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Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrP Sc ) and purified prion fibrils were heated or pressurized at 800 megapascals and 60°C for 2 h in different buffers and in water. Prion proteins (PrP) were analyzed for their proteinase K resistance in immunoblots and for their infectivity in hamster bioassays. A notable decrease in the proteinase K resistance of unpurified prion proteins, probably because of pressure-induced changes in the protein conformation of native PrP Sc or the N-truncated PrP-(27-30), could be demonstrated when pressurized at initially neutral conditions in several buffers and in water but not in a slightly acidic pH. A subsequent 6 -7 log 10 reduction of infectious units/g in phosphate-buffered saline buffer, pH 7.4, was found. The proteinase K-resistant core was also not detectable after purification of prions extracted from pressurized samples, confirming pressure effects at the level of the secondary structure of prion proteins. However, opposite results were found after pressurizing purified prions, arguing for the existence of pressure-sensitive ␤-structures (PrP Sc ⌬Psen ) and extremely pressure-resistant ␤-structures (PrP Sc ⌬Pres ). Remarkably, after the first centrifugation step at 540,000 ؋ g during isolation, prions remained proteinase K-resistant when pressurized in all tested buffers and in water. It is known that purified fibrils retain infectivity, but the isolated protein (full and N-truncated) behaved differently from native PrP Sc under pressure, suggesting a kind of semicrystalline polymer structure. Transmissible spongiform encephalopathies (TSEs)1 are associated with the accumulation of an isoform of the cellular prion protein, designated PrP Sc in brain. Evidence indicates that misfolding of the cellular isoform into a ␤-sheet-rich aggregated pathogenic self-propagating multimer is the main step of infection (1). Even though detailed studies on the structure of the cellular prion protein (PrP C ) have been performed (e.g. with NMR (2)) and the amino acid sequence is known to be preserved in both isoforms, many features of prions are intriguing because PrP Sc -causing TSE is known to be insoluble. Up to now, studies of the PrP C structure after expression of recombinant cellular prions (2), the purification of native PrP Sc after immunoaffinity chromatography and elution at basic pH (3), or the isolation of proteinase K-resistant materials (iPrP res ) (4) have been prerequisites for performing studies on the structure of infectious prions. Thus, existent models are mainly derived from spectroscopic data from studies of such structures. Moreover, treatments with heat, salts, denaturants, and extreme pH revealed new conformational states of prions that were different from native. And so far, when a conformational change leads to a reduction in infectivity, inactivation is irreversible (5).The ␤-rich secondary structure most likely provides infectious prions with a tremendous resistance to conventional autoclaving, whi...

-The aim of this study was to determine the immunomodulatory effects of IL-12, IL-18 and CD154 (CD40 ligand, CD40L) in DNA-vaccination against the classical swine fever virus. Four recombinant plasmids were constructed including the CSFV coding region for the glycoprotein gp55/E2 alone or together with porcine IL-12, IL-18 or CD154 genes. Five groups of four pigs each were immunized intramuscularly (i.m.) three times with the respective constructs. The control group was inoculated with empty plasmid DNA. Eighteen days after the final immunization, the pigs were challenged with a lethal dose of CSFV strain Eystrup and monitored for a further 16 days. This study showed that co-delivery of IL-18 and CD154 induced an earlier appearance of serum antibodies, reduced B-cell deficiency after infection and protected pigs against a lethal CSFV infection. In contrast, co-delivery of IL-12 led to a reduced titer of neutralizing antibodies and protection against a lethal CSFV challenge in comparison to the other pigs and to pigs that were immunized with a gp55/E2 plasmid alone.

High hydrostatic pressure is a mild technology compared with high temperatures and is commonly used for food pasteurization. Crude brain homogenates of terminally diseased hamsters infected with scrapie 263K strain were heated at 60 6C and/or pressurized up to 1000 MPa for 2 h. Prion proteins were analysed for their proteinase K sensitivity using a Western blot technique. PrP Sc pressurized with 500 MPa or above proved to be proteinase K sensitive. To test the remaining infectivity of the pressurized material, hamsters were infected intracerebrally. Results showed a greatly delayed onset of disease (from 80 up to 153 days) when samples had been pressurized at 500 MPa and above. An increase in the survival rate was also observed: 47 % survival over 180 days was seen following infection with homogenates pressurized at 700-1000 MPa.Prion diseases are associated with the accumulation of an isoform of the cellular prion protein, designated PrP Sc , after misfolding in a b-rich aggregated pathogenic multimer, which seems to be the main component of the transmissible form (Prusiner, 1991). The transmissible agent resists conventional autoclaving (Brown et al., 2000). Thus, the European Directive of 1996 recommends the use of at least 133 uC for 20 min to achieve effective inactivation during sterilization processes or rendering. Other suitable treatments, such as the combination of alkali and heat, or the use of hypochlorite solutions, although efficient, are aggressive, with a consequent loss of quality or texture in the treated tissues. Therefore, an interest in assessing the effects of unconventional milder technologies on prion stability and infectivity is growing. Milder processes would provide alternative sterilization procedures for at-risk materials and may reduce economic losses in the rendering industry. Here we report the effects of treatment with high hydrostatic pressure at 60 uC on the proteinase K (PK) sensitivity of hamster PrP Sc and the in vivo infectivity of the highpressure-treated samples.These experiments were performed with a single brain pool of the 263K strain of hamster-adapted scrapie agent. Brains containing PrP Sc were homogenized in a 1 : 10 dilution of PBS, pH 7?4, in a FastPrep cell disrupter (Qbiogene). Sets of duplicate samples were heated at 60 uC and/or pressurized up to 1000 MPa for 2 h independently. Total volumes of 250 ml of homogenate (10 %, w/v) were pressurized in a hydraulic press, U 101 (Polish Academy of Sciences, Warsaw). U 101 is a manually operated twin-piston hydraulic press (100 mm piston length, 80 mm piston movement). The vessel is a cylinder made of steel, with an inside diameter of 16 mm and 150 mm in height. The piston position is monitored with a linear transformer transducer and the pressure-measuring unit is an in-vessel manganin pressure gauge; both are digitally displayed. The pressure-transmitting medium was a 7 : 3 mixture of petroleum ether (boiling point 80-100 uC) and hydraulic oil (SAE 32). Samples were pressurized in polyethylene caps hermetically clos...

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