12 research outputs found
Detection of prions in blood from patients with variant Creutzfeldt-Jakob disease
Prions can be detected in blood from patients with variant Creutzfeldt-Jakob disease with high sensitivity and specificity.</jats:p
Development of a methodology for large-scale production of prions for biological and structural studies
Prion diseases are a group of infectious neurodegenerative diseases produced by the conversion of the normal prion protein (PrPC) into the disease-associated form (PrPSc). Extensive evidence indicate that the main or sole component of the infectious agent is PrPSc, which can replicate in affected individuals in the absence of nucleic acids. However, the mechanism of PrPC-to-PrPSc conversion remains elusive, which has been attributed to the lack of sufficient structural information of infectious PrPSc and a reliable system to study prion replication in vitro. In this article we adapted the Protein Misfolding Cyclic Amplification (PMCA) technology for rapid and efficient generation of highly infectious prions in large-scale. Murine prions of the RML strain were efficiently propagated in volumes up to 1,000-fold larger than conventional PMCA. The large-scale PMCA (LS-PMCA) procedure enabled to produce highly infectious prions, which maintain the strain properties of the seed used to begin the reaction. LS-PMCA was shown to work with various species and strains of prions, including mouse RML and 301C strains, hamster Hyper prion, cervid CWD prions, including a rare Norwegian CWD prion, and human CJD prions. We further improved the LS-PMCA into a bioreactor format that can operate under industry-mimicking conditions for continuous and unlimited production of PrPSc without the need to keep adding brain-derived prions. In our estimation, this bioreactor can produce in 1d an amount of prions equivalent to that present in 25 infected animals at the terminal stage of the disease. Our LS-PMCA technology may provide a valuable tool to produce large quantities of well-defined and homogeneous infectious prions for biological and structural studies
Quantitative Detection and Biological Propagation of Scrapie Seeding Activity In Vitro Facilitate Use of Prions as Model Pathogens for Disinfection
Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants
Transmission, Strain Diversity, and Zoonotic Potential of Chronic Wasting Disease
Chronic wasting disease (CWD) is a prion disease affecting several species of captive and free-ranging cervids. In the past few decades, CWD has been spreading uncontrollably, mostly in North America, resulting in a high increase of CWD incidence but also a substantially higher number of geographical regions affected. The massive increase in CWD poses risks at several levels, including contamination of the environment, transmission to animals cohabiting with cervids, and more importantly, a putative transmission to humans. In this review, I will describe the mechanisms and routes responsible for the efficient transmission of CWD, the strain diversity of natural CWD, its spillover and zoonotic potential and strategies to minimize the CWD threat
A superior alternative to titrations of scrapie infectivity in animals
Prionen verursachen transmissible spongiforme Enzephalopathien wie Scrapie,
die bovine spongiforme Enzephalopathie oder die Creutzfeldt-Jakob-Krankheit
und bestehen aus fehlgefaltetem, aggregierten Prionprotein (PrP). Aufgrund
ihrer hohen Toleranz gegenüber üblichen Desinfektionsverfahren stellen sie
sowohl eine besondere Herausforderung als auch ein exzellentes Paradigma für
die Aufbereitung von chirurgischen Instrumenten dar. Tatsächlich haben sich
Prionen bereits als aussagekräftige Modellpathogene für die Entwicklung neuer
Formulierungen zur hochwirksamen Desinfektion auch von Bakterien, Viren und
Pilzen bewährt. Um Prionen zukünftig in größerem Umfang als Testerreger zur
Entwicklung neuer Mittel und Verfahren für die Breitspektrumdesinfektion ohne
Rückgriff auf Tierversuche nutzen zu können, wurden in dieser Arbeit in vitro-
Ersatzmethoden für Prion-Bioassays in Nagetieren aufgebaut und validiert. Zur
quantitativen biochemischen Bestimmung der PrP-konvertierenden Keimaktivität
des Scrapie-assoziierten Prionproteins wurde ein spezifisch adaptierter
Protein Misfolding Cyclic Amplification (PMCA)-Assay etabliert. Mit hamster-
adaptierten Scrapie-Prionen (Stamm 263K) kontaminierte Stahlstifte wurden
verschiedenen Desinfektionsbehandlungen und anschließend einer quantitativen
PMCA unterzogen. Die Ergebnisse des PMCA-Assays wurden dann mit bereits früher
erhobenen Bioassay-Daten zur Reduktion infektiöser Priontiter durch die
angewandten Desinfektionsverfahren verglichen. Zusätzlich dazu wurde ein
Zellkulturassay mit primären Gliazellen aus Hamstern zum biologischen in
vitro-Nachweis der Keimaktivität des Prionproteins entwickelt. Der Nachweis
von Scrapie-assoziierter Keimaktivität an Stahlstiftprüfkörpern in der
quantitativen PMCA übertraf die Sensitivität des Nachweises von
Prioninfektiosität im Tierversuch um mindestens eine Größenordnung. Dabei
konnte die Reduktion der Prioninfektiosität an Stahlstiftprüfkörpern durch 16
verschiedene Desinfektions-verfahren mit Titerreduktionen von weniger als
101-fach bis zu größer oder gleich 105,5-fach durchgängig korrekt abgeschätzt
werden. Mittels PMCA biochemisch nachgewiesene Keimaktivität ließ sich auch
biologisch in der Zellkultur detektieren. Konzeptionell deuten die Befunde
dieser Arbeit darauf hin, dass die proteinöse Keimaktivität und pathogene
Infektiosität von 263K Scrapie-Prionen biochemische und biologische
Manifestationen desselben molekularen Prinzips darstellen. In praktischer
Hinsicht steht mit der Gliazellkultur-gekoppelten PMCA potentiell eine
gleichwertige oder sogar überlegene Alternative zum Prion-Bioassay in Tieren
zur Verfügung.Prions are misfolded, aggregated prion proteins (PrP) and the causative agents
of transmissible spongiform encephalopathies such as scrapie, bovine
spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease. Prions are
pathogens with an unusually high tolerance to inactivation and constitute a
complex challenge as well as an informative paradigm for the re-processing of
surgical instruments. They have been exploited successfully as model pathogens
for the development of novel disinfectants with simultaneous activity also
against bacteria, viruses and fungi. In order to further facilitate the use of
prions as model agents in the search for novel broad-range disinfectants
without the use of animal experiments we established and validated an
experimental platform for the sensitive quantitative measurement and
biological detection of scrapie seeding activity in vitro. We used
specifically adapted serial protein misfolding cyclic amplification (PMCA) for
the quantitative biochemical detection of the seeding activity of scrapie
associated prion protein. Steel wires contaminated with 263K scrapie prions
were subjected to different decontamination procedures and subsequently
analysed by quantitative PMCA. The results of the PMCA assay were validated by
comparison to bioassay data previously established for similar treatments of
steel wires. Furthermore a novel cell culture assay of cerebral glial cell
cultures from hamsters was established for the biological detection of 263K
scrapie seeding activity. The detection limit for scrapie seeding activity on
steel wires in the PMCA assay exceeded that for prion infectivity detected by
bioassay at least tenfold. The residual scrapie infectivity’s on test wires
treated with 16 different disinfection procedures yielding scrapie titre
reductions of ≤101- to ≥105.5-fold could be correctly deduced, for all
examined procedures, from our quantitative seeding activity assay. In
addition, we found that scrapie seeding activity biochemically detected by
PMCA could be also demonstrated biologically in the cell culture assay.
Conceptually, the findings from this work suggested the seeding activity and
infectivity of 263K scrapie prions as corresponding biochemical and biological
manifestations of the same replicative molecular principle. In practical
terms, our biochemical and biological in vitro assays, when combined,
potentially provide a coequal or even superior alternative to titrations of
scrapie infectivity in animals. In vitro-detection of the seeding activity of
scrapie associated prion protein: A superior alternative to titrations of
scrapie infectivity in animal
Grass Plants Bind, Retain, Uptake, and Transport Infectious Prions
Prions are the protein-based infectious agents responsible for prion diseases. Environmental prion contamination has been implicated in disease transmission. Here, we analyzed the binding and retention of infectious prion protein (PrPSc) to plants. Small quantities of PrPSc contained in diluted brain homogenate or in excretory materials (urine and feces) can bind to wheat grass roots and leaves. Wild-type hamsters were efficiently infected by ingestion of prion-contaminated plants. The prion-plant interaction occurs with prions from diverse origins, including chronic wasting disease. Furthermore, leaves contaminated by spraying with a prion-containing preparation retained PrPSc for several weeks in the living plant. Finally, plants can uptake prions from contaminated soil and transport them to aerial parts of the plant (stem and leaves). These findings demonstrate that plants can efficiently bind infectious prions and act as carriers of infectivity, suggesting a possible role of environmental prion contamination in the horizontal transmission of the disease