathological conversion of PrP. To this end we developed HPLC-MS approach
to analyze PrPSc accumulation in the brain of experimentally infected voles. We will investigate if the longer survival times
observed in 109Met/Ile voles could be explained by a lesser propensity of the 109Ile allotype to pathological conversion.
DIS-11 EFFICIENT TRANSMISSION OF SCRAPIE IN
TRANSGENIC MICE EXPRESSING SHEEP PRION PROTEIN PRP
IS CONDITIONED BY HIGH LEVEL OF PRPC EXPRESSION
DOMINIQUE BÉTEMPS, ANNA BENCSIK, CAROLE CROZET, LATIFA CHOUAF-LAKHDAR, SÉBASTIEN DECHAVANNE,
JACQUES GRASSI, THIERRY BARON
Dominique Bétemps, Anna Bencsik, Latifa Chouaf-Lakhdar, Sébastien Dechavanne, Thierry Baron: Afssa Lyon,
France. Carole Crozet. CNRS/Institut de Génétique Humaine Jacques Grassi.CEA Saclay
Compared to wild-type mice, transmission of animal prion diseases is facilitated in transgenic mice expressing the prion
protein of the host of the disease. We produced transgenic mice expressing the sheep prion protein (allele A136 R154
Q171) under the control of the neuron specific enolase promoter (TgOvPrP4). Here, we first describe the results
obtained following transmission of 2 scrapie experimental isolates, SSBP/1 and CH1641, isolated from british scrapie
cases. It is noteworthy that the latter had failed to transmit to any wild-type mouse line in previous studies. These
experiments also allowed us to clarify the finding that a number of challenged transgenic mice did not show any
detectable accumulation of abnormal PrP (PrP Sc), at the end of their life. Using ELISA techniques, all these mice were
found to express lower levels of ovine cellular PrP (PrPc) than those found in sheep brain stem. By contrast, normal
uninfected mice from this mouse line typically express 3 fold higher levels of ovine PrP C compared to those found in the
sheep brain stem. These results could suggest that the genetic background may have a major influence on the expression,
at least in this particular mouse line, since the expected numbers of copies of the transgene was found, whatever the
expression level of ovine PrPc. However, overall, there was a complete correlation between the absence of PrPsc and the
low expression of PrPc in the brain of transgenic mice. We then describe the transmission of a series of natural French
scrapie isolates, showing that all these scrapie isolates, including sheep of various genotypes but also from one goat,
successfully transmitted to this ovine transgenic mouse line.
101
Poster Session 1
DIS-12 POSSIBLE HIGHER VIRULENCE OF THE BSE AGENT
AFTER PASSING TO AN INTERMEDIATE HOST
JEAN-YVES MADEC1, CORINNE IDA LASMÉZAS2, JEAN-PHILIPPE DESLYS2, THIERRY BARON1
1: AFSSA 2: CEA France
Transmissible Spongiform Encephalopathies (TSEs) are believed to be caused by an infectious agent, which is partially,
if not entirely, composed of a pathological form (PrPsc)of a host-encoded PrP protein. This PrPsc protein displays
molecular features such as insolubility in detergents and resistance to proteases (PrPres protein) and is generally
demonstrated in the brain of TSE-infected animals at the clinical stage of the disease.
However, the capability of the BSE agent to be experimentally transmitted from cattle to C57Bl/6 mice without a detectable
cerebral accumulation of the PK-resistant prion protein (PrPres) has been described (Lasmézas et al (1997) Science 275:
402-405) questioning the relationship between the formation of PrPres and the infectious agent. Using the same mouse line,
we evaluated the PrPres accumulation in the brain of 378 mice at first passage of BSE from 12 different cattle BSE isolates.
In each BSE experiment, we always distinguished two sub-populations of the inoculated animals exhibiting neurological
symptoms, with (46% of the mice, 172/378) or without (54% of the mice, 206/378) detectable PrPres in the brain. When
detectable, PrPres accumulated at a similar level among all the animals.
Interestingly, compared to mice inoculated with cattle material, PrPres was always present in the brain of C57Bl/6 mice
inoculated with the BSE agent that had first been transmitted experimentally or accidentally to sheep, cheetah, macaques
and humans and the incubation periods were shorter. These results pinpoint the higher capacity of the bovine agent to cross
subsequent species barriers once it has passed from cattle to another intermediate host. Thus, our study suggests that should
the BSE agent have transmitted to the sheep population, it might harbour a greater virulence to humans than cattle BSE.
DIS-13 CLINICO-PATHOLOGICAL AND MOLECULAR
CHARACTERIZATION OF NOVEL HUMAN SPORADIC TSE
PHENOTYPES.
PIERO PARCHI, ROSARIA STRAMMIELLO, SABINA CAPELLARI, SILVIO NOTARI, ARMIN GIESE, JAMES POWERS, INGA ZERR,
DENNIS W. DICKSON, MAURIZIO POCCHIARI, HANS A. KRETZSCHMAR, PIERLUIGI GAMBETTI, BERNARDINO GHETTI.
Department of Neurological Sciences, University of Bologna, Italy; Institut fuer Neuropatologie, Ludwig-MaximiliansUniversitaet Muenchen, Germany; National Prion Disease Pathology Surveillance Center, Case Western Reserve
University, USA; Department of Neurology, Georg-August University, Goettingen; Dipartimento di Biologia Cellulare e
Neuroscienze, Istituto Superiore di Sanità, Roma, Italy.
102
Phenotypic and molecular analyses of hundreds of cases have, to date, identified 6 sporadic human TSE subtypes
(Parchi et al. Ann Neurol, 1999). We report 4 subjects showing atypical biochemical and pathological features. Subject
1: A 67 year-old male died after a 2.5 year course of ataxia, upper limb dystonia, pyramidal signs, and late dementia.
Neuropathologic examination showed mild spongiform changes, mainly involving occipital cortex and cerebellum. There
were also small, unicentric plaque-like PrP deposits with unique morphologic and topographic features. The PrP-res
profile on Western blot comprised a type 2 fragment (19kDa), a 12kDa N-terminally truncated glycosylated peptide and
a 8 kDa unglycosylated internal fragment. PRNP codon 129 was Met/Met. Subject 2: A 50 year-old woman died after a
3 year course of progressive dementia, spasticity and seizures. Pathologic features included widespread spongiform
degeneration and PrP-positive amyloid plaques. PrP-res was of type 2 but showed a unique glycoform ratio. Codon 129
was Met/Val. Subject 3: a 66 year-old woman died after a 6 month course of progressive dementia, unsteady gait, and
myoclonus. Subject 4: a 59 year-old male died after a 5 month course of progressive dementia, ataxic gait, and
myoclonus. Pathological and molecular features were very similar in subjects 3 and 4. Spongiform degeneration mainly
affected the cerebral cortex and the striatum. No PrP-positive plaques were seen. The PrP-res profile on western blot
comprised 3 major bands of 26, 21, and 17 kDa, respectively. The lower band was best seen with antibodies against the
C-terminus, whereas it was only weakly stained by 3F4. Codon 129 was Val/Val in both subjects. These 4 cases
demonstrate the existence of previously uncharacterized rare phenotypes of human sporadic TSE.
Poster Session 1
DIS-14 THE PHENOTYPE OF DISEASE-SPECIFIC PRP
ACCUMULATION IN THE BRAIN OF BOVINE SPONGIFORM
ENCEPHALOPATHY AFFECTED SHEEP
LORENZO GONZALEZ, STUART MARTIN, FIONA HOUSTON, NORA HUNTER, COLIN MACALDOWIE4AND MARTIN
JEFFREY
1Veterinary Laboratories Agency (VLA-Lasswade), Pentlands Science Park, 2Institute of Animal Health, UK; 3
Institute of Animal Health Neuropathogenesis Unit, Edinburgh UK; 4Moredun Research Institute, Pentlands Science
Park, UK.
In view of the established link between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt Jacob disease
and of the susceptibility of sheep to experimental BSE, the detection of potential cases of naturally occurring BSE in
sheep has become of great importance.
We have determined the immunohistochemical phenotype of disease-associated prion protein (PrPd) accumulation in the
brain of 64 sheep of various breeds and PrP genotypes that had developed neurological disease after experimental BSE
challenge with different inocula by a range of routes.
Sheep BSE was characterized by neuron-associated intra- and extracellular PrPd aggregates and by conspicuous and
consistent deposits in the cytoplasm of microglia-like cells. The stellate PrPd type was also prominent in most brain areas
and marked linear deposits in the striatum and midbrain were distinctive. Sheep of the ARR/ARR and ARQ/AHQ
genotypes displayed lower levels of PrPd than other sheep and the intracerebral injection of BSE inoculum resulted in
higher levels of PrPd accumulating in the brain when compared with other routes. The PrP genotype and the route of
challenge also appeared to have an effect on the incubation period of the disease giving rise to complex combinations of
magnitude of PrPd accumulation and incubation period.
We conclude that the characteristic phenotype of PrPd accumulation in the brain of BSE affected sheep can help the
identification of potential naturally occurring ovine BSE. The low levels of PrPd detected in the brain of some sheep
raise questions about its significance to clinical disease and about the dynamics of PrP conversion into abnormal forms.
H-01 THE NATIONAL ANONYMOUS TONSIL ARCHIVE:
A RESOURCE TO STUDY THE PREVALENCE OF ABNORMAL
PRION PROTEIN
KELLY C, BATEY K, CLEWLEY JP, DABAGHIAN AH, MORTIMER PP, CONNOR N, MOLESWORTH AM, GILL N
CJD Team, Communicable Disease Surveillance Centre and TSE Unit, Specialist and Reference Microbiology
Division, Health Protection Agency, UK.
An archive of tonsil tissue is being established by the Health Protection Agency for the purpose of estimating the number
of people who might be incubating vCJD in Britain. The tonsil tissues will be tested for the pathological form of the
prion protein, and any other appropriate markers of CJD. Tonsils will be sent from hospitals following routine surgery.
One tonsil will arrive in the laboratory in formalin and the other will be frozen. A piece of the formalin fixed tonsil will
be embedded in paraffin wax and used for immuno-histopathological studies. An aliquot of the frozen tonsil will be
tested by Western blotting, or other suitable and available serological test. Any screen positives will be referred to expert
laboratories for confirmatory testing. The codon 126 M/V genotype of PrPSc positive specimens will be determined.
The contribution of P. Horby, F. Lever and C. Lawson to this project is acknowledged.
103
Poster Session 1
H-02 POLYMORPHISM OF CODON 129 IN THE PRION GENE
IN THE ICELANDIC POPULATION
STEFANIA THORGEIRSDOTTIR*, THORDUR TRYGGVASON*, SVEINN GUDMUNDSSON** AND GUDMUNDUR GEORGSSON*
*Institute for Experimental Pathology, University of Iceland, Keldur, Iceland, **The Blood Bank, University Clinics,
Iceland.
The purpose of this study was to determine the frequency of various genotypes of codon 129 in the prion gene of healthy
Icelandic people. Studies have shown that the phenotype of and susceptibility to Creutzfeldt-Jakob disease (CJD) is
related to certain polymorphism within the prion gene. Thus all cases of variant CJD (vCJD) have been homozygous
for methionine (M) at codon 129, and the majority of cases of sporadic CJD (sCJD) that have been studied are either
homozygous for methionine or valine at this codon. By providing new basic knowledge about genetic markers of the
normal Icelandic population, the study of this risk factor could give a clue to the prediction of infection with the
infectious prion-protein (PrPSc) in the future, for example of possible risk of contracting CJD by consumption of
products of BSE-infected cattle or even sheep. This study is a part of a multi-national research program, supported by
the EU, as the comparison of polymorphism at codon 129 could contribute to explaining different frequency of CJD in
various countries.
We collected blood samples from 208 healthy Icelandic individuals (blood donors), 104 males and 104 females, isolated
DNA, amplified the prion gene by PCR and determined polymorphism at codon 129 by restriction fragment length
polymorphism analysis. Our results showed that 46.6 % of the individuals tested were homozygous M/M at codon 129,
44.7 % were heterozygous; M/valine (V) and 8.7 % were V/V homozygous. The Icelandic nation seems to have a similar
distribution of genotypes at this codon as some of our neighbouring countries where this polymorphism has been
studied. The frequency of the M/M genotype is 10 % higher in Iceland than in the UK, but although not statistically
different (p>0.05), this difference could theoretically make the Icelandic nation more susceptible towards vCJD.
H-03 THE NORMAL POPULATION DISTRIBUTION OF THE MET
ALLELE AT THE PRNP129 POLYMORPHISM IN VARIOUS
REGIONS OF FRANCE AND IN WEST-EUROPE
LUCOTTE, G. AND MERCIER, G.
Center of Molecular Neurogenetics, France
104
The present study was conducted to know allelic variation of codon 129 at the prion gene in France. Six French
populations have been studied (Paris, Rennes, Chambéry, Grasse and Perpignan), totalizing a number of 1374 normal
subjects. Mean heterozygosity in France = 46.5%, and the mean Met allele (a high risk factor for Creutzfeldt-Jacob
disease) = 0.674. There is a genetic heterogeneity (X2=38.44) between the six populations compared, and Met allele
frequencies are inversely correlated with latitude. Such a correlation with latitude
(r = -0.78) was also found when Met allele frequencies in france are compared to those already published in five other
European countries and in Turkey.
Poster Session 1
H-04 EFFECT OF APPENDECECTOMY OR TONSILLECTOMY ON
THE RISK OF CJD IN PATIENTS TREATED WITH HUMAN
EXTRACTIVE GROWTH HORMONE
BRANDEL JP, HAIK S, DELASNERIE-LAUPRÍTRE N, WELARATNE A, AGID Y, CHAUSSAIN JL, ALPÉROVITCH A
INSERM unité 360. Hôpital de la SalpÍtrière, Paris (Brandel JP, Delasnerie-LauprÍtre N, Alpérovitch A) CNR MCJ
iatrogène. Hôpital de la SalpÍtrière (Brandel JP, Haik S, Welaratne A, Agid Y) Service de Pédiatrie. Hôpital SaintVincent de Paul, Paris (Chaussain JL)
A recent European case-control study has shown that appendecectomy or tonsillectomy were less frequent in patients
with sporadic Creutzfeldt-Jakob (CJD) compared to controls (Ward et al., 2002). This suggests a possible involvement
of lymphoreticular system in the pathogenesis of the disease. Such prion replication in lymphoid tissues is well-known in
natural and experimental scrapie and in variant CJD patients. Recently PrPsc accumulation was surprisingly evidenced
in the spleen of some sporadic CJD cases. The aim of the study was to determine if appendecectomy or tonsillectomy
modified the risk of CJD in the French patients treated with human extractive growth hormone (hGH). We sent 1316
questionnaires to the family of patients treated with hGH and non suspect of having CJD (controls) and 90 to the
family of patients died with CJD (iCJD). We have received 453 and 61 answers for the controls and the iCJD group
respectively. The majority of questionnaires were filled by parents of the patients. The frequency of appendecectomy or
tonsillectomy was not different in both groups. This result is in agreement with the absence of PrPsc in lymphoid organs
of iCJD patients. It cannot be excluded that the “protective” effect observed in sporadic CJD was linked to a bias of
answer : a relative of the patient was interviewed in CJD group but controls answered directly.
References
Ward HJT, Everington D, Croes EA, Alperovitch A, Delasnerie-LauprÍtre N, Zerr I, Poser S, van Duijn CM, for the
European Union (EU) Collaborative Study Group of Creutzfeldt-Jakob Disease. Sporadic Creutzfeldt-Jakob disease
and surgery. A case-control study using community controls. Neurology 2002; 59:543-548.
H-05 RISK FACTORS ASSOCIATED WITH CJD IN SLOVENIANS
AND A REAL-TIME PCR METHOD FOR PRNP GENE CODON
129 SNP
VESNA GALVANI, RUTH RUPREHT, VLADKA CURIN SERBEC, BLANKA VIDAN-JERAS
Blood Transfusion Centre of Slovenia, Slovenia
Prion protein (PrP) gene (PRNP) has been the most informative genetic marker for predisposition to new variant
Creutzfeld-Jacob disease (vCJD) so far. All victims of vCJD carried M at position 129 of PrP. It has been suggested
that prions travel through the immune system to get from the gut to the brain and hypothesised that HLA could be
involved in this carriage with HLA-DQ7 being less efficient. Contradictory reports have raised the question of the
influence of sampling in population studies.
We developed a rapid and reliable real-time PCR for codon 129 SNP screening using 7900HT ABI PRISM. Two
differentially sampled groups of Slovenians, namely Slovenian residents (n=270) and Slovenian population (n=100) were
analysed and compared. The comparison of both groups with other populations served for the estimation of the risk for
the development of vCJD in Slovenians.
The frequencies at the codon 129 SNP in the Slovenian population (43.3% M, 45.4% M/V, 11.3% V) differed from the
frequencies of the same types among the Slovenian residents (51.4% M, 38.2% M/V, 10.4% V) although the differences
were not statistically significant. The difference, although not statistically significant, in the frequencies of HLA-DQ7
between Slovenian population (31%) and Slovenian residents (39%) adds support to the discrepancies in frequencies of
HLA-DQ7 in other populations and shows the importance of sampling criteria. Analysing the adequacy of HLA-DQ7 as
a possible predictive factor for developing CJD by case-control studies could be improved with exact and equal sampling
of groups of patients and controls. To be well aware of the influence of CJD genetic risk factors in the Slovenians, which
is slightly higher than in British, might help establishing blood transfusion and transplantation strategy.
105
Poster Session 1
H-06 ULTRASTRUCTURE OF KURU PLAQUES RETRIEVED
FROM PARAFFIN-EMBEDDED BLOCKS
LIBERSKI P.P.(1), KOVACS G.(2), SIKORSKA B. (1), PAUL BROWN (3) AND BUDKA H (2).
(1) Department of Molecular Pathology and Neuropathology, Medical University Lodz, Lodz, Poland, (2) Neurological
Institute, University of Vienna, Vienna, Austria and (3) National Institutes of Health, Bethesda, USA
Kuru was the first human prion disease discovered in 1957 by Gibbs and Zigas. Recently, the interest in kuru came
back because of the epidemics of variant Creutzfeldt-Jakob disease and the presence of vast number of different forms
of plaques in the latter disease. In 1997, we reported a detailed immunohistochemical study of well preserved kuru brain
(Brain Pathol., 1997; 7: 547-554). Here, we report an ultrastructural study of kuru plaques retrieved from formalinfixed paraffin-embedded blocks. PrP amyloid was very well preserved in paraffin blocks; probably, removal of most of
the neuropil made amyloid fibrils easier to find, Different forms of plaques were readily seen. The most prominent were
large aggregates of fibrils without any definitive shapes. Typical compact kuru plaques were seen but less frequently.
Robust astrocytic reaction and contacts of plaques with microglial cells were also visible. In conclusion, we
demonstrated that PrP amyloid is well preserved in formalin-fixed paraffin-embedded blocks. This is also, surprisingly,
the first detailed study of kuru plaques at the level of electron microscopy. Of note, there are much more fibrillar forms
of amyloid in the brain than previously reported.
H-07 PROTEOLYTIC CLEAVAGE OF MAMMAL AND HUMAN
BRAIN PRPC
I. LAFFONT1, R. HÄSSIG1, S. HAIK2, V. SAZDOVITCH2, J.J. HAUW2, B.A. FAUCHEUX2 AND K. L. MOYA1
1CEA-CNRS URA 2210, Service Hospitalier Frédéric Joliot, France. 2INSERM U.360, Laboratoire de
Neuropathologie R. Escourolle, Hôpital de la Salpetrière, France
106
Human brain PrPc is cleaved within its neurotoxic domain at aa 110/111-112. This cleavage generates a highly stable Cterm fragment (C1) anchored to the plasma membrane. To determine if the major form of mammalian brain PrPc is fulllength or truncated we compared different species by Western blotting. The results showed that the major form of
rodent brain PrPc is the full-length form and that in baboon and cattle a truncated form is present in high proportions.
In extracts from 4 human brains, a PrPc full-length signal was detected, along with, depending on the individual case, a
stronger or a weaker C1 signal. These results reveal important inter-individual variations in terms of PrPc truncation in
human brain. To exclude the possibility that C1 was the result of postmortem lysis, we incubated brain homogenates at
37°C. We found no artifactual in vitro truncation of PrPc in our experimental conditions. To study possible sources for
the striking variability in PrPc cleavage we examined a larger series of human brain samples that varied in terms of age
and postmortem interval. The results showed a low level of C1 in these cases, and no obvious variation in C1 related to
age. When the results were analyzed with respect to postmortem interval, there was a modest -but not significanttendency for C1 to increase with this parameter. Thus, neither age nor postmortem interval appears to fully explain the
large variability observed in the first series of human brain samples analyzed. To further examine mechanisms that may
contribute to the cleavage of PrPc, we studied the protease ADAM10 which has been reported to cleave PrPc in vitro.
We examined our series of human samples for the presence of the active form of the enzyme. Interestingly, the human
brain samples that contained the most C1 also had a much higher level of active ADAM10, results that are consistent
with a possible role for ADAM10 in PrPc cleavage.
Poster Session 1
H-08 METHIONINE 129 VARIANT OF HUMAN PRION PROTEIN
OLIGOMERIZES MORE RAPIDLY THAN THE VALINE 129
VARIANT: IMPLICATIONS FOR DISEASE SUSCEPTIBILITY TO CJD
ABDESSAMAD TAHIRI-ALAOUI1, ANDREW C. GILL2, PETRA DISTERER1 AND WILLIAM JAMES1
1Sir William Dunn School of Pathology, University of Oxford, UK. 2Institute for Animal Health, Compton, UK.
The human PrP gene (PRNP) has two common alleles that encode either methionine or valine at codon 129. This
polymorphism modulates disease susceptibility and phenotype of human transmissible spongiform encyphalopathies
(TSEs) but the molecular mechanism by which these effects are mediated remains unclear. Here, we compared the
misfolding pathway that leads to the formation of b-sheet-rich oligomeric isoforms of the methionine 129 variant of PrP
to that of the valine 129 variant. We provide evidence for differences in the folding behaviour between the two variants
at the early stages of oligomer formation. We show that Met129 has a higher propensity to form b-sheet-rich oligomers
whereas Val129 has a higher tendency to fold into a-helical-rich monomers. We provide evidence that the oligomers of
both variants are initially a mixture of a-rich and b-rich conformers that evolve with time to an increasingly
homogeneous b-rich form. This maturation process, which involves no further change in proteinase-K resistance, occurs
more rapidly in the Met129 form than the Val129 form. Although the involvement of such b-rich oligomers in prion
pathogenesis is speculative, the misfolding behaviour could, in part, explain the higher susceptibility of individuals that
are methionine homozygote to both sporadic and variant CJD.
H-09 GEOGRAPHICAL DISTRIBUTION OF SPORADIC
CREUTZFELDT-JAKOB DISEASE IN THE UK BY SUB-TYPEIS IT RANDOM?
WARD HJT(1), BURLINGTON K(1), EVERINGTON D(1), HEAD MW(1), BISHOP M(1), KNIGHT RSG(1), COUSENS
S(2), SMITH PG(2), WILL RG(1)
1 National Creutzfeldt-Jakob Disease Surveillance Unit (NCJDSU), Edinburgh, UK. 2 London School of Hygiene &
Tropical Medicine, London, UK
Creutzfeldt - Jakob disease (CJD) is one of the transmissible spongiform encephalopathies. The origin of the most
common form, sporadic CJD (sCJD), is unknown. There are various hypotheses to explain the occurrence of sCJD,
ranging from normal prion protein (PrP) spontaneously misfolding and aggregating as an abnormal form of the prion
protein (PrPSc), through rare spontaneous somatic mutations of prion protein gene, to low level contamination events.
With the spread of bovine spongiform encephalopathy (BSE) from cattle to humans as variant CJD, there has been
renewed interest in the possibility of sCJD being caused by transmission from an environmental source such as animals
or by as yet unidentified iatrogenic mechanisms.
Epidemiological studies have attempted to elucidate the role of environmental factors in the aetiology of sCJD.
Hypotheses investigated have included that cases were infected through diet, occupation, surgery, contact with animals
and contact with other cases. Despite these efforts, the mode of transmission, if any, of sCJD remains unknown.
Clusters or areas of high incidence of sCJD cases have been reported in various countries, for example, England,
Australia, France, Japan and USA, however, these are extremely difficult to interpret.
This study examines the geographical distribution of residence at death of sCJD in the UK according to the Parchi &
Gambetti sub-classification. Maps of cases plotted by sub-type appear to show differences in geographical distribution.
These will be presented together with statistical and epidemiological analyses of these maps and of potential risk factors
linking the cases within sub-types by geographical place of residence.
107
Poster Session 1
H-10 IDENTIFICATION OF PREDISPOSING POLYMORPHISMS IN
CANDIDATE GENES OF SPORADIC CREUTZFELDT-JAKOB
DISEASE BY SNP GENOTYPING AND ASSOCIATION ANALYSIS
W. XIANG1, C. VOLLMERT2, O. WINDL1, A. ROSENBERGER4, I. WESTNER1, I. ZERR3, H.-E. WICHMANN2, H.
BICKEBOLLER4, S. POSER3, T. ILLIG2 AND H. A. KRETZSCHMAR1
1 Institute of Neuropathology, Ludwig-Maximilians-University Munich 2 Institute of Epidemiology, GSF-National
Research Center of Environment and Health, Neuherberg, Germany 3 Department of Neurology, University of
Gottingen, Germany 4 Department of Genetic Epidemiology, University of Gottingen, Germany
Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form of human prion disease worldwide. The cause of this
disease is unclear. Up to now, epidemiological studies have not revealed any environmental factor being responsible. One
genetic factor predisposing to sCJD is known being a common polymorphism in the coding region of the PRNP gene at the
codon 129 (M129V). However, the major predisposing factor(s) for sporadic CJD are still unknown. The aim of this study
was to identify predisposing polymorphisms in several candidate genes using a large population based association study.
In addition to the PRNP gene, we selected several candidate genes which were proven to be differentially regulated during
prion diseases or to interact with prion proteins by previously published studies or our own expression profiling analysis.
Thus their involvement in prion diseases is highly probable. These candidate genes included proteases, apolipoproteins as
well as metal-binding proteins.
By means of MALDI-TOF MS technique (Mass Array System, Sequenom, San Diego) we genotyped 30 SNPs in eight
candidate genes. Genotyping was performed in 584 German sCJD cases and 749 healthy controls matched for age and
gender which were taken from a large population based study performed in the city and region of Augsburg, Germany
(KORA Survey 2000). The role of these SNPs as possible additional risk factors to the PRNP_129 polymorphism was
evaluated. Using this unique patient sample, we could evaluate published data in PRNP and other genes which were derived
from much smaller cohorts and produced conflicting data.
H-11 GEOGRAPHIC LOCALISATION OF VARIANT CJD IN THE
UNITED KINGDOM: LOCALLY ELEVATED RISK OR WHOLE
POPULATION EXPOSURES?
MOLESWORTH AM[1], GILL ON[1], COUSENS S[2], WARD HJT[3]
[1]Health Protection Agency, Communicable Disease Surveillance Centre, London, UK [2]London School of Hygiene
and Tropical Medicine, London, UK [3]National CJD Surveillance Unit, Edinburgh, UK
108
The geographic distribution of variant CJD (vCJD) cases may reflect how they became infected, whether the level of
exposure is similar over wide areas or there are local factors that augment transmission of the vCJD agent in small areas
resulting in cases that are geographically close.
In 2000 a cluster of 5 cases of vCJD was investigated in a rural area in Leicestershire, UK. Enquiries revealed they might have
acquired their infection through eating beef purchased from local butchers where there was a risk of cross-contamination of
carcass meat with BSE-infected bovine brain. Following this investigation a standard public health approach to the
investigation of any geographically localised cases of vCJD has been adopted, that seeks to identify primarily any local dietary
or iatrogenic exposures to the vCJD agent that might explain the cases’ local occurrence.
By the start of March 2004, 35 cases of vCJD had been involved, with their families’ consent, in a further 12 investigations
throughout the UK. In each location cases were associated through geographic proximity of residence at some time since 1980.
In some locations transmission of the BSE agent may have occurred through the consumption of unfit beef, or of beef certified
as fit for human consumption but contaminated with infectious material as a result of slaughter, butchery or other processing
practices. However for none of the investigations was the evidence considered sufficient to consider it a likely explanation for
the local occurrence of cases. Nowhere has any plausible evidence that vCJD infection was acquired through some aspect of
healthcare been identified. We cannot exclude the possibility that local factors have raised the risk of acquiring vCJD in other
parts of the UK than Leicestershire, but have found no compelling evidence to support the phenomenon of locally elevated
risks. Chance remains a plausible explanation for the geographically associated cases we have observed and investigated.
Poster Session 1
H-12 PRP DEPOSITS, DENSITY OF NEURONS AND ACTIVATED
MICROGLIAL CELLS IN THE GRANULAR LAYER OF THE
CEREBELLUM IN SPORADIC CREUTZFELDT-JAKOB DISEASE.
BAPTISTE A. FAUCHEUX1,2, STÉPHANE HAIK1,2, VÉRONIQUE SAZDOVITCH1,2, NICOLAS PRIVAT1,2, AGUEDA
MATOS2, JEAN-PHILIPPE BRANDEL1, CHARLES DUYCKAERTS2, CLAUDE-ALAIN MAURAGE3, ANNE VITAL4,
JEAN-LOUIS LAPLANCHE5, JEAN-JACQUES HAUW1,2.
1 INSERM U360, 2 Laboratoire de Neuropathologie R. Escourolle, Hôpital de la SalpÍtrière, Paris; 3 INSERM
U422, Service d’Anatomie et Cytologie pathologiques A, Hôpital Roger Salengro, Lille; 4 Laboratoire de
Neuropathologie, Université de Bordeaux II, Bordeaux; 5 Service de Biochimie-Biologie Moléculaire, Hôpital
Lariboisière, Paris, France.
In human prion diseases, neurodegeneration varies according to the forms of the disease, brain regions and neuronal
populations. In some forms of sporadic Creutzfeldt-Jakob disease (sCJD), patients are affected by a severe loss of
neurons located in the granular layer of the cerebellum. We therefore investigated granula cells depletion and its
relationships with factors that have been proposed to contribute to the degenerative process, such as microglial activation
and PrPres accumulation. The density of granule cells was estimated with a computer-assisted image analysis system and
neuropathological lesions (spongiosis, gliosis) were scored. Activated microglia was detected using immunohistochemistry. We studied the location and patterns of the prion protein deposits as shown by immuno-histochemistry,
the biochemical characteristics of proteinase K resistant prion protein (PrPres type), and the polymorphism at codon 129
of the PRNP gene as deduced from genomic DNA sequencing. Neuronal density was associated with the PrPres type
characterized by Western blotting of cerebellum tissue (p<0.0001), spongiosis (p<0.001), the density of activated
microglial cells (p<0.01) and patient’s genotype at codon 129 (p<0.001). Our findings clearly indicate relationships
between some characteristics of the host, the type of PrPres present in the tissue, and microglial activation. Some other
determinants remain to be elucidated, however.
R-01 MODELLING STUDIES ON BSE OCCURENCE TO ASSIST
IN THE AGE RAISING OF SYSTEMATIC TESTS IN FRANCE
VIRGINIE SUPERVIE AND DOMINIQUE COSTAGLIOLA
INSERM EMI 0214, France
In France, since July 2001, all cattle aged 24 months and over sent to fallen stock plants or slaughtered in abattoirs have
to be tested for BSE. The objective of this study was to contribute to a risk assessment to review the age of systematic
tests. From the estimated mean number of BSE-infected cattle[1], and 95% confidence interval values, we ascertained
the number and age distribution of BSE cases detected in abattoirs by systematic tests during last and future six-month
periods of BSE surveillance. As in our preceding study the BSE infection incidence was only estimated until June 1997,
several schemes were assumed to extrapolate this incidence from July 1997. From these dynamics, we simulated the
future of BSE-infected animals by randomly assigning, to each infected animal, an infection age, an incubation time, and
a lifetime from available or estimated data[1]. Varying assumptions regarding the test sensitivity and the BSE case
proportion detected in abattoirs were explored. The scheme that minimized the mean square error between the predicted
and observed BSE cases in abattoirs from January 2001 to June 2003 was considered as the most likely.
The best-fitting was obtained under the following assumptions: mean estimate of the number of BSE-infected cattle,
linear decrease of the BSE infection number from July 1997 to December 1998 and then no infection, 30% of BSE cases
are detected in abattoirs and systematic tests detect BSE cases within 6 months of clinical onset. Under this scheme, no
BSE case aged of less than 36 months should be observed since 2001, and from 2004 in the worst case scenario.
No BSE case aged of less than 42 months has been detected in France since February 2001, which confirms our results.
Also, it would be possible to increase the test age in France at 30 months in abattoirs and in fallen-stock plants without
identifiable impact for the public health.
1. Supervie & Costagliola. The unrecognised French BSE epidemic.Vet Res, in press.
109
Poster Session 1
R-02 NOVEL CELL-FREE CONVERSION SYSTEM:
APPLICATIONS FOR HIGH THROUGHPUT SCREENING OF
DRUGS, AND FOR ASSESSING THE RISK OF CROSS-SPECIES
TSE TRANSMISSION
ILIA BASKAKOV
Medical Biotechnology Center, University of Maryland Biotechnology Institute, USA
Recently we developed a novel experimental procedure for cell-free conversion of recombinant PrP 90-231 (recPrP) to
the amyloid rods in the absence of PrPSc. Cell-free conversion occurs via an autocatalytic mechanism and displays a
species barrier, two key aspects of prion propagation (Baskakov, 2004 JBC). The amyloid rods formed by recPrP have
physical properties that are similar to those of PrPSc, as judged by EM, FTIR, stability toward denaturation, and epitope
presentation. Furthermore, the PK-resistant fragments of the amyloid rods were identical to those observed upon PKdigestion of the novel subpopulation of PrPSc recently identified in patients with sporadic CJD (Zou et al. 2003 JBC).
Here I present development of a semi-automated system for cell-free conversion of full length recPrP 23-231 into
amyloid form using 96-well plates. In its current setup the conversion reactions are monitored automatically and require
only 0.5-5 microgram of recPrP per well (0.002-0.020 mg/ml). This range of concentrations is at least 100-fold lower
than the concentrations used before for generating amyloid from PrP-derived peptides and other amyloidogenic proteins.
Potential applications of novel cell-free conversion will be discussed. In particular, automated conversion system offers
remarkable advantages for rapid high throughput screening of potential anti-prion drugs. Novel conversion system can
also be used as a rapid assay for assessing the intrinsic propensities of TSE transmission between different species.
R-03 IS THE DRIVEN HEAVY METAL PUSH-PULL PRION
THEORY A GOOD MODEL FOR IRREVERSIBILITY OF THE
PRPSC TRANSCONFORMATION?
YVES CHAPRON*, ALAIN PORQUET§, JEAN-MICHEL PEYRIN° AND LAURENT CHARLET¥
*AIED La Terrasse, F, §CABE Geneva University, CH, °EA3545 Université Paris Sud, F, ¥LGIT University of
Grenoble, F.
110
Although it has been demonstrated that PrPc is a copper binding protein, recent evidences states that PrP can also bind
other divalent cations such as nickel or manganese. Interestingly it has been reported that Manganese chelation by
recombinant PrP may trigger PrP transconformation into a protease resistant form. Using Molecular dynamics (MD)
we study the consequences of manganese chelation by the N terminal domain of PrPc. Our previous quantum
calculations have shown that Mn2+ can take the position of Cu2+ in 4 sites of the 58-91 octorepeat peptide of PrPc.
Although Mn2+/ Cu2+ exchange is not favoured due to the comparative high prion affinity for Cu2+, the exchange is
made probably irreversible in condition when Mn2+ is oxidized into Mn3+, by dissolved oxygen which then leads to a
shrinkage of the coordination cage. The further change of coordination of the central metal ion leads to a cascade of local
modification of the backbone conformation, extending to the PrPc core fold. Thus in this first "pushing" step, Mn pushes
Cu out of the coordination cage and acquire a more stable structure, once it is oxidized.
Once this change in prion heading part (PrP58-91) has taken place, PrP is further transformed to PrPsc by a "pulling"
step mechanism. To study this reaction step, the beta-helix part was modelled according to the F. Cohen lab (thanks
C.Govearts). Our MD simulations show that Mn2+ ions is trapped for a relatively long time into the beta-helix. In
contrast, Mn2+ ions that are present nearby the beta-helix will not be able to get into the helix, and will thus remain
untrapped. Thus these trajectories show that the Mn2+ ions trapped into the beta-helix formation will further "pull" the
transformation of PrPc into PrPsc. Therefore in our "Push-Pull Prion Theory", Mn2+ acts twice to transform PrP into
PrPsc: it first pushes Cu2+ out the PrP complexation cage, and second, it pulls the beta-helix together. A movie will
illustrate movement of Mn2+ into the solvated beta-helix.
Poster Session 1
R-04 DEGRADATION OF TSE-PRIONS BY MICROORGANISMS
OF THE GASTROINTESTINAL TRACT OF CATTLE
C. SCHERBEL (1), R. PICHNER (1), S. MULLER-HELLWIG (2) , S. SCHERER (2), M. GROSCHUP (3), R. DIETRICH (4), E.
MÄRTLBAUER (4), M. GAREIS (1)
(1) Institute for Microbiology and Toxicology, Federal Research Centre for Nutrition and Food, Germany. (2) Institute
for Microbiology, ZIEL, Germany. (3) Institute for Novel and Emerging Diseases, Federal Research Centre for Viral
Diseases of Animals, Riems, Germany. (4) Institute for Hygiene and Technology of Milk, Veterinary Faculty, LudwigMaximilians-University, Munich, Germany
Thus far less is known about the ability of microorganisms of the gastrointestinal tract to degrade or inactivate TSEprions. Proteins in foodstuff are degraded almost completely by the polygastric digestion systems of ruminants. Due to
the polypotent metabolic activity of the complex microflora in the gastrointestinal tract this also should apply to the
protein structure of prions.
The aim of this study was to investigate the degradation of PrPSc by the complex ruminal and intestinal microflora. For
that purpose samples were taken from contents of rumen and Colon ascendens of healthy adult cattle immediately after
slaughtering, and incubated together with homogenized brains of scrapie (strain 263K) infected hamsters for up to 20
hours under anaerobic and aerobic conditions. Anaerobic incubation both with rumen and colon contents for 20 hours at
37 °C resulted in a clear decrease of PrPSc signal after Western blotting and immunodetection. A loss of PrPSc signal
below the detection limit of Western blot could be observed within an aerobic incubation of colon contents for 20 hours
at 37 °C. In contrast PrPSc signal remains stable after aerobic incubation of rumen contents for 20 hours.
These results indicate the high ability of the complex microflora of the gastrointestinal tract to degrade PrPSc.
Confirmation of these results by the use of bioassays are in progress.
R-05 PROBABILISTIC MODELING OF BSE IN GREAT BRITAIN:
RESPECTIVE ROLE OF THE TRANSMISSIONS AND THE
SLAUGHTERING IN THE LONG-TERM EVOLUTION OF THE
DISEASE
CHRISTINE JACOB AND PIERRE MAGAL
Unité de Mathématiques et Informatique Appliquées, INRA, France. Département de Mathématiques, Faculté des
Sciences et Techniques, France
We propose a dynamic modeling of the evolution of bovine spongiform encephalopathy in Great Britain allowing a longterm prediction of the evolution of the disease. The modelled quantity is the probability for an animal of being in each
health state: S (Susceptible), E (Contaminated but not yet infectious), I (Infectious (the last months of incubation and
clinical state)), and R (Removed from the population, i.e. slaughtered). The model is in discrete time with a step of one
year. We suppose two age groups: the less than one year old, and others. The transmissions may be "vertical" coming
from the mother for the young animals, or "horizontal" from artificial food or contaminated environment for both groups.
The assumptions concerning the horizontal transmissions are very general: the probability of not being contaminated for
a youn animal is supposed of convex form according to the percentage of infectives of the previous year. And for the
other animals, this probability may be convex or concave-convex. The convex form (e.g. exponential) is suitable when
the contamination may take place for any quantity of absorbed infectious material whereas the concave-convex form
supposes that the contamination needs a minimum threshold of absorbed infectious material. We study mathematically
the long-term evolution of the disease. We obtain a condition for endemicity versus extinction which is function of the
transmission probabilities and the slaughtering probabilities: one maximizes the chances for extinction when the
infectious animal and her calf are both slaughtered. But this measurement does not guarantee the extinction. Simulations
illustrate the results.
111
Poster Session 1
R-06 PRION PROTEIN GENOTYPING OF MINOR ITALIAN
OVINE BREEDS
SBAIZ L., PELETTO S., RIINA M.V., MIRISOLA A., MANIACI M.G., RU G., BOSSERS A.*, MARONI A.°, MODA G.§,
CARAMELLI M., ACUTIS P.L
CEA (Centro di referenza per le Encefalopatie Animali) Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e
Valle d'Aosta, Turin, Italy: * Central Institute for animal Disease Control (CIDC-Lelystad), Lelystad, The Netherlands.
° Ministero della Salute, Rome, Italy. § Regione Piemonte, Direzione di Sanità Pubblica, Turin, Italy.
According to Commission Decision 2003/100/EC, each Member State has to introduce a TSE resistance breeding
program in all sheep breeds. This could have an impact mostly in minor breeds, leading to excessive inbreeding or loss of
productive traits.Aim of this study was to investigate prion protein (PrP) allele and genotype frequencies of five minor
Italian sheep breeds in order to support the development of selection programmes.PrP polymorphisms at codons 136,
154, 171 of 1675 pure-bred and cross-bred rams, belonging to Biellese (n=1207), Delle Langhe (n=188), Frabosana
(n=115), Sambucana (n=129) and Saltasassi (n=36) breed were detected by direct sequencing and pyrosequencing. A
high frequency of the resistant ARR allele (34,5-36,4%) was detected in Delle Langhe, Frabosana and Sambucana
breeds. The resistant ARR/ARR genotype had a frequency of 10,6-11,6%. In Biellese and Saltasassi breeds the ARR
allele had lower frequencies (8.3% and 15,3%, respectively) and only 1.4% and 2,8% of the examined animals had the
ARR/ARR genotype.Statistical analysis was performed for Biellese breed comparing PrP allele frequencies between
pure-bred and cross-bred animals. The data showed that ARR allele was significantly lower in pure-bred
rams.Furthermore, comparison of PrP allele frequencies between pure-bred rams over and below 18 months of age
showed that in breeding rams over 18 months of age there was a significant decrease of ARR allele.As for Delle Langhe,
Frabosana and Sambucana breeds, the relatively high frequency of resistant and semi-resistant genotypes allows the
implementation of selection programmes without increasing the extinction risk of these minor breeds. Nevertheless our
results showed that breeding for scrapie resistance in Biellese and Saltasassi breed will have to consider the low
frequency of ARR allele, which in Biellese also seems to be submitted to a negative selection by farmers.
This research was supported by Ministry of Health grant IZSPLV001/2002
R-07 REGIONAL BSE RISK ASSESSMENT
ALINE A. DE KOEIJER
Quantitative Veterinary Epidemiology (QVE), Department of Infectious Diseases, Animal Sciences Group, Wageningen
University and Research Center, The Netherlands.
112
The EU has been very active in the last 6 years, in assessing the BSE risk of various countries and regions for BSE risk.
Over the recent years, this method has proven itself, by pointing out that many countries which claim to be free from
BSE, should find BSE, when they would actively look for it. Although the method is not quantitative, it has still proven
to be the best BSE risk assessment method for countries so far. Various quantitative methods have been put forward, but
none has been tested as extensively as the EU GBR method. Obviously, at present this method is running into the
limitations of the qualitative system, and further quantification is inevitable for continuation.
Poster Session 1
R-08 TRANSMISSION OF BSE TO NON-HUMAN PRIMATES
DIRK MOTZKUS (*), ANDREAS W. STUKE (*), WALTER SCHULZ-SCHAEFFER (#) AND GERHARD HUNSMANN (*)
(*) Deutsches Primatenzentrum GmbH, Dept. Virology and Immunology, Germany. (#) Institut fur Neuropathologie,
Germany.
The pathologies of bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans
appear remarkably similar regarding spongiformity, gliosis and the occurrence of Proteinase K resistant Prion protein (PrP)
plaques in the central nervous system. Most likely, the infective agent causing BSE has crossed the species barrier and
transmitted a new variant of Creutzfeldt-Jakob disease (vCJD) to man.
In cooperation with five European Primate Centers a quantitative study for the transmission of the BSE agent to non-human
primates was initiated to assess the risk of vCJD infection in humans. BSE brain homogenate was serially diluted and orally or
intracerebrally administrated to cynomolgus macaques (Macaca fascicularis). In our facility clinical symptoms of simian
spongiform encephalopathy were first observed in the group receiving the highest intracerebral dose 931 days post infection.
Transmission of BSE to macaques resulted in increased anxiety, reduced water uptake, temporal itchiness as well as extremities
and trunk ataxia. Duration of clinical symptoms showed a high variability, ranging from 90-100 days for two and 17 days for
one macaque until the final stage of disease, when the animals were sacrificed. Immunohistological deposition of Prion protein
was detected in the gyrus cinguli surrounding spongiform neuronal tissue. Cloning and sequencing of the PrP genes of the
macaques revealed wild-type sequences for all individuals. One group of six M. fascicularis receiving the same dose of BSE
homogenate via the oral route do not show clinical symptoms yet. Comparison of incubation period, clinical symptoms and
systematic biochemical analysis of pathological material will be used to determine the minimal infective dose of the used BSE
homogenate. Our study provides an essential contribution to the quantitative assessment of the risk of BSE transmission
through consumption of cattle-derived products in man. This study was supported by the EU grant QLK1-2001-01096.
R-09 FREQUENCIES OF PRION PROTEIN (PRP) GENE
OCTAPEPTIDE REPEATS IN CATTLE BREEDS AND BSE
AFFECTED CATTLE IN ITALY.
RIINA M.V., LEONE P.*, SBAIZ L., PELETTO S., ZACCARIA B., BALSAMO A., MUTINELLI F.°, PONGOLINI S.§, MAZZA M.,
CASALONE C., CARAMELLI M., ACUTIS P.L.
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy. * C.N.R. - Istituto di Biologia e
Biotecnologia Agraria, Segrate (Milan), Italy. ° Istituto Zooprofilattico Sperimentale delle Venezie, Padova, Italy: §
Istituto Zooprofilattico Sperimentale delle Lombardia e dell'Emilia-Romagna, Modena, Italy
In the bovine PrP gene only one polymorphism in the octapeptide repeat region, containing either 5, 6, or 7 copies of a
motif of 8/9 aminoacids has been identified to date. This polymorphism was not shown to have an effect on susceptibility
to Bovine Spongiform Encephalopathy (BSE). In this study we investigated such polymorphism in Italian BSE affected
cattle and in a representative sample of each of the corresponding breeds.Eighty-three out of the 118 so far identified
Italian BSE cases were examined, belonging to Italian Friesian (FI)(n=54), Bruna (BR)(n=18), Pezzata Rossa (PR)(n=9),
Piedmontese (PI)(n=1), and Rendena (RE)(n=1) breed.A group of cattle randomly chosen from commercial herds of the
same breeds FI(n=96), BR(n=96), PR(n=74), PI(n=51), and RN(n=48) was also analyzed.The PrP gene fragment coding
the octapeptide region was amplified and the PCR products were analyzed by electrophoresis in 3% ethidium bromidestained agarose gel to determine the number of copies. One sample representative of each homozygous animal was verified
by automatic sequencing.Three alleles were identified in both groups characterised by 5,6, and 7 copies of the octapeptide
sequence, but the allele with seven repeats was present only in the Bruna breed. All the six genotypes were found, except
5:5 genotype which was not present in the BSE affected cattle group. The six copy allele was most frequent in both
groups and in all the breeds except the Bruna. In this breed the allele with 7 copies prevailed in the representative sample,
while the 6 copy allele had the highest frequency in the BSE affected group. “Chi square” analysis comparing allele
frequencies in the BSE affected group versus the representative sample group did not show any significant difference for
Friesian and Pezzata Rossa breed. In Bruna breed the 6 copy allele was significantly associated to the BSE affected cattle
(p- value=0,0006) suggesting a possible higher susceptibility to BSE given by this allele.
113
Poster Session 1
R-10 HIGH SCRAPIE INCIDENCE INDUCED BY REPEATED
INJECTION CHALLENGE WITH SUB-INFECTIOUS PRION DOSES.
JACQUEMOT CATHERINE1, HONTEBEYRIE MIREILLE2, RYBNER CATHERINE1, DORMONT DOMINIQUE3*, LAZARINI
FRANÇOISE1*
1 Neurovirologie et Régénération du Système Nerveux, Dpt Neurosciences, Institut Pasteur, France. 2. Repliement et
Modélisation des Protéines, Dpt Biologie Structurale et Chimie, Institut Pasteur, France. 3. Service de Neurovirologie,
CEA,Fontenay-aux-Roses
As natural or iatrogenic exposures to prions are likely to occur throughout repeated challenges, we examined the effects
of repeated injections of low or sub-infectious scrapie prion doses in mice.
Twelve groups of 24 mice were inoculated by the intra-peritoneal route at intervals of 1, 2 and 5 days per week during
200 days with the mouse-adapted C506M3 scrapie strain. The injected prion doses varied from 10-5 (2.5 10-3 mg of
brain) to 10-8 dilutions of the scrapie brain homogenate used as inoculum. The data presented here were from the first
490 days after beginning the inoculations.
Of mice injected in a single challenge with scrapie inoculum of 10-4, 10-5, 10-6 dilution, 1/10, 1/10 and 0/10 animals
developed scrapie at 421 and 357 days post-inoculation (pi), respectively. Of mice injected with scrapie inoculum of 10-5
dilution at intervals of 1, 2 and 5 days per week during 200 days, 18/18, 24/24 and 24/24 animals died of scrapie,
respectively. Of mice injected with scrapie inoculum of 10-6 dilution at intervals of 1, 2 and 5 days per week during 200
days, 21/23, 22/23 and 15/24 animals died of scrapie, respectively. To date, of mice injected with repeated scrapie
inoculum doses of 10-7 or 10-8 dilution, approximately one third and one quarter of animals died of scrapie, respectively.
For all given doses, we found that incidence of scrapie is higher when the total dose was injected as multiple challenges
than as a single one. Animals alive at 700 days pi will be sacrificed and examinated biochemically and histopathologically.
In conclusion, a high incidence of scrapie cases was observed in mice receiving repeated doses of low infectivity, whereas
there was no disease in mice that were injected once with the same doses. The risk of infection with prion disease may
thus increase with repeated doses.
Acknowledgments - We thank Karim Sebastien for animal care and collection of tissues. This work was supported by
GIS ‘infections à prions’ and Pasteur Institute.
* These authors contribute equally to this work
114
Poster
Session 2
Poster Session 2
DEC-01 THE ASSESSMENT OF EMERGING AND TRADITIONAL
CLEANING STRATEGIES IN THE DECONTAMINATION OF
SURGICAL STAINLESS STEEL
I.P. LIPSCOMB, R. HAYS, V.H. PERRY, C.W. KEEVIL
The ability of traditional cleaning methods such as enzymatic, detergent and surfactant cleaning solutions in removing
dried on surface contamination is difficult to accurately ascertain. Current methods for the detection of proteins or
infectious organisms on surfaces largely depend on protocols that involve the sampling of the area of interest, with a
swab or wipe, and the subsequent detection of any contamination on the collection material. This detection may involve
traditional tissue culture techniques, or the application of other methods such as protein chemistry, enzyme immunoassay
(EIA) or quantitative PCR (qPCR). However it is readily apparent that such approaches are inefficient and are unlikely
to detect very low levels of contamination. Indeed, the detection of low levels of prion protein contamination on the
highly convoluted surfaces of surgical instruments, varying from forceps to endoscopes, poses particular problems. Here
we have applied both commercially available and new cleaning solutions with or without the application of sonication to
examine their cleaning efficacy. We have also taken advantage of new developments in microscopy: utilising Episcopic
Differential Interference Contrast / Epi-Fluorescence (EDIC/EF) techniques and the use of sensitive fluorescent dyes
for the detection of sub-picogram amounts of protein and prion agent contamination on surgical metal surfaces to
validate cleaning.
DEC-02 PRION PROTEIN DECONTAMINATION OF SURGICAL
INSTRUMENTS USING GAS PLASMAS
M.P. WILLIS, I.P. LIPSCOMB & C.W. KEEVIL
Environmental Healthcare Unit, University of Southampton, Biomedical Sciences Building, UK
According to the “protein hypothesis” a conformational isomer of the host protein PrP is the infectious agent causing
Transmissible Spongiform Encephalopathies (TSEs) and accumulates in the central nervous system. This agent is
recognised as being particularly resistant to standard methods of inactivation and decontamination such as autoclaving at
121oC for 15 minutes, UV irradiation or many gaseous disinfectants. Steel instruments can retain TSE infectivity even
after formaldehyde treatment. Reports indicate a strong affinity of the protein and conventional sterilisation often
ineffectually removes the material. The risk of transmitting the prion protein iatrogenically via surgical instruments is of
great current concern. Here we hypothesise the use of commercial gas plasmas for effective cleaning of surgical
instruments. Initial pilot studies using contaminated coupons of stainless steel (Grade 316L) indicate gas plasmas may be
able to destroy the protein at the atomic level with no hazardous chemical emissions. Reports suggest plasma cleaning
leaves no organic residue, and it exhibits no surface tension constraints which affect standard aqueous cleaners. Plasmas
also exhibit a short processing time and no waste disposal, indicating them as an efficient and cost effective alternative
for standard cleaners. The use of a specially developed Episcopic DIC microscope along with sensitive protein staining
appears to suggest plasma cleaning offers an effective and harmless method for the sterilisation of stainless steel
instruments.
117
Poster Session 2
DEC-03 EFFICACY OF CHEMICAL DISINFECTANTS,
DETERGENTS AND STERILIZATION CYCLES WITH REGARD TO
ELIMINATE PROTEINS CONTAINED IN MOUSE BRAIN
C. VADROT*, B. BRANCHU**, M. SINEGRE** AND J-C DARBORD*
* AGEPS, LABORATOIRE DES ESSAIS, PARIS, FRANCE. ** HOPITAL BEAUJON, SERVICE
PHARMACIE, FRANCE
The persistence of biological soils on medical device surfaces is the main cause of disinfection or sterilization failures.All
biological soils are likely to mask the presence of infectious contaminations, and prevent further treatments by reducing
efficacy.The prion protein itself is extremely resistant to disinfection and sterilization methods.The aim of this study was to
compare the efficacy of different products used as detergent (DTT),disinfectant or sterilant using bovine serum(BS) and
mouse brain(MB) proteins as a model.
We have used undiluted BS or a mixture made of equal part of BS (1/5 dilution-v/v) and grinded MB as biological
soils.Glass tube or aspiration canula received 120 µl of the experimental soil before drying.Each support was (1)soaked in
a chemical agent, followed by triple rinsing in distillated water and dried or (2)sterilized in autoclave. Colorimetric method
was used to detect residual proteins (Biorad DC ref 500-0116).The principle of this test is based on Lowry's reaction
proposed by the prEN ISO 15883 standard.The results for MB are expressed in percentages of residual protein.
Glutaraldehyde 2%-20min
1
Peracetic acid 0,15%-20min
0
DTT-1 0,5%-15min
8
NaOH 0,5M-15min/1M-15 min
0/0
NaOCl 0,25%-15min/0,5%-15 min
1/0
Sterilization 105°C-30 min/134°C-18 min
13/28
The commercial DTT-1 effect is disappointing in comparison with NaOH and NaOCl, which are able to eliminitate
biological soils at the time-concentration recommended by french regulations.It would be also possible to reduce the
effective contact times NaOH (1M-5min),NaOCl (2%-15min).The sterilization results were foreseeable: this treatment
has an action on protein denaturation and not on detergency,and this undesirable action is increased by high
temperature.These preliminary results will be completed by tests using PrPres MB as contaminated soils.
This work was allowed by the financial support of the French GIS-Prion-program.
DEC-04 APPLICATION OF TITANIUM DIOXIDE
PHOTOCATALYSIS TO INACTIVATE PRIONS
IOANNIS PASPALTSIS1, KONSTANTINA KOTTA 1, ROZA LAGOUDAKI 2, NIKOLAOS GRIGORIADIS 2, IOANNIS POULIOS 3
AND THEODOROS SKLAVIADIS 1 PRESENTING : SPYRO PETRAKIS
1 Prion Disease Research Group, Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, 54124,
Thessaloniki, Greece. 2 B’ Neurological Clinic, AHEPA University Hospital, 54124, Thessaloniki, Greece. 3 Laboratory of Physical
Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece
118
The first instance of transmission of the TSE pathogen through neurosurgical instruments was reported in 1977. The
information about TSE pathogens then was minimal, as was knowledge of their nature. Today, our knowledge of prion diseases
is far greater. Despite this improved understanding, inactivation of prions remains problematic due to their resistance to
conventional methods of decontamination. Experimentally effective methods are inadequate for routine use in daily practice for
several reasons. In some cases their utility is limited to only a few applications in the broad range of fields where prions may
occur. In others they are impractical or too costly on a large scale. Thus, iatrogenic cases of TSE disease remain a problem. As
recently as the year 2000 in Australia, there was a report that “nine hospital patients may have been exposed to CJD”.
In this report we present data supporting the use of heterogeneous titanium dioxide photocatalysis for prion inactivation. The
potential of titanium dioxide to oxidize organic pollutants is already known. It utilizes the energy of the UV-A light to produce
free .OH radicals, which serve as oxidizing agents. When the reaction goes to completion the final products are CO2 and H2O.
After treatment of infected brain homogenates with titanium dioxide according our methodology, the PrPSc content of 3mg
brain equivalent is no longer detectable by western blot. These results are independent of the species used as PrPSc source. In a
bioassay 263K hamster scrapie homogenate was treated with TiO2. Hamsters which received the titanium dioxide-treated
material showed a very significant increase in time of disease onset compared to the positive control hamsters. Optimization of
the treatment conditions could lead to total PrPSc inactivation. The method proposed here may be applicable in small surgery,
dental or ophthalmological practices, and could be scaled up for use in hospitals, research facilities or even abbatoirs.
Poster Session 2
DEC-05 PROTEOLYTIC INACTIVATION OF TSES;
CORRELATION BETWEEN LOSS OF SPECIFIC
IMMUNOREACTIVE MATERIAL AND INFECTIVITY
DICKINSON J, MCLEOD A, HALL G, DENNIS M, MURDOCH H, SUTTON JM AND RAVEN NH.
Health Protection Agency - Porton Down, CAMR, UK
The potential for iatrogenic spread of Creutzfeldt-Jakob disease (CJD) during routine surgery has been well described
in clinical cases and animal models. Current methods of prion decontamination include pro-longed autoclave cycles and
harsh chemical treatments, which are impractical and hazardous both to operators and the environment. This study
reports a novel biological method that offers a practical solution to the problems of TSE decontamination.
Thermostable proteases were assessed for their ability to digest PrPSc from BSE (301V) infected mouse brain
homogenate (MBH). Proteases that were able to demonstrate no immunoreactive bands on Western blot were assessed
by bioassay. Using a BSE (301V) VM mouse model we have demonstrated that the removal of all immunoreactive
material, recognised by the commercial anti-prion antibody 6H4, results in a significant but incomplete reduction in
infectivity.
Using our own antibody reagents we have identified a series of protease resistant molecules of 40-70 kDa in infectious
prion material. Their elimination results in a greater than 7-log reduction in infectious dose with many mice surviving 18
months post inoculation. We propose that these may be novel isoforms of the prion protein and are responsible for the
residual levels of infectivity that remain when the PrPSc monomer bands are digested.
These results will be discussed in relation to the development of a practical method for prion inactivation that can be
easily employed in a healthcare setting.
DEC-06 AUTOMATED DECONTAMINATION OF SURFACEBOUND PRIONS
ANDI SCHMITT, INGO M. WESTNER, LUKAS REZNICEK, WINFRIED MICHELS, GERDA W¸NSCH, WEI XIANG AND HANS
A. KRETZSCHMAR
I. Westner, A. Schmitt, L. Reznicek, G. W¸nsch, W. Xiang, H. Kretzschmar : Institut fur Neuropathologie der
Universität Munchen, Feodor-Lynen-Strasse 23, 81377 Munchen. Dr. Winfried Michels: Miele & Cie. KG Professional,
tersloh.
A long known, but only recently more investigated characteristic of the prion infection is its transmissibility using metalbound PrPSc. Prions are resistant to conventional chemical and thermal decontamination and therefore pose a
significant risk of transmission using non-disposable surgical instruments, i.e. reprocessing the instruments. In recent
studies these observations have been reproduced in animal experiments and in cultured cells.
Together with Miele Professional we evaluate automated decontamination procedures using a modified
washer/dishwasher for routine use in central sterile supply departments (CSSD). The main chemical compounds used in
this study are active oxygen generated from hydrogen peroxide by alkalization and sodium hypochlorite at process
temperatures of 55 or 60°C. As an infectivity assay we use murine neuroblastoma cells which had been selected for
prion-sensitivity for metal-bound PrPSc. Further studies using indicator mice are planned.
In contrast to conventional methods for prion inactivation (e.g. sodium hydroxide or high temperature autoclaving), this
procedure might be even applicable for sensitive instruments such as minimally invasive surgical instruments inclusively
the optics.
119
Poster Session 2
DEC-07 DECONTAMINATION METHODS FOR HEALTHCARE
AND RESEARCH ENVIRONMENTS
FERNIE, K., HAMILTON, S., O’FLYNN, M. AND SOMERVILLE, R.A.
Neuropathogenesis Unit, Institute for Animal Health, Edinburgh,UK
Agents responsible for the TSEs have been shown to be very resistant to inactivation by methods which reliably
inactivate conventional micro-organisms. It has now been shown that infectivity is present in many tissues in the preclinical phase of these diseases and concern has been expressed about the effectiveness of sterilisation of surgical
instruments but also about decontamination methods for wider use in healthcare environments and other situations
where TSE infectivity may be present. Extended periods of autoclaving at 132-138°C have been shown to be unreliable.
Results of an examination of the UK recommendation for sterilization of surgical instruments of exposure to 134-137°C
for 3 minutes in a porous load autoclave will be presented.
Sodium hydroxide produces a reduction in titre but does not completely inactivate, however, exposure to sodium
hydroxide when combined with autoclaving has been shown to be effective. Development of treatments which combine
heat and hydroxide is underway to find a sterilisation process that can be applied to surgical instruments. A combination
of hydroxide and heat can however be extremely corrosive. Part of this study examines possible damage to surgical
instruments exposed to such treatments and compares them to instruments treated by conventional methods in the
Sterile Services Department of a hospital. Initial results show that the use of heat/hydroxide combinations may be
practical. Infectivity studies using various combinations of heat and hydroxide treatments are now underway to find a
practical decontamination method which successfully inactivates the TSE agents but leaves surgical instruments
undamaged and fit for future use.
Exposure to sodium hypochlorite containing 20,000 ppm available chlorine is widely recommended as an effective
decontamination method. Unlike previous studies, our recent studies show significant amounts of infectivity sometimes
remain after treatment.
DEC-08 THERMOSTABILITY OF PRION RODS
IS RAISED BY LIPIDS
MULLER, HENRIK; RIESNER, DETLEV
Institute of Physical Biology, Dusseldorf, Germany.
120
To determine prion degradation factors at different temperatures and in presence or absence of lipids an inactivation
system established by Appel et al. (J Gen Virol. 2001;82:465-73) was successfully adapted for application of a 50 ml
pressure reactor. The reactor vessel was loaded with prion rods or recombinant prion protein rPrP(90-231) from Syrian
hamster with or without bovine edible tallow and heated to a temperature between 35 °C and 180 °C. Independently of
the solution volume and mixture all temperatures were reached within 25 min. To recover and separate the amount of
undegraded PrP, a purification method published earlier by Wessel & Fl¸gge (Anal Biochem. 1984;138:141-143) was
optimised especially for the quantitative analysis of small amounts of PrP in the presence of a large excess of lipids. A
minimal recovery rate of 95 % was achieved. Protein sticking to the inner reactor surface turned out to be of no
significance. The amount of undegraded PrP was quantitatively detected by an optimised immunoblot with a sensitivity
threshold of 50 pg. It was confirmed that lipids have a profound capacity to protect prion rods against heat degradation.
In the present study the lipid water ratio and the solution volume in the reactor were varied. In respect to the volume
effect, in 10 g tallow PrP was degraded completely at a temperature of 70 °C. In contrast, using 30 g tallow PrP
immunoreactivity was still detectable at least up to a temperature of 140 °C. At higher temperatures in addition to the
PrP monomer bands an increased tendency to form aggregates of higher molecular mass was observed. The usage of a
plastic vessel instead of the stainless steel made reactor vessel also increased the content of undegraded PrP after
treatment under otherwise identical conditions. Consequently, a degradation-enhancing effect of the inner reactor surface
was measurable as well. The results will be interpreted also with respect to the safety of oleochemical production
processes.
Poster Session 2
DEC-09 WESTERN BLOT ASSESSMENT OF PRION
INACTIVATION BY ALKALI TREATMENT IN THE PROCESS OF
HORTICULTURE FERTILIZER PRODUCTION FROM MEAT MEAL
TAKASHI YOKOYAMA, KIMI SHIMADA, YUICHI TAGAWA, YUKO USHIKI, YOSHIFUMI IWAMARUA,HIROKO HAYASHI,
MORIKAZU SHINAGAWA
National Institute of Animal Health, Prion Disease Research Center, Japan.
Western blot detection of abnormal isoform of prion protein (PrPSc) is used to assess prion inactivation during the
manufacturing process, which involves heating under alkaline conditions, in order to produce horticulture fertilizer from
meat meal. PrPSc was detected in the sample prepared from horticulture fertilizer spiked with a 0.25 microgram
equivalent of scrapie-infected mouse brain. On the other hand, PrPSc was not detected in a sample containing 0.25 g
brain equivalent prepared from a small-scale processed mixture of scrapie-infected mouse brains and the meat meal by
the same method used to produce the horticulture fertilizer. This finding indicates that PrPSc decreased to at least
1/10^6 when processed by heating under alkaline conditions. The loss of infectivity in the processed mixture was
consistent with reduction of PrPSc detected in the Western blot.
DEC-10 THE PRODUCTION OF INSECT CELL DERIVED
RECOMBINANT PRP - A GLYCOSYLATED AND MEMBRANE
ANCHORED REAGENT
IAN D SYLVESTER, SHALU PATEL AND IGOR B BRONSTEIN, INSTITUTE FOR ANIMAL HEALTH, COMPTON, UK
The production of recombinant proteins has revolutionised the way we diagnose and investigate the mechanisms of
disease. This has led to better therapeutics and more effective preventative measures. Prokaryotic systems are relatively
cheap and easy to culture, and allow for convenient manipulation of the protein sequences expressed. However, they are
unable to perform post-translational modifications such glycosylation and the expressed proteins often have to be
artificially folded in vitro. Eukaryotic expression systems allowed the production of post-translationally modified and in
vivo folded protein. However, whilst this protein is a more representative model of the native protein, it is expensive to
produce, it can be inconvenient to manipulate the expressed sequence and may require specialised production facilities.
We have developed a recombinant PrP expression system based on insect cell culture. Like prokaryotic systems these
cultures are cheap and easy to manipulate and the expressed protein sequence can be readily manipulated. However,
unlike prokaryotic systems, we produce an in vivo folded protein which possesses post-translational modifications such
as glycosylation and the addition of glycosylphoshatidylinositol (GPI) moiety. In addition we can alter the nature of the
glycosylation using different transgenic host cells and produce a soluble form of PrP lacking the GPI anchor. Such
proteins represent invaluable reagents in investigation of prion diseases. We intend to use them to study the function
and cellular processing of PrP in normal and diseased cells, to investigate the formation and clearance of amyloid fibrils
and the interaction of PrP with biological (membranes) and artificial (stainless steel) surfaces.
121
Poster Session 2
DIA-01 THE OCTAPEPTIDE REPEATS IN MAMMALIAN PRION
PROTEIN CONSTITUTE A PH-DEPENDENT AGGREGATION SITE
RALPH ZAHN
Institut fur Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zurich, CH-8093 Zurich,
Switzerland
Structural studies of mammalian prion protein at pH values between 4.5 and 5.5 established that the N-terminal 100residue domain is flexibly disordered. Here we show that at pH values between 6.5 and 7.8, i.e., the pH at the cell
membrane, the octapeptide repeats in recombinant human prion protein hPrP(23-230) encompassing the highly
conserved sequence PHGGGWGQ are structured. The nuclear magnetic resonance solution structure of the octapeptide
repeats at pH 6.2 reveals a new structural motif that causes a reversible pH-dependent PrP oligomerization into
macromolecular aggregates. Comparison with the crystal structure of HGGGW-Cu2+ indicates that the binding of
copper ions induces a conformational transition that presumably modulates PrP aggregation. The immobilization of the
cellular prion protein to the cell surface along with these results suggests a functional role in homophylic cell adhesion.
The structured octapeptide repeats represent a new target for prion diagnostics and treatment of prion diseases.
DIA-02 THE UK TSE ARCHIVE
MAURICE BARDSLEY
Veterinary Laboratories Agency, Weybridge, Surrey.
The TSE Archive at the UK Veterinary Laboratories Agency houses the largest collection of animal TSE material in the
world. Defra funding is provided to the Archive group to receive, store, and supply tissues and fluids to the
international research community. Mostly cattle and sheep, the Archive freezers/'/ contents derive from natural field case
suspects and some VLA experimental programmes, some control material is also available. In excess of one hundred and
fifty freezers are maintained at -80C, and are supported by back-up power, online monitoring and staff on overnight
standby. They are located at the Weybridge site in a purpose built, air-conditioned warehouse with secure access
control. Some 700,000 samples are held although approximately 40% are not available for release because of VLA
project restrictions.
It is possible that a prospective collection service may be offered to researchers, if a request cannot be serviced from
frozen stock. This would be resourced by the Neuropathology group on an opportunity basis, and acting as a supplier to
the Archive. Both groups operate to ISO9001:2000 certification.
All incoming requests are subject to review and approval by a Defra managed expert group that includes representation
from UK, France and Germany. The VLA requires successful recipients to sign a Material Transfer Agreement to
safeguard the interests of Defra, and charges are levied to commercial customers.
Request documentation may be found at the TSE Archive website
"http://www.defra.gov.uk/corporate/vla/science/science-tse.htm".
122
Poster Session 2
DIA-03 CHARACTERIZATION OF THE CAPRINE PRP GENE.
STUDY OF NEW POLYMORPHISMS AND RELATIONSHIP WITH
THE RESISTANCE/SUSCEPTIBILITY TO THE SCRAPIE DISEASE
ACIN, C.1, MARTIN-BURRIEL, I.1,2, MONLEON, E.1, RODELLAR, C.2, BADIOLA J.J.1 AND ZARAGOZA, P.2
1 National Reference Centre for TSEs in Spain. University of Zaragoza. Veterinary Faculty, Spain. 2 Biochemical
Genetics Laboratory. University of Zaragoza. Veterinary Faculty. Spain.
Scrapie is a neurodegenerative disease of the central nervous system (CNS) of sheep (Claridge, 1795) and goats (Chelle,
1942). At present, the knowledge of the genetic control of the scrapie disease in goats is limited, and there is not available
data of PrP polymorphisms in Spanish goat. The first PrP variability results were obtained by Goldmann et al. (1996),
which found polymorphisms in the 42, 138, 142, 143 and 240 codons. This group has also described variations in the
number of repeated octapeptides in the PrP gene (Goldmann et al., 1998) and a new polymorphism in the 102 codon.
The studies of Billinis et al. (2002) showed the polymorphisms at 143 and 240 codons, nine new polymorphisms in the
21, 23, 49, 107, 154, 168, 207 and 220.
We report here the first PrP sequencing study of two Spanish native breeds (Pirenaica and Moncaina). Moreover,
natural scrapie was detected in a herd constituted with Saanen and Alpine goats, this herd was also analysed and the
allelic frequencies for the detected polymorphisms were compared with those obtained from healthy Saanen and Alpina
herds. Although the number of animals and herds have been limited, this first study indicates that a high number of
polymorphic sites can be found in the goat PrP gene (18, 127, 142, 154, 211, 219, 222 and 232), however, the frequencies
for the rare allele are low. Whereas the 142, 154 and 211 codons were polymorphic in the Moncaina and Pirenaica
breeds, the polymorphism on 18 codon was observed in Moncaina. In the scrapie-infected animals the Alpine goat was
homozygous 142II, the Saanen was 142MI showing that the fact of being heterozygous in this codon not excludes the
development of the disease.
In summary, the first description of allelic frequencies for the PrP gene in Spanish goat populations is reported. We
describe the new polymorphisms observed in the native and world-wide extend breeds as well as a possible PrP allele
related to scrapie susceptibility in goats (211Q).
DIA-04 CHARACTERIZATION OF THE PORCINE
PRION PROTEIN
MARIA. E HERVA, JOAQUIN CASTILLA, MONICA MORALES , BEATRIZ PARRA , JOSE RODRIGUEZ, AROA RELANO, Y
JUAN. M TORRES.
Centro de Investigacion en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid
Recent evidences from our group suggest that BSE prion might propagate through porcine species: Porcine PrP (PoPrP) transgenic mice were susceptible to BSE infection but showing a strong species barrier. The new porcine prion
generate after BSE inoculation in Po-PrP transgenic mice is being further characterized.
In this work we study some biochemical characteristic of both Po-PrPSc and Po-PrPC as well as their cellular
distribution. For that, we use Po-PrP obtained from: i) a porcine cell line, PK15 (porcine kidney 15); ii) Po-PrP
transgenic mice or iii) from pigs. The results showed that porcine PrPc has some peculiarity in relation to PrPc from
other species. Surprisingly, porcine PrPc exhibits a considerable degree of insolubility in non-denaturing detergents.
Finally, we analyze the capacity of the PK15 line to support the propagation of a pathogenic form of the porcine PrPc,
which has been generated by several passages of BSE in transgenic mice expressing porcine PrP.
123
Poster Session 2
DIA-05 ‘COMPLEX’ PRP GENOTYPES REVEALED DURING
LARGE-SCALE SCRAPIE-SUSCEPTIBILITY GENOTYPING WITHIN
THE UK NATIONAL SCRAPIE PLAN
B MCKEOWN, P. ROWAN AND J. GREENHAM
Orchid BioSciences (Europe) Ltd, Abingdon, UK
The ovine TSE scrapie is distinguished from other prion diseases which impact human health in that it might be tackled
through a program of selective breeding, increasing the frequency of genotypes known to be more resistant to the disease
through normal oral routes of infection. Despite the lack of evidence for a direct human health concern, there is a finite
chance that natural scrapie is masking BSE in sheep populations which were historically fed MBM supplements. These
supplements are implicated in the creation of the BSE epidemic in UK cattle of the eighties and early nineties.
In order to eliminate this possibility, the European Commission has endorsed a proposal mandating that member States
establish scrapie-susceptibility genotyping and controlled breeding schemes to eliminate the most susceptible genotypes
and increase flock resistance by increasing the most refractory genotypes.
To date we have genotyped almost one million individual animals, as the major genotyping service-provider of the UK
National Scrapie Plan. We have observed a small number of the tested animals (~0.1%) that reproducibly display
genotypes which appear to be imbalanced, or simply impossible, given our current knowledge of confirmed haplotypes
and a diploid genome. These imbalanced profiles are primarily identified in our fluorescent chain-terminating primer
extension assay as heterozygous SNPs in which the ratio of incorporation of one terminator to the other is significantly
different from what is normally observed.
We postulate that the underlying reason for the majority of these imbalanced profiles may be some degree of blood
chimerism, possibly due to twin-twin transfusion in utero. The apparent imbalance is most prevalent in the blood but
does extend, at a reduced rate, to other tissues. Implications for breeding will be discussed.
DIA-06 BSE & SCRAPIE TESTING IN BELGIUM
S. ROELS 1,3, H. DE BOSSCHERE 1, R. GEEROMS 1, C. SAEGERMAN2, P. DECHAMPS 2
1 National Reference Labaroratory for Veterinary TSE (Belgium & Luxemburg), Veterinary Agrochemical Research
Centre (CODA/CERVA), Department of Biocontrol Groeselenberg 99, Belgium. 2 Federal Agency for Safety of the
Food Chain, Belgium. 3 Presenting author (stroe@var.fgov.be).
124
Belgium is a country with about ten million inhabitants and approximately 3,000,000 cattle, 188,000 sheep, 39,000 goat
and 15,000 deer. From 1990 on, bovine spongiform encephalopathy (BSE) was controlled in a passive surveillance
scheme. In 2001, an active surveillance (based on EU regulation 999/2001) was added and since then much more cases
were detected. Till end 2003, 118 cases of BSE in cattle and 57 cases, including 13 primary outbreaks, of TSE/scrapie
were diagnosed in sheep. No cases of TSE in goats or deer were detected yet. Since the introduction of the rapid testing
in Belgium, using the Biorad sandwich ELISA, we have controlled 1.128.725 healthy slaughterhouse samples, 1030
suspected samples, 7925 eradication samples and 83.700 fallen stock samples of bovines added with 4641 healthy
slaughterhouse samples, 491 suspected samples, 632 eradication samples and 1276 fallen stock samples of sheep (20012003). Since january 2002, we also use the Bovine Western blot of Biorad as a confirmatory test. So, since then we use,
as a routine, 4 confirmatory tests, including histopathology, immunohistochemistry, electron microscopy (SAFs) and the
Western blotting. In Belgium we have a homogeneous surveillance scheme with 18 laboratories performing the first
screening of the slaughterhouse and fallen stock samples all using the Biorad sandwich ELISA. The National Reference
Laboratory (CODA/CERVA) must confirm all suspected samples. We present the analysis of the results of this
surveillance scheme, including occurrence of false positives and influences of sampling procedures.
Reference :
Roels S, De Bosschere H, Saegerman C, Dechamps P, Vanopdenbosch E (2004). BSE : Surveillance and testing in
Belgium. New Food, issue 1, 36-40
Poster Session 2
DIA-07 NEW MONOCLONAL ANTIBODY 14D11 FOR
IMMUNOHISTOCHEMICAL STAINING OF PRPSC IN NERVOUS
TISSUE OF HUMAN, CATTLE AND SHEEP
INGOLF LACHMANN2, MICHAEL HARDT1, CARMEN NAVARRO3, HERMANN NIEPER1, ANDREA KONRATH1,
AWAD A. OSMAN2
1Landesuntersuchungsanstalt fur Gesundheits- und Veterinärwesen, Germany. 2Roboscreen Gesellschaft fur molekulare
Biotechnologie mbH, Germany. 3Department of Pathology and Neuropathology, Hispital Meixoeiro, Spain.
Immunohistochemical detection of the pathological form of prion protein (PrPsc) in brain and spinal cord sections
confirms the diagnosis of bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD) and Scrapie.
Specific monoclonal antibodies needed for such applications are rare. Therefore, we developed a monoclonal antibody
14D11, raised against recombinant human prion protein, for the immunohistological detection of PrPsc accumulation in
the nervous tissue of different species. The tissue sections used in this study were pretreated with proteinase K and
hydrated by autoclaving. The immunhistological analysis of this pretreated sections using 14D11 (1 µg - 0.1 µg/ml) as
primary antibody followed by anti-mouse IgG antibody and streptavidin-biotin peroxidase incubation showed a highly
sensitive detection of PrPsc. The observed immunoreactivity of this antibody clearly differentiated between pathological
changed tissue and healthy controls and the results did not differ from that observed after staining with well-known
monoclonal antibodies 3F4 and L42.
DIA-08 IMMUNISATION WITH RECOMBINANT ALPHA- AND
BETA-PRP LEADS TO PRODUCTION OF MONOCLONAL
ANTIBODIES WITH DIFFERENT SPECIFICITIES FOR NATIVE
PRPC AND PRPSC
A. KHALILI-SHIRAZI, S. HAWKE, G. MALLINSON, M. TAYEBI, A. R. CLARKE, G. S. JACKSON, AND J. COLLINGE.
MRC Prion Unit, Department of Neurodegenerative Diseases, Institute of Neurology, National Hospital,UK
Prion diseases involve conversion of normal cellular prion protein (PrPC), a largely alpha-helical structure, to an
abnormal conformational isoform (PrPSc) that shows an increase in beta-sheet content. Recombinant PrP91-231 in
physiological conditions adopts a largely alpha-helical structure (alpha-PrP) while at low pH, following reduction of the
native disulphide bond, this helical region is converted to beta-sheet to produce beta-PrP. The in vitro conversion of
recombinant alpha-PrP to beta-PrP might mimic the recruitment of PrPC to PrPSc during prion pathogenesis.
We immunised Prnp0/0 mice with the alpha-PrP91-231 and beta-PrP91-231 and raised monoclonal antibodies (Mabs)
to elucidate the immune responses to these two protein conformers.
Although Mabs raised against both alpha-PrP and beta-PrP recognised recombinant alpha-PrP and beta-PrP by ELISA
and denatured PrPC and PrPSc by western blotting, their specificities were different. Mabs raised against alpha-PrP
(e.g. ICSM18) were efficient in immunoprecipitating native PrPC but could only weakly immunoprecipitate PrPSc. In
contrast, Mabs raised to beta-PrP (like ICSM35) efficiently immunoprecipitated native PrPSc but were less effective
against PrPC.
The epitopes of these Mabs were also different. Most of those raised against beta-PrP were directed to PrP epitopes
between residues 93-105, indicating for the first time, that this region is exposed in both human vCJD and mouse RML
prions. In contrast, none of the Mabs raised against alpha-PrP Mabs recognised this region and mostly recognised amino
acid residues 142-153. In addition, the Mabs raised against alpha-PrP could differentiate between PrP glycoforms (like
ICSM4 and ICSM10), a property not shared by those raised against beta-PrP.
We have shown that alpha-PrP and beta-PrP are suitable antigens to produce Mabs for studying of PrPC and PrPSc.
These alternatively folded isoforms of PrP are perceived differently by the immune system, a difference that may be
exploitable for diagnostic and therapeutic purposes.
125
Poster Session 2
DIA-09 NO PRP-NULL MICE ARE NEEDED TO OBTAIN SPECIFIC
MONOCLONAL ANTIBODIES AGAINST RECOMBINANT BOVINE PRP
MAJA CERNILEC1, TANJA VRANAC1, RUTH RUPREHT1, MARA POPOVI2, POLONA JUNTES3 AND VLADKA CURIN
SERBEC1
1 Blood Transfusion Centre of Slovenia, älajmerjeva 6, Slovenia. 2 School of Medicine, Institute of Pathology, Korytkova
2, Slovenia. 3 Veterinary Faculty, University of Ljubljana, Gerbièeva 60, Slovenia
Since prion protein (PrP) is very conserved among different animal species, it is believed that, if used as an antigen for
immunisation, it provokes no or only poor immune response. Therefore, by using recombinant PrP as antigen, Prnp0/0
mice are prerequisite for the production of anti-PrP monoclonal antibodies.
The aim of our study was to test the immune response of BALB/c mice upon immunisation with recombinant bovine PrP
(recBoPrP) and to compare it with the immune response in Prnp0/0 mice. Mouse antiserum, tested with indirect ELISA,
showed a high anti-recBoPrP titer, even slightly better in case of BALB/c mice. After the fusion of spleen cells of BALB/c
mouse, having the highest titer, with mouse mieloma NS1 cells, several hybridoma clones producing anti-PrP specific
antibodies were obtained. A monoclonal antibody (mAb) E12/2 of sub-class IgG1, showing especially high anti-recBoPrP
affinity, was selected and tested on non-BSE and BSE infected bovine as well as normal and CJD human brain tissue by
immunohistochemistry and Western blot. The results show that the specificity of E12/2 is comparable to commercially
available anti-bovine PrP mAb 6H4, obtained upon immunisation with recBoPrP in Prnp0/0 mice.
The immune response of our BALB/c lineage could have been the consequence of a mutation in Prnp gene. To exclude
this possibility, oligonucleotide primers were designed and open reading frame of the third exon of the prion protein was
sequenced. The sequence was compared to published Prnp sequences of other mice strains and no variations were found.
The expression of PrP protein was confirmed by Western blot of mice brain tissue homogenate, using 6H4 for detection.
Our results show that Prnp0/0 mice are not necessary to produce mAbs to recBoPrP. We speculate that minor differences
between self protein and immunising antigen could be crucial for the generation of immune response.
E12/2 might prove to be useful for diagnostic purposes.
DIA-10 PANEL OF MONOCLONAL ANTIBODIES
DISCRIMINATES BETWEEN HUMAN AND BOVINE BRAIN
RECOMBINANT PRPS AND GLYCOSILATION PATTERNS
PRP,
KATRINA PRETNAR HARTMAN, ANJA VENTURINI, VESNA GALVANI, SIMON KOREN, RUTH RUPREHT, VLADKA CURIN
SERBEC
Blood Transfusion Centre of Slovenia, älajmerjeva 6, Slovenia
126
Prion diseases, such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle, are
fatal neuro-degenerative disorders. Beside approved diagnostic procedures, vaccination and therapy are current goals in the field.
Since the prion protein (PrP) is evolutionary very conserved, there is no or very little immune response after an individual
encounters an antigen from other species. As it seems, the key answer is the right choice of antigen for immunisation. On the
other hand monoclonal antibodies (mAbs) against all forms of PrP are essential tools for research in this field as well as for
therapy.
In our experiments we chose recombinant human PrP (recHuPrP) protein for immunisation of BALB/c and Prnp0/0 mice.
Both strains developed antibodies against antigen. After fusion of lymphocytes from spleen and NS-1 mieloma cells, hybridoma
cell lines from knocked out mice produced higher antibody titer than those from BALB/c, as expected. Further investigation
revealed also different selectivity.
Selected mAbs were tested on recHuPrP, recBoPrP and on samples of normal human, murine (both strains) and bovine brain
homogenates. Antibodies' sensitivity was tried also in peripheral blood, cells and serum of healthy individuals with known codon
129 polymorphism. Reaction pattern was unique for each set of MAbs. Some of them selectively bound normal bovine PrP,
others normal human PrP. Some antibodies bound all PrPs, inclusive recombinant proteins. The affinity for di-, mono- and
unglycosilated form of PrPs was also different.
Immunisation with recHuPrP as an antigen initiated the production of broad panel of different MAbs, which could be used for
discrimination between different PrPs and also for identification of different glycosilation forms.
Poster Session 2
DIA-11 DNA APTAMERS AS A NEW TOOL IN DIAGNOSTICS
TIBOR HIANIK, VERONIKA OSTATNA, TOMAS SAMUELY, ZUZANA ZAJACOVA
Department of Biophysics and Chemical Physics, Comenius University, Slovak Republic
DNA aptamers are single stranded nucleic acids with high affinity to proteins or other low and macromolecular
compounds, that is comparable with affinity of antibodies. The aptamers can bind not only to a target protein, but can
recognize its individual structural elements. DNA aptamers can be chemically modified by thiol groups or biotin, that
allowing them to attach to the solid surface. This system can be used as a biosensor for detection protein in complex
biological liquids using gravimetric method based on quartz crystal microbalance, optical methods based on surface
plasmon resonance technique or electrochemical methods based on application of electrochemical indicators. Aptamer
based biosensors can be used for detection prions and their pathologic form typical for BSE. On the model system
composed of 32-mer DNA aptamer modified by biotin at its 3’ end and specific for thrombin, we demonstrated the
ability of this system to detect thrombin using QCM method and electrochemical indicator method. By means of AFM
technique, we studied the topology of aptamers on a amphiphilic surface and the aptamer-thrombin complexes. The
influence of buffer composition on the binding of proteins by aptamer as well as non specific interaction of proteins with
aptamers were studied. The application of aptamer based biosensors in diagnostics of various diseases are discussed.
DIA-12 HIGH AFFINITY PRP-BINDING SEQUENCES
ISOLATED FROM RANDOM PEPTIDE PHAGE DISPLAYED
LIBRARIES
J. MONTAG, A. STROM AND A.W. STUKE
German Primate Centre (DPZ), Department of Virology and Immunology, Germany.
Transmissible spongiform encephalopathies (TSE) are neuron degenerative diseases accompanied by conversion of the
cellular prion protein (PrP) into a pathogenic isoform. For diagnosis of TSEs, antibodies against both isoforms are
needed. Several approaches to develop monoclonal antibodies (mab) led to the isolation of isoform unspecific mabs.
Here we report the use of the rapidly developing recombinant antibody technology to generate antibodies against PrP.
We isolated fifty recombinant phage antibodies (pab) via phage display and analysed them. Phage from pSKAN and
Ph.D.7/-12 libraries were selected against divers PrP antigens: peptides spanning the PrP, recombinant PrP and PrP
overexpressed on the cell surface of fibroblasts.The phage were tested for their binding avidity, affinity and specifcity in
ELISAs. To create reproduceable and evaluable results different approaches were accomplished. Phage and mab signals
were adjusted. The avidity of the selected phage were compared to those of mabs in ELISAs. By using the adjustment of
signals it was found that phage binding avidity is comparable to that of mabs. We conclude the pab can be used for TSE
diagnosis. Still problems of reproduceability with the phage ELISAs have to be further improved. Blocking methods in
ELISA measurement were optimized to reagent to assure the phage do not unspecifically bind to the surface of the
plates. By using several blocking reagents in different concentrations 1.5% BSA showed the best blocking effect.
The binding sequences were acquired and the corresponding peptides were synthesized. Currently the peptides are
tested in an in vitro cell culture system for their ability to prevent PrP conversion. In order to find common binding
motifs homology comparisions were performed. Some of the peptides showed significant homologies. The sequences and
motifs were compared by BLAST to find putative cellular receptors of PrP. Several proteins were identified that will be
further inquired.
127
Poster Session 2
DIA-13 A NOVEL BLOOD BASED TSE DIAGNOSTIC TEST
ZWALD, D., KUHN, F., P¸RRO, M., SCHMID, J., OESCH, B. AND A.J. RAEBER
Prionics AG, Schlieren-Zürich, Switzerland
There is an urgent need to develop sensitive blood based assays for ante mortem diagnosis of transmissible spongiform
encephalopathies (TSE). We have developed immunochemical assays for the detection of PrPSc in blood and fractions
thereof using the monoclonal antibody 15B3 which exclusively recognizes the pathological isoform of the prion protein,
PrPSc, but not the normal isoform PrPC. Because 15B3 is an IgM subtype it has been speculated that the specificity to
PrPSc is due to high avidity effects conferred by the multimeric structure of the antibody. Therefore, we cloned the
heavy and light chain variable regions of 15B3 into a human IgG framework and expressed the antibody transiently in
eukaryotic cells. Immunoprecipitations with the bivalent 15B3 IgG antibody coupled to magnetic beads showed identical
specificity towards PrPSc as the original IgM subtype suggesting that the specificity is not dependent on the pentameric
structure. In addition to the detection by immunoprecipitation followed by Western blot analysis, we developed a
sandwich ELISA using 15B3 as a capture antibody and an N-terminal PrP specific antibody for detection. To determine
the capability of 15B3 to detect PrPSc in plasma as a matrix for an ante mortem diagnostic test, we performed spiking
experiments of BSE brain homogenates into normal bovine plasma. Using the ELISA detection system, the 15B3
antibody but not a control antibody specifically captured PrPSc spiked into plasma. We investigated whether 15B3 is
also able to detect PrPSc in blood fractions of TSE infected animals. The ELISA platform was used for the analysis of
scrapie positive and normal sheep plasma samples and we show that scrapie positive plasma samples result in higher
ELISA values compared to negative plasma samples.
DIA-14 DETECTION OF PRPSC IN THE TONGUE OF SHEEP
AFFECTED BY NATURAL SCRAPIE.
C.CORONA, C.CASALONE, P.L. ACUTIS, M.I.CRESCIO, F. MARTUCCI, B.IULINI, C.FLORIO, G. PERFETTI§, C. BONA, G.
RU, M.CARAMELLI
CEA-Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D'Aosta, Torino, Italy. §Istituto Zooprofilattico
del Lazio e della Toscana, Siena, Italy
128
Previous studies in rodents (1, 2) showed tongue involvement after either oral or intracerebral challenge with several
strains of Scrapie. Aim of this study was to investigate the presence of PrPSc in tongues of natural Scrapie affected
sheep, analysed by immunohistochemistry (IHC) and Western blot (WB).
We considered ten sheep positive at rapid test, six of which showing clinical signs, coming from two different outbreaks
and six negative adult sheep from unaffected flocks.
Brain, lymphoid tissue and tongue were collected from each animal and examined by IHC and WB. The tongue was
examined by both methods in specular areas at the level of the apex and the corpus linguae
The brain of positive animals was studied by histology, IHC and WB and confirmed as positive. The last two methods
resulted also positive for lymphoid tissue.
PrPSc was detected by WB and IHC in both areas of the tongues of seven animals out of ten, including only three of the
animals with clinical signs. No immunoreactions were observed in brain, peripheral lymphoid tissue and tongue of
negative controls.
This is, to our knowledge, the first demonstration of PrPSc accumulation in tongue. Further work is required to
establish at which stage of pathogenesis the tongue is involved and to assess possible implications for public health,
considering the fact that this organ is not included in the Specified Risk Material list.
This study was supported by Ministry of Health grant IZSPLV 004/01.
1. Bartz JC, Kincaid AE, Bessen RA. J Virol 2003; 77: 583-91
2. Thomzig A, Kratzel C, Lenz G, Kruger D, Beekes M. EMBO rep 2003; 5: 530-33
Poster Session 2
DIA-15 SCREENING LYMPHOID TISSUE OF SHEEP FOR A TSE
INFECTION BY WESTERN BLOTTING.
LANGEVELD JPM, JACOBS JG, VAN KEULEN LJM, BOSSERS A, AND VAN ZIJDERVELD FG
Central Institute for Animal Disease Control Lelystad (CIDC-Lelystad), The Netherlands, jan.langeveld@wur.nl
In a large fraction of the sheep population, lymphoid tissue is infected early in the pre-clinical phase, allowing live testing
on tonsillar tissue. During slaughter of sheep a possibility exists to perform testing before carcasses are being further
processed for human consumption. Early detection of TSEs is important for enhancement of safety measures and
consumers’ confidence.
A practical method which can be applied in routine control testing or rapid testing programs was developed. It consists
of a modification of the Western blotting technique generally used for screening BSE in cattle. Adaptation of tissue
treatment conditions and choice of PrP-specific antibody lead to a high reliabillity of the procedure of PrPres detection
in lymphoid material, also from precilinical animals as early as the third month of life. The gold standard method,
immunohistochemistry, supported these findings, though this technique can even detect scrapie a month earlier, at least
in VRQ/VRQ animals. The easy location and their size make retro-pharyngeal lymph nodes a practical target for such
preclinical testing in slaughterline material.
DIA-16 IMMUNOHISTOCHEMICAL LOCALIZATION OF PRION
PROTEIN IN SIMIAN EYES.
NICOLE SALÈS, JEAN-GUY FOURNIER, SOPHIE FREIRE, MYRIAM BUREL, JEAN-PHILIPPE DESLYS, CORINNE I.
LASMÉZAS.
CEA, DRM, Fontenay-aux-Roses, France.
Due to the resistance of the causative agent to many decontamination procedures, the iatrogenic prion transmission is a
major public health concern. This is particularly important for ophthalmology since the eyes are part of the central
nervous system and are frequently submitted to surgical procedures, particularly in elderly patients. Therefore, it is
important to determine the tissular and cellular localization of PrPres in areas subjected to ophtalmologic surgery. We
used immunohistochemical procedures at the optical and ultrastructural levels to perform this localization in the eyes of
Cynomolgus macaques infected by various routes with several CJD and BSE strains.
We show that:
1) in accordance with previous observations in human and rodent eyes, the retina of monkeys at the terminal stage of
TSEs is strongly immunoreactive for PrPres,
2) this immunoreactivity is observed, though at different levels, for all the strains and after all the routes of infection
tested,
3) double-labelling experiments for PrPres and for several synaptic proteins show that, at the optical level, the
localization of PrPres is clearly synaptic restricted specific zones,
4) at the electron microscopy level, a presynaptic localization of PrPres is observed in the complex triad synapses;
5) in our conditions, no PrPres could be detected in the cornea.
In conclusion, the cornea, which had been reported to be infectious in transmission experiments, contains very low (if
any) amounts of PrPres, contrasting with its huge accumulation in the retina. Hence the potential risk associated with
clinical procedures on a normal cornea, such as tonometry, is likely to be very small compared with that of surgery
involving inner parts of the eye. Besides their implications for human health, our observations reinforce the concept of
prion diseases being primarily a pathology of synapses and they open the way to a better understanding of
pathophysiological aspects of these diseases.
129
Poster Session 2
DIA-17 AN ANALYSIS OF MUSCLE SAMPLES FROM CASES OF
SPORADIC AND VARIANT CREUTZFELDT-JAKOB DISEASE FOR
THE PRESENCE OF PRP(SC)
AH PEDEN, M GLATZEL, DL RITCHIE, MW HEAD, A AGUZZI, JW IRONSIDE.
National CJD Surveillance Unit,, Bryan Matthews Building, University of Edinburgh, Western General Hospital,
Edinburgh, UK. For A Aguzzi and M Glatzel : Institute of Neuropathology, University Hospital of Zurich, Switzerland
Variant Creutzfeldt-Jakob disease (vCJD) is thought to differ from other human prion diseases in that PrP(Sc) can be
detected at extraneural sites throughout the body, principally in the lymphoid tissues. However, a recent report shows
PrP(Sc) at very low levels in skeletal muscle and/or spleen from a third of Swiss patients with sporadic CreutzfeldtJakob disease (sCJD). This may represent centrifugal spread from the CNS or may indicate something particular about
these specific Swiss cases. In order to address this issue we have used the same high-sensitivity Western blot technique,
involving precipitation with sodium phosphotungstic acid, to screen sCJD and vCJD muscle samples of UK origin at
the National CJD Surveillance Unit. These studies have led to the identification of at least one positive muscle sample
from a case of sCJD. PET blot analysis confirmed this result and showed a heterogeneous distribution of PrP(Sc) in
muscle fibres. These findings highlight the importance of taking multiple samples and employing high sensitivity
techniques to determine whether PrP(Sc) is present in different tissues. The positive muscle sample came from a case of
sCJD with MV1 subtype and was atypical in a number of respects including an unusually long clinical phase (4 years).
These results suggest that the presence of PrP(Sc) in muscle is not an exclusive characteristic of current Swiss cases and
that accumulation of PrP(Sc) in extraneural tissues in sCJD may be a function of disease duration.
DIA-18 REGULATION OF PRP EXPRESSION TO
CHARACTERISE PRIONS FROM SEVERAL SPECIES
LARS HEINIG, DIRK MOTZKUS AND ANDREAS W. STUKE
German Primate Centre, Dept. Virology and Immunolgy, Germany. LHeing@dpz.gwdg.de
130
Available bioassays in animals for the detection and characterization of prion diseases need several months to receive
conclusive results. In contrast, cell culture systems are easy to manipulated and allow a higher number of experiments in
between days.
We designed a ex vivo amplification modell in cells with an inducible PrP expression. It was created for the human PrP.
The expression is controlled by a tetracycline (Tc) - regulated element. We have engineered a vector by exchanging the
Prnp gene from human against the Prnp genes form non-human primates (rhesus monkey and crab-eating macaque),
human primates (gorilla and pygmy chimp) and further mammals (mice, great tumblers, sheep and cow). Selected
vectors were stably integrated into murine 3T3 Tet-Off fibroblast cells and transiently in PrP knock-out cells. Murine
3T3 fibroblasts have a basic PrPC expression level. We can regulated the stimulation of the PrP expression in the cells
by different Tc concentration to adjust the optimal concentration for the PrP conversion.
PrPC is nesserary for the conversion of cellular PrPC into endogenous PrPSc by stimulation with non-endogenous
PrPSc. The cell lines will be infected with exogenous PrPSc from mice, bovine, monkey and human. In the first
experiments we have incubated the cells with the pathogenic bovine PrP. The level of the different PrP isoforms will be
examine by proteinase K digestion. In the created cell lines we can monitor the conversion also by spectroscopy. This
cellular system will be used to investigate in the mechanisms underlying the species barrier.
Poster Session 2
DIA-19 LONGITUDINAL ANALYSIS OF PRPC EXPRESSION
PATTERNS ON BLOOD CELLS FROM EXPERIMENTALLY BSEINOCULATED CYNOMOLGUS MONKEYS
BARBARA YUTZY1, E. HOLZNAGEL1, C. COULIBALY1, M. TORNER1, J.-P. DESLYS2, AND J. LOWER1
1 Paul-Ehrlich-Institut (PEI), Langen (Germany). 2 Commissariat à l’Energie Atomique, Département de Recherche
Médicale (CEA), France
It was shown by others that the cellular prion protein (PrPc) expression pattern on blood cells is different in humans
suffering from sporadic Creutzfeldt-Jakob-Disease. We applied this FACS analysis test in experimentally BSEinoculated cynomolgus monkeys to examine whether this phenomenon also occurs in the simian animal model for variant
CJD during the asymptomatic phase of infection. Animals with clinical symptoms and/or positive 14-3-3 protein
cerebrospinal fluid tests were excluded from statistical analysis.
Cynomolgus monkeys (n = 6) were intracerebrally inoculated with BSE. Non-inoculated age- and sex-matched monkeys
(n = 8) served as controls. The first cases of simian vCJD were observed from week 153 post inoculation onwards. The
testing of PrPc fluorescence intensities started at week 104 p.i. During the incubation period (104th - 149th week p.i.),
we detected statistically significant group differences in only 2/13 time points.
In contrast to the published cross-sectional sCJD study in symptomatic humans, we observed an increase rather than a
decrease in the PrPc mean fluorescence intensity ratios of asymptomatic simian vCJD carriers. However, the observance
of PrPc expression on blood cells during the asymptomatic phase of simian vCJD has a very low diagnostic value. This
may be due to the high individual variation of PrPc MFIRs and/or technical difficulties in analysing PrPc on blood cells.
Acknowledgments. This work is supported by a grant from the European Union (QLK1-2002-01096). We would like to
thank Gerhard Hunsmann (German Primate Centre, Gottingen, Germany) who is the EU-coordinator of the BSE-inprimate-consortium, and the other members of the consortium in Sweden (Pär Bierke et al., Swedish Institute for
Infectious Disease Control, Stockholm), Italy (Maurizio Pocciari et al., Instituto di Sanità, Rome) and France (Corinne
Laszémas et al., CEA, Fontenay-aux-Roses) for their help.
DIA-20 CIRCADIAN RHYTHM OF PRION PROTEIN
UTA HEINEMANN, MARIO BARTL, MONIKA BODEMER, KATHARINA STOECK, DANIELA VARGES, GISELA FELDMANN,
INGA ZERR
University hospital, dept. neurology, Goettingen.
Human prion diseases include sporadic Creutzfeldt-Jacob-Disease (sCJD), infectious disorders (variant CJD, Kuru)
and genetic forms (familial CJD, fatal familial insomnia FFI, Gerstmann-Straeussler-Scheinker-Syndrom GSS). The
common pathogenesis of these disorders is characterized by the prion hypothesis, whereby the physiological prion
protein PrPc is folded into a beta-helical structure protein called PrPsc. The reasons for this process are not known so
far. For better understanding the development of these disorders it is necessary to analyze the features and functions of
the physiological prion protein. Various studies suggest a role of PrPc in circadian rhythm: Many patients with prion
disorders show interferences in day-night-regulation, especially in FFI. Further a circadian expression of PrP mRNA in
cell culture models was demonstrated. This study analyzes the concentration of PrPc in serum by ELISA (BioRad) in 6
healthy test persons of different age group and both sex. Blood samples were taken partly every 2 hours, partly every 6
hours over a period of 24 hours. This resulted in individual curves of circadian distribution. Statistically significant
elevation in most samples were found at 8 p.m. Statistically significant reduction in most samples were found at 2 p.m.
and 4 p.m. Thus we found further hints for a role of the prion protein within circadian rhythm by individual specific
patterns of distribution and interindividual reproducible peaks.
131
Poster Session 2
DIA-21 WIDESPREAD DISTRIBUTION OF PRPRES IN
PERIPHERAL TISSUES INCLUDING SKELETAL MUSCLE IN AN
ITALIAN PATIENT WITH VARIANT CREUTZFELDT-JAKOB
DISEASE
LIMIDO L, GIACCONE G, MANGIERI M, CAPOBIANCO R, SUARDI S, DI FEDE G, ZERBI P*, FOCIANI P*, BUGIANI O,
TAGLIAVINI F
Istituto Nazionale Neurologico Carlo Besta and *Università di Milano - Ospedale Luigi Sacco, Milano, Italy.
We report the neuropathological and biochemical studies of the Italian patient with vCJD. This 26-year-old woman was
homozygous for methionine at PRNP codon 129 and died 26 months after the onset of neurological symptoms. Cerebral
and peripheral tissues were examined by immunohistochemistry and Western blot to determine the distribution and the
biochemical properties of PrPres. The neuropathologic picture was characterized by extreme and widespread neuronal
loss, astrogliosis and microglial activation and by the presence of PrPres-immunoreactivity, under the form of amyloid
cores surrounded by vacuoles of spongiosis (florid plaques). In many locations, such as caudate nucleus, putamen and
spinal cord, florid plaques were absent, but PrP-immunoreactivity was abundant under the form of diffuse and focal
deposits. PrP plaques were also numerous in the white matter. Western blot analysis of brain tissue showed a PrPres
profile marked by the preponderance of the diglycosylated species, with the non-glycosylated form migrating at 19 kd.
PrPres was also detected in the retina, optic nerve, pituitary gland, dura mater, peripheral nerves, skeletal muscles and
lymphoreticular tissue (tonsil, lymphonodes, spleen, appendix and Peyer’s patches). In some tissues the PrPres
glycoforms appeared equally represented, while in others the predominance of the diglycosylated species was striking.
The results indicate that the involvement of peripheral tissues in vCJD may be more widespread than previously
reported and that the glycoform ratio of PrPres depends on the tissue in which it accumulates.
DIA-22 PURIFICATION AND DETECTION OF PRPBSE AND
PRPSC WITHOUT PK-DIGESTION
EVA BIRKMANN, OLIVER SCHAEFER, NICOLE WEINMANN, ANDREAS WILM AND DETLEV RIESNER
Institut fur Physikalische Biologie, Heinrich-Heine-Universität Dusseldorf, Germany
132
One of the characteristic steps of transmissible spongiform encephalopathies is the accumulation of a pathogenic isoform
(PrPSc) of the host encoded prion protein. PrPSc forms aggregates and is partially resistant to protease digestion (PK).
These properties distinguish PrPSc from PrPC as a disease-specific marker in the central nervous system and peripheral
tissues of infected animals and are used in all tests commercially available so far. Studies from Safar et al (1998) indicate
that in infected hamster different amounts of PK-sensitive PrPSc (sPrP) depending on the strains occur. The same effect
is discussed in respect to BSE. In our studies we show that the amount of PK resistant PrPBSE (rPrP) varies to a great
extend. Thus purification of BSE-prions without PK-digestion was inevitable. The sample preparation (Safar et al 2002,
Lee et al 2000) was modified so that PK-digestion could be avoided. Accordingly not only rPrP but also sPrP, i.e.
probably early states of the pathogenic PrP could be detected. The system was applied to nervous tissue of Scrapie
infected hamsters and BSE infected cattle. To detect partially purified prion particles we made use of Dual-Colour
Fluorescence-Correlation-Spectroscopy (FCS). The advantage of FCS compared to conventional systems is that FCS
allows analysis of aggregates irrespective of PK-resistance of the prion protein. Aggregates were fluorescence labelled
with specific antibodies, which must be able to recognize native PrPSc respectively PrPBSE. Here R1 and D13 for
Scrapie and 12F10 and Saf32 for BSE showed the best results. The principle was successfully applied to the detection of
Afl-petides in CSF of AD-patients (Pitschke et al 1998) and prion aggregates in CSF of CJD-patients (Bieschke et al
2000). Further experiments determining and enhancing the sensitivity in earlier states of the disease are in progress.
Therefore we try to enhance the sensitivity by amplification of the prion particles (Saborio et al 2001).
Poster Session 2
DIA-23 ORAL AND INTRACEREBRAL CHALLENGE OF SARDA
BREED SHEEP WITH SCRAPIE AND ANALYSIS OF SEQUENTIAL
PRPSC ACCUMULATION IN LYMPHOID AND NERVOUS TISSUES
G. VACCARI°, C. DÍAGOSTINO°, S. MARCON°, L. DE GROSSI*, F. GIORDANO*, R. BORRONI°, F. ROSONE*, M. DI
BARI°, R. NONNO°, D. CACIOLO*, F. ACOCELLA, R. BRIZIOLI* AND U. AGRIMI°
° Istituto Superiore di Sanità, Dep. Food Safety and Animal Health, Rome, Italy. * Istituto Zooprofilattico Sperimentale
del Lazio e della Toscana, Rome, Italy. § Cerusico s.a.s., Milan, Italy
The pathogenesis of natural scrapie shows considerable variation in sheep, in part related to PrP genotype and strain of
agent. In order to study both the susceptibility and the pathogenesis of scrapie in Sarda breed sheep with different
genotypes we used a double strategy, inoculating sheep both by the intracerebral (IC) or oral (OR) ruote. Inoculum
comprised a pool of the entire brains of 8 ARQ/ARQ Sarda breed sheep from two scrapie outbreaks. This pool has been
characterised by molecular and biological strain typing. Overall, 162 sheep were included in the study. The susceptibility
of sheep in relation to PrP genotypes was studied by the challenge of sheep with susceptible (ARQ/ARQ, ARQ/AHQ,
AHQ/AHQ; n=13), semi-resistant (ARR/ARQ, ARH/ARQ, ARR/AHQ, ARH/AHQ; n=22) and resistant (ARR/ARR,
n=6) genotypes by the most efficient IC route. At the time of writing, five out of six ARQ/ARQ sheep died with survival
times ranging from 420 to 462 days post inoculation (p.i.). All sheep with the others genotypes are still healthy.
The pathogenesis was studied in orally-dosed sheep by analysing the sequential accumulation of PrPSc in sheep
sacrificed at different times p.i. Fourthy-three sheep with the susceptible, 38 with the semi-resistant and 19 with the
resistant genotypes were challenged, along with 21 control sheep dosed with normal brain. No clinical sign of scrapie has
been observed after 19 months post-infection. A large variety of frozen and formalin-fixed tissues is being collected from
these sheep. The analysis of the collected tissues is still ongoing; however, in sheep with susceptible genotypes sacrificed
up to now, accumulation of PrPSc was detected by western blot in lymphoid tissues and in the intestine starting at 9
months post-challenge. Sheep with the others genotypes and control sheep were negative in all tissues examined.
DIA-24 A SIMPLE PRECIPITATION METHOD FOR INCREASING
THE SENSITIVITY OF PRPSC TESTING.
METTE HANSEN, PETER M. H. HEEGAARD, PETER LIND
Danish Institute for Food and Veterinary Research, Department of Veterinary Diagnostics and Research, Copenhagen
V, Denmark
Transmissible spongiform encephalopathies (TSEs) are associated with conversion of the normal cellular prion protein
(PrPC) into a protease-resistant abnormal isoform (PrPSc). Detection of PrPSc in brain tissue is used for the diagnosis
of TSEs. The aim of the study was to develop a simple PrPSc enrichment method to be used in connection with western
blot analysis to increase the sensitivity of the test. We developed a simple method comprising homogenisation, proteinase
K digestion and ethanol precipitation increasing the sensitivity of the test 5-10 times.
The method was tested on brain samples from scrapie-infected sheep, cerebral cortex samples from BSE-positive cows
and 263K-infected hamster brains and spleens. Sheep scrapie brain homogenate was diluted in normal sheep brain
homogenate in a dilution series and our method was compared to the Prionics®-Check procedure. The band intensity
measured by densitometry at dilution 1:40 for the Prionics samples was similar to dilution 1:400 for our method.
Cerebral cortex samples from BSE-positive cows that had earlier been tested as part of a ring trial test at diagnostic
laboratories in Denmark (Prionics®-Check, Enfer BSE test or PLATELIA BSE ELISA, BioRad) where 16-17 of 20
samples were found positive, were also tested by our protocol which found all 20 samples positive. Hamster brain and
spleen homogenates were tested with the Prionics-Check procedure and our method and the result was an increase in
band intensity. It was shown that our method concentrated PrPSc without altering the glycoform pattern, which makes
this protocol suitable for glycotype analysis for strain identification of PrPSc. The major advantages of the procedure
include speed, low cost, simplicity and reproducibility unaffected by species origin and applicability to other types of
analyses. This should make it a method of choice for detecting low amounts of PrPSc in both neural and non-neural
tissue samples, as would be present in preclinical cases.
133
Poster Session 2
DIA-25 PATIENTS WITH CREUTZFELDT-JAKOB DISEASE AND
CO-OCCURRENCE OF DIFFERENT PRPRES TYPES: A CLINICOPATHOLOGICAL AND BIOCHEMICAL STUDY
S HAIK, BA FAUCHEUX, D MARCÉ, V SAZDOVITCH, JP BRANDEL, N PRIVAT, N DELASNERIE-LAUPRÉTRE, MB
DELISLE, A LAQUERRIÈRE, JF PELLISSIER, JL LAPLANCHE, D DORMONT, JP DESLYS AND JJ HAUW
INSERM U360 (SH, BAF, VS, JPB, NP, NDL and JJH), Laboratoire de Neuropathologie R. Escourolle (SH, BAF, VS, NP and
JJH), Cellule Nationale de Référence des Maladies de Creutzfeldt-Jakob (SH and JPB), Hôpital de la Salpétrière, France;
Neurovirologie, CEA, Fontenay-aux-Roses (DM, DD, JPD), INSERM U466, CHU Toulouse III-Rangueil (MBD), Service
d’Anatomie et de Cytologie Pathologiques, Hôpital Charles Nicolle, Rouen (AA); Service d’Anatomie Pathologique et de
Neuropathologie, Hôpital de La Timone, Marseille (JFP); Laboratoire Central de Biochimie, Hôpital Lariboisière, Paris (JLL), France
The biochemical properties of the protease resistant isoform (PrPres) of the host encoded prion protein (PrPc) have
been thought to reflect prion strain information. The co-occurrence of different isoforms of PrPres in the same brain, as
shown by Western blotting, has been recently reported in sporadic Creutzfeldt-Jakob disease (sCJD) but its incidence
and significance remain unknown. This raises important questions concerning human strains of prion and the
classification of sCJD variants. Moreover, the presence of PrPres diversity in the same brain could be interpreted as a
co-infection by several prion that produce different PrPres types. In a series of 65 sCJD cases that was representative of
the French definite sCJD population, we studied the PrPres types in five-brain areas. The severity of lesions and the
PrPres immunohistochemical pattern were analysed in the same areas. In this group, eight cases (12%) presented two
distinct types of PrPres in the same region or in different regions of the brain. This co-occurrence of PrPres types was
not related to age at death, clinical course of the disease or neuropathological phenotype. The spatial distribution of
PrPres types was correlated with the genotype at codon 129. In these patients, comparisons of PrPres denaturation
transition suggest the presence of an intermediate isoform of PrPres that can be seen as type 1 or type 2 depending on
the brain region and codon 129 status. The presence of PrPres heterogeneity did not correlate with a distinctive cliniconeuropathological phenotype and may reflect regional variation in prion protein metabolism between individuals. Our
study confirms however that sCJD cases can be classified into two main groups and shows the requirement of a
systematic study of PrPres in different brain regions to affirm the presence of minor variants of sporadic CJD.
DIA-26 IN VITRO AMPLIFICATION OF THE PATHOLOGICAL
PRION PROTEIN IN BSE
JEAN-NOEL ARSAC1, ISABELLE LEPARC-GOFFART2, THIERRY BARON1, JEAN-YVES MADEC1
1 : AFSSA Lyon, France 2: AFSSAPS Lyon, France
134
An indisputable hallmark of prion diseases, like Bovine Spongiform Encephalopathy (BSE), is the accumulation in infected tissues of
an abnormal isoform of a cellular prion protein (PrPc), that displays insolubility in detergents and partial resistance to proteases when
conformed in a pathological way (PrPsc or PrPres). However, particularly in BSE, the late-stage accumulation of PrPsc in tissues
may often preclude the confirmatory diagnosis of the disease using the currently available methods. Several in vitro conversion
systems of the prion protein were already published, one of them involving a cyclic amplification of the PrP protein (Saborio et al,
Nature (2001) : 411, 810-813) and these strategies may also contribute in future to bring undetectable PrPsc to detectable level.
On the basis of the approach mentioned above (Saborio et al, 2001), we focused our work on the signal amplification of the BSE PrPsc
following long-term incubation of BSE PrPsc together with a large excess of cellular PrP protein and without any sonication step. At first
using a mouse model of BSE, the conversion of murine PrPc appeared dramatically more efficient when triggered by murine BSE PrPsc
(60-fold amplification factor) compared to several other murine scrapie PrPsc (4-fold amplification factor). BSE PrPsc amplification was
then assayed by diluting and incubating brain homogenates from BSE positive cattle in normal cattle brain homogenates and the efficacy
of amplification, albeit significant, appeared 15-fold inferior to the one obtained with the same strain in the murine model.
All together, our results indicate that the long-term incubation of PrPsc linked to the BSE strain with normal homologous PrP allows
the amplification of the PrPsc signal detected by immunoblotting and may thus open new prospects in future for the diagnosis of
BSE.
Poster Session 2
DIA-27 H-FABP AS DIAGNOSTIC MARKER FOR
NEURODEGENERATIVE DISEASES
STEINACKER P. (1), MOLLENHAUER B. (1), BIBL M. (2), CEPEK L. (1), POSER S. (1), KRETZSCHMAR H.A. (3) OTTO M.
(1)
1 Georg-August-University, Department of Neurology, Gottingen, Germany. 2 Georg-August-University, Department
of Psychiatry, Gottingen, Germany. 3 Ludwig-Maximilian-University, Department of Neuropathology, Munich,
Germany
Recently, by 2D-PAGE analysis of cerebrospinal fluid (CSF) and subsequent sequencing, H-FABP (heart-fatty acid
binding protein) was shown to be a potential biomarker for Creutzfeldt-Jakob disease (CJD). In a small sample size of
patients elevated levels of H-FABP were detected in plasma and CSF by ELISA measurement in comparison to nondemented control patients and a group of patients with other dementias, respectively (Guillaume et al., Proteomics
3(8):1495-9, 2003).
The aim of our study was to evaluate if H-FABP may be a potential biomarker for the differential diagnosis of
dementias. Therefore we measured H-FABP in CSF and serum of patients having CJD (n=14), Alzheimer’s disease
(AD, n=18), Dementia with Lewy-Bodies (DLB, n=16) Parkinson’s disease Dementia (PDD, n=5) and in non-demented
control patients (NDC, n=16).
We confirm the finding, that H-FABP levels in CSF (p<0.0001) and serum (p=0.002) of CJD patients are increased
compared to non-demented controls. Levels of H-FABP were significantly different between CJD, AD and DLB in
CSF. However, discrimination between CJD and AD was not possible in serum. Interestingly highest levels of H-FABP
were found in serum of DLB patients.
The results of this study suggest that H-FABP could be a useful biomarker for the examined dementias if levels in CSF
and serum are determined in parallel.
DIA-28 INCREASED EXPRESSION OF PRESYNAPTIC PROTEINS
IN SCRAPIE-INFECTED GT1-1 CELLS.
SANDBERG M. (1), LOW P. (1), TARABOULOS A. (2) & KRISTENSSON K. (1)
(1) Dept. of Neuroscience, Karolinska Institutet, Stockholm, Sweden. (2) Dept. of Mol. Biology, Hadassah Medical
School, Jerusalem, Israel
Prions are transmissible pathogens that cause neurodegenerative diseases but the mechanisms involved in the nervous
system dysfunctions are unclear. To investigate neuronal dysfunction during prion infection, we use GT1-1 cells
(neuronal cell line derived from mouse hypothalamus) infected with the scrapie RML strain. We have earlier described a
disturbance in voltage-dependent N-type calcium channels in prion-infected GT1-1 (ScGT1-1) cells. Since different
proteins in the SNARE complex partly can regulate the activity of voltage-dependent N-type calcium channels, the
expression of presynaptic proteins was investigated in ScGT1-1 cells and control GT1-1 cells by western blot and
immunohistochemistry. The most distinguished finding was an increase in the protein level of SNAP-25 in ScGT1-1
cells. When Real-time PCR was used to determine the increase at the mRNA level both SNAP-25a and SNAP-25b were
equally increased in ScGT1-1 cells when compared to control cells. The difference in expression of SNAP-25 in ScGT11 and uninfected GT1-1 cells was also determined following treatment with db-cAMP / IBMX that induce
differentiation of these cells. These results indicate changes in the expression of presynaptic proteins that might directly,
or indirectly, contribute to the disturbances of N-type calcium channels in prion-infected GT1-1 cells.
This research was supported by US Army med. res. acq. act. grant DAMD17-03-102288
135
Poster Session 2
DIA-30 METABOTROPIC GLUTAMATE RECEPTOR SIGNALING
IN CREUTZFELD-JAKOB DISEASE
AGUSTIN RODRIGUEZ(1), MERITXELL FREIXES(1), BERTA PUIG(1), ISIDRE FERRER(1,2).
(1)Institut de Neurpatologia. Servei Anatomia Patologica. Hospital de Bellvitge. (2)Universitat de Barcelona. Hospitalet
de Llobregat. Spain. iferrer@csub.scs.es
CJD is a human transmissible spongiform encephalopaty associated with a dysfunction in membrane syaloglycoprotein,
PrP, wich is converted into an abnormal isoform with a predominant beta-sheet structure that is partially resistant to
protease digestion, has a different solubility and, more important, becomes pathogenic. Glutamate is the main excitatory
neurotransmiter in the cerebral cortex. Altered glutamatergic transmission has been suggested to have a central role in
many neurodegenerative diseases. Metabotropic glutamate receptors (mGluRs) are coupled to intracellular signal
transduction via G proteins and mediate slower responses. In present work, we studied possible alterations in group I, II
and III of mGluRs. Group I are positively coupled to phospholipase Cbeta1 (PLCbeta1), while both group II and group
III are negatively coupled to the activity of adenylyl cyclase (AC). PLCbeta1 and AC expression was examined in the
brain of ten cases with sporadic CJD (sCJD) met/met at codon 129 and four age-matched controls by means of
electrophoresis and Western blotting of total cortical homogenates. Significant decreased levels about 88% of PLCbeta1,
revealed by quantitative densitometry of the bands, and significant increased levels about 300% of AC, with statistically
significant difference at the 99% and 99'9% confidence level respectively, when compared sCJD cases with controls.
PLCbeta1 and AC solubility were also examined. No modifications in PLCbeta1 and AC solubility in PBS-,
deoxycholate-, and SDS-soluble fractions were observed in sCJD samples when compared with controls. The present
results suggest a possible alteration of mGluRs in sCJD cases, wich is, apparently, not dependent on abnormal
interactions between proteins studied and PrP.
DIA-31 IDENTIFICATION AND EVALUATION OF SPECIFIC
SURROGATE MARKERS FOR PRION DIAGNOSIS
V LEBLANC, F MOUTHON, C PICOLI, A STURNY, V NOUVEL
DSV/DRM/GIDTIP CEA Fontenay-aux-Roses, France
136
AND
JP DESLYS.
In order to identify new TSE surrogate markers that can be used alone or in combination to facilitate rapid early specific
diagnosis of CJD and TSE in a non invasive manner, we developed the Representational Differential Analysis (RDA) to
reveal modified gene expression pattern during a prion infection. This approach allowed to identify 80 disregulated
sequences in a scGT-1 cellular model (cf abstract F. Mouthon et al). In this study we have evaluated by RT-PCR the
relevance of these sequences in vivo in several mouse models. We have performed kinetic studies (with sequential
sacrifice of the animals in triplicates at days 6, 45, 90, 150, and 195 or 260 depending on the route of inoculation) with
two different prion strains (scrapie or BSE) inoculated by either intracerebral or intraperitoneal route. Brain, spleen and
blood were harvested at each time point, the whole representing more than 12000 points of RT-PCR.
Specific patterns of gene disregulation have been observed in both strains at different phases of the kinetic in the spleen
of BSE and scrapie infected animals versus mock infected controls (few days after infection, at day 45 and in the late
phase of the disease). Different levels of gene modulation were observed ranging from 4 to 30 fold. Variations were
observed that were specific to the scrapie strain.
The modulation of the gene expression observed in the brain was less important. This apparent limitation could be linked
to a dilution effect as neurons represent less than 10% of the brain cells.
These data strongly suggest that multiparametric tests with several surrogate markers could allow non invasive early in
vivo diagnosis of prion diseases. Ongoing experiments encompass preselection of target cells and in situ hybridization.
Poster Session 2
DIA-32 CLUSTERIN SOLUBILITY AND AGGREGATION IN
CREUTZFELD-JAKOB DISEASE
M. FREIXES(1), B. PUIG(1), A. RODRIGUEZ(1), B. TORREJON-ESCRIBANO(3), R. BLANCO(1), I. FERRER(1,2)
(1) Institut de Neuropatologia, Servei Anatomia Patològica, Hospital de Bellvitge;(2) Unitat de Neuropatologia,
Departament de Biologia Cellular i Anatomia Patològica, Universitat de Barcelona; and (3) Serveis Cientifico-Tècnics,
Unitat de Biologia de Bellvitge; 08907 Hospitalet de Llobregat; Spain
Prion protein(PrPC)is a glycolipid-anchored cell membrane syaloglycoprotein that localises in presynaptic
membranes.Prion protein has the property of aggregating into amyloid fibrils and being deposited in the brains of
transmissible encephalopathies(TSEs) when PrPC is converted into abnormal protease-resistant PrP
(PrPRES).Clusterin is a heterodimeric glycoprotein which expression in astrocytes is enhanced in association with
punctate-type PrPRES deposits during TSE progresion.In addition,clusterin co-localizes in PrPRES plaques in several
human TSEs,including Creutzfeld-Jakob disease(CJD).Clusterin expression was examined in eight sporadic cases of
CJD and three age-matched controls by immunohistochemistry,Western blotting(WB)and solubility aggregation.Single
and double-labelling immunohistochemistry disclosed clusterin localization in PrPRES plaques.Moreover,clusterin in
plaques was resistant to protease digestion,as revealed in tissue sections pre-incubated with
proteinaseK.Therefore,clusterin in CJD,but not in control brains,was partially resistant to protease digestion, a feature
also demonstrated in WB of total brain homogenates immunostained with anti-clusterin antibodies which were processed
in parallel with WB to PrP, without and with pre-incubation with proteinaseK.Protein aggregates were analyzed in brain
homogenates subjected to several solvents.PrP was recovered in the deoxycholate fraction in control and CJD cases,but
in the SDS fraction only in CJD,thus indicating differences in PrP solubility between CJD and controls.Clusterin was
recovered in the cytosolic,deoxycholate and SDS fracion in both CJD and control cases,but only clusterin form CJD
was recovered in the urea-soluble fraction and, especially, in the remaining pellet.These findings demonstrate the
capacity of clusterin to form aggregates and interact with PrP aggregates and it can be suggested that clusterin
participates in PrP clustering thus modificating PrP toxicity in CJD.
DIA-33 NOVEL, HIGHLY SENSITIVE, RAPID TECHNIQUES, FOR
THE DIAGNOSIS OF PRION PROTEIN CONTAMINATION
I.P. LIPSCOMB, D. BOCHE, V.H. PERRY, J. GRASSI, S. CLARK, C.W. KEEVIL
This recently commissioned project aims to integrate the use of novel fluorescent marker techniques, monoclonal
antibodies and advanced microscopy methods to produce a highly sensitive protocol for prion protein detection. This
work involves the collaboration with an internationally recognised French group who have developed new panels of
monoclonal antibodies and a leading UK diagnostics company. Analysis has initially concentrated on a generic rapid
staining protocol for ‚ pleated sheet amyloid using fluorescent markers, to identify prion deposits in suspect biopsied
material. This rapid pre-screen is used to select specimens for subsequent confirmation of the prion diagnosis using the
new monoclonal antibodies with either chromagen or fluorescent labels. This approach also shows excellent possibilities
in being extended to the contamination of metal surfaces, such as surgical instruments and work surfaces, by the
inclusion of the advanced light microscopy technique of episcopic differential interference contrast (EDIC) microscopy,
allowing sub-micron, sub-picogram detection of PrPsc in both brain and spleen.
137
Poster Session 2
DIA-34 APPLICATION OF THE IMPROVED CONFORMATION
DEPENDENT IMMUNOASSAY FOR THE TESTING OF
DIFFERENT ORGANS FROM CJD PATIENTS
BELLON A.1,2, GLATZEL M. 3, LAUDE H. 2, GRONER A. 1, AGUZZI A. 3 AND VEY M. 1
1 Department of Virology, Aventis Behring GmbH, Germany. 2 Prion team, VIM, INRA, Jouy en Josas, France. 3
Institute of neuropathology, University of Zurich, Zurich, Switzerland.
Diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) can only be confirmed after the death of the
patient so far. It involves the detection of the abnormal isoform PrPSc in the patient’s brain. The sandwich Conformation
Dependent Immunoassay (sCDI) is a newly introduced method which appears more sensitive than current tests which
are based on WB or ELISA technology. The increased sensitivity is mainly due to prion protein concentration on the
plate by capture via a novel monoclonal anti prion antibody, 1120-64-9. Moreover, in sCDI, similar to direct CDI, the
PK digestion step can be avoided, thus enabling the detection of PK-sensitive PrPSc species. As already reported (Bellon
et al. JGV, 2003), sCDI allows the high sensitivity detection of brain CJD PrPSc spiked into normal plasma, including
purified PrPSc and microsomes, derived from three different human prion strains, i.e. sCJD type 1 and 2, and vCJD.
Here we report that prions can be detected with this method in several organs of sCJD patients, which were recently
reported to be positive for PrPSc (Glatzel et al., NEJM, 2003). In our study, spleen and muscle from sCJD patients
were tested. We were able to detect PrPSc in 3 of the 4 sCJD spleens tested and in 1 of the 2 sCJD muscles tested.
PrPSc titers in the infected spleens seem to be 1000 times lower than in positive brains. Furthermore, sCDI allows
detection of PrPSc in less than 10 mg spleen material. From these data, we conclude that mab 1120-64-9 represents a
powerful tool to detect and quantify PrPSc in extraneural tissues from sCJD patients with the option to integrate into
high throughput screening tests such as CDI or ELISA.
DIA-35 DEVELOPMENT OF A NOVEL IMMUNOASSAY FOR THE
DETECTION OF TSE AGENTS ON SURGICAL INSTRUMENTS
AND OTHER BIOLOGICAL SAMPLES
MURDOCH H, TAYLOR D, O’BRIEN S, CONLAN B, CARR J, DICKINSON J, SUTTON JM AND RAVEN N
HPA- Porton Down, Porton Down, UK
138
Routine surgery and transplant / transfusion remain potential routes for the transmission of Creuztfeldt-Jakob disease
(CJD) and its new variant form (vCJD). To reduce the likelihood of this happening new methods are required to detect
the presence of prion material in complex biological samples, such as tissue or blood, and on the surface of surgical
instruments. The method described here has been designed as a flexible format for the detection of prion material in such
samples.
The method uses antibody conjugated to an adenylate kinase (AK) marker enzyme to generate ATP. The ATP generated
is then coupled with luciferin/luciferase reagents to provide an ultrasensitive bioluminescent detection method. A
thermostable AK was cloned, expressed and purified. The enzyme was conjugated via a cleavable linker to the
commercially available anti-prion antibody 6H4 and a model assay developed for recombinant PrP. Cleavage of the AK
after antibody binding allows transfer of the remainder of the assay to standard laboratory equipment. This enables the
method to be applied to large surgical equipment without the need for complex apparatus. The assay format uses heat
denaturation steps to eliminate any contaminating enzymatic activity by utilising the thermostability of the AK. We have
been able to detect recPrP at a concentration of 6pM in a background of mouse brain homogenate. We have also
demonstrated that the assay can detect recPrP in complex biological samples such as blood, and in a model of prion
contamination on surgical steel. The potential of the assay to allow ultrasensitive detection of TSE agents in a variety of
samples will be discussed.
Poster Session 2
DIA-36 CJD PRION PROTEIN DETECTION BY IMMUNOQUANTITATIVE PCR
STÉPHANIE GOFFLOT1, BENAISSA EL MOUALIJ1, MANUEL DEPREZ2, JEAN-FRANÇOIS THONNART1,
JACQUES GRASSI3, ERNST HEINEN1 AND WILLY ZORZI1.
1 Service d’Histologie Humaine-Centre de Recherche sur les Protéines Prions (CRPP), Université de Liège-CHU, Tour de
Pharmacie, Belgium. 2 Laboratoire de Neuropathologie, Tour de Pathologie, Belgium. 3 CEA, Service de Pharmacologie et
d’Immunologie, Saclay, France.
Prion diseases are unique transmissible neurodegenerative disorders affecting humans and animals. The most common
human prion disorder is Creutzfeldt-Jakob disease, classified as sporadic, familial, iatrogenic and variant. The spectrum
of neurological and neuropsychological conditions associated with these diseases is expanding due to improved detection
of prion protein strains, gene mutations and allelic polymorphism. The recent observation of a raise of sporadic CJD
incidence and reports of a possible transmission of variant Creutzfeldt-Jakob disease by the blood are strong incentive
for further development of highly sensitive methods of detection of prion protein.
Immuno-PCR is an extremely sensitive detection method combining the specificity of antibody detection and the
sensitivity of PCR. We have developed an immuno-quantitative PCR (iqPCR*) exploiting real-time PCR technology in
order to improve current immuno-detection method. We have already applied this new technology to the detection of
resistant prion protein from bovine brain. The iqPCR revealed to be highly sensitive. Moreover, our test was proved to
be more sensitive than a routine Platelia (BioRad) diagnosis test (Gofflot S., 2004).
The present work evaluated the immuno quantitative PCR in experiments aimed at detecting the resistant form of prion
protein in human brain extract. The iqPCR technique proved to be highly sensitive, so it could be proposed as a useful
tool for the biopsy diagnosis of early prion disease. This technique is available with a high throughput screening and
could be applied in a clinical preventive analysis strategy.
* Patent WO0131056, 2001-05-03: Detection method by PCR, Zorzi Willy (BE); El Moualij Benaissa (BE); Zorzi
Danièle (BE); Heinen Ernst (BE); Melen Laurence (BE).
Gofflot S. et al. (2004) J. Immunoassay, in press.
This work is supported by the ‘Région Wallonne’ : contract 14531, iPCRq.
DIA-37 MULTIPLEX IMMUNO-QUANTITATIVE PCR AS A NEW
AND ORIGINAL APPROACH TO DETECT SEVERAL EPITOPES OF
THE PRION PROTEIN IN THE SAME WELL.
STÉPHANIE GOFFLOT1, BENAISSA EL MOUALIJ1, DANIÈLE ZORZI1, JACQUES GRASSI2, ERNST HEINEN1
AND WILLY ZORZI1.
1Centre de Recherche sur les Protéines Prions (CRPP) - Histologie Humaine, Université de Liège-CHU, Tour de
Pharmacie, Belgium. 2 CEA, Service de Pharmacologie et d’Immunologie, Saclay, France.
Immuno-quantitative PCR is a detection method which allows sensitive detection of antigens* (Gofflot S., 2004). We
have developed a multiplex immuno-quantitative PCR exploiting ‘double dye’ probes real-time PCR technology in order
to reveal different antigens in the same well.
On one hand, we have synthesized two different reporter DNA molecules, designed probes using the “Primer Express” software
and have realized real-time PCR. By plotting the distinct fluorescence versus the cycle number of PCR (FAM and YaKima Yellow
“Eurogentec SA”), we have shown that it is possible to amplify and to detect two different DNA molecules in the same well.
On the other hand, we have applied this multiplex-technique to the immuno-detection of the bovine recombinant prion protein by
using two monoclonal antibodies raised against two distinct epitopes of this protein: the first one recognizing the carboxyterminal
side (142-160) and the other one, the amino terminus (79-92). The detection was carried out with these antibodies covalently
coupled one to DNA1 and the other to a streptavidin-DNA2 complex. The DNAs were amplified by PCR in presence of the two
probes with the two different dyes. The results were analyzed during the exponential phase of the amplification curves.
In the present work we have demonstrated that the multiplex immuno-quantitative PCR is possible and allows the
detection of two or more different antigens in the same well.
* Patent WO0131056, 2001-05-03: Detection method by PCR, Zorzi Willy (BE); El Moualij Benaissa (BE); Zorzi
Danièle (BE); Heinen Ernst (BE); Melen Laurence (BE).
Gofflot S. et al. (2004) J. Immunoassay, in press.
This work is supported by the ‘Région Wallonne’ : contract 14531, iPCRq.
139
Poster Session 2
DIA-38 BSE SCREENING KIT WITH SIMPLIFIED PREPARATION
METHOD FOR EIA SAMPLE
TAKUJI YAMAMOTO, YUKO USHIKI, SHUNJI HATTORI, YUICHI TAGAWA, HIROE TSUKAGOSHI-NAGAI, NORIAKI
KINOSHITA, AND SHINKICHI IRIE
Senjyu midorityou 1-1-1,adachiku,tokyo,Japan
INTRODUCTION
In Japan, all of slaughtered and died bovine were tested for BSE infection. The primary screening test was undertaken
by the meat inspection office or live stock hygiene service center in each prefecture. In this circumstance, BSE kit
adapted for relatively small number of samples is required. Here we developed the new BSE screening system which has
simpler and safer protocol for sample preparation steps for EIA.
THE DESIGN OF THE ASSAY SYSTEM (named as Nippibl BSE Assay)
1. Nippibl BSE ASSAY PRETREATMENT KIT
First step is the homogenization of the bovine's brain and enzyme treatment. We developed new apparatus for
homogenization by passing porous rigid polypropylene filter which named Bio Masher. In our enzyme treatment step,
purification step is not contained. It means that the frequency of opening the sample tube can be reduced (safer) and
shorten the process of sample preparation. At first process, the bovine's brain (100mg) were homogenized using Bio
Masher, and homogenate was suspended in the buffer (1ml) containing the enzyme, and incubate the tube at 56 degree.
After heat inactivation the enzyme at 100 degree, sample is ready for ELISA assay.
2. Nippibl BSE ASSAY KIT
Second step is detection of PrP by sandwich ELISA. We selected a pair of highly sensitive antibodies. The dynamic
range for recombinant prion protein with our kit was 31.3 -2,000 pg/ml, and the sensitivity was 2.8 pg/ml. The detection
kit was sufficiently stable more than 6 months.
CONCLUSION
Here we developed the new BSE assay system. There are many merits in the system.
1. The pretreatment of a sample could be processed in a short time and risk of the infection nature during sample
processing could be reduced. 2. No extra apparatus is necessary, so it is more economical. 3. The result showed very
sufficient sensitivity and reproducibility.
It is expected that the Nippibl BSE assay system would be widely useful in many laboratories.
DIA-39 PRP IMMUNOHISTOCHEMISTRY IN HUMAN PRION
DISEASES: FROM ANTIBODY SCREENING TO STANDARDIZED
FAST IMMUNODIAGNOSIS USING AUTOMATION
NICOLAS PRIVAT, STÉPHANE HAIK, FRANÇOISE FIERVILLE, VÉRONIQUE SAZDOVITCH, YVELINE FROBERT, BAPTISTE A.
FAUCHEUX, JACQUES GRASSI, JEAN-JACQUES HAUW.
INSERM U360 (NP, SH, VS, BAF and JJH) and Laboratoire de Neuropathologie R. Escourolle (NP, SH, FF, VS, BAF and JJH),
Hôpital de la Salpétrière, France. Service de Pharmacologie et d’Immunologie (YF and JG), CEA Saclay, Gif-sur-Yvette, France.
140
Demonstration of PrPsc accumulation in the CNS is required to affirm the diagnosis of prion diseases. This is usually achieved
using PrPsc immunohistochemistry in paraffin-embedded tissue that requires multiple epitope retrieval and denaturing
pretreatments. Such protocols are not only time-consuming but also induce tissue alterations that preclude fine morphological
analysis. The aim of this study was to increase the sensitivity and to simplify the procedure of PrPsc immunohistochemistry in
human pathology. We studied a panel of more than 50 monoclonal antibodies using the same pre-treatments to pre-select the
most promising one. Antibodies produced using different immunogens (human and bovine recombinant PrP, PrP peptides,
scrapie-associated fibrils from 263K-infected Syrian hamsters) were directed against different epitopes along the PrP sequence.
They were tested using a panel of different forms of genetic, infectious and sporadic human diseases. Highly efficient antibodies
were then used in different simplified procedures and checked for their efficacy at 37°C working temperature. The last step was
to establish a standardized and reliable immunostaining procedure using an automated diagnostic system for
immunohistochemistry (Ventana Medical Systems). Using this strategy, we identified rare monoclonal antibodies allowing a high
specific and fast immunodiagnosis with very limited denaturing pretreatments. Our automated method is more sensitive than
other procedure using the reference 3F4 monoclonal antibody that is not suitable in this automated system. Our automated
method is adapted to PrPsc detection in the CNS and also lymphoid tissue such as tonsil biopsy from human patients.
Poster Session 2
DIA-40 SOME RAPID TESTS MAY BE NOT SUITABLE FOR THE
DIAGNOSIS OF NOR98 SCRAPIE ISOLATES
SARRADIN P.(1), BRATBERG B.(2), LANTIER F.(1) , BENESTAD S.L.(2)
1.Unité de Pathologie infectieuse et Immunologie, INRA, France. 2.National Veterinary Institute, Oslo, Norway
A new type of scrapie, designated Nor98, has been diagnosed in 35 Norwegian sheep since 1998. They differ from
classical scrapie cases by i) their unusual epidemiological and clinical features, ii) the absence of histopathological lesions
and PrPSc, as demonstrated by IHC, in the DMVN at the level of the obex, and iii) their genotype. Indeed, 20 of the 35
Nor98 cases carried at least one AHQ allele of the Prnp gene, genotypes rarely associated with scrapie. No PrPSc was
either detectable by IHC and ELISA in the lymphoid tissues investigated.
In this study, we present the results obtained by two laboratories using routinely TSE rapid tests on sheep brain samples
from 2 negative, 3 classical scrapie and 20 Nor98 cases. The rapid tests were the two most widely used: a western-blot
(Prionics Check WesternÆ) and an ELISA (Bio-Rad Platelia/TeSeEÆ).
According to the ELISA results, the 25 isolates could be sorted as follows: negative samples (OD<0.040), Nor98 samples
(0.230<OD<3.2) and classical scrapie samples (OD>3.3).
The rapid western blot identified clearly the classical scrapie samples as they showed the 3 bands of the typical PrPres
profile and no band was detectable for the negative isolates. Regarding the Nor98 samples, all positive with the ELISA,
9 of them were clearly negative, while 11 gave results difficult to interpret, showing a range of strong to very weak
signals, all of them at a molecular weight corresponding to that of undigested PrP, according to the level of the positive
control and the PK band.
Nine of the 11 samples with an OD under 1.7 were negative by the western-blot rapid test, indicating a clear difference
in sensitivity between the two rapid tests. These results confirm that, likely due to a lower concentration of PrPSc
compared to classical cases, a concentration step of the protein is required for the diagnosis of the Nor98 scrapie cases
and that some of the rapid tests may be not suitable for this purpose.
DIA-41 A SIMPLE AND RAPID POSTMORTEM EIA ASSAY
FOR THE DETECTION OF TRANSMISSIBLE SPONGIFORM
ENCEPHALOPATHIES
L. ESTEY, K. VELEK, S. SOLLER, L. PLOURDE, R. TOOMIK, V.LEATHERS, C. WONG, S. WILSON, C. STANLEY, AND Q. TONELLI
Production Animal Services Research and Development, IDEXX Laboratories, Inc. Westbrook, USA
Misfolding of the normal host prion protein (PrPC) and its subsequent accumulationas a protease-resistant conformer
(PrPSc) is a well-documented correlate oftransmissible spongiform encephalopathies (TSEs). Proteinase K resistance
iscommonly leveraged as a method for distinguishing PrPSc from PrPC in mostTSE diagnostics on the market. IDEXX
has developed a TSE diagnostic that does not require proteinase K digestion, but instead uses Seprion-capture
technologyapplied to a microtiter plate format. This method utilizes a nonbiologicalPrPSc-specific ligand that selectively
binds PrPSc in the presence of excess PrPC.Captured PrPSc is then detected using an anti-PrP antibody-HRPO
conjugate.Sample preparation is limited to homogenization of tissue and the addition ofdiluent; no other processing is
required before applying samples to the assayplate. Assay run time is 3-4 hours, depending on the tissue type under
evaluation.The IDEXX assay has been used to detect PrPSc in characterized samples frombovine and ovine brains, as
well as cervid lymph nodes. The specificity of theIDEXX assay was 100% in all species and tissues tested. Sensitivity
was greaterthan 99% for non-obex tissue from bovines (versus immunohistochemistry [IHC]conducted on obex tissue)
and 100% for a small population of IHC-positivecervid lymph nodes. A 100% correlation between the IDEXX assay and
anEU-approved bovine spongiform encephalopathy assay was observed for bovinesamples. Field evaluations are
continuing to test IDEXX kit performance on largepopulations of bovine, scrapie and cervid samples. The IDEXX
assay, with itsabsence of a proteinase K digestion step and minimal handling during samplepreparation, provides a
sensitive, rapid and easy-to-use method for identifyingTSE-positive samples. The simplicity of the method allows
straightforwardadaptation to automation, making it an ideal tool for screening large numbersof samples.
141
Poster Session 2
DIA-42 DIFFUSION WEIGHTED IMAGING (DWI) AND
FLAIR IN JAKOB-CREUTZFELDT DISEASE (CJD): HIGH
SENSITIVITY AND SPECIFICITY FOR DIAGNOSIS
MICHAEL D. GESCHWIND, GEOFFREY S. YOUNG, NANCY J. FISCHBEIN, JENNIFER L. MARTINDALE, ROLAND G. HENRY,
JAMES E. CALDWELL, SONGLING LIU, YING LU, STEPHEN WONG, HONG LIU, BRUCE L. MILLER, WILLIAM P. DILLON
From the Departments of Radiology (G.S.Y, N.J.F, S.L., Y.L. R.G.H., S.W., H.L., and W.P.L), Neurology (M.D.G.,
J.L.M, and B.L.M.) and Anesthesiology (J.E.C.), University of California, San Francisco, California, U.S.A.
Background and Purpose: Abnormal findings on diffusion and FLAIR MRI sequences have been reported in patients
with Jakob-Creutzfeldt disease (CJD), however no large formal study has been conducted to determine if these
sequences are useful diagnostic tools for CJD. We conducted a blinded evaluation of the sensitivity and specificity of
diffusion weighted (DWI) and FLAIR MRI for CJD.
Methods: A mix of hard copy and digital DWI and FLAIR images from 40 patients with probable or definite CJD and
53 controls with other forms of dementia were retrospectively evaluated by two blinded readers who rated the likelihood
of CJD based on the imaging findings.
Results: FLAIR and DWI MRI demonstrated 91% sensitivity, 95% specificity, and 94% accuracy in detecting CJD.
Inter-rater reliability was high (kappa=0.93). Sensitivity was higher for DWI than FLAIR images. Typical findings,
highly specific features, anatomic distribution of abnormality, and pitfalls in interpretation are presented. Critical
elements for successful differentiation of CJD from other dementias included narrow window soft-copy review of
artifact-free DWI and FLAIR sequences, familiarity with the typical patterns of gray matter abnormality seen in CJD
and recognition of the normal variation in the signal intensity of cortex on DWI and FLAIR.
Conclusion: Specific patterns of abnormality on FLAIR and particularly on DWI MRI are highly sensitive and specific
for CJD, and these sequences should be obtained whenever this diagnosis is suspected.
DIA-43 A NEW SENSITIVE ASSAY FOR THE DETECTION OF
OVINE AND CAPRINE PRPRES TESEEÔ SHEEP / GOAT
FEYSSAGUET M.1, MORIZE J.L.1, COMPOINT A.1, NESPOULOUS G.1, BILHEUDE J.M.1 , BOURGEOIS J.P.1 AND GRASSI J.2
1 Bio-Rad - 3 boulevard Raymond Poincaré - Marnes la Coquette – France. 2 CEA - Service de Pharmacologie et
d'Immunologie - Gif sur Yvette - France
142
TeSeE™ Sheep / Goat is a new rapid assay developped by Bio-Rad for the post-mortem diagnosis of TSEs in small
ruminants. This new assay is based on the same assay procedure than the current TeSeE™ kit with a combination of
monoclonal antibodies selected for their high affinity and specificity to sheep and goat PrPsc.
Internal and external evaluation conducted in nervous tissues (obex, brainstem, spinal cord) and in peripheral tissues
(lymph node, tonsil, spleen, ileum...) from naturally or experimentally infected sheep and goat clearly demonstrate the
very high sensitivity of this new test.
Specifitiy studies conducted on the semi-automatically format (Bio-Rad NSP system) with fresh sample of nervous or
peripheral tissues collected from slaughterhouse show that TeSeE™ is well adapted for screening large numbers of
samples.
TeSeE™ Sheep / Goat represents a promising tool to achieve a more precise overview of the scrapie prevalence which
was seriously underestimated by the use of most of Rapid test BSE approved.
Industrial kit lot of TeSeE™ Sheep / Goat is under evaluation by the EU (2004, "EU scrapie test evaluation").
Poster Session 2
DIA-44 OVINE TSE CONFIRMATORY TESTING WITH THE
NEW TESEE™ SHEEP / GOAT WESTERN BLOT ASSAY
BENESTAD S.L.1, GRASSI J.2, BILHEUDE J.M.3 , BOURGEOIS J.P.3 AND BRATBERG B.1
1 National Veterinary Institute - Oslo – Norway. 2 CEA, Service de Pharmacologie et Immunologie, DRM/DSV Saclay,
Gif-sur-Yvette, France. 3 Bio-Rad - R&D TSE - Marnes la Coquette - France
The Bio-Rad TeSeE™ screening rapid assay is one of the 5 rapid tests that have been evaluated and approved by the
European Commission for diagnosis of BSE in cattle. It has also been accepted for TSE testing in sheep and goats.
Since 1998, a new and atypical type of scrapie, designated Nor98, has been diagnosed in 35 Norwegian sheep, all
positive by the Bio-Rad ELISA. According to the EU regulations, positive results obtained by rapid tests must be
confirmed by the demonstration of typical spongiform changes by histopathology or by the detection of abnormal PrP by
immunohistochemistry (IHC) or Western blot (WB), or by the detection of Scrapie Associated Fibrils (SAFs).
Contrary to most classical scrapie cases, Nor98 cases demonstrate no histopathological changes at the level of the obex.
The PrPSc IHC shows either no, or atypical staining in this area. These features render their confirmation by standard
methods difficult.
To overcome these problems, Bio-Rad has developed a new confirmatory procedure based on the page/WB technique
combining the initial purification, including concentration step, of the Bio-Rad TeSeE™ rapid assay with an improved
Western Blotting detection system using monoclonal antibodies selected for their high affinity to PrP.
Data will be presented demonstrating that TeSeE™ Sheep and Goat Western Blot efficiently provides the confirmation
of Nor98 cases. In addition, it allows their identification by showing clearly their characteristics glycoprofile with a
marked lower band at ca 12KD.
DIA-45 BIO-RAD EIA TSE KITS AS RAPID SCREENING TEST
FOR DETECTION OF CHRONIC WASTING DISEASE (CWD) IN
BRAIN AND LYMPHOÏD TISSUES
FEYSSAGUET M. 1, BILHEUDE J.M. 1, ALLAIN G. 1, SAUVAGE N. 1, HENAUX S. 1, NESPOULOUS G. 1 AND BOURGEOIS J.P.
1 Bio-Rad - Marnes la Coquette - France
CWD is recognized as an important prion disease of native North American cervids. It has been observed in Mule Deer
(Odocoileus hemionus), while Tailed Deer (Odocoileus virginianus) and Rocky Mountain Elk (Cervus elaphus nelsoni).
The disease affects both free- ranging species and captive animals. PrPres accumulates early in the incubation period in
lymphoïd tissue of the alimentary tract prior to detection in the central nervous system. Currently diagnosis of CWD is
confirmed by identification of PrPres in brain and lymphoÔd tissues of affected animals by Immunohistochemistry
(IHC).
Preliminary evaluations of the Bio-Rad rapid assay TeSeE™ for CWD diagnosis were performed in US states on
collection of frozen samples. A total of 483 deer and 146 elks were tested on 1/2 and/or 3 tissues : brain tissues and/or
tonsil and/or retro-pharyngeal lymph node tissues. The results show a very good correlation with IHC for the 3 types of
tissues and underline that early detection is obtained when assays are performed on lymph node tissues or tonsils.
Field validation studies on lymph node tissues from 597 deer and elk show that IHC and Bio-Rad EIA Rapid test
TeSeE™ appear to be equally sensitive for CWD detection.
These studies also demonstrated that the Bio-Rad EIA Rapid test with TeSeE™ NSP system is well adapted for the
screening of large numbers of samples from deer and elk population.
143
Poster Session 2
DIA-46 APPROACHES TO SCRAPIE DIAGNOSIS: A COMPARATIVE
STUDY AMONG IMMUNOHISTOCHEMISTRY AND PRIONICS® RAPID
TESTS ON CENTRAL NERVOUS AND LYMPHORETICULAR SYSTEM
MONLEON E, HORTELLS P, BOLEA R, ACIN C, VARGAS A, BADIOLA JJ, MONZON M.
National Reference Centre for TSE. Faculty of Veterinary, Zaragoza, Spain.
The capability of different rapid tests for detecting PrPsc in CNS tissue and LRS tissues in sheep, even before the onset
of clinical symptoms in some scrapie cases, has been previously assessed; however its comparison with confirmatory tests
are not numerous in the literature. The main objective of this study was to compare the ability for PrPsc detection of two
currently applied rapid tests (Western blot and Check-LIA, Prionics®) using CNS as well as LRS samples
corresponding to clinical and preclinical field cases from naturally infected animals.
Thirty four scrapie positive sheep of Rasa Aragonesa breed and ARQ/ARQ genotype were included in this study.
Twenty seven animals presented clinical signs of the disease. The following tissues were collected: the whole brain,
tonsils and the retropharyngeal lymph node (RPLN). The half portion of each of them were immediately fixed (in
formalin 10%) for histopathological processing and the remaining sample stored at - 70°C for rapid tests developing.
All samples were analysed by three tests: IHC, WB and LIA. All three could detect all positive cases even when PrPsc
accumulation in CNS (preclinical animals) was not observed, although IHC showed a higher sensitivity.
On the other hand, PrPsc presence in the CNS was confirmed by the routine IHC methodology in all 34 sheep studied
except in three preclinical animals which were considered scrapie positive cases by tonsil and RPLN IHC. Meanwhile, in
two sheep at the terminal stage tonsil and RPLN IHC showed negative results but PrPsc accumulation was evidenced by the
analysis of the brain. Results about PrPsc presence / absence in the two LRS tissues (tonsil and RPLN) differed in no cases.
Therefore, a remarkable conclusion that can be drawn from this work is that all animals involved in the study could be
diagnosed, regardless the test applied (rapid as well as confirmatory assays) only in case that CNS and tonsil / RPLN
analysis were considered.
DIA-47 EVALUATION OF PRIONICS CHECK - LIA TEST FOR
THE SCREENING OF PRPSC IN THE LYMPHORETICULAR
SYSTEM OF SHEEP
NAPPI R., CARAMELLI M., MARTUCCI F., MAZZA M., ACUTIS P.L., PINTO L., OESCH B.*., BOZZETTA E.
CEA- Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta; *Prionics AG, Schlieren, Switzerland
144
The aim of the study was to investigate the presence of PrPSc in the lymphoreticular system (LRS) of italian Scrapie
infected sheep.For this purpose, we tested the performance of a luminescence immunoassay (Prionics check LIA), already
approved from EU for the active surveillance of TSE, on lymphoid tissue. We first applied this test to lymph nodes, positive
for PrPsc with a highly sensitive Western blot (WB) based on NaPTA precipitation,of six sheep coming from different
Italian Scrapie outbreaks. All animals were positive by confirmatory tests performed on central nervous tissue (CNS).
Subsequently, the Prionics check LIA was used for the screening of a Piedmont scrapie outbreak, comparing the results
obtained with NaPTA WB. The trial was carried out on 98 Biellese sheep with ARQ/ARQ (73), ARR/ARQ (11),
ARQ/AHQ (9), ARQ/VRQ (4) genotypes, four of which Scrapie positive. For the screening of the LRS lymph nodes,
tonsil, spleen, ileum and ileumcaecal valve were analyzed. To warrant comparable results, the samples of spleen and ileum
were cut in small pieces by two scalpels without any buffer, untill the tissue appeared homogeneous.The resulting
homogenate was split in two aliquots for the two methods, while lymph nodes and tonsils were simply cut in half part and
used at random.The results showed a complete agreement between Prionics-Check LIA and NaPTA WB. LRS positivity
and CNS negativity, due to a preclinical status of the animals, was never detected while; among four sheep positive at CNS
(genotype ARQ/ARQ), only 3 resulted positive to LRS.The test demonstrated the same sensitivity of NaPTA WB (that can
reach an analytical sensitivity 3 logs more then routine WB) and the LIA. It was however noted that critical points on
phases of homogenation and resuspension of pellet can imply a decrease in luminescence unit values.Our results show that
Prionics-Check LIA test is a reliable tool for the screening of the disease-specific form of PrP in ovine lymphoid tissue.
Poster Session 2
DIA-48 COMPARATIVE EVALUATION OF THE BIO-RAD
TESEE™ AND PLATELIA® ASSAY FORMAT
BILHEUDE J.M. 1, ALLAIN G. 1, SAUVAGE N. 1, HENAUX S. 1, NESPOULOUS G. 1, FEYSSAGUET M. 1 AND BOURGEOIS J.P
1 Bio-Rad - Marnes la Coquette - France
The Bio-Rad PLATELIA® BSE Rapid test was one of the first test evaluated and approved by the European
Commission. Today, the Bio-Rad rapid assay is used in many countries where post-mortem screening of slaughtered
animals has been implemented.
In 2002, the practicability of the test procedure was improved in order to facilitate the automation of the purification
concentration steps using in Deepwell Microplate format and TeSeE™ NSP system.
This new TeSeE™ assay format was compared to the PLATELIA® BSE assay and demonstrated similar results in term
of sensitivity when tested with panel of diluted positive samples. Two bovine panels and one ovine panel with dilutions
given results near to the cut-off value were used for this validation.
The specificity was evaluated on a population of 5343 negative bovine samples. No first intention false positive results
were observed when testing the samples with TeSeE™ NSP system. The OD distribution of the negative samples was
also improved. On a second population of 1058 samples manually tested with both Platelia® and TeSeE™ assays,
similar results and OD distribution were observed.
This comparative evaluation clearly demonstrated that the performances were equivalent between the TeSeE™ and the
PLATELIA® BSE kits. Based on the data obtained during the evaluation, it was also clear that the performance level
was equal when testing the samples manually or with the TeSeE™ NSP system.
The data presented in this poster clearly indicates that the TeSeE™ assay couple with the TeSeE™ NSP system is well
adapted for routine surveillance of regularly slaughtered cattle.
DIA-49 BSE CONFIRMATORY TESTING WITH THE NEW
TESEE™ BOVINE WESTERN BLOT ASSAY
BILHEUDE J.M.1, GRASSI J.2 ROELS S.3, VANOPDENBOSHE E.3, JACKMAN R.4, JENKINS A.4, EVEREST S.4, STACK
M.4, CHAPLIN M.4, NESPOULOUS G.1 AND BOURGEOIS J.P.1.
1 Bio-Rad - Marnes la Coquette – France. 2 CEA - Service de Pharmacologie et d'Immunologie - Gif sur Yvette –
France. 3 Veterinary and Agrochemical Research Centre (VAR) - Brussels – Belgium. 4 Veterinary Laboratories Agency
- New Haw - Addlestone - Surrey - UK
Bovine Spongiform Encephalopathy (BSE) is now under control in the European Community by implementation of
measures including systematic testing of cattle over 24 or 30 months. Positive samples identified during this surveillance
are systematically confirmed by OIE-approved methods (IHC, SAFs, histopathology) as BSE-affected. These methods
require a high level of interpretative expertise and are in general time consuming and expensive.
Western Blot technique can also be used for rapid confirmatory testing. It allows qualitative analysis of the PrPsc in term
of molecular weight after a proteolytic treatment with a clear evidence of N-Terminus truncation of the disease
associated PrPsc when compared to the unprocessed PrPsc.
However, most of Western Blots suffer from a lack of sensitivity. To overcome this problem, Bio-Rad has optimized a
new confirmatory test based on a western blot initially developed at the CEA (Comoy, Auvré, Marcé and Deslys) with
the same PrPsc purification and concentration steps already used in the Bio-Rad EIA test and a new combination of
monoclonal antibodies.
Comparative studies performed in National Reference Laboratories with two of the CE approved rapid tests in 1999 on
20 brain homogenates and subsequent dilutions, show that TeSeE™ Bovine Western Blot assay is at least as sensitive as
the most sensitive rapid test.
Results obtained also on series of 316 brain samples collected in Belgium in the framework of passive and active
surveillance demonstrate the capacity of this new Western Blot assay to confirm cases identified in the field.
145
Poster Session 2
DIA-50 A PITFALL IN DIAGNOSIS OF HUMAN PRION
DISEASES USING DETECTION OF PROTEASE-RESISTANT PRION
PROTEIN IN URINE.
HISAKO FURUKAWA, KATSUMI DOH-URA, RYO OKUWAKI, SUSUMU SHIRABE, KAZUO YAMAMOTO, HEIICHIRO UDONO,
TAKASHI ITO, SHIGERU KATAMINE, MASAMI NIWA
Dept. Pharmacology1(HF,MN),Dept. Molecular Microbiology and Immunology(RO,SK),The First Department of
Internal Medicine(SS), Division of Cytekine Signaling(KY), Dept.Biochemistry(TI), Nagasaki University Graduate
School of Biomedical Sciences. Dept. of Prion research, Tohoku University Graduate School of Medicine(KD).
Laboratory for Immunochaperones, RIKEN Yokohama Institute(HU).
Since a definite diagnosis of prion diseases relies on the detection of abnormal isoform of prion protein (PrPSc), it has
been urgently necessary to establish a non-invasive diagnostic test to detect PrPSc in human prion diseases. To evaluate
diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed
proteinase K (PK)-resistant protein in patients affected with prion diseases and the control subjects by Western blot, a
coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant
signal migrating around 32 kDa previously reported by Shaked et al. was not observed in this study. Instead, discrete
protein bands with an apparent molecular mass of approximately 37 kDa were detected in the urine of many patients
affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western
blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by the
incubation of secondary antibodies alone, i.e., in the absence of the primary anti-PrP antibodies. Mass spectrometry and
N-terminal protein sequencing analysis revealed that majority of the PK-resistant 37 kDa proteins in patients’ urine were
outer membrane proteins (OMPs) of Enterobacterial species. OMPs isolated from these bacteria were resistant to PK
and the PK-resistant OMPs from Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrPSc. These findings caution that bacterial
contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial
species should be excluded in the immunological tests for PrPSc in clinical samples, in particular, urine.
DIA-51 PRION URINE COMPRISES A GAG-PROTEIN COMPLEX
THAT CAN BE STAINED BY CONGO RED
MICHELE HALIMI, TEHILA MEYER, YAEL DAYAN , YAEL LEVI AND RUTH GABIZON
Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah University Hospital,
Jerusalem, Israel.
146
An in-vivo early test for prion diseases in accessible tissues is the key for the development of prophylactic treatments in
at risk individuals or for the follow up of treatments in CJD patients. We have published previously that prion urine can
be distinguished from other samples by the presence of a protease resistant urine PrP isoform. Here we show that prion
urine comprises an array of disease specific molecules. Among them Light chain IgG (LC), a beta sheet peptide, becomes
protease resistant in prion urine, as opposed to LC in diseases such as multiple myeloma, were LC is believed to be
amyloidotic. In addition, different tests indicate that prion urine samples, as opposed to AD samples, comprise an excess
of GAGs, one of them heparan sulfate. Based on these findings, and since amyloidotic proteins and GAGs are the main
components of amyloid plaques, we developed a Congo Red dot blot assay for prion urine. The binding of Congo Red
to prion urine fractions seems to depend on the presence of both GAGs and proteins, since digestion of denatured
proteins from prion urine fractions significantly reduced the signal. In addition, Congo Red did not bind to similarly
prepared urine fractions originating from SanFillipo patients, a genetic disease in which heparan sulfate is released into
urine at large quantities. These findings are consistent with the possibility that an amyloid seed is present of in prion
urine, which can subsequently incorporate amyloidotic urine proteins, such as LC.
Poster Session 2
DIA-52 BOVINE URINARY PRP LACKS THE N-TERMINAL TAIL
MEHL, M., OESCH, B. AND A.J. RAEBER
Prionics AG, Schlieren-Zurich, Switzerland
Urine is an attractive substrate for an ante mortem diagnostic test for transmissible spongiform encephalopathies (TSE).
Because currently used immunological detection methods are not sensitive enough to detect prion proteins (PrP) in
urine, we purified and characterized urinary PrP by a procedure consisting of 1) a 200 fold concentration of bovine
urine with 10 kDa size exclusion filtration 2) immune affinity chromatography using the monoclonal antibody 6H4
coupled to sepharose and 3) a purification step using reversed phase chromatography. Purified PrP was mapped by
Western blotting using horseradish peroxidase-conjugated monoclonal antibodies raised against different parts of the
prion protein. Purified urinary PrP showed a smear in the 20-30 kDa molecular weight range on Western blot. The
protein was detected with antibodies directed against amino acids 157 - 218 of the bovine PrP sequence. However,
antibodies that recognize the N-terminal amino acids 25 - 112 failed to show signals on Western blot suggesting that the
N-terminus of urinary PrP is truncated. To determine the size of the unglycosylated protein, we performed
deglycosylation of purified PrP with PNGaseF. Deglycosylated PrP showed a signal with an apparent molecular weight
of 20 kDa. These findings show that PrP in urine is glycosylated and lacks the N-terminal tail - possibly as a result of a
proteolytic process. Using ELISA and Western blot analysis we estimated the concentration of PrP in bovine urine to be
between 10 - 100 pg/ml which is about two orders of magnitude lower than that reported for blood. Furthermore, we
found that the sampling procedure is crucial to achieve reproducible results. The identification of urinary prion protein is
an important preliminary step for the development of a prion protein based urine ante mortem test.
DIA-53 PRION PROTEIN/ZAP-70 INTERACTION WITHIN
MICRODOMAINS DURING T CELL ACTIVATION
MATTEI V., DI PROSPERO L., CIARLO L., MANGANELLI V., TASCIOTTI V., PUPO S., PAVAN A., SORICE M.
Viale Regina Elena N. 324, 00161 Rome, Italy
In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of
lymphocytic cells. PrPc was distributed in small clusters on the plasma membrane, as revealed by both scanning confocal
and immunoelectron microscopy.
In order to evaluate its possible role in tyrosine signaling pathway triggered by glycosphingolipid enriched microdomains
(GEM), we analyzed PrPc presence in microdomains and its association with gangliosides, using anti-GM3 MoAb for
identification of GEM in lymphoblastoid T cells. In this cells scanning confocal microscopical analysis revealed a
consistent colocalization between PrPc and GM3 despite an uneven distribution of both on the cell surface, indicating
the existence of PrPc-enriched microdomains. In addition, PrPc was present in the Triton-insoluble fractions,
corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrPc-GM3 interaction was derived
from the results of TLC immunostaining, showing that prion protein was associated with GM3 and GM1 in PrPc
immunoprecipitates.
Then, we analyzed the interaction of prion protein PrPc with signaling components of glycosphingolipid-enriched
microdomains in lymphoblastoid T cells.
A strict association between PrPc and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation
experiments. The phosphorylation syk protein ZAP-70 was immunoprecipitated by anti-PrP after T cell activation.
These results demonstrate that PrPc interacts with ZAP-70, suggesting that PrPc is a component of the multimolecular
signaling complex within microdomains involved in T cell activation.
147
Poster Session 2
DIA-54 SCRAPIE PATHOGENESIS REVEALED BY MAGNETIC
RESONANCE IMAGING AND SPECTROSCOPY
J BARR, 1., Y-L CHUNG 2, E L THOMAS 2, J D BELL 2, J R FRASER 1
1.Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, UK. 2.MR Unit, MRC CSC, Imperial College
Faculty of Medicine, London, 2CRC MR 3Research Group, St George’s Hospital Medical School, London, UK
Scrapie is a neurodegenerative disease affecting both sheep and goats and belongs to an extended family of diseases, the
transmissible spongiform encephalopathies (TSEs) affecting both humans and animals. The pathological features of
disease include vacuolation of the neuropil, gliosis and the deposition of an abnormal protein PrP. Neuronal loss is
known to occur however the actual extent of this loss throughout the brain is difficult to assess by microscopy alone. The
use of magnetic resonance spectroscopy and imaging could provide an alternative method for assessing this and other
pathological features of TSE disease both in vivo and in vitro studies.
In this study we evaluate the use of magnetic resonance imaging and spectroscopy to detect anatomical and biochemical
changes in a murine model of scrapie.
An ME7 murine scrapie model was studied throughout the course of disease progression by magnetic resonance (MR)
imaging and spectroscopy. We determined the :a) earliest time when MR changes could be observed, b)time and extent
of BBB disruption, and c) relationship between severity of scrapie pathology(by histology and western blots) and
detectable MR changes.
Results show that MR can be used to detect pathological changes in the scrapie infected animals well before clinical
signs are apparent.
DIA-55 VERTICAL TRANSMISSION OF BSE IN BO-PRP
TRANSGENIC MICE AFTER INTRA-CEREBRAL INOCULATION
J. CASTILLA, A. BRUN,F. DIAZ, J. SALGUERO, M.J. CANO, A. GUTIERREZ, B. PINTADO, JM TORRES
Centro de Investigacion en Sanidad Animal (CISA-INIA), Carretera de Algete a El Casar, s/n. Madrid (Spain)
To test the efficiency of transmission of BSE prions from infected mothers to their offspring we set a series of experiment
in transgenic mice expressing bovine PrP.
In this work we show substantial evidence of maternal transmission of BSE in Bo-PrP transgenic mice after intracerebral inoculation. PrPres was detected in brains of newborns from infected mothers only when mating was carried
out at times post-inoculation in which brain PrPres deposition was readily detected. No infectivity could be found in
colostrum/milk after reinoculation in boTg mice. The results showed here suggest the ability of BSE prions to spread
centrifugally from the Central Nervous System to peripheral tissues in a mouse model.
148
Poster Session 2
DIS-15 DISCRIMINATION OF SHEEP Q171 CARRIERS
(SCRAPIE SUSCEPTIBLE) WITH 2A11 ANTIBODY
A BRUN, A RELANO-GINÉS, SF MARTIN, JC ESPINOSA AND JM TORRES
Center of Animal Health Investigation, National Institute of Agricultural Technology and Investigation, Valdeolmos,
Madrid, Spain
Scrapie is an infectious neurodegenerative fatal disease of sheep and goats belonging to the group of Transmisible
Spongiform Encephalopathies (TSEs). The incidence of scrapie (and the phenotypic expression) is strongly influenced
by alterations in the host gene that encodes the prion protein (PrP), which is the protein accumulated as an abnormal
isoform in TSEs. There are several polymorphisms in the open reading frame of PrP described. From them, the
polymorphisms at codons 136, 154 and 171 are very important, with maximum resistance to prion infection with R at
position 171, while Q at the same position is related to the highest susceptibility (reviewed by Hunter N, The Genetics of
Sheep, pp. 225-240, 1997). Selective breeding for resistant genotypes is being extensively performed, and friendly
detection tools of these genotypes are required for an extensive use. Here, we describe the selective detection of sheep
PrP harbouring a Q at 171 position that would allow to discriminate them from R171 carriers, in such a way that Q171
carriers could be rejected as breed parents. For this, the 2A11 antibody (Brun A et al., Neurosci Res 48(1): 75-83, 2004)
which recognises an epitope within residues 163-171 of ovine PrP is used. It is shown that the replacement of Q171 by
R171 avoid the recognition of prion protein by 2A11, in such a way that the application of 2A11 for the detection of
Q171 carriers is proposed. Current work is being done for the application of this antibody to tests for the discrimination
of 171 position.
DIS-16 MOLECULAR PROFILING AND COMPARISON OF FIELD
TSE CASES DIAGNOSED IN CATALUNYA; INSIGHTS IN THE BINDING
OF 6H4 AND P4 MABS TO THE SCRAPIE AND BSE PRION.
E.VIDAL (1), M.MARQUEZ (1,3), C.COSTA (3), R.TORTOSA (3), A.DOMONECH (3) AND M.PUMAROLA (1,2,3)
1 - Laboratori PRIOCAT, CReSA Foundation, UAB, Barcelona, SPAIN. 2 - Animal Tissue Bank of Catalunya (BTAC),
Institut de Neurociències, UAB, Barcelona, SPAIN. 3 - Medicine and Surgery Department, Veterinary Faculty, UAB,
Barcelona, SPAIN.
Molecular profiling of the prion protein (PrP) is a technique which has been lately applied to the characterization of
TSE strains. The possibility to differentiate between BSE and Scrapie infection in sheep, at the moment only at an
experimental level (Stack et Al. 2002, Thuring et Al. 2004), or the atypical BSE cases described in France (Biacabe et
Al. 2003) are very interesting examples of such an application.
The 26 BSE cases and 2 scrapie cases from the active TSE surveillance program diagnosed in our lab (PRIOCAT
Laboratory, CReSA Foundation, Reference TSE lab in Catalunya) have been compared using the western blotting
technique. Molecular profiling has been achieved by means of:
- Comparing the glycosilation pattern (that is the relative proportion of the different glycoforms, assessed by
densitometry).
- The molecular weight of the pK digested resistant form of PrP (deglicosilation with PGNase has been perfomed to
easily recognize differences in molecular weight).
- Binding of the digested and undigested PrP to 6H4 and P4 monoclonal antibodies.
All molecular data reported here is supported by the histopathological and immunohistochemical study performed on the
CNS tissues of those animals.
Analyzing the results obtained during the characterization of our field cases altogether with the published results we
discus in this poster the specificity of P4 MAb for scrapie prions (vs BSE prions). Although P4MAb is for sure going to
be a useful tool to distinguish between the sources of infection in field cases, some controversial results need to be
discussed.
Besides, the hypothesis of whether both kinds of agents can be found simultaneously (and distinguished) in a single case
is also discussed.
149
Poster Session 2
DIS-17 MOLECULAR ANALYSIS OF
PATHOLOGICAL PRION PROTEIN (PRPSC) IN
ITALIAN SCRAPIE OUTBREAKS.
M. MAZZA, F. MARTUCCI, S. NODARI, F. INGRAVALLE, C. MAURELLA, C. LIGIOS*, F. SCHOLL°, F. VITALE§, S. BAROCCI†,
M. CARAMELLI, P. L. ACUTIS
CEA- Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle díAosta, Torino – Italy. * Istituto
Zooprofilattico Sperimentale della Sardegna, Sassari – Italy. ° Istituto Zooprofilattico Sperimentale delle Regioni Lazio e
Toscana, Roma – Italy. §Istituto Zooprofilattico Sperimentale della Sicilia, Palermo – Italy. † Istituto Zooprofilattico
Sperimentale dell'Umbria e delle Marche, Fermo - Italy
Several molecular studies based on glycoform ratio and molecular mass of PrPsc were carried out to discriminate between
natural scrapie in sheep, experimental BSE in sheep and natural BSE in cattle.
Molecular mass of the unglycosylated band was found to be different, decreasing in the following order: natural scrapie >
bovine BSE > experimental sheep BSE. The glycoform ratio of PrPsc was not always able to distinguish between these
isolates, as different authors reported conflicting results. A reproducible and rapid Western blot (WB) method was developed
by Stack et al. (2002), to evidence BSE entered in sheep population, combining molecular mass analysis, glycoform profiling
and differential staining characteristics using the two different monoclonal antibodies 6H4 and P4. The 6H4 can detect both
BSE and scrapie while the P4 shows a strong affinity for PrPsc isolated from scrapie samples but not from BSE both in cattle
and in sheep. On the basis of the valid results obtained, we applied this WB to analyse 40 ovine TSE cases of known breed
and PrP genotype, from different Italian outbreaks, and three Italian bovine BSE cases. PrPsc was extracted from each
brainstem sample by a modified Prionics technique and the blots were incubated with 6H4 and P4 antibodies. PrPsc glycotype
was determined analysing by a densitometer at least 8 runs of each sample. Our results showed a similar molecular weight of
the unglycosylated PrPsc band in all scrapie cases, always higher than BSE. As reported by Stack, the glycoform analysis
couldn't discriminate between BSE and scrapie samples. All the scrapie cases were well stained by P4 and 6H4, while BSE, as
expected, showed a very weak signal with P4. Molecular characterization of PrPsc showed that all the examined Italian scrapie
cases were similar and different from BSE. This research was supported by the Ministry of Health (1AA/F3).
DIS-18 DISTINCT MOLECULAR VARIANTS IN BOVINE PRION
DISEASES IN EUROPE
A.-G. BIACABE1, C. CASALONE2, P.-L. ACUTIS2, F. VAN ZIJDERVELD3, A. DAVIDSE3, J. LANGEVELD3, M. CARAMELLI2,
T. BARON1
1 Afssa-Lyon, Unité ATNC, France. 2 CEA -Istituto Zooprofilattico, Turino, Italy. 3 Central Institute for Animal
Disease Control, The Netherlands.
150
Bovine spongiform encephalopathy (BSE) in cattle is thought to be caused by a unique infectious agent, with stable and uniform
features, even when transmitted to other species. Recently, two novel molecular signatures of proteinase K-resistant fragment of
the bovine prion protein (PrPres) have been reported (1) (2). Indeed, Western blot analysis showed a PrPres pattern with a
higher or a lower molecular mass of the unglycosylated form in French and Italian cases respectively. In both cases a less
predominance of the biglycosylated form than in typical BSE was found.
More recently, other similar atypical cases have been identified in France (4 cases) and in Netherlands (1case). Three of the four
novel French atypical cases, born before 1995, exhibited a PrPres pattern similar to the 3 cases published before (1). A 7th
French atypical case, 8 years of age, was found similar to the 2 atypical Italian cases (2). The Dutch atypical case seems similar
to the French cases with higher molecular mass of the unglycosylated PrPres, 13 years of age, discovered in slaughterhouse.
Here, we report comparative and detailed Western Blot molecular analysis of these new cases using different antibodies.
While such cases could have a different origin compared to typical BSE, they remind the occurrence of sporadic
Creutzfeldt-Jakob disease in human with different molecular types. We propose to provisionally refer the cattle TSE
molecular types as type 1 (high molecular mass) and type 2 (low molecular mass) by analogy with the human disease.
1. Biacabe, A.-G., Laplanche, J.-L., Ryder, S. & Baron, T. (2004) EMBO Reports 5, 110-115.
2. Casalone, C., Zanusso, G., Acutis, P., Ferrari, S., Capucci, L., Tagliavini, F., Monaco, S. & Caramelli, M. (2004) Proc
Natl Acad Sci USA.
Poster Session 2
DIS-19 A NEW ANIMAL MODEL HIGHLY SUSCEPTIBLE TO
DIFFERENT HUMAN PRIONS
°AGRIMI U., °VACCARI G., °DI BARI M.A., #BRUCE M., #SUTTIE A., #BOYLE A., °FAZZI P., *CARDONE F., *POCCHIARI
M., °SIMSON S., °FRASSANITO P., °NONNO R.
° Istituto Superiore di Sanità, Dep. Food Safety and Animal Health, Rome, Italy. # Institute for Animal Health,
Neuropathogenesis Unit, Edinburgh, UK. * Istituto Superiore di Sanità, Dep. Cell Biology and Neuroscience, Rome,
Italy
Efficient transmission of humans prions to animal models represents a valuable tool for characterising human isolates. To
improve the traditional mouse-based model, we carried out transmission studies of human prions in the bank vole (C.
glareolus), a wild rodent species which proved to be highly susceptible to different prion sources, irrespective of PrP
sequence homology with the donor species.
A panel of 8 sporadic and genetic cases of human prion diseases were studied after transmission to mice and bank voles.
In voles, very efficient transmission was observed with MM1 (188±22 days survival time) and MV1 (179±10) sCJD
types, as well as with genetic CJD with the E200K (158±13) and V210I (159±7) mutations. Less transmission efficiency
was seen with MM2 (408±100), while voles inoculated with MV2, VV2 and P102L GSS are still healthy 350 days post
inoculation.
Molecular analysis of PK-treated PrPSc from affected voles showed that the molecular weight was maintained unaltered
compared to that of the human cases, with types 1 being higher of type 2; an increase of di-glycosylated isoform of
PrPSc occurred after transmission in voles.
Lesion profiles performed on the brain of affected voles strengthened the distinction of types 1 and type 2, being types 1
and genetic CJD cases very similar each others but different from type 2.
For comparison, the ME7 scrapie strain was also inoculated in voles, showing clear differences in survival time,
glycotype and lesion profile from all human isolates.
The same human prions transmitted very inefficiently to C3H mice; however, bank voles inoculated with mousepassaged MM1 and MV1 sCJD reproduced survival times, glycotypes and lesion profiles identical to those observed
after direct inoculation of the human cases.
The bank vole appears a suitable model for human prions transmission and, being susceptible to a range of animal
prions, would represent a unique tools for comparing isolates from different host species.
151
Poster Session 2
DIS-20 MOLECULAR ANALYSIS OF SCRAPIE ISOLATES FROM
DIFFERENT EUROPEAN COUNTRIES AND COMPARISON WITH
EXPERIMENTAL BSE IN SHEEP
R. NONNO°, E. ESPOSITO°, S. MARCON°, G. VACCARI°, M. CONTE°, T. BARON+, M. BRUCE#, P.L. ACUTIS*, C. LIGIOS§,
U. AGRIMI°
° Istituto Superiore di Sanità, Dep. Food Safety and Animal Health, Rome, Italy. # Institute for Animal Health, Neuropathogenesis Unit,
Edinburgh, UK. *CEA - Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy. § Istituto Zooprofilattico
Sperimentale della Sardegna, Sassari, Italy. + Agence Francaise de Securite Sanitaire des Aliments , Unité Virologie-ATNC, Lyon, France.
Despite the important results recently obtained in the TSE field, several questions remain unanswered and continue to affect
the ability of health Authorities to cope with such diseases. One of the most important concerns the availability of standardised
systems for discriminating TSE strains. The appearance of BSE has indeed reminded that there are several TSE strains with
different pathogenic properties, which may affect both animals and human beings. Our current knowledge about the extent of
the variability of TSE strains circulating in Europe is limited. All the above highlights the urgency of technical approaches for
the surveillance of TSE strains in ruminants which are able to characterise the most common strains circulating in Europe and
allow the identification of new emerging ones. The biochemical characterization of PrPSc appears as a promising tool for
screening TSE cases in small ruminants to check for the presence of BSE. In fact, PrPSc from cattle and sheep affected by
BSE seems to have distinctive properties from those of PrPSc from scrapie cases. Recently we analysed the molecular
characteristics of several italian scrapie cases, showing that they are remarkably similar and well distinguishable from sheep
BSE. With the aim to investigate the molecular variability of scrapie cases in different European countries, we analysed by
western blot 15 additional italian scrapie cases, 10 scrapie cases from UK and 4 from France. By means of glycotype analysis
and selective binding by mAb P4, we show that these scrapie isolates can be grouped into different categories, irrespective of
sheep PrP genotype. Furthermore, they are all distinguishable from sheep BSE. These findings confirm the usefulness of
PrPSc molecular analysis for scrapie and BSE discrimination and suggest that it represents a possible approach for a first step
characterization of scrapie strains. Transmission studies are in progress to corroborate these findings by biological strain typing.
DIS-21 IMMUNOHISTOCHEMICAL DIFFERENTIATION OF
(PRE)-CLINICAL BSE AND SCRAPIE INFECTION IN SHEEP
C.M.A. THURING (1,2), L.J.M. VAN KEULEN (1), J.P.M. LANGEVELD (1), M.E.W. VROMANS (1), F.G. VAN ZIJDERVELD
(1), AND T. SWEENEY (2)
1 TSE section, Central Institute for animal Disease Control (CIDC), Lelystad, the Netherlands. 2 Department of Animal Husbandry
and Production, Faculty of Veterinary Medicine, University College Dublin, Ireland
152
Sheep have been shown to be susceptible to BSE either by intracerebral or by oral infection. Since the clinical signs of
BSE in sheep are indistinguishable from those of natural scrapie, laboratory tests are needed that can discriminate BSE
from scrapie infection in sheep. The objective of this study was to determine whether i) there is PrPSc accumulation in
tonsil and/or third eyelid biopsies of BSE infected sheep prior to clinical disease and ii) whether there are differences in
PrPSc accumulations in these biopsies between BSE and scrapie infected sheep.
These differences could then be used for a “live” test to differentiate pre-clinical BSE from scrapie infection.
Homozygous ARQ sheep (n=10) were orally dosed at 4-5 months of age with a brain homogenate from BSE infected cattle.
Third eyelid and tonsil biopsies were taken with a maximum of 6 monthly intervals post infection and immunohistochemically
examined for PrPSc. While third eyelid protuberances were difficult to identify resulting in many unsuitable biopsies, all
third eyelid biopsies that did contain lymphoid follicles were negative for PrPSc. In contrast, tonsil biopsies were positive for
PrPSc from as early as 11 months in one animal ranging to 20 months post infection in the other animals.
Consistent differences in the morphology of PrPSc granules in tingible body macrophages (TBM’s) of BSE and scrapie
infected sheep were detected using anti-peptide antibodies directed towards amino acids 93-106 of the ovine prion
protein: PrPSc appeared as single granules in TBM’s of tonsil sections from BSE infected sheep, whereas clusters of
PrPSc granules were visualised within TBM’s in tonsils of scrapie infected sheep. In contrast, antibodies directed
towards epitopes N and C-terminally from the 93-106 region of the ovine prion protein did not reveal any differences in
morphology of PrPSc granules in TBM’s between the BSE and scrapie infected sheep.
Poster
Session 3
Poster Session 3
THE-01 CHARACTERIZATION OF PRION PROTEIN-BINDING
PEPTIDES
IANNIS PASPALTSIS1, MARCUS GEISSEN2, JAN LANGEVELD3, MARTIN GROSCHUP2 , CYNTHIA PANAGIOTIDIS1 AND
THEODOROS SKLAVIADIS1.
1Laboratory of Pharmacology, School of Pharmaceutical Sciences, Aristotle University of Thessaloniki, Thessaloniki,
Greece. 2Institute of Novel and Emerging Infectious Diseases, Federal Research Centre for Virus Diseases of Animals,
Germany. 3 Division of Infectious Diseases and Chain Quality, Institute for Animal Science and Health, The
Netherlands.
Pathological prion protein [PrP(Sc)]has been linked to a number of neurodegenerative diseases. The exact mechanism
by which PrP(Sc) leads to such devastating illnesses remains unknown. A possible explanation that has been proposed
is that PrP(Sc)comes in contact with physiological prion protein [PrP(C)], initiating its conversion to the pathological
form. The identification of amino acid sequences that interact with PrP(C) could be useful in understanding the
mechanism of pathogenesis of prion diseases. Furthermore, this interaction may lead to an induction of inhibition of
aggregation of prion protein that would make such peptides good candidate drugs for the inhibition of prion aggregation.
We describe here the results of our work to identify and characterize peptides that interact with PrP(C).
To identify concensus amino acid sequences that interact with PrP(C), bacterially expressed human recombinant prion
protein was used as the target molecule to pan an M13 phage display library of random heptamers. After five rounds of
screening, groups of heptamers with consensus sequences were evident. Preliminary PEPSCAN analysis with
individual phage displayed peptides revealed that the peptides recognize specific epitopes on the prion protein molecule.
Individual heptamers have been synthesized to facilitate further characterization of their interactions with PrP(C). We
present results showing that in an in vivo conversion assay several of the heptamers are able to inhibit aggregation of
PrP(C). These findings suggest that some of the identified PrP-binding peptides might be useful therapeutically to
inhibit prion protein aggregation.
THE-02 INTERFERENCE OF PENTOSAN POLYSULFATE AND
HEPARAN SULFATE MIMETICS WITH THE BINDING OF THE
MOUSE SCRAPIE PRION PROTEIN TO MOLRP-EXPRESSING
BHK CELLS
SABINE GAUCZYSNKI1, SUSANNE EL-GOGO1, EMILIA KLANKKI1, DENIS BARRITAULT2 , CORINNE I. LASMÉZAS3 AND
STEFAN WEISS1
1 Laboratorium fur Molekulare Biologie - Genzentrum-Institut fur Biochemie der LMU Munchen, Munchen, Germany.
2 Laboratoire CRRET/CNRS FRE 2412, Université Paris XII-Val de Marne, Créteil, France. 3 CEA Laboratory for
Prion Pathogenesis, Service de Neurovirologie, DRM/DSV, Fontenay-aux-Roses, France
Recently, we identified the PrP interacting 37 kDa/67 kDa laminin receptor (LRP/LR) (1) as the cell surface receptor
for the cellular prion protein (PrPc) (2) and heparan sulfate proteoglycans (HSPGs) as co-factors within the PrPLRP/LR binding complex (3). Furthermore, it has been shown that LRP/LR is required for PrPSc propagation in
neuronal cells (4).
Here we investigated the binding of the proteinase K-digested mouse scrapie prion protein (moPrP27-30) to mammalian
cells via the Semliki-Forest-Virus system. Enhanced binding of moPrP27-30 to BHK cells was observed when
moLRP::FLAG was overexpressed to the cell surface and LRP/LR specific antibodies totally blocked the binding
reaction suggesting that LRP/LR might act as a receptor for PrPSc. Expression of rec. moPrP to the cell surface also
increased the binding of moPrP27-30 deducing that PrPc might contribute to the cell binding of its infectious
counterpart.
Sulfated glycans such as pentosan polysulfate (SP54) and synthetic heparan sulfate mimetics (HMs) have a therapeutic
and prophylactic potential in TSEs (5-7). Here we show the inhibitory effect of SP54, its derivative PS3, HM5004 and
HM2602 on the binding of moPrP27-30 to moLRP::FLAG expressing BHK cells suggesting that these substances might
interfere with scrapie prion propagation by blocking the moPrP27-30-LRP/LR interaction on the cell surface. Here
HMs might act as competitors of endogenous heparan sulfates known as co-receptors for the binding of PrPc to its
receptor LRP/LR. Since HMs revealed a higher anti-prion potential than SP54 and PS3, they represent promising
reagents for the treatment of TSEs.
1. Rieger, R. et al. (1997) Nat Med, 3, 1383-8.
2. Gauczynski, S. et al. (2001) EMBO J, 20, 5863-75.
3. Hundt, C. et al. (2001) EMBO J, 20, 5876-86.
4. Leucht, C. et al. (2003) EMBO Rep 4, 290-5.
5. Farquhar, C. et al. (1999) Lancet, 353, 117-24
6. Adjou, K. T. et al. (2003) J Gen Virol, 84, 2595-603
7. Schonberger, O. et al. (2003) BBRC, 312, 473-9
155
Poster Session 3
THE-03 INDUCTION OF
PRION PROTEIN-SPECIFIC
ANTIBODY RESPONSES USING PRP-RETROPARTICLES
PATRICIA BACH, DAPHNE NIKLES, FABIO MONTRASIO, FRANK L. HEPPNER, ADRIANO AGUZZI, CHRISTIAN J.
BUCHHOLZ, ULRICH KALINKE
Paul-Ehrlich-Institut, 63225 Langen, Germany
Passive immunization with antibodies directed against the cellular form of the prion protein (PrP) can protect against
prion disease. However, so far active immunization with recombinant PrP failed to induce in vivo protective antibody
responses. Reasoning that on the surface of virus particles the recombinant protein would be presented in an ideal way
to serve as B cell antigen, for immunization we used PrP-retroparticles displaying amino acids 121 to 231 of PrP on the
envelope of murine leukemia virus like particles (for details see contribution by Nikles et al.). Indeed, PrP-retroparticle
preparations devoid of adjuvant were able to induce high antibody titers in PrP deficient (Prnp-/-) mice, as evidenced by
ELISA and FACS methods. Already 7 days after priming of Prnp-/- mice with PrP-retroparticle preparations containing
approximately 1010 particles, high PrP-specific titers were induced. Upon boosting, PrP-specific titers were slightly
enhanced and the prevalence of PrP-specific immunoglobulin of the G subclass (IgG) was further increased. The titers
remained elevated for about 4 weeks and then declined slowly. In wild type mice low but significant IgM and IgG titers
were induced within 7 days that then rapidly declined. Currently, various immunization protocols are being analyzed
involving adjuvant and different dendritic cell subsets as antigen presenting cells to induce high PrP-specific serum titers
in wild type mice. In a next step, the in vitro protective capacity of PrP-specific sera will be tested.
THE-04 GENERATION OF PRION PROTEIN DISPLAYING
RETROPARTICLES
DAPHNE NIKLES, PATRICIA BACH, KLAUS BOLLER, CHRISTOPH MERTEN, FABIO MONTRASIO, KLAUS CICHUTEK,
ULRICH KALINKE, CHRISTIAN J. BUCHHOLZ
Paul-Ehrlich-Institut, 63225 Langen, Germany
156
Passive immunization with antibodies directed against the cellular form of the prion protein can protect against prion
disease. However, so far active immunization with recombinant prion protein failed to induce in vivo protective prion
protein (PrP) specific antibody responses.
Reasoning that on the surface of virus particles the recombinant protein would be presented in an ideal way to serve as
B cell antigen, a retroviral display system for the murine PrP was established. The full length PrP or the C-terminal
domain (aa 121-231) were fused to the transmembrane domain of the platelet derived growth factor receptor (PDGFR)
or to the N-terminus of the murine leukaemia virus (MLV) Env protein. The constructs were transfected into cells
expressing the MLV or the HIV gag/pol genes coding for the viral capsid proteins. The PrP-PDGFR as well as the
PrP110-PDGFR and the PrP110-Env proteins were effectively incorporated and showed the typical glycosylation
pattern. Incorporation efficiency was highest with the PrP110-PDGFR construct, yielding particle numbers of above
1011/ml in concentrated stocks. Immuno-gold electron microscopy showed a high density of gold particles in the
membranes of particles with a typical retroviral shape. Upon intravenous injection, PrP-retroparticle preparations devoid
of adjuvant were able to induce high antibody titers in PrP deficient mice, as evidenced by ELISA and FACS assays.
Already 7 days after priming with approximately 1010 particles, high PrP-specific titers were induced. Upon boosting,
PrP-specific titers were slightly enhanced and the prevalence of PrP-specific immunoglobulin of the G subclass was
further increased.
The data demonstrate that the PrP molecule can be displayed on retroviral particles thus allowing investigations about
the oligomerisation state of PrP and the development of a novel vaccination strategy against TSE.
Poster Session 3
THE-05 ANTI-PRP ANTIBODIES BLOCK PRPSC REPLICATION
IN PRION INFECTED CELL CULTURES BY ACCELERATING
PRPC DEGRADATION
PERRIER VÉRONIQUE1, CROZET CAROLE1, FROBERT YVELINE2, MOURTON-GILLES CHANTAL 3, GRASSI JACQUES2 &
LEHMANN SYLVAIN1
1 Institut de Génétique Humaine, Montpellier, France. 2 CEA Saclay, DRM/SPI, France. 3 CNRS UMR 5094, Faculté
de Pharmacie, Montpellier, France
The usage of anti-PrP antibodies represents one of the most promising strategy for the treatment of prion diseases. In the
present study, we screened various anti-PrP antibodies, with the aim to identify those that will block PrPSc replication in
prion infected cell culture. Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein and
SAF61 directed against PrP amino acid residues (144-152), not only inhibited PrPSc formation in prion-infected
neuroblastoma cells but also decreased the PrPC levels in non infected N2a cells. In addition, treatment with both
SAF34 and SAF61 antibodies decreased the PrPC and the PrPSc levels in the cells, synergistically. In presence of both
antibodies, our results showed that the mode of action which leads to the disappearance of the PrPSc in cells is directly
coupled to PrPC degradation by reducing the half-life of the PrPC protein.
THE-06 TREATMENT OF SCN2A CELLS WITH PRIONSPECIFIC YYR ANTIBODIES
MARTY T. LEHTO, DWAYNE A. ASHMAN AND NEIL R. CASHMAN
Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada
Monoclonal antibodies directed against the tyrosine-tyrosine-arginine (YYR) repeat motif are able to recognize the
pathological form of the prion protein (PrPSc) but not the normal form (PrPC). These antibodies have been able to
immunoprecipitate PrPSc in tissues from all TSE’s tested including CJD, vCJD, GSS, BSE, sheep scrapie and CWD.
The YYR antibodies may prove useful in immunotherapy or immunoprophylaxis of prion diseases. To this end, we have
treated scrapie infected mouse neuroblastoma cells (ScN2a) with YYR antibodies. Results indicate that the IgM mAbs
12B1 and 28B1, and the IgG mAb 4C2, reduce the cell content of protease-resistant PrP of ScN2a cells in a time and
concentration-dependent manner. A proportion of PrPSc is retained in treated cells, suggesting that some prion protein
conversion can occur in a cell compartment inaccessible to mAbs in the culture supernatant. (Supported by Caprion
Pharmaceuticals, McDonald's Corporation, and the Canadian Institutes of Health Research)
157
Poster Session 3
THE-07 CELL-FREE CONVERSION OF BACTERIAL PRION
PROTEIN
MARTIN EIDEN, LEILA KUPFER, KATHLEEN GARNER AND MARTIN H. GROSCHUP
Federal Research Centre of Virus Diseases of Animals, Institute for Novel and Emerging Infectious Diseases, Germany
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases of man and
animals which are transmissible via natural and experimental routes. During disease development normal proteasesensitive prion protein (PrPC) converts into the abnormal protease-resistent PrPSc isoform by a postranslational process
involving profound changes in the secondary and tertiary structure. This conformational change in protein structure
appears to be a fundamental event in the propagation of the causative agent and the pathogenesis of TSEs. Cell-free
reactions have provided evidence that PrPSc itself can induce the conversion of PrPC to proteinase K resistant PrP
(PrPres) in a selective manner. This supports the ‘protein-only’ hypothesis for the TSE-agent. Basically, cell-free
conversion reactions require only the mixing of purified or recombinant PrPC with isolated PrPSc from TSE affected
brains to generate newly formed PrPres.
Here we present a cell-free system utilizing PrPC produced by E.coli. We used a non-radioactive system for detection of
PrPres which is based on antibodies, which discriminate between PrPSc and newly formed PrPres. Visualization was
carried out by an enhanced chemiluminescence approach. This cell-free system allows the molecular characterization of
mouse passaged scrapie and BSE strains which exhibit specific conversion properties. Moreover, by using a chemical
compound library, we could find inhibitors of the cell-free conversion process. Such inhibitors are good candidates which
should be tested further to determine their use as therapeutic drugs against prion diseases.
THE-08 INTERCELLULAR TRANSFER OF THE PRION
PROTEIN : A POTENTIAL THERAPEUTIC STRATEGY TO
AFFECT PRION PROPAGATION.
MOUTHON F., STURNY A. , NOUVEL V, DESLYS J.P.
CEA/DSV/DRM/GIDTIP, Fontenay-aux-Roses, France
158
Prion diseases are fatal neurodegenerative diseases that involve misfolding of the prion protein (PrP). Normal host PrP
is bound to the external surface of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Although the
mechanism of PrP misfolding is not clear, this process may occure at the cell surface or during the endocytic trafficking.
We have studied the membrane properties of the normal PrP anchored by a GPI. Using a cell line overexpressing PrP
and flow cytometry techniques, we have demonstrated the intercellular transfer of this GPI protein during direct cell to
cell contact from the outer layer of the plasma membrane of one cell to another cell. This energy dependant intercellular
transfer is specific of GPI anchorage and is inhibited by an enzymatic treatment which removes the GPI anchor.
Currently available data suggest that PrPc might be implicated in neuronal architecture and survival but its function
remains unclear. By using PrPc defective cell lines and combining them with PrP-GPI purified protein, we can
reconstitute, in trans, at the membrane surface the wild type expression of PrPc. Acquisition of PrP-GPI confers to PrPc
defective cells, protection from specific oxidative stress expositions and apoptotic treatment.
Several data suggest a role for rafts in formation of PrPres and recent studies indicated a modification of raft
organization by amphotericin B treatment, which was previously described to interfere with prion replication. In our
experiment, we observed a stabilisation of the PrP anchored at the plasma membrane after amphotericin B treatment in
a dose dependant manner and a decrease of its availability for intercellular exchanges. The confirmation of this effect in
Prion infected cell lines are currently in progress.
This phenomenon of intercellular transfer of the PrP constitutes a new insight in the molecular biology of this protein.
The use of engineered PrP-GPI offers new perspectives for therapeutic strategies.
Poster Session 3
THE-09 PRION PROTEIN MISFOLDING AND ASSEMBLY INTO
AMYLOID UNDER PRESSURE
JOAN TORRENT *, MARIA TERESA ALVAREZ-MARTINEZ §, JEAN-PIERRE LIAUTARD §, AND REINHARD LANGE *
* Département Biologie-Santé, Université de Montpellier 2, Montpellier, France. § INSERM U431, IFR 122,
Montpellier, France
Protein folding is essential for the translation of genetic information into biological activity. In the case of cellular prion
protein (PrPC), a failure in this process causes several neurodegenerative diseases, by provoking cell damage and
ultimately death. An aberrant isoform (PrPSc) emerges as the essential constituent implicated in the pathogenesis of
these diseases. Despite intensive research, the mechanism of the structural transition between the normal and
pathological isoforms remains enigmatic. Even the “protein only hypothesis” is not yet experimentally proven. To gain
insight into the question of why PrP folds into two different conformational states, we have studied the pressure-induced
unfolding/refolding of recombinant SHaPrP90-231 and several variant forms. Using this original approach, an
alternative route of misfolding was unraveled, leading to a stable amyloidogenic conformer and to mature amyloid fibrils.
The two pressure-induced aberrant structures share certain features with the infectious isoform. One or both of them
may be situated on the pathogenic pathway. This result has important implications, not only for ultimately proving the
protein-only hypothesis and for understanding the mechanism of the disease, but also for designing molecular templates
which may prove useful in the search of compounds preventing PrPSc-like misfolding and aggregation.
THE-10 IDENTIFICATION AND CHARACTERIZATION OF TWO
PRECURSOR OLIGOMERIC STATES IN PRP AMYLOIDOGENESIS
HUMAN REZAEI1, FRÉDÉRIC EGHIAIAN2, JAVIER PEREZ3, DELPHINE RAPP4. JEAN-PAUL CHAPEL4,
BÉNÉDICTE DOUBLET1, YVAN CHOISET1, THOMAS HAERTLE1, JEANNE GROSCLAUDE1.
1 Institut National de Recherche Agronomique (INRA), France ; 2 Laboratoire d’Enzymologie et Biochimie
Structurales, CNRS Gif sur Yvette ; 3 Synchrotron SOLEIL,Saint-Aubin ; 4 Laboratoire des Matériaux Polymères et
des Biomatériaux, UMR CNRS 5627, université Claude Bernard - Lyon I, FRANCE
Recently it was shown that in pathologies due to protein misassembly, low oligomeric state of the misfolded protein
rather than large aggregates play an important role. In prion diseases the lethal evolution of the pathology is associated
with the formation of PrPsc which present amyloidic properties after purification. However the cell toxicity mechanism
of PrPsc is still unclear and the amyloid deposit hypothesis as the causal agent responsible of cellular death is subject of
debate.
To get better insight into the molecular mechanisms of PrPc/PrPsc conversion, we studied the kinetic pathway of heat
induced amyloidogenesis of the full length recombinant ovine PrP at pH 4.0. In contrast with previously reported data,
our results suggest a sequential irrevesible process in which the limiting step in the conversion is the formation of a PrP
trimer from an unfolded state. This trimer rapidly condenses sequentially into a 12-mer and then in a 36-mer of PrP with
an increase in the beta sheet content compared to the monomer. Furthermore the structural analysis of these purified
oligomers by small angle X ray scattering and atomic forces microscopy revealed a quasi ovoid shape for the 12-mers
and 36-mers. Limited proteolysis and peptide analysis of 12-mers and 36-mers show a difference in the accessibility of
the C-terminal domain of these two oligomers allowing the identification of regions undergoing a structural change
during the conversion process. The existence, during the conversion process, of at least two kinds of distinct oligomers
with specific structural and physico-chemical properties, can account for the close relation between PrPsc and strain
phenomena in the protein only hypothesis. Finally, the identification of early events during the PrPc/PrPsc conversion as
well as their characterization, can constitute as much as targets for therapeutic compounds.
159
Poster Session 3
THE-11 ROLE OF INTRACELLULAR TRAFFICKING IN THE
PATHOLOGICAL CONVERSION OF PRION PROTEIN AND IN THE
IDENTIFICATION OF NOVEL THERAPEUTIC TARGETS
VINCENZA CAMPANA (AB), SIMONA PALADINO (AB), DANIELA SARNATARO (B) AND CHIARA ZURZOLO (AB).
a Department de Biologie Cellulaire et Infection, Institut Pasteur, Paris-France. b Dipartimento di Biologia e Patologia
Cellulare e Molecolare, Università degli Studi di Napoli “Federico II”, Napoli-Italy
The prion protein (PrPC) is a GPI-anchored protein endogenously expressed on the surface of neurons and other
peripheral tissues. Its conversion into a pathological isoform, PrPSc, is thought to be responsible for some variants of
“Spongiform Encephalopathies”. The molecular basis of PrPC- PrPSc conversion and the intracellular compartment
where it occurs have not been yet clarified. We believe that alterations in PrP trafficking have a role in the pathogenesis
of the disease. Therefore, understanding the mechanisms of intracellular trafficking of PrPC and of some pathogenic
mutants will be important for the identification of novel molecular targets for TSE therapy in vivo.
We transfected two polarized epithelial cells (FRT and MDCK), well characterized for GPI-protein trafficking, with
cDNA constructs encoding wtPrP or T182A, a mutant associated with genetic CJD. Our data indicate that ER
retention and impairment of the ERAD degradation pathway might have a predominant role in the pathogenic
mechanisms.
We found that T182A mutant is retained in the ER and it is highly associated with detergent insoluble microdomains
(rafts). Interestingly, rafts have not been yet shown in the ER. However, while raft-association of mature isoform of
wtPrP is affected by sphingolipid-perturbant drugs, rafts-association of the mutant is higly reduced by cholesterolperturbant drugs in both cell lines. These data indicate that the nature of ER and post-ER rafts is different. Therefore
wtPrP and its pathological mutants might be associated with different raft types. To test this hypothesis, we are currently
analysing by TLC the lipid composition of microdomains immunoprecipitated with wtPrP and T182A after sucrose
density gradient of cellular lysates metabolically labelled by using 3H-sphinganine or 3H-cholesterol.
Furthermore, using GFP labelled version of wtPrP and T182A we are analysing the membrane mobility of these
proteins by fluoscerence recovery after photobleaching (FRAP).
THE-12 EFFECT OF METAL IONS ON DE NOVO
AGGREGATION OF FULL-LENGTH PRION PROTEIN
ARMIN GIESE, JOHANNES LEVIN, UWE BERTSCH, HANS KRETZSCHMAR
Institute of Neuropathology, Ludwig-Maximilians-Universität Munchen, Munchen, Germany
160
It is well-established that the N-terminal part of prion protein contains metal ion binding sites with specificity for copper.
In addition, disturbances in the levels of copper and manganese have been described in TSE-affected brain tissue, and
changes in copper levels have been suggested to influence incubation time following experimental infection. Therefore,
we studied the effect of various heavy metal ions (Cu++, Mn++, Ni++, Co++, Zn++) on aggregation and conversion of
recombinant prion protein in an in vitro model developed by the group of D. Riesner that utilises changes in the
concentration of SDS to induce structural conversion and aggregation of PrP. To enable efficient monitoring of protein
aggregation, we used fluorescently labelled PrP and cross-correlation analysis as well as SIFT (Scanning for intensely
fluorescent targets) in a confocal single molecule detection system to quantify and characterize PrP aggregates. We
found a specific strong pro-aggregatory effect of Mn++ at low micromolar concentrations that could be reversed by
addition of EDTA. Moreover, the effect of Mn++ could be blocked by nanomolar concentration of Cu++. These findings
suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo.
Poster Session 3
THE-13 KINETICS OF PRP 82-146 FIBRIL FORMATION
DETERMINED BY SURFACE RESONANCE
MARCO GOBBI*, LAURA COLOMBO*, TIZIANA MENNINI*, ELENA ACCARDO§, MARCO VANONI§, FABRIZIO TAGLIAVINI+,
GIANLUIGI FORLONI* AND MARIO SALMONA*
*Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy; §Dipartimento di Biotecnologie e Bioscienze,
Università di Milano-Bicocca, Milano, Italy; +Istituto Nazionale Neurologico "Carlo Besta", Milano, Italy
Prion protein (PrP) amyloid formation is a central feature of prion diseases such as Gerstmann-Sträussler-Scheinker
disease (GSS) and variant Creutzfeldt-Jakob disease. Biochemical studies have shown that amyloid fibrils purified from
GSS brain contain a major PrP fragment spanning residues 81-82 to 144-153 of PrP. We have recently described the
physicochemical properties of the synthetic fragment spanning residues 82-146 (PrP 82-146), showing that it formed
aggregates consisting of 9.8-nm-diameter fibrils with a parallel cross-betastructure. To have a deeper insight in the
polymerization process, surface plasmon resonance technique was used for real time monitoring of PrP 82-146 fibril
extension. The nature of oligomers present in peptide solutions was preliminarily determined by SDS/PAGE following
photo-induced cross-linking of unmodified peptide. PrP82-146 fibrils were covalently bound on sensor chips and the
binding of low-order oligomers (monomers through trimers) was measured by flowing peptide solutions onto the chip. As
expected for an elongation process, the association phase did not show saturation and followed a first order kinetics. In
fact a linear correlation among the association rates, the density of immobilized fibrils and peptide concentration (from 3 to
40 micromolar) was always observed. The exposure of chips to 20 micromolar peptide for 2 min increased fibril length of
about 30%, indicating the occurrence of a very rapid elongation process. Only a fraction of bound peptide dissociated
(half-times 0.1-20 min), whereas about 70% remained irreversibly bound. This proportion increased to above 90% when
the association phase was prolonged from 2 to 5 min. Our data unveil the presence of a “dock-and-lock” mechanism
during PrP 82-146 fibril formation, suggesting that the initial binding step (“docking”) is followed by a “locking” step,
likely due to the isomerization of bound peptide forming a very stable complex and generating new binding sites.
THE-14 DE NOVO GENERATION OF MAMMALIAN PRIONS
G. LEGNAME1,2, I.V. BASKAKOV6, H.-O.B. NGUYEN1, D. REISNER7, F.E. COHEN1,3,4, S.J. DEARMOND1,5, AND S.B.
PRUSINER1,2,4
1Institute for Neurodegenerative Diseases and Departments of 2Neurology. 3Cellular and Molecular Pharmacology,
4Biochemistry and Biophysics, 5Pathology, University of California at San Francisco, San Francisco, CA, USA;
6University of Maryland Biotechnology Institute, Baltimore, MD, USA; 7Institut fur Physikalische Biologie, HeinrichHeine Universitat, Dusseldorf, Germany
Despite a wealth of persuasive data, there remains some resistance to the postulate that prions are infectious proteins
(Prusiner 1982). While the P101L mutation in a synthetic peptide was able to induce prion infectivity in transgenic mice
expressing PrP(P101L), the resulting PrPSc(P101L) was only weakly resistant to proteolysis. In parallel to those studies, we
pursued an alternative approach using wild-type (wt) recombinant (rec) PrP produced in E.coli. Since neither recPrP in an
alpha-helical nor beta-sheet rich conformation induced prion disease in mice, we folded N-terminally truncated MoPrP(89230) into amyloid (Baskakov, Legname et al. 2002) and bioassayed the samples in Tg mice expressing MoPrP(89-230)
(Supattapone, Muramoto et al. 2001). All of these Tg mice developed neurologic dysfunction between 350 and 500 days
after inoculation. Western blotting of brain extracts showed protease-resistant PrPSc; serial transmission of the brain extracts
to wt FVB and Tg(MoPrP)4053 mice resulted in incubation times of 200 and 100 days, respectively. Comparative analysis of
the prions generated de novo and RML, a well characterized mouse-passaged scrapie strain, indicates that at least two new
strains with different biochemical, biophysical and neuropathological characteristics were created. Our results support of the
propositions that prion are infectious proteins and that sporadic prion disease requires only the spontaneous conversion of
PrPC into PrPSc.
References : Baskakov, I. V., G. Legname, et al. (2002). "Pathway complexity of prion protein assembly into amyloid." J.
Biol. Chem. 277: 21140-21148.
Prusiner, S. B. (1982). "Novel proteinaceous infectious particles cause scrapie." Science 216: 136-144.
Supattapone, S., T. Muramoto, et al. (2001). "Identification of two prion protein regions that modify scrapie incubation
time." J. Virol. 75: 1408-1413.
161
Poster Session 3
THE-15 CRYSTAL STRUCTURES OF ANTIBODY-BOUND
OVINE PRION SCRAPIE-SUSCEPTIBILITY VARIANTS PROVIDE
A MECHANISTIC BASIS FOR THE DESIGN OF ANTI-TSE
DRUGS
FRÉDÉRIC EGHIAIAN1, JEANNE GROSCLAUDE2, STÉPHANIE LESCEU3, PASCALE DEBEY4, BÉNÉDICTE DOUBLET2,
HUMAN REZAEI2 AND MARCEL KNOSSOW1
1Laboratoire d'Enzymologie et de Biochimie Structurales, Gif sur Yvette, France. 2Virologie et Immunologie
Moléculaires (VIM), INRA, Jouy-en-Josas, France. 3Institut Pourquier, Montpellier. 4Muséum National d'Histoire
Naturelle, Paris, France.
In the light of our present knowledge of TSE, the design of inhibitors of the PrPSc formation still constitutes a
promising strategy for prion disease therapy. For this approach to be efficient, an in-depth understanding of PrPSc
formation is required, on both a structural and mechanistic viewpoint: to date, neither the molecular details of the PrPC>PrPSc conversion nor the mechanism by which mutations in the prnp gene control this process are known. We solved
the 2.5 Å resolution crystal structures of three scrapie-susceptibility sheep PrP (OvPrP) variants complexed with an
antibody that binds to PrPC and to PrPSc. The structures reveal two important features of the PrPC->PrPSc
conversion. Firstly, the epitope of the antibody is mainly located in the last two turns of OvPrP second alpha-helix and is
exposed in PrPC and PrPSc. Taking our results together with biochemical data, we propose that the conformational
change responsible of the PrPC->PrPSc conversion is restricted to residues 100-174 of the prion protein. Further similar
studies on PrPSc structure will help define precisely PrPC regions that undergo transconformation as well as PrPSc
interaction domains: compounds interacting with those parts of PrP could have an anti-prion effect. Secondly,
comparison of the structures of sheep scrapie-sensitivity variants defines local changes in distant parts of the protein that
account for the observed differences of PrPC stability, resistant variants being destabilized compared to sensitive ones.
Additive contributions of these sensitivity-modulating mutations to resistance suggest a causal relationship between
scrapie resistance and lowered stability of the PrP protein. A screening of substances decreasing PrPC stability could
therefore be the starting point for the conception of a prion disease therapy.
This work was partly supported by the GIS "Infections à prions"
THE-16 CRYSTAL STRUCTURE OF THE GLOBULAR DOMAIN
OF SHEEP PRP.
L.F.HAIRE(1) , S.M.WHYTE(1) , N.VASISHT(1) , A.C.GILL(2), C.VERMA(3,4), E.J.DODSON(3), AND
G.G.DODSON(1,3).
PRESENTING : P.M.BAYLEY1
1 Structural Biology Group, National Institute for Medical Research, London, UK. 2 TSE Division, Institute for
Animal Health, Compton, UK. 3 York Structural Biology Laboratory, University of York, UK. 4 Bioinformatics
Institute, Biopolis Way, Matrix,Singapore.
162
The transformation of the prion protein PrP from a normal cellular conformation to that of an aggregated form,
characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and
Bovine Spongiform Encephalopathy, is a fundamental step in neuropathogenesis. PrP is therefore a potential target for
therapeutic intervention. We report the crystal structure, at 2Å resolution, of residues 123-230 of the C-terminal globular
domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single monomeric molecule
whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian
species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2
and the N-terminal portion of helix-3, bounded by the intra-molecular disulphide bond, 179-214. The loop 164-177,
between beta-2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the
sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these
comprises the beta strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand
is involved in lattice contacts about a crystal dyad to generate a 4-stranded intermolecular beta-sheet between
neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a
partial alpha/beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give
rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional
intermolecular beta-structure.
Poster Session 3
THE-17 TOWARDS AN ASSESSMENT OF MEMBRANE
TOPOLOGY OF MUTANT VERSIONS OF PRION PROTEIN WITH
MICRO-DELETIONS IN THE TRANSMEMBRANE DOMAIN 1
JENS LUTZ, CHRISTINE BRABECK, ALEXANDER BURKLE
Molecular Toxicology Group, Dept of Biology, Univ of Konstanz, D-78457 Konstanz, Germany. jens.lutz@unikonstanz.de
The transmembrane domain-1 (TM-1) of prion protein (PrP) comprises the most highly conserved region of this protein
in mammalians. A missense mutation in TM-1 has been implicated in some cases of Gerstmann-Sträussler-Scheinker
syndrome (GSS), resulting in an increased probability of transmembrane topology of PrPc (CtmPrP), which has been
postulated to be a neurotoxic factor in prion diseases besides PrPSc (1, 2). Interestingly, a mutant of murine PrP, lacking
8 amino acids within TM-1, inhibits accumulation of PrPSc in scrapie-infected Neuro2a cells in a trans-dominant fashion
(4). Deletion the entire TM-1 domain has already been shown to cause the complete loss of transmembrane topology
(3). In order better to understand the possible physiological and pathophysiological functions of CtmPrP, we are
currently establishing an in vitro assay to assess differences in the membrane topology of a series of mutants carrying
micro-deletions from two to eight amino acids within the most highly conserved part of TM-1, i.e. between codons 114
and 121. Our current data will be presented.
1. Hegde, R. S., et al., 1998 Science 279:827-34.
2. Hegde, R. S., et al., 1999. Nature 402:822-6.
3. Hˆlscher, C., et al., 2001. J Biol Chem 276:13388-94.
4. Hˆlscher, C., et al., 1998. J Virol 72:1153-9.
THE-18 IN VIVO CYTOTOXICITY: RELATION STRUCTUREFUNCTION OF P106-126 VARIANTS AND SCREENING OF
NEUROPROTECTIVE DRUGS.
ANN-LOUISE BERGSTROM, NICOLE ZSURGER, PETER HEEGAARD, PETER LIND AND JOELLE CHABRY
Institut de Pharmacologie Moléculaire et Celluaire, Valbonne, France
Neuronal cell death is the main hallmark of transmissible spongiform encephalopathies (TSEs). Recently, we developed
an in vivo assay to study the neurotoxicity mechanisms using intra-ocular injections of synthetic peptides of PrP. The
damages on neuronal cells were assayed first by measurement of the electroretinogram and second by histology studies
on retinal sections. Eye is a relevant model in the context of TSEs since it is a natural route for scrapie infection
particularly in sheep and human cornea transplants may cause Creutzfeldt-Jakob disease. Moreover, retina is highly
susceptible to prion diseases.
Two approaches are currently followed: First, the structure/function relationships between neurotoxicity and amyloidforming ability and second, the screening of new therapeutic drugs.
a- The synthetic peptide PrP106-126 has been described as amyloidogenic, neurotoxic and partially proteinase K
resistant. In contrast to the soluble, the acid form of “aged” PrP106-126 was found to be neurotoxic in wild-type mice. In
PrP-deficient mice, no toxic effect was found with both “aged” and soluble PrP106-126. A structure-relaxed variant of
PrP106-126 with an N-termimal addition of 2 arginines and 4 glycines residues (RG2-variant) was found to be nontoxic. Thioflavine T assay showed PrP106-126 to form amyloid fibrils after aging, whereas the amidated and RG2variant had a reduced ability to form amyloid fibrils. In summary, the in vivo neurotoxicity of these peptides is strongly
correlated with their amyloid-forming abilities.
b- The in vivo model described above will also be used to screen drugs or compounds with neuroprotective properties
capable to thwart the neuronal death induced by PrP106-126. This project will be a simple and rapid complementary
approach to assay in vivo neuroprotective properties of molecules. To date, no prophylactic or therapeutic treatment
against prion diseases is available.
163
Poster Session 3
THE-19 INTERACTION OF PRPC WITH NRAGE, A PROTEIN
INVOLVED IN NEURONAL APOPTOSIS.
BIRKIR THOR BRAGASON AND ASTRIDUR PALSDOTTIR
Institute for Experimental Pathology, Department of Virology and Molecular Biology, University of Iceland, Iceland.
Several studies indicate that PrPC is subject to proteasomal breakdown after retrograde transport from the ER. When
exposed to the cytoplasm, PrPC has been shown to display biochemical characteristics similar to PrPSc, e.g. protease
resistance and detergent insolubility, and it can accumulate in aggresomes. Some studies indicate that this cytoplasmic
PrPC is neurotoxic and causes apoptosis, others that it is neuroprotective and inhibits apoptosis.
We isolated a cytoplasmic PrPC binding protein, NRAGE (neurotrophin receptor-interacting MAGE homologue), in a
yeast two-hybrid (YTH) screen of a rat brain cDNA library with an ovine PrPC construct. Other studies show that, in
neurons, NRAGE interacts with the cytoplasmic region of p75NTR, and facilitates apoptosis through the JNK pathway,
characterised by cytosolic accumulation of cytochrome C, activation of caspases-3, -9, and 7, and phosphorylation and
accumulation of c-jun.
Binding to NRAGE in the YTH system was conserved when ovine PrPC was exchanged for rat PrPC. Rat PrPC was
used in experiments thereafter. The YTH interaction was confirmed by GST-pulldown and co-immunoprecipitation (coIP). The co-IPs, and YTH experiments with truncated rat-PrPC constructs, indicate that NRAGE binds to the Cterminus of PrPC. An NRAGE antibody was produced. We examined NRAGE distribution in an adult rat brain by
immunohistochemistry. The NRAGE antibody stained, for example, the cytoplasm of cortical neurons and choroid
plexus epithelia, glial cells did not stain. When the proteasome in PC12 (rat) cells was inhibited with lactacystin,
endogenous NRAGE formed perinuclear aggregates. The aggregates were vimentin positive, suggesting that they are
aggresomes. When NRAGE and PrPC-EGFP were co-expressed in COS7 cells they co-localized in perinuclear
aggregates after lactacystin treatment. The periphery of these aggregates stains with hsc70 suggesting that they are
aggresomes.
THE-20 BRAIN SEROTONIN AS A PREFERENTIAL TARGET IN
BSE NEUROPATHOLOGY
VIDAL C (1), HERZOG C (2), LAUNAIS JM (3), LAPLANCHE JL (3), LEFEBVRE-ROQUE M (2), DORMONT D (2),
LASMÉZAS C (2).
(1) Institut Pasteur, Paris; (2) SNV, CEA, Fontenay-aux-Roses; (3) Hopital Lariboisière, Paris.
164
Little is known on neurodegenerating processes in prion diseases. The mechanisms underlying neuronal targeting and
the role of neurotransmitters and receptors in the susceptibility to infection remain to be elucidated. Several clinical and
experimental data have indicated disturbances in the metabolism of brain monoamines and particularly of serotonin. The
present study was aimed to evaluate the extent of serotonin alterations and their functional consequences in a mouse
model of BSE. We used behavioral tests designed to evaluate serotonergic functions : light/dark boxes (anxiety), hotplate test (pain), activity boxes (circadian rhythm). Mice infected ic with a BSE strain (6BP1) showed behavioral
abnormalities i.e. changes in circadian activity, anxiolytic-like behavior and hyperalgesia preceding clinical motor
symptoms by 15/20 days. Biochemical analysis of these brains by HPLC revealed a significant reduction of serotonin
concentration and an increased turnover rate, suggesting a defect of synthesis probably due to a loss of serotonergic
neurons. This hypothesis was confirmed by comparing the behavioral deficits of BSE mice to those observed in noninfected mice subjected to selective lesion of serotonergic neurons via icv injection of the neurotoxin 5, 7dihydroxytryptamine. The latter mice exhibited clear cut signs of anxiolytic-like behavior and increased nocturnal
activity similar to BSE infected animals. These results provide evidence for a preferential damage of the serotonergic
system in the BSE mouse model, starting at early stages of infection. Changes in this system may account for several
symptoms of CJD, particularly in respect of fluctuating mood and arousal common in vCJD patients. We suggest that
serotonergic neurons constitute a specific cell subset for the propagation and accumulation of prion agents, thus
designating a promissing target for the development of diagnostic and neuroprotective tools.
Supported by GIS Prion 2003, France.
Poster Session 3
THE-21 OVER-EXPRESSION OF PRP-C PROMOTES
NEURONAL SURVIVAL IN IN VITRO AND IN VIVO MODELS.
COULPIER M., MESSIAEN S., HAMEL R., ARRABAL S., ELOIT M.
UMR 1161 de Virologie INRA-AFSSA-ENVA, Ecole Vétérinaire de Maisons-Alfort, France
Neuronal death is a well-known figure of prion diseases but the mechanisms of this death are largely unknown. Whereas
it has been demonstrated that the cellular form of the prion protein (PrP-c) into neurons plays a role in this process, its
physiological function remains unknown. In vitro, both anti-apoptotic and pro-apoptotic role have been suggested for
PrP-c. Here, we used in vitro and in vivo models in order to investigate the role of PrP-c on neuronal survival. In vitro,
we show that over-expression of PrP-c protects neuroblastoma cells from staurosporine-induced death. In vivo, overexpression of PrP-c in transgenic TGa20 mice protects facial motoneurons from axotomy-induced death in neonates. 4
days after transection of the facial nerve 25 to 30 % of facial motoneurons appeared protected compared to wild-type
mice. On the contrary, facial motoneurons were not affected in PrP-c -/- mice suggesting either the existence of
compensatory mechanisms or the absence of physiological role for PrP-c in the axotomy-induced motoneuron death
model. These results argue in favor of an anti-apoptotic role for PrP-c and show for the first time an anti-apoptotic role
in vivo. They suggest that an alteration of PrP-c could have, in some cases, significant consequences on neuronal
survival.
THE-22 CYTOSOLIC (PRP) IS NOT TOXIC IN N2A CELLS
AND PRIMARY NEURONS EXPRESSING PATHOGENIC PRP
MUTATIONS
LUANA FIORITI1,2, SARA DOSSENA1,2, LEANNE R. STEWART3, DAVID A. HARRIS3, GIANLUIGI FORLONI2, AND
ROBERTO CHIESA1,2.
Dulbecco Telethon Institute (DTI) and Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy
Familial prion diseases are linked to mutations in the prion protein (PrP) gene, which are thought to favor
conformational conversion of PrP into a pathogenic isoform. The cellular mechanisms by which mutant PrP causes
neurological dysfunction are poorly understood. It has been proposed that a highly toxic form of PrP may accumulate in
the cytosol of neurons due to impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from
the endoplasmic reticulum (ER) (Ma et al., Science, 298:1781-1785, 2002). To test whether this neurotoxic mechanism is
operative in familial prion diseases, we have evaluated the effect of proteasome inhibitors on the viability of N2a and
cerebellar granule neurons expressing mouse PrP homologues of the D178N and nine-octapeptide mutations. We found
that the inhibitors MG132 and ALLN caused accumulation of an unglycosylated, aggregated form of PrP in transfected
N2a cells overexpressing either wild-type or mutant PrP, but not in untransfected N2a. This form contained an
uncleaved signal peptide, suggesting that it represented PrP chains that had failed to translocate into the ER lumen
during synthesis, rather than retrogradely translocated PrP (Drisaldi et al., J Biol. Chem. 278: 21732-21743, 2003).
Quantification of N2a viability after treatment with proteasome inhibitors did not reveal significant differences between
untransfected and transfected cells, indicating that accumulation of untranslocated, cytosolic PrP is not toxic.
Proteasome inhibitors (MG132, ALLN, clasto-Lactacystin beta-lactone, epoxomicin) did not induce accumulation of
unglycosylated, insoluble PrP in cerebellar granule neurons expressing either wild-type or mutant PrP. Moreover, no
difference in susceptibility to proteasome inhibitors toxicity was found between PrP knockout neurons and neurons
expressing PrP. These data argue that formation of cytosolic PrP is unlikely to represent a pathogenic event in familial
prion diseases
165
Poster Session 3
THE-23 IDENTIFICATION OF PROAPOPTOTIC MARKERS OF
DISEASE IN MURINE SCRAPIE
D.A. BROWN, J.W. IRONSIDE AND J.R. FRASER
D.A. Brown and J.R. Fraser :Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, UK. J.W. Ironside :
NCJD Surveillance Unit Western General Hospital, Edinburgh, UK.
Transmissible spongiform encephalopathies (TSEs) provoke a spectrum of degeneration in the CNS, which is initiated
long before clinical disease can be identified. One of the characteristic pathological changes observed in these diseases is
neuronal loss. Studies in experimental mouse models suggest that the neuronal cell death observed in TSEs is through an
apoptotic mechanism. However there are many apoptotic pathways, and it is not known which ones operate in the
TSEs. Identification of a TSE specific pathway could help in the development of therapeutic strategies for the treatment
of TSEs.
In this study two scrapie mouse models which differ in their sequence of pathology observed in the hippocampus were
used : the ME7/CV model in which neuronal loss is targeted to CA1 and the 87V/VM model where the pathology
targets CA2 of the hippocampus. The involvement of the caspase-dependant pathway of apoptosis was investigated in
these models. Using immunocytochemical and Western blot techniques the up-regulation of proapoptotic markers Fas,
caspase-3, whole and cleaved poly(ADP-ribose) polymerase (PARP) were investigated. TUNEL labelling was
performed to identify DNA strand breaks.
Active caspase-3, whole and cleaved PARP were shown to be upregulated in both mouse models. Fas receptor
expression was observed in astrocytes surrounding the lesioned area of the brain. TUNEL labelling was observed in the
brains of mice in both models, which correlated with active caspase-3 labelling.
These results indicate a caspase-dependent apoptotic mechanism of cell death in TSEs. Further studies analysing early
events in the neurodegenerative process are required to determine whether caspase inhibitors could play a role in
therapeutic intervention in TSE diseases.
THE-24 ROLE OF THE CELLULAR PRION PROTEIN IN
APOPTOSIS
S.F. MARTIN, B. PARRA, A. RELANO AND J.M. TORRES
Center of Animal Health Investigation, National Institute of Agricultural Technology and Investigation, Spain
166
Prion protein (PrPC) is a cell membrane glycoprotein that can change to structurally modified form (PrPSc). The PrPC
function is not well known. Actually, the reported data related to PrPC functions could be assembled in three groups: i)
a possible synaptic role where copper-binding protein could regulate the copper content of the synaptic cleft and at
intracellular free calcium levels the synapse; ii) a role in cellular resistance to oxidative stress either as inductor or as
possessor of superoxide dismutase activity; iii) a possible apoptotic inductor. However, this proapoptotic function has
been related with PrPSc and/or neurotoxic peptide fragment corresponding to amino acids 106-126 of human prion
protein (PrP106-126). These forms induced apoptosis by toxic and degenerative effects on cultured cells and affected
tissues. This toxicity of PrPSc and PrP fragments appears to depend on PrPC neuronal expression and on microglial
activation. In addition, neuronal cell death is a salient feature in the pathology of prion diseases where histological and
electron microscopical studies have shown that cell death in prion disease occurs by apoptosis. For a better
understanding of the role of PrPC in apoptosis, we examined the influence of cellular prion protein PrPC in the control
of apoptosis in cultured cells. We used different apoptotic models: i) specific stimuli (TNF-alpha), ii) nonspecific stimuli
(serum deprivation and senescence) and cytotoxic stimuli (ceramide). In all models, we observed a PrPC accumulation,
which is directly dependent of apoptotic levels on cells. Therefore, these data suggest that PrPC accumulation can act
either as an inductor or as a protector on apoptosis in cultured cells.
Poster Session 3
THE-25 PRION PROTEIN PREVENTS BREAST CARCINOMA
CELLS FROM APOPTOSIS
MARYAM DIARRA-MEHRPOUR, SAMUEL ARRABAL, ABDELALI JALIL, XAVIER PINSON, HUGUES RIPOCHE, DOMINIQUE
DORMONT (1) AND SALEM CHOUAIB
Laboratoire de Cytokines et Immunologie des tumeurs Humaines, INSERM U-487, Institut Gustave Roussy PR1 and
IFR 54, France [M. D-M., A. J., X.P, S. C.]. UMR 8125 CNRS, Institut Gustave Roussy PR2, France [H. R.]. CEA,
Service de Neurovirologie, CRSSA, EPHE, IPSC, Université Paris XI, Fontenay-aux-Roses, France [D. D.]. UMR
1161 de Virologie INRA AFSSA ENVA Maisons Alfort, France [.S. A.].
In order to define genetic determinants of tumor cell resistance to the cytotoxic action of TNF, we have applied cDNA
microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. We
found a great number of differentially expressed genes involved in the PI3K/Akt signalling pathway. More notably, we
found that the PRNP gene coding for the cellular prion protein (PrPc), was over-expressed in TNF resisatnt cells as
compared to MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining indicating
that PrPc was over-expressed at both mRNA and protein levels in TNF-resistant derivative. Further investigations,
involving recombinant adenoviruses expressing the human PrPc indicated that PrPc over-expression converted TNF
sensitive MCF7 cells into TNF resistant, at least in part by a mechanism involving alteration of cytochrome c release
from mitochondria and nuclear condensation.
(1) In memoriam, to Pr.Dominique Dormont, who was a pioneer in Prion research and who showed to the scientific
THE-26 CATHEPSIN B AND L ARE INVOLVED IN
DEGRADATION OF PRIONS IN GT1- NEURONAL CELLS
ELIN K NORDSTROM 1, KATARINA M LUHR 1, ALBERT TARABOULOS 2, PETER LOW 1 AND KRISTER KRISTENSSON 1
1. Department of Neuroscience B2:5, Karolinska Institutet, Stockholm, Sweden. 2. Department of Molecular Biology,
Hebrew University Hadassah Medical School, Jerusalem, Israel.
The abnormal isoform of the prion protein, PrPSc, accumulates in late endosomes and lysosomes in scrapie-infected
cells. We have here analyzed the involvement of cathepsin B and L in cellular processing of PrPSc in immortalized
neuronal gonadotropin-releasing hormone cells (GT1-1) infected with scrapie. Treatment with inhibitors of either
cathepsin B or L resulted in accumulation of PrPSc. Such an increased accumulation also occurred when the expression
of both cathepsins were inhibited using RNA interference. We conclude that cathepsin B and L are involved in the
degradation of PrPSc in scrapie-infected GT1-1 cells and that they can compensate for each other’s functions. This study
shows that specific proteases, abundantly present in neurons, have the capacity to degrade PrPSc. These findings may
increase the therapeutic possibilities in affecting the balance of PrPSc accumulation and degradation.
This research was supported by EUQLK2-CT-2002.81628.
167
Poster Session 3
THE-27 EFFECTS OF INNATE IMMUNE RESPONSE
MOLECULES ON PROTEASE RESISTANT PRION PROTEIN
(PRPSC) DEGRADATION
KATARINA LUHR, PETER LOW AND KRISTER KRISTENSSON.
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
We have previously observed that specific cellular proteases, cathepsin B and L, can degrade protease resistant prion
protein (PrPSc) in scrapie-infected GT1-1 cells (ScGT1-1). In the present study we investigate how cathepsin levels and
activities may be regulated during a prion infection.
First, we analyzed the expression of GT1-1 cell-derived cathepsins, before and during a scrapie-infection. No change in
the RNA transcripts of the cysteine proteases cathepsin B and L (expressed at high levels in the GT1-1 cells) or
cathepsin H and S (expressed at low levels in the cells) could be seen in ScGT1-1 cells compared to their non-infected
controls.
Second, we analyzed whether molecules released by innate immune responses to a neuronal infection can affect
cathepsin expression and cathepsin-induced degradation of PrPSc. In other studies, certain cytokines have been shown
to influence the activity of cathepsins. We here exposed ScGT1-1 cells to interferon-gamma, tumor necrosis factor-alpha,
or nitric oxide and studied the effects on expression and activity of cathepsin B and L as well as the effect on the levels of
PrPSc. The identification of molecules regulating the accumulation or degradation of PrPSc in a cell may be of
importance for future manipulations of the uptake and spread of prions to the CNS and might elucidate mechanisms
involved in clearance of PrPSc during prion infections.
This research was supported by EC grant QLK2-CT-2002-81628 and the Swedish Society for Medical Research.
THE-28 ISOLATION OF ANTI-PRION DRUGS USING YEAST
BASED ASSAY
BACH STEPHANE$, TALAREK NICOLAS*, DHINTILLAC AGNÈS*, BLONDEL MARC$, CULLIN CHRISTOPHE*
* Hérédité Structurale et Prions, I.B.G.C., Bordeaux, France. $ Cell cycle laboratory, CNRS, Station biologique,
Roscoff, France.
168
The yeast Saccharomyces cerevisiae posses several cytoplasmic heritable elements that exhibit prion-like properties. The
two most studied prion phenotypes are [PSI] and [URE3]. These phenotypes are due to the partial inactivation of
Sup35p and Ure2p respectively. These inactivation propagated through the interactions of the normal protein with the
abnormal form that confers its “abnormality” in a snow ball mechanism.
This mechanism requires a functional HSP104 chaperone protein as well as a precise balance between several
chaperones. The guanidium salts are known in yeast to eliminate (cure) the prions probably by inhibiting the Hsp104
activity. We took advantage of the yeast genetic facilities to engineering new strains in which the prion phenotypes
([PSI] as well as [URE3]) may be easily scored and cured by guanidium hydrochloride. These strains were used to
isolate new molecules effective against yeast prions. Remarkably, these drugs are also efficient to promote the clearance
of PrpSc in a mammalian assay. A remakable corelation between the molecules efficient in yeast or mammalian cells
exists, proving the existence of a common mecanism conserved through the evolution and making the yeast-based assay
even more interesting.
Poster Session 3
THE-29 IN VIVO EFFICACY OF QUINACRINE IN ANIMAL
MODELS OF PRION DISEASE
C. RYOU1,2, P. LESSARD1, Y. FREYMAN1, B. J. GUGLIELMO3, L. YUNG3, M. A. BALDWIN1,2,4, J. C. CRAIG4, S. J.
DEARMOND1,5, B. C. H. MAY1,2, F. E, COHEN1,6,7, E. T. LIN8, Y. HUANG8, S. B. PRUSINER1,2,7, AND G. LEGNAME1,2.
1Institute of Neurodegenerative Diseases, Departments of 2Neurology, 3Clinical Pharmacy, 4Pharmaceutical Chemistry,
5Pathology, 6Cellular and Molecular Pharmacology, 7Biochemistry and Biophysics, 8Biopharmaceutical Sciences,
University of California, San Francisco, California.
Prion diseases of humans and animals are neurodegenerative disorders that often exhibit a relatively rapid clinical course
and are uniformly fatal. Currently, no effective therapy for prion diseases exists. One of the most potent drugs found to
inhibit prion formation in neuroblastoma cells is quinacrine [K. Doh-ura, T. Iwaki, B. Caughey, J. Virol. 74, 4894-4897
(2000), C. Korth, B. C. H. May, F. E. Cohen, S. B. Prusiner, Proc. Natl. Acad. Sci. USA 98, 9836-9841 (2001)] but
attempts to show therapeutic efficacy in vivo have been disappointing [S. J. Collins et al., Ann Neurol 52, 503-506
(2002), A. Barret et al., J Virol 77, 8462-8469 (2003)]. To study the pharmacological efficacy of quinacrine in vivo, we
measured alterations in the incubation times of prion-infected mice given quinacrine. Wild-type FVB and CD-1 mice
were inoculated intracerebrally with RML prions and treated with various doses of quinacrine. Oral administration of
quinacrine began after 50% or 75% of the incubation period had elapsed; quinacrine was given for 30 d, 60 d or until
death. We found that quinacrine is effective in delaying the onset of neurologic dysfunction in both FVB and CD-1 mice
when administration of the drug was initiated at the mid-point (50%) of the incubation peroid. A regimen of 37.5
mg/kg/day for 30 days was successful in extending the survival of the mice approimately three weeks. At the time the
quinacrine treated mice developed neurologic deficits, we found a reduction in PrPSc levels, a prevention of neocortical
shrinkage, and a delay in the development of astrocytic gliosis compared to untreated, prion-infeccted mice. Toxicity
studies showed that the regimens for quinacrine treatment were well tolerated in the mice and pharmacokinetic
measurements showed high quinacrine levels in brain. Our results demonstrate that an antiprion therapeutic can be
administered long after prion infection is initiated and produce a significant prolongation of life in mice.
THE-30 FUNCTIONAL PRP KNOCKOUT BY EXPRESSION OF
ENDOPLASMIC RETICULUM-ANCHORED ANTI-PRP SINGLECHAIN ANTIBODIES
REZA DABAGHIAN1,2, ALEXANDER GIEBEL1, YONGHUA ZHANG1,2, SASCHA BENEKE2,3, CHRISTINE BRABECK2,3,
WALTER BODEMER4, SUSANNE KRASEMANN4, GERHARD HUNSMANN4, ANDREAS HUNZIKER2, FRANK BREITLING2,
ALEXANDER BURKLE1,2,3
1 SCMS Gerontology, Institute for Ageing and Health, University of Newcastle upon Tyne, UK. 2 Deutsches
Krebsforschungszentrum, Heidelberg, Germany . 3 Department of Biology, University of Konstanz, Konstanz,
Germany. 4 Deutsches Primatenzentrum, Gottingen, Germany . alexander.buerkle@uni-konstanz.de
We have recently created a set of single-chain antibodies reactive with mouse PrP, based on previously described
hybridomas secreting monoclonal PrP antibodies [1] and phage display technology. The coding sequences of single-chain
antibodies that proved to bind their cognate peptide antigen successfully in vitro were subcloned into the eukaryotic
expression vector pCMV-myc-ER, thus leading to the expression, in transfected Neuro2a cells, of a fusion protein
comprising an ER leader sequence, the coding sequence for the respective single-chain antibody, a c-myc epitope for
immunodetection as well as an ER retention signal (KDEL). This fusion enables formation of “intrabodies”, i.e.
translocation to the nascent single-chain antibodies to the lumen of the endoplasmic reticulum and retention in this
compartment. Co-transfection of such expression constructs for PrP-specific intrabodies with an expression construct for
PrPc (pCMV-PrP; [2]) revealed that the PrP-specific intrabodies decreased the level of PrPc whereas an intrabody
recognising an irrelevant antigen did not. The effect of PrP-specific intrabodies could be prevented by administration of
the proteasome inhibitor ALLN, strongly suggesting that PrP-specific intrabodies induced enhanced proteasomal
degradation of PrPc. Future experiments will show if such ‘phenotypic knockout’ of PrPc can protect cell cultures against
prion infection. If so, this could form the basis for a novel concept of gene therapy / prophylaxis for prion diseases.
[1] Krasemann S et al., Mol Med 1996;2:725-34
[2] Holscher et al., J Virol. 1998;72:1153-9
169
Poster Session 3
THE-31 DEVELOPMENT OF NOVEL THERAPEUTIC AGENTS
FOR PRION DISEASE
BUMPASS DC, LELEAN J, KIRBY E, RAVEN NH AND SUTTON JM
Health Protection Agency, Porton Down, Salisbury, UK.
Variant Creutzfeldt-Jacob disease is a fatal progressive neurodegenerative disorder for which no therapy is currently
available. Although the total number of cases is currently low, the number of individuals presently incubating the
condition is unknown and predicting the future scale of vCJD is extremely difficult. Therefore it is imperative that
therapies for the treatment of vCJD are developed.
The focus of our research is the production of therapeutic agents for the treatment of vCJD, targeting the maturation
process in the conversion of PrPc to PrPsc. A phage display approach is being utilised to generate peptides that inhibit
the interaction between PrPc and PrPsc. These peptides will be tested for their ability to interfere with PrPsc
accumulation in a cell culture model of prion infection.
To date we have selected pools of phage from two different phage display libraries (New England BioLabs) which
exhibit specificity for PrPc in ELISA studies when compared with a BSA-coated plastic control. The specificity of these
phage pools for PrPc and/or PrPSc will be further investigated. The preliminary data from this study is promising and
future work will assess the potential of individual peptides to be developed into therapeutic agents.
THE-32 LATEST ADVANCES IN PRION THERAPEUTIC BY
HEPARAN SULPHATE MIMETICS
C. LARRAMENDY (1), A. BARRET (1), E. DAUDIGEOS (1), E. MATHIEU (1), C. RIFFET (2), E. PETIT (3), D. PAPY-GARCIA
(2,3), D. BARRITAULT (3), C. LASMÉZAS (1), N. SALÈS (1) AND JP. DESLYS (1).
1: CEA, DSV/DRM, Groupe d'Innovation Diagnostique et Thérapeutique sur les Infections à Prions, Fontenay aux
Roses, France. 2: Laboratoire CRRET, CNRS FRE24-12, Université Paris XII-Val de Marne, France. 3: OTR3 sarl,
Créteil, France
170
A novel generation of heparan sulfate mimetics has recently been described for the treatment of prion diseases*.
However as our lead molecule possessed a benzene residue, new series of derivatives devoid of toxic chemical groups
were studied for their anti-prion activities. The two more potent inhibitors, CR29 and CR36, hampered PrPres
accumulation in the ScGT1 cell model at doses 10 times lower or equal to the previous molecules (10ug/ml) respectively.
In the same experimental conditions, Pentosan Polysulphate (PPS) was not efficient even up to a concentration of
100ug/ml.
To investigate their potency in vivo after neuroinvasion, mice infected by intracerebral route were treated twice a week
from the beginning of clinical signs until death and the survival times were measured. No effect on survival was observed
with either the new derivatives or PPS. This suggests that despite their theoretical greater ability to cross the blood brain
barrier, only a direct injection of the molecules in the brain might create an effect as proposed with PPS for humans.
In BSE and scrapie intraperitoneally-infected mice, both CR29 and CR36 significantly reduced PrPres accumulation in
the spleen. CR36 turned out to be more efficient than PPS in the BSE model, even when the latter was used at a higher
toxic dose. Preliminary results indicate that these early treatments (before the neuroinvasion phase) also increase
survival.
Our conclusion is that these new heparan mimetics can constitute a very interesting treatment before neuroinvasion by
interfering with replication of TSE agents in peripheral organs and lymphoid tissues. In situ studies by immunohistology
are in progress to investigate structural or cellular effects of these compounds in relation to their activity.
* : K.T. Adjou et al., J Gen Virol, 2003
Poster Session 3
THE-33 INHIBITION OF PRP EXPRESSION WITH SMALL
INTERFERING RNA OLIGONUCLEOTIDES IN NEURO-2A
CELLS
MARCO PIETRELLA, BETTINA S. FRANZ, JOHANNES LˆWER, FABIO MONTRASIO
Paul-Ehrlich-Institut, Prion Research Group Pr1, Langen, Germany
Prion protein expression is necessary although not sufficient to enable prion replication. Furthermore, in vivo models
showed that both the incubation time and the progression to terminal disease are inversely related to PrPC expression
levels. As possible “therapeutical” approach for prion diseases it is therefore conceivable to aim at the reduction of the
total levels of expressed PrPC. Small interfering RNAs (siRNAs) have been successfully used in eukaryotic systems to
achieve post-transcriptional gene silencing. The aim of our study was to first assess whether siRNAs could be used to
downregulate PrPC expression levels in a cell culture system and, in a second step, to asses whether post-transcriptional
PrP silencing can interfere with prion replication and accumulation.
Neuro-2a cells were either transiently transfected with four synthetic siRNAs molecules targeted to different sequences
within the murine PrP coding region or stably transfected with a siRNA-expression vector harboring either a PrPspecific siRNA sequence or a scrambled control sequence. PrPC-expression levels were than tested by FACS, Western
Blot and immunofluorescence analysis.
Transient transfection of Neuro-2a with anti-PrP siRNAs determined a significant reduction of the total amount of PrPC
as compared to the controls. The efficacy in gene-silencing of the four tested siRNAs differed and was not enhanced by
higher concentrations or combinations of them as shown by FACS analysis. Stably transfected Neuro-2a cells showed an
evident reduction of PrPC levels for more than four months.
Having shown that siRNA technology can be successfully used to decrease PrPC expression levels, it remains to be
determined whether it is possible to interfere with prion replication and accumulation on a long-term basis by the gene
silencing approach.
THE-34 TRANSGLUTAMINASE AND PRION: NEW MECHANISM
AND POSSIBLE THERAPEUTIC APPROACH.
KUNTAEK LIM, HYUNJUNG OH, SEONG SOO A. AN
Department of Research and Development, PeopleBio Inc., Republic of Korea. seongaan@peoplebio.net
Transglutaminase (TG) and its activities are related to many neurological diseases, such as Alzheimer’s disease. The
interactions between prion protein (PrP) and TG have not investigated. Here, we reported that recombinant human 23231 PrP (rH23) was cross-linked to fibrinogen (Fbg) and Casein (Csn) by both TG (porcine pancreas) and plasma
Factor XIIIa. The cross-linked rH23 on Csn coated plate by TG was detected by using mouse anti-prion antibody,
which detected the recombinant, native and PrPres PK-PrP, with HRP-conjugated goat-anti-mouse antibody. Next, we
tested whether the HRP-conjugated Fbg could be conjugated to coated plates with both cellular and reduced/blocked
rH23 proteins by TG. Experiments were repeated with FXIIIa after addition of thrombin and Ca2+, and it also could
facilitate cross-linking of cellular and reduced/blocked rH23 to Fbg and Csn.
Afterwards, for isolating the specific TG cross-linking site in prion protein, we synthesized peptides in various lengths
from the several potential TG interacting sites in prion protein. Each peptide was conjugated with HRP for the
detection, and TG and FXIIIa was used to test whether these peptides could be cross-linked to the coated plates with
Fbg, Csn, cellular and reduced/blocked rH23 proteins. One peptide from the C-terminus had the best TG substrate
specificity than others in its ability to be cross-linked to the above rH23 proteins plates.
Interestingly, thiol-blocking agents, such as N-ethylmaleimide (NEM), blocked the PrP propagation, which suggested
that free thiol group is involved. TG has the free thiol in its active site. NEM and putrescine inhibited the cross-linking
reaction by TG or FXIIIa in our system.
Therefore, TG or FXIIIa have to potential to interact with PrP as protein X and to participate in the propagation of PrP
in the prion diseases. TG and its inhibitors could open new possible therapeutic approaches in prion disease.
171
Poster Session 3
THE-35 DISCOVERY OF A NEW DRUG CAPABLE OF
INHIBITING PRP* AND PRPRES FORMATION
KAZUO KUWATA*, NORIYUKI NISHIDA, SUSUMU SHIRABE AND SHIGERU KATAMINE
*Department of Biochemistry and Biophysics, Gifu University, School of Medicine, Gifu, *Department of Molecular
Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki.
The conformational conversion of the normal form of prion protein (PrPC) to a scrapie form (PrPSc) plays a central
role in the pathogenesis of prion diseases. Although the molecular mechanism for the process is still unknown, an
intermediate form of the protein, designated as PrP*, between the normal and the pathological may exist and play an
important role in the process. Based on the structural information of molecular dynamics in amino-acid residues of PrP,
we conducted in silico ligand screening focused on a binding site corresponding to the unstable region, and came up with
59 candidates from over 320,000 substances, which we then tested in vitro. Among the candidates we discovered a
substance, named GN8420, which strongly prevented PrPSc production in the prion-infected GTFK-1 cell-line. Thermal
unfolding experiments showed that GN8420 decreases the population of the intermediate conformer and also the fibrillike aggregates. These findings suggest that the PrP* is obligatory for the PrPC to PrPSc conversion process and an offpathway intermediate.
THE-36 SYNTHESIS AND ACTIVITY OF CONGO RED
ANALOGUES AGAINST THE TRANSMISSIBLE SPONGIFORM
ENCEPHALOPATHIES
S. SELLARAJAH, T. LEKISHVILI, C. BOWRING, A. THOMPSETT, H. RUDYK, S. VASILJEVIC, C.R. BIRKETT, D.R. BROWN,
I.H. GILBERT
Welsh School of Pharmacy, Cardiff University, UK. Department of Biology and Biochemistry, University of Bath, Bath,
UK. Institute for Animal Health, Compton, Newbury, Berkshire, UK.
Congo Red 1, a sulfonated diazo dye, shows activity in a scrapie model, the SMB cell-line which persistently expresses
PrPSc.
Congo Red, the lead molecule, however, is carcinogenic and too polar a molecule to cross the blood-brain barrier.
The goal of our research is to carry out structure activity relationship studies:
1.to improve activity
2.to improve pharmacokinetic properties such as blood-brain barrier permeability
A number of analogues have been prepared and assayed for their activity in curing persistently infected SMB cells.
Some of the data will be presented here
172
Poster Session 3
THE-37 PROTEOMIC INSIGHT INTO SCRAPIE GT1-7 CELLS
TREATED BY AN ANTI-PRION DRUG ACTIVE IN VIVO
JEAN-FRANÇOIS CHICH1, VALÉRIE LABAS2, FRANCK MOUTHON3, JEAN-PHILIPPE DESLYS3, JOELLE VINH2 AND
JEANNE GROSCLAUDE1
1 Biologie Physico-Chimique des Prions, VIM, INRA, Jouy-en-Josas, France. 2 Neurobiologie et diversité cellulaire,
CNRS UMR 7637, ESPCI, Paris, France. 3 GIDTIP, Department of Medical Research, Atomic Energy Commission,
Fontenay-aux-Roses, France.
To date a few anti-prion drugs are candidates for in vivo reversion of prion infections. The conversion of prion protein
into its pathological form is central to the infectious process. Nevertheless a number of cellular partners are likely to be
involved and their interactions constitute as many targets for drug action. An heparan sulfate mimetic was shown (data
not published) to hamper infection progression in scrapie or BSE infected mice and to cure GT1-7 cells derived from a
murine teratocarcinoma. In order to identify cellular networks involved in the infection and the cure processes we have
undertook a proteomic approach on this in vitro model, which allowed to obtain large amounts of homogeneous
biological material in a quick and reproducible way.
Non-infected, non-infected-treated, infected and infected-treated cells were compared by 2D electrophoresis. Patterns
were analysed statistically (6 fold repetitions for each treatment) to identify significant expression differences. More than
800 spots were detected and around 50 exhibited significant quantitative variations. Spot identifications were performed
by MS-MS spectrometry and analysis of metabolic pathways implicated were investigated. Interestingly, we designated
several proteins of the cytoskeleton and key molecules in the oxidative stress pathways with an expression level modified
during infection and treatment. Correlations with transcriptomic approach performed on the same cells (Patent 0205392;
April 29th, 2002) are currently analysed.
THE-38 CXCR3 - A TARGET FOR THERAPEUTIC
INTERVENTION?
C. RIEMER, J. SCHULTZ, A. SCHWARZ, M. BURWINKEL, M. BAIER
Robert-Koch-Institut, Berlin, Germany.
Among the chemokines highly and early upregulated during scrapie infections are ligands of the CXC-receptor 3
(CXCR3), which is expressed on microglia, astrocytes and neurons.
To investigate the role of CXCR3 ligands in the pathogenesis of a neurodegenerative amyloidosis CXCR3-deficient
(CXCR3-/-) mice were infected intracerebrally with the scrapie strain 139A and analysed in comparison to similarly
infected age matched wild-type controls.The CXCR3-/- mice showed a delayed disease onset and significantly prolonged
survival times. Immunohistochemistry revealed a strongly reduced activation of microglia cells in the terminal stage of
the disease in comparison to wild-type mice. In contrast, staining for GFAP showed an increased astrocytosis in the
CXCR3-/- mice. Both, PET-blot and Western- blot analysis revealed a significantly increased accumulation of PrPSc in
the CXCR3-/- mice compared to the wild-type controls. Furthermore, despite their prolonged survival times, infectivity
levels were at least-100 fold higher in brains of CXCR3-/- mice in the asymptomatic stage of the disease.
The results points toward an involvement of CXCR3 in glia activation, glia/neuronal interactions, and prion-replication
in infected brain tissue.
173
Poster Session 3
THE-39 ESSENTIAL ROLE OF COPPER BINDING SITE AND
STRESS-INDUCIBLE PROTEIN1 BINDING SITE OF PRION
PROTEIN FOR ANTI-APOPTOTIC AND ANTI-OXIDATIVE
ACTIVITY
AKIKAZU SAKUDO, DEUG-CHAN LEE, TAKUYA NISHIMURA, TOYOO NAKAMURA, YOSHITSUGU MATSUMOTO, SHIGEYOSHI
ITOHARA, KEIICHI SAEKI, AND TAKASHI ONODERA
Department of Molecular Immunology, School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan
The function of cellular prion protein (PrPC), which contains two evolutionarily conserved domains among mammals;
viz., the octapeptide repeat region (OR) (amino acid residues 51-90) and the hydrophobic region (HR) (amino acid
residues 112-145), remains unclear. To understand the biological functions of PrPC, a line of prion protein (PrP) gene
(Prnp)-deficient immortalized hippocampal neuronal cells has therefore been established. Removal of serum from the
cell culture causes apoptosis in Prnp-/- cells. Transfection of Prnp suppresses the apoptosis of Prnp-/- cells under serumfree conditions. These results suggest that PrPC has an anti-apoptotic function. In this study, to further examine
requirement of the specific regions of PrPC for anti-apoptotic function, we evaluated the anti-apoptotic activity of
various deletion mutants of PrPC in Prnp-/- cells. Removal of amino acid residues 53-94 within the OR enhances
apoptosis and decreases SOD activity. Deletion of amino acid residues 95-132 within the HR abolishes its ability to
activate SOD and to prevent apoptosis, whereas deletion of residues 124-146 of PrPC has little effect on the antiapoptotic activity and SOD activation. We further examined apoptosis of Prnp-/- cells expressing Dpl fused to various
lengths of the N-terminal region of PrPC to investigate the required regions of PrPC for anti-apoptotic function. Dpl
fused to PrP(1-124) containing OR and HR showed anti-apoptotic function, whereas Dpl fused to PrP(1-95) containing
OR did not rescue Prnp-/- cells from apoptotic cell death. As the interaction site of PrPC-binding protein stressinducible protein 1 (STI1) is mapped to amino acid residues 113-128 within the HR, both PrPC-copper binding and
PrPC-STI1 binding may be required for anti-apoptotic and anti-oxidative activity of PrPC.
THE-40 FUNCTIONAL ROLE OF PRPC IN NEURONAL
CALCIUM SIGNALLING
MARTIN FUHRMANN, GERDA W¸NSCH, HANS KRETZSCHMAR AND JOCHEN HERMS
Zentrum fur Neuropathologie und Prionforschung, Munchen, Germany.
174
PrPc is highly expressed in neurons and plays a critical role in prion diseases, however its physiological function remains
unclear. Previous neurophysiologic evaluations of PrP deficient mice showed a significant reduction of slow
afterhyperpolarizing currents (sIAHP) in hippocampal CA1 pyramidal cells suggesting a direct role for PrP in the
modulation of neuronal excitability.
Here we aim to dissect the contribution of calcium or K+-currents to this phenotype. For that purpose we perform patch
clamp and confocal calcium imaging studies in hippocampal slice preparations of PrP deficient mice compared to
littermate controls to directly measure the calcium transient and its contribution to the sIAHP.
In Prnp0/0 mice we observed a significant reduction of the maximal amplitude of the calcium transient that directly
follows the depolarisation of CA1 neurons. However no alterations were observed in the time course of the calcium
decay between Prnp0/0 and littermate controls.
The calcium transient was found to follow a single exponential decay of 3.4 seconds (+/- 0.3 sec; n=29) in Prnp0/0
compared to 3.2 (+/- 0.25 sec; n=31) in wildtype. Moreover simultaneous patch-clamp measurements of the calcium
dependent K+-currents did not reveal significant alterations in Prnp0/0 CA1 neurons. The mean amplitude of both the
IAHP, 100ms after the end of the stimulus as well as the sIAHP with a maximum at around 700 ms after the end of the
stimulus was very similar in Prnp0/0 and wildtype neurons (IAHP: WT=101.9 pA +/- 12.9; Prnp0/0=103.4 pA +/- 13.3;
sIAHP: WT=19.3 pA +/- 2.7; Prnp0/0=17.8 pA +/- 2.6).
These results indicate that loss of PrPc affects the calcium influx through the plasma membrane rather than mechanisms
that modulate the time course of the calcium decay, like calcium release from intracellular stores, intracellular calcium
buffers or calcium extrusion mechanisms.
Poster Session 3
THE-41 ABERRANT METABOLISM OF GAGS IS PART OF
PRION DISEASE PATHOGENESIS
TEHILA MAYER*, MARSHA ZEIGLER** AND RUTH GABIZON*
Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah University Hospital,
Hdassah, Jerusalem, Israel.
Many lines of evidence suggest a connection between prion disease pathogenesis and GAGs. PrPC has been shown
repeatedly to bind specifically to heparin. GAGs have been shown to be a component of plaques in several prion
diseases. GAGs related molecules such as pentosan sulfate decrease the accumulation of PrPSc in cell culture and
heparin like molecules have been tested successfully in prion in-vivo systems. In ScN2a cells, enzymes and chemicals
that reduce GAGs content also inhibit the accumulation of PrPSc. In this work, we investigated further the role of
GAGs in prion disease pathogenesis. We show here that a complex of GAGs and proteins is excreted in prion disease
urine, as opposed to normal controls. In addition, the total concentration of GAGs is significantly larger in scrapie
infected brains, as compared to normal brains. Interestingly, the activity of several lysosomal GAG degrading enzymes,
such as hexosaminidase and alpha-L-iduronidase, were significantly elevated in scrapie infected brains as well as in
several other organs. We suggest that GAG accumulation, which may result from the aberrant binding of PrPSc to
these molecules, plays an important role in prion disease pathogenesis. Indeed, genetic diseases in which GAGs are
accumulated as a result of deficient GAG degrading enzymes are characterized by brain degeneration and excretion of
GAGs in urine. Increasing the activity of GAGs degrading enzymes may be a self protecting mechanism against prion
infection.
THE-42 NEURONS AND ASTROCYTES RESPOND TO PRION
INFECTION BY INDUCING MICROGLIA RECRUITMENT
MATHIEU MARELLA AND JOELLE CHABRY
Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 6097, Centre National de la Recherche
Scientifique 660, Valbonne, France
The accumulation and activation of microglial cells at sites of amyloid prion deposits or plaques have been documented
extensively. Here, we investigate the in vivo recruitment of microglial cells soon after intraocular injection of scrapieinfected cell homogenate (hgtsc+) using immunohistochemistry on retinal sections. A population of CD11b/CD45positive microglia was specifically detected within the ganglion and internal plexiform retinal cell layers by 2 d after
intravitreal injection of hgtsc+. Whereas no chemotactism properties were ascribed to hgtsc+ alone, a massive migration
of microglial cells was observed by incubating primary cultured neurons and astrocytes with hgtsc+ in a time- and
concentration-dependent manner. hgtsc+ triggered the recruitment of microglial cells by interacting with both neurons
and astrocytes by upregulation of the expression levels of a broad spectrum of neuronal and glial chemokines. We show
that, in vitro and in vivo, the microglia migration is at least partly under the control of chemokine receptor-5 (CCR-5)
activation, because highly specific CCR-5 antagonist TAK-779 significantly reduced the migration rate of microglia.
Activated microglia recruited in the vicinity of prion may, in turn, cause neuronal cell damage by inducing apoptosis.
These findings provide insight into the understanding of the cell-cell communication that takes place during the
development of prion diseases.
175
Poster Session 3
THE-43 IN VIVO NEUROPROTECTION BY THE PRION
PROTEIN
FRIGG, R., WENZEL, A., WARIWODA, H., OESCH, B., RAEBER, A.J., REMÉ, C. AND C. GRIMM
Prionics AG, Wagistrasse 27A, 8952 Schlieren-Zurich, Switzerland. Laboratory of Retinal Cell Biology, Department of
Ophthalmology, University Hospital Zurich, Zurich, Switzerland.
Neurodegeneration characterizes disorders such as Alzheimer’s disease, age-related macular degeneration, or ischemiainduced nervous tissue damage. A common feature of neurodegenerative disorders is given by the apoptotic nature of
acute or progressive loss of neurons. Different animal models serve to elucidate underlying pathogenetic mechanisms and
to develop therapeutic strategies. Light-induced retinal degeneration represents an easily accessible in vivo model system
to study acute neuronal cell demise. Dissection of this apoptotic pathway showed involvement of activator protein 1
complex (AP-1) and caspase-1 activation. Since the pathological isoform of the prion protein (PrP) is associated with
neurodegeneration, we analyzed the influence of different levels of PrP expression in the light damage model. Here we
show that mice lacking PrP expression are more susceptible to light damage and that overexpression of PrP strongly
protects against light toxicity. Light-induced stress leads to irreversible apoptotic photoreceptor damage involving upregulation of AP-1 and caspase-1. PrP-overexpressing mice did not show caspase-1 activation, whereas AP-1 induction
was observed in all types of mice. Analysis of phosphorylation of different factors involved in signal transduction and
transcription revealed different phosphorylation patterns of Erk1/2, Jak2, and STAT-1 in light damage-resistant PrPoverexpressing mice. These data suggest that PrP acts as a neuroprotective agent downstream of AP-1 by interfering
with tyrosine phosphorylation. The function of the normal PrP, a highly conserved protein expressed predominantly in
the nervous system, has remained enigmatic as of yet. Our results establish PrP as a neuroprotective/anti-apoptotic
factor in vivo leading to new insights in the molecular mechanisms involved in neurodegeneration as well as to novel
approaches towards neuroprotective therapeutics.
THE-44 LOOKING FOR MARKERS OF BSE INFECTION: AN
INVESTIGATION OF THE IMMUNE SYSTEM FROM BSE
INFECTED MICE AND CATTLE
EMMA B. BORTHWICK, AURIOL C. WIILIAMS, JOHN L. WILLIAMS
Department of Genomics and Bioinformatics, Roslin Institute, Roslin
176
The diagnosis of transmissible spongiform encephalopathy (TSE) still relies on the detection of the disease-specific
isoform of the host prion protein, termed PRPsc, which accumulates in the tissues, particularly in the central nervous
system (CNS) of infected animals at late stages of incubation of the disease. Several studies have attempted to
characterise differential gene expression in CNS tissues during disease pathogenesis (Duguid, 1989; Dandoy-Dron,
1998). To date there are no proven methods of diagnosis using readily accessible tissues such as blood or lymphoidal
tissues. Recent work performed at the Roslin Institute identified decreased levels of erythroid differentiation-related
factor (EDRF) in the spleen of TSE infected mice, which is reflected in erythroid cells in the blood. This observation
gives rise to the possibility of using differential gene expression in peripheral tissues as an early diagnostic test for TSE
infection (Meile, 2001).
Following oral transmission, infectivity passes from the gastrointestinal system to the CNS via the lymporeticular system
(LRS). In mice PRPsc has been detected in the Peyers Patch (distal ileum) and the spleen within 3 months of BSE
infection (Maignien, 1999). However in cattle, PRPsc is only detected in the Peyers Patch 6 months post infection
(Terry, 2003) and cannot be detected in the spleen. This lack of BSE infective agent implies a host-dependent and
species-specific infection in the LRS.
Gene expression, during early infection is being investigated in both the spleen and white blood cells to detect genes that
are differentially expressed between normal and TSE infected individuals. Parallel projects in mouse (BXD12ty) and
cattle (Holstein) are investigating gene expression during a time course of BSE infection using Microarray technology to
simultaneously examine the expression of a large number of genes. The results of the study may lead to early markers
for BSE infection.
Poster Session 3
THE-45 VIMENTIN AND HSP25 EXPRESSION IN
ASTROCYTES DURING RODENT EXPERIMENTAL SCRAPIE.
NICOLE SALÈS 1, KARIM T. ADJOU 2, DIMITAR KADIYSKY 1, FABIEN AUBRY 1, CHRISTOPHE CRÉMINON 3, THOMAS
MAIGNIEN 1 , CORINNE I. LASMÉZAS 1 , AND JEAN-PHILIPPE DESLYS 1
1. CEA, DRM, Fontenay-aux-Roses, France. 2. Ecole Nationale Vétérinaire Maison-Alfort, service de Pathologie du
Bétail, France. 3. CEA Saclay, SPI, Gif-sur-Yvette, France.
In prion diseases, like in other neurodegenerative diseases, the clinical signs and the fatal issue are related to a massive
neuronal death. Another hallmark of the disease, an extensive reaction of the glial cells, can be easily evidenced in
astrocytes by an increased expression of the mature intermediary filament GFAP. However, contradictory results have
been reported regarding the role of glial cells as support for PrP conversion and as rescue for neurons. In an attempt to
clarify the role of the different glial cell populations during prion diseases, we have looked for proteins which are
expressed in other deleterious conditions, focusing on cytoskeletal and stress proteins.
In three different models of rodent scrapie, we show that vimentin, a juvenile cytoskeleton protein which is re-expressed
after various insults to the brain, is strongly expressed in non-dividing astrocytes. This expression is spatially and
temporally correlated with the accumulation of PrPres and with the development of brain lesions, contrasting with the
progressive and diffuse overexpression of GFAP. Concerning the two major heat shock proteins which are expressed in
the brain in stressful conditions (mainly HSP 70 in neurons and HP25 in astrocytes) we found that, contrasting with the
apparent lack of HSP70 immunoreactivity and with non-specific effects on other glial cells, the sub-population of
vimentin-possitive astrocytes specifically express HSP25 during scrapie neuroinvasion. This induced expression may be
related to the role of chaperone of small HSPs which could be directed towards astrocytic intermediary filaments and/or
towards adjacent aggregates of misfolded PrPres. Alternatively, anti-apoptotic functions recently described for HSP25
might play a role in tentative neuronal rescue. Astrocytes should then be considered as key players potentially able to
revert or at least to stop neurodegenerative lesions provided that PrPres accumulation could be pharmacologically
inhibited.
THE-46 PRPSC BINDING TO CELLS REQUIRES ENDOGENOUS
GAG EXPRESSION
NUHA HIJAZI AND RUTH GABIZON
Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Kiryat Hadassah, Jerusalem, Israel
Many lines of evidence suggest an interaction between GAGs and the PrP proteins, as well as a possible role for GAGs
in prion disease pathogenesis. In this work, we set to determine whether the PrP-GAG interaction affects the binding
and internalization of PrPSc to normal cells. Such binding may be the first step in prion disease pathogenesis. To this
effect, we incubated PK digested scrapie brain homogenates with several lines of CHO cells, in the presence or absence
of heparin. Our results show that the binding of PrPSc to control CHO cells, which express PrPC poorly, was very
similar to the binding of PrPSc to PrP overexpressing CHO cells. Interestingly , the binding of PrPSc to both cell lines
was mostly inhibited by the presence of heparin before or during the incubation of the homogenate with the cells. To
the contrary, PrPSc binding to CHO cells in which either total GAGs or only heparan sulfate expression was abolished
was very poor, and was nor reduced by the presence of heparin. To assess whether the PrPSc can bind to cells in the
absence of PrPC, we incubated fibroblasts lines derived from PrP0/0, control FVB and MHU2M -FVB mice with
scrapie brain homogenates in the presence or absence of heparin. We show that although the PrP0/0 cells do bind
PrPSc , such binding could not be reduced by heparin.
In summary, the heparin dependent binding of PrPSc to cells requires both PrPC and GAG expression in the target
cells.
177
Poster Session 3
THE-47 PRION INTERCELLULAR TRANSFER
PAQUET S. 1, FÉVRIER B. 2, ARCHER F. 1, RAPOSO G. 2, LAUDE H. 1, VILETTE D. 1
1 Unité de Virologie Immunologie Moléculaires, INRA, Jouy-en-Josas, France. 2 Unité Mixte de Recherche 144 Centre
National de la Recherche Scientifique Institut Curie, Paris, France.
Although a critical aspect of prion propagation in vivo is the ability of infected cells to infected new target cells, the
mechanisms underlying such processes are poorly documented. Rov and Mov are epithelial and neuroglial cells,
respectively, engineered to express ovine PrPc, and permissive to sheep prion multiplication (Vilette et al., 2001; Archer
et al., 2004). Following infection of these lines, a progressive increase in abnormal PrP levels is observed, suggesting that
new cells may become infected with time and successive subpassages.
To study the mechanisms underlying prion dissemination, we developed a co-culture system using already infected cells
and recipient uninfected cells. We studied the accumulation and the distribution of abnormal PrP in these co-cultures by
biochemical techniques and by immunofluorescence microscopy. We found that the efficiency of the spread of sheep
prions differs greatly among Rov and Mov cultures. We showed that transfer of infectivity can be obtained through
direct cell contacts, in agreement with earlier findings obtained in a mouse cell culture model (Kanu et al., 2002).
However, our results also suggested that spread of prions can occur through cellular secretions. In that respect, we found
that infected cultures released prions in association with exosome-like vesicles. The respective contribution of these 2
mechanisms is under study. The standardization of such co-cultures may offer an attractive tool for the screening and the
identification of drugs interfering specifically with the cell-to-cell spreading of prions.
THE-48 CU(II) AFFINITY OF MEMBRANE-BOUND PRPC:
BRAIN REGION AND PH DEPENDENCIES
GEMA ELVIRA(1), REINERIO GONZALEZ-IGLESIAS(1), ISIDRO FERRER (2), JORDI YAG¸E (3), MARIA GASSET (1)
1. IQFR-CSIC, Madrid, Spain. 2. UB-Banco de Tejidos, Barcelona, Spain. 3. H. Clinic, Barcelona, Spain
Cu(II) affinity of PrPC from CJD-control human brain cortex membrane lysates has been probed by Cu(II)IMAC.Imidazol elution profiles probed in parallel with 3F4 and 6H4 showed the absence of non-interacting and high
affinity PrP species, independently of 129 polymorphism. When compared with hamster brain membranes,
Cu(II)affinity appeared as a function of brain region. Moreover, pH-dependency of Cu(II)-IMAC elution profiles
suggested the engangement of a glycosaminoglycan-containg partner as a modulator of the interaction strength.
178
Poster Session 3
THE-49 CELLULAR PRION PROTEIN (PRPC) LOCALIZES AT
THE INTERCELLULAR JUNCTIONS OF BRAIN ENDOTHELIAL
CELLS
PEDRO VIEGAS, NATHALIE CHAVEROT, NICOLAS PERRIÈRE, FRANÇOISE ROUX, PIERRE-OLIVIER COURAUD, SYLVIE
CAZAUBON
Département de Biologie Cellulaire, Institut Cochin, INSERM, CNRS UMR8104, IFR 116, Paris, France and
INSERM U26, Hôpital Fernand Widal, Paris, France
The normal isoform of the human prion protein (PrPC) is a glycosylphosphatidylinositol-anchored protein shown
necessary for the propagation of prion infection. Although it has been implicated in copper metabolism and protection
from oxidative stress and shown to interact with several proteins, its physiological function remains elusive. The prospect
of oral infectivity and the expression of PrPC in several blood cell types lead us to investigate its function at the BloodBrain Barrier (BBB), a possible point of entry of the prion infection from blood to the brain.
By confocal immunofluorescence microscopy and immunoblot analyses we show that PrPC is expressed in freshly
isolated rat brain capillaries, primary cultures of rat and mouse cerebral endothelial cells, and several brain endothelial
cell lines. In addition, we observe that PrPC is highly enriched at cell-cell contacts in capillaries as well as in confluent
endothelial monolayers, suggesting a role in cellular adhesion. Immunofluorescence microscopy of a coculture of wild
type and PrP-/- brain endothelial cells suggests that PrPC establishes homophilic interactions between molecules on
adjacent cells as described for cell adhesion molecules (CAMs). Interestingly PrPC seems to co-localize with PECAM-1,
the major CAM expressed in endothelial cells: both molecules share the same cell confluence-dependent behavior, being
re-localized from a diffuse distribution in isolated cells to the cell junctions in confluent cells, and are co-enriched in
caveolin-rich raft microdomains. On-going experiments aim at assessing whether PrPC and PECAM-1 directly interact
at the endothelial intercellular junctions and whether PrPC might interfere with cell adhesion and BBB function.
THE-50 PRP(C) CO-IMMUNOPRECIPITATES WITH NICOTINIC
ACETYLCHOLINE RECEPTOR BETA 4 SUBUNIT IN NORMAL
HUMAN BRAIN
SPYROS PETRAKIS 1, IOANNIS PASPALTSIS1, JON LINDSTROM2 AND THEODOROS SKLAVIADIS 1
1 Prion Disease Research Group, Laboratory of Pharmacology, School of Pharmacy, Aristotle University of
Thessaloniki, Thessaloniki, Greece. 2 Department of Neuroscience, University of Pennsylvania, Philadelphia, USA
The identification of proteins that interact with PrP(C) has been the aim of many studies owing to the importance of
understanding both the physiological role of PrP(C) and the mechanism of its conversion to the pathological isoform,
PrP(Sc). To date, prion protein has been shown to interact with structural protein complexes of the cell membrane,
components of the cytoskeleton, and intracellular proteins involved in signal transduction. In this study we investigated
an association of PrP(C) with the beta 4 subunit of nicotinic aceytlcholine receptors in normal human brain. Nicotinic
aceytlcholine receptors (nAChRs) are members of the ligand-gated ion channel superfamily that are formed by the
pentameric association of alpha and beta subunits. They are present in both the central and peripheral nervous systems.
In performing phage display experiments to isolate peptides with high affinity to recombinant human prion protein we
identified a peptide with very close homology to the murine and human beta 4 subunit of nAChR. Immunoprecipitation
experiments with specific antibodies against prion protein and alpha 3,beta 4 subunits showed that PrPC co-precipitates
with both the nAChR subunits from normal brain homogenates. These data are in agreement with previous publications
reporting co-localization of PrP and nAChR in the neuromuscular junction and they suggest that PrP may play a role in
signal transduction in postsynaptic membranes.
179
Poster Session 3
THE-51 INHIBITION OF PRION REPLICATION BY LENTIVIRAL
GENE TRANSFER OF DOMINANT NEGATIVE PRPS MUTANTS
CAROLE CROZET 1, YEA-LIH LIN 2, CLÉMENT METTLING 2, PIERRE CORBEAU 2, SYLVAIN LEHMANN 1,3, AND
VÉRONIQUE PERRIER 1
1Laboratoire de Biologie des Encéphalopathies Spongiformes - Institut de Génétique Humaine, CNRS Montpellier,
France. 2Laboratoire Lentivirus et Transfert de Gènes - Institut de Génétique Humaine, CNRS Montpellier, France.
3Laboratoire de Biochimie. Hopital St Eloi, Montpellier, France
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders, which include Creutzfeldt-Jakob
disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle. Currently there is no
treatment to cure Transmissible Spongiform Encephalopathies. Since lentivirus mediated gene transfer can transduce both
dividing and non dividing cells as neurons in a stable and long term fashion, they provide a potentially useful tool for
therapeutic strategies. By taking advantage of the prion ‘resistant’ polymorphisms Q171R and E219K that naturally exist
in sheep and humans, respectively, we evaluated a lentiviral gene transfer therapeutic approach. Here we show that VSVG (Vesicular Stomatitis Virus) pseudotyped FIV (Feline Immunodeficiency Virus) derived vectors carrying the mouse
Prnp gene in which these mutations have been inserted, are able to inhibit prion replication in chronically prion infected
cells. Therefore, this result presents an option for the development of gene or cell therapy approaches to prion disease.
THE-52 PRE-CLINICAL IMPROVEMENT OF COMBINED
IMMUNOTHERAPY AND CHEMOTHERAPY FOR THE NEW
VARIANT CREUTZFELDT-JAKOB DISEASE
SUE GODSAVE 1, BEA KRENN 1, CHIARA ZURZOLO 2, VINCENZA CAMPANA 2, ALBERT TARABOULOS 3, KRISTER
KRISTENSSON 4, DAVID PERETZ 5, STAN PRUSINER 5 AND PETER PETERS1
1.Netherlands Cancer Inst. (NKI-AVL), Amsterdam, The Netherlands 2.Dip. Biol e Patol Cell e Mol, Università
"Federico II" di Napoli, Italy/Pasteur Institute, Paris, France. 3.Dept. Mol. Biol.,Hebrew University-Hadassah Medical
School, Jerusalem, Israel. 4.Dept.of Neuroscience, Karolinska Inst. Stockholm, Sweden. 5. Inst. for Neurodegenerative
Diseases, Univ. of California, San Francisco,USA/InPro
180
There is currently no effective therapy for prion diseases. Recent reports have shown that anti-PrPc antibodies reduce
PrPSc in prion-infected cells. The effects of passive immunotherapy using Fab-fragments (D18 and D13) of prion
protein antibodies on prion conversion in infected mice are now analyzed within our EU consortium. Parameters as
clinical signs, incubation time, neuropathology and antibody penetration are studied. Routinely a 28 day perfusion of 2
mg D18 (recognizing residues 133 to 157 of PrPc) or D13 (recognizing the 95 to 105 region of PrPc) using an Alzet
pump is performed.
Using monovalent Fab fragments of D13 MAbs we observed induction of neuronal apoptosis in vivo at 7-10 days postinjection. Solforosi et al.(1) recently observed apoptosis with D13 IgG already by 48 h, but found no adverse effect of
Fab-D13. D13 is thus toxic with high mortality and demonstrates the high risk of using antibodies against particular
PrPc epitopes for immunotherapy.
Recently, it was shown that Fab-D18 antibodies inhibit prion propagation and clear cell cultures of prion infectivity. In
vivo treatment with Fab-D18 between days 40-68 (post inoculation) had a small beneficial effect on survival (10-12
days), while treatment instituted between day 80-108 and 100-125 had no effect. A problem with this approach may be
penetration throughout the brain parenchyma even for Fabs.
Effective chimeric anti-PrPc antibodies potentially able to cross the blood-brain barrier have been constructed. These
antibodies react both with PrPc and with receptors involved in trancytosis of the blood-brain endothelium, hoping for
increased brain penetration from the circulation. The anti-prion efficacy of the bispecific antibodies is currently under
examination. Immunotherapy combined with chemotherapy will be examined in scrapie-infected animals. Drugs
including chlorpromazine-quinacrine are promising.
(Supported by EU Grant QLK2-CT-2002-81628)
1-Solforosi et al, Science 2004;303,1514-6.
Poster Session 3
THE-53 INVESTIGATION OF FLUPIRTINE AND DAPSONE ON
COGNITIVE FUNCTION IN PATIENTS WITH CJD
MARKUS OTTO MD (1), LUKAS CEPEK MD (1), BRIT- MOLLENHAUER (1), PETER RATZKA MD (1), EVA IRLE PHD (2),
GABRIELA PERGANDE PHD (3), BARBARA ELLERS-LENZ MA (4), OTTO WINDL PHD (5), HANS A. KRETZSCHMAR MD
(6), SIGRID POSER MD (1), HILMAR PRANGE MD (1)
(1) Department of Neurology, University of Goettingen, Germany (2) Department of Psychiatry, University Goettingen,
Germany (3) ASTA Medica AG since 1.11.01 Baxter Oncology GmbH, Frankfurt/Main, Germany (4) VIATRIS
GmbH & Co. KG, Frankfurt/Main, since 1.3.03 Omega Mediation, Offenbach. Germany (5) Veterinary Laboratories
Agency - Weybridge, United Kindom (6) Institute of Neuropathology, University Munich, Germany
Background: In cell culture experiments, flupirtine maleate (FLU), a triaminopyridine compound, was able to protect
neuronal cells from apoptotic cell death induced by prion protein fragments. In a double-blind study flupirtine was able to
delay progression of cognitive changes for a short time. In one study dapsone was shown to delay onset of symptoms of
infected animals.
Methods: Twenty-eight patients with CJD were randomized to an oral treatment with either FLU (n=13) or matching
placebo (PLA; n=15). For inclusion and continuing the study the patients had to achieve at least 50% in two of the sub-scales
of the dementia tests employed. A battery of standardized questionnaires was employed to monitor the progression of the
disease. The main outcome variable was the cognitive part of the Alzheimer’s disease assessment scale (ADAScog); the
difference between baseline (BL) and the best score under treatment was defined as the primary efficacy variable for
hypothesis testing. These results were compared to eight patients who were treated with a combination of FLU and dapsone
according to the same protocol.
Results and Discussion: CJD types were homogeneously distributed among the treatment groups. Patients treated with FLU
showed significantly less deterioration in the dementia tests than patients treated with PLA. The mean change in ADAScog
(BL to best) was +8.4 (±15.3) in the FLU group and +20.6 (±15.1) in the PLA group (p = 0.02, 1-sided t-test). Patients who
were treated with a combination of dapsone and FLU did also worsen. First results will be presented and discussed.
THE-54 DOWN REGULATION OF CELL SURFACE PRION
PROTEIN: AN INTRABODY-MEDIATED ANTI-PRION STRATEGY.
ALESSIO CARDINALE (1), ILARIA FILESI (1), VITO VETRUGNO (2), MAN-SUN SY (3) AND SILVIA BIOCCA (1)
1. University of Tor Vergata, Department of Neuroscience,Rome,Italy. 2. Istituto Superiore di Sanità, Department of
Cell Biology and Neurosciences, Rome, Italy. 3. Institute of Pathology, Case Western Reserve University, School of
Medicine,Cleveland, Ohio, USA
Prion diseases are fatal, transmissible neurodegenerative disorders characterized by the conversion of the cellular prion
protein (PrPc) into the abnormal scrapie isoform (PrPsc). The pathogenic mechanisms leading to the in vivo conversion
of the cellular isoform into PrPsc is not yet understood. The notion that PrP-PrPsc conversion could take place in a
specific membranous compartment and that altered ER-based quality control mechanisms could be involved in prion
pathogenesis, has potential for a protecting approach whereby the normal traffic of the PrPc protein is perturbed.
In order to study the biological consequences of knocking down the cell surface PrP by its forced retention into the
endoplasmic reticulum (ER), we have developed an intrabody-based strategy. Here we describe the generation of two
anti-prion single chain antibody fragments (scFv), tagged with the ER retention signal KDEL, directed against different
epitopes of PrP and their expression in different mammalian cell lines. The stable expression of the ER-retained
intrabodies in PC12 cells induces the following permanent alterations: a) a marked surface depletion of PrP and its
retention in the ER and b) the impairment of PrP maturation with the appearance of a new aberrant endoH-sensitive
glycosylated form. The cellular effects of prion accumulation in the ER and the efficacy of this approach on the
inhibition of scrapie propagation will be discussed.
Intracellular antibody-mediated diverting of PrP from its localisation prospects a new powerful tool to study the
biogenesis, the intracellular traffic, the site of conversion and the mechanisms of pathology of the prion protein, a
prerequisite for developing effective anti-prion strategies useful for therapy.
181
Poster Session 3
THE-55 THE IMMUNE RESPONSE TO PRION PROTEIN
PEPTIDE, EXPRESSED AS A FUSION PROTEIN
TANJA VRANAC1, IVA HAFNER2, MARA POPOVI3, ROMAN JERALA2 AND VLADKA CURIN SERBEC1
1Blood Transfusion Centre of Slovenia, Slajmerjeva 6, 1000 Ljubljana, Slovenia. 2National Institute of Chemistry,
Hajdrihova 19, 1000 Ljubljana, Slovenia. 3University of Ljubljana, School of Medicine, Institute of Pathology
The induction of the immune response by immunisation with prion protein (PrP) poses a significant problem due to
similarity among mammal PrPs. To overcome tolerance, different strategies have been used. For obvious reasons, the use
of Prnp0/0 mouse model as well as immunisation with PrPSc rich brain extracts, the methods used for production of
diagnostic anti-PrP monoclonal antibodies (mAbs) in the past, are unsuitable for vaccine development. Immunisation
with PrP peptides, on the other hand, seems a promising approach. Small peptides are poorly immunogenic and easily
degraded and are usually linked to a carrier protein. Large carriers, like BSA or KLH, elicit high immune responses, but
the majority of Abs is directed against carriers, which is an unwanted feature for a vaccine.
We therefore used a small, sparingly soluble protein ketosteroid isomerase (KSI) as a carrier for C-terminal peptide
fragment P1 from human PrP. Covalent conjugate of P1 with carrier KLH has already been proven to be highly
immunogenic and suitable for preparation of mAbs in our previous studies. Fusion protein was prepared by ligation of
the P1 coding sequence into pET31b(+) expression vector downstream of KSI gene. A P1-KSI fusion protein was
overproduced in E. coli. BALB/c mice were immunised following standard immunisation protocol. The polyclonal sera
were tested by ELISA. The results showed, that a larger part of the response had been against P1 and not against KSI.
Immunohistochemistry on the brain tissue sample of the patient with sporadic Creutzfeldt-Jakob disease showed strong
staining of amiloid plaques. We therefore concluded that the ability of P1 to induce PrPSc specific response, observed
after immunisation with P1-KLH, was retained also when P1 was fused to a completely different carrier. Last, but not
least, the described approach allows production of high quantities of the immunogen at low costs.
THE-56 PROTEOMIC ANALYSIS OF NEURODEGENERATION IN
A TRANSGENIC MOUSE MODEL OF AN INHERITED PRION
DISEASE
EMILIANO BIASINI1,2, TANIA MASSIGNAN1,2, LUANA FIORITI1,2, MARIO SALMONA2, GIANLUIGI FORLONI2, VALENTINA
BONETTO1,2 AND ROBERTO CHIESA1,2
182
1Dulbecco Telethon Institute (DTI) and 2Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy.
Transgenic (Tg) mice that express a mutant prion protein containing nine octapeptide repeats (PG14), whose human
homologue is associated with an inherited prion disease, accumulate in their brains an insoluble and weakly proteaseresistant form of the mutant protein that resembles PrPSc. As this form accumulates in the brain, Tg(PG14) mice
develop a progressive neurological syndrome characterized clinically by ataxia, and neuropathologically by cerebellar
atrophy with massive apoptotic degeneration of granule neurons (Chiesa et al., PNAS 97:5574-5579, 2000). To
investigate the molecular events responsible for the cerebellar pathology observed in this model, we have carried out a
differential proteomic analysis of nervous tissue from Tg(PG14) mice at different stages of their neurological illness.
Detergent extracts of cerebella from Tg(PG14) mice at the presymptomatic, initial and terminal stages of the disease
were subjected to 2-dimensional (2D) gel electrophoresis. The 2D protein maps were compared with those obtained
from age-matched Tg mice expressing wild-type PrP by quantitative densitometry using the Progenesis software
(Nonlinear Dynamics). The differentially represented proteins were identified by peptide mass fingerprinting using
MALDI mass spectrometry. We identified 25 proteins, whose levels were significantly modified in at least one set of
Tg(PG14) mice compared with age-matched controls. These proteins belong to three major functional groups: structural
proteins, proteins involved in oxidative stress, and in Ca2+ metabolism. The expression level of some of these proteins
was characterized by Northern and Western blot analyses of brain tissue. We are currently investigating the role of the
identified proteins in the neurological dysfunction of the Tg(PG14) mice.
Poster Session 3
THE-57 INTERMEDIATE STATES AND IMPLICATIONS FOR THE
SPECIES BARRIER OF THE HUMAN PRION PROTEIN REVEALED
BY HIGH PRESSURE NMR
WERNER KREMER#, NORMAN KACHEL#, RALPH ZAHN§, HANS ROBERT KALBITZER#
#Institute for Biophysics and Physical Biochemistry; Universität Regensburg, Regensburg, Germany. §Institute of
Molecular Biology and Biophysics, ETH Honggerberg, Zurich, Switzerland.
Prions as causative agents of transmissible spongiform encephalopathies in humans and animals are principally
composed of the infectious isomer, PrPSc, of the cellular prion protein, PrPc. The conversion and thus the propensity of
PrPc to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool
to study the physico-chemical properties of proteins, especially folding, as well as the dynamics and structure of folding
intermediates[1;2]. Here we combine pressure with multidimensional NMR spectroscopy to characterise the physicochemical properties of the human prion protein PrPc. Spectra at variable pressure and temperature show that the
pressure stabilized intermediate is very similar to the acid-induced A-state of huPrPc which was shown to form fibrils by
electron microscopy[3]. The application of pressure is reversible and we see virtually no difference between huPrPc(121230) and huPrPc(23-230). The most pressure-sensitive region is the loop between strand 1 and helix 1, indicating that
this region is the first entry point for the infectious conformer to convert the cellular protein. Importantly residues I139,
H140, and F141 exhibit a cluster of very low dG values and seem to be the most unstable part of the protein.
Interestingly I139 was shown to be the residue responsible for the mouse/hamster species barrier. The highest dG values
are observed in helix III very close to the disulfide bridge in accordance with known hydrogen protection patterns. The
results indicate that the species barrier as well as the first entry-point of the scrapie isomer is determined in addition to
surface charge differences through contributions of residue-biased and thus species-specific subpopulations of
conformers as seen in prion strains with differing infectivity.
[1] Kuwata et al.(2002) Biochemistry, 41, 12277-12283.
[2] Inoue et al.(2000) Nat. Struct. Biol., 7, 547-550.
[3] Jackson et al. (1999) Science, 283, 1935-1937.
THE-58 EFFECT OF PLASMINOGEN DISRUPTION IN THE
DEVELOPMENT OF THE SCRAPIE IN MICE
MARIO SALMONA*, LAURA COLOMBO*, ADA DE LUIGI*, MARIA BENEDETTA DONATIS§, RAFFAELLA CAPOBIANCO+,
MICHELA MANGERI+, ELENA QUAGLIO*, ROBERTO CHIESA*, FABRIZIO TAGLIANI+ AND GIANLUIGI FORLONI*
*Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy, §Consorzio "Mario Negri-Sud", Santa Maria
Imbaro, Chieti, Italy , +Istituto Neurologico "Carlo Besta", Milano, Italy.
Plasminogen binds selectively to the pathogenic isoform of prion protein (PrPSc), but not to the cellular isoform (PrPC).
To clarify what part plasminogen plays in scrapie infection we inoculated plasminogen-deficient (plg0/0), heterozygous
plasminogen-deficient (plg+/-) and wild-type mice (plg+/+) with scrapie by either intracerebral or intraperitoneal
injection and monitored the onset of neurological symptoms, survival and the brain accumulation of PrPSc. After
intracerebral injection these parameters were all virtually the same in the three groups. After intraperitoneal injection
Plg0/0 survived for a mean (± s.e.m.) of 224±8 days compared with respectively 220±4 and 215±5 days for Plg+/- and
Plg+/+ mice. A significant difference was attained (p<0.05) between the survival of Plg0/0 and Plg+/+ mice. To determine
whether the absence of plasminogen affected the cerebral accumulation of PrPSc the amount of protease K-resistant PrP
in the brains of the mice was evaluated by Western blot analysis and immunohistochemistry when the first signs of
scrapie appeared in plg+/+ animals (~175 days) or when they reached the terminal phase of the illness. At the onset of
the disease PrPSc accumulation was remarkably higher in plg+/+ and plg+/- than in plg0/0 mice, and this difference
was paralleled by a difference in the severity of spongiform changes and astrogliosis in the cerebral cortex and
subcortical gray structures. Once the disease progressed and mice began to die, the differences in PrPSc accumulation,
spongiform changes and astrogliosis were no longer apparent. In conclusion, our data indicate that plasminogen plays a
role in the early propagation and spread of PrPSc.
183
Poster Session 3
THE-59 PRP 82-146 OLIGOMERIZATION DETERMINED BY
PHOTO-INDUCED CROSS-LINKING
LAURA COLOMBO*, CLAUDIA MANZONI*, MARCO GOBBI*, MICHELA MORBIN+, GIANLUIGI FORLONI*, FABRIZIO
TAGLIAVINI+ AND MARIO SALMONA*
*Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy; +Istituto Nazionale Neurologico "Carlo Besta",
Milano, Italy
The major component of Gerstmann-Sträussler-Scheinker disease (GSS) amyloid is a prion protein (PrP)) fragment
spanning residues ~82-146. Electron microscopy analysis of synthetic PrP 82-146 solutions showed that in the first 24 h
peptide assemblies comprised two fibril populations having a diameter of 5.5 and 9.5 nm, respectively. The former
prevailed after 1 h incubation while the latter became the predominant structure after 1 day and was the only form at
later stages. Moreover, examination of pellets obtained from peptide suspensions after 7-day incubation indicated that
the morphology of fibrils was very similar to human GSS amyloid plaques cores. This suggested that PrP 82-146
aggregation is a stepwise process starting with formation of protofilaments which combine to produce the amyloid fibril.
We have applied the photoinduced cross-linking technique of unmodified peptide to study PrP 82-146 oligomerization
with the aim to unveil the nature and the distribution of oligomers formed in the initial stages of fibril formation.
Peptide solutions were dissolved in 5 mM phosphate buffer, pH 7.4 at the concentration of 100 µM, incubated at 37 °C
for 2-18 h and subjected to light irradiation from 1/30 to 10 seconds. The reaction was quenched by the addition of 500
mM TRIS-HCl buffer, pH 6.8 containing glycerol, SDS and dithiothreitol. The samples were boiled for 5 minutes and
analyzed by SDS-PAGE. A population of small oligomers in equilibrium with the monomer was observed after short
incubation and light irradiation times as indicated by the presence of significant amounts of monomer, dimer and traces
of trimer. At longer exposure times the abundance of oligomers increased with particular regards to trimer, tetramer,
pentamer and oligomers with an apparent molecular weight above 45.000 kDa. These data indicate that a dynamic
equilibrium among different oligomers precedes PrP 82-146 aggregation.
THE-60 DIFFERENTIAL EXPRESSION OF BAX AND BCL-2 IN
NATURAL SCRAPIE INFECTED SHEEP.
J LYAHYAI1, R BOLEA2, P ZARAGOZA1, JJ BADIOLA2, I MARTIN-BURRIEL2.
1 Biochemical Genetics and Blood Groups Laboratory. University of Zaragoza. Zaragoza. Spain. 2 National Reference
Centre for TSEs in Spain.
184
Although apoptosis has been implicated in the neuronal loss of prion diseases, its role in these disorders has received
limited attention. There have been several reports describing neuronal apoptosis in the brain of scrapie-infected sheep,
the brain and retinae of mice infected with scrapie, and the brain of human Creutzfeldt-Jakob disease patients.
Research into programmed cell death have identified a large number of genes and pathways that control and influence
the progression of apoptosis from the initial death trigger to the final demise of the cell. The development of strategies
aimed at reducing apoptosis is receiving growing interest in other neurodegenerative diseases.
Bcl-2 family members are key regulators of apoptosis. This family includes proteins, which can promote either cell
survival, such as Bcl-2, Bcl-XL, Mcl-1, A1, Bcl-W or cell death, like Bax, Bak, Bcl-XS, Bad. The relative amounts or
equilibrium between these pro- and anti-apoptotic proteins influence the susceptibility of cells to a death signal.
Bcl-2 family plays a pivotal role in caspases activation, deciding whether a cell will live or die.
In the present study, we have examined the expression of Bcl-2 related genes in natural scrapie affected and control
sheep brain. Transcript levels were quantified using Real Time RT-PCR. On the other hand, we have used
immunohistochemical techniques to identify the tissue areas where Bcl-2 related proteins as well as caspase-3 were overexpressed.
We have detected an increase of the ratio Bax/Bcl-2 transcripts in the scrapie infected animals. In addition, the upregulation of Bax was confirmed by immunohistochemical staining. Finally, the accumulation of caspase-3 was also
detected in scrapie brains. These findings demonstrate the involvement of the pro-apoptotic protein Bax in the neuronal
death observed in scrapie sheep and open a new way for anti-apoptotic therapies in prion diseases.
Poster Session 3
THE-61 DIFFERENTIAL RNA EXPRESSION IN SCRAPIE GT1 TREATED BY ANTI-PRION DRUGS.
MOUTHON F. (1,2), LEBLANC V. (1,2), PICOLI C. (1), NOUVEL V. (1), DESLYS J.P. (1)
1)CEA/DSV/DRM/GIDTIP, Fontenay-aux-Roses, France. 2) BIO-RAD, Marnes-la-coquette, France
The pathological hallmarks of prion infected brain tissues are PrPsc accumulation and neurodegeneration. Nevertheless ,
the pathological mechanisms and the number of cellular partners implicated are still poorly understood. To elucidate that,
we have adapted a Representational Difference Analysis approach for total RNA comparaison between scrapie GT-1
cells (murine hypothalamic cell line) and uninfected control. RDA is a powerfull and sensitive approch combining PCR
amplification and substractive hybridization to detect nucleic acid sequence differences between complex genomes.
This screening allowed to find 80 sequences disregulated in infected cells versus controls out of which 44 were upregulated and 36 down regulated. Database screening step and sequence analysis allowed to identify 50 sequences with
known messenger homologues and 30 sequences without homology to any other known messenger sequence.
The verification by cDNA Real Time PCR quantification (Bio Rad) allowed to confirm modifications of expression over
5 fold for 90% of the sequences previously identified.
The relevance of these markers has then been explored on infected cells versus cells cured with various treatments
including quinacrine and three heparan mimetics. We confirmed the pattern of differential expression for 11 sequences
(5 of which correspond to identified genes) in infected cells with a return to a quasi-normal level in treated cells only
with heparan sulfate mimetics treatment but not with quinacrine. This normalisation of the pattern of expression
corresponded to the disappearance of PrPres in treated cells. Correlations with proteomic approach are currently under
scrutiny (cf abstract Chich et al) and in vivo analysis are in progress (cf abstract Leblanc et al).
The disregulated gene products are currently under investigation for their interest for their relevance as new therapeutic
targets and for the development of tests based on the combination of surrogate markers.
THE-62 LOOKING FOR MARKERS OF BSE INFECTION: AN
INVESTIGATION OF THE IMMUNE SYSTEM FROM BSE
INFECTED MICE AND CATTLE.
E.B BORTHWICK, A.C WILLIAMS, J.L WILLIAMS
Roslin Institute, Roslin, Midlothian
The diagnosis of transmissible spongiform encephalopathy (TSE) still relies on the detection of the disease-specific isoform
of the host prion protein, termed PRPsc, which accumulates in the tissues, particularly in the central nervous system (CNS)
of infected animals at late stages of incubation of the disease. Several studies have attempted to characterise differential
gene expression in CNS tissues during disease pathogenesis (Duguid J.R, 1989 and Dandoy-Fron F, 1998). To date there
are no proven methods of diagnosis using readily accessible tissues such as blood or lymphoidal tissues. Recent work
performed at the Roslin Institute identified decreased levels of erythroid differentiation-related factor (EDRF) in the spleen
of TSE infected mice, which is reflected in erythroid cells in the blood. This observation gives rise to the possibility of using
differential gene expression in peripheral tissues as an early diagnostic test for TSE infection (Meile G, 2001).
Following oral transmission, infectivity passes from the gastrointestinal system to the CNS via the lymporeticular system
(LRS). In mice PRPsc has been detected in the Peyers Patch (distal ileum) and the spleen within 3 months of BSE
infection (Maignien T, 1999). However in cattle, PRPsc is only detected in the Peyers Patch 6 months post infection (Terry
L.A, 2003) and cannot be detected in the spleen. This lack of BSE infective agent implies a host-dependent and speciesspecific infection in the LRS.
Gene expression, during early infection is being investigated in both the spleen and white blood cells to detect genes that are
differentially expressed between normal and TSE infected individuals. Parallel projects in mouse (BXD12ty) and cattle
(Holstein) are investigating gene expression over a time course of BSE infection using microarray technology to
simultaneously examine the expression of a large number of genes. The results of the study may lead to early markers for
BSE infection.
185
Poster Session 3
THE-63 EXCESSIVE COPPER BINDING RENDERS PRION
PROTEINS RESISTANT TO PROTEOLYTIC DEGRADATION
THORSTEN KUCZIUS(1), ANNE BUSCHMANN(2), WENLAN ZHANG(1), HELGE KARCH(1), MARTIN H. GROSCHUP(2)
1)Institute for Hygiene, University Hospital Munster, Munster, Germany. 2)Institute for Novel and Emerging Infectious
Diseases, Federal Research Centre for Virus Disdeases of Animals, Greifswald - Insel Riems, Germany
Transmissible spongiform encephalopathies are characterized by conversion of the cellular prion protein (PrPC) into its
pathological isoform (PrPSc) which is partial resistant to proteinase K (PK) digestion. There are several evidences
suggesting interactions of PrPC and copper ions which bind to the highly conserved octapeptide region. This binding
modifies the stability of PrPC and the molecular mass of PrPSc after PK treatment and changes its detergent solubility.
In this study the influence of copper and other metal ions on the proteolytic activity of PK as well as on the hydrolysis of
ovine PrPC and ovine and murine PrPSc was analysed. We found that pre-incubation with > 200 µM copper sulphate
prior to proteolytic degradation had a clear inhibitory effect on the proteolytic activity of PK thus selectively stabilizes
PrPC and PrPSc. Binding of copper could stabilize substrates in general. In the investigations reported here the
substrates ovalbumin, N-benzoyl-L-tyrosine ethyl ester (BTEE) and PrP became resistant to PK digestion after addition
of copper to the reaction. The influence of metal binding to the substrate was analyzed after removing unbound copper
ions from the sample. Ova was completely hydrolysed, while PrP remained resistant under these conditions pointing at
strong interactions of copper with PrP. The stabilization of PrP by copper was highly specific and not as much present
with other divalent cations. The binding of copper to PrP may change the tertiary structure of the protein making it
resistant to PK hydrolysis. This interaction could represent a physiological state of PrPC. On the other hand this
observation indicates unexpected interactions of PrP molecules with itself and/or divalent cations which may
misinterpreted as de-novo PrP amplification if experiments are not carefully controlled. However, the large majority of
copper ions are bound to proteins such as PrPC and only trace amounts are accessible in the cell.
THE-64 RADIATION-INDUCED PROTECTION FROM PRION
DISEASE
J. DAVID KNOX (1), RON E. MITCHEL (2), DOUGLAS R. BOREHAM (3)
1. Canadian Science Centre of Human and Animal, Health, Division of Host Genetics and Prion Diseases, Winnipeg,
Manitoba , Canada. 2. Radiation Biology and Health Physics Branch, Chalk River Laboratories, Atomic Energy of
Canada Limited, Chalk River, Ontario Canada.3. Medical Physics and Applied Radiation, Sciences Unit, McMaster
Institute of Applied Radiation Sciences (McIARS), Hamilton, Ontario, Canada.
186
To date compounds that have been shown to prolong the incubation period in animal models, including sulfated
polyanions and tetrapyrroles, need to be administered at or before the time of infection in order to be effective. This
limits their usefulness in treating the disease and suggests that they act by inhibiting the uptake of the infectious agent.
Here we demonstrate that in C57/BL6 mice intracranially infected with the prion strain ME7, exposure to 60Co-g
radiation at a low dose rate (0.5 mGy/min) up to 50 days post inoculation significantly delayed the onset of clinical signs
and the time to death. A wide range of ionizing radiation doses as well as hyperthermia treatments, all administered at
three different time points during the preclinical phase of the disease, were tested. A significant dose response was
observed with the radiation treatments, but the hyperthermia treatment had no effect. The results support the hypothesis
that the induction of endogenous host systems in response to a mild stress can slow the progression of prion diseases and
suggest that whole body radiation exposure may be therapeutic much later than 50 days post-infection.
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Westfälische WilhelmsUniversity, University Hospital
Münster, Germany
+49 251 8352217
tkuczius@uni-muenster.de
51 ALEXANDRE GALO,
Laboratorio Nacional de
Investigacao Veterinaria,
Portugal
+351 21 7115217
dir@lniv.min-agricultura.pt
52 GIANLUIGI ZANUSSO,
University of Verona, Italy
+39458074461
gianluigi.zanusso@univr.it
Participant
List
Participant List
ACIN CRISTINA C
University of Zaragoza
Animal Pathology
Veterinary Faculty
C/ Miguel Servet 177
50013 Zaragoza Spain
Tel. : 0034 976 76 28 19
Fax. : 0034 976 76 16 08
crisacin@unizar.es
ACUTIS PIER LUIGI
Istituto Zooprofilattico del Piemonte
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686245
Fax. : 39-0112686322
cea@izsto.it
ADAMS CRAIG W
Beckman Coulter
Molecular Markers
4300 N. Harbor
CA92834-3100 Fullerton USA
Tel. : 714-773-7865
Fax. : 714-773-8512
cwadams@beckman.com
ADJOU KARIM
Ecole nationale vétérinaire d'Alfort
Pathologie du bétail
7, avenue du Général de Gaulle
94704 Maisons-Alfort France
Tel. : 01 43 96 71 24
Fax. : 01 43 96 70 55
kadjou@vet-alfort.fr
AGRIMI UMBERTO
Istituto Superiore di Sanità
Food Safety and Animal Health
Viale Regina Elena 299
161 Rome Italy
Tel. : 39-0649902462
Fax. : 39-0649387077
agrimi@iss.it
ALLIX SÉBASTIEN
7, avenue du Général de Gaulle
Tel. : 01 43 96 72 53
Fax. : 01 43 96 71 39
sallix@vet-alfort.fr
AN SEONG
PeopleBio Inc.
R&D
116 Yonsei Engineering Research Center
134 Sinchon-dong, Seodaemun-gu
120-749 Seoul South Korea
Tel. : 82-2-6447-7825
Fax. : 82-2-6447-7826
seongaan@peoplebio.net
ANDREOLETTI OLIVIER
INRA-ENVT
SA
23 Chemin des capelles
31076 Toulouse France
Tel. : 33-5 61 19 38 95
Fax. : 33-5 61 19 38 34
o.andreoletti@envt.fr
ANDRIEU THIBAULT
CEA
SNV
18, route du panorama
92265 Fontenay-aux-roses France
Tel. : 01 46 54 94 77
Fax. :
andrieu@dsvidf.cea.fr
ANDRIEVSKAIA OLGA
Canadian Food Inspection Agency
Animal Disease Research Institute
3851 Fallowfield Rd
OntarioK2H 8P9 Ottawa Canada
Tel. : 613-228-6698
Fax. :
andrievskaiao@inspection.gc.ca
ARRABAL SAMUEL
AFSSA
UERB
27-31 avenue du Général Leclerc
94701 Maisons-Alfort Cedex France
Tel. : 33.1.49.77.27.93
s.arrabal@dg.afssa.fr
ARSAC JEAN-NOËL
AFSSA
ATNC (Agent Transmissible Non
Conventionnel)
31 avenue Tony Garnier
69364 Lyon France
Tel. : 04-78-72-65-43
jn.arsac@lyon.afssa.fr
AUBIN JEAN-THIERRY
LFB
Research & Development-BSU
3 avenue des Tropiques
BP 305
91958 Courtaboeuf cedex France
Tel. : 33 1 69 82 71 3
Fax. : 33 1 69 82 72 6
AUBIN@lfb.fr
BADIOLA JUAN J
TSE National Reference Center
Miguel Servet, 177
Tel. : 00 34 976 76201
Fax. : 00 34 976 76160
badiola@unizar.es
BAIER MICHAEL
Robert-Koch-Institut
Neurodegenerative Diseases
Nordufer 20
13353 Berlin Germany
Tel. : +30-45472230
Fax. : +30-45472609
baierm@rki.de
BAILLY YANNICK
Neurotransmission et Sécrétion
Neuroendocrine
UPR2356
5, rue Blaise Pascal
67084 Strasbourg France
Tel. : 33.3.88.45.66.36
Fax. : 33.3.88.60.16.64
byan@neurochem.u-strasbg.fr
BALACHANDRAN ARU
National Reference Laboratory for
scrapie and CWD
3851, Fallowfield Road
OntarioK2H 8P9 Ottawa Canada
Tel. : 613-228-6698
Fax. : 613-228-6103
BalachandranA@inspection.gc.ca
BARBARO KATIA
Istituto Zooprofilattico Sperimentale
IZS - Lazio e Toscana
via Appia Nuova 1411
178 Rome Italy
Tel. : +39.06.79099468
Fax. : +39.06.79340724
bseroma@rm.izs.it
BARDON JAN
State Veterinary Institute
Departement of Special Microbiology
Jakoubka ze Stribra .1
779 00 Olomouc Czech Pepublic
Tel. : 00-420 585 557111
Fax. : 00-420 585 222394
jbardon@svuol.cz
BARDSLEY MAURICE I
VLA
Pathology Department
New Haw
KT15 3NB Addlestone UK
Tel. : +44 (0)1932 357
Fax. : +44 (0) 1932 35
m.bardsley@vla.defra.gsi.gov.uk
BARENCO MONTRASIO MARIA GRAZIA
Paul-Ehrlich Institute
Prion Research Group Pr1
Paul-Ehrlich-Str. 51-59
63225 Langen Germany
Tel. : 00496103/774017
barma@pei.de
BARIZZONE FULVIO
Istituto Zooprofilattico del Piemonte,
Liguria e V
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : +39-0112686296
Fax. : +39-0112686322
cea@izsto.it
BAROCCI SIMONE
Umbria-March
Lab. TSE
C.da San Martino, 6/a
63023 Fermo Italy
Tel. : +39-0734-621489
Fax. : +39-0734-623449
s.barocci@pg.izs.it
193
Participant List
BARON HENRY
ZLB Behring SA
External Affairs
46 Quai de la Rapée
75601 Cedex 12 Paris France
Tel. : +33-1 55 71 57 09
Fax. : +33-1 55 71 57 00
henry.baron@zlbbehring.com
BATTIONI PIERRETTE
Université Paris 5, UMR 8601
Laboratoire de Chimie et de Biochimie
45 rue des St Peres
75270 Paris Cedex 06 France
Tel. : 01 42 86 21 84
Fax. : 01 42 86 04 02
Pierrette.Battioni@univ-paris5.fr
BARON THIERRY GM
AFSSA-Lyon
Unité ATNC
31, avenue Tony Garnier
69364 Lyon France
Tel. : 04-78-69-68-33
Fax. : 04-78-61-91-45
t.baron@lyon.afssa.fr
BAYLEY PETER M
National Institute for Medical Research
Physical Biochemistry
The Ridgeway
Mill Hill
NW7 1AA London UK
Tel. : 0044-20 8816 2085
Fax. : 0044-20 8906 4477
pbayley@nimr.mrc.ac.uk
BARR JANICE B
Institute for Animal Health
Pathology
Ogsten Building
West Mains Road
EH9 3JF Edinburgh Scotland
Tel. : 44-1316675204
janice.barr@bbsrc.ac.uk
BARRET AGNÈS
Commissariat à l'Energie Atomique
DSV/GIDTIP
18 route du Panorama
92265 Fontenay aux Roses France
Tel. : 01 46 54 82 83
Fax. : 01 46 54 94 19
barret@dsvidf.cea.fr
BARRITAULT DENIS S
University Paris 12
Laboratoire CRRET, CNRS
Ave Gal de Gaulle
94000 Créteil France
Tel. : 08 70 72 44 52
Fax. : 01 42 33 44 53
barritault@univ-paris12.fr
BARRON RONA M
NPU
Ogston Building
Kings Buildings
West Mains Road
EH9 3JF Edinburgh UK
Tel. : 44-131667520
Fax. : 44-131668387
rona.barron@bbsrc.ac.uk
194
BAYLIS MATTHEW
Compton Laboratory
Compton
RG20 7NN Newbury UK
Tel. : 44-1635578411
Fax. : 44-1635577237
matthew.baylis@bbsrc.ac.uk
BEDECS KATARINA
Stockholm University
Biochemistry and Biophysics
Svante Arrheniusväg 10-12
10691 Stockholm Sweden
Tel. : +46-8-8162110
Fax. : +46-8-153679
coco@dbb.su.se
BEEKES MICHAEL
Robert Koch-Institut
P26 - Pathogenesis and Diagnostics of
TSEs
Nordufer 20
Tel. : +49-30 4547-2396
Fax. : +49-30 4547-2267
BeekesM@rki.de
BELLON ANNE
INRA
Virology
domaine de Vilvert
78350 Jouy en Josas France
Tel. : +33 1 34 65 26 11
Fax. : +33 1 34 65 26 21
abellon@jouy.inra.fr
BARTAK PAVEL
State Veterinary Institute Jihlava
Virology
Na Harfe 9
190 00 Prague 9 Czech Republic
j.kaspar@oks.cz
BENCSIK ANNA
AFSSA
Prion Unit
69364 Lyon France
a.bencsik@lyon.afssa.fr
BASKAKOV ILIA V
University of Maryland Biotechnology
Inst.
Medical Biotechnology Center
725 W. Lombard St
MBC
MD21201 Baltimore USA
Tel. : 1 (410) 706-4562
Fax. : 1 (410) 706-8184
baskakov@umbi.umd.edu
BENESTAD SYLVIE
National Veterinary Institute
Department of Pathologie
Box 8156 Dep.
0033 Oslo Norway
Tel. : +47 23 21 63 23
Fax. : +47 23 21 63 03
sylvie.benestad@vetinst.no
BENSAUDE FABRICE F
CEA
GIDTIP
18 rue du panorama
92260 Fontenay aux roses France
Tel. : 01 46 54 82 83
fbensaude@free.fr
BERGOT ANNE SOPHIE
INSERM E209
Immunology
184 rue du Fbg St Antoine
75012 Paris France
Tel. : 01 43 44 34 42
bergot@st-antoine.inserm.fr
BERINGUE VINCENT
INRA
Virologie & Immunologie Moléculaires
Domaine de Vilvert
Tel. : 01 34 65 26 16
Fax. : 01 34 65 26 21
beringue@jouy.inra.fr
BERTSCH UWE
Ludwig-Maximilians-Universitaet
Zentrum f. Neuropathologie u.
Prionforschung
Feodor-Lynen-Strasse 23
D-81377 Muenchen Germany
Tel. : 089/2180-78026
Fax. : 089/2180-78037
uwe.bertsch@med.uni-muenchen.de
BÉTEMPS DOMINIQUE
AFSSA Lyon
ATNC
69364 Lyon Cedex 07 France
Tel. : 04.78.72.65.43
Fax. : 04.78.61.91.45
d.betemps@lyon.afssa.fr
BIACABE ANNE-GAËLLE AG
AFSSA-Lyon
Virology-ATNC
69364 France France
Tel. : 04-78-72-65-43
Fax. : 04-78-61-91-45
ag.biacabe@lyon.afssa.fr
BIASINI EMILIANO
Dulbecco Telethon Institute and Istituto
di Ricerc
Neuroscience
Via Eritrea 62
20157 Milano Italy
Tel. : 39-0239014428
Fax. : 39-023546277
biasini@marionegri.it
BIERKE P.
Swedish Institute for Infectious Disease
Control
ADM
Nobels Väg 18
171 82 Solna Sweden
Tel. : +46-8457 25 60
Fax. : +46-8 278519
par.bierke@smi.ki.se
Participant List
BILHEUDE JEAN-MARC
Bio-Rad
Food Science Division
3 boulevard Raymond Poincaré
92430 Marnes la Coquette France
Tel. : 01 47 95 60 14
Fax. : 01 47 95 61 11
sandrine_villette@bio-rad.com
BIRKMANN EVA
Heinrich-Heine-Universitaet Duesseldorf
Institut fuer physikalische Biologie
Universitaetstr. 1
Geb. 26.12.U1
40225 Duesseldorf Germany
Tel. : 49-2118115314
Fax. : 49-2118115167
birkmann@uni-duesseldorf.de
BISHOP MATTHEW T
UK CJD Surveillance Unit
Genetics Laboratory
Crewe Road
EH4 2XU Edinburgh UK
Tel. : 0131 537 2962
Fax. : 0131 343 1404
m.bishop@ed.ac.uk
BLOBEL GÜNTER
The Rockefeller University
Laboratory of Cell Biology
1230 York Avenue
NY10021 New York USA
Tel. : (212)-327-8096
Fax. : (212)- 327-7880
blobel@rockefeller.edu
BOCHE DELPHINE
CNS Inflammation Group
Southampton Neuroscience Group
School of Biological Sciences
University of Southampton
SO16 7PX Southampton UK
Tel. : 00-44 (0)2380 594 187
Fax. : 00-44 (0)2380 592 711
D.Boche@soton.ac.uk
BOCHOT CONSTANCE
Université Paris 5, UMR 8601
Laboratoire de Chimie et de Biochimie
45 rue des saints pères
75270 Paris cedex 06 France
Tel. : 01 42 86 21 84
Fax. : 01 42 86 04 02
constance.bochot@univ-paris5.fr
BOECKING DETLEF H
DLR (German Aerospace Center)
PT (Project managment agency)
Suedstr. 125
53175 Bonn Germany
detlef.boecking@dlr.de
BONA CRISTINA
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686296
Fax. : 39-0112686322
cea@izsto.it
BORRONI RENATA
Istituto Superiore Sanità
Laboratorio Medicina Veterinaria
viale Regina Elena,299
161 Rome Italy
Tel. : 39-3482931723
borroni@iss.it
BORTHWICK E. B.
Roslin Institute
Genomics and Bioinformatics
Roslin
EH25 9PS Edinburgh Scotland
Tel. : 44-13105274450
emma.borthwick@bbsrc.ac.uk
BOSCOBOINIK DANIEL
Prionics AG
Area Management
Wagistr. 27 A
8952 Schlieren Switzerland
Tel. : 0041 44 200 2000
Fax. : 0041 44 200 2010
daniel.boscoboinik@prionics.ch
BOSSERS ALEX
CIDC-Lelystad
Bacteriology and TSEs
PObox 2004
8203 AA Lelystad The Netherlands
Tel. : +31-320-238273
Fax. : +31-320-238153
alex.bossers@wur.nl
BOURGEOIS JEAN-PIERRE
Bio-Rad
Tel. : 01 47 95 61 24
Fax. : 01 47 95 61 11
BOUTAL HERVÉ H
CEA de Saclay
DRM SPI
Bât 136
DSV DRM SPI LERI
91191 Gif sur Yvette France
Tel. : 01 69 08 77 04
herve.boutal@cea.fr
BOUZAMONDO-BERNSTEIN ESSIA
University of California San Francisco
IND & Neuropathology
513 Parnassus Avenue HSW 430
CA94143 San Francisco USA
Tel. : 1-415-476-5238
Fax. : 1-415-476-7963
zizaboy@itsa.ucsf.edu
BOVE DANIELA
Istituto zooprofilattico
Food analysis
Via Salute 2 Portici
80055 Portici Italy
Tel. : 0039.81.7865208
Fax. : 0039.81.7763125
d.bove@izsmportici.it
BRAGASON BIRKIR T
Keldur Institute for experimental
pathology
Molecular biology
Vesturlandsvegur
112 Reykjavik Iceland
Tel. : +354-567-4700
Fax. : +354-567-3979
birkirbr@hi.is
BRANDEL J-P
INSERM 360 / cellule de référence des
MCJ
Groupe hospitalier Pitié-Salpétrière
47-83, boulevard de l'hôpital
75651 Cedex 13 Paris France
Tel. : 01 42 16 26 26
Fax. : 01 42 16 26 25
jean-philippe.brandel@psl.ap-hop-paris.fr
BROWN DEBBIE A
Pathology
Ogston Building
West Mains Road
Tel. : 0131-319-8212
Fax. : 0131-668-3872
debbie.brown@bbsrc.ac.uk
BROWN PAUL W
NIH
Lab CNS studies
bldg 36 rm 4a19 msc4123
Maryland20892 Bethesda USA
Tel. : 301-496-5292
brownp@ninds.nih.gov
BRUCE MOIRA E
Neuropathogenesis Unit
Ogston Building
West Mains Road
Tel. : 44-131667520
moira.bruce@bbsrc.ac.uk
BRUGERE-PICOUX JEANNE
ENVA
7 av Gl de Gaulle
94704 Maisons Alfort France
Tel. : +33 1 43 96 71 22
jbrugere-picoux@vet-alfort.fr
BUDKA HERBERT
Medical University of Vienna
Institute of Neurology
AKH 4J
Währinger Gürtel 18-20
A- 1097 Vienna Austria
Tel. : +43 1 40 400 5500
Fax. : +43 1 40 400 5511
h.budka@akh-wien.ac.at
BUERKLE ALEXANDER
University of Konstanz
Chair of Molecular Toxicology, Box X911
Universität strasse 10
D-78457 Konstanz Germany
Tel. : +49-7531-884035
Fax. : +49-7531-884033
alexander.buerkle@uni-konstanz.de
195
Participant List
BUMPASS DONNA C
HPA Porton Down
TSE research
Porton Down
SP4 0JG Salisbury UK
Tel. : 44-198061200
donna.bumpass@hpa.org.uk
CARDINALE ALESSIO
University of Tor Vergata
Department of Neuroscience
Via di Montpellier 1
133 Rome Italy
Tel. : +39 6 7259 6411
Fax. : +39 6 7259 6407
Cardinal@uniroma2.it
CAZEAU GÉRALDINE
AFSSA
Epidemiology
31 avenue tony garnier
69364 cedex 7 Lyon France
Tel. : 04 78 72 65 43
Fax. : 04 78 61 91 45
g.cazeau@lyon.afssa.fr
BUSCHMANN ANNE
Federal Research Centre for Virus
Diseases of Anim
Institute for Novel and Emerging
Infectious Diseas
Boddenblick 5a
17493 Greifswald / Insel Riems Germany
Tel. : 0049-38351-7161
Fax. : 0049-38351-7192
anne.buschmann@rie.bfav.de
CARDONE FRANCO F
Department of Cellular Biology and
Neurosciences
viale Regina Elena 299
181 Rome Italy
Tel. : 39-6-49903313
Fax. : 39-6-49903012
cardone@iss.it
CERNILEC MAJA
Blood Transfusion Centre of Slovenia
Department for the production of
diagnostic reagen
Slajmerjeva 6
1000 Ljubljana Slovenia
Tel. : +386.01 5438 199
Fax. : +386.01 302 224
maja.cernilec@ztm.si
CAREDDU MARIA ELENA
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686320
Fax. : 39-0112686322
cea@izsto.it
CHABRY JOELLE
INSERM
Molecular and Cellular Pharmacology
660 route des lucioles
6560 Valbonne France
Tel. : 33.4 93 95 77 67
Fax. : 33.4 93 95 77 08
chabry@ipmc.cnrs.fr
CADO SYLVIE
Laboratoire Pasteur-Cerba
Santé animale
rue de l'Equerre
95066 Cergy Pontoise cedex 9 France
Tel. : 01 34 40 20 71
scado@pasteur-cerba.com
CALAVAS DIDIER D
AFSSA
Site de Lyon
31 av Tony Garnier
69346 Lyon cedex 7 France
Tel. : +33 (0) 4 78 69 68 21
Fax. : +33 ( 0)4 78 61 91 45
d.calavas@lyon.afssa.fr
CALIGIURI VINCENZO V
Istituto zooprofilattico sperimentale del
mezzogio
Epidemiology
via salute , 2 - Portici
80055 Portici Italy
Tel. : 0039.81.7865270
Fax. : 0033.81.7865267
v.caligiuri@izsmportici.it
196
CARTONI CLAUDIA
Viale Regina Elena, 299
161 Rome Italy
Tel. : 0039-06-49902848
Fax. : 0039-06-49387077
cartoni@iss.it
CASAGRANDE FABRICE
CEA
SNV
Tel. : 01 46 54 87 38
casagrande@dsvidf.cea.fr
CAMPANA VINCENZA
Institut Pasteur
Biologie cellulaire et infection
25-28 rue de docteur Roux
75724 Paris Cedex France
Tel. : 0033-01-4438955
Fax. : 0033-01-4061323
vcampana@pasteur.fr
CASALONE CRISTINA
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : +39-0112686341
Fax. : +39-0112686322
cea@izsto.it
CANO BENITO MARIA JESUS MJ
INIA-CISA
BMCP
Ctra. Algete a El Casar Km 8.100
28130 Valdeolmos Spain
Tel. : 916202300
Fax. : 916202247
cano@inia.es
CATHALA FRANCOISE
68 Bd Saint Michel
75006 Paris France
Tel. : +33 1 43 54 34 26
Fax. : +33 1 43 54 34 26
no
CARAMELLI MARIA
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686296
Fax. : 39-0112686322
cea@izsto.it
CAUGHEY BYRON
NIH Rocky Mountain Labs
Lab. of Persistent Viral Diseases
903 S. 4th St.
Montana59840 Hamilton USA
Tel. : 1-406-363-9264
Fax. : 1-406-363-9286
bcaughey@nih.gov
CHAPRON YVES
Alpine Institute of Environmental
Dynamics
Biophysics
108 rue du Puy
38660 La Terrasse France
Tel. : 04 76 08 20 86
yves.chapron@wanadoo.fr
CHIANINI FRANCESCA
Moredun Research Institute
Virology
Pentland Science Park
Bush Loan
Penicuik
EH26 0PZ Midlothian Scotland
Tel. : +44 (0) 131 445 511
Fax. : +44 (0) 131 445 611
Chiaf@mri.sari.ac.uk
CHICH JEAN-FRANÇOIS
I.N.R.A.
Biologie Physico-Chimique des Prions
Virologie et Immunologie Moléculaires
Bâtiment 440
Domaine de Vilvert
78352 Jouy-en-Josas France
Tel. : 01 34 65 29 12
chichATjouy.inra.fr
CHIESA ROBERTO
di Ricerc
Neuroscience
Via Eritrea 62
20157 Milano Italy
Tel. : 39-0239014428
Fax. : 39-023546277
chiesa@marionegri.it
Participant List
CLEWLEY JONATHAN
Health Protection Agency
SBVL
Central Public Health Laboratory
61 Colindale Avenue
NW9 5HT London United Kingdom
Tel. : +44 (0) 20 8200 4400 ext 3245
Fax. : +44 (0) 20 8200 1569
jonathan.clewley@hpa.org.uk
COULPIER MURIEL
INRA-AFSSA-ENVA, UMR 1161
Animal Health
7 Av du Gal de Gaulle
Tel. : 01 43 96 70 46
Fax. : 01 43 96 71 31
mcoulpier@vet-alfort.fr
KOEIJER ALINE
Animal Sciences Group, Wageningen UR
Quantitative Veterinary Epidemiology
(QVE), Depart
P.O.Box 65
8200 AB 8200 AB Lelystad The
Netherlands
Tel. : 31-320238321
Fax. : 31-320238961
aline.dekoeijer@wur.nl
DE
CLOUX JEAN-LUC
Immunotech Beckman Coulter
Prion department
Avenue De lattre de Tassigny 130
13276 Marseille France
Tel. : +33-491 17 35 34
Fax. : +33-491 17 27 40
jcloux@beckman.com
CRESCIO MARIA INES
Liguria e V
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686261
Fax. : 39-0112686322
cea@izsto.it
COLLINGE JOHN
Medical Research Council
Prion Unit
Queen Square
WC1N 3BG London England
Tel. : 2 078 374 888
Fax. : 2 078 378 047
pacollinge@prion.ucl.ac.uk
CRONIER SABRINA
INRA
VIM
Domaine de Vilvert
Batiment 440
Tel. : 01.34.65.26.13
cronier@jouy.inra.fr
COMOY EMMANUEL
CEA
DSV
92265 Fontenay-aux-Roses France
Tel. : 01 46 54 90 05
Fax. : 01 46 54 93 19
emmanuel.comoy@cea.fr
CROVILLE SANDRINE
Danone
Food Safety Center
RD 128
91767 Palaiseau France
Tel. : +33-1 69 35 74 15
Fax. : +33-1 69 35 76 97
sandrine.croville@danone.com
DELISLE MARIE BERNADETTE
CHU Rangueil
Pathology
1, Avenue J. Pouhles
Tel. : 33 (0)5 61 32 29 51
Fax. : 33 (0)5 61 32 21 27
delisle.b@chu-toulouse.fr
COMTET LOÏC
Institut Pourquier
R&D
326, rue de la Galéra
34090 Montpellier France
Tel. : 00 33 (0)4 99 2
Fax. : 00 33 (0)4 67 0
loic.comtet@institut-pourquier.fr
CROZET CAROLE A
CNRS
Herault
434 avenue de la Cardonille
Tel. : 04 99 61 99 30
Fax. : 04 99 61 99 01
ccrozet@igh.cnrs.fr
DESLYS JEAN-PHILIPPE
CEA/DSV/DRM/GIDTIP
Département de Recherche Médicale
18, route du Panorama
Tel. : +33-1 46 54 82 79
Fax. : +33-1 46 54 93 19
jpdeslys@cea.fr
CORDIER CÉLINE
AFSSA-Lyon
ATNC
69364 Lyon cedex 07 France
Tel. : (33)4 78 72 65
Fax. : (33)4 78 61 91
c.cordier@lyon.afssa.fr
CULLIN CHRISTOPHE
IBGC CNRS
HSP
3 rue Camille Saint Saens
33000 Bordeaux France
Tel. : 33 (0) 556 999 017
Fax. : 33 (0) 556 999 017
cullin@ibgc.u-bordeaux2.fr
DI
CORONA CRISTIANO
Liguria e V
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686261
Fax. : 39-0112686322
cea@izsto.it
CZUB STEFANIE
National Centre for Foreign Animal
Disease
1015 Arlington Street
ManitobaR3E 3M4 Winnipeg Canada
Tel. : (204) 789-2021
Fax. : (204) 789-2038
czubs@inspection.gc.ca
DI
COSTE HANDAN F
Institute for Diagnosis and Animal
Health (IDAH)
Molecular Biology
Dr. Staicovici street, no. 63
sector 5
76202 Bucharest Romania
Tel. : 00-40-21-410.13.90
Fax. : 00-40-21-411.33.94
office@idah.ro; coste.handan@idah.ro
DE
GROSSI LUIGI
Ist. zooprofilattico reg. Lazio e Toscana
LAZIO
Strada Terme 4A
1100 Viterbo Italy
Tel. : +39-0761250147
ldegrossi@rm.izs.it
DEBECKER DANNY
Bio-Rad
Food Science
Begoniastraat 5
9810 Nazareth Belgium
Tel. : +32-9 382 7384
Fax. : +32-9 385 65 54
Danny_Debecker@bio-rad.com
RIO V.J.
Veterinary Laboratories Agency
CERA
New Haw
kt15 3nb Addlestone United Kingdom
Tel. : 44-1932357621
v.delriovilas@vla.defra.gsi.gov.uk
DEL
GIAMBERARDINO LUIGI
GIS PRION
N.A.
102 rue Didot
75014 Paris France
Tel. : 33 1 58 14 05 10
Fax. : 33 1 58 14 05 11
gisprion@infobiogen.fr
SARNO ALESSANDRA
Istituto zooprofilattico
Diagnostic
via Salute 2 Portici
80055 Portici Italy
Tel. : 0039.81.7865206
Fax. : 0039.81.7751377
adisarno@izsmportici.it
DIARRA-MEHRPOU MARYAM
INSERM u 487
IGR
39 rue camille desmoulins
94805 Villejuif France
Tel. : 01 42 11 46 50
mehrpour@igr.fr
197
Participant List
DICKINSON JOANNE
HPA - Porton Down
Research
CAMR
Tel. : +44 (0) 1980 612100
Fax. : +44 (0) 1980 612731
joanne.dickinson@hpa.org.uk
DOBSON CHRISTOPHER M
University of Cambridge
Department of Chemistry
Lensfield Road
CB2 1EW Cambridge United Kingdom
Tel. : (44) (0)1223 763070
Fax. : (44) (0)1223 763418
acb53@cam.ac.uk
DODET BETTY
Dodet Bioscience
Communication
66 cours Charlemagne
69002 Lyon France
Tel. : 33 (0)4 72 41 17 05
Fax. : 33 (0)4 72 41 17 14
betty.dodet@dodetbioscience.com
DOSSENA SARA
di Ricerc
Neuroscience
Via Eritrea 62
20157 Milano Italy
Tel. : 39-0239014428
Fax. : 39-023546277
dossena@marionegri.it
DREXLER JAROSLAV
Nad prehradou 404
109 00 Praha 10 Czech Republic
Tel. : 00-420-274869731
Fax. : 00-420-267292233
DUCROT CHRISTIAN
INRA
Santé Animale
Unité d'Epidémiologie Animale
Centre de recherche de Theix
63122 Saint Genes Champanelle France
Tel. : (33) 4 73 62 42 63
Fax. : (33) 4 73 62 45 48
ducrot@clermont.inra.fr
DUHEM KOENRAAD
CNIEL
Sécurité Alimentaire
42 rue de Châteaudun
75009 Paris France
Tel. : 01 49 70 71 19
Fax. : 01 42 80 63 45
kduhem@cniel.com
198
DUMPITAK CHRISTIAN
Heinrich-Heine-Universität Düsseldorf
Institut für Physikalische Biologie
Universitätsstr. 1
Geb. 26.12.U1
D-40225 Düsseldorf Germany
Tel. : 49-211-81-15314
Fax. : 49-211-81-15167
dumpitak@biophys.uni-duesseldorf.de
DUPIN MARILYNE
Biomerieux SA
RHONE
Chemin de l'Orme
69280 Marcy-l'Etoile France
marilyne.dupin@eu.biomerieux.com
DYRBYE HENRIK
Rigshospitalet
Dept. of Neuropathology, 6301
Blegdamsvej 9
DK 2100 Copenhagen Denmark
Tel. : +45-35 45 63 35
Fax. : +45-35 45 63 23
Dyrbye@rh.dk
EATON SAMANTHA L
Moredun Research Institute
Virology
Pentlands Science Park
Bush Loan
Eh26 0PZ Penicuik Scotland
Tel. : 44-1314455111
Fax. :
eatos@mri.sari.ac.uk
ECROYD HEATH W
Agronomique, INR
Gamètes Males et Fertilité
UMR 6175 INRA-CNRS
Station de Physiologie de la Reproduction
et des C
37380 Monnaie France
Tel. : +33 (0)2 47 42 78 03
Fax. : +33 (0)2 47 42 77 43
ecroyd@tours.inra.fr
EGHIAIAN FRÉDÉRIC
INRA
Virologie Immunologie Moléculaires
Biologie Physico-chimique des Prions
INRA, Domaine de Vilvert
Tel. : 01 34 65 27 89
Fax. : 01 34 65 26 21
feghiaia@jouy.inra.fr
EIDEN MARTIN
Federal Research Centre of Virus
Diseases of Anima
Institute for Novel and Emerging
Infectious Diseas
Boddenblick 5a
17489 Greifswald Greifswald
Tel. : +49 (0)38351 71
martin.eiden@rie.bfav.de
EL HACHIMI KHALID
EPHE/INSERM
U 289
47 bd de l'Hôpital
Hôpital de la Salpétrière
75013 Paris France
Tel. : 01 42 16 26 78
Fax. : 01 42 16 26 78
hachimi@infobiogen.fr
ELOIT MARC
Alfort National Veterinary School
UMR1161 ENVA-INRA-AFSSA
7 av Gal de Gaulle
94704 Maisons Alfort France
Tel. : 33 1 43 96 70 0
eloit@vet-alfort.fr
ELVIRA GEMA
CSIC
IQFR
Serrano 119
E-28006 Madrid Spain
Tel. : 34-915619400 ex
Fax. : 34-915642431
gelvse@yahoo.es
ESPINOSA JUAN CARLOS
CISA-INIA
BMCP
Carretera de Algete a El Casar de
Talamanca
Tel. : 34 91 620 23 00
Fax. : 34 91 620 22 47
espinosa@inia.es
EVERINGTON DAWN
University of Edinburgh
National CJD Surveillance Unit (UK)
Western General Hospital
Crewe Road
Tel. : 44-1315373104
d.everington@ed.ac.uk
EWALD FRANCOIS
CNAM
26 Bd Haussmann
75311 Paris cedex 09 France
Tel. : 33-1-42479381
Fax. : 33-1-42479123
ewald@cnam.fr
FABRE DE LOYE AGNÈS
Veterinary College
Physiology
7,avenue du général de gaulle
94704 Cedex Maisons-Alfort France
Tel. : 01 43 96 71 37
Fax. : 01 43 96 71 39
franck.labonne@agriculture.gouv.fr
FAUCHEUX BAPTISTE A
INSERM
Research Unit U.360
Hopital de la Salpetriere
47 Blvd de l'Hopital
75013 Paris France
Tel. : 33-1-4216-2568
Fax. : 33-1-4423-9828
baptiste.faucheux@chups.jussieu.fr
FERAUDET CÉCILE C
CEA-Saclay
Service de Pharmacologie et
d'Immunologie / DRM
Bâtiment 136
CEA Saclay
Tel. : 01 69 08 77 04
Fax. : 01 69 08 59 07
feraudet@cea.fr
Participant List
FERNIE KAREN
TSE Inactivation
Ogston Building
West Mains Road
Tel. : 44-1313198210
Fax. : 44-1316683872
karen.fernie@bbsrc.ac.uk
FOSTER JAMES D
Ogston Building
Kings Buildings
West Mains Road
MidlothianEH9 3JF Edinburgh UK
Tel. : 0044-0131 319 8
Jim.Foster@BBSRC.AC.UK
FUZI MIKLOS
Natl. Centre for Epidemiology
Bacteriology
2-6 Gyali ut
1097 Budapest Hungary
Tel. : 36-1-476.1118
Fax. : 36-1-476.1243
fuzim@oek.antsz.hu
FEYSSAGUET MURIEL
Bio-Rad
Tel. : 01 47 95 61 31
Fax. : 01 47 95 61 11
FOURNIER JEAN-GUY
CEA
DSV/DRM/GIDTIP
18 rue Panorama
Tel. : 01 46 54 98 96
Fax. : 01 46 54 77 26
fournier@dsvidf.cea.fr
GABIZON RUTH
Hadassah University Medical Centers
Laboratory of Experimental Neurology
Kiryat Hadassah
P.O.B. 12000
91120 Jerusalem Israel
Tel. : 972-2-6777858
Fax. : 972-2-6429441
gabizonr@hadassah.org.il
FICHET GUILLAUME
CEA
GIDTIP
Tel. : 33 1 46 54 82 83
fichet@dsvidf.cea.fr
FRASER CAROL A
Dept of Neurodegenerative Diseases
Queen Square House
Queen Square
WC1N 3BG London UK
Tel. : +44-207 8373 61
c.fraser@prion.ucl.ac.uk
GAGNA CARLA
Liguria e V
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686320
Fax. : 39-0112686322
cea@izsto.it
FIESCHI JACQUES
Immunotech Beckman Coulter
Prion Unit
130 avenue de Lattre de Tassigny
13276 Marseille France
Tel. : 33 (0) 491172733
Fax. : 33 (0) 491172740
jfieschi@beckman.com
FIORITI LUANA
di Ricerc
Neuroscience
Via Eritrea , 62
20157 Milan Italy
Tel. : 39-0239014428
Fax. : 39-023546277
fioriti@marionegri.it
FLAN BENOÎT
LFB
BSU
3 avenue des Tropiques
BP 305
91958 Courtaboeuf cedex France
Tel. : 33 1 69 82 71 3
Fax. : 33 1 69 82 72 6
FLAN@lfb.fr
FORLONI GIANLUIGI
Istituto di recerche farmacologiche Mario
Negri
Neuroscience Department
Via Eritrea 62
20157 Milano Italy
Tel. : 39 02 39014462
Fax. : 39 02 3546277
forloni@marionegri.it
FREIXES MERITXELL
University of Barcelona
Institut of Neuropathology. Cellular
Biology and P
C/ Feixa Llarga sn
Barcelona8907 Hospitalet de Llobregat
Spain
Tel. : 932607459/ 934035808
Fax. : 934035810
merifreixes@yahoo.com
FREYMUTH FRANÇOIS
CHU Caen
Laboratoire de virologie humaine et
moléculaire
Avenue Georges Clemenceau
14033 Caen Cedex France
Tel. : 02 31 27 25 54
Fax. : 02 31 27 25 57
freymuth-f@chu-caen.fr
FRIGG RICO
Prionics AG
Research
Wagistr. 27 a
Tel. : 0041 44 200 2000
Fax. : 0041 44 200 2010
rico.frigg@prionics.ch
FURUKAWA HISAKO
Nagasaki University Graduate School of
Biomedical
Pharmacology 1
1-12-4 Sakamoto
852-8523 Nagasaki Japan
Tel. : +81-95-849-7043
Fax. : +81-95-849-7044
hisako@net.nagasaki-u.ac.jp
GAJDUSEK CARLTON D
University of Tromsoe
Dep. of Experimental Pathology
N-9037 Tromsoe Norway
Tel. : 47-77644685
Fax. : 47-77645391
GASSET MARIA
Insto Quimica-Fisica "Rocasolano"
CSIC
Serrano 119
E-28006 Madrid Spain
Tel. : 34-91-5619400 Ext 1308
Fax. : 34-91-5642431
mgasset@iqfr.csic.es
GATTI JEAN-LUC
INRA
Physiology of Reproductiion
PRC
INRA-Nouzilly
37380 Nouzilly France
Tel. : 02 47 42 78 03
Fax. : 02 47 42 77 43
gatti@tours.inra.fr
GAUCZYNSKI SABINE
Gene Center
Prion Research Laboratory
Feodor-Lynen-Str. 25
81377 Munich Germany
Tel. : 49-89-2180-769
Fax. : 49-89-2180-769
gzynski@lmb.uni-muenchen.de
GAVIER-WIDEN DOLORES
National Veterinary Institute (SVA)
Sweden
Wildlife Diseases
S-75189 Uppsala Sweden
Tel. : +46-(O)18 674215
Fax. : +46-(O)18 309162
dolores@sva.se
199
Participant List
GEFFROY BERTELLA
Ministere de la Justice
Ile de france
5 rue des italiens
75009 Paris France
Tel. : 01 44 32 62 33
Fax. : 01 44 32 98 78
marie-odile.bertella-geffroy@justice.fr
GESCHWIND MICHAEL D
University of California, San Francisco
Neurology
UCSF Memory & Aging Center
350 Parnassus Ave., Suite 706
CA94117-1207 San Francisco USA
Tel. : 415-476-8613
Fax. : 415-476-4800
mgeschwind@memory.ucsf.edu
GIESE ARMIN
LMU
Institute of Neuropathology
81377 München Germany
Tel. : +49-89-2180-78048
Fax. : +49-89-2180-78037
Armin.Giese@med.uni-muenchen.de
GIRONES ROSINA
Microbiology
Diagonal, 645
8028 Barcelona Spain
Tel. : 34934021483
Fax. : 34934110592
rgirones@ub.edu
GOFFLOT STÉPHANIE
University of Liège
CRPP
1 Avenue de l Hopital B36
4000 Liège Belgique
Tel. : +32 (0)4 366 43 28
Fax. : +32 (0)4 366 43 21
sgofflot@ulg.ac.be
GOLDBERG MICHEL E
Institut Pasteur
Structural Biology and Chemistry
28 rue du Dr Roux
Tel. : 33 1 45 68 83 8
Fax. : 33 1 40 61 30 4
goldberg@pasteur.fr
GOLDMANN WILFRED
Neuropathogenesis Unit, TSE Division
Ogston Building
West Mains Road
EH9 3JF Edinburgh United Kingdom
wilfred.goldmann@bbsrc.ac.uk
200
GONATAS NICHOLAS K
University of Pennsylvania, School of
Medicine
Pathology and Laboratory Medicine
609 Stellar Chance Laboratories
422 Curie Boulevard
Pennsylvania19104-6100 Philadelphia
USA
Tel. : 215-662-6695
Fax. : 215-573-2058
gonatasn@mail.med.upenn.edu
GONZALEZ LORENZO
VLA Lasswade
R&D
Pentlands science park
Bush Loan
Penicuik
EH26 0PZ Midlothian UK
Tel. : 44-1314456169
l.gonzalez@vla.defra.gsi.gov.uk
GOODRICH ANDREW K
Abbott Laboratories
Diagnostics Division
100 Abbott Park Road
Dept 09L4, Bldg AP6C-4
IL60064 Abbott Park USA
Tel. : 847-937-3351
Fax. : 847-937-2776
andrew.goodrich@abbott.com
GOODSIR CAROLINE
VLA Lasswade
R&D
Bush Loan
Penicuik
Tel. : 44-1314456169
c.goodsir@vla.defra.gsi.gov.uk
GRASSI JACQUES
CEA
Service de Pharmacologie et
d'Immunologie
Bâtiment 136
CEA/Saclay
91191 Gif sur Yvette cedex France
Tel. : 33-1-69-08-28-7
Fax. : 33-1-69-08-59-0
jacques.grassi@cea.fr
GROSCLAUDE JEANNE
INRA
Virologie et Immunologie Moléculaires
Domaine de Vilvert
Tel. : 33-1-34-65-26-28
Fax. : 33-1-34-65-26-21
jngroscl@jouy.inra.fr
GRUBENBECHER STEPHANIE
Heinrich-Heine-University Düsseldorf
Institute f. Neuropathology
Moorenstr. 5
Uni-Kliniken
Building 14.79
40225 Düsseldorf Germany
Tel. : 0049-211-8118653
Fax. : 0049-211-8117804
grubenbecher@uni-duesseldorf.de
GUARNIERI FRANCK
ARMINES
Cindyniques
BP 207 rue claude daunesse
6904 Sophia Antipolis France
Tel. : 33-4-93-95-74-72
Fax. : 33-4-93-65-40-32
franck.guarnieri@ensmp.fr
GYLLBERG HANNA
Stockholm university
Biochemistry and Biophysics
Svante Arrhenius väg 12
S-10691 Stockholm Sweden
Tel. : 46-8-162487
hanna.gyllberg@dbb.su.se
HAGENAARS THOMAS J
Animal Sciences Group, Wageningen UR
Quantitative Veterinary Epidemiology
(QVE), ID-Lel
P.O.Box 65
8200 AB Lelystad The Netherlands
Tel. : +31-320 238398
Fax. : +31-320 238961
thomas.hagenaars@wur.nl
HAÏK STÉPHANE
INSERM
U360
Salpétrière Hospital
47, Bd de l'Hôpital
75013 Paris France
Tel. : 01 42 16 18 81
Fax. : 01 44 23 98 28
haik@chups.jussieu.fr
HALIMI MICHELE MH
Hadassa Hebrew University Medical
Centers
Kiryat Hadassah POB 12000
Tel. : 972-2-6777858
Fax. : 972-2-6429441
michelehal@hotmail.com
HANSEN METTE
Danish Institute of Food and Veterinary
Research
Department of Veterinary Diagnostics
and Research
27, Bülowsvej
1790 Copenhagen V Denmark
Tel. : 00-45 72346236
Fax. : 00-45 72346001
mse@dfvf.dk
HASENJAEGER OLAF
Freie Universität Berlin
FB Biologie
Sparrstrasse 9
App. 41 802
Tel. : 49-3083202003
tcbtcb@web.de
HASSIG RAYMONDE
CEA
SHFJ-DRM-DSV
4, place du GÈnÈral Leclerc
91401 Orsay France
Tel. : 01-69-86-77-62
Fax. : 01-69-86-77-45
hassig@shfj.cea.fr
HAUW JEAN-JACQUES
Hôpital de la Salpétrière
Neuropathologie
47 Bd de l'Hôpital
75013 Paris France
Tel. : (33)142161881
Fax. : (33)144239828
jean-jacques.hauw@psl.ap-hop-paris.fr
Participant List
HAY ROSIE
Environmental Healthcare Unit
Biomedical sciences building
Bassett Crescent East
SO16 7PX Southampton Great Britain
Tel. : 02380-592034
Fax. : 02380-594459
rosiehay282@hotmail.com
HEAD MARK W
National CJD Surveillance Unit
Bryan Matthews Building
Crewe Road
EH4 2XU Edinburgh United Kingdom
Tel. : +44 1 31 537 3103
Fax. : +44 1 31 537 3056
m.w.head@ed.ac.uk
HEEGAARD PETER MH
Danish Institute for Food and Veterinary
Research
Veterinary Diagnostics and Research
27, Bülowsvej
1790 V Copenhagen Denmark
Tel. : 45-72346241
Fax. : 45-72346000
pmhh@dfvf.dk
HEINEMANN UTA
university hospital
Neurology
robert-koch-str 40
37075 Goettingen Germany
Tel. : 49-551-398401
Fax. : 49-551-397020
uta.heinemann@med.uni-goettingen.de
HEINEN ERNST
Medecine
Rue de Pitteurs 20
4020 Liège Belgium
Tel. : 00-3243665170
Fax. : 00-3243665173
eheinen@ulg.ac.be
HERMS JOCHEN J
LMU Munich
Neuropathology
Feodor-Lynen Str. 23
Tel. : 49-89-2180-78
Fax. : 49-89-2180-78
jochen.herms@med.uni-muenchen.de
HERVA MOYANO MARIA EUGENIA
CISA
Molecular and Cellular Prion Biology
Carrereta de Valdeolmos a El Casar
Tel. : 916202300
Fax. : 916202247
eherva@inia.es
HERZOG CHRISTIAN
Commissariat à l'Energie Atomique
Service de Neurovirologie
BP 6
Tel. : +33 1 46 54 82
Fax. : +33 1 46 54 93
herzog@dsvidf.cea.fr
HIANIK TIBOR
Comenius University
Biophysics and Chemical Physics
Mlynska dolina F1
84248 Bratislava Slovak Republic
Tel. : 421-2-60295683
Fax. : 421-2-65426774
hianik@fmph.uniba.sk
HIJAZI NUHA
Hadassah-Hebrew University Medical
Center
Kiryat Hadassah P.O.B 12000
Tel. : 972-2-6777858
Fax. : 972-2-6429441
nuha@md.huji.ac.il
HEINIG LARS
German Primate Centre
Immunology and Virology
Kellerweg 4
37077 Göttingen Germany
Tel. : +49-551 3851 147
Fax. : +49-551 3851 184
lheinig@dpz.gwdg.de
HILLS ROBERT A
TSE Secretariate
Health Canada
Rm. 0189, Building No. 7
AL/LP 0700B5
Tunney's Pasture
OntarioK1A 0L2 Ottawa Canada
Tel. : 1-613-957-9005
Fax. : 1-613-946-4589
Bob_Hills@hc-sc.gc.ca
HERES LOURENS
CIDC-Lelystad
Bacteriology and TSE
Edelhertweg 15
PO BOX 2004
8203 AA Lelystad The Netherlands
Tel. : 31 320 238 583
Fax. : 31 320 238 153
lourens.heres@wur.nl
HIRSCHBERGER THOMAS
University of Munich
Biophysics
Oettingenstr. 67
80538 Muenchen Germany
Tel. : +49 89 2180 9232
Fax. : +49 89 2180 9202
Thomas.Hirschberger@physik.unimuenchen.de
HOFF-JOERGENSEN RIKKE
Danish Institute for Food- and Veterinary
Research
Section for Biological Products
Bülowsvej 27
DK-1790 Copenhagen V Denmark
Tel. : +45-72 34 60 00
Fax. : +45-72 34 60 01
rhj@dfvf.dk
HOLZNAGEL EDGAR
Federal Agency for Sera and Vaccines
(Paul-Ehrlich
Transmissible Spongiform
Encephalopathies Pr1
Paul-Ehrlich-Str 51
D-63225 Langen Germany
Tel. : (06103) 77 73 00
Fax. : (06103) 77 12 34
holed@pei.de
HOPE JAMES
Veterinary Laboratory Agency
CERA
Pentlands Science Park
Bush Loan
EH26 0PZ Pencuik United Kingdom
Tel. : 44-131-445-617
Fax. : 44-131-445-616
j.hope@vla.defra.gsi.gov.uk
HORROCKS CLAIRE
Neuropathology
Woodham Lane
New Haw
KT15 3NB Addlestone United Kingdom
Tel. : +44(0)1932 3577
c.horrocks@vla.defra.gsi.gov.uk
HUNDT CHRISTOPH
Roche Diagnostics
Roche Applied Science
Nonnenwald 2
82372 Penzberg Germany
Tel. : +49 8856-60 6933
christoph.hundt@roche.com
HUNSMANN GERHARD
Deutsches Primatenzentrum
Virology and Immunology
Kellnerweg 4
D-37077 Goettingen Germany
Tel. : 49-551-3851155
Fax. : 49-551-3851184
ghunsma@gwdg.de
HUNTER NORA
West Mains Road
Tel. : + 44 (0) 131 66
nora.hunter@bbsrc.ac.uk
HURAUX JEAN-MARIE
Hôpital Pitié-Saplétrière
Laboratoire de Virologie CERVI
83 Bd de l'hôpital
75013 Paris France
Tel. : 01 42 17 74 01
jean-marie.huraux@psl.ap-hop-paris.fr
201
Participant List
IMAMURA MORIIKAZU M
Prion Disease Research Center
Kannondai 3-1-5
305-0856 Tsukuba Japan
Tel. : +81-29-838-7757
imamuram@affrc.go.jp
IRONSIDE JAMES W
Crewe Road
Tel. : 0131.537.1980
Fax. : 0131.537.3056
james.ironside@ed.ac.uk
KNOX J. DAVID
Health Canada
Host Genetics and Prion Diseases
1015 Arlington St.
ManitobaR3E 3R2 Winnipeg Canada
Tel. : 204-789-6083
Fax. : 204-789-5021
david_knox@hc-sc.gc.ca
KAIMANN TINA KATHARINA
Heinrich-Heine-University
Institut of Physical Biology
Universitätsstr. 1
Tel. : +49 211 81 1531
kaimann@biophys.uni-duesseldorf.de
KOFFI YAO
IMTIX-SANGSTAT
Manufacturing
1541 av Marcel Mérieux
69280 Marcy l'Etoile France
Tel. : 04 37 22 58 08
Fax. : 04 37 22 58 98
yao.koffi@genzyme.com
IULINI BARBARA
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : +39-0112686261
Fax. : +39-0112686322
cea@izsto.it
KAO CLARA
Danone
Centre de Sécurité des Aliments
RD 128
91767 Palaiseau France
Tel. : +33 1 69 35 74 64
Fax. : +33 1 69 35 76 97
clara.kao@danone.com
JACKMAN ROY
TSE Molecular Biology
New Haw
Tel. : +44 (0)1932 357
Fax. : +44 (0)1932 357
r.jackman@vla.defra.gsi.gov.uk
KARIV-INBAL ZEHAVIT
Hadassah Hebrew University Medical
Center
Kiryat Hadassah P.O.B. 12000
Tel. : 972-2-6777858
Fax. : 972-2-6429441
zehavitinbal@bezeqint.net
JACOB CHRISTINE
INRA
Mathématiques et Informatique
appliquées
Domaine de Vilvert
Tel. : 01 34 65 22 25
Fax. : 01 34 65 22 17
christine.jacob@jouy.inra.fr
202
KAESERMANN FABIAN
University of Bern
Department of Biochemistry and
Chemistry
Freiestrasse 3
3012 Bern Switzerland
Tel. : +41-31-6314887
kaesermann@ibc.unibe.ch
KASPAR JAN
O.K. Servis BioPro, s.r.o.
Diagnostics
Na Harfe 9
190 00 Prague Prague 9
j.kaspar@oks.cz
JARRIGE NATHALIE
AFSSA
Epidemiology
69364 Lyon France
Tel. : 04 78 72 65 43
n.jarrige@lyon.afssa.fr
KAZUO TSUKUI
Tokyo Metropolitan Red Cross Blood
Center
Laboratory Department
03/01/1927
Hiroo
150-0012 Shibuya-ku Japan
Tel. : 81-03-5485-6013
Fax. : 81-03-3406-7892
ka-tsukui@tokyo.bc.jrc.or.jp
JAT PARMJIT P
Department of Neurodegenerative
Disease
National hospital for Neurology and
Neurosurgery
Queen Square
WC1N 3BG London England
Tel. : 44-20 7837 3611 x 4452
f.vaughan@prion.ucl.ac.uk
KHALILI AZY
Institute of Neurology, Univeersity
College London
Neurodegenerative Diseases, MRC Prion
Unit
Queen Square
WC1N 3BG London UK
Tel. : (44)(207)(676-2180)
Fax. : (44)(207)(676-2187)
a.khalili@prion.ucl.ac.uk
JEFFREY MARTIN
VLA Lasswade
R&D
Bush Loan
Penicuik
Tel. : 44-1314456169
m.jeffrey@vla.defra.gsi.gov.uk
KLASS MICHAEL R
Abbott Laboratories
D09PT
AP20
Illinois60064-6013 Abbott Park USA
Tel. : 847-937-2148
Fax. : 847-935-4182
michael.klass@abbott.com
KORTH CARSTEN
University of Duesseldorf
Neuropathology
Moorenstrasse 5
Tel. : +49-211-811 6153
Fax. : +49-211-811 7804
korth@med.uni-duesseldorf.de
KOVACS GABOR GEZA
National Institute of Psychiatry and
Neurology
Neuropathology
Huvosvolgyi ut 116.
H-1021 Budapest Hungary
Tel. : +36-1-391-5409
Fax. : +36-1-391-5409
kovacsgg@opni.hu
KREMER WERNER
University of Regensburg
Institute for Biophysics
Universitaetsstr. 33
93053 Regensburg Germany
Tel. : +49-941-9432285
Fax. : +49-941-9432479
norman.kachel@biologie.uniregensburg.de
KRENN BEA
Dutch Cancer Institute
Tumorbiology
Plesmanlaan 121-H4
1066 CX Amsterdam Netherlands
Tel. : +31-20 512 2017
Fax. : +31-20 512 2029
b.krenn@nki.nl
KRISTENSSON KRISTER S
Karolinska Institutet
Dept Neuroscience
Retzius vaeg 8
SE-171 77 Stockholm Sweden
Tel. : 46-8-524 87825
Fax. : 46-8-325 325
krister.kristensson@neuro.ki.se
KUCZIUS THORSTEN
Institute for Hygiene
University Hospital Muenster
Robert Koch Strasse 41
48149 Muenster Germany
Tel. : +49-251-8352217
Fax. : +49-251-8357175
tkuczius@uni-muenster.de
Participant List
LARRAMENDY CLAIRE
CEA
DSV/DRM/GIDTIP
18 route du panorama
Tel. : 01 46 54 82 83
larramendy@dsvidf.cea.fr
LECLERE EDWIGE
University Claude Bernard
69
12 rue des pierres plantées
69001 Lyon France
Tel. : 06 87 91 37 78
el.lf@laposte.net
LACHMANN INGOLF
Roboscreen GmbH
Cell Culture & Antibody Production
Delitzscher Str. 135
4129 Leipzig Germany
Tel. : 49-3419725970
Fax. : 49-3419725979
ilachmann@roboscreen.com
LASMÈZAS CORINNE I
CEA
DRM
BP6
Tel. : 33 1 46 54 78 05
Fax. : 33 1 46 54 77 26
lasmezas@cea.fr
LECROIX STÉPHANIE
Bio-Rad
92430 Marnes la Coquette france
Tel. : 01 47 95 69 58
Fax. : 01 47 95 61 11
LAFFONT ISABELLE
CNRS-CEA URA2210
SHFJ-DRM-DSV
4, place du Général Leclerc
91401 Orsay France
Tel. : 01-69-86-77-62
Fax. : 01-69-86-77-45
laffont@shfj.cea.fr
LAUDE HUBERT
INRA
Domaine de Vilvert
78350 Jouy en josas France
Tel. : 01 34 65 26 00
Fax. : 01 34 65 26 21
laude@biotec.jouy.inra.fr
LANE AMIN
Microsens Biotechnologies
Technical
2 Royal College Street
NW1 0TU London UK
Tel. : +44-20 7691 214
roger.rosedale@microsens.co.uk
LAURSEN HENNING
Rigshospitalet
Neuropathology 6301
Blegdamsvej 9
DK-2100 Copenhagen Denmark
Tel. : +45 35 45 6321
Fax. : +45 35 45 6323
hlaursen@rh.dk
LA BONNARDIÈRE CLAUDE
INRA
Virologie & Immunologie Moléculaires
Domaine de Vilvert
Tel. : 01 34 65 26 43
Fax. : 01 34 65 26 21
clb@jouy.inra.fr
LANGE MARC
Bio-Rad
FSD
marc_lange@bio-rad.com
LANGE REINHARD
Université Montpellier 2
Biologie-Santé, cc 105
Place Eugène Bataillon
Tel. : 04 67 14 33 85
Fax. : 04 67 14 33 86
lange@montp.inserm.fr
LANGEVELD JAN PM
CIDC-Lelystad
TSEs, Mycobacteria and Brucellosis
Edelhertweg 15
8219 PH Lelystad The Netherlands
Tel. : 0320-237217
Fax. : 0320-238050
jan.langeveld@wur.nl
LAPLANCHE JEAN-LOUIS
Université Paris 5
Biologie Cellulaire
Faculté de Pharmacie
4 avenue de l'observatoire
laplanch@pharmacie.univ-paris5.fr
LAZARINI F.
Institut Pasteur
biologie structurale et chimie
25 rue du dr roux
75015 Paris France
Tel. : 01 45 68 87 15
lazarini@pasteur.fr
LE PROVOST FABIENNE
INRA
Animal genetic
LGBC-INRA
Tel. : 01 34 65 25 69
Fax. : 01 34 65 24 78
Fabienne.LeProvost@jouy.inra.fr
LEBLANC VIRGINIE
CEA
GIDTIP
route du panorama
Tel. : 01-46-54-81-05
leblanc@dsvidf.cea.fr
LEBON PIERRE
Hôpital Saint Vincent de Paul
Service de Bacteriologie-Virologie
74-82 Avenue Denfert Rochereau
75014 Paris France
Tel. : 33 1 40 48 82 43
Fax. : 33 1 40 48 83 51
p.lebon@svp.ap-hop-paris.fr
LEDOUX JEAN-MARIE
Vétérinaire
17 rue Jules Guesde
59390 Lys-Lez-Lannoy France
Tel. : 03 20 75 91 85
ledoux.jean-marie@wanadoo.fr
LEFEBVRE-ROQUE MAXIME
CEA
SNV
18 route du panorama
Tel. : 01 46 54 82 83
Roque@dsvidf.cea.fr
LEGNAME GIUSEPPE A
IND-UCSF
Neurology
513 Parnassus Avenue
Tel. : 415-514-1580
Fax. : 415-476-8386
legname@itsa.ucsf.edu
LEHMANN SYLVAIN
CNRS
Intitut of Human Genetics
141, rue de la Cardonille
Tel. : +33-4 99 61 99 31
Sylvain.Lehmann@igh.cnrs.fr
LEHTO MARTY T
University of Toronto
CRND
Rm 328 Tanz Neuroscience Bldg.
6 Queen's Park Crescent West
OntarioM5S 3H2 Toronto Canada
Tel. : 416-978-0772
Fax. : 416-978-1878
marty.lehto@utoronto.ca
LELIVELD RUTGER
Heinrich-Heine University
Neuropathology
Moorenstrasse 5
Tel. : 0049-211-8118653
Fax. : 0049-211-8117804
rutger.leliveld@uni-duesseldorf.de
203
Participant List
LOMBARD MICHEL F
IABs
Veterinary Vice Presidency
22, rue Crillon
69006 Lyon France
Tel. : + 33 4 78 93 90 89
lombard.family@wanadoo.fr
MADEC JEAN-YVES
AFSSA
Prion team
F-69364 Lyon France
jy.madec@lyon.afssa.fr
LUCOTTE GERARD
Center of Molecular Neurogenetics
Biology
44, rue Monge
75005 Paris France
Tel. : 01 39 72 71 06
Fax. : 01 39 72 79 27
lucotte@hotmail.com
MAHAL SUKHVIR P
MRC Prion Unit
Neurodegenerative Diseases
Queen Square
WD3 2GP London UK
Tel. : 44-2076762187
Fax. : 44-2076762180
s.mahal@prion.ucl.ac.uk
LUHR KATARINA M
Neuroscience
Retzius väg 8
Tel. : 46-8-52487879
Fax. : 46-8-325325
katarina.luhr@neuro.ki.se
MAJTENYI KATALIN
Institute of Psychiatry and Neurology
Neuropathology
Huvosvolgyi ut 116.
H-1021 Budapest Hungary
Tel. : 36-1-391-5400
Fax. : 36-1-391-5409
majtenyi@opni.hu
LIBERSKI PAWEL
Medical University of Lodz
Molecular Pathology and
Neuropathology
Czechoslowacka 8/10
92216 Lodz Poland
Tel. : 48 42 679 14 77
Fax. : 48 42 679 14 77
ppliber@csk.am.lodz.pl
LULEY SANDRA
Abbott GmbH & Co. KG
Area Business Development
Max-Planck-Ring 2
65205 Wiesbaden Germany
Tel. : 49-15114038960
Fax. : 49-6122581668
sandra.luley@abbott.com
MALLINSON GARY
International Blood Group Reference
Laboratory
Prion
Southmead Road
BS10 5ND Bristol UK
Tel. : 44-7711447241
g.mallinson@prion.ucl.ac.uk
LIMIDO LUCIA
Istituto Nazionale Neurologico
U.O. Neuropatologia
Via Celoria, 11
20133 Milano Italy
Tel. : 39/022394260
Fax. : 39/0270638217
llimido@istituto-besta.it
LUTZ JENS
University of Konstanz
Molecular Toxicology Group
Jacob Burckhardt Str. 31
D-78464 Konstanz Germany
Tel. : +49-7531 884058
Fax. : +49-7531 884033
jens.lutz@uni-konstanz.de
MANSON JEAN
Ogston Building
West Mains Road
EH9 3JF Edinburgh United Kingdom
Tel. : 44-1316675204
Fax. : 44-1316683872
jean.manson@bbsrc.ac.uk
LINNE TOMMY
Swedish University of Agricultural
Sciences
Molecular Biosciences
Box 588, Biomedical Centre
S-75 123 Uppsal Uppsala Sweden
Tel. : 46184714036
linnet@bmc.uu.se
Lyahyai Jaber
Universidad de Zaragoza
Anatomie, Embriologie and Animal
Genetic
Miguel Servet 177
Tel. : (34) 976 76 16 00 Ext 4203
Fax. : (34) 976 76 16 12
508223@docto.unizar.es
MANUELIDIS LAURA
Yale medical school
Surgery
333 Cedar St, room FMB11
CT6510 New Haven USA
Tel. : 203-785-4442
laura.manuelidis@yale.edu
LIPSOMB IAN IP
SO16 7PX Southampton Great Britain
Tel. : 00-44 (0) 2380592034
Fax. : 00-44 (0) 2380594459
I.Lipscomb@soton.ac.uk
LYNHAM BERNADETTE
University College Dublin
Department of Zoology
Belfield
Dublin 4 Dublin Ireland
Tel. : 00353 1 716 208
bernadette.lynam@ucd.ie
LOEWER JOHANNES
Paul-Ehrlich-Institut
President
Paul-Ehrlich-Street 51-59
Tel. : +49 (0) 6103 77 1008
Fax. : +49 (0) 6103 77 1240
joeme@pei.de
MACDIARMID STUART C
New Zealand Food Safety Authority
Programme Development Group
PO Box 2385
South Tower
86 Jervois Quay
6000 Wellington New Zealand
Tel. : +64-4-463 2500
Fax. : +64-4-463 2530
stuart.macdiarmid@nzfsa.govt.nz
LEONE PAOLO
Consiglio Nazionale delle Ricerche
Ist. Biologia e Biotecnologia Agraria
Via F.lli Cervi 93
20090 Segrate Italy
Tel. : +39-02 21 01 35 07
Fax. : +39-02 26 41 21 35
leone@ibba.cnr.it
LESCEU STÉPHANIE
Institut Pourquier
R&D
326 rue de la Galéra
stephanie.lesceu@institut-pourquier.fr
LEUTENEGGER THOMAS
Prionics AG
Area Management
Wagistr 27 A
Tel. : 0041 44 200 2000
Fax. : 0041 44 200 2010
thomas.leutenegger@prionics.ch
204
MARELLA MATHIEU
IPMC
CNRS
660 route des lucioles
6560 Valbonne France
Tel. : 04 93 95 77 67
marella@ipmc.cnrs.fr
MARQUEZ MERCEDES
Autonomous University of Barcelona
Animal Tissue Bank of Catalunya
(BTAC)
Veterinary Faculty
UAB Campus
8193 Bellaterra Spain
Tel. : +34 93 581 12 35
Fax. : +34 93 581 31 42
mercedes.marquez@uab.es
Participant List
MARTIN STUART
VLA Lasswade
R&D
Bush Loan
Penicuik
Tel. : 44-1314456169
s.martin@vla.defra.gsi.gov.uk
MAYER- SONNENFELD TEHILA
Hadassah- Hebrew University medical
center
Kiryat Hadassah P.O.B 12000
Tel. : 972-2-6777858
Fax. : 972-2-6429441
tehila@md.huji.ac.il
MOLESWORTH ANNA M
Communicable Disease Surveillance
Centre
61 Colindale Avenue
NW9 5EQ London UK
Tel. : +44-(0)20 8200 6868
Fax. : +44-(0)20 8200 7868
anna.molesworth@hpa.org.uk
MARTIN TREVOR C
VLA
TSE molecular biology
New Haw
Addlestone
KT15 3NB SURREY U.K.
Tel. : 44-1932357566
Fax. : 44-1932357239
t.c.martin@vla.defra.gsi.gov.uk
MCDONNELL GERALD E
STERIS
Technical Affairs
Steris House
Jays Close
Viables
RG22 4AX Basingstoke UK
Tel. : 44-1256866560
Fax. : 44-1256866503
gerry_mcdonnell@steris.com
MONLEÒN EVA
Animal Pathology
Facultad de Veterinaria
Miguel Servet 177
Tel. : 0034976761000 e
Fax. : 34976761608
emonleon@unizar.es
MARTÌN SERGIO FRANCISCO
INIA-CISA
BMCP
Ctra. de Algete a El Casar, km. 8.100
28130 Madrid Spain
Tel. : 916202300 ext 1
Fax. : 916202247
mgonzalez@inia.es
MARTIN-BURRIEL INMACULADA
National Reference Centre for TSEs
Miguel Servet 177
Tel. : 34 976 761 622
Fax. : 34 976 761 612
minma@unizar.es
MASTERS COLIN L
The University of Melbourne
Department of Pathology
Parkville
3010 Victoria Australia
Tel. : +61 38 344 5868
Fax. : +61 38 344 4004
c.masters@unimelb.edu.au
MATEJ RADOSLAV
Czech Ntional Reference lab of human
TSE
Pathology
MATTEI VINCENZO
University La Sapienza of Rome
Department of experimental medicine
and pathology
Viale regina elena 324
161 Rome Italy
Tel. : 00 -39-0649972675
Fax. : 00 -39-064454820
vincenzo.mattei@libero.it
MATTHEWS DANNY
Centre for Epidemiology and Risk
Analysis
New Haw
Addlestone
KT15 3NB Addlestone United Kingdom
Tel. : 44-1932-359512
Fax. : 44-1932-354929
d.matthews@vla.defra.gsi.gov.uk
MCGOVERN GILLIAN
VLA Lasswade
R&D
Bush Loan
Penicuik
Tel. : 44-1314456169
g.mcgovern@vla.defra.gsi.gov.uk
MCINTYRE MARIE
Compton Laboratory
Compton
RG20 7NN Newbury UK
Tel. : 44-1635578411
Fax. : 44-1635577237
marie.mcintyre@bbsrc.ac.uk
MEHL MARTIN
Prionics AG
Research
Wagistr. 27 a
CH-8952 Schlieren Switzerland
Tel. : +41 44 200 2000
Fax. : +41 44 200 2010
martin.mehl@prionics.ch
MILLER BRIAN J
Provincial Government
Agriculture / Food Safety
O.S. Longman Laboratory
6909-116 Street
AlbertaT6H 4P2 Edmonton Canada
Tel. : (780) 427-8201
Fax. : (780) 422-3438
brian.miller@gov.ab.ca
MITROV EVA, JUDITA E
Slovak Medical University
Department of prion diseases
Limbov 12
841 04 Bratislava Slovak Republic
Tel. : 421 2 59 36 9
Fax. : 421 2 59 36 95
mitrova@upkm.sk
MOJZIS MARTIN
State Veterinary Institute Zvolen
Virology
Na Harfe 9
j.kaspar@oks.cz
MONTAG JUDITH
Immunology and Virology
Kellnerweg 4
Tel. : +49-551-3851-147
jmontag@dpz.gwdg.de
MONTRASIO FABIO
Paul-Ehrlich-Institute
Prion Research Group, Pr1
Paul-Ehrlich-Strasse 51-59
Tel. : +49-6103-77 4018
Fax. : +49-6103-77 1234
monfa@pei.de
MORALES CAMARZANA MONICA
CISA-INIA
BMCP
Ctra. Algete a El Casar km 8.100
Tel. : 916202300
Fax. : 916202247
morales@inia.es
MOREL NATHALIE
CEA
DRM/SPI
Bat. 136, CEA saclay
Tel. : 01 69 08 77 04
Fax. : 01 69 08 59 07
nathalie.morel@cea.fr
MORENO-ROMIEUX CAROLE
INRA
Genetique animale
SAGA BP 27
31326 Castanet Tolosan cedex France
Tel. : 0033.5.61.28.51.91
Fax. : 0033.5.61.28.53.53
moreno@toulouse.inra.fr
MORIGNAT ERIC
AFSSA
Epidemiology
69364 Lyon France
Tel. : 04 78 69 68 40
e.morignat@lyon.afssa.fr
205
Participant List
MOTZKUS DIRK
Deutsches Primatenzentrum (DPZ)
Virology and Immunology
Kellnerweg 4
37077 Göttingen Lower Saxony
dmotzkus@dpz.gwdg.de
NAGL IVAN
State Veterinary Institute Prague
Pathology
Na Harfe 9
j.kaspar@oks.cz
MOUDJOU MOHAMMED
INRA
Virologie et Immonologie Moléculaires
Domaine de Vilvert
Tel. : 33-1 34 65 26 42
Fax. : 33-1 34 65 26 21
moudjou@jouy.inra.fr
NANDI PRADIP K
Agronomique
Pathologie Infectieuse et Immunologie
Route National 44
Tel. : 33 2 47 42 78 87
Fax. : 33 2 47 42 77 79
nandi@tours.inra.fr
MOUILLET-RICHARD SOPHIE
Ministère de l'agriculture
Direction générale de l'alimentation
251, rue de Vaugirard
75732 cedex 15 Paris France
Tel. : 01 49 55 58 94
sophie.mouillet@agriculture.gouv.fr
MOUTHON FRANCK
CEA/DSV/DRM/GIDTIP
DRM
Route du panorama
Tel. : 33 1 46 54 82 83
mouthon@dsvidf.cea.fr
MOYA KENNETH L
CNRS
Departement de Recherche Medicale
CEA-CNRA URA 2210
SHFJ
4, place du General Leclerc
91406 Orsay France
Tel. : 01 69 86 77 11
moya@shfj.cea.fr
MÜLLER HENRIK
Heinrich Heine - University
Institute of Physical Biology
Universitätsstrafle 1
Tel. : +49211/81-15314
Fax. : +49211/81-15167
mueller@biophys.uni-duesseldorf.de
MURDOCH HEATHER
HPA-Porton Down
TSE Research
Porton Down
Tel. : +44 (0) 1980 612100
Fax. : +44 (0) 1980 612731
heather.murdoch@hpa.org.uk
206
MURRAY DEIRDRE
Armines / Ecole des Mines de Paris
Pole Cindyniques
BP 207
Rue Claude Daunesse
6904 Sophia Antipolis cedex France
Fax. : 04 93 95 75 81
deirdre.murray@cindy.ensmp.fr
NAPPI RAFFAELLA
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686266
Fax. : 39-0112686322
cea@izsto.it
NASH ANGUS
National institute for medical research
Physical biochemistry
The Ridgeway
NW7 1AA London UK
Tel. : 44 20 8816 2089
PBAYLEY@NIMR.MRC.AC.UK
NETHERWOOD TRUDY
Food Standards Agency
BSE Division - Research branch
Aviation House
Room 315C
125, Kingsway
WC2B 6NH London UK
Tel. : +44(0)207276832
Fax. : +44(0)207276836
trudy.netherwood@foodstandards.gsi.gov.
uk
NGUYEN HAI CHI
Laboratoire de pathologie du bétail
7 Avenue du Général de Gaulle
Tel. : 01 43 96 72 35
Fax. : 01 43 96 70 55
cecilechi@yahoo.com
NICOLAE ALEXANDRU
Health (IDAH)
IDAH
sector 5
Tel. : 00-40-21-410.13.90
Fax. : 00-40-21-411.33.94
office@idah.ro; alexandru.nicolae@idah.ro
NDEDE PAUL K
DEFRA
VTSED
1A page street
AREA 205
SW1P 4PQ London UK
paul.ndede@defra.gsi.gov.uk
NICOLAE STEFAN M
Health (IDAH)
IDAH
sector 5
Tel. : 00-40-21-410.13.90
Fax. : 00-40-21-411.33.94
office@idah.ro; nicolae.stefan@idah.ro
NEARY CHRIS
Beckman Coulter, Inc.
Prion Business Center
4300 N. Harbor Blvd.
CA92835 Fullerton USA
Tel. : 714-773-6600
Fax. : 714-773-8698
cwneary@beckman.com
NIKLES DAPHNE
FG 6/1
Paul-Ehrlich-Straße 51-59
Tel. : +49 6103 77 40 14
Fax. : +49 6103 77 1255
strda@pei.de
NENNESMO INGER
Dept. of Laboratory Medicine, Division
of Patholog
Karolinska University Hospital/
Huddinge
F46
14186 Stockholm Sweden
Tel. : +46-8-58587846
Fax. : +46-8-58581020
inger.nennesmo@kus.se
NISHINA KOREN A
Dartmouth Medical School
Department of Biochemistry
7200 Vail Building, Room 302
NH03755-3844 Hanover USA
Tel. : 603-650-1194
Fax. : 603-650-1128
Koren.A.Nishina@dartmouth.edu
NESPOULOUS GILLES
Bio-Rad
3 boulevard Raymond PoincarÈ
Tel. : 01 47 95 61 89
Fax. : 01 47 95 61 11
NIXON RANDAL
Oregon Health & Sciences University
Pathology
3181 SW Sam Jackson Park Rd
OR97239 Portland USA
Tel. : 503-494-6758
Fax. : 503-494-2845
nixonr@ohsu.edu
Participant List
NONNO ROMOLO
Viale Regina Elena, 299
161 Rome Italy
Tel. : 0039-06-49902854
Fax. : 0039-06-49387077
romolo.nonno@iss.it
OSMAN AWAD
Roboscreen GmbH
Protein Research
Delitzscher Strasse 135
4129 Leipzig Germany
Tel. : 49341972597
Fax. : 49341972597
aosman@roboscreen.com
PARRA ARRONDO BEATRIZ
CISA-INIA
BMCP
Ctra. Algete a EL Casar km 8.100
Madrid28130 Valdeolmos Spain
Tel. : 916202300
Fax. : 916202247
parra@inia.es
NORDSTROM ELIN K
Dept Neuroscience
Retzius vaeg 8, B2:5
Dept Neuroscience
Tel. : +46-8-524 878 10
Fax. : +46-8-325 235
elin.nordstrom@neuro.ki.se
OTTO MARKUS
University of Goettingen
Department of Neurology
Robert-Koch Str. 40
37070 Goettingen Germany
Tel. : 49-551-398404
Fax. : 49-551-3914449
motto@gwdg.de
PASTORE MANUELA
CNRS
Institut de Génétique humaine
141 rue de la Cardonille
Tel. : 0033-4 99 61 99 30
Fax. : 0033-4 99 61 99 30
mxp82@cwru.edu
PAGAT ANNE-MARIE
Aventis Pasteur
Development
1541 avenue Marcel Mérieux
69280 Marcy l'Etoile France
anne-marie.pagat@aventis.com
PEDEN ALEXANDER H
Crewe Road
Tel. : 0131-537-1980
Fax. : 0131-537-1404
A.Peden@ed.ac.uk
NOVAK MICHAL
Institute of Neuroimmunology
Neurosciences
Dubravska No9
845 10 Bratislava Slovakia
Tel. : +421-905 609 558
Fax. : +421-2 5477 4276
Michal.Novak@savba.sk
NUGIER JÉRÔME
CEA
DRM/SPI
Bâtiment 136
Tel. : 01.69.08.77.04
Fax. : 01.69.08.59.07
nugier@cea.fr
O'CONNELL DAVID
University College Dublin
Department of Zoology
Belfield
Dublin 4 Dublin Ireland
Tel. : (01) 716 2089
Fax. : (01) 706 1152
david.oconnell@ucd.ie
OESCH BRUNO
Prionics AG
R&D
Wagistr. 27a
Tel. : +41 44 200 20 00
Fax. : +41 44 200 20 10
bruno.oesch@prionics.ch
OLIVER JO
Technical
NW1 0TU London UK
Tel. : +44 20 7691 214
ONODERA TAKASHI
University of Tokio
Molecular immunology
YAYOI 1-1-1, BUNKYO-KU
113-8657 Tokyo Japan
Tel. : 81-3-5841-5196
Fax. : 81-3-5841-8020
aonoder@mail.ecc.u-tokyo.ac.jp
PAISLEY LARRY G
Danish Institute for Food and Veterinary
Research
Epidemiology and Risk Analysis
M¯rkh¯j Bygade 19
DK-2860 S¯borg Denmark
Tel. : 45-7234-7330
Fax. : 45-7234-7001
lpa@dfvf.dk
PALSDOTTIR A.
Institute for Experimental Pathology
Molecular Biology
Vesturlandsvegur
IS-112 Reykjavik Iceland
Tel. : 354-5674700
Fax. : 354-5674714
astripal@hi.is
PANAGIOTIDIS CYNTHIA H
Aristotle University of Thessaloniki
Pharmacology/Pharmacy
Paneptistemopoli
54124 Thessaloniki Greece
Tel. : 30-2310997637
Fax. : 30-2310997645
cpanagi@pharm.auth.gr
PAQUET SOPHIE
INRA
virologie et immunologie moléculaires
domaine de Vilvert
Tel. : 01.34.65.26.11
Fax. : 01.34.65.26.21
spaquet@jouy.inra.fr
PARCHI PIERO
University of Bologna
Department of Neurological Sciences
Via Foscolo 7
40123 Bologna Italy
Tel. : +39-051-6442151
Fax. : +39-051-6442214
parchi@neuro.unibo.it
PELETTO SIMONE
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : +39-0112686245
Fax. : +39-0112686322
cea@izsto.it
PEOC'H KATELL
Faculté des Sciences Pharmaceutiques et
Biologique
Laboratoire de Biologie Cellulaire, EA
3621
4 Avenue de l'Observatoire
75006 Paris France
Tel. : 01 49 95 64 39
katell.peoch@lrb.ap-hop-paris.fr
PERETZ DAVID
Chiron
Protein Chemistry
4560 Horton Street
California94608-2916 Emeryville USA
Tel. : (510)923-4432
DavidPeretz@chiron.com
PERRIER VÉRONIQUE
Institut de Génétique Humaine
UPR1142 du CNRS
141 rue de la Cardonille
Tel. : 04 99 61 99 30
Fax. : 04 99 61 99 01
vperrier@igh.cnrs.fr
PETERS PETER J
Dutch Cancer Institute-A. van
Leeuwenhoek Hospital
Tumorbiology
Plesmanlaan 121-H4
Tel. : 31 (0)20 512 2031
Fax. : 31 (0)20 512 2029
p.peters@nki.nl
207
Participant List
PETRAKIS SPYROS I
Paneptistemiopoli
Tel. : +30-2310-997637
Fax. : +30-2310-997645
spyrospetrakis@hotmail.com
POURQUIER JÉRÔME
Institut Pourquier
R&D
326, rue de la GalÈra
Tel. : 00 33 (0)4 99 23 24 25
Fax. : 00 33 (0)4 67 04 20 25
j.pourquier@institut-pourquier.fr
RAZGA JAKUB
Bio-Rad
Life Sciences
Nad ostrovem 1119
147 00 Prague Czech Republic
Tel. : +420-2 4143 0532
Fax. : +420-2 4143 1642
jakub_razga@bio-rad.com
PEYRIN JEAN-MICHEL
Université Paris Sud
Biotechnology
5 rue Jean-Baptiste Clément
92296 Chatenay Malabry France
Tel. : 01 46 83 58 65
jean-michel.peyrin@cep.u-psud.fr
POURQUIER PHILIPPE
Institut Pourquier
R&D
326, rue de la Galéra
Tel. : 00 33 (0)4 99 23 24 25
Fax. : 00 33 (0)4 67 04 20 25
philippe.pourquier@institut-pourquier.fr
RELAÒ GINES AROA
CISA-INIA
BMCP
Ctra. Algete a El Casar km 8.100
Tel. : 916202300
Fax. : 916202247
rgines@inia.es
PREUSSER MATTHIAS
Medical University Vienna
Institute of Neurology, AKH 4J, POB 48
Waehringer Guertel 18-20
A-1097 Vienna Austria
Tel. : +43-1-40400-55
Fax. : +43-1-40400-55
matthias.preusser@akh-wien.ac.at
RES PIETER
Netherlands Cancer Institute
Tumorbiology
Plesmanlaan 121
Tel. : 31-20-5122015
p.res@nki.nl
PRIVAT NICOLAS
INSERM
U360
Salpétrière Hospital
47, Bd de l'Hôpital
75013 Paris France
Tel. : 01 42 16 18 81
Fax. : 01 44 23 98 28
nicolas.privat1@libertysurf.fr
REZAEI H.
Agronomique (INRA)
Unité de Virologie et Immunologie
Moléculaire
Centre de recherche de Jouy en Josas
Domain de Vilvert
Tel. : 33 1 34 65 27 89
Fax. : 33 1 34 65 26 21
rezaei@jouy.inra.fr
PRUSINER STANLEY B
University of California, San Francisco
Institute for Neurodegenerative Diseases
Box 0518
CA 94143-0518 San Francisco USA
Tel. : 415.476.4482
Fax. : 415.476.8386
stanley@ind.ucsf.edu
REZNICEK LUKAS
Ludwig-Maximilians-Universität
Zentrum für Neuropathologie
81377 München Deutschland
Tel. : 0172/7384836
Lukas-Reznicek@gmx.de
RAEBER ALEX
Prionics AG
Research
Wagistr. 27 a
Tel. : 0041 44 200 2000
Fax. : 0041 44 200 2010
alex.raeber@prionics.ch
RICKETTS MAURA N
Health Canada
CIDPC
Building 6 Tunney's Pasture
Address Locator: 0601E2
OntarioK1A 0K9 Ottawa Canada
Tel. : 001 613 946-5072
Fax. : 001 613 952-6668
maura_ricketts@hc-sc.gc.ca
RAMOS AZEVEDO
Laboratòrio Nacional de Investigação
Veterinària
Pathology
Rua dos Lagidos
Lugar da Madalena
4485-655 Vairão Portugal
Tel. : 00.351.252.660.600
Fax. : 00.351.252.660.695
azevedo.ramos@lniv.min-agricultura.pt porto.di
RIESNER DETLEV
Heinrich-Heine-Universität D¸sseldorf
Universitätsstr. 1
Geb. 26.12.U1
D-40225 Düsseldorf Germany
Tel. : +49-211-81-1484
Fax. : +49-211-81-1516
riesner@biophys.uni-duesseldorf.de
PHILIPPE SANDRINE
AFSSA
Epidemiology
69364 Lyon France
Tel. : 33 (0)4 78 72 6
Fax. : 33 (0)4 78 61 9
s.philippe@lyon.afssa.fr
PIENING NIKLAS
Ludwig-Maximilians-Universität
M¸nchen
Zentrum für Neuropathologie und
Prionforschung
Feodor-Lynen-Strasse 23
Tel. : 49-89-2180-78042
Fax. : 49-89-2180-78037
Niklas.Piening@med.uni-muenchen.de
PIETRELLA MARCO
Prion research group Pr1
Paul-Ehrlich-Str 51-59
piema@pei.de
POCCHIARI MAURIZIO
Cell Biology and Neuroscience
161 Rome Italy
Tel. : 39-649903202
Fax. : 39-649903012
pocchia@iss.it
PONTE MAYA L
UCSF
Medical Anthropology
2614 41st st. NW #4
DC20007 Washington USA
Tel. : (202) 965-7770
mponte@itsa.ucsf.edu
208
POTTGIEflER CHRISTINE
Federal Research Centre for Virus
Diseases of Animals
Institute for Epidemiology
Seestr. 55
16868 Wusterhausen Germany
Tel. : 49-3397980174
Fax. : 49-3397980200
christine.pottgiesser@wus.bfav.de
RAYON FRÉDÉRIC
Abbott GmbH & Co. KG
Area Business Development
Max-Planck-Ring 2
65205 Wiesbaden Germany
Tel. : +49-15114038960
Fax. : +49-6122581668
sandra.luley@abbott.com
RIGOU PEGGY
INRA
Biologie physico chimique des prions,
VIM
domaine de Vilvert
78350 Jouy en josas France
Tel. : 01 34 65 26 03
prigou@jouy.inra.fr
Participant List
RIINA MARIA VITTORIA
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-3480652562
mariavittoriariina@yahoo.it
SALA MARCELLO
IZS - Lazio e Toscana
178 Rome Italy
Tel. : +39.06.79099473
Fax. : +39.06.79099462
msala@rm.izs.it
SCHELCHER FRANÇOIS
National Veterinary School
Animal Health
23 chemin des Capelles
Tel. : 05.61.19.38.37
Fax. : 05.61.19.38.34
f.schelcher@envt.fr
RODRIGUEZ AGUSTIN
Institut de Neuropatologia. Departament
de Biologi
C/Feixa llarga sn
8907 Hospitalet de Llobregat Spain
Tel. : 93 260 74 59
cortofuegos@hotmail.com
SALËS NICOLE
CEA
SNV
BP6
60 avenue du Général Leclerc
batiment 02, room 406d
Tel. : 01 46 54 78 20
Fax. : 01 46 54 77 26
NSales@dsvidf.cea.fr
SCHELL JENS
CEA
DRM/GIDTIP
18, Route du Panorama
92265 Paris France
Tel. : 33 1 46 54 95 86
jens_schell@web.de
RODRÌGUEZ BENITO JOSE ANTONIO
CISA - INIA
BMCP
Carretera Algete el casar s/n
Tel. : 916202300
Fax. : 916202246
rbenito@inia.es
ROELS STEFAN
CODA/CERVA (Veterinary and
Agrochemical Research Centre
Biocontrol - Pathology
Groeselenberg 99
1180 Brussels Belgium
Tel. : 0032.2.379.05.47
Fax. : 0032.2.379.04.79
stroe@var.fgov.be
ROSEDALE ROGER J
Management
NW1 0TU London UK
Tel. : +44-20 7691 214
ROSSET MARTINE
INSERM
E 209
Hopital St Antoine
Bat Kourislky
184 rue du Fbg St Antoine
75012 Paris France
Tel. : 01 43 44 34 42
Fax. : 01 43 40 17 48
rosset@st-antoine.inserm.fr
RYBNER CATHERINE
Institut Pasteur
Biologie Structurale et Chimie
28 rue du Docteur Roux
75015 Paris France
Tel. : 01.45.68.87.15
Fax. : 01.45.68.87.68
crybner@pasteur.fr
RYOU CHONGSUK
University of California San Francisco
513 Parnassus Ave. HSE-774
Box 0518
Tel. : 415-502-1948
cryou@itsa.ucsf.edu
SALMONA MARIO
Mario Negri Institute
Molecular Biochemistry and
Pharmacology
Via Eritrea 62
20157 Milan Italy
Tel. : 39/02/39014.1
Fax. : 39/2/3546277
salmona@marionegri.it
SANDBERG MALIN K
Dept. of Neuroscience
Retzius väg 8 B2:5
S-171 77 Stockholm Sweden
Tel. : +46-8-524 87810
Fax. : +46-8-325 325
malin.sandberg@neuro.ki.se
SARRADIN PIERRE
INRA
UR
PII
Tel. : 02 47 42 76 68
Fax. : 02 47 42 75 23
psarrad@tours.inra.fr
SAUNIER ROZENN
EFSA
Biological Hazard
rue de Genève 10
1140 Brussels Belgium
Tel. : 00.32.2.337.22.86
Fax. : 00.32.2.726.69.63
rozenn.saunier@efsa.eu.int
SBAIZ LUCA
Liguria
CEA
Via Bologna, 148
10154 Turin Italy
Tel. : 39-0112686245
Fax. : 39-0112686322
cea@izsto.it
SCHÄTZL HERMANN M
Technical University of Munich
Institute of Virology
Biedersteinerstrasse 29
D-80802 Munich Germany
Tel. : 089-4140-3240
Fax. : 089-4140-3243
schaetzl@lrz.tum.de
SCHERBEL CHRISTINA
Federal Research Centre for Nutrition
and Food
Institute for Microbiology and Toxicology
E.-C.-Baumann-Str. 20
95326 Kulmbach Germany
Tel. : 09221-803227
m-scherbel@baff-kulmbach.de
SCHMITT ANDREAS
LMU Muenchen
ZNP-Muenchen
Feodor-Lynen-Str.23
81377 München Germany
Tel. : 089/2180-78013
aschmitt@inp.med.uni-muenchen.de
SCHNEIDER MICHAEL
University of Munich
German TSE Resarch Platform
D-81377 München Germany
Tel. : 0049-89-2180-78020
Fax. : 0049-89-2180-78037
Michael1.Schneider@med.unimuenchen.de
SCHOLL FRANCESCO
IZS- Lazio e Toscana
178 Rome Italy
Tel. : 39.06.79099424
Fax. : 39.06.79340724
fscholl@rm.izs.it
SCHUMACHER CAROLIN
Merial
Grandes Prophylaxies
69007 Lyon FRANCE
Tel. : (33) 4 72 72 30 00
Fax. : (33) 4 72 72 31 81
carolin.schumacher@merial.com
SELLARAJAH SHANE
Cardiff University
Welsh School of Pharmacy
King Edward VII Avenue
CF10 3XF Cardiff UK
Tel. : 44-2920874000
sellarajahs@cf.ac.uk
209
Participant List
210
SHIMADA KIMI K
National institute of Animal Health
Prion disease research center
Kannondai
Tel. : 81-29-838-7757
Fax. : 81-29-838-7757
kimishim@affrc.go.jp
SOLBRIG-LEBUHN HEIKE
Orchid BioSciences Europe Ltd
Business Development
Kempener Allee 112 c
47803 Krefeld Germany
Tel. : +49 (0) 2151 976656
Fax. : +49 (0) 2151 976657
hsolbrig@orchid-deutschland.de
STOECK KATHARINA
University Hospital of Zürich
Neuropathology
Schmelzbergstr. 12
8091 Zürich Switzerland
Tel. : 0041-1-255 2107
Fax. : 0041-1-255 4402
Katharina.Stoeck@usz.ch
SIHVONEN LIISA HELENA
EELA
Department of Virology
Hameentie 57, P.O.Box 45
00581 Helsinki Helsinki Finland
Tel. : +358-9 3931856
Fax. : +358-9 3931711
liisa.sihvonen@eela.fi
SOMERVILLE ROBERT A
West Mains Road
Tel. : 44-131319823
Fax. : 44-131668387
robert.somerville@bbsrc.ac.uk
STOEHR JAN
University of Duesseldorf
Dep. Physical Biology
Universitaetsstr.1
Tel. : +49-211 81 14703
Fax. : +49-211 81 15167
stoehr@biophys.uni-duesseldorf.de
SIKORSKA BEATA
Medical University of Lodz
Molecular Pathology and
Neuropathology
Czechoslowacka 8/10
92216 Lodz Poland
Tel. : 48 42 6791477
Fax. : 48 42 6791477
elmo@csk.am.lodz.pl
SOUIL E.
Institut Cochin
Plate-forme Histo.petit animal
Institut Cochin/Hop.Cochin
Pavillon G.Roussy
27 rue du Fbg ST Jacques
75014 Paris France
Tel. : 01.40.51.64.52
souil@cochin.inserm.fr
STRAPPATELLI MARIA RITA
Prionics Italia S.R.L.
Head Office
Via prestinari, 2
20158 Milano Italy
Tel. : 39-02-39314357
Fax. : 39-02-39310036
info@prionics.it
SIMMONS MARION M
VLA Weybridge
Pathology
Woodham Lane
Tel. : 44-1932 35756
Fax. : 44-1932 35780
m.m.simmons@vla.defra.gsi.gov.uk
SPIROPOULOS JOHN
Neuropathology
Woodham Lane
New Haw
Addlestone
KT15 3 NB Weybridge England
Tel. : 44-1932357795
j.spiropoulos@vla.defra.gsi.gov.uk
SIMON STEPHANIE S
CEA
DRM/SPI
bat136, CEA Saclay
Tel. : 01 69 08 77 04
Fax. : 01 69 08 59 07
stephanie.simon@cea.fr
STANLEY CHRISTOPHER J
Management
NW1 0TU London UK
Tel. : 44-20 7691 214
christopher.stanley@microsens.co.uk
SIMONEAU STEVE
CEA
GIDTIP
Tel. : 01 46 54 84 87
steve.simoneau@cea.fr
STEINACKER PETRA
University of Göttingen
Neurology
von-Siebold-Str. 5
Tel. : 551399794
psteina@gwdg.de
SKLAVIADIS THEODOROS K
Paneptistemiopoli
Tel. : 30-2310997615
Fax. : 30-2310997645
sklaviad@auth.gr
STEINBACH FALKO
IZW
FG 3
Alfred-Kowalke Str. 17
Tel. : 49/30/5168-206
Fax. : 49/30/5126-104
steinbach@izw-berlin.de
SLIVARICHOVA DANA
Institute of preventive and clinical
medicine
Dep. of prion diseases
Limbov 12
833 03 Bratislava Slovakia
Tel. : +421-2 59369567
Fax. : +421-2 59369585
slivarichova@upkm.sk
STEVERLYNCK CÉLINE C
INSERM
U538
Faculté de Médecine St Antoine
27 rue de Chaligny
75012 Paris France
Tel. : 01.40.01.13.30
Fax. : 01 40 01 13 90
c.steverlynck@wanadoo.fr
STRUCKMEYER THOMAS
Prionics AG
Marketing and Sales
Wagistr. 27 A
Tel. : 0041 44 200 2000
Fax. : 0041 44 200 2010
thomas.struckmeyer@prionics.ch
STUKE ANDREAS W
Virology
Kellnerweg 4
Tel. : 49-5513851153
Fax. : 49-5513851153
astuke@dpz.gwdg.de
STUMPF ROBERT
Abbott Laboratories
ADD OEM Operations
Dept. 081D, Building AP31-3
Illinois60064-6197 Abbott Park United
States
Tel. : 847-937-2441
Fax. : 847-937-9805
Robert.Stumpf@abbott.com
SUGIURA KATSUAKI
Food Safety Commission Secretariat
Information and Emergency Response
Division
2-13-10 Prudential Tower 6F,
Nagata-cho, Chiyoda-ku
100-8989 Tokyo Japan
Tel. : 81-3-5251-9178
Fax. : 81-3-3591-2236
katsuaki.sugiura@op.cao.go.jp
Participant List
SUPERVIE VIRGINIE
INSERM
EMI 0214
56 Boulevard Vincent Auriol
BP 335
75013 Paris France
Tel. : 01 42 16 42 57
Fax. : 01 42 16 42 61
virginie.supervie@ccde.chups.jussieu.fr
SUTTON J. MARK
TSE Research
Porton Down,
Tel. : 00-44 (0) 1980 612649
Fax. : 00-44 (0) 1980 611310
mark.sutton@hpa.org.uk
TAYEBI MOURAD
MRC prion unit
Department of Neurodegerative diseases
3rd floor, Queen Square House
Queen Square
W1CN 3BG London United Kingdom
Tel. : 2073714934
m.tayebi@prion.ucl.ac.uk
TURNO PASQUALE
ABBOTT SPA
SALES
VIA MAR DELLA CINA 262
144 Roma Italy
Tel. : 0039-06529911
Fax. : 0039-069073105
pasquale.turno@abbott.com
THORGEIRSDOTTIR STEFANIA
Institute for Experimental Pathology
Molecular Biology
Keldur v/Vesturlandsveg
IS-112 Reykjavik Iceland
Tel. : 354-5674700
Fax. : 354-5674714
stef@hi.is
ULVUND MARTHA J
Norwegian School of Veterinary Science
(NVH)
Section for Small Ruminant Research
Kyrkjevegen 332-334
4325 Sandnes Norway
Tel. : +47 51 60 35 11
Fax. : +47 51 60 35 09
martha.ulvund@veths.no
SYLVESTER IAN D
TSEs
Compton
RG20 7NN Newbury England
Tel. : 44-1635577259
ian.sylvester@bbsrc.ac.uk
TILLY GAËLLE
INRA
Animal genetic
LGBC-INRA
Tel. : 01 34 65 25 76
Fax. : 01 34 65 24 78
tilly@jouy.inra.fr
TABOURET GUILLAUME
INRA
Animal Health
Ecole nationale vétérinaire
23, chemin des Capelles
Tel. : 05.61.19.38.88
Fax. : 05.61.19.38.34
g.tabouret@envt.fr
TONGUE SUSAN C
Veterinary Laboratories Agency (VLA) Weybridge
TSE Workgroup (CERA)
Woodham Lane
New Haw
s.tongue@vla.defra.gsi.gov.uk
TAGLIAVINI FABRIZIO
Istituto Nazionale Neurologico Carlo
Besta
Neuroscienze Cliniche
via Celoria 11
20133 Milano Italy
Tel. : 39-022394384
Fax. : 39-0270638217
ftagliavini@istituto-besta.it
TAHIRI-ALAOUI ABDESSAMAD
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
OX1 3RE Oxford UK
Tel. : (44) 1865 27554
Fax. : (44)1865 285756
alaoui@molbiol.ox.ac.uk
TAPIOVAARA HANNELE
National Veterinary and Food Research
Institute
Virology
P.O.B. 45
Hämeentie 57
00581 Helsinki Helsinki Finland
Tel. : +358-9 3931 938
Fax. : +358-9 3931 772
hannele.tapiovaara@eela.fi
TARABOULOS ALBERT
Hebrew University of Jerusalem
Molecular Biology
Ein Kerem
Tel. : 972-2-6757085
Fax. : 972-2-6757086
taraboul@cc.huji.ac.il
TORRENT I MAS JOAN
Université Montpellier 2
Biologie-Santé, CC105
Place Eugène Bataillon
Tel. : 33.4.67.14.33.85
Fax. : 33.4.67.14.33.86
torrent@montp.inserm.fr
TORRES JUAN MARIA
Instituto Nacional de Investigacion y
Tecnologia Animal
CISA-INIA
Carretera de Algete a El Casar s/n
Tel. : 34-91 620 23 00
Fax. : 34-91 620 22 47
jmtorres@inia.es
TOUZEAU SUZANNE
INRA
Mathématiques et Informatique
Appliquées
Unité BIA
Domaine de Vilvert
78352 Jouy-en-Josas Cedex France
Tel. : +33 (0)1.34.65.22.38
Fax. : +33 (0)1.34.65.22.17
Suzanne.Touzeau@jouy.inra.fr
TRUCHOT LYDIE
Hôpital Neurologique
Service de Biochimie
59 Bd Pinel
69003 Lyon France
Tel. : 04 72 35 76 83
Fax. : 04 72 35 76 88
armand.perret-liaudet@chu-lyon.fr
VACCARI GABRIELE
161 Rome Italy
Tel. : +39-0649902848
Fax. : +39-0649387077
gvaccari@iss.it
VADROT CYRIL
AGEPS
Paris
7 rue du fer moulin
75005 Paris France
Tel. : 01 46 69 13 13 poste 1864
Fax. : 01 46 69 15 14
cyril.vadrot@eps.ap-hop-paris.fr
VAN POUCKE MARIO
Ghent University
Laboratory for Animal Genetics and
Breeding
Heidestraat 19
9820 Merelbeke Belgium
Tel. : 00-32-(0)9-264.78.06
Fax. : 00-32-(0)9-264.78.49
Mario.VanPoucke@UGent.be
VAUCLARE ELISABETH
IDEXX SARL
95614
BP 232
95614 Cergy Pontoise France
Tel. : (33) 1 34 32 62 00
elisabeth-vauclare@idexx.com
VETRUGNO VITO
Cell biology and Neurosciences
Viale regina elena 299
161 Roma Italy
Tel. : +39-06-4990-2742
Fax. : +39-06-4990-3012
vetrugno@iss.it
VIDAL CATHERINE
CEA
Neurovirology
vidal@dsvidf.cea.fr
211
Participant List
VIDAL ENRIC
CRESA
PRIOCAT Lab
Veterinary Faculty
UAB Campus
8193 Bellaterra Spain
Tel. : 34 935 811 235
Fax. : 34 935 813 142
enric.vidal.barba@uab.es
WEAR ANGUS R
VLA Newcastle
Whitley Road
Longbenton
NE12 9SE Newcastle upon Tyne
England
Tel. : 44-1912662292
Fax. : 44-1912663605
a.wear@vla.defra.gsi.gov.uk
VIEGAS PEDRO
Institut Cochin
Département Biologie Cellulaire
22 Rue Méchain
75014 Paris France
Tel. : 01 40 51 64 23
Fax. : O1 40 51 64 23
pedro.viegas@cochin.inserm.fr
WEBER LAURA
Prionics AG
M&S
Wagistr 27 a
Tel. : 0041 44 400 20 00
daniel.boscoboinik@prionics.ch
VILOTTE JEAN-LUC
INRA
Animal genetic
LGBC-INRA
Tel. : 01 34 65 25 76
Fax. : 01 34 65 24 78
vilotte@jouy.inra.fr
WEINMANN NICOLE
Heinrich-Heine-Universität D¸sseldorf
Universitätsstrasse 1
Geb. 26.12.U1
Tel. : +49-(0)211-8114703
Fax. : +49-(0)211-8115167
weinmann@biophys.uni-duesseldorf.de
VINDEL ELISABETH
CNIEL
Sécurité Alimentaire
42 rue de Châteaudun
75009 Paris France
Tel. : 01 49 70 71 11
Fax. : 01 42 80 63 45
evindel@cniel.com
VRANAC TANJA
Blood Transfusion centre of Slovenia
Dept. for the production of diagnostic
reagents
Alajmerjeva 6
1000 Ljubljana Slovenia
Tel. : +386.01 5438199
Fax. : +386.01 302 224
tanja.vranac@ztm.si
VUIDEPOT ANNE-LISE
Nestlé
Quality and Safety
PO box 44
1026 Lausanne Switzerland
Tel. : 00-41 21 785 9224
Fax. : 00-41 21 785 8553
anne-lise.vuidepot@rdls.nestle.com
WARD HESTER JT
National CJD Surveillance Unit (UK)
Crewe Road South
Tel. : 00.44.131.537.3091
Fax. : 00.44.131.343.1404
h.ward@ed.ac.uk
212
WEISS STEFAN
Gene Center
Prion Research Laboratory
D-81377 Munich Germany
Tel. : 49-89-2180-76951
Fax. : 49-89-2180-76999
Weiss@lmb.uni-muenchen.de
WESTAWAY DAVID
University of Toronto
CRND
Tanz Building,
6 Queens' Park Crescent West
OntarioM5S 3H2 Toronto Canada
Tel. : 416-978-1556
Fax. : 416-978-1878
david.westaway@utoronto.ca
WESTNER INGO M
Zentrum für Neuropathologie und
Prionforschung
CJD-Surveillance Germany
81377 München Deutschland
Tel. : 49-89-21807801
Fax. : 49-89-21807803
ingo.westner@med.uni-muenchen.de
WETZEL HARTMUT
HCS Health Care Service
Consulting
Rue Rigolette 14
CH 1266 Duillier Switzerland
Tel. : 41-22 361 0756
Fax. : 41-22 361 0740
hwetzel2003@yahoo.com
WILL ROBERT
Crewe Road
Tel. : +44-131 537 2128
r.g.will@ed.ac.uk
WILLE HOLGER
University of California at San Francisco
513 Parnassus Avenue
CA94143-0518 San Francisco USA
Tel. : +1 415 502-1949
Fax. : +1 415 476-8386
wille@cgl.ucsf.edu
WILLIAMS ALUN
Royal Veterinary College
Department of Pathology and Infectious
Diseases
Hawkshead Lane
North Mymms
AL9 7TA Hatfield UK
Tel. : 44-1707666572
Fax. : 44-1707666298
alunwilliams@rvc.ac.uk
WILLIAMS AURIOL C
Roslin Institute
Roslin Institute
EH25 9PS Roslin UK
Tel. : 44-1315274450
Fax. : 44-1314400434
auriol.williams@bbsrc.ac.uk
WILLIAMS JOHN L
Roslin Institute
Roslin Biocentre
EH25 9PS Roslin Scotland
Tel. : 44-131527431
Fax. : 44-131440043
john.williams@bbsrc.ac.uk
WILLIAMSON ANTHONY
Scripps Research Institute
Department of Immunology (IMM2)
10550, North Torrey Pines Road
CA92037 La Jolla United States
Tel. : 858-784-8620
Fax. : 858-784-8360
anthony@scripps.edu
WILLIS MATTHEW P
SO16 7PX Southampton UK
Tel. : 00-44 (0) 2380592034
Fax. : 00-44 (0) 2380594459
m.willis@soton.ac.uk
WILSON STUART M
Microsens
None
LBIC, 2 Royal College Street
NW1 0TU London UK
Tel. : +44 (0)20 7691 2147
Fax. : +44 (0)20 7691 2036
stuart.wilson@microsens.co.uk
Participant List
WINDL OTTO
TSE Molecular Biology
Woodham Lane
KT15 3NB New Haw, Addlestone UK
Tel. : 0044-(0)1932-35
Fax. : 0044-(0)1932-35
o.windl@vla.defra.gsi.gov.uk
YUTZY BARBARA
Pr 1
Paul-Ehrlich-Str. 51-59
Tel. : +49(0)6103-7054
Fax. : +49(0)6103-1234
yutba@pei.de
WISEMAN FRANCES F K
Institute of Animal Health-NPU
Mouse Genetics
Ogston Buliding
West Mains Road
Tel. : 44-1316675204
Fax. : 44-1316683872
frances.wiseman@bbsrc.ac.uk
Zahn Ralph
ALICON AG
Biotop
Wagistrasse 23
Tel. : 41-434950567
Fax. : 41-434950569
info@alicon.ch
WITSCHI URSULA
Federal Office of Public Health
FOOD SAFETY
Schwarzenburgstrasse 165
3097 Liebefeld Switzerland
Tel. : 0041-31 323 44 31
Fax. : 0041-31 322 95 74
ursula.witschi@bag.admin.ch
ZANUSSO GIANLUIGI
University of Verona
Neurological and Visual Sciences
P.le L.A. Scuro, 10
37134 Verona Italy
Tel. : 39-45-8074461
Fax. : 39-45-585933
gianluigi.zanusso@univr.it
WÜTHRICH KURT
ETH Zürich
Institute for Molecular Biology &
Biophysics
Rämistrasse 101
wuthrich@mol.biol.ethz.ch
ZOLLINGER ERNST
Prionics AG
Marketing and Sales
Wagistr. 27 A
Tel. : 0041 44 200 2000
Fax. : 0041 44 200 2010
ernst.zollinger@prionics.ch
XIANG WEI
Institute of Neuropathology
Ludwig-Maximilians-University Munich
Feodor-Lynenstr. 23
Tel. : 0049-89-2180-78
Fax. : 0049-89-2180-78
wxiang@med.uni-muenchen.de
ZORZI WILLY
CRPP
1 Avenue de l'hôpital B36
4000 Liège Belgique
Tel. : +32-4-366.43.27
Fax. : +32-4-366.43.21
Willy.Zorzi@ulg.ac.be
YAMAKAWA YOSHIO
National Institute of Infectious Diseases
Biochemistry and Cell Biology
Toyama 1-23-1
162-8640 Shinjuku-ku Japan
Tel. : +81-3-5285-1111 ext.2127
Fax. : +81-3-5285-1157
yamakawa@nih.go.jp
ZUCCON FABIO
Istituto Zooprofilattico
Sperimentale della Sardeg
Nuoro
Via F.lli Kennedy, 2
8100 Nuoro Italy
Tel. : 39-078430331
zucconfm@yahoo.com
YAMAMOTO TAKUJI
Nippi corporation
Research Institute of BIOMATRIX
Senjyumidorityou
120-0044 Adachiku Japan
Tel. : 81-3-3888-5189
Fax. : 81-3-3888-5180
t-yamamoto@nippi-inc.co.jp
ZURZOLO CHIARA
Institut Pasteur
Cell biology and Infection
25 Rue du Dr Roux
zurzolo@pasteur.fr
YOKOYAMA TAKASHI
National Institute of Animal Health
Prion Disease Research Center
3-1-5 Kannondai
Tel. : 81-29-838-7757
Fax. : 81-29-838-7757
tyoko@affrc.go.jp
ZWALD DANIEL
Prionics AG
Research
Wagistr. 27 a
Tel. : 0041 44 200 2000
Fax. : 0041 44 200 2010
daniel.zwald@prionics.ch
213
Index
Index
NAME
ORAL/POSTER - PAGE
A
Abrial, D
P-84
Accardo, E
P-161
Acin, C
P-98, P-123, P-144
Acocella, F
P-133
Acutis, PL O-45, P-100, P-112, P-113,
P-128, P-144, P-150, P-150, P-152
Adjou, KT
O-75, P-82, P-91, P-177
Agard, D
O-65
Agid, Y
P-105
Agrimi, U
P-88, P-101, P-133,
P-151, P-152
Aguzzi, A
P-130, P-138, P-156
Ahrens, R
O-68
Aitchison, L
O-42, P-98, P-99
Ali, M
P-91
Allain, G
P-143, P-145
Allix, S
P-92, P-91
Alpérovitch, A
P-105
Alvarez-Martinez, MT
P-159
An, SSA
P-171
Andréoletti, O
O-48, P-97
Antloga, K
O-62
Appel, TR
O-60
Archer, F
P-178
Arrabal, S
P-165, P-167
Arsac, JN
O-47, P-134
Ashman, DA
P-157
Aubry, F
P-177
Aucouturier, P
O-76
Auvre, F
O-49
B
Bach, P
P-156, P-156
Bach, S
P-168
Bacilieri, L
P-86
Backer, S
P-82
Badiola, JJ P-98, P-123, P-144, P-184
Baier, M
P-173
Bailly, Y
P-93
Baldwin, MA
P-169
Ballerini, C
O-76
Balsamo, A
P-113
Bardsley, M
P-122
Barizzone, F
P-92
Barnicle, D
O-46
Barocci, S
P-150
Baron, GS
O-63
Baron, H
O-59
Baron, T
O-43, O-44, O-47, P-83,
P-101, P-102, P-134, P-150, P-152
Barr, J
P-148
Barret, A
O-71, P-170
Barritault, D
O-75, P-155, P-170
Barron, R
O-42, P-99
Bartl, M
P-131
Baskakov, I
P-110, P-161
Bate, C
O-78
Batey, K
P-103
NAME
ORAL/POSTER - PAGE
Baybutt, H
Bayley, PM
Baylis, M
Bell, JD
Bell, K
Bellon, A
Bencsik, A
Beneke, S
Benestad, SL
O-42, P-98, P-99, P-99
P-162
O-35
P-148
P-100
P-138
O-44, O-47, P-83, P-101
P-169
O-48, P-94, P-96,
P-141, P-143
Benetta Donatos, M
P-183
Ben-Hur, T
O-55
Bergot, AS
O-76
Bergstrom, AL
P-163
Beringue, V
O-43, P-97
Bertsch, U
O-74, P-88, P-160
Besnard, N
O-43
Bétemps, D
P-101
Beyreuther, K
O-57
Biacabe, AG
P-83, P-150
Biasini, E
P-182
Bibl, M
P-135
Bickeboller, H
P-108
Bieschke, J
O-74
Bilheude, JM
P-142, P-143,
P-143, P-145, P-145
Biocca, S
P-181
Birkett, CR
P-172
Birkmann, E
P-132
Bishop, M
O-42, P-98, P-107
Blanco, R
P-137
Blanquet-Grossard, F
P-93
Blasche, T
P-87
Blondel, M
P-168
Boche, D
P-81, P-137
Bodemer, M
P-131
Bodemer, W
P-169
Bolea, R
P-144, P-184
Boller, K
P-156
Bona, C
P-92, P-93, P-128
Bonetto, V
P-182
Boreham, DR
P-186
Borroni, R
P-133
Borthwick, E
P-176, P-185
Bossers, A
P-112, P-129
Bottero, P
P-93
Bourgeois, JP
P-142, P-143,
P-143, P-145, P-145
Bouzamondo-Bernstein, E
P-94
Bowring, C
P-172
Boyle, A
O-37, P-151
Bozzetta, E
P-144
Brabeck, C
P-163, P-169
Bradford, B
O-42
Bragason, BT
P-88, P-164
Branchu, B
P-118
Brandel, JP
P-105, P-109, P-134
Bratberg, B
O-48, P-96, P-141, P-143
Breitling, F
P-169
Brewer, J
O-78
Brizioli, R
P-133
Bronstein, IB
P-121
NAME
ORAL/POSTER - PAGE
Brown, DA
Brown, DR
Brown, P
Bruce, M
Brugère, H
Brugère-Picoux, J
Bruley Rosset, M
Brun, A
Bruns, C
Bruzzone, MG
Buchholz, CJ
Budka, H
Bugiani, O
Bumpass, DC
Burel, M
Burgess, S
Burkle, A
Burlington, K
Burwinkel, M
Buschmann, A
Busk, N
P-166
P-172
P-106
O-37, P-151, P-152
P-82, P-91
P-82, P-91
O-76
P-148, P-149
O-77
O-45
P-156, P-156
O-64, P-106
P-132
P-170
P-129
O-35
P-163, P-169
P-107
P-173
O-47, O-52, P-186
O-34
C
Caciolo, D
Calavas, D
Caldwell, JE
Campana, V
Cancellotti, E
Cano, MJ
Capellari, S
Capobianco, R
Capucci, L
Caramelli, M
Cardinale, A
Cardone, F
Carlsson, U
Carr, J
Carredu, M
Cartoni, C
Casalone, C
Cashman, NR
Castellani, RJ
Castilla, J
Caughey, B
Cazaubon, S
Cazeau, G
Cepek, L
Cernilec, M
Cesbron, JY
Chabry, J
Chapel, JP
Chaplin, M
Chapron, Y
Charbonnier, A
P-133
P-83, P-84, P-84
P-142
P-160, P-180
O-42, P-99
P-148
P-102
P-132
O-45, P-100
O-45, P-87, P-92,
P-92, P-93, P-100,
P-112, P-113, P-128,
P-144, P-150, P-150
P-181
P-95, P-101, P-151
P-96
P-138
P-87, P-93
P-101
O-45, P-100,
P-113, P-128, P-150
P-157
P-100
P-123, P-148
O-63
P-179
P-83, P-84
P-135, P-181
P-126
P-93
P-163, P-175
P-159
P-145
P-110
O-62
217
Index
NAME
218
ORAL/POSTER - PAGE
Charley, L
P-110
Chaussain, JL
P-105
Chaverot, N
P-179
Chen, S
P-100
Chi Nguyen, TH
P-91
Chianini, F
P-86
Chich, JF
P-173
Chiesa, R
O-69, P-165, P-182, P-183
Chin, S
P-100
Chishti, MA
O-68
Choiset, Y
P-159
Chong, A
P-83, P-96
Chouaf-Lakhdar, L
O-44, P-101
Chouaib, S
P-167
Chung, YL
P-148
Ciarlo, L
P-147
Cichutek, K
P-156
Clark, S
P-137
Clarke, AR
P-125
Clewley, JP
P-103
Cohen, FE
O-65, P-161, P-169
Collin, E
P-84
Collinge, J
O-72, P-125
Colombo, L
P-161, P-183, P-184
Colucci, M
P-100
Comoy, EE
O-49, O-62, P-91
Compoint, A
P-142
Comte, J
O-66
Conlan, B
P-138
Connor, M
P-103
Conte, M
P-101, P-152
Conti, B
O-67
Coomaraswamy, J
O-68
Coopers, S
O-40
Copabianco, R
O-45, P-183
Corbeau, P
P-180
Cornuejols, MJ
P-82, P-91
Corona, C
O-45, P-128
Correia, E
O-49
Costa, C
P-149
Costagliola, D
P-109
Coulibaly, M
P-131
Coulpier, M
P-165
Couquets, C
P-82, P-91
Couraud, PO
P-179
Cousens, S
O-40, P-107, P-108
Covaerts, C
O-65
Craig, JC
P-169
Creminon, C
O-53, P-177
Crescio, MI
P-100, P-128
Criado, J
O-67
Crozet, C
O-44, P-101, P-157, P-180
Cullin, C
P-168
Cunningham, C
P-81
Curin Serbec, V
P-105, P-126,
P-126, P-182
Czub, S
P-85
NAME
ORAL/POSTER - PAGE
NAME
ORAL/POSTER - PAGE
D
E
Dabaghian, AH
P-103
Dabaghian, R
P-169
Dacheux, JL
P-82
D'Angelo, A
P-87, P-93
Darbord, JC
P-118
Daudigeos, E
P-170
Davidse, A
P-150
Dayan, Y
P-146
De Bosschere, H
P-81, P-94, P-124
De Grossi, L
P-133
De Koeijer, A
P-90, P-112
De Luigi, A
P-183
Deacon, RMJ
P-81
Dealler, S
O-51
Dearmond, S
P-94, P-161, P-169
Debey, P
P-162
Dechamps, P
P-124
Dechavanne, S
P-101
Dehen, C
O-62
Del Rio Vilas, V
P-91
Delasnerie-Laupritre, N
P-105, P-134
Delisle, MB
P-134
Dennis, M
P-119
Deprez, M
P-139
Desjouis, G
P-84
Deslys, JP
O-41, O-49, O-54,
O-62, O-71, O-75, P-82, P-91,
P-102, P-129, P-131, P-134,
P-136, P-158, P-170,
P-173, P-177, P-185
Dhintillac, A
P-168
Di Bari, M
P-101, P-133, P-151
Di Fede, G
P-132
Di Giamberardino, L
P-95
Di Prospero, L
P-147
Diagostino, C
P-133
Diarra-Mehrpour, M
P-167
Diaz, F
P-148
Dickinson, J
P-119, P-138
Dickson, D
P-102
Dietrich, R
P-111
Dillon, WP
P-142
Disterer, P
P-107
Dobson, CM
O-58
Dodson, EJ
P-162
Dodson, GG
P-162
Doh-Ura, K
P-146
Domonech, A
P-149
Donnelly, CA
O-33
Dormont, D O-75, P-114, P-134, P-167
Dossena, S
O-69, P-165
Doublet, B
P-159, P-162
Drisaldi, B
O-68
Drummond, D
P-83
Ducrot, C
P-83, P-84, P-84
Dugas, M
P-90
Duval, E
O-62
Duyckaerts, C
P-109
Eaton, S
Ecroyd, H
Edwards, J
Eghiaian, F
Eiden, M
El Hachimi, K
El Moualij, B
El-Gogo, S
Ellers-Lenz, B
Eloit, M
Elvander, M
Elvira, G
Erhardt, G
Ertmer, A
Espinosa, JC
Esposito, E
Estey, L
Etchegaray, N
Everest, S
Everington, D
P-83, P-86
P-82
O-46
P-159, P-162
P-158
P-82, P-91
P-139, P-139
P-155
P-181
P-165
P-96
P-178
O-47
O-77
P-149
P-152
P-141
O-41, O-54
O-46, P-145
O-40, P-107
F
Faucheux, BAP-106, P-109, P-134, P-140
Fazzi, P
P-151
Feldmann, G
P-131
Ferer, I
P-137
Ferguson, NM
O-33
Fernandez, H
P-89
Fernandez, J
P-89
Fernie, K
O-61, P-83, P-120
Ferrer, I
P-136, P-178
Février, B
P-178
Feyssaguet, M
P-142, P-143, P-145
Fichet, G
O-62
Fierville, F
P-140
Filesi, I
P-181
Fioriti, L
P-165, P-182
Fischbein, NJ
P-142
Florio, C
P-128
Fociani, P
P-132
Forestier, L
O-71
Forloni, G
P-161, P-165, P-182,
P-183, P-184
Foster, J
O-35, O-36, P-83
Fournier, JG
P-129
Franz, BS
P-171
Fraser, JR
P-148, P-166
Fraser, PE
O-68
Frassanito, P
P-151
Freire, S
O-49, P-129
Freixes, M
P-136, P-137
Frémont, A
P-91
Freyman, Y
P-169
Frigg, R
P-176
Frobert, Y
O-53, P-140, P-157
Frölich, K
P-87
Fuhrmann, M
P-174
Furukawa, H
P-146
Index
NAME
ORAL/POSTER - PAGE
NAME
ORAL/POSTER - PAGE
G
H
Gabizon, R O-55, P-146, P-175, P-177
Gagna, C
P-92, P-92
Gagna, C
P-92
Gall, E
O-42, P-98
Gallet, PF
O-71
Galvani, V
P-105, P-126
Gambetti, P
P-100, P-102
Gareis, M
P-111
Garner, K
P-158
Gasset, M
P-178
Gatti, JL
P-82
Gauczysnki, S
P-155
Gavier-Widen, D
P-95, P-96
Gazzuola, P
P-100
Geeroms, R
P-81, P-124
Geissen, M
P-155
Gelmetti, D
O-45
Gentles, N
O-61
Georgsson, G
P-104
Geschwind, MD
P-142
Ghetti, B
O-69, P-102
Giaccone, G
P-132
Giebel, A
P-169
Giese, A
O-74, P-102, P-160
Gilbert, IH
P-172
Gilch, S
O-77
Gill, AC
P-107, P-162
Gill, N
P-103
Gill, ON
P-108
Giordano, F
P-133
Glatzel, M
P-130, P-138
Gobbi, M
P-161, P-184
Godsave, S
P-180
Gofflot, S
P-139, P-139
Goldmann, W
O-35, P-83
Gonzales, L
P-86, P-96, P-103
Gonzalez-Iglesias, R
P-178
Grassi, J
O-53, P-101, P-137,
P-139, P-139, P-140,
P-142, P-143, P-145, P-157
Greenham, J
P-124
Grigoriadis, N
O-55, P-118
Grimm, C
P-176
Groner, A
P-138
Groschup, MH
O-47, O-60,
P-111, P-186
Grosclaude, J
O-66, P-159,
P-162, P-173
Groshup, M
P-155, P-158
Guarnieri, F
O-31,
Gubbins, S
O-34
Gudmundsson, S
P-104
Guglielmo, BJ
P-169
Gutierrez, A
P-148
Haeberlé, AM
Haertle, T
Hafner, I
Hagenaars, TJ
Hagiwara, K
Haik, S
P-93
P-159
P-182
O-33
P-85
P-105, P-106, P-109,
P-134, P-140
Haire, LF
P-162
Halimi, M
P-146
Hall, G
P-119
Hamel, R
P-165
Hamilton, S
P-120
Hansen, M
P-133
Hard Av Segerstad, C
P-96
Hardti, M
P-125
Harris, DA
O-69, P-165
Hart, P
O-42, P-98
Hässig, R
P-106
Hattori, S
P-140
Hauw, JJ
P-106, P-109, P-134, P-140
Hawke, S
P-125
Hayashi, H
P-89, P-121
Hays, R
P-117
Head, M
O-37
Head, MW
P-107, P-130
Heath, C
O-40
Heegaard, P
P-133, P-163
Heinemann, U
P-131
Heinen, E
P-139, P-139
Heinig, L
P-130
Henaux, S
P-143, P-145
Henry, RG
P-142
Heppner, FL
P-156
Herms, J
P-174
Herva, ME
P-123
Herzog, C
O-41, O-54, P-164
Hianik, T
P-127
Higuchi, Y
P-85
Hijazi, N
P-177
Hirschberger, T
O-74
Holznagel,
P-131
Hontebeyrie, M
P-114
Hopkins, S
P-94
Horrocks, C
P-97
Hortells, P
P-98, P-144
Hostrup-Pedersen, J
O-32
Houston, F
O-35, P-96, P-103
Hua, Z
P-100
Huang, Y
P-169
Hunsmann, G
P-113, P-169
Hunter, N O-35, O-36, P-83, P-96, P-103
Hunziker, A
P-169
I
Illig, T
Imamura, M
Ingravalle, F
Irie, S
P-108
P-89
P-92, P-100, P-150
P-140
NAME
ORAL/POSTER - PAGE
Irle, E
P-181
Ironside, J
O-37, P-99, P-130, P-166
Iswanti-Stanek, D
O-52
Ito, T
P-146
Itohara, S
P-174
Iulini, B
P-128
Iwamaru, Y
P-89, P-121
J
Jackman, R
Jackson, GS
Jacob, C
Jacobs, JG
Jacquemot, C
Jalil, A
James, W
Jamieson, L
Jarrige, N
Jayat-Vignolles, C
Jeffrey, M
Jenkins, A
Jerala, R
Julien, R
Juntes, P
O-46, P-145
P-125
P-111
P-129
P-114
P-167
P-107
O-42
P-84
O-71
P-86, P-96, P-103
P-145
P-182
O-71
P-126
K
Kachel, N
P-183
Kadiysky, D
P-177
Kalbitzer, HR
P-183
Kalinke, U
P-156, P-156
Kaps, I
P-87
Karch, H
P-186
Kariv-Inbal, Z
O-55
Katamine, S
P-146, P-172
Keevil, CW
P-117, P-117, P-137
Kehler, C
O-77
Kelly, C
P-103
Khalili-Shirazi, A
P-125
King, D
O-42
Kinoshita, N
P-140
Kirby, E
P-170
Klankki, E
P-155
Klingenstein, R
O-50
Knight, RSG
O-40, P-107
Knossow, M
P-162
Knox, JD
P-186
Kocisko, D
O-63
Kohlmann, A
P-90
Konrath, A
P-125
Konstantina, K
P-118
Koren, S
P-126
Korth, C
O-50
Kovacs, GG
O-39, O-64, P-106
Krasemann, S
P-169
Kremer, W
P-183
Krenn, B
P-180
Kretzschmar, H
O-68, O-74, P-88,
P-90, P-102, P-108, P-119,
P-135, P-160, P-174, P-181
219
Index
NAME
ORAL/POSTER - PAGE
Kristensson, K
Kub-ler, E
Kuczius, T
Kuhn, F
Kupfer, L
Kuwata, K
P-135, P-167,
P-168, P-180
O-52
P-186
P-128
P-158
P-172
L
Labas, V
Labonnardière, C
Lachmann, I
Laffont, I
Lagoudaki, R
Lai, TL
Laisne, A
Lamoury, F
Lane, A
Lange, R
Langeveld, J
220
P-173
P-84
P-125
P-106
P-118
O-43
O-43
O-75
O-51
P-159
P-129, P-150,
P-152, P-155
Lantier, F
O-48, P-141
Laplanche, JL
P-109, P-134, P-164
Laquerrière, A
P-134
Larramendy, C
P-170
Lasmézas, C
O-41, O-54, O-62,
O-66, O-75, P-1O2, P-129,
P-155, P-164, P-170, P-177
Latawiec, D
P-94
Laude, H
O-43, O-48, P-97,
P-138, P-178
Launais, JM
P-164
Lazarini, F
P-114
Le Dur, A
O-43, P-97
Leathers, V
P-141
Leblanc, V
P-136, P-185
Ledur, A
O-48
Lee, DC
P-174
Lefebvre-Roque, M
O-66, P-164
Legname, G
P-161, P-169
Lehmann, S
O-73, P-157, P-180
Lehto, MT
P-157
Leitch, M
O-40
Lekishvili, T
P-172
LeLean, J
P-170
Lemaire-Vieille, C
P-93
Leone, P
P-113
Leparc-Goffart, I
P-134
Lesceu, S
P-162
Lessard, P
P-169
Levi, Y
P-146
Levin, J
P-160
Lewer, J
P-171
Lezmi, S
O-44
Liautard, JP
P-159
Liberski, PP
P-106
Ligios, C
P-150, P-152
Lim, K
P-171
Limido, L
O-45, P-132
Lin, ET
P-169
NAME
ORAL/POSTER - PAGE
Lin, YL
Lind, P
Lindstrom, J
Lingappa, VR
Lipscomb, IP
Liu, H
Liu, S
Low, P
Lower, J
Lu, M
Lu, Y
Lucassen, R
Lucotte, G
Luhken, G
Luhr, KM
Lutz, J
Lyahyai, J
Lynam, B
P-180
P-133, P-163
P-179
O-50
P-117, P-117, P-137
P-142
P-142
P-135, P-167, P-168
P-131
P-95
P-142
O-60
P-104
O-47
P-167, P-168
P-163
P-184
P-86
M
Maas, E
O-77
Macaldowie, C
P-103
Madec, JY
O-47, P-83, P-102, P-134
Magal, P
P-111
Magalhes, AC
O-63
Maignien, T
P-177
Majtenyi, K
O-39
Mallinson, G
P-125
Manganelli, V
P-147
Mangieri, M
O-45, P-132, P-183
Maniaci, MG
P-112
Manson, J
O-42, P-98, P-99
Manuelidis, L
O-56
Manzoni, C
P-184
Marcé, D
O-49, P-134
Marcon, S
P-133, P-152
Marella, M
P-175
Maroni, A
P-112
Marquez, M
P-89, P-149
Martin, S
P-96, P-103
Martin, SF
P-149, P-166
Martin-Burriel, I
P-123, P-184
Martindale, JL
P-142
Märtlbauer, E
P-111
Martucci, F
P-128, P-144, P-150
Massignan, T
P-182
Masters, CL
O-57
Mastrangelo, P
O-68
Maté, C
P-89
Mathieu, E
P-170
Matos, A
P-109
Matsumoto, Y
P-174
Mattei, V
P-147
Maurage, CA
P-109
Maurella, C
P-87, P-92, P-92,
P-93, P-100, P-150
May, BCH
P-169
Mayer, T
P-175
Mazza, M
P-100, P-113, P-144, P-150
McConnell, I
O-37
NAME
ORAL/POSTER - PAGE
McCormack, J
McDonnell, G
McKenzie, C
McKeown, B
McLeod, A
Mehl, M
Meier, M
Meissner, K
Mennini, T
Mercier, G
Merten, C
Messiaen, S
Mettling, C
Meussdoerffer, F
Meyer, T
Michels, W
Miller, BL
Mirisola, A
Mitchel, RE
Moda, G
Moleswoth, AM
Mollenhauer, B
Monaco, S
Monleon, E
Montag, J
Montrasio, F
Monzon, M
Morales, M
Morbin, M
Morel, N
Morignat, E
Morize, JL
Mortimer, PP
Motzkus, D
Mount, HTJ
Mourton-Gilles, C
Mouthon, F
Moya, KL
Muller, H
Muller-Hellwig, S
Murdoch, H
Murray, D
Mutinelli, F
Mylne, MJA
P-99
O-62
P-83
P-124
P-119
P-147
O-52
O-52
P-161
P-104
P-156
P-165
P-180
P-87
P-146
P-119
P-142
P-112
P-186
P-112
P-103, P-108
P-135, P-181
O-45
P-98, P-123, P-144
P-127
P-156, P-156, P-171
P-98, P-144
P-123
P-184
O-53
P-83, P-84
P-142
P-103
P-113, P-130
O-68
O-53, P-157
P-136, P-158,
P-173, P-185
P-106
P-120
P-111
P-119, P-138
O-31
P-113
P-83
N
Nakamura, T
Nakamura, Y
Nappi, R
Navarro, C
Nayki, I
Negro, A
Nespoulous, G
Nguyen, HC
Nguyen, HOB
Nickles, D
Nieper, H
Nishida, N
P-174
P-85
P-144
P-125
O-52
O-53
P-142, P-143,
P-145, P-145
P-82
P-161
P-156, P-156
P-125
P-172
Index
NAME
ORAL/POSTER - PAGE
Nishimura, T
P-174
Niwa, M
P-146
Nodari, S
P-150
Nohtomi, K
P-85
Nonno, R
P-101, P-133, P-151, P-152
Nordstrom, EK
P-167
Noremark, M
P-96
Notari, S
P-102
Nouvel, V
P-136, P-158, P-185
Nowoslawski, L
O-69
Nunziante, M
O-77
O
Oboznaya, M
O'Brien, S
O'Connell, D
Oesch, B
O'Flynn, M
Oh, H
Okuwaki, R
Oldstone, M
Oliver, J
O'Neill, G
Onodera, T
Osman, A
Ostatna, V
Otto, M
O-77
P-138
P-86
O-52, P-128,
P-144, P-147, P-176
P-120
P-171
P-146
O-67
O-51
O-35
P-174
P-125
P-127
P-135, P-181
ORAL/POSTER - PAGE
Philippe, S
Piccardo, P
Pichner, R
Picoli, C
Pienig, N
Pietrella, M
Pinson, X
Pintado, B
Pinto, L
Plourde, L
Pocchiari, M
Pongolini, S
Popovi, M
Porquet, A
Poser, S
Poulios, I
Powers, J
Prado, MA
Prange, H
Pretnar Hartman, K
Preusser, M
Price, P
Privat, N
Prusiner, SB
Puig, B
Pumarola, M
Pupo, S
Pürro, M
O-44
O-69
P-111
P-136, P-185
P-88
P-171
P-167
P-148
P-144
P-141
P-95, P-102, P-151
P-113
P-126, P-182
P-110
P-108, P-135, P-181
P-118
P-102
O-63
P-181
P-126
O-64
O-52
P-134, P-109, P-140
O-65, P-94, P-161,
P-169, P-180
P-136, P-137
P-89, P-149
P-147
P-128
Q
P
Paisley, LG
Paladino, S
Palsdottir, A
Panagiotidis, C
Papy-Garcia, D
Paquet, S
Parchi, P
Parnham, D
Parra, B
Paspaltsis, I
Pastore, M
Patel, S
Pavan, A
Peden, AH
Peelman, L
Peletto, S
Pellisseir, JF
Peretz, D
Perez, J
Perfetti, G
Pergande, G
Perrier, V
Perrière, N
Perry, VH
Peters, P
Petit, E
Petrakis, S
Peyrin, JM
NAME
O-32
P-160
P-88, P-164
P-155
O-75, P-170
P-178
P-102
P-83
P-123, P-166
P-118, P-155, P-179
P-100
P-121
P-147
P-130
P-81
P-112, P-113
P-134
P-180
P-159
P-128
P-181
P-157, P-180
P-179
P-81, P-117, P-137
P-180
P-170
P-118, P-179
P-110
Quaglio, E
Quaterman, E
P-183
O-51
R
Raeber, AJ
Ralston, HJ
Raposo, G
Rapp, D
Ratzka, P
Raven, NH
Rawlins, JNP
Reid, HW
Reisner, D
Relano, A
Relano-Ginès, A
Remé, C
Renardi, C
Renstrom, L
Rezaei, H
Reznicek, L
Riemer, C
Riesner, D
Riffet, C
Riina, MV
Ripoche, H
Ritchie, D
Riviere, J
P-128, P-147, P-176
P-94
P-178
P-159
P-181
P-119, P-138, P-170
P-81
P-86
P-161
P-123, P-166
P-149
P-176
P-81
P-96
O-66, P-159, P-162
P-119
P-173
O-60, P-120, P-132
P-170
P-112, P-113
P-167
O-37, P-130
O-41, O-54
NAME
ORAL/POSTER - PAGE
Rocchi, M
P-86
Rodellar, C
P-123
Rodriguez, A
P-136, P-137
Rodriguez, J
P-123
Roels, S
P-81, P-94, P-124, P-145
Rogers, M
P-86
Rosenberger, A
P-108
Rosone, F
P-133
Roth, KA
O-69
Roux, F
P-179
Rowan, P
P-124
Ru, G
P-87, P-92, P-92, P-93,
P-100, P-112, P-128
Rudyk, H
P-172
Rupreht, R
P-105, P-126, P-126
Rutger Leliveld, S
O-50
Rybner, C
P-114
Ryder, S
P-97
Ryou, C
P-169
S
Saegerman, C
Saeki, K
Safar, J
Sakudo, A
Salès, N
P-124
P-174
P-94
P-174
O-41, O-54, O-75,
P-129, P-170, P-177
Salguero, J
P-148
Salmona, M P-161, P-182, P-183, P-184
Samarut, J
O-44
Samuely, T
P-127
Sandberg, M
P-135
Sarnataro, D
P-160
Sarradin, P
O-48, P-82, P-141
Sata, T
P-85
Sato, Y
P-85
Sauvage, N
P-143, P-145
Sazdovitch, V
P-106, P-109,
P-134, P-140
Sbaiz, L
P-112, P-113
Schaefer, O
P-132
Schätzl, HM
O-77
Scherbel, C
P-111
Scherer, S
P-111
Schettler, E
P-87, P-95
Schinina, ME
P-101
Schmerr, MJ
P-89
Schmid, J
P-128
Schmitt, A
P-119
Scholl, F
P-150
Schötzl, H
O-66
Schultz, J
P-173
Schulz, G
O-77
Schulz-Schaeffer, W
P-113
Schwartz, A
P-173
Sellarajah, S
P-172
Shimida, K
P-89, P-121
Shinagawa, M
P-89, P-121
Shirabe, S
P-146, P-172
Sidler, M
O-52
221
Index
NAME
ORAL/POSTER - PAGE
Sikorska, B
Simmons, MM
Simon, S
Simoneau, S
Simson, S
Sinegre, M
Sklaviadis, T
Smith, PG
Smith-Bathgate, B
Solforosi, L
Soller, S
Somerville, RA
Somerville, RA
Sorgato, C
Sorice, M
Spilman, P
Spiropoulos, J
Stack, M
Stadelmann, M
Stanley, C
Steinacker, P
Steinbach, F
Stewart, LR
Stoeck, K
Strammiello, R
Strohschneider, M
Strom, A
Strome, B
Stuke, A
Sturny, A
Suardi, S
Supervie, V
Suttie, A
Sutton, JM
Sweeney, T
Sy, MS
Sylvester, ID
P-106
O-34, P-96
O-53
O-66, O-75
P-151
P-118
P-179, P-118, P-155
O-40, P-107
O-40
O-67
P-141
O-61, P-120
P-120
O-53
P-147
P-94
P-97
P-145
O-52
O-51, P-141
P-135
P-87
P-165
P-131
P-102
O-64
P-127
O-68
P-113, P-130, P-127
P-136, P-158
O-45, P-132
P-109
P-151
P-119, P-138, P-170
P-152
P-181
P-121
T
222
Tagawa, Y
P-121, P-140
Tagliani, F
P-183
Tagliavini, F O-45, P-132, P-161, P-184
Tahiri-Alaoui, A
P-107
Talarek, N
P-168
Taraboulos, A
P-135, P-167, P-180
Tasciotti, V
P-147
Tatzelt, J
O-74
Tavan, P
O-74
Tayebi, M
P-125
Taylor, D
P-138
Thimon, V
P-82
Thomas, EL
P-148
Thompsett, A
P-172
Thomson, V
P-99
Thonnart, JF
P-139
Thorgeirsdottir, S
P-104
Thorne, L
O-46
Thuring, CMA
P-152
Tilly, G
O-43
Tonelli, Q
P-141
NAME
ORAL/POSTER - PAGE
Tongue, SC
O-34
Toomik, R
P-141
Torero-Ibad, R
O-76
Torner, M
P-131
Torrejon-Escribano, B
P-137
Torrent, J
P-159
Torres, JM P-123, P-148, P-149, P-166
Tortosa, R
P-149
Tryggvason, T
P-104
Tsukagoshi-Nagai, H
P-140
Tuzi, N
O-42, P-98, P-99
Tuzy, N
P-99
U
Udono, H
Ushiki, Y
Ushiri, Y
Uyehara-Lock, J
P-146
P-140
P-121
P-94
V
Vaccari, G P-101, P-133, P-151, P-152
Vadrot, C
P-118
Van Keulen, LJM
P-129, P-152
Van Poucke, M
P-81
Van Zijderveld, FG P-129, P-150, P-152
Vanoni, M
P-161
Vanopdenbosch, E
P-81, P-94, P-145
Vargas, A
P-144
Varges, D
P-131
Vasiljevic, S
P-172
Vasisht, N
P-162
Velek, K
P-141
Venturini, A
P-126
Verma, C
P-162
Vetrugno, V
P-181
Vey, M
P-138
Vidal, C
P-164
Vidal, E
P-149
Vidan-Jeras, B
P-105
Viegas, P
P-179
Vilette, D
O-43, P-178
Vilotte, JL
O-43
Vinard, JL
P-83
Vinh, J
P-173
Vital, A
P-109
Vitale, F
P-150
Voigtlaender, T
O-64
Volland, H
O-53
Vollmert, C
P-108
Vorberg, I
O-77
Vranac, T
P-126, P-182
Vromans, MEW
P-152
W
Ward, HJT
Wariwoda, H
O-40, P-107, P-108
P-176
NAME
ORAL/POSTER - PAGE
Webb, P
O-34
Weber, P
O-74
Weinmann, N
P-132
Weiss, S
O-70, P-155
Welaratne, A
P-105
Wenzel, A
P-176
Westaway, D
O-68
Westner, I
P-90, P-108, P-119
Whyte, SM
P-162
Wichmann, HE
P-108
Will, RG
O-38, O-40, P-98, P-107
Wille, H
O-65
Williams, A
O-78
Williams, AC
P-176, P-185
Williams, JL
P-176, P-185
Williamson, AR
O-67
Willis, MP
P-117
Wilm, A
P-132
Wilson, R
O-78
Wilson, S
O-51, P-141
Windl, O
O-68, P-90, P-108, P-181
Winklhofer, K
O-74
Wiseman, F
P-99
Wong, C
P-141
Wong, S
P-142
Wopfner, F
O-66, O-77
Wünsch, G
P-90, P-119, P-174
X-Y
Xiang, W
Yagüe, J
Yamakawa, Y
Yamamoto, K
Yamamoto, T
Yang, J
Yokoyama, T
Young, GS
Yung, L
Yutzy, B
P-90, P-108, P-119
P-178
P-85
P-146
P-140
O-68
P-89, P-121
P-142
P-169
P-131
Z
Zaccaria, B
Zahn, R
Zajacova, Z
Zanini, A
Zanusso, G
Zaragoza, P
Zeigler, M
Zerbi, P
Zerr, I
Zhang, W
Zhang, Y
Ziegler, U
Zorzi, W
Zou, W
Zsurger, N
Zurzulo, C
Zwald, D
P-113
P-122, P-183
P-127
P-87
O-45, P-100
P-123, P-184
P-175
P-132
P-102, P-108, P-131
P-186
P-169
O-47
P-139, P-139
P-100
P-163
P-160, P-180
P-128
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