scholarly journals Detection of bovine spongiform encephalopathy, ovine scrapie prion-related protein (PrPSc) and normal PrPc by monoclonal antibodies raised to copper-refolded prion protein

2003 ◽  
Vol 370 (1) ◽  
pp. 81-90 ◽  
Author(s):  
Alana M. THACKRAY ◽  
Jean-Yves MADEC ◽  
Edmond WONG ◽  
Robert MORGAN-WARREN ◽  
David R. BROWN ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Bovine Spongiform Encephalopathy ◽  
Surface Protein ◽  
Lymphoid Cells ◽  
Spongiform Encephalopathy ◽  
Related Protein ◽  
Allelic Form ◽  
Tissue Samples ◽  
Electrophoretic Mobilities

Prion-related protein (PrP) is a glycosylphosphatidylinositol-linked cell-surface protein expressed by a wide variety of cells, including those of the nervous system and the immune system. Several functions of normal cellular PrP (PrPc) have been proposed that may be associated with the capacity of this protein to bind copper. In the present study, we describe the generation of a panel of monoclonal antibodies raised to copper-refolded PrP, which may be used to analyse the normal and disease-associated forms of this protein. The anti-PrP monoclonal antibodies were reactive by Western blot and ELISA with recombinant murine PrPc refolded in the presence or absence of either copper or manganese, and with the disease-susceptible allelic form V136R154Q171 ('VRQ'; where single-letter amino-acid notation has been used) and disease-resistant allelic form A136R154R171 ('ARR') of recombinant ovine PrPc. FACS analysis of lymphoid cells using these monoclonal antibodies showed that wild-type non-activated mouse lymphocytes expressed little, if any, PrPc. These monoclonal antibodies were shown to react with the unglycosylated and monoglycosylated forms of PrPSc (abnormal disease-specific conformation of PrP) in prion-infected tissue samples from all of the different species tested by Western blot. In addition, this analysis allowed one to make a distinction between bovine spongiform encephalopathy ('BSE') and scrapie PrPSc isolates from experimentally infected sheep on the basis of their different electrophoretic mobilities.

scholarly journals A Comparison of Rapid Bovine Spongiform Encephalopathy Testing Methods on Autolyzed Bovine Brain Tissue

2005 ◽  
Vol 17 (2) ◽  
pp. 99-102 ◽  
Author(s):  
Angus Wear ◽  
Kirstine Henderson ◽  
Kath Webster ◽  
Indu Patel
Keyword(s):  
Western Blot ◽  
Bovine Spongiform Encephalopathy ◽  
Bovine Brain ◽  
Spongiform Encephalopathy ◽  
The European Union ◽  
Routine Testing ◽  
New Methods ◽  
Surveillance Programs ◽  
The Eu ◽  
Do So

In 1999, the European Union (EU) approved 3 rapid methods for the testing of bovine brain samples for the presence of bovine spongiform encephalopathy (BSE). The evaluation that led to the approval did not include an analysis of autolyzed material. Member states of the EU have active surveillance programs for BSE, which target fallen stock as well as other categories of cattle. Autolysis is a common feature of fallen stock samples because there can be a considerable delay between death and collection of samples. Therefore, it is important to know whether these tests perform optimally on autolyzed samples. The Veterinary Laboratories Agency (VLA) selected 250 positive fallen stock samples. These had been detected during routine testing using the Prionics®-Check Western blot and confirmed as BSE cases by immunohistochemistry or electron microscopy. Samples were graded according to the degree of autolysis and then tested by the 3 methods: Prionics®-Check Western blot, Platelia test, and Enfer test. All 3 methods correctly classified the samples as positive BSE cases, therefore alleviating doubt about their ability to do so. Subsequent EU validation exercises, such as those conducted in 2002–2003, have included the testing of autolyzed material. It is important that all new methods be evaluated on autolyzed tissue before approval for official use.

scholarly journals Resistance of Domestic Cats to a US Sheep Scrapie Agent by Intracerebral Route

2002 ◽  
Vol 14 (5) ◽  
pp. 444-445 ◽  
Author(s):  
Amir N. Hamir ◽  
Wilber W. Clark ◽  
Diane L. Sutton ◽  
Janice M. Miller ◽  
Mick J. Stack ◽  
...  
Keyword(s):  
Western Blot ◽  
Bovine Spongiform Encephalopathy ◽  
Extended Period ◽  
Spongiform Encephalopathy ◽  
Neurologic Disease ◽  
Domestic Cats ◽  
Species Barrier ◽  
Scrapie Agent ◽  
Abnormal Form ◽  
Sheep Scrapie

Feline spongiform encephalopathy (FSE) is thought to have resulted from consumption of food contaminated with bovine spongiform encephalopathy and the latter is believed to result from the consumption of food contaminated with scrapie. However, no direct experimental documentation exists to indicate that the scrapie agent is capable of amplifying in cats, and, therefore, crossing the species barrier. During 1979, 6 cats ranging in age from 3.5 to 18 months were intracerebrally inoculated with sheep scrapie (inoculum G-639-PP) and were observed for an extended period. Inoculated cats did not develop neurologic disease, and microscopic lesions of spongiform encephalopathy were not evident. Immunohistochemistry and Western blot techniques failed to detect the abnormal form of prion protein (PrPres). These results indicate that the sheep scrapie agent (G-639-PP) used in this study was not capable of amplifying in cats and therefore was unable to cross the species barrier to produce FSE.

scholarly journals Infectious titres of sheep scrapie and bovine spongiform encephalopathy agents cannot be accurately predicted from quantitative laboratory test results

2012 ◽  
Vol 93 (11) ◽  
pp. 2518-2527 ◽  
Author(s):  
Lorenzo González ◽  
Leigh Thorne ◽  
Martin Jeffrey ◽  
Stuart Martin ◽  
John Spiropoulos ◽  
...  
Keyword(s):  
Western Blot ◽  
Bovine Spongiform Encephalopathy ◽  
Surrogate Marker ◽  
Inverse Correlation ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Test Results ◽  
Biochemical Tests ◽  
Spongiform Encephalopathies ◽  
Laboratory Test Results

It is widely accepted that abnormal forms of the prion protein (PrP) are the best surrogate marker for the infectious agent of prion diseases and, in practice, the detection of such disease-associated (PrPd) and/or protease-resistant (PrPres) forms of PrP is the cornerstone of diagnosis and surveillance of the transmissible spongiform encephalopathies (TSEs). Nevertheless, some studies question the consistent association between infectivity and abnormal PrP detection. To address this discrepancy, 11 brain samples of sheep affected with natural scrapie or experimental bovine spongiform encephalopathy were selected on the basis of the magnitude and predominant types of PrPd accumulation, as shown by immunohistochemical (IHC) examination; contra-lateral hemi-brain samples were inoculated at three different dilutions into transgenic mice overexpressing ovine PrP and were also subjected to quantitative analysis by three biochemical tests (BCTs). Six samples gave ‘low’ infectious titres (106.5 to 106.7 LD50 g−1) and five gave ‘high titres’ (108.1 to ≥108.7 LD50 g−1) and, with the exception of the Western blot analysis, those two groups tended to correspond with samples with lower PrPd/PrPres results by IHC/BCTs. However, no statistical association could be confirmed due to high individual sample variability. It is concluded that although detection of abnormal forms of PrP by laboratory methods remains useful to confirm TSE infection, infectivity titres cannot be predicted from quantitative test results, at least for the TSE sources and host PRNP genotypes used in this study. Furthermore, the near inverse correlation between infectious titres and Western blot results (high protease pre-treatment) argues for a dissociation between infectivity and PrPres.

Retrospective Neuropathological Review of Prion Disease in UK Haemophilic Patients

1998 ◽  
Vol 80 (12) ◽  
pp. 909-911 ◽  
Author(s):  
James Ironside ◽  
Jeanne Bell ◽  
Paul Giangrande ◽  
Christopher Ludlam ◽  
Margaret M. Esiri ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
Prion Disease ◽  
Clotting Factor ◽  
Spongiform Encephalopathy ◽  
Post Mortem ◽  
Blood Products ◽  
Factor Concentrate ◽  
Donor Source

SummaryIn 1996, the CJD surveillance unit in Edinburgh, UK described nvCJD which was thought to be the human equivalent of bovine spongiform encephalopathy (BSE). The identification of prion protein in the tonsil of an affected individual has raised the question of transmission of nvCJD via blood products. This study examines the post mortem brains of 33 patients who were treated with clotting factor concentrate of predominately UK donor source during the years 1962-1995. The brains were examined by conventional histological methods and also for the prion protein using monoclonal antibodies KG9 and 3F4. No evidence of spongiform encephalopathy was found and the immunocytochemistry was negative for PrP in all cases. It is concluded that, at present, there is no evidence for the transmission of nvCJD via clotting factor concentrate to patients with haemophilia.

scholarly journals Properties of L-Type Bovine Spongiform Encephalopathy in Intraspecies Passages

2011 ◽  
Vol 49 (5) ◽  
pp. 819-823 ◽  
Author(s):  
H. Okada ◽  
Y. Iwamaru ◽  
M. Kakizaki ◽  
K. Masujin ◽  
M. Imamura ◽  
...  
Keyword(s):  
Western Blot ◽  
Bovine Spongiform Encephalopathy ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Brain Homogenate ◽  
Spongiform Encephalopathy ◽  
Biochemical Characteristics ◽  
Atypical Bovine Spongiform Encephalopathy ◽  
Single Host ◽  
The Brain

The origin and transmission routes of atypical bovine spongiform encephalopathy (BSE) remain unclear. To assess whether the biological and biochemical characteristics of atypical L-type BSE detected in Japanese cattle (BSE/JP24) are conserved during serial passages within a single host, 3 calves were inoculated intracerebrally with a brain homogenate prepared from first-passaged BSE/JP24-affected cattle. Detailed immunohistochemical and neuropathologic analysis of the brains of second-passaged animals, which had developed the disease and survived for an average of 16 months after inoculation, revealed distribution of spongiform changes and disease-associated prion protein (PrPSc) throughout the brain. Although immunolabeled PrPSc obtained from brain tissue was characterized by the presence of PrP plaques and diffuse synaptic granular accumulations, no stellate-type deposits were detected. Western blot analysis suggested no obvious differences in PrPSc molecular mass or glycoform pattern in the brains of first- and second-passaged cattle. These findings suggest failures to identify differences in mean incubation period and biochemical and neuropathologic properties of the BSE/JP24 prion between the first and second passages in cattle.

scholarly journals Molecular Analysis of the Abnormal Prion Protein during Coinfection of Mice by Bovine Spongiform Encephalopathy and a Scrapie Agent

2001 ◽  
Vol 75 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Thierry G. M. Baron ◽  
Anne-Gaelle Biacabe
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
Proteinase K ◽  
Spongiform Encephalopathy ◽  
Molecular Features ◽  
Electrophoretic Mobilities ◽  
Scrapie Agent ◽  
Scrapie Strain ◽  
Sheep Scrapie

ABSTRACT Molecular features of the proteinase K-resistant prion protein (PrP res) may discriminate among prion strains, and a specific signature could be found during infection by the infectious agent causing bovine spongiform encephalopathy (BSE). To investigate the molecular basis of BSE adaptation and selection, we established a model of coinfection of mice by both BSE and a sheep scrapie strain (C506M3). We now show that the PrP res features in these mice, characterized by glycoform ratios and electrophoretic mobilities, may be undistinguishable from those found in mice infected with scrapie only, including when mice were inoculated by both strains at the same time and by the same intracerebral inoculation route. Western blot analysis using different antibodies against sequences near the putative N-terminal end of PrP res also demonstrated differences in the main proteinase K cleavage sites between mice showing either the BSE or scrapie PrP res profile. These results, which may be linked to higher levels of PrP res associated with infection by scrapie, were similar following a challenge by a higher dose of the BSE agent during coinfection by both strains intracerebrally. Whereas PrP res extraction methods used allowed us to distinguish type 1 and type 2 PrP res, differing, like BSE and scrapie, by their electrophoretic mobilities, in the same brain region of some patients with Creutzfeldt-Jakob disease, analysis of in vitro mixtures of BSE and scrapie brain homogenates did not allow us to distinguish BSE and scrapie PrP res. These results suggest that the BSE agent, the origin of which remains unknown so far but which may have arisen from a sheep scrapie agent, may be hidden by a scrapie strain during attempts to identify it by molecular studies and following transmission of the disease in mice.

Lymphoid signal transduction mechanisms linked to cellular prion protein

2005 ◽  
Vol 83 (5) ◽  
pp. 644-653 ◽  
Author(s):  
I E Mazzoni ◽  
H C Ledebur, Jr. ◽  
E Paramithiotis ◽  
N Cashman
Keyword(s):  
Protein Kinase ◽  
Prion Protein ◽  
Surface Protein ◽  
Cellular Prion Protein ◽  
Lymphoid Cells ◽  
Mitogen Activated Protein Kinase ◽  
Spongiform Encephalopathy ◽  
Wild Type ◽  
Con A ◽  
Calcium Dependent

The normal cellular isoform of the prion protein (PrPC) is a glycosylphosphatidylinositol-anchored cell surface protein that is expressed widely, including in lymphoid cells. We compared lectin-induced mitogenesis and selected cell signaling pathways in splenocytes from wild-type BALB/c mice and Zrch Prnp0/0(PrP0/0) mice bred on a BALB/c background for more than 10 generations.3H-thymidine incorporation induced by concanavalin A (Con A) or phytohemagglutinin (PHA) was significantly reduced in PrP0/0splenocytes, most prominently early in activation (24 and 48 h). Con A activation in PrP0/0splenocytes was associated with differences in the phosphorylation (P) patterns of protein kinase C (PKC α/β, but not δ) and the PKC downstream effectors p44/42MAPK (mitogen-activated protein kinase). P-PKC and P-MAPK profiles were similar in wild-type and PrP0/0splenocytes following PMA treatment, indicating that the ability of these 2 enzymes to be phosphorylated is not impaired in the absence of PrPC. Con A-induced calcium fluxes, monitored by indo-1 fluorescence, were equivalent in PrP0/0and PrP+/+splenocytes, suggesting that calcium-dependent mechanisms are not directly implicated in the differential phosphorylation patterns or mitotic responses. Our data indicate that PrP0/0splenocytes display defects in upstream or downstream mechanism(s) that modulate PKCα/β phosphorylation, which in turn affects its capacity to regulate splenocyte mitosis, consistent with a role for PrPCin immune function.Key words: PKC, MAPK, mitosis, bovine spongiform encephalopathy, Creutzfeldt-Jacob disease.

scholarly journals Detection of PrPSc in peripheral tissues of clinically affected cattle after oral challenge with bovine spongiform encephalopathy

2012 ◽  
Vol 93 (12) ◽  
pp. 2740-2748 ◽  
Author(s):  
Martin Franz ◽  
Martin Eiden ◽  
Anne Balkema-Buschmann ◽  
Justin Greenlee ◽  
Hermann Schatzl ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Protein Misfolding ◽  
Brain Homogenate ◽  
Diagnostic Methods ◽  
Spongiform Encephalopathy ◽  
Variant Form ◽  
Mesenteric Lymph Nodes ◽  
Tissue Samples ◽  
Peripheral Tissues ◽  
Resistant Disease

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease that mainly affects cattle. Transmission of BSE to humans caused a variant form of Creutzfeldt–Jakob disease. Following infection, the protease-resistant, disease-associated isoform of prion protein (PrPSc) accumulates in the central nervous system and in other tissues. Many countries have defined bovine tissues that may contain prions as specified risk materials, which must not enter the human or animal food chains and therefore must be discarded. Ultrasensitive techniques such as protein misfolding cyclic amplification (PMCA) have been developed to detect PrPSc when present in minuscule amounts that are not readily detected by other diagnostic methods such as immunohistochemistry or Western blotting. This study was conducted to determine when and where PrPSc can be found by PMCA in cattle orally challenged with BSE. A total of 48 different tissue samples from four cattle infected orally with BSE at various clinical stages of disease were examined using a standardized PMCA protocol. The protocol used brain homogenate from bovine PrP transgenic mice (Tgbov XV) as substrate and three consecutive rounds of PMCA. Using this protocol, PrPSc was found in the brain, spinal cord, nerve ganglia, optic nerve and Peyer’s patches. The presence of PrPSc was confirmed in adrenal glands, as well as in mesenteric lymph nodes – a finding that was reported recently by another group. Interestingly, additional positive results were obtained for the first time in the oesophagus, abomasum, rumen and rectum of clinically affected cattle.

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