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SID 4


Annual/Interim Project
Report for Period 01/08-12/08
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Project details
2. Project title
Application of the Transient Scrapie Cell Assay (TraSCA) for in
vitro detection of ovine and bovine prions.
3. Defra Project Manager
4. Name and
address of
contractor
5. Contractor’s Project Manager
6. Project:
Lesley Columbine
Prof. Adriano Aguzzi
Institute of Neuropathology
University Hospital Zürich
Schmelzbergstr. 12
Zürich, Switzerland
Postcode 8091
Prof. Adriano Aguzzi
start date .................
January 2006
end date ..................
December 2008
This form is in Word format
and boxes may be expanded
or reduced, as appropriate.
SID 4 (Rev. 3/06)
SE2001
1. Defra Project code
Page 1 of 13
Scientific objectives
7.
Please list the scientific objectives as set out in the contract. If necessary these can be expressed in an
abbreviated form. Indicate where amendments have been agreed with the Defra Project Manager, giving the
date of amendment.
Recent results by various groups indicate that body fluids like urine, milk or sputum contain prion infectivity
and may efficiently contribute to horizontal prion transmission. Up to now, the detection of prion infectivity
in animal tissues and body fluids was very difficult and time consuming. Traditionally it relies on murine
bioassays (MBA) and thus is significantly time consuming, expensive and involves the use of large
numbers of mice. Further, this assay suffers from an inexact quantification of the amount of prion
infectivity in the various tissues or body fluids due to genetic differences within the animal cohorts of PrP
overexpressing tga20 mice.
An in vitro assay (Scrapie Cell Assay “SCA”) for determination of prion infectivity, as sensitive as the MBA
has recently been developed and is currently used by various groups including our group. However, the
assay is still time consuming and detection is so far restricted to mouse-adapted scrapie strains therefore, limiting the use only to scientific questions related to work with mice.
At present we are in the process of successfully improving the SCA as originally published and developing
of a murine Transient Scrapie Cell Assay (TraSCA). The advantage of TraSCA technology is the
circumvention of time consuming passaging procedures, therefore allowing for dramatic shortening in
assay time.
We further plan to extend the applicability of the TraSCA and develop the ovine and bovine TraSCA for
the rapid measurement of prion infectivity in ovine and bovine tissues and fluids. Epitope-tagged PrPC
allows detection of de novo formed PrPSc within 24 hours post exposure to prions. We will therefore
develop a modification of the SCA, forthwith denominated Transient Scrapie Cell Assay (TraSCA), utilising
murine and ovine cell lines stably expressing tagged ovine and bovine PrPs. Discrimination of tagged
PrPSc from tagged PrPC requires a proteinase K digest step in the TraSCA. To avoid this we also intend to
investigate the feasibility of utilising motif-grafted antibodies specifically recognizing PrPSc and luminescent
conjugated polymers, compounds staining PrPSc with high sensitivity and differentiating between prion
strains (Sigurdson, 2007). Success of such technology may allow for high-throughput fluorescence
activated cell sorting (FACS)-based platforms to be used for readout of the TraSCA.
It is anticipated that this project will allow for the use of the TraSCA to determine prion infectivity in tissue
homogenates and fluids of sheep and cows. A cell-based infectivity assay such as the TraSCA for ovine
and bovine prions will allow for sensitive high throughput screening of risk material. In addition, TraSCA
technology will not only dramatically reduce the number of mice used in prion research but also
significantly accelerate experiments and analysis of prion infectivity in ovine and bovine tissues and fluids,
thereby greatly advancing research on TSEs.
Further, our plan was and is to implement new direct methodologies for the detection of PrPSc. For this
reason we have established the staining of prions by luminescent conjugated Polymers (LCPs) (Sigurdson
et al., Nat. Methods, 2007) that could be used directly on cells as well as organotypic brain slices (Falsig
et al., Nat. Neuroscience, 2008).
The objectives of this project were to significantly improve the currently available scrapie cell assay for the
detection of prion infectivity in tissues or fluids in two respects:

to significantly decrease assay time by using heterologous tagged PrP variants or novel detection
SID 4 (Rev. 3/06)
Page 2 of 13
methods for PrPSc (transient scrapie cell assay - TraSCA)

to make the scrapie cell assay available for the detection of ovine and bovine prions – on cell
culture based models and if possible - on organotypic brain slices.
Successful implementation of TraSCA will allow for high-throughput automated screening of ovine and
bovine tissues and fluids using cell cultures, organotypic slice culture as well as tools that differentiate
between prions and PrPC.
This research project therefore addressed questions directly related to Defra’s goals:

improve the health and welfare of kept animals and to protect society from impact of animal
diseases

to reduce the number of animals used in research and to pursue the development of rapid,
inexpensive alternatives to mouse bioassays for TSE infectivity.
Summary of Progress
8.
Please summarise, in layperson’s terms, scientific progress since the last report/start of the project and
how this relates to the objectives. Please provide information on actual results where possible rather
than merely a description of activities.
(1) Significantly decrease assay time by novel detection methods for PrPSc (transient scrapie cell assay TraSCA) by using various methods including the use of luminescent conjugated polymers (LCPs) or
organotypic slice cultures.
(2) To make the scrapie cell assay available for the detection of ovine and bovine prions by using various
methods including the use of luminescent conjugated polymers (LCPs) or organotypic slice cultures.
Ad (1): So far there is no reagent available that can distinguish prion infected from uninfected cells in vivo.
Methods to discriminate PrPC from PrPSc rely either on a PK digestion step or treatment of cells with
GuanidiumHCl which gives a punctuate staining with anti PrP antibodies in infected cells whereas
uninfected cells show a uniform weak immunopositivity. Whereas methods relying on PK digestion render
in vivo analysis like FACS impossible, approaches using “epitope exposure” with GdnHCl do not allow
high-throughput assays due to the impracticability for standardisation. Therefore, a compound enabling to
differentiate reliably and without the need for PK treatment is urgently needed.
As outlined in last years report we first focused on alternative detection methods for PrPSc in cell lines.
Here, we pursued a dual strategy: First, we exploited the capacity of PrPSc specific antibodies (Moroncini,
2004; Moroncini, 2006) in the detection of infected cells. Second, we assessed the potential of
luminescent conjugated polymers (LCPs; Sigurdson, 2007) to discriminate between infected and
uninfected cells as well as between infected or non-infected slice culutres.
In our previous report we showed that the motif-grafted antibodies 89-112 and 136-158 developed by
Gianluca Moroncini (Moroncini, 2004; Moroncini, 2006) also can be used to specifically recognize PrPSc in
cell culture homogenates. Furthermore, these antibodies were shown to specifically stain PrP deposits in
human prion diseases (Carnoy`s Fixed tissue) without PK treatment. Due to the fact that these antibodies
recognize PrPSc in its native conformation without binding to PrPC, we assumed that life staining of
infected cells could be possible with these immunoreagents without the use of PK or GdnHCl. We tested
different staining procedures (short-term at 37°C vs. over night at 4°C, different concentrations of primary
and secondary antibodies) and achieved to differentiate N2aPK1RML (neuroblastoma cells chronically
infected with the prion strain RML6) cells and N2aPK1Mock (N2aPK1 cells treated with healthy brain
homogenate) cells (Figure 1).
SID 4 (Rev. 3/06)
Page 3 of 13
Figure 1: Specific labelling of N2aPK1RML cells with motif-grafted
antibodies. N2aPK1 cells chronically infected with RML (N2aPK1RML)
and controls (N2aPK1Mock) were labelled with biotinylated anti-PrP-IgG
89-112, which was detected with streptavidin-FITC. Panels on the right
show the corresponding section by light microscopy (LM).
Although these first experiments looked highly promising,
repetitions with N2aPK1 cells freshly infected with RML or another
cell line chronically infected with RML (CADRML) gave inconclusive
results. Right now, we are in the process to further optimize the
staining procedures to reliably differentiate between prion infected
and uninfected cells.
Next, we tested the potential of these antibodies to detect prion
infected cells by FACS analysis (Figure 2). By now, we were not
able to identify conditions under which the motif-grafted
antibodies discriminated between prion infected and uninfected
cells.
b12
89-112
Mock
RM
136-158
Mock
RM
Mock
RM
RML
and control cells after labelling with motif-grafted antibodies. N2aPK1
Figure 2: FACS analysis of N2aPK1
cells chronically infected with RML (N2aPK1RML) and controls (N2aPK1Mock) were labelled with biotinylated anti-PrPIgG 89-112, which was detected with streptavidin-FITC. As a further negative control served antibody b12 which
contains a scrambled PrP motif (please refer to Moroncini et al. for details on antibodies).
At present, we are in the process of evaluating staining conditions and plan to extend our analysis to
additional cell lines and prion strains as depicted below.
Progress in the detection of prion infected cells by luminescent conjugated polymers (LCPs)
In a second approach we investigated the potential of LCPs to differentiate prion infected from uninfected
cells without the need for PK treatment (Figure 3). This approach was based on our experience to use
LCPs as a tool to biophysically characterize prion protein aggregates (Sigurdson, 2007). In contrast to
sterically rigid amyloidotropic dyes such as thioflavin T and Congo red, the LCPs used here contain
swiveling thiophene backbones whose geometry modulates their fluorescence. Noncovalent binding to
proteins, including amyloids, constrains the rotational freedom of LCPs and thus alters their spectral
properties in a conformation-dependent manner. LCP reactivity and emission spectra of brain sections
discriminated among four immunohistochemically indistinguishable, serially mouse-passaged prion strains
derived from sheep scrapie, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE),
SID 4 (Rev. 3/06)
Page 4 of 13
and mouse-adapted Rocky Mountain Laboratory scrapie prions (Sigurdson, 2007). Furthermore, using
LCPs we differentiated between field isolates of BSE and bovine amyloidotic spongiform encephalopathy,
and identified noncongophilic deposits in prion-infected deer and sheep (Sigurdson, 2007). These findings
led to the conclusion that it may be possible to also detect prions in cell lines using LCPs.
A
B
Figure 3: LCP staining patterns of CADRML and control cells after Carnoy`s fixation. CAD cells chronically
RML
Mock
) were stained with
infected with RML (CAD ) and controls treated with uninfected brain homogenate (CAD
PAMT.
Although we can detect differences in staining patterns and emitted light between infected and uninfected
controls in some preparations, we further optimized the staining procedure to achieve reproducibility and
therefore a stable read out. Furthermore we tested additional cell lines and prion strains in this setup. In
the end we anticipated the usage of LCPs also for FACS analysis of prion infected cells – which will be
done on the basis of the following data:
LCPs were developed as a tool to detect and biologically characterized amyloids. In contrast to commonly
used amyloidotropic dyes such as Thioflavin T (ThT) and Congo red (CR), LCPs are polymers consisting
of thiophene backbone with conjugated system of double and single bonds. This allows rotation of
molecule upon binding to amyloids and thus alters its spectral properties in conformation dependent
manner. Another advantage compared to ThT and CR is the possibility to easily alter its polar character by
the modification of the side chains (R) of LCP. The Luminescent conjugated polymers (LCPs) were shown
to specifically stain protein aggregates in ex vivo tissue sections of various PrPSc strains (Sigurdson C, et
al., Nat. Methods; 2007). The unique spectral properties of LCPs have been also successfully used to
distinguish protein aggregates associated with distinct murine prion strains (Sigurdson C et al., Nat.
Methods; 2007). In addition, LCPs have been shown to identify protein aggregates negative for Congo red
and ThT staining. LCP were also shown to allow discrimination of different conformation of Aβ 1-42 fibrils
generated in vitro and resolve conformational heterogeneity of amyloid deposits in vivo. Thus LCP seem
to be a sensitive method for detection of prion/amyloid aggregates and also as a tool for studying the
mechanism of fibril formation. Here we used PTAA (polythiophene acetic acid) to distinguish PrPSc
infected cells.
Figure 4: PrPSc infected cell discrimination using luminescent conjugated polymers (LCPs).
A) 100 000 PrPSc infected (RML or 22L PrP strain) LD9 cells were stained with PTAA (polythiphene acid;
5µg/ml PTAA diluted in PBS; 15 minutes at 37°C) and incubated for another 24h in standard growth
medium (without PTAA). After EtOH/HAc (3:0; 10 minutes at RT), cell nuclei were additionally stain DAPI.
Fluorescent images showing positive PTAA staining in 22L, RML infected LD9cells. Weak PTAA signal
was observed also in some uninfected LD9cells.
SID 4 (Rev. 3/06)
Page 5 of 13
B) Spectral shift is one of the main characteristic features of LCPs. Therefore we analyze PTAA emission
spectra of cells infected with 22L PrP strain uninfected cells using SpectraMax Software which showed
slight spectral shift in case of 22L infected cells.
C) Quantification of PTAA positive cells. 100 cells from 3 independent experiments were counted and
PTAA positive cells were plotted as a percentage of total number of cells counted.
D) Suspension of 500 000 LD9 cells were stained with PTAA (5µg/ml PTAA diluted in PBS; 15 minutes at
37°C). Unbound PTAA was removed from the cells by centrifugation and washing with PBS. Afterwards
cells were resuspended in PBS and emission spectra were analyzing using plate reader (excitation 448nm). Signal intensity of 22L infected cells is significantly higher compared to uninfected cells. Free
PTAA (1µg/ml) was used as a positive control for spectral analysis. Experiment was done with 3
independent samples.
E) Western blot confirmed Proteinase K resistant material in 22L and RML infected LD9 cells. 100µg of
proteins were treated with 20µg/ml of proteinase K for 30minutes at 37°C. Control gel (without PK
digestion) was performed with 50µg of proteins. Both blots were analyzed with POM1 antibody.
SID 4 (Rev. 3/06)
Page 6 of 13
Ad (2) Identification of assays for the detection of ovine and bovine prions
In addition to our efforts to identify cell lines susceptible to ovine and bovine prions we also collaborated
with the laboratory of Charles Weissmann to understand how cells differ in their susceptibility to prion
strains (Mahal, 2007). Here, Mahal et al. assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5,
which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows
their discrimination in vitro within two weeks, using the standard scrapie cell assay (SCA). Within this
collaboration it became very clear that a cloned murine neuroblastoma cell population, N2a-PK1, is highly
heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may
show very different relative susceptibilities to the two strains, indicating that the responses can vary
independently.
Given that we are far from understanding the factors modulating susceptibility to different prion strains, we
looked for alternative possibilities to measure prion infectivity in vitro. Therefore, we developed the prion
organotypic slice culture assay (POSCA) that allows prion amplification and titration of infectivity ex vivo
under conditions that closely resemble intracerebral infection (Falsig J, 2008; Falsig, 2008).
The POSCA allows amplification and detection of prions in 35 days which is fivefold faster than
conventional mouse bioassay. Furthermore, the POSCA detected replication of prion strains from
disparate sources, including bovines and ovines, with variable detection efficiency by using e.g. the LCPs.
To further improve the detection of additional prion strains we are right now in the process of testing
different tissue donors for the POSCA. We also consider mice expressing a transgene encoding the Prnp
sequence of bank voles as promising since bank voles are susceptible to Scrapie and CJD prions (Nonno,
2006).
Luminescent conjugated polymers (LCPs)1 are molecules with electron-rich backbone and variable sidechains that display different reactivity and conformation-dependent fluorescence spectra upon binding to
protein aggregates. Fibrils with distinct morphologies generated from chemically identical recombinant PrP
yielded unique LCP spectra, suggesting that spectral characteristic differences resulted from distinct
supramolecular PrP structures. Thus, LCPs may help to detect structural differences among discrete
protein aggregates and to link protein conformational features with disease phenotypes. In addition, LCPbinding to protein aggregates might affect prion conversion and thus yield useful molecules for therapy or
strain typing.
The prion organotypic slice culture assay (POSCA) allows for an efficient and rapid amplification of PrPSc
in brain slices after exposure to prions. We have used this assay to replicate various mouse-adapted prion
strains, yielding prion aggregates with different growth rates, morphology and deposition patterns in tga20
brain slices (prepared from mice over-expressing PrPC). Moreover, fluorescence emission spectra upon
staining of different prion strains by LCPs in organotypic brain slices appeared to differ in spectral
composition. This provides a means to differentiate prion strains on the basis of the supra-molecular
structure of their aggregates. Therefore, these two technologies may allow for the investigation, in a fast
manner, of prion strain-related phenomena that remain unexplained with a focus on the underlying
molecular mechanisms.
One aim of the present project is and was to develop a methodological tool using the POSCA and the
LCPs for discriminating murine, ovine and bovine prion strains on the basis of their respective LCP spectra
in organotypic brain slices.
SID 4 (Rev. 3/06)
Page 7 of 13
Organotypic cerebellar slice cultures were prepared and infected with mouse-adapted prion strains like
previously reported2. PrPSc associated with different strains replicate with different efficiencies (Fig. 5a,
b). We identified 4 strains (that were originally derived from scrapie infected species) with high efficiency
of prion replication (RML, 22L, 139A, 79A) and 3 strains with low efficiency (301C, ME7, natural scrapie) in
cerebellar slices. Staining with an LCP (polythiophene acetic acid, PTAA) was performed like previously
reported1. At 3 weeks post-infection a subgroup of prion strains showed a diffuse deposition pattern
mainly located in the molecular cell layer (RML, 22L, 139A, 79A). After 5 weeks, dense aggregates in the
vicinity of blood vessels could also be observed with RML (Fig. 5d). However the 3 other strains yielded
only dense aggregates, generally localized in the meningeal region (ME7, NS, 301C, Fig. 5e).
Fig. 5: Detection of different prion strains in organotypic cerebellar slice cultures. (a, b) Immunoblots from
cultures from 10-d-old tga20+/+ and Prnpo/o pups, inoculated with various prion strains and harvested after 35 d.
Mock, noninfectious brain homogenate. (a) Transmission of mouse-adapted scrapie strains RML, 139A, 79A and
Mock (b) Western blotting performed under less stringent conditions on 30 mg protein digested with 25 mg ml–1
proteinase K (PK) (+) or 10 mg undigested protein (–) and detected with POM1. Cultures were inoculated with 0.1 mg
of brain homogenate and cultured for 35 d. Sc: terminal brain. 5193/1, 5192/2: inocula deriving from 2 different
passages of the strain “NS” in Bl/6 mice. (c, d, e, f) Fluorescence microscopy images from infected slice cultures after
21 days (c) or 35 days (d, e, f) of culturing. (c, d) RML-infected slice. (e, f) ME7-infected slice. (f) High magnification of
the dense aggregate shown in (e). PTAA stains prion deposits, IsolectinB4 stains microglia and epithelial cells.
We generally observed that the fast replicating strains yield aggregates with a mainly diffuse pattern of
deposition in the molecular layer whereas strains that replicate slower yield mainly dense prion aggregates
in the meningeal region (Table 1). Fluorescence spectra yielded by PTAA upon binding to aggregates
from four mouse-adapted prion strains in cerebellar slices seemed to differ in composition of wavelengths
(Fig. 6a). A correlation diagram is obtained by calculating the ratios of light intensity at different
wavelengths, illustrating structural differences of fibrils originating from different prion strains (Fig. 6b).
In order to develop a methodological tool for discriminating prion strains on the basis of their respective
LCP spectra in organotypic brain slices further experiments will aim at optimizing the staining protocol for
PTAA as well as H7A, a newly designed LCP with flexible backbone. Additionally the genotype of brain
slices may broaden the number of prion strains that can be used in this setup. To this effect we are in the
process of characterizing tgbankvolePrP mice (over-expressor of the Clethrionomys glareolus PrPC) that
may be susceptible to natural cases of ovine, bovine and human prions strains, which would allow for
studying strain adaptation in brain slices.
SID 4 (Rev. 3/06)
Page 8 of 13
Fig. 6: Spectral signature of prion strains in brain slices stained with PTAA. (a) Spectrum of PTAA upon binding
to aggregates from four mouse-adapted prion strains. Fluorescent light emission has been normalized to the most
intensely emitted wavelength (602nm). (b) "Correlation diagram": Alternative representation of the data from (a),
providing information about how PTAA binds to the fibrils.
The following table summarizes the efficacy and characteristics of the vearious prion strains propagated in
the organo-typic slice cultures.
Table 1: Seven different prion strains segregate into 2 groups according to their efficiency of replication and
deposition pattern of aggregates in organotypic brain slices. NS: “Natural Scrapie” (original inoculum obtained from a
scrapie-sick sheep from Colorado, U.S.A. and adapted to wild-type mice in our laboratory by C. Sigurdson).
SID 4 (Rev. 3/06)
Page 9 of 13
References
Falsig J, Julius C, Margalith I, Schwarz P, Heppner F, Aguzzi A (2008) A versatile prion replication assay
in organotypic brain slices. Nature Neurosci 11:109-117.
Falsig J, Aguzzi A (2008) The prion organotypic slice culture assay - POSCA. Nat Protoc In Press.
Mahal SP, Baker CA, Demczyk CA, Smith EW, Julius C, Weissmann C (2007) Prion strain discrimination
in cell culture: The Cell Panel Assay. Proc Natl Acad Sci U S A 104:20908-20913.
Moroncini G, Mangieri M, Morbin M, Mazzoleni G, Ghetti B, Gabrielli A, Williamson RA, Giaccone G,
Tagliavini F (2006) Pathologic prion protein is specifically recognized in situ by a novel PrP
conformational antibody. Neurobiol Dis 23:717-724.
Moroncini G, Kanu N, Solforosi L, Abalos G, Telling GC, Head M, Ironside J, Brockes JP, Burton DR,
Williamson RA (2004) Motif-grafted antibodies containing the replicative interface of cellular PrP
are specific for PrPSc. Proc Natl Acad Sci U S A 101:10404-10409.
Nonno R, Bari MA, Cardone F, Vaccari G, Fazzi P, Dell'omo G, Cartoni C, Ingrosso L, Boyle A, Galeno R,
Sbriccoli M, Lipp HP, Bruce M, Pocchiari M, Agrimi U (2006) Efficient transmission and
characterization of creutzfeldt-jakob disease strains in bank voles. PLoS Pathog 2:e12.
Sigurdson CJ, Nilsson KPR, Hornemann S, Manco G, Polymenidou M, Schwarz P, Hammarström P,
Wüthrich K, Aguzzi A (2007) Prion strain discrimination using luminscent conjugated polymers.
Nature Methods 4(12):1023-30.
Amendments to project
9.
Are the current scientific objectives appropriate for the remainder of the project? .................YES
SID 4 (Rev. 3/06)
Page 10 of 13
NO
If NO, explain the reasons for any change giving the financial, staff and time implications.
Contractors cannot alter scientific objectives without the agreement of the Defra Project Manager.
Progress in relation to targets
10. (a) List the agreed milestones for the year/period under report as set out in the contract or any agreed
contract variation.
It is the responsibility of the contractor to check fully that all milestones have been met and to
provide a detailed explanation when they have not been achieved.
Milestone
Number
Milestones met
Target date
Title
In full
01/01
Generation of tagged ovine and bovine
PrPs
month 7
01/02
Identification of susceptible cell lines
month 7
02/01
Assessment of properties of tagged
PrPs
month 16
02/02
Cloning of highly susceptible sublines
month 16
02/03
Generation of cell lines stably
expressing tagged PrPs
month 26
In
progress
03/01
Assessment of generated cell lines in
TraSCA
month 30
In
progress
03/02
Assessment of application of
technology to measurement of prion
infectivity in fluids and tissues
month 36
Note:
As stated in the scientific progress report above and last years report,
rather than go straight into cloning, expressing and deriving all
possible tagged PrPs in ovine and bovine cell lines we have opted
first to acquire knowledge on susceptibility and performance of cell
lines and alternative detection methods (POSCA) for ovine and
bovine prions. This does not preclude generation of tagged versions
of ovine and bovine PrPs, but due to experience obtained with the
murine system by now we consider this approach more efficient in
terms of allocation of research resources.
Since the investigation of alternative detection methods for PrPSc
(motif-grafted antibodies and luminescent conjugated polymers) yield
promising first results we propose to focus our efforts on these
methods.
The application of the various culture systems (cell culture;
organotypic slice culture) is currently tested as a tool to investigate
prion infectivity in fluids and tissues e.g. derived from scrapie sick
sheep.
See note
below
Yes
See note
below
Yes
(b) Do the remaining milestones look realistic?.....................................................................YES
If you have answered NO, please provide an explanation.
SID 4 (Rev. 3/06)
Page 11 of 13
On time
Yes
NO
Publications and other outputs
11. (a) Please give details of any outputs, e.g. published papers/presentations, meetings attended during this
reporting period.
Falsig J, Julius C, Margalith I, Schwarz P, Heppner F, Aguzzi A (2008) A versatile prion replication
assay in organotypic brain slices. Nature Neurosci 11:109-117.
(b) Have opportunities for exploiting Intellectual
Property arising out of this work been identified? ............................................................YES
If YES, please give details.
(c) Has any other action been taken to initiate Knowledge Transfer?...................................YES
If YES, please give details.
NO
NO
Future work
12. Please comment briefly on any new scientific opportunities which may arise from the project.
We further consider the concentration of our efforts on the detection of de novo formed PrPSc by motifgrafted antibodies, specifically recognizing PrPSc (Moroncini et al., 2004 and this work) and luminescent
conjugated polymers. As shown in this report we have established in vitro tools that appear to be
applicable for various prions strains - also derived from e.g naturally scrapie sick sheep - in cell lines as
well as organotypic slice cultures
We have expanded our investigations on additional cell lines and prion strains and we have optimized the
staining and readout procedures in order to achieve reproducible assessment of infected cells e.g. by
FACS sorting. Furthermore, we will investigate the applicability of the POSCA to assess ovine and bovine
prions and identify cell lines susceptible to ovine and bovine prions.
The combination of highly susceptible cell lines and organotypic slice cultures with the detection
opportunities enabled by the luminescent conjugated polyelectrolyte probes or motif-grafted
antibodies allow sensitive and specific high-throughput screening of ovine and bovine tissues and
fluids and tissue.
SID 4 (Rev. 3/06)
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Declaration
13. I declare that the information I have given is correct to the best of my knowledge and belief.
Name
Position held
Adriano Aguzzi
MD, PhD hc, FRCP, FRCPath
Chairman, Department of Pathology
University Hospital Zürich
SID 4 (Rev. 3/06)
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Date
03.05.2009