Biomedical subjects

Alex Bossers

Publications and source records attributed to Alex Bossers.

4 recordsLinked to original sources

PubMed2006

Rapid and discriminatory diagnosis of scrapie and BSE in retro-pharyngeal lymph nodes of sheep.

BACKGROUND: Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrPres. RESULTS: Optimal PrPres detection in RLN by WB was achieved by proper tissue processing, antibody choice and inclusion of a step for PrPresconcentration. The analyses were performed on three different sheep sources. Firstly, in a study with preclinical scrapie cases, WB of RLN from infected sheep of VRQ/VRQ genotype--VRQ represents, respectively, polymorphic PrP amino acids 136, 154, and 171--allowed a diagnosis 14 mo earlier compared to WB of brain stem. Secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002-2003 yielded positive results depending on genotype: all sheep with genotypes ARH/VRQ, VRQ/VRQ, and ARQ/VRQ scored positive for PrPres, but ARQ/ARQ and ARR/VRQ were not all positive. Thirdly, in an experimental BSE study, detection of PrPres in all 11 ARQ/ARQ sheep, including 7 preclinical cases, was possible. In all instances, WB and IHC were almost as sensitive. Moreover, BSE infection could be discriminated from scrapie infection by faster electrophoretic migration of the PrPres bands. Using dual antibody staining with selected monoclonal antibodies like 12B2 and L42, these differences in migration could be employed for an unequivocal differentiation between BSE and scrapie. With respect to glycosylation of PrPres, BSE cases exhibited a greater diglycosylated fraction than scrapie cases. Furthermore, a slight time dependent increase of diglycosylated PrPres was noted between individual sheep, which was remarkable in that it occurred in both scrapie and BSE study. CONCLUSION: The present data indicate that, used in conjunction with testing in brain, WB of RLN can be a sensitive tool for improving surveillance of scrapie and BSE, allowing early detection of BSE and scrapie and thereby ensuring safer sheep and goat products.

Animals↗

PubMed2005

Sheep scrapie susceptibility-linked polymorphisms do not modulate the initial binding of cellular to disease-associated prion protein prior to conversion.

Conversion of the host-encoded protease-sensitive cellular prion protein (PrPC) into the scrapie-associated protease-resistant isoform (PrPSc) of prion protein (PrP) is the central event in transmissible spongiform encephalopathies or prion diseases. Differences in transmissibility and susceptibility are largely determined by polymorphisms in PrP, but the exact molecular mechanism behind PrP conversion and the modulation by disease-associated polymorphisms is still unclear. To assess whether the polymorphisms in either PrPC or PrPSc modulate the initial binding of PrPC to PrPSc, several naturally occurring allelic variants of sheep PrPC and PrPSc that are associated with differential scrapie susceptibility and transmissibility [the phylogenetic wild-type (ARQ), the codon 136Val variant (VRQ) and the codon 171Arg variant (ARR)] were used. Under cell-free PrP conversion conditions known to reproduce the observed in vivo differential scrapie susceptibility, it was found that the relative amounts of PrPC allelic variants bound by various allelic PrPSc variants are PrP-specific and have comparable binding efficiencies. Therefore, the differential rate-limiting step in conversion of sheep PrP variants is not determined by the initial PrPC-PrPSc-binding efficiency, but seems to be an intrinsic property of PrPC itself. Consequently, a second step after PrPC-PrPSc-binding should determine the observed differences in PrP conversion efficiencies. Further study of this second step may provide a future tool to determine the mechanism underlying refolding of PrPC into PrPSc and supports the use of conversion-resistant polymorphic PrPC variants as a potential therapeutic approach to interfere with PrP conversion in transmissible spongiform encephalopathy development.

Animals↗

PubMed2003

Enzymatic degradation of prion protein in brain stem from infected cattle and sheep.

Prions-infectious agents involved in transmissible spongiform encephalopathies-normally survive proteolytic and mild protein-destructive processes. Using bacterial keratinase produced by Bacillus licheniformis strain PWD-1, we tested conditions to accomplish the full degradation of prion protein (PrP) in brain-stem tissue from animals with bovine spongiform encephalopathy and scrapie. The detection of PrPSc, the disease-associated isoform of PrP, in homogenates was done by Western blotting and various antibodies. The results indicated that only in the presence of detergents did heat pretreatment at >100 degrees C allow the extensive enzymatic breakdown of PrPSc to a state where it is immunochemically undetectable. Proteinase K and 2 other subtilisin proteases, but not trypsin and pepsin, were also effective. This enzymatic process could lead to the development of a method for the decontamination of medical and laboratory equipment. The ultimate effectiveness of this method of prion inactivation has to be tested in mouse bioassays.

Animals↗

PubMed2003

In vitro conversion of normal prion protein into pathologic isoforms.

The in vitro conversion techniques in cell-free and cell culture systems have provided tools to adequately study the underlying mechanism of TSEs, namely PrP conversion. These systems also have provided tools that make it easier to study the interspecies and intraspecies transmissibilities of TSEs. Finally, these systems also may assist in the discovery of TSE therapeutic strategies and in the development of extremely sensitive TSE detection techniques. In vivo TSE transmission studies are limited to (transgenic) animals (mostly mice). Although the cell culture systems also are restricted in their species-range (mostly mouse), the currently used cell-free systems. Allow studying almost all possible species barriers (including the potential transmission of various TSEs to humans). One advantage of the cell culture systems, however, is that they generate do novo TSE infectivity. Studies using cell cultures also take into account several cofactors in addition to PrP that might be involved in replication the TSE agent. Although the in vitro systems provide accurate tools to study TSE agent parameters, they mainly or only focus on the molecular processes of PrP conversion. Other factors (i.e., host genetic factors [99]) that, for example, determine the differential uptake of the TSE agent from the environment, might play an additional role in determining the susceptibility of hosts for TSEs and on the transmission of the disease among individuals.

Animals↗