RESUMO
The link between a new variant form of Creutzfeldt-Jakob disease (vCJD) and the consumption of prion contaminated cattle meat as well as recent findings showing that vCJD can be transmitted by blood transfusion have raised public health concerns. Currently, a reliable test to identify prions in blood samples is not available. The purpose of this study was to evaluate the possibility to remove scrapie prion protein (PrP(Sc)) and infectivity from red blood cell (RBC) suspensions by a simple washing procedure using a cell separation and washing device. The extent of prion removal was assessed by Western blot, PMCA and infectivity bioassays. Our results revealed a substantial removal of infectious prions (3 logs of infectivity) by all techniques used. These data suggest that a significant amount of infectivity present in RBC preparations can be removed by a simple washing procedure. This technology may lead to increased safety of blood products and reduce the risk of further propagation of prion diseases.
Assuntos
Transfusão de Sangue , Separação Celular/métodos , Síndrome de Creutzfeldt-Jakob/prevenção & controle , Eritrócitos/química , Proteínas PrPSc/isolamento & purificação , Animais , Cricetinae , Proteínas PrPSc/patogenicidadeRESUMO
Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60 degrees C for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.
Assuntos
Endopeptidase K/química , Pressão Hidrostática , Proteínas PrPSc/química , Animais , Encéfalo/metabolismo , Soluções Tampão , Fenômenos Químicos , Físico-Química , Cricetinae , Endopeptidase K/metabolismo , Concentração de Íons de Hidrogênio , Proteínas PrPSc/metabolismo , Proteínas PrPSc/patogenicidade , Fatores de TempoRESUMO
Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60°C for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.
Assuntos
Animais , Cricetinae , Endopeptidase K/química , Pressão Hidrostática , Proteínas PrPSc/química , Soluções Tampão , Encéfalo/metabolismo , Físico-Química , Endopeptidase K/metabolismo , Concentração de Íons de Hidrogênio , Proteínas PrPSc/metabolismo , Proteínas PrPSc/patogenicidade , Fatores de TempoRESUMO
Transmissible spongiform encephalopathies (TSE) are attributed to the conversion of the cellular prion protein (PrP(c)) into an abnormal isoform (PrP(sc)). This can be caused by the invasion of living organisms by infectious particles, or be inherited due to mutations on the PrP(c) gene. One of the most intriguing problems of prion biology is the inability to generate the infectious agent in vitro. This argues strongly that other cellular proteins besides those added in test tubes or found in cellular preparations are necessary for infection. Despite recent progress in the understanding of prion pathology, the subcellular compartments in which the interaction and conversion of PrP(c) into PrP(sc) take place are still controversial. PrP(c) interacts with various macromolecules at the cell membrane, in endocytic compartments and in the secretory pathway, all of which may play specific roles in the internalisation of PrP(sc) and conversion of PrP(c). A specific interacting protein required for the propagation of prions was originally proposed as a prion receptor, and later referred to as a ligand, a cofactor, protein X, or a partner. However, current studies indicate that PrP(c) associates with multi-molecular complexes, which mediate a variety of functions in distinct cellular compartments. It is proposed that a deeper understanding of the mechanics of such interactions, coupled to a better knowledge of the corresponding signalling pathways and ensuing cellular responses, will have a major impact on the prevention and treatment of TSE.