Poster Session PPo8: Diagnostics, Therapeutics and Decontamination

PPo8-1: Performances of Three Rapid Post Mortem Tests for Active Surveillance of TSE in Goats

Daniela Meloni,¹ Elsa Manzardo,¹ Maria C. Cavarretta,¹ Simone Peletto,¹ Umberto Agrimi,¹ Jan Langeveldt,¹ Alex Bossers,² Silvia Colussi,² Pier Luigi Acutis,² Francesco Ingravalle² and Elena Bozzetta²

¹CEA; Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta; Torino, Italy; Istituto Superiore di Sanità; Roma, Italy; ²Central Veterinary Institute of Wageningen UR; Lelystad, The Netherlands

Key words: Goat TSE, post mortem test, sensitivity

Introduction. Seven rapid tests were approved according to the Commission Regulation (EC) 162/2009 for active surveillance of TSE in sheep and goat, however EFSA Scientific Opinion on Analytical Sensitivity of Approved TSE rapid tests – 2009 recommends only three of those tests: Bio-Rad TeSeE SAP (A), Bio-Rad TeSeE Sheep/Goat (B) and Idexx HerdChek Antigen Test Kit EIA (C). Aim of the study was to provide a direct comparison of the performances of the three rapid tests when applied to goat brainstem.

Results. Differences among the three systems were observed in terms of diagnostic sensitivity, test C being the most sensitive (94,7%, 95%CI:82,3–99,4%) while all three rapid tests displayed 100% specificity. The agreement in reproducibility and repeatability was very good.

Patients. A sample size of 73 confirmed positive and negative samples, including both classical and atypical field cases, experimentally inoculated goats and regularly slaughtered animals, were tested.

Conclusion. Whenever all three tests displayed good performance, according to results achieved on diagnostic sensitivity a difference in the efficacy of TSE active surveillance system in goat populations, based on the test adopted, can be expected.

Acknowledgments

This work was funded by EU project GoatBSE (FOOD-CT-2006-36353).

PPo8-2: Dimerization of PrP^Sc by Thienyl Pyrimidine Compounds Trap Prion Infectivity

Adeline Ayrolles-Torro, Karine Toupet, Joan Torrent, Ilia Baskakov, Guillaume Poncet-Montange, Thibaut Imberdis, Sylvain Lehmann, Didier Rognan, Martine Pugnière, Jean-Michel Verdier and Véronique Perrier

Recent studies have reported variant Creutzfeldt-Jakob disease (vCJD) cases resulting from transfusion of prion-contaminated blood, raising concerns about the safety of blood product, as well as the possibility of a second epidemic of vCJD. It has become urgent to develop an innovative therapeutic strategy. The emerging concept, not only for prion diseases, but also for other neurodegenerative disorders, is that amyloid fibrils, rather than being pathological, might be the result of a protective process to sequester more toxic and infectious soluble oligomers, that are key intermediates in the replication cycle of prions.

Therefore we developed a rational drug design approach to search for chemical molecules that could trap small PrP^Sc oligomers through stabilization of dimers, trimers, or small oligomeric entities, thus preventing their recycle into the pathological process. By virtual screening followed by cellular screening, we identified a family of thienyl pyrimidine derivatives that specifically trigger oligomerization of PrP^Sc and not of PrP^C. Bioassays performed in prion-infected cells and brain homogenates showed that these compounds diminish prion infectivity. These results indicate that trapping prions in small oligomeric forms with thienyl pyrimidine compounds represents a new promising approach for the development of therapeutics, not only for prion diseases, but also for other neurodegenerative proteinopathies, such as Alzheimer’s disease.

PPo8-3: Application of Epifluorescence Scanning for Monitoring the Efficacy of Protein Removal by RF Gas-plasma Decontamination

Helen C. Baxter, Anita C. Jones and Robert L. Baxter

School of Chemistry; University of Edinburgh; Kings Buildings; Edinburgh, Scotland UK

Key words: RF, gas plasma decontamination, surface monitoring, EFSCAN, TSE

A variety of gas-plasma techniques are capable of destroying both small organic molecules and larger biomolecular structures adhering to, or adsorbed on, a variety of surfaces without causing significant damage to the surfaces themselves. This has led to a large number of applications in the medical field including prion decontamination. However, it would be true to say that, while the technique is capable of very significant reduction of the surface load of contamination, the development of methods to quantify the efficacy of particular treatments lags far behind the development of the plasma processes themselves.

The requirement for a quantitative routine method for analysis of surface-bound protein molecules, suitable for measurement of sub-nanogram mm-2 levels, which could be employed as a routine method for contamination quantification both before and after decontamination procedures, has led us to develop a more general approach, epifluorescence scanning (EFSCAN).¹ This involves in situ covalent derivatization of the amino and thiol groups of surface-bound protein molecules with fluorescein-based fluorophores and development of a surface scanning fluorimeter capable of measuring the resultant fluorescence of labelled surfaces-bound proteins.

We will report the use of this technology for monitoring, (a) stainless steel wire insert contamination procedures for TSE bioassays, (b) residual contamination on reprocessed surgical instruments and (c) the efficacy of RF gas-plasma decontamination procedures of surgical instruments and of TSE contaminated wire inserts.

References

1. Baxter, et al. Application of epifluorescence scanning for monitoring the efficacy of protein removal by RF gas–plasma decontamination. NJP 2009; 11:115028.

PPo8-4: Temporal Evolution of Creutzfeldt-jakob Disease Monitored by 3-tesla Proton Magnetic Resonance Spectroscopy

Koji Fujita,¹ Masafumi Harada,² Tatsuhiko Yuasa,³ Makoto Sasaki,⁴ Yuishin Izumi¹ and Ryuji Kaji¹

¹Department of Clinical Neuroscience; Institute of Health Biosciences; ²Department of Medical Imaging; Institute of Health Biosciences; The University of Tokushima Graduate School; Tokushima, Japan; ³Department of Neurology; Kamagaya-Chiba Medical Center for Intractable Neurological Disease (KC-MIND) Kamagaya General Hospital; Kamagaya, Japan; ⁴Advanced Medical Science Center; Iwate Medical University; Morioka, Japan

Key words: prion disease, Creutzfeldt-Jakob disease, magnetic resonance imaging, magnetic resonance spectroscopy

Introduction. We investigated the propagation of neuronal and glial alterations in vivo in Creutzfeldt-Jakob disease (CJD) using 3-Tesla proton magnetic resonance spectroscopy (MRS).

Results. In cases 1 and 2, successive elevations of the Cho to NAA ratio indicated progressive neuronal dysfunction and gliosis. In case 3, a substantial increase in myo-Inositol with relatively preserved NAA, compared to disease controls, indicated astrocytosis without massive neuronal damage during this period, and agrees with data from studies of other types of CJD. A positive lactate peak in every session of all three patients suggested persistent anaerobic glycolysis or glial proliferation early following onset.

Patients and Methods. Three patients with CJD underwent 3-Tesla MRI and MRS (with single or multi voxel methods, or both) between 12 and 24 weeks following onset. Two patients had sporadic CJD (cases 1 [probable] and 2 [definite]), and one patient had familial CJD with a methionine to arginine substitution at codon 232 of the prion protein gene (case 3). The neurochemical compounds, N-acetyl aspartate (NAA), choline-containing compounds (Cho), creatine and phosphocreatine, myo-Inositol, and lactate were evaluated.

Conclusion. Our 3-Tesla MRS studies demonstrate in vivo that altered glial cells can precede neuronal damage, and propagate during the course of CJD. Three-Tesla MRS is a useful technique for monitoring in vivo biochemical changes that reflect the progression of glial and neuronal damage in the brains of CJD patients.

PPo8-5: Receptor-associated Protein (RAP) Inhibits Generation of Disease-associated Prion Protein (PrPd) in Cell Cultures

Larisa Cervenakova,¹ Oksana Yakovleva,¹ Irina Vasilyeva,¹ Sergey Akimov,^1,2 Irina Mikhailenko³ and Dudley K. Strickland³

¹American Red Cross Holland Laboratory; Rockville, Maryland USA; ²Johns Hopkins University School of Medicine; Baltimore, MD USA, ³University of Maryland School of Medicine; Baltimore, Maryland USA

Key words: transmissible spongiform encephalopathy (TSE), prion protein (PrP), cell culture, fukuoka-1, mouse-adapted variant Creutzfeldt-Jakob disease (mo-vCJD), receptor associated protein (RAP), treatment

Introduction. The low-density lipoprotein (LDL) receptor-related protein (LRP1), member of the LDL receptor superfamily, interacts with number of ligands, including cellular prion protein (PrPC). RAP is a 39–44 kDa protein found in the rough endoplasmic reticulum that binds to LDL-receptor family members (LDL-RFMs) and functions as a specialized chaperone assisting in their folding and preventing association of nascent LDL-RFMs with ligands during transport to the cell surface. RAP is expressed in various organs and tissues, including the brain, and is efficiently transferred across the blood-brain barrier. A decrease in RAP levels enhances amyloid deposition in mouse model of Alzheimer’s disease. We hypothesized that RAP may also affect the formation or stability of PrPd.

Results. At rRAP concentrations between 20 and 250 nM, PrPd in cell cultures was significantly reduced within 12 to 24 hours. Cells treated six times with 250 nM rRAP at 8, 12, 24, 36, 48, 60 hours showed complete absence of PrPd through at least 20 passages after the treatment was stopped.

Materials and Methods. Murine spleen- and bone marrow-derived cell cultures, infected with either mouse-adapted vCJD or Fukuoka-1 agents, were treated with various concentrations of human rRAP for various periods of time. The formation of PrPd was then assessed by testing for the PrPres, the core product of PrPd after the treatment with proteinase K, by western blotting.

Conclusion. In vitro, rRAP inhibits formation and/or promotes the clearance of PrPd in cell cultures infected with TSE agents directly or through interaction with other protein(s), such as LDL-RFMs.

PPo8-6: Evaluation of a Sandwich ELISA for the Gamma-isoform of 14-3-3 Proteins for Laboratory Diagnosis of Creutzfeldt-Jakob Disease

Yuki Matsui,¹ Katsuya Satoh,² Toshiaki Miyazaki,³ Susumu Shirabe,⁴ Ryuichiro Atarashi,² Yasufumi Kataoka¹ and Noriyuki Nishida²

¹Department of Pharmaceutical Care and Health Sciences; Faculty of Pharmaceutical Sciences; Fukuoka University; Fukuoka, Japan; ²Department of Molecular Microbiology and Immunology; Graduate School of Biomedical Science; Nagasaki University; Nagasaki, Japan; ³Cyclex Co., Ltd.; Nagano, Japan; ⁴Center for Community and Campus Health; Nagasaki University; Bunkyo, Nagasaki Japan

Key words: 14-3-3 protein, diagnosis, ELISA, CJD

Introduction. Gamma-isoform of 14-3-3 proteins (14-3-3g) is expressed in neurons and could be a specific marker for neuronal damages. The protein has been reported to be a detectable biomarker especially in CSF of CJD patients by either western blotting (WB) or Enzyme linked immunosorbent assay (ELISA). However, WB for 14-3-3g is not sensitive enough and data are diverse among reports. ELISA kit specific for 14-3-3g has not been available and the standard procedure and criteria are needed for clinical use. Here, we developed a new sandwich ELISA kit for 14-3-3g and evaluated whether or not the kit can be useful for laboratory differential diagnosis of variety of neurological diseases.

Results. Sensitivity of the sandwich ELISA kit for 14-3-3g was 95.6% and specificity was 71.2% as well as ELISA for total tau protein. 7 CJD cases was negative for the WB but positive using the ELISA for 14-3-3g, indicating the ELISA is much more sensitive for CJD.

Patients and Methods. 124 CJD patients (114 cases of sporadic CJD patients, 7 familial CJD and 3 iatrogenic CJD) and 99 patients with other neurodegenerative diseases were subjected in this study. CSF samples obtained from these cases were analyzed by WB for 14-3-3g and by ELISA for total tau protein. We evaluated sensitivity and specificity of the sandwich ELISA kit for 14-3-3g.

Conclusion. The sandwich ELISA kit for 14-3-3g was much more sensitive test than conventional WB for 14-3-3g and was useful for laboratory diagnosis of CJD similar to ELISA for tau protein.

PPo8-7: Prion Diagnostics by Single Particle Counting

O. Bannach, F. Henke, E. Reinartz, D. Willbold, D. Riesner and E. Birkmann

Institut für Physikalische Biologie; Heinrich-Heine-Universität Düsseldorf; Düsseldorf, Germany

Key words: blood plasma diagnosis

Prion diseases can be transmitted via body fluids and excretions, such as urine, feces, saliva and blood. Among other findings, the transmission of four cases of variant CJD by blood transfusion raised serious concerns about blood product safety, and emphasizes the need of a robust test system to detect prions in living humans or animals. We present optimization and application of an ultra-sensitive diagnostic assay based on surface-FIDA (fluorescence intensity distribution analysis) that allows for counting single prion particles. Prion aggregates are immobilized via a capture antibody to a chip surface, and are labeled with two fluorescent antibodies. Sample preparation is based on detergent and lipase treatment followed by particle enrichment via precipitation with phosphotungstic acid. Particle binding was enhanced by introducing a centrifugation step to sediment aggregates to the capture surface. Two laser beams scan the surface and distribution of prion particles can be visualized. Images from two channels are co-localized and evaluated with respect to particle number, size and intensity. The method was successfully applied to brain tissue from humans and cattle, as well as to blood plasma from sheep. We deliver proof-of-principle that our diagnostic approach is capable to detect single particles in blood, and is therefore a potential ante mortem test for prion diseases.

PPo8-8: Detection and Characterization of Prions Using Fluorescent-labeled PrP Peptides

Kazuo Kasai,¹ Akiyoshi Hitara,² Takafumi Ohyama,² Kiyoshi Nokihara,² Yuichi Matsuura,¹ Shunsuke Yajima,³ Takashi Yokoyama¹ and Shirou Mohri¹

¹Prion Disease Research Center; National Institute of Animal Health; Tsukuba, Ibaraki ,Japan; ²HiPep Laboratories; Kamigyo-ku, Kyoto, Japan; ³Faculty of Applied Bioscience; Tokyo University of Agriculture; Setagaya-ku, Tokyo, Japan

Key words: fluorescent-peptide, prion-detection, characterization

Although the pathogenesis of prion diseases has not been completely elucidated, the conversion of the cellular isoform of the prion protein (PrP^C) to prion-associated isoform (PrP^Sc) is the central event in the prion pathogenesis of prion diseases. We attempted to discriminate PrP^Sc from PrP^C through the protein-peptide interaction studies. We synthesized 25 polypeptides, each consisting of 15 amino acid residues and shifted by 10 amino acids; these peptides covered the entire human PrP sequence. The N-termini of the peptides were attached a fluorescent dye. The peptides were mixed with the brain homogenates of the animals, and their fluorescence intensities were measured. We selected the peptides that exhibited different fluorescence intensities between the prion-affected animal samples (mouse scrapie, sheep scrapie and cattle BSE) and those of healthy animals. Two peptides corresponding to the hydrophilic reion (PrP21-35) and the octapeptide-repeat domain (PrP51-65) were distinct. Pull-down assays revealed the binding of these peptides to PrP^Sc of scrapie and BSE, despite the alterations of fluorescence intensities were differed related to the prion strains and/or animal species. These results suggest that the fluorescence change was attributed to the conformational differences of the PrP^Sc. This difference was not detected by the pull-down assays. Thus, this novel approach may provide a new insight into the conformational analysis of the PrP^Sc of different prion strains.

PPo8-9: A Data Driven Methodology for the Determination of Novel Protein Biomarkers in BSE

Janice B. Barr, David Waddington and Rona M. Barron

The Roslin Institute & R(D)VS; University of Edinburgh; Roslin, Scotland UK

Key words: SELDI-TOF, BSE, proteins

Following a successful murine study establishing that differential protein expression profiling using SELDI -TOF MS technology could distinguish between scrapie infected and non-infected brain tissue homogenates,¹ we sought in the present study to apply this methodology to samples from out-bred large animals in the field.

Bovine brain tissue samples (26 BSE & 25 non-BSE) were obtained from the VLA, TSE Archive, UK for the determination of disease status by differential protein expression profiling. The sample homogenates were analysed on ProteinChip arrays using two surface chemistries (CM10 & IMAC-Cu). Analysis of the resulting spectral data using statistical learning and linear discriminant analysis techniques, established protein markers of disease with good predictive power (AUC = 0.88). Future work will include identification of the differentially expressed proteins and further testing of additional samples including other neurological conditions to determine specificity for TSE infection.

References

1. Barr JB, et al. Differential protein profiling as a potential multi-marker approach for TSE diagnosis. BMC Infect Dis 2009; 9:188.

PPo8-10: Liposome-siRNA-peptide Complexes Protect and Deliver: PrP siRNA across the Blood-Brain Barrier to Neuronal Cells and Cure Prion Infection in vitro

Mark D. Zabel and Bruce Pulford

Department of Microbiology, Immunology and Pathology; College of Veterinary Medicine and Biomedical Sciences; Colorado State University; Fort Collins, CO USA

Key words: prions, siRNA, liposomes, peptide

Recent advances toward an effective therapy for prion diseases employ RNA interference to suppress PrP^C expression and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with host PrP^CPrP^C expression levels. However, delivery of lentivirus encoding PrP shRNA has demonstrated only modest efficacy in vivo. Here we describe a new siRNA delivery system incorporating a small peptide that binds siRNA and acetylcholine receptors (AchRs), acting as a molecular messenger for delivery to neurons, and cationic liposomes that protect siRNA-peptide complexes from serum degradation. These liposome-siRNA-peptide complexes (LSPCs) delivered PrP siRNA specifically to AchR-expressing cells, suppressed PrP^C expression and cured prion infection in vitro. LSPCs injected intravenously into mice resisted serum degradation and delivered PrP siRNA throughout the brain to AchR and PrP^C-expressing neurons. These data promote LSPCs as effective vehicles for delivery of PrP and other siRNAs specifically to neurons to treat prion and other neuropathological diseases.

PPo8-11: A One Step Triplex Immunofluorometric Assay Enables Differential Diagnosis of BSE, Classical and Atypical Scrapie

Yue Tang,¹ Jorg G. Jacobs,² Jan Langeveld² and Maurice J. Sauer¹

¹Department of Molecular Pathogenesis and Genetics; Veterinary Laboratories Agency-Weybridge; New Haw; Addlestone, Surrey UK; ²Department of Bacteriology and TSEs; Central Veterinary Institute of Wageningen UR; Lelystad, The Netherlands

Introduction. There remains a need for simple differential diagnostic methods to facilitate cost effective TSE surveillance aimed at protecting the food chain and animal welfare. Differential methods generally require application of parallel multiple tests. We previously described a multiplex immunofluorometric assay, based on a bead array flow cytometry technology, which distinguished, in a single assay, BSE (bovine and ovine) and classical scrapie (Tang et al. 2010). Recent developments have enabled this assay to incorporate atypical scrapie diagnosis.

Results and conclusions. Analysis of sheep PrPres from blinded brain stem samples indicated that whereas PrPres from classical scrapie bound to 12B2, 9A2 and 94B4, PrPres from BSE and atypical scrapie bound predominantly to 9A2 and 94B4 and to 12B2 and 94B4, respectively. Furthermore, analysis of serially diluted samples indicated that the assay was several fold more sensitive for atypical scrapie than a leading commercially available test. These data suggest that this multiplex approach will enable appreciable simplification of TSE diagnosis, providing a platform suitable for both screening and differential diagnosis.

Methods. The assay principle is based on distinguishing differences in the extent of PK digestion of prion protein derived from each TSE type. This is achieved by establishing the extent of binding of capture antibodies 12B2, 9A2 and 94B4 to regionally distinct epitopes of the product (PrPres); each capture mAb is attached to a distinct bead array type. A biotinylated reporter antibody (Sha31), used in conjunction with streptavidin-phycoerythrin, enabled flow cytometric quantification of the extent of each interaction and thus classification.

PPo8-12: Distinct Multiplex Diagnosis of Seven TSE-types from Cattle and Sheep

Jorg G. Jacobs,¹ Lucien J.M. van Keulen,¹ Maurice J. Sauer,² Yue Tang,² Alex Bossers¹ and Jan P.M. Langeveld¹

¹Central Veterinary Institute of Wageningen UR; Lelystad, The Netherlands; ²Department of Molecular Pathogenesis and Genetics; Veterinary Laboratories Agency-Weybridge; New Haw; Addlestone, Surrey UK

Key words: multiplex, immunoblot, differential diagnosis, TSE, ruminant

In cattle and sheep seven main types of TSE can be discriminated, which upon typing in mice, bank voles or hamsters might yield ultimately one or more strains. There is a need for quick and effective isolate typing methods, especially for safety decisions on the presence or absence of BSE in animals other than cattle. western blotting will yield detailed information on the molecular aspects of PrP^Sc which often leads to direct recognition of the kind of strain to be expected. Until now the technique was used by running in parallel different blots for each antibody. But there is a demand for a faster quantitative multiplex test. Here, we used a single incubation multiplexed blot with three PrP specific antibodies. The antibodies were pre-labeled with fluorescent Alexa dye conjugated anti-mouse Fab’s. Detection was with a Typhoon Trio imager with three lasers. The combination of three antibodies directed towards three PrP epitopes enabled differential diagnosis of all seven main TSE types: atypical scrapie, classical scrapie, CH1641 scrapie and BSE in sheep, as well as the BSE types C, H and L in cattle. Due to a combination of a special N-terminal feature (different PK cleavage) and dualistic antibody dependent glycoprofile, for the first time CH1641 could be reliably discriminated from the other TSEs in sheep. This application of three differential antibodies in a single incubation step on a single blot represents an effective approach for analysing PrPres: it exhibits excellent linear correlation, and allows robust quantitation of N-terminal cleavage differences and glycoprofiles.

PPo8-13: Degradation of Pathogenic Prion Protein and Prion Infectivity by Lichens

Christopher J. Johnson,¹ James P. Bennett,¹ Steven M. Biro,^1,2 Cynthia M. Rodriguez,^1,2 Richard A. Bessen³ and Tonie E. Rocke¹

¹USGS National Wildlife Health Center; ²Department of Bacteriology; University of Wisconsin, Madison; ³Department of Veterinary Molecular Biology; Montana State University; Bozeman, MT USA

Key words: prion, lichen, bioassay, protease, degradation

Few biological systems have been identified that degrade the transmissible spongiform encephalopathy (TSE)-associated form of the prion protein (PrPTSE) and TSE infectivity. Stability of the TSE agent allows scrapie and chronic wasting disease agents to persist in the environment and cause disease for years. Naturally-occurring or engineered processes that reduce infectivity in the environment could aid in limiting environmental TSE transmission. We have previously identified that species of at least three lichens, unusual, symbiotic organisms formed from a fungus and photosynthetic partner, contain a serine protease capable of degrading PrPTSE under gentle conditions. We tested the hypothesis that lichen extracts from these three species reduce TSE infectivity by treating infected brain homogenate with extracts and examining infectivity in mice. We found lichen extracts diminished TSE infectious titer by factors of 100 to 1,000 and that reductions in infectivity were not well-correlated with the extent of PrPTSE degradation observed by immunoblotting. For example, treatment of brain homogenate with Cladonia rangiferina extract caused <100-fold reduction in PrP immunoreactivity but ∼1,000-fold decrease in infectivity, suggesting that some PrPTSE remaining after extract treatment was rendered uninfectious or that the lichen protease favors more infectious forms of PrPTSE. Our data also indicate that lichen species closely related to those with prion-degrading protease activity do not necessarily degrade PrPTSE. Characterization of the lichen species-specificity of PrPTSE degradation within the genera Cladonia and Usnea and comparison with known lichen phylogeny has yielded clusters of species on which to focus searches for anti-prion agents.

PPo8-14: Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil

Samuel E. Saunders,¹ Jason C. Bartz,² Kurt C. Vercauteren³ and Shannon L. Bartelt-Hunt¹

¹Department of Civil Engineering; University of Nebraska-Lincoln; Peter Kiewit Institute; Omaha, Nebraska USA; ²Department of Medical Microbiology and Immunology; Creighton University; Omaha, Nebraska USA; ³USDA; Animal and Plant Health Inspection Service; Wildlife Services; National Wildlife Research Center; Fort Collins, CO USA

Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). Serial PMCA of treated soil samples suggests a greater than 6-log decrease in infectious titer compared with controls. We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity.

PPo8-15: Humanized PrP^Sc-specific Antibody Fragments–A Step Towards Human Prion Disease Therapy

Nives Škrlj,¹ Tanja Vranac,² Mara Popovi,³ Vladka urin Šerbec^1,2 and Marko Dolinar¹

¹Biochemistry Chair; Faculty of Chemistry and Chemical Technology; University of Ljubljana; Jamova, Ljubljana Slovenia; ²Department for Production of Diagnostic Reagents and Research; Blood Transfusion Centre of Slovenia; Šlajmerjeva, Ljubljana Slovenia; ³Institute of Pathology; Faculty of Medicine; University of Ljubljana; Korytkova, Ljubljana Slovenia

Key words: prion, antibody humanization,scFv, antibody fragment

Murine monoclonal antibody V5B2 which specifically recognizes the disease-associated form of the prion protein (PrP^Sc) represents a potentially valuable tool for prion diseases diagnostics and therapy. For easier biotechnological production, we first prepared single-chain antibody fragments (scFv) with retained specificity of the parent antibody. Since human immune system reacts to murine antibodies, application of murine mAbs is not suitable for therapeutic intervention in human prion diseases. We thus constructed a murine scFv with two opposing arrangements of variable domain segments connected by a flexible linker. Next, we performed humanization by variable domain resurfacing approach guided by computer modelling. Based on sequence alignments and on a computer model, a humanized version bearing 13 mutations was designed.

Humanized scFv was constructed, expressed and purified using an in-house bacterial vector system. Unaltered binding affinity to the antigen was demonstrated by ELISA and maintained binding specificity was demonstrated by western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, our humanized scFv specific for PrP^Sc could represent a potential therapeutic reagent.

PPo8-16: FTIR Microspectroscopy: A Multiple-screening Platform for Investigating Single-cell Biochemical Perturbations upon Prion Infection

Alessandro Didonna,^1,* Lisa Vaccari,^2,* Alpan Bek³ and Giuseppe Legname^1,2,4,*

*These authors contributed equally to the work

¹Neurobiology Sector; Scuola Internazionale Superiore di Studi Avanzati; International School for Advanced Studies (SISSA-ISAS); Trieste, Italy; ²ELETTRA Laboratory; Sincrotrone Trieste S.C.p.A.; ³CBM S.c.r.l.; Consorzio per il Centro di Biomedicina Molecolare; Center for Molecular Biomedicine; and ⁴Italian Institute of Technology; SISSA-ISAS Unit; Bonomea, Trieste, Italy

Key words: prion, PrP^Sc, infrared microspectroscopy (IRMS), synchrotron radiation (SR), functional group mapping, cluster analysis

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrP^Sc) possesses the capability to replicate into nascent PrP^Sc from the host-encoded cellular isoform of the prion protein. The present work aims at providing novel insight into cellular response upon prion infection, as evidenced by Synchrotron Radiation InfraRed MicroSpectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations occurring in prion infected mouse hypothalamic GT1-1 cells at cellular and sub-cellular levels. Monitoring spectral differences allowed to discriminate between infected and uninfected cells, introducing IRMS as a robust diagnostic tool to detect prions in biological samples. By comparing the average spectra of infected and un-infected cells, an increased number of lysosomes in infected cells was postulated, and further confirmed by SR-IRMS at sub-cellular spatial resolution and by fluorescent microscopy. The purpose of this work, therefore, consists in proposing IRMS as a powerful multi-screening platform, drawing on the synergy with conventional biochemical assays to increase the accuracy of investigations performed at the single cell level.

PPo8-17: Characteristics of a New Microbial Protease that Degrades the Prion Protein

Jack O’Sullivan, Mary Murphy and Hilary E.M. McMahon

UCD School of Biomolecular and Biomedical Science; University College Dublin; Belfield, Dublin Ireland

Key words: prion, protease, decontamination

Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders affecting animals and humans. The prion protein (PrP^Sc) is the agent responsible for these disorders and it is renowned for its stability and resistance to standard sterilisation practices. Currently there are concerns over the existence of TSE infected asymptomatic individuals within the population. To restrict iatrogenic spread a range of methods hazardous to both surgical equipment and operators have been proposed. Some methods proposed to eliminate prion contamination, including exposure to NaOH (1 M), sodium hypochlorite solution (20,000 ppm of available chlorine) or high temperature porous load autoclave, are not suitable for many delicate devices. Alkaline cleaners (pH 12.2) along with mixes of 0.2% SDS and 0.3% NaOH (pH 12.8) were reported to completely remove 263 scrapie infectivity. For delicate equipment there is a need for harmless approaches lacking alkaline processing. The search for milder approaches that can be used in a safe and routine manner to limit the spread of TSEs has lead to the screening of microorganisms for proteases capable of degrading PrP^Sc. We have isolated a new microbial isolate which produces a protease capable of degrading PrP^Sc at pH 7.0 and under mild conditions. This protease could have the potential to be adapted for decontamination practices.

PPo8-18: Coated Nanogolds as Novel Potent Anti-prion Compounds

Hoang Ngoc Ai Tran,^1,† Fernanda Sousa,^2,† Fabio Moda,^3,† Subhra Mandal,⁴ Munish Chanana,⁵ Chiara Vimercati,³ Ruggerone Margherita,³ Campagnani Ilaria,³ Michela Morbin,³ Silke Krol,² Fabrizio Tagliavini³ and Giuseppe Legname^1,6,

¹Neurobiology Sector; Scuola Internazionale Superiore di Studi Avanzati; International School for Advanced Studies (SISSA-ISAS); Bonomea, Trieste Italy; ²NanoBioMed Laboratory at LANADA; CBM; Cluster in Biomedicine; Basovizza, Trieste Italy; ³Division of Neurology and Neuropathology; “Carlo Besta” National Neurological Institute; Milano, Italy; ⁴SISSA-ISAS; Bonomea, Trieste Italy; ⁵Max-Planck Institute of Colloids and Interfaces; Wissenschaftspark Golm, Potsdam, Germany; ⁶ELETTRA Laboratory; Sincrotrone Trieste S.C.p.A.; Basovizza, Trieste Italy

^†These authors equally contributed to this work.

Key words: prion protein, scrapie prion replication, nanoparticles, anti-prion compounds

Prion diseases uniquely manifest as spontaneous, inherited and infectious, resulting in invariably fatal brain disorders. In this study, nanogold particles with layer-wise deposition of oppositely charged polyelectrolytes, such as polyallylamine hydrochloride (PAH) and polystyrenesulfonate (PSS), were produced. Different coatings, finishing with a positive or a negative layer, were tested, and different numbers of layers were investigated for their ability to interact and reduce the accumulation of the disease-causing prion protein (PrP^Sc) in scrapie prion infected mouse hypothalamic GT1 (ScGT1), and neuroblastoma N2a (ScN2a) cells. The efficiency of the particles in inhibiting prion replication was found to be dependent upon the outermost layer, and the number of layers. The best results in terms of cell survival and efficiency were obtained for particles coated with two layers, and PAH as outermost layers. The particles efficiently hampered the accumulation of PrP^Sc in ScN2a cells, and showed curing effects on ScGT1 cells with a nanoparticle concentration in the picomolar range. Finally, incubation periods of prion-infected mice treated with nanomolar concentrations of nanogold particles were significantly longer compared to untreated controls. Our findings, therefore, define a novel class of potent anti-prion compounds.

PPo8-19: Probing Prion Interactions with Small Molecule Human Metabolites

Rolando Perez-Pineiro, Trent C. Bjorndahl, Ying W. Dong, Mark V, Berjanskii, David S. Wishart

Department of Biological Sciences; University of Alberta

Key words: prion, ligand binding, human metabolites

After more than thirty years of study, the exact physiological function of the prion protein remains controversial. A number of independent studies have examined possible protein function using knock-out strains, in silico modeling of sequence and structural homology, and yeast two-hybrid screening. While these methods are useful for identifying macromolecular binding interactions and piecing together biological pathways, they do not address possible small molecule binding partners. Thousands of potential ligands for PrP have been examined, however, these studies have usually focused on potential treatments for prion disease or the inhibition of fibril formation rather than probing protein function. In this study, a directed approach was taken by screening a subset of small molecules from the Human Metabolite Database (http://www.hmdb.ca). We focused on a subset of 153 water soluble compounds present in human cerebrospinal fluid (CSF) on the basis that the CSF is a likely location for small-molecule binding partners for the prion protein, which is highly expressed in the brain. The initial screening was performed using 1D-NMR by comparing mixtures of metabolites in the presence and absence of recombinant Syrian hamster prion protein. Through this analysis, four compounds were identified as possible binding partners based on chemical shift perturbations of the ligand. Among these “hits”, thiamine HCl and cytidine show quenching of the fluorescence of the prion protein at 280 and 295 nm with estimated Kd’s in the micromolar range. Follow up 2D NMR experiments have been performed to determine the binding sites of these ligands.

PPo8-20: The Anti-prion Activity of Soil Organic Compounds Humic and Fulvic Acids

Joanna Narkiewicz,^1,2 Ai H.N. Tran,¹ Gabriele Giachin,¹ Liviana Leita² and Giuseppe Legname^1,

¹Neurobiology Sector; Scuola Internazionale Superiore di Studi Avanzati; International School for Advanced Studies; Bonomea, Trieste Italy; ²Agricultural Research Council (CRA); Research Centre for Soil-Plant System; Trieste, Gorizia Italy

A notable feature of prion diseases, as scrapie in sheep and chronic wasting disease in mule deer and elk, is horizontal transmission between grazing animals, suggesting that contaminated environment may contribute significantly to disease transmission. Increasing evidence suggests that soil may present natural reservoir of prion infectivity. Recent studies have shown that prions may persist in contaminated soil and remain infectious for years. As the mechanism of prion retention and persistence in soil is unknown, it is necessary to understand which soil components may interact with prions and thus contribute to disease transmission. Several reports indicate that prion have potential to interact with soil minerals, however the contribution of soil organic fraction in adsorption to prions has been neglected.

Here, we present strong evidence for soil humic substances (HS) interaction with prions. We show that two HS, classified as humic and fulvic acids, interact with recombinant prion proteins in vitro. Moreover, we show that both HS possess anti-prion activity, both in vivo and in vitro. Both compounds induced elimination of PrP^Sc from chronically scrapie-infected GT1 mouse hypothalamic cells (ScGT1) in a dose-dependent manner. ScGT1 cells treatment with HS at concentration of 20μg/mL eliminated more than 95% of PrP^Sc and did not affect cell viability. Moreover, both HS induced inhibition of prion fibril formation in vitro, as determined by thioflavin T assay. Our results suggest that HS may contribute significantly to prion inactivation in natural soil environments.

PPo8-21: Detection of PrPCWD in Rocky Mountain Elk Feces Using Protein Misfolding Cyclic Amplification

Bruce E Pulford,¹ Terry Spraker,¹ Jenny Powers,² Margaret Wild² and Mark D. Zabel¹

¹Department of Microbiology; Immunology and Pathology; College of Veterinary Medicine and Biomedical Sciences; Colorado State University; ²Biological Resource Management Division; United States National Park Service; CO, USA

Key words: CWD, feces, PMCA, elk

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting cervids, including mule and white-tailed deer (Odocoileus hemionus and virginianus), elk (Cervus elaphus nelsoni) and moose (Alces alces shirasi). The method of CWD transmission between hosts is unclear, though there is evidence that feces excreted by infected animals may play a role. Recently, CWD prions was detected in feces using bioassays in cervidized mice, which took many months to produce results. In this study, we use a more rapid procedure, protein misfolding cyclic amplification (PMCA), to test elk feces for the presence of PK-resistant cervid PrP (PrPCWD). Feces were collected from symptomatic and asymptomatic elk in several northern Colorado locations, homogenized, mixed with normal brain homogenate from Tg5037 mice (expressing cervid PrP) and subjected to up to 9 rounds of PMCA (1 round = 40 secs sonication/30 mins at 70% maximum power, 24 hours). Western blots were used to detect PrPCWD using BAR-224 anti-PrP antibody. Rectal and CNS tissue from the elk were IHC-labeled and examined for the presence of PrPCWD. Fecal samples from symptomatic and asymptomatic elk that tested positive by IHC showed characteristic PrPCWD bands on western blots following PMCA. In addition, PMCA detected PrPCWD in 25% of fecal samples from IHC-negative animals. These data suggest that PMCA may (1) prove useful as a non-invasive method to supplement or even replace IHC testing of cervids for CWD, and (2) identify additional asymptomatic carriers of CWD, the prevalence of which may be underestimated using IHC.

PPo8-22: Studies on the DEGs (Differential Expression Genes) Between CWD-infected and Normal Tg elk Mouse Brain

Min-Jeong Kim, Dong-Seob Tark, Hyun-Joo Sohn, Yoon-Hee Lee, Hyo-Jin Kim, Won-Yong Lee, Chang-Hee Kweon and In-Soo Cho

Foreign Animal Disease Division; National Veterinary Research and Quarantine Service; Anyang, Korea

Key words: differential expression genes, CWD, Tg elk mouse brain, genefishing, real time PCR

Prion diseases are a class of transmissible fatal disorders. In order to uncover alterations associated with their pathogenesis, several studies were based on differential gene expression analysis using cDNA libraries, mRNA differential displaies and gene microarrays. These genomic approaches are potentially very useful to identify genes differentially expressed during prion diseases, which may participate directly or indirectly to the pathogenesis. In this study, we compared the gene expression profiles of normal and CWD-infected Tg mouse with GeneFishing synthesis and real-time PCR analysis. DEGs were screened by ACP-based PCR method using the GeneFishing synthesis according to the instructions (Seegene, Korea). The amplified PCR products were analyzed with Agilent DNA 1000 kit and 2100 Expert Software for quantificaiton. To validate candidate genes, we applied quantitative real-time PCR. The PCR were performed with Taqman probes and Gene expression master mix (ABI). These reactions were conducted by an Applied Biosystem 7500 real-time PCR system and the ABI Prism SDS 2.1 software. We identified eleven DEGs that appeared to be differentially expressed between CWD-infected and normal Tg elk mouse. We discovered five out of eleven DEGs strongly expressed and the other three genes downregulated in infected mouse. Furthermore, we observed that these DEGs show similar patterns of up and downregulation by Agilent DNA analyzer and Taqman real time PCR in infected brains. The newly identified DEGs from this study could be used in diagnosis and studying the pathogenesis of prion disease through the advanced research.

PPo8-23: An Improved Assay for Titration of Variant CJD Infectivity using Human PrP Transgenic Mice

S. Halliez,¹ F. Reine,¹ L. Herzog,¹ E. Jaumain,¹ A. Le Dur,¹ J.L. Vilotte,² H. Laude¹ and V. Béringue¹

¹INRA; UR892; Virologie Immunologie Moléculaires; ²UMR1313; Génétique Animale et Biologie Intégrative; Jouy-en-Josas, France

Key words: bioassay, variant CJD, human PrP transgenic mouse, spleen

A sensitive and reliable bioassay method to titrate variant CJD infectivity in biological fluids and tissues of infected individuals or to validate decontamination and inactivation procedures is crucially needed. We have developed a bioassay model based on the use of the humanized transgenic mouse line designated tg650, which overexpress human PrPMET129. Its potential in terms of quantification of variant CJD infectivity in serial-tenfold brain dilutions has been evaluated by two different methods: (i) the “classical” bioassay, based on the appearance of clinical symptoms and the detection of PrPres in the brain of the inoculated mouse, (ii) a shortened bioassay based on the detection of PrPres in the mouse spleen. Both methods allow detection of infectivity after inoculation of <1 ng of variant CJD infected brain (i.e., 10-8 brain dilution). Depending on the method, a time-schedule of 2.5-year and ∼1-year, respectively, was necessary to determine the ID₅₀.

These data suggest that the tg650 humanized model may provide 4 orders of magnitude-improved sensitivity as compared to the RIII model currently employed for the titration of BSE-related prions infectivity. It also indicates that detection of lymphotropic prions accumulation in the spleens may be quicker than and as sensitive as detection in the brains.

PPo8-24: Formalin Fixed Paraffin Embedded Tissue as a Starting Point for PrP^Sc Detection by ELISA

Eric M. Nicholson

Virus and Prion Research Unit; National Animal Disease Center; ARS; USDA; Ames, IA USA

Key words: formalin fixed prion, detection ELISA

Introduction. Formalin fixed paraffin embedded tissue are regularly employed in TSE diagnosis by IHC, the standard by which all other diagnostic protocols are currently judged. While IHC affords advantages over diagnostic approaches that typically utilize fresh or frozen tissue, such as western blot and ELISA, the process of fixing, staining, and analysis of individual sections by hand does not readily allow for either rapid nor high though put screening. However, preservation of tissues in formalin is not dependent upon the availability of refrigeration.

Results. Samples containing as little as one 4 µm section can be used to readily discriminate positive from negative samples.

Discussion. This approach cannot replace IHC but may be used with IHC approach as both a more rapid and readily high through put screen where fresh or frozen tissues are not available or not practical. Applicability to different TSEs and time in formalin are important considerations and are discussed.

Materials and Methods. Formalin fixed paraffin embedded tissues resulting from TSE transmission studies at the National Animal Disease Center were cut at 4 µm thickness. The samples were boiled to allow manual removal of paraffin, sonicated to disrupt the tissue and analyzed using a commercial ELISA test kit.

PPo8-25: Intervention with Pentosan Polysulphate and Agent Clearance in a Murine Model of TSE Contaminated Transfusion

Christine F. Farquhar,¹ Simon Cumming,¹ Fraser Laing,¹ Irene McConnell,¹ Marc L. Turner² and Jean C. Manson¹

¹Neuropathogenesis Division; The Roslin Institute and Royal (Dick) School of Veterinary Studies; University of Edinburgh; and ²The Scottish Centre for Regenerative Medicine; University of Edinburgh; and The Scottish National Blood Transfusion Service

Key words: transmissible spongiform encephalopathy, scrapie, therapeutics, pentosan polysulphate, transfusion

We have shown that the polyanion, pentosan polysulphate (PPS), a heparin analogue with anti-coagulant, -thrombotic, -inflammatory and -viral activity, can increase survival time if given parenterally soon after parenteral scrapie inoculation into rodents. Efficacy is transmissible spongiform encephalopathy (TSE) model specific, and drug tolerance is generally good but varies with host species. We established a low dose, 100% incidence, intravenous model of ME7 scrapie in C57BL/Dk mice to test potentially clinically relevant intervention strategies for individuals exposed to vCJD through blood or blood products. Drug given parenterally only when the animals became symptomatic produced a small increase in survival time and some evidence for localised neuroprotection. Early interventions in this model can clear infection completely in some animals but not others. We show that reliance on analysis by routine pathology or PrP immunohistochemistry, would under report continued infection as determined by bioassay, and leave a potential transmission risk. Intervention can deplete, or clear, the peripheral tissues of infectivity but if neuroinvasion has occurred replication can produce high titres in the brain. Our data analysing the growth kinetics of infection by spleen and brain bioassay at intervals throughout the time course of this model, and the effect of different treatment regimen will be presented.

Financial Disclosure

UK Department of Health, Biotechnology and Biological Sciences Research Council and Medical Research Council; European Union “Neuroprion FP6”.

PPo8-26: Wet versus Dry: Do Environmental Conditions have an Effect on Prion Decontamination?

Thomas J. Secker, Rodolphe Hervéa and C. William Keevil

Environmental Healthcare Unit; School of Biological Sciences; University of Southampton; Southampton, UK

Key words: prion, amyloid, decontamination, environmental conditions

Introduction. Transmissible spongiform encephalopathies (TSE) are a group of rare fatal neurodegenerative disorders that affect both animals and humans. The infectious agent is thought to be a protease resistant isoform (PrPSc) of the protease sensitive cellular prion protein (PrPC). Iatrogenic CJD has become a risk especially throughout neurosurgery due to the resistance of the infectious PrPSc to many chemical and enzymatic detergents and its strong adhesive properties to stainless steel. The environmental conditions that surgical instruments are exposed to post surgery can make a vast difference in the level of prion attachment to surgical stainless steel and subsequent decontamination efficacy.

Results. Increases in drying time showed a considerable rise in prion contamination and a decrease in cleaning chemistry efficiency. A moist environment greatly slowed the adhesion of protein and prion amyloid over 24 hours and increased cleaning chemistry efficacy at refrigerated temperatures; however a further improvement was observed at room temperature.

Methods. Surgical stainless steel was contaminated with 1 µg of 10% ME7-infected brain homogenate and dried at both room and refrigerated temperatures in moist and dry environments. Remaining PrPSc amyloid and general protein contamination, before and after treatment in enzymatic cleaning chemistries was visualised with Thioflavin T and SYPRO Ruby and analysed using episcopic differential interference contrast/epifluorescence microscopy.

Conclusion. Simple improvements in the handling of surgical instruments post surgery could greatly decrease both tissue protein and prion amyloid contamination and improve the efficiency of commonly used cleaning chemistries in sterile service departments, reducing the risk of iatrogenic prion transmission.

PPo8-27: Novel Abnormal Prion Protein (PrP^Sc)-Specific Epitopes in the N-terminal Region of PrP

Kentaro Masujin,¹ Yuko Ushiki-Kaku,² Hiroyuki Okada,¹ Yoshihisa Shimizu,¹ Kazuo Kasai,¹ Yoshifumi Iwamaru,¹ Morikazu Imamura,¹ Shirou Mohri¹ and Takashi Yokoyama¹

¹Prion Disease Research Center; National Institute of Animal Health; Tsukuba, Ibaraki Japan; ²Nippi Research Institute of Biomatrix/Toride; Ibaraki, Japan

Key words: PrP^Sc, antibody

It has been known that the conformation of PrP^Sc differs from that of PrP^C, which is a host encoded-cellular protein. However, the precise characteristics of PrP^Sc remain to be elucidated. We attempted to generate novel PrP^Sc-specific monoclonal antibodies (mAbs) to clarify the PrP^Sc properties.

Intact PrP^Sc was purified from the brain of bovine spongiform encephalopathy (BSE)-affected mice; further, PrP gene-ablated mice were immunized with this purified PrP^Sc. Hybridomas were screened using enzyme-linked immunosorbent assay (ELISA). The PrP^Sc purified from the BSE-affected mice was treated with (denatured) or without (native) guanidine thiocyanate (GdnSCN), and then used as an ELISA antigen. We selected mAbs, which were preferentially reacted with the native PrP^Sc. The immunoreactivity of the generated mAbs was examined using immunoprecipitation and immunohistochemical analysis.

ELISA revealed two mAbs (6A12 and 8D5) that exhibited stronger immunoreactivity against the native PrP^Sc than that of denatured PrP^Sc. The results of the immunoprecipitation assay confirmed their specific immunoreactivity against PrP^Sc. In the frozen section of the brain, PrP^Sc was detected using PrP^Sc-specific mAbs without antigen retrieval. Peptide spot assay revealed that the epitopes for the 6A12 and 8D5 were located at PrP 31–39 and PrP 41–48, respectively. This finding suggests the presence of PrP^Sc-specific epitopes in the N-terminal region; this structure corresponds to the primary amino acid sequence of PrP. The generated PrP^Sc-specific mAbs are expected to be powerful analytical tools to elucidate the properties of native and intact PrP^Sc.

PPo8-28: Ultrasensitive Human Prion Detection in Cerebrospinal Fluids by Real-time Quaking-Induced Conversion

Ryuichiro Atarashi,¹ Katsuya Satoh,² Kazunori Sano,¹ Takayuki Fuse,¹ Hitoki Yamanaka,¹ Naohiro Yamaguchi,¹ Daisuke Ishibashi,¹ Takehiro Matsubara,¹ Takehiro Nakagaki,¹ Masahito Yamada,³ Hidehiro Mizusawa,⁴ Tetsuyuki Kitamoto,⁵ Amelia McGlade,⁶ Steven John Collins,⁶ Susumu Shirabe,² Shigeru Katamine¹ and Noriyuki Nishida¹

¹Department of Molecular Microbiology and Immunology; ²First Department of Internal Medicine; Nagasaki University Graduate School of Biomedical Sciences; Japan; ³Department of Neurology; Kanazawa University Graduate School of Medical Science; Japan; ⁴Department of Neurology and Neurological Science; Graduate School; Tokyo Medical and Dental University; Japan; ⁵Division of CJD Science and Technology; Department of Prion Research; Tohoku University Graduate School of Medicine; Tohoku University Graduate School of Medicine; Japan; ⁶Department of Pathology; Australian National Creutzfeldt-Jakob Disease Registry; The University of Melbourne; Australia

Key words: QUIC(quaking-induced conversion), CJD(Creutzfeldt-Jakob disease), CSF(cerebrospinal fluid) diagnosis

The development of in vitro amplification technology for abnormal forms of prion protein (PrP^Sc) has generated the possibility for developing a novel diagnostic test for prion diseases. However, ultrasensitive PrP^Sc detection in easily accessible specimens such as cerebrospinal fluid (CSF) has not yet been successful in human prion diseases. Earlier, we developed a new PrP^Sc amplification assay, designated quaking-induced conversion (QUIC)¹, which uses soluble recombinant PrP (rPrP-sen) as a substrate along with intermittent automated shaking. To further improve the rapidity and practicality of this method, we combined QUIC technology with thioflavin T (ThT) fluorescence dye, used to monitor amyloid fibril formation. This assay, termed “real-time QUIC (RT-QUIC)”, allows for the detection within 48 h of <1 fg of PrP^Sc in diluted sporadic Creutzfeldt-Jakob disease (sCJD) brain homogenate. Moreover, we performed a blind study which achieved 87.5% sensitivity (14 out of 16 definite sCJD-CSF samples) and 100% specificity (0 out of 14 non-CJD CSF samples). These findings indicate that the real-time QUIC will be of great use in the antemortem diagnosis of sCJD.

References

1. Atarashi R, Wilham JM, Christensen L, Hughson AG, Moore RA, Johnson LM, et al. Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking. Nature Methods 2008; 5:211-2.

PPo8-29: Degradation of Abnormal Prion Protein by a New Protease from a Hyperthermophile

Yuichi Koga,¹ Shun-ichi Tanaka,² Akikazu Sakudo,³ Kazuyoshi Ikuta,¹ Kazufumi Takano^1,4 and Shigenori Kanaya¹

¹Osaka University; Osaka, Japan; ²Amano Enzyme Inc; Nagoya, Japan; ³University of the Ryukyus; Nishihara, Japan; ⁴Japan Science and Technology Agency (JST); Suita, Japan

Key words: decontamination, protease

Background. The abnormal prion protein (PrPSc) which is a protease resistant isoform of normal prion protein (PrPC), thought to be an infectious agent of transmissible spongiform encephalopathies (TSE). Decontamination of PrPSc from clinical equipment is important issue to avoid iatrogenic transmission of prion diseases. Several reagents or physical procedures are available to inactivate PrPSc but they cannot apply to some equipment such as endoscope. Therefore there is a demand for effective enzymatic agents to remove contaminated PrPSc in mild condition. This study is intended to develop novel medical cleaning agents using a thermo-stable protease.

Results and Discussion. In this work, we revealed that the Tk-subtilisin can degrade abnormal prion to a level undetectable by western-blot analysis while Proteinase K cannot degrade it under the same condition. Furthermore, we also found that the Tk-subtilisin can degrade proteins in the presence of surfactants including SDS. Therefore, it is a promising enzyme that can be developed as a detergent additive to decrease the secondary infection risk of TSE.

Methods. Tk-subtilisin, a highly stable protease isolated from a hyperthermophile, can maintain its proteolytic activity under protein-denaturing conditions. Abnormal prion protein (Chandler strain and Obihiro strain) accumulated in scrapie infected-mouse brain homogenate were degraded with Tk-subtilisin or Proteinase K at varied conditions, and detected by western-blot analysis.

PPo8-30: BSE Samples with Non Conclusive Results

Rocío Sarasa,¹ Eva Monleón,¹ Antonia Vargas,¹ Martin Groschup,² Dietmar Becher,³ Juan J. Badiola¹ and Marta Monzón¹

¹Research Centre for Encephalopathies and Transmissible Emerging Diseases; University of Zaragoza; Zaragoza, Spain; ²Friedrich-Loeffler-Institut; Insel Riems, Germany; ³Micromun, Greifswald Germany

Key words: BSE, diagnosis, non conclusive samples

Due to the intensification of the Bovine Spongiform Enceph-alopathy (BSE) surveillance program, some samples whose result is non-conclusive according to the OIE confirmatory testing continue to be found and therefore, problems with their diagnosis continue to exist since Transmissible Spongiform Encephalopathies are not possible to be discarded nor to be confirmed in them.

In this study, we present five samples from bovine nervous tissue presenting non conclusive signals with conventional confirmatory techniques besides a very advanced grade of autolysis to set up and establish different diagnostic techniques to be applied on those samples whose results are not conclusive was intended here.

With this aim, immunohistochemical and molecular techniques were applied. In addition, a ultrastructural study by electron microscopy and an experimental infection in murine model have been developed.

Electron microscopy provided quite conclusive results.The inoculation in the murine model was successful just in a scarce proportion, maybe due to a very low PrP^Sc concentration in samples, but it was useful for confirming the positive results demonstrated by SAF illustration. Furthermore, an immunohistochemical protocol was necessary to be modified in order to detect PrP^Sc in mice because of the very low content of PrP^Sc.

Otherwise, an immunocytochemical protocol for liquid fractions showed specific immunostaining which supported the results provided by the rest of techniques applied here.

Acknowledgement

This work was funded by a grant from the Spanish Science and Education Department (AGL2006-08467).

PPo8-31: Plasma Proteome Differences Between Sporadic Creutzfeldt-Jakob Disease and Alzheimer’s Disease Patients

Franco Cardone, Serena Principe, Federica Fratini, Anna Ladogana, Marco D’Alessandro, Anna Poleggi, Paola Piscopo, Hanin Abdel-Haq, Silvia Graziano, Angelina Valanzano, Angela De Pascalis, Edmondo Campisi, Daniela Biondo, Maria Puopolo, Annamaria Confaloni, Marco Crescenzi and Maurizio Pocchiari

Department of Cell Biology and Neurosciences; Istituto Superiore di Sanità; viale Regina Elena, Rome Italy

Key words: plasma proteome, Creutzfeldt-Jakob disease, Alzheimer’s disease, mass spectrometry

In human prion disorders the development of methods for the identification of infected individuals is urgently required to increase the safety of medical procedures involving potentially infectious tissues. These methods should be characterized by an adequate level of sensitivity and specificity, and preferably applicable in easily accessible tissues and fluids such as blood. Molecular biomarkers in blood of individuals affected by neurological disorders have been poorly investigated, but there are data suggesting that pathological events taking place in the brain might influence peripheral processes, thus becoming accessible through the analysis of blood. In this framework, within the FP6 project “Prionscreen”, we compared three pools of plasma samples obtained from sporadic Creutzfeldt-Jakob disease (CJD) and Alzheimer’s (AD) disease patients, and from healthy controls, which were subjected to depletion of the most abundant proteins, trypsin digestion, and “tandem mass tag” labeling of the obtained peptides. Mixtures of labeled peptides were subjected to LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis for quantitatively comparing peptides obtained from the three plasma pools. This approach was able to detect a panel of functionally related proteins whose levels were significantly higher in CJD plasma samples compared to healthy controls and AD patients. Further studies on a larger panel of CJD patients and controls are in progress to validate the diagnostic utility and the pathogenetic significance of this CJD-specific plasma proteome pattern.

PPo8-32: Production of Monoclonal Antibodies Against Glycated Prion Protein

Eva Dvoráková,¹ Hana Glierová,¹ Marek Prouza,² Radoslav Matěj³ and Karel Holada¹

¹Institute of Immunology and Microbiology; First Faculty of Medicine; Charles University Prague; Czech Republic; ²EXBIO Praha; a.s.; Vestec, Czech Republic; ³National Reference Laboratory for Diagnostics of Human Prion Disorders; Department of Pathology and Molecular Medicine; Thomayer Teaching Hospital; Prague, Czech Republic

Key words: glycated prion protein, caboxymethyl lysine, monoclonal antibody EM-31

Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. As deposits of PrPTSE remain in the body long time, there is substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have potential to serve as diagnostic marker. The aim of this study was to prepare monoclonal antibodies specific for glycated prion protein. Recombinant human prion protein (rhPrP) was isolated from transformed E. coli BL21 (DE3) and purified on Cu resin (TALON) by affinity chromatography. rhPrP was modified by glyoxylic acid, that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of 9 mice. Splenic cells from mice producing highest titers of PrP antibodies were used for fusion with myeloma cell line resulting in production of 960 hybridoma cells. Clones were primary screened by ELISA for reactivity to rhPrP and rhPrP-CML. Further screening resulted in the selection of 19 promising clones. One of them (EM-31) strongly reacts with rhPrP-CML, but not with unmodified rhPrP or PrP^C. Importantly after the CML modification of human brain homogenate the EM-31 antibody recognizes only PrP^C-CML on Western blot, but no other CML modified protein. At the present time the testing of antibodies on tissues of CJD patients is in progress.

Acknowledgement

Supported by grants IGA MZ NS10335-3 and GACR 203/07/1517.

PPo8-33: Proteomics-based Development of Biomarker for Prion Diseases

Bo-Yeong Choi,¹ Su Yeon Kim,¹ Chi-Kyeong Kim,¹ Seong Soo An,² Soo Jae Lee³ and Young Ran Ju¹

¹Department of Arboviruses; Center for Immunology and Pathology; National Institute of Health; Korea Centers for Disease Control & Prevention; Seoul, Republic of Korea; ²Nano Systems Major; College of BioNano Technology; Kyungwon University; Gyeonggi-do, Republic of Korea; ³ProBiond Co.; Information Center for Admissions; Konkuk University; Seoul, Korea

Key words: prion disease, proteomics, biomarker

Introduction. Creutzfeldt-Jakob disease (CJD) is one of the transmissible spongiform encephalopathies (TSEs) caused neurodegerative disease. The several protein markers, including 14-3-3 protein, S-100 protein, neuron-specific enolase (NSE), and Tau protein have been reported to be useful for diagnosis of CJD. Among these CSF biomarkers, the 14-3-3 protein assay has a high specificity and sensitivity, but the assay of the high false-positive rates for CJD. We profiled proteome of ME7 scrapie infected mouse brain and analyzed their interaction and function to facilitate the differential diagnosis of CJD as use high sensitive and specific novel biomarker.

Results. We found significant changes in the levels of many proteins in the ME7 scrapie infected mice. This analysis resulted in the identification of 43 candidate proteins with high peptide scores and ratio values (p value <0.05). We identified 36 potential candidate proteins that were upregulated biological processes, and seven potential candidate proteins that were downregulated biological processes. Of these, the differentially expressed novel candidate protein was confirmed in ME7 scrapie infected mice brain compared to control groups.

Materials and Methods. In order to detect quantitative differences in brain protein expression between control and infected groups, a comparative proteomic study was performed. C57BL/6 mice were inoculated intracerebrally with 10% brain homogenate of ME7 scrapie infected mouse. Mice were monitored for clinical symptoms for up to 171 days post inoculation (dpi), and we analyzed infected host cell using 1D-gel electrophoresis, LC-MS/MS, SEQUEST, Cytoscape, GO mapping and immunoblot.

Conclusions. We consider that our proteomic approach is useful to find biomarkers for CJD with high diagnostic specificity and sensitivity. These proteins are useful targets for pre-mortem diagnostic, prognostic, and therapeutic development. Thus, further study is required to confirm the candidate biomarker that may apply to diagnosis of pre-mortem for CJD patients.

PPo8-34: Titration of Buffy Coat Infectivity in Sheep Experimentally-infected with 127S Strain Using an in vitro Cell Culture Assay

Aude Arzel,¹ Christiane Segarra,² Bruno You,¹ Anne-Laure Jacquot,¹ Daisy Bougard,² Steve Simoneau,¹ Hubert Laude,³ Olivier Andreoletti,⁴ Joliette Coste and Benoît Flan

¹Laboratoire du Fractionnement et des Biotechnologies; Les Ulis, France; ²Etablissement Français du Sang de Pyrénées-Méditerranée; France; ³Institut National de la Recherche Agronomique; Paris, France; ⁴École Nationale Vétérinaire de Toulouse; Toulouse, France

Key words: TCIA, blood infectivity, sheep, prion, PMCA, buffy coat

Recent advances in culturing prions in cellular models have opened up new alternatives to bioassays. Indeed, we recently developed an in vitro infectivity titration method, Tissue Culture Infectivity Assay (TCIA) and showed using infected brain homogenate that its sensitivity is nearly equivalent to the bioassay (You et al. 2009). We have now applied this technique successfully to the detection and titration of infectivity in 127S orally-infected sheep blood (buffy coat) collected at 120 days post-inoculation. The titre obtained was 4.36 TCID₅₀/ml corresponding to approximately 22 LD₅₀/ml (1 TCID₅₀ = 5 LD₅₀). In an attempt to improve the sensitivity, we combined this technique with two rounds of Protein Misfolding Cyclic Amplification (TCIAPMCA) for amplification of the PrP^Sc from infected cells before western blot detection. Using this modified assay, we detected more infected wells with a resulting titre of about 17 TCIDPMCA50/ml. Therefore as expected, performing two rounds of PMCA after carrying out the TCIA, resulted in an increase in sensitivity now equivalent to the bioassay. Moreover, PMCA may also contribute to an optimization of the TCIA, i.e., by reducing the number of passages which are required to detect infected cells.

In conclusion we show that our in vitro assays (TCIA and TCIAPMCA) can detect infectivity in different biological matrices ranging from brain homogenate containing massive quantities of infectivity to blood components with very low infectivity with a sensitivity equivalent to the bioassay. To our knowledge this constitutes the first description of blood infectivity detection and quantification using a cell based assay.

Financial Disclosure

Fondation Alliance BioSecure.

References

1. You, et al. J Virol Meth 2010; 164:1-6.

PPo8-35: Single-chain Fragments of Variable Region Antibodies against the Bovine Prion Protein from Chicken

Inbeen Yim, Jeongmin Lee, Sangho Choi and Hee-Jong Woo

College of Veterinary Medicine; Seoul National University; Seoul, Korea

Key words: bovin prion protein, monoclonal antibody, phage display

As prion protein is highly conserved in mammalian, antibody to the infectious prion protein (PrP^Sc) is not easy to get. Phage display has become one of the approach methods for monoclonal antibodies. Selection of the monoclonal antibodies by this approach is more rapid than conventional cell fusion. Chicken has been, also, regarded as a useful animal for development of the specific antibodies against conserved proteins of mammals. To obtain recombinant monoclonal antibodies to PrP, we have immunized chickens with the two kinds of short peptides conjugated by keyhole limpet hemocyanin (KLH). The one is located in first α-helix region and the other is in C-terminus. After isolation of mRNA of heavy-variable region (VH) and light-chain variable region (VL) from the spleen of the immunized mouse, DNA of VH and VL were obtained by RT-PCR and joined by a overlap extension PCR encoding peptide (Gly4Ser)3 as a scFv DNA fragments. We have got 20 clones of scFv gene, and then accomplishing screening of high affinity scFv molecules. For further study, we expect these antibodies are adopted for the functional and analytical approach for prion protein.

PPo8-36: Lentiviral Delivery of Dual MicroRNA Targeting Prion Protein for Therapeutic Application

Sang-Gyun Kang,¹ Yu-Mi Roh,² Allen Herbst,¹ Camilo Duque-Velasquez,¹ Han Sang Yoo,² Debbie McKenzie¹ and Judd Aiken¹

¹Centre for Prions and Protein Folding Diseases; University of Alberta; Edmonton, AB Canada; ²Department of Infectious Diseases; College of Veterinary Medicine; BK21 for Veterinary Science and KRF Zoonotic Priority Research Institute; Seoul National University; Seoul, Korea

Key words: lentiviral delivery, artificial microRNA

Knock-out strategies have been successfully applied to reduce PrP^C expression, however, these are not practical for therapeutic use. RNA interference (RNAi) is a well-established, sequence- specific, post-transcriptional gene silencing mechanism. RNAi rapidly depletes mRNAs by introducing a double-strand RNA homologous to a specific message. We have previously demonstrated that dual microRNAs (miRNA) expressed, in a tandem, on a long primary transcript enhanced knock-down efficacy more than three-fold compared to a single miRNA knock-down. The next step for therapeutic application is to design a delivery system with a high transduction efficiency to introduce the miRNAs into non-dividing neurons. Primary miRNAs targeting PrP^C expression were designed and cloned into an single expression cassette by linking two different miRNAs (miRNAdual). The primary miRNAdual cluster was cloned into a viral expression vector and packaged into lentiviral particles by transfection of 293FT host cells. western blot analysis indicated that PrP^C significantly declined in the N2a cells transduced with the lentiviral particles. We are currently developing methods to enhance the transduction efficiency in primary neurons. The dual targeting RNAi and lentiviral delivery used in this study may provide a therapeutic approach for prion diseases as well as other neurodegenerative disorders.

PPo8-37: Cold Atmospheric Plasma for the Decontamination of Reusable Surgical Instruments

Rodolphe Hervé,¹ Mike Kong,² Emmanuel Comoy,³ Jean-Philippe Deslys³ and Bill Keevil¹

¹Environmental Healthcare Unit; School of Biological Sciences; University of Southampton; UK; ²School of Engineering, University of Loughborough; UK; ³Centre à l’Energie Atomique; Fontenay-aux-Roses, France

Key words: prion contamination, reusable surgical instruments, infectivity

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases generally believed to be transmitted simply by the absorption or ingestion of self-aggregating protease-resistant prion proteins (PrP^Sc), which are particularly resilient to classic methods of decontamination used in healthcare facilities (enzymatic washing and steam sterilization). With the emergence of variant Creutzfeldt-Jakob disease (vCJD), an acquired TSE with a relatively long asymptomatic incubation period, there is a need to develop new techniques for the decontamination of reusable surgical instruments to prevent the spreading of the disease through surgical interventions on apparently healthy individuals unknowingly carrying the infectious agent.

Cold atmospheric plasma (CAP) is a new technology which achieved satisfying levels of decontamination in preliminary evaluations. To further assess the efficacy of this technology, we spiked surgical surfaces with 263K-infected brain homogenates, and treated these with CAP. We then used very sensitive episcopic differential interference contrast microscopy combined with fluorescent dyes (EDIC/EF) to assess the amount of residual PrP^Sc and other proteins on these surfaces. We are also using the intracranial wire implant model to evaluate to what extent CAP treatment can reduce the prion infectivity potentially present on the contaminated surfaces of surgical instruments. Our data shows that CAP has the potential to achieve over 6-log reduction in residual proteinaceous contamination, with an initial load of only 1 microgram. An animal infectivity assay is currently in progress, and results shall reveal how infectivity correlates with residual contamination and if CAP treatment can also reduce significantly the risk of transmission of PrP^Sc during surgery.

PPo8-38: Quantitative Proteomic Analysis of Prion Infected Mouse Plasma

Allen Herbst,^1,2 Xin Wei,³ Di Ma,⁴ Lingjun Li^3,4 and Judd Aiken^1,2

¹Centre for Prions and Protein Folding Diseases; ²Agriculture Food and Nutritional Sciences; University of Alberta; Edmonton, AB, Canada; ³Department of Chemistry; University of Wisconsin; Madison, WI USA; ⁴School of Pharmacy; University of Wisconsin; Madison, WI USA

Key words: biomarkers, proteomics, plasma, diagnostics

Definitive TSE diagnosis is currently limited to post-mortem assays for protease-resistance prion protein. Utilizing a quantitative proteomics approach, we analyzed prion-infected mouse plasma to identify surrogate markers of prion disease. C57/Bl6 mice were experimentally infected with the RML strain of mouse-adapted scrapie by intraperitoneal inoculation. Age-matched uninfected animals served as controls. Blood was collected from infected and control animals at 108, 158 and 198 (end-stage clinical disease) days post-inoculation. Plasma from each time point was purified by centrifugation and individual samples were pooled. Infected and control pools from each time point were fractionated using lectin affinity chromatography. Purified glycoproteins were trypsinized and isotopically labeled using hydrogenated or deuterated formaldehyde. The labeled infected and control peptides were then mixed and fractionated using two separate dimensions (high and low pH) of reverse phase chromatography. The abundance and identity of individual peptide pairs (light and heavy isotope labels) in each fraction was determined by mass spectrometry using a nanoESI LTQ mass spectrometer. Peptides enriched in the serum of infected animals relative to control were considered candidate biomarkers. Validation of biomarkers was performed by immunological detection using western blot on individual samples. The identification of surrogate markers will assist with the development of ante-mortem prion disease diagnostics as well as provide insight to disease progression and pathology.

PPo8-39: Expansion of the BSE Surrogate Biomarker Inventory

Sharon LR. Simon,¹ Lise Lamoureux,¹ Margot Plews,¹ Stefanie Czub,² Catherine Graham² and J. David Knox¹

¹Prion Disease Program; Public Health Agency of Canada; Winnipeg, Canada; ²Animal Diseases Research Institute; Canadian Food Inspection Agency; Lethbridge, Canada

Key words: BSE, biomarker, proteomics

Neurodegeneration in suspected cases of bovine spongiform encepalopathy (BSE) causes symptoms such as nervous/aggressive behaviour and lack of coordination. Diagnosis of BSE, however, is only confirmed by the post-mortem detection of PrPd in brain tissue. For assessing the health of breeding stock or the efficacy of therapeutic interventions post-mortem testing is not an option generating continued demand for an ante mortem test. In order to meet these needs such a test must be based on a sample that is easily accessible and permits repeated sampling such as blood or urine. Previously, in a small cohort of Fleckvieh-Simmental cows we used two dimensional differential gel electrophoresis (2D-DIGE) to identify proteins in bovine urine that were able to discriminate between control and BSE infected cattle with a high degree of accuracy. To determine whether the results obtained from the Fleckvieh-Simmental cows were a typical bovine response to BSE infection a second cohort of bovine urine was obtained. In this study 2D-DIGE was used to analyze the urine obtained from four BSE-infected and four age-matched neutered males that were either Friesian or Friesian/Holstein crosses at nine different time points throughout the course of the disease. As previously, we were able to identify proteins that enabled us to discriminate between control and infected cattle with a high degree of accuracy throughout the disease course. A comparison of the classifying proteins will be presented as well as a demonstration of the influence of confounding factors such as breed, age and gender on the urine proteome.

PPo8-40: Quantification of Infectious Titres Using a Generalized Linear Model

Rocio Castro-Seoane,¹ Holger Hummerich,¹ Trevor Sweeting,² Malin Sandberg,¹ John Collinge¹ and Peter-Christian Klöhn¹

¹MRC Prion Unit; Department of Neurodegenerative Diseases; UCL Institute of Neurology; Queen Square, London UK; ²Department of Statistical Science; University College London; London UK

The recent development of an in-vitro infectivity assay, the Scrapie cell assay, not only allows investigation of molecular events that lead to prion propagation, but is also instrumental to a fast prion titre determination of mouse-prion infected specimen. At limiting dilutions of prions this assay results in PrP^Sc-negative and -positive wells and the number of positive wells in ni independent infections at the ith dilution follows a binomial distribution with parameters ni and Pi. If the number scrapie-infected cells is assumed to have a Poisson distribution then the proportion of negative wells (1-Pi) is equal to exp(-mci), where m is the mean number of infectious units per volume and ci the dilution. A complementary log-log transformation converts this equation to

log(-log (1-Pi) = log m + log ci

Thus if the experimental data follows a Poisson distribution then a complementary log-log transformation is linear in log ci with a slope of one. To test this hypothesis we prepared multiple dilution series of brain homogenate, infected susceptible cells and determined the number of negative wells. Linear regression analysis resulted in an estimate of the slope β of 1.06 ± 0.20, in agreement with the assumption of an underlying Poisson distribution for the number of infected cells. This prompted us to use a generalized linear model (GLM), for which a flexible iterative method for maximum likelihood estimation is available to calculate infectious titres. We illustrate the advantages of using a GLM and present statistical data from comparative in vitro and in vivo experiments.

PPo8-41: Brain MRI Activity and Serum Biochemical Markers for Evaluating Blood-Brain Barrier Function in Creutzfeldt-Jakob Disease

Katsuya Satoh,^1,2 Kazuo Mutsukura,¹ Ryuichiro Atarashi,² Susumu Shirabe,³ Yuki Matsui,⁴ Hitaru Kishida,⁵ Yoshiyuki Kuroiwa,⁵ Nobuo Sanjo,⁶ Hidehiro Mizusawa⁶ and Noriyuki Nishida²

¹First Department of Internal Medicine; ²Department of Molecular Microbiology and Immunology; Graduate School of Biomedical Science; Nagasaki University; ³Organization of Rural Medicine and Residency Education; Nagasaki University Hospital; Nagasaki, Japan; ⁴Department of Neurology; Graduate School of Medicine; Yokohama City University; Yokohama, Japan; ⁵Department of Neurology and Neurological Science; Graduate School; Tokyo Medical and Dental University; Tokyo, Japan

Background. The appearance of 14-3-3 protein in the CSF is a reflect of neuronal damage in the patients with Creutzfeldt-Jakob disease (CJD), and has been reported to be the most sensitive and specific markers in the clinical diagnosis of sporadic CJD. However, this protein is occasionally detected in the CSF of prion-unrelated neurological conditions. Recent studies attempted to diagnosis for CJD by the detection of peripheral circulating proteins derived from brain tissue. To aim of our study was to evaluate whether MMP-9 can be useful serum marker for differential diagnosis of rapidly progressive dementia.

Results. Serum MMP-9 concentration of CJD patients was lower than AD and IND samples. There were no significant differences between CJD and healthy controls.

Serum MMP-9/TIMP-1 ratio was higher value in AD and IND than CJD, but MMP-2/TIMP-2 ratio in CJD patients was higher than that of health subjects. The levels of S100b were significantly higher in CJD patients compared to disease controls.

Methods. We measured serum concentration of biochemical markers (MMP-9, MMP-2,TIMP-1, TIMP-2 and S100b) known as peripheral marker of BBB permeability obtained from 54 patients with sporadic CJD by ELISA. Eighteen healthy controls and disease controls included 16 patients with Alzheimer disease (AD), 36 patients with inflammatory neurological disease (IND) were used for comparison.

Conclusion. Our results showed that serum MMP-9/TIMP-1 ratio may be useful in the differential diagnosis CJD, AD and IND. These proteins are first useful peripheral markers for the diagnosis of CJD.

PPo8-42: Analysis of Eight Rating Scales Supports the use of Functional Outcome Measures in Prion Disease Clinical Trials: Experience from the PRION-1 trial and the National Prion Monitoring Cohort

Simon Mead,¹ Andrew Thompson,¹ Gosala Gopalakrishnan,¹ Michael Ranopa,² Peter Rudge,¹ Stephen Wroe,^1,* Fleur Hudson,² Angus MacKay,³ Janet Darbyshire,² John Collinge^1, and Sarah Walker²

¹MRC Prion Unit; Department of Neurodegenerative Disease; UCL Institute of Neurology; and ²MRC Clinical Trials Unit; London; ³NHS Highland Mental Health Services; Argyll and Bute Hospital; Lochgilphead, Argyll

PRION-1, the largest clinical trial in prion disease to date, showed no effect of the experimental therapeutic quinacrine on survival. Here we report analyses showing that quinacrine had no demonstrable benefit (p > 0.4) when assessed by a range of rating scales. These included neurocognitive (Mini-Mental State Examination, ADAScog), psychiatric (Brief Psychiatric Rating Scale), global (Rankin and Global Impression of Change), clinician-rated (Clinician’s Dementia Rating (CDR)) and functional (Barthel) scales. These rating scales have several potential benefits over survival as an outcome measure: here we assess their validity and performance over 77 person-years follow-up in 101 symptomatic patients in PRION-1. Overall, across a range of models applied, we found that the Barthel and CDR scales were most robust to the difficulties posed by a prion disease clinical trial. A combination of selected subcomponents from these two scales gave increased power to detect clinically relevant effects in a future clinical trial of feasible size, compared to use of survival alone. We also discuss our work refining the use of these scales in the National Prion Monitoring Cohort, an ongoing prospective observational study of all types of human prion disease in the UK, and how this will inform the planning of future therapeutic trials.

PPo8-43: A Distinct Proteinase K Resistant Prion Protein Fragment Challenges the Diagnosis of Prion Diseases in Goats

Torsten Seuberlich,¹ Florian Lörtscher,^1,* Ilias G. Bouzalas,^2,* Anna Oevermann,¹ Jan P.M. Langeveld,³ Chrysostomos I. Dovas,² Maria Papanastassopoulou² and Andreas Zurbriggen¹

¹NeuroCentre; National and OIE Reference Laboratories for BSE and Scrapie; Vetsuisse Faculty; University of Berne; Switzerland; ²Laboratory of Microbiology and Infectious Diseases; Faculty of Veterinary Medicine; Aristotle University of Thessaloniki; Greece; ³Department of Bacteriology and TSEs Central Veterinary Institute; Lelystad, The Netherlands

*These authors equally contributed to this work

Key words: scrapie, TSE, prion protein, goats, surveillance

Many efforts have been directed towards establishing molecular diagnostic tools that reliably identify and discriminate the pathological prion protein in BSE, classical and atypical scrapie in small ruminants. While considerable work has been done in sheep TSEs, little is known about the molecular disease phenotype in goats. In the present study we report the in depth laboratory analysis of a scrapie outbreak in a Greek goat herd. In total 30 out of 93 adult animals turned out as TSE positive. In these animals the molecular profile of the proteinase K resistant prion protein core fragment (PrPres) in western blot was uniform and similar to the one in classical scrapie in sheep. However, in ten additional goats we identified a single band of a molecular mass of ∼12 kDa. Comparative epitope mapping studies indicated that this peptide is a C- and N-terminally truncated PrPres fragment, but does not include the amino acids 148–155 of the caprine prion protein, which are commonly targeted by monoclonal antibodies in confirmatory diagnostic procedures. This peptide is therefore reminiscent of but not identical to the predominant one observed in atypical scrapie. Noteworthy, these animals where clinically healthy and negative in the screening ELISA. At present, the significance of these findings with regard to the TSE outbreak in this flock remains uncertain. Yet, this situation challenges current strategies of laboratory disease confirmation and point to the requirement to gain deeper insights into the diversity of prion disease phenotypes in goats.

PPo8-44: Cell-based Quantification of Chronic Wasting Disease Prions

Vadim Khaychuck,² Jifeng Bian,¹ Dana Napier,¹ Rachel Angers,^2,† Catherine Graham⁴ and Glenn Telling^1-3

¹Sanders Brown Center on Aging; University of Kentucky Medical Center; Lexington, KY USA; ²Department of Microbiology, Immunology and Molecular Genetics; ³Department of Neurology; University of Kentucky Medical Center; Lexington, KY USA; ⁴Canadian Food Inspection Agency; Lethbridge, Alberta, Canada

^†Present address: Medical Research Council Laboratory of Molecular Biology; Cambridge, UK

Bioassay in susceptible animals has, until recently, been the sole means of assessing prion infectivity. The scrapie cell assay (SCA), while a substantial advance, has been limited to the detection of mouse-adapted scrapie prions. We generated cells susceptible to chronic wasting disease (CWD) prions by ex vivo transgenesis of elk prion protein (PrP) in rabbit epithelial kidney RK13 cells. We show that expression of retroviral Gag and selection of susceptible clones was critical for susceptibility. Having isolated cell cultures with susceptibility to prions from diseased elk, we describe a modification of the SCA allowing evaluation of CWD prions. We compare this cervid prion cell assay (CPCA) to bioassays in transgenic (Tg) mice, the only other existing method for quantification. Bioassay is conservatively >100-fold more expensive and takes >16-fold longer than CPCA. Endpoint titration in Tg mice produced elk CWD titers ∼10⁷, and titers from CPCA ranged from ∼10⁶ to 10^6.5. Our findings also suggest the possibility of distinguishing cervid prion strains by adapting the CPCA in a cell panel assay format, analogous to the scrapie cell panel assay.

Ppo8-45: Identification of DNA Patterns from Circulating Nucleic Acids related to Bovine Spongiform Encephalopathy (BSE)

Maria Sensen¹, Paul M.K. Gordon¹, Ekkehard Schütz^2,3, Julia Beck², Howard B. Urnovitz², Bertram Brenig³, Martin H. Groschup⁴, Ted Sutton⁵, Robert B. Church⁶ and Christoph W. Sensen¹

¹University of Calgary; Faculty of Medicine; Department of Biochemistry and Molecular Biology; Sun Center of Excellence for Visual Genomics; Calgary, Alberta Canada; ²Chronix Biomedical GmbH; Göttingen, Germany; ³Institute of Veterinary Medicine; Georg-August-Universität; Göttingen, Germany; ⁴Institute for Novel and Emerging Infectious Diseases at the Friedrich-Loeffler-Institut; Federal Research Institute for Animal Health; Greifswald–Insel Riems, Germany; ⁵Cornerpost Consulting Ltd.; Vermilion, Alberta Canada; ⁶University of Calgary; Faculty of Medicine; Calgary, Alberta Canada

The diagnostic value of serum DNA is well established in both malignant and chronic inflammatory diseases. In a completed proof of principle study using high-throughput DNA sequencing technologies, we were able to demonstrate that elk infected with Chronic Wasting Disease (funded by the Alberta Prion Research Institute, APRI) and cattle infected with BSE (funded by German Ministry for Education and Research, BMBF) show specific nucleotide patterns in their circulating nucleic acids (CNA) during the progress of the disease. We have recently shown in a study of 575 individuals that serum DNA from either breast or prostate cancer patients can be traced back to a limited number of chromosomal hotspot regions. These disease-specific CNAs appear to be the result of apoptotic cells. A study on a large BSE infected cohort was initiated (funded by Alberta Lifestock and Meat Agency, ALMA) to extend these findings and validate the earlier TSE data. We are characterizing the serum DNA patterns derived from blood samples of BSE infected cattle and controls to determine the chromosomal origin of these apoptotic sequences. The objectives of this study are to use TSE’s as model for determining the role of apoptosis in neurological diseases and to establish the earliest time-point in BSE infection by which the presence of the disease can be detected. The results could lead to a cost effective DNA-based testing approach for early BSE. Such a test could be applied to all cattle prior to entering the human food chain.