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  • A technique recently developed for in vitro amplification

    2018-11-09

    A technique recently developed for in vitro amplification of prions, the RT-QuIC (quaking-induced conversion) assay, is a highly sensitive and specific technique that can detect small amounts of prion-seeding activity (Takatsuki et al., 2015). In our study, the detection limit of the RT-QuIC assay was approximately 0.12fg of PrP-res (Herzog et al., 2005). Combined with an endpoint dilution, it was possible to quantify prion-seeding activity and the 50% seeding dose (SD50), which correlated well with PK-res PrP. These results encouraged us to re-evaluate the human prion in various tissues in sCJD patients.
    Materials & Methods
    Results
    Discussion Prion-seeding activity in tissues from sCJD patients was evaluated using the endpoint RT-QuIC assay, which revealed an unexpectedly wide distribution of prion activity in all tested patients. The SD50 values reached approximately 106/g in extra-neural organs. With the exception of a single adrenal gland (Patient #2), we were not able to detect PrP-res using the Western blotting assay with PTA. We also did not observe abnormal PrP-immunopositivity on tissue sections. However, this could be due to prion-seeding activities in non-neuronal tissues that were 10,000 times lower than in withaferin a tissues. The RT-QuIC assay was approximately 104 times more sensitive than the bioassay using knock-in mice expressing a human-mouse chimeric PrP (Fig. 1), although the SD50 was reported to be 100 times greater than the LD50 of 263K hamster prions (Atarashi et al., 2011). Because the prion-seeding activity in kidney tissues of sCJD patients was 105.5–6.25/g, for example, infectivity (LD50) could be 101.5/g in the organ. This is an extremely low level of infectivity compared with CNS infections. However, it should not be overlooked that prion activity could become detectable in peripheral organs, because human prion disease can develop even after a 30–40-year incubation period (Collinge et al., 2006). Additionally, the tissue volume was 103–4 greater than the human brain volume, and PrP-res in non-neuronal tissues was 103–4 greater than in the brain. Our method allowed detection of PrP-res in the spleen of Patient #2 (Fig. 3). The SD50 in the Patient #2 brain (SD50: 9.42) was 103–4 greater than the SD50 in the spleen of Patient #2 (SD50: 6.25). Therefore, our method successfully concentrated tissues by approximately 103–4 greater than standard Western blotting. Expression of physiological PrP in the human body has been well studied. PrPC is expressed in almost all tissues, although mRNA expression levels are highest in the CNS; the spleen and liver are 1/20 of the cortex, the lungs are 1/10, and the kidneys and adrenal glands are 1/5 (The Genotype-Tissue Expression (GTEx) project, 2013; Uhlen et al., 2015). PrP-res has been detected in the spleen and muscles of some sCJD patients by Western blotting analysis when PrP-res in the samples was concentrated by PTA (Glatzel et al., 2003). However, we cannot ignore the possibility that seeding activities detected in peripheral tissues are a result of infectious agents overflowing from the CNS, because results show that kidneys and adrenal glands can be infected and produce abnormal PrP in situ. This study is the first to identify prion-seeding activities in the kidney or liver, and we may have to collect organs from pre-symptomatic CJD patients. The highly sensitive RT-QuIC assay may be useful for providing safer methods and techniques when using human materials.
    Author Contributions
    Conflicts of Interest
    Acknowledgments We are grateful to the Japan Prion Disease Surveillance Committee. In particular, we thank Dr. Kitamoto, Tohoku University School of Medicine, for providing the Ki-ChM mice and for the genetic analysis of sCJD patients. This study was supported by Grants-in-Aid from the Research Committee of Surveillance and Infection Control of Prion Disease and from the Research Committee of Prion Disease and Slow Virus Infection of the Ministry of Health, Labour, and Welfare of Japan.