title: Heparin enhances the cell-protein misfolding cyclic amplification efficiency of variant Creutzfeldt-Jakob disease.
abstract: Highly sensitive in vitro screening tests are required to prevent the iatrogenic spread of variant Creutzfeldt-Jakob disease (vCJD). Protein misfolding cyclic amplification (PMCA) is a candidate for such a test, but the sensitivity of this method is insufficient. Polyanions were reported to enhance PMCA efficiency, but their effects on vCJD are unclear. We developed a cell-PMCA of vCJD, wherein cell lysate containing exogenously expressed human PrP was used as substrates, to investigate the effects of various sulfated polysaccharides on amplification efficiency. PrP(res) amounts after cell-PMCA were analyzed by western blotting. Heparin, dermatan sulfate, and dextran sulfate (average molecular weight [MW] 1400kDa) enhanced efficiency, but dextran sulfate (average MW 8kDa) and a heparin pentasaccharide analog had no effect. Pentosan polysulfate inhibited cell-PMCA reaction. The amplification efficiency of cell-PMCA of vCJD increased to >100-fold per round with heparin. The enhancing effects of heparin on cell-PMCA were seed dependent: it was high for vCJD, low for sporadic Creutzfeldt-Jakob disease, and low to negligible for hamster-adapted scrapie-derived 263K. In multi-round PMCA, signals were detected at earlier rounds with heparin than without heparin, and PrP(Sc) in 10(-10) diluted vCJD brain was detected by the sixth round. Heparin-assisted cell-PMCA of vCJD represents a significant step toward detecting very minute amounts of PrP(Sc) in the body fluids of asymptomatic vCJD patients.
authors.affiliation: Research and Development Division, Benesis Corporation, c/o Division of Neurological Science, Department of Prion Research, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokoyama
authors.forename: Atsuko
authors.initials: A
authors.lastname: Takeuchi
authors.forename: Miyuki
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Tetsuyuki
authors.initials: T
authors.lastname: Kitamoto
authors.forename: James W
authors.initials: JW
authors.lastname: Ironside
authors.forename: Masanori
authors.initials: M
authors.lastname: Morita
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002474:Cell-Free System; D007562:Creutzfeldt-Jakob Syndrome / Q000378:metabolism*; D006224:Cricetinae; D003871:Dermatan Sulfate / Q000494:pharmacology; D016264:Dextran Sulfate / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008647:Mesocricetus; D051379:Mice; D008970:Molecular Weight; D010426:Pentosan Sulfuric Polyester / Q000494:pharmacology; D018620:PrPSc Proteins / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects*; D012608:Scrapie / Q000378:metabolism; D013045:Species Specificity
chemical_list: D018620:PrPSc Proteins; D003871:Dermatan Sulfate; D010426:Pentosan Sulfuric Polyester; D006493:Heparin; D016264:Dextran Sulfate