In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc
National CJD Surveillance Unit, School of Molecular and Clinical Medicine (Pathology), University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK. The Journal of Pathology
(Impact Factor: 7.43).
09/2007; 213(1):21-6. DOI: 10.1002/path.2204
Variant Creutzfeldt-Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals is therefore of major importance. Following infection, it is assumed that minute amounts of disease-associated prion protein (PrP(Sc)) replicate by conversion of the host cellular prion protein (PrP(C)). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic amplification (PMCA), can amplify vCJD PrP(Sc) from human brain tissue, and that the degree of amplification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and amplified PrP(Sc) can be detected using a conformation-dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrP(Sc).
Available from: Maurizio Pocchiari
- "The protein-misfolding cyclic amplification (PMCA) was originally developed using hamster brain homogenate and has since been shown to be an efficient method for the amplification of brain PrP TSE of other species including mouse, sheep, cattle, bank voles, cervids, and humans        . In human samples, the amplification of PrP TSE is strongly influenced by the correct matching of methionine/valine in the 129 residue of PrP, suggesting that this polymorphic site of the protein is important for the amplification of PrP misfolding by the PMCA assay   . Ten cycles of sonication are sufficient to increase the sensitivity of standard western blots from 6–12 picograms to 0.3–0.5 picograms of brain PrP TSE and, with an improved automated protocol which enables a substantial increase in the number of amplification cycles, up to femtogram levels . "
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ABSTRACT: Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal rare neurodegenerative disorders affecting man and animals and caused by a transmissible infectious agent. TSE diseases are characterized by spongiform brain lesions with neuronal loss and the abnormal deposition in the CNS, and to less extent in other tissues, of an insoluble and protease resistant form of the cellular prion protein (PrP(C)), named PrP(TSE). In man, TSE diseases affect usually people over 60 years of age with no evident disease-associated risk factors. In some cases, however, TSE diseases are unequivocally linked to infectious episodes related to the use of prion-contaminated medicines, medical devices, or meat products as in the variant Creutzfeldt-Jakob disease (CJD). Clinical signs occur months or years after infection, and during this silent period PrP(TSE), the only reliable marker of infection, is not easily measurable in blood or other accessible tissues or body fluids causing public health concerns. To overcome the limit of PrP(TSE) detection, several highly sensitive assays have been developed, but attempts to apply these techniques to blood of infected hosts have been unsuccessful or not yet validated. An update on the latest advances for the detection of misfolded prion protein in body fluids is provided.
Available from: onlinelibrary.wiley.com
- "Using substrates prepared from normal human brain, human platelets and the transgenic mice expressing human PrP produced by Bishop et al. (2006), Jones et al. (2009) showed that PrP Sc from vCJD patients was amplified efficiently in 129 MM substrate, to a lesser degree in 129 heterozygous substrate and weakly (if at all) in 129 VV substrate. Similar results were obtained when BSE PrP Sc and substrates from transgenic mice were used (Jones et al. 2007, 2008, 2009). Extrapolating from these results, one would suggest that the replication of the bovine agent is variously slowed down when MV PrP is expressed, and more dramatically slowed when VV PrP is expressed. "
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ABSTRACT: J. Neurochem. (2011) 119, 251–261.
Prions, the agents responsible for transmissible spongiform encephalopathies, are infectious proteins consisting primarily of scrapie prion protein (PrPSc), a misfolded, β-sheet enriched and aggregated form of the host-encoded cellular prion protein (PrPC). Their propagation is based on an autocatalytic PrP conversion process. Despite the lack of a nucleic acid genome, different prion strains have been isolated from animal diseases. Increasing evidence supports the view that strain-specific properties may be enciphered within conformational variations of PrPSc. In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most frequent form of prion diseases and has demonstrated a wide phenotypic and molecular spectrum. In contrast, variant Creutzfeldt-Jakob disease (vCJD), which results from oral exposure to the agent of bovine spongiform encephalopathy, is a highly stereotyped disease, that, until now, has only occurred in patients who are methionine homozygous at codon 129 of the PrP gene. Recent research has provided consistent evidence of strain diversity in sCJD and also, unexpectedly enough, in vCJD. Here, we discuss the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.
Available from: Leigh Thorne
- "PMCA was pioneered by Soto et al.  in a rodent model of scrapie and this is an in vitro technique that amplifies trace amounts of PrPSc within a test sample during iterative rounds of sonication and incubation at 37°C. Importantly, this technique has been adapted for the high level amplification of PrPSc from natural sources of prion infection including ovine scrapie , bovine BSE , human CJD  and cervid CWD . "
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ABSTRACT: ABSTRACT: Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrPSc in 7 of 15 and 14 of 14 sheep respectively. However PrPSc was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.
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