NR ATGN
AU Weissmann,C.; Kloehn,P.; Mahal,S.P.; Baker,C.A.; Deleault,N.; Supattapone,S.
TI Transmission of prions
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Oral sessions ORAL-44
PT Konferenz-Vortrag
AB We isolated variants of a neuroblastoma-derived cell line that are highly susceptible to mouse prion infection and others that are resistant. The susceptible line (N2aPK1) was used to develop a novel, in vitro assay for prion infectivity (Standard Scrapie Cell Assay or SSCA) that is 10 times faster than the mouse bioassay and at least as sensitive. The sensitivity can be further extended by the SCA in endpoint format (SCEPA). Using SCA, we found that chronically infected N2aPK1 cells secrete about 3000 LD50 units of prions/hr/ 106 cells. Infectivity from conditioned medium passed through a filter with a pore size of 1000 kDa but not 300 kDa and had a buoyant density of 1.15 g/cm3, corresponding to the density of exosomes. Experiments are in progress to investigate whether infectivity is associated with virus-like particles. We have analysed prion production by single cells of chronically infected N2a cell populations by SCEPA and have determined that a subset of cells with particular properties are the most prolific prion producers. Using SCA we determined that in vitro incubation of PrPc (provided by normal mouse brain homogenate) with a small amount of PrPsc (provided by scrapie-infected brain homogenate) generates within 8 hours a ~6 fold increase in infectivity, accompanied by a ~9 fold increase in PrPsc, in a time- and substrate-dependent reaction, showing that prion replication can take place rapidly in a cell-free system.
AD C.Weissmann, S.P.Mahal, C.A.Baker, Department of Infectology, Scripps Florida, 5353 Parkside Drive, Jupiter, FL 33458. USA; P.Kloehn, MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, Queen Square, London WC1N 3BG, UK; N.Deleault, S.Supattapone, Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA
SP englisch
PO Deutschland