In Vitro Prion Amplification Methodology for Inhibitor Screening.

Prion (PrPC) is an endogenous protein found mainly in the nervous system, and its misfolded isoform (PrPSc) is associated with a group of neurodegenerative disorders known as transmissible spongiform encephalopathies, or simply prion diseases. The PrPSc isoform shows an intriguing ability to self-perpetuate, acting as template for PrPC misfolding and consequent aggregation. Aggregation in vitro and in vivo follows a fibrillation processes that is associated with neurodegeneration. Therefore, it is important to investigate and understand the molecular mechanisms involved in this process; such understanding also allows investigation of the action of possible candidate molecules to inhibit this process. Here, we highlight useful in vitro methodologies and analyses that were developed using PrP as a protein model but that, as other amyloid proteins also exhibit the same behavior, may be applied to understand other "prion-like" diseases such as Alzheimer's and Parkinson's disease.

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells.

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population.

A solvent/detergent-treated and 15-nm filtered factor VIII: a new safety standard for plasma-derived coagulation factor concentrates.

BACKGROUND: Since the early 1990 s the Committee for Proprietary Medicinal Products has set the mandatory requirement that all manufacturing processes for blood products include two virus removal/inactivation steps that are complementary in their action. OBJECTIVES: The objective was to develop a manufacturing process for factor VIII (FVIII) including two complementary steps of viral inactivation/elimination. METHODS: A 35-15 nm nanofiltration step was added to a former FVIII manufacturing process that included solvent/detergent (S/D) treatment to generate a new FVIII concentrate called Factane. The impact of nanofiltration on the structural and functional characteristics of FVIII, as well as virus/transmissible spongiform encephalopathy reduction factors were assessed. RESULTS: Using an innovative approach, FVIII was successfully nanofiltered at 35-15 nm, while the biological properties of the active substance were unmodified. FVIII coagulant and antigen content for Factane and previous S/D-treated FVIII (FVIII-LFB, commercialized as Facteur VIII-LFB) were comparable. The FVIII one-stage chromogenic and coagulant/antigen ratios confirmed that nanofiltered FVIII was not activated. After nanofiltration, the copurified von Willebrand factor (vWF) was reduced but vWF/FVIII binding properties were unaffected. Phospholipid binding and thrombin proteolysis studies displayed no differences between Factane and FVIII-LFB. The rate of factor Xa generation was slightly lower for Factane when compared to FVIII-LFB. Viral validation studies with different viruses showed no detectable virus in the filtrate. CONCLUSIONS: Nanofiltration of FVIII at 15 nm is feasible despite the large molecular weight of FVIII and vWF. Nanofiltration has been proven to be highly effective at removing infectious agents while preserving the structural and functional integrity of FVIII.

Natural scrapie: detection of fibrils in extracts from the central nervous system of sheep.

Extracts from the cervical spinal cord and from the medulla, thalamus, cerebellum and cerebral cortex of the brains of 10 sheep, histopathologically confirmed as cases of scrapie, were examined by electron microscopy for the presence of scrapie-associated fibrils. Characteristic fibrils were observed in all the extracts except for that from the thalamus of one sheep. No fibrils were found in any extracts from three control sheep. A comparison of these results with a similar study of 22 cases of bovine spongiform encephalopathy (BSE) suggests that in cases of scrapie the area of the brain chosen for the detection of fibrils is less critical than in cases of BSE, in which fibrils are more readily extracted from areas of the brain stem.

Transmissible mink encephalopathy (TME) in Chinese hamsters: identification of two strains of TME and comparisons with scrapie.

TME from a single source was transmitted by intracerebral injection to Chinese hamsters, producing clinical disease in all seven animals after incubation periods of over 600 days. The brain from each of the primary cases was used to establish separate intracerebral passage-lines of TME and this led to the isolation of two different strains of agent, designated 333K and 333W. These strains were easily distinguished by the incubation periods they produced (about 130 and 230 days, respectively) under standard conditions of infection, and by the characteristic profiles of vacuolation seen in different regions of the brain. Comparisons were made with a strain of scrapie passaged in Chinese hamsters, designated 34W, which could be distinguished from both strains of TME. Nevertheless the properties of the scrapie and TME strains overlapped, with one of the TME strains (333K) resembling the 34W strain of scrapie in Chinese hamsters more closely than the other TME strain (333W). These similarities strengthen the view that TME and scrapie are caused by a similar type of infectious agent. The very large 'species barrier effect' on transmitting TME to Chinese hamsters was in marked contrast to the minimal effect seen with scrapie and an explanation for this is suggested. Two interesting pathological features of the study were (a) the severe loss of pyramidal cells produced in the hippocampus by the 34W strain of scrapie, and (b) the focal, symmetrical vacuolation of the thalamus caused by 333K TME.