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

Genome-wide association study identifies susceptibility loci for acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we perform a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identify a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10^{-8}; KMT5B). We also identify a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N = 1287) at 6p21.32 (rs3916765; P = 1.51 × 10^{−10}; HLA). Our results inform on AML etiology and identify putative functional genes operating in histone methylation (KMT5B) and immune function (HLA)

Towards further reduction and replacement of animal bioassays in prion research by cell and protein misfolding cyclic amplification assays

Laboratory animals have long since been used extensively in bioassays for prions in order to quantify, usually in terms of median infective doses [ID50], how infectious these pathogens are in vivo. The identification of aberrant prion protein as the main component and self-replicating principle of prions has given rise to alternative approaches for prion titration. Such approaches often use protein misfolding cyclic amplification (PMCA) for the cell-free biochemical measurement of prion-associated seeding activity, or cell assays for the titration of in vitro infectivity. However, median seeding and cell culture infective doses (SD50 and CCID50, respectively) of prions are neither formally congruent nor definitely representative for ID50 titres in animals and can be therefore only tentatively translated into the latter. This may potentially impede the acceptance and use of alternative methods to animal bioassays in prion research. Thus, we suggest performing PMCA and cell assays jointly, and to check whether these profoundly different test principles deliver consistent results in order to strengthen the reliability and credibility of prion ID50 assessments by in vitro methods. With regard to this rationale, we describe three pairs of PMCA and glial cell assays for different hamster-adapted prion agents (the frequently used 263K scrapie strain, and 22A-H scrapie and BSE-H). In addition, we report on the adaptation of quantitative PMCA to human variant Creutzfeldt-Jakob disease (vCJD) prions on steel wires for prion disinfection studies. Our rationale and methodology can be systematically extended to other types of prions and used to further reduce or replace prion bioassays in rodents