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Volume 23, Number 6—June 2017


Distribution and Quantitative Estimates of Variant Creutzfeldt-Jakob Disease Prions in Tissues of Clinical and Asymptomatic Patients

Jean Y. Douet, Caroline Lacroux, Naima Aron, Mark W. Head, Séverine Lugan, Cécile Tillier, Alvina Huor, Hervé Cassard, Mark Arnold, Vincent Beringue, James W. Ironside, and Olivier AndréolettiComments to Author 
Author affiliations: Institut National de la Recherche Agronomique, Toulouse, France (J.Y. Douet, C. Lacroux, N. Aron, S. Lugan, C. Tillier, A. Huor, H. Cassard, O. Andréoletti); University of Edinburgh, Edinburgh, Scotland, UK (M.W. Head, J.W. Ironside); Animal and Plant Health Agency, Loughborough, UK (M. Arnold); Institut National de la Recherche Agronomique, Jouy-en-Josas, France (V. Beringue)

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Table 4

Protein misfolding cyclic amplification reactions seeded with tissue homogenate from vCJD and control patients*

Tissue Clinical vCJD patients, Met129/Met129
Preclinical vCJD patient, Met129/Val129
Non–vCJD controls, Met129/Met129
vCJD-1 vCJD-2 vCJD-3 vCJD-4 vCJD-A NC-1 NC-2
Frontal cortex 10−8 10−8 10−8 10−8
Pituitary gland NA NA NA NA 10−2
Trigeminal ganglia NA NA NA NA
Dorsal root ganglia NA NA NA NA
Cervical lymph node 10−5 10−4 10−4 10−3 10−4 NA NA
Tonsil 10−3 10−4 10−6 10−3 10−3 NA
Appendix 10−4 10−4 10−4 10−3 10−2
Distal Ileum 10−3 10−5 10−5 10−2 10−3
Spleen 10−4 10−4 10−5 10−4 10−3
Thymus NA 10−3 10−2 10−2 10−2 NA NA
Lung 10−2 10−2 10−3
Heart 10−2 10−2
Liver 10−4 10−2 10−2 10−4 10−2
Kidney 10−2 10−3 10−3
Salivary gland 10−4 10−3 10−2 10−3 10−2
Pancreas 10−2 10−2 10−4
Thyroid 10−2 10−2 10−2
Adrenal gland 10−3 10−3 10−3 10−4
Bone marrow 10−4 10−5 10−3 10−4
Skeletal muscle 10−4 10−2 NA
Testis NA 10−3 NA NA NA
Ovary NA 10−4 NA NA NA NA NA

*PMCA reactions were seeded with 10-fold serial dilutions of 10% tissues homogenates (10−2–10−9) that had been collected postmortem from 4 symptomatic vCJD patients (vCJD-1–vCJD-4) or an asymptomatic vCJD-infected person (vCJD-A). At least 4 replicates of each sample dilution were tested in 2 independent PMCA experiments. Prions from patients vCJD-1–vCJD-4 were homozygous for methionine at codon 129 of the PRNP gene. Prion from patient vCJD-A was heterozygous (methionine/valine) at codon 129 of the PRNP gene. PMCA substrate was prepared by using brains from transgenic mice overexpressing the ARQ variant of sheep prion protein. Reactions seeded with tissues from 2 non–vCJD-infected control patients (NC-1 and NC-2) were included as negative controls. PMCAs were subjected to 4 rounds of amplification, each composed of 96 cycles (sonication for 10 s and incubation for 14.5 min at 39.5°C) in a Qsonica700 Sonicator (Qsonica LLC, Newtown, CT, USA). PMCA reactions were analyzed by Western blotting for proteinase K–resistant PrP by using Sha31 antibody epitope YEDRYYRE. Values are the highest dilution that resulted in a positive Western blot result in >50% of the tested replicates after 4 PMCA amplification rounds. NA, not applicable; PMCA, protein misfolding cyclic amplification; PrP, prion protein; ­vCJD, variant Creutzfeldt-Jakob disease; –, negative.

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