72.733.200) (see Results). == Additional procedures to minimise contamination == Access to the laboratory was restricted to authorised personnel and standard operating procedures were adopted for the use of laboratory equipment and sample manipulation. posterioribiochemical and biological analyses ofde novogenerated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observede novoappearance of PrPScin unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrPScin unseeded negative controls suggested that the most likely explanation for the appearance ofde novoPrPScwas the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical KL-1 points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrPScto be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitivein vitroprion replication and warn on cautious interpretation when assessing the spontaneous appearance of prionsin vitro. == Author Summary == In an attempt to transpose to anin vitrosystem the particular sensitivity of the vole model to human and animal Transmissible Spongiform Encephalopathies (TSEs), we first explored the suitability of vole brain homogenate as a substrate for PMCA. As well as observing the highly efficient replication of a variety of prion sources, we also found preliminary evidence ofde novoprion generation in unseeded reactions. Careful analysis of the PMCA procedure, undertaken further to investigate these findings, showed, however, that they were the result of cross-contamination with seeded samples. We next identified and investigated the critical points of this procedure that are potentially responsible for cross-contamination. Our results suggest thatin vitrosystems Rabbit Polyclonal to CKLF2 for prion amplification could be more prone to cross-contamination than previously thought, particularly when harsh procedures, such as sonication, are involved. Experimental conditions able to reproduce spontaneous prion formation in a simple and easily reproduciblein vitrosystem would be of crucial interest for understanding TSEs and other important neurodegenerative disorders. Given thatin vitromethods are increasingly used in this field, our results emphasise the possible drawbacks of such approaches and call for the use of rigorously controlled conditions and cautious interpretation of data. == Introduction == Transmissible Spongiform Encephalopathies (TSEs) are progressive and fatal neurodegenerative disorders that include scrapie of sheep, bovine spongiform encephalopathy (BSE) of cattle and Creutzfeldt-Jakob disease (CJD) of humans[1]. The nature of the causal agent of TSEs has long been a matter of intense scientific debate. The prion hypothesis postulates that KL-1 the causal agent, the prion, consists only of proteins without nucleic acid genome[1]. Alternative hypotheses postulate the presence of a small nucleic acids genome[2], although evidences for this are still lacking. The virino hypothesis proposes that the causal agent is an informational hybrid between the agent genome and host conformationally altered PrP[3]. Recently, new evidences were brought in support of the prion hypothesis, although a fundamental role of non proteinaceous cofactors could not be definitively excluded[4],[5],[6],[7],[8]. The accumulation in the central nervous system of a post-translationally altered isoform (PrPSc) of the cellular prion protein (PrPC) is the key event in TSE pathogenesis[1], Nevertheless the relationships between PrPScand infectivity are not definitively clear and evidences for high titers of TSE infectivity associated with extremely low levels of PrPSchave been reported[9]. The modification of PrPCinvolves mostly unknown conformational changes during which an increase in the amount of -sheet of the normal protein and a decrease in its -helical content is observed[10],[11]. According to the prion theory, PrPScthus acquires, via a template-based mechanism, the ability to trigger the conversion of PrPCinto new PrPSc. The process proceeds thereafter in an autocatalytic manner, leading PrPScaggregates to grow by including new PrPCmonomers[1],[12]. The severe outbreak of BSE, first detected in the UK in 1986, and the announcement in 1996 that the BSE agent was responsible for a newly recognised form of TSE in humans, named variant CJD, created enormous concern among European consumers and prompted health authorities to promote. KL-1
