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ABSTRACT: It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 10³ID₅₀ as measured by intracerebral inoculation of tg338 mice (ID₅₀ IC in tg338). This was consistent with a whole blood titer greater than 10³·⁶ID₅₀ IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation.
PLoS Pathogens 06/2012; 8(6):e1002782. · 9.13 Impact Factor
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Caroline Lacroux, Didier Vilette,
Natalia Fernández-Borges,
Claire Litaise,
Séverine Lugan,
Nathalie Morel,
Fabien Corbière,
Stéphanie Simon,
Hugh Simmons,
Pierrette Costes,
Jean-Louis Weisbecker,
Isabelle Lantier,
Frederic Lantier,
François Schelcher,
Jacques Grassi,
Joaquin Castilla,
Olivier Andréoletti
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ABSTRACT: The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.
Journal of Virology 12/2011; 86(4):2056-66. · 5.40 Impact Factor
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ABSTRACT: Protein misfolding is central to the pathogenesis of several neurodegenerative disorders. Among these disorders, prion diseases are unique because they are transmissible. The conversion of the host-encoded GPI-anchored PrP protein into a structurally altered form is crucially associated with the infectious and neurotoxic properties of the resulting abnormal PrP. Many lines of evidence indicate that distinct aggregated forms with different size and protease resistance are produced during prion multiplication. The recent isolation of various subsets of abnormal PrP, along with the improved biochemical tools and infectivity detection assays have shed light on the diversity of abnormal PrP protein and may give insights into the features of the more infectious subsets of abnormal PrP.
Prion 04/2011; 5(2):84-7. · 2.85 Impact Factor
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ABSTRACT: Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Although large aggregates of proteinase
K-resistant PrP (PrPres) are infectious, the precise relationships between aggregation state and infectivity remain to be established. In this study,
we have fractionated detergent lysates from prion-infected cultured cells by differential ultracentrifugation and ultrafiltration
and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but,
in contrast to typical aggregated PrPres, remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cutoff. Cell-based
assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious
PrPres aggregates can be recovered in the absence of strong in vitro denaturating treatments now gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity
and/or the multiplication cycle of prions.
Journal of Biological Chemistry 03/2011; 286(10):8141-8148. · 4.77 Impact Factor
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ABSTRACT: Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Although large aggregates of proteinase K-resistant PrP (PrP(res)) are infectious, the precise relationships between aggregation state and infectivity remain to be established. In this study, we have fractionated detergent lysates from prion-infected cultured cells by differential ultracentrifugation and ultrafiltration and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but, in contrast to typical aggregated PrP(res), remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cutoff. Cell-based assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious PrP(res) aggregates can be recovered in the absence of strong in vitro denaturating treatments now gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity and/or the multiplication cycle of prions.
Journal of Biological Chemistry 01/2011; 286(10):8141-8. · 4.77 Impact Factor
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ABSTRACT: Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.
PLoS ONE 01/2011; 6(5):e20563. · 4.09 Impact Factor
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ABSTRACT: Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.
Journal of Virology 03/2010; 84(5):2444-52. · 5.40 Impact Factor
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ABSTRACT: Prion-based diseases are incurable transmissible neurodegenerative disorders affecting animals and humans.
Here we report the discovery of the in vivo antiprion activity of Guanabenz (GA), an agonist of alpha2-adrenergic receptors routinely used in human medicine as an antihypertensive drug. We isolated GA in a screen for drugs active in vivo against two different yeast prions using a previously described yeast-based two steps assay. GA was then shown to promote ovine PrP(Sc) clearance in a cell-based assay. These effects are very specific as evidenced by the lack of activity of some GA analogues that we generated. GA antiprion activity does not involve its agonist activity on alpha2-adrenergic receptors as other chemically close anti-hypertensive agents possessing related mechanism of action were found inactive against prions. Finally, GA showed activity in a transgenic mouse-based in vivo assay for ovine prion propagation, prolonging slightly but significantly the survival of treated animals.
GA thus adds to the short list of compounds active in vivo in animal models for the treatment of prion-based diseases. Because it has been administrated for many years to treat hypertension on a daily basis, without major side-effects, our results suggest that it could be evaluated in human as a potential treatment for prion-based diseases.
PLoS ONE 02/2008; 3(4):e1981. · 4.09 Impact Factor
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Déborah Tribouillard-Tanvier,
Suzana Dos Reis,
Fabienne Gug,
Cécile Voisset,
Vincent Béringue,
Raimon Sabate,
Ema Kikovska,
Nicolas Talarek,
Stéphane Bach,
Chenhui Huang,
Nathalie Desban,
Sven J Saupe,
Surachai Supattapone,
Jean-Yves Thuret,
Stéphane Chédin, Didier Vilette,
Hervé Galons,
Suparna Sanyal,
Marc Blondel
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ABSTRACT: 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases.
Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome.
6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.
PLoS ONE 02/2008; 3(5):e2174. · 4.09 Impact Factor
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ABSTRACT: We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface.
Journal of Virology 11/2007; 81(19):10786-91. · 5.40 Impact Factor
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ABSTRACT: Despite circumstantial evidence that prions can be found extracellularly or at the surface of infected cells, little is known about how these infectious agents spread from cell to cell. In order to gain better insight into this critical issue, this study used two different cell lines (neuroglial MovS and epithelial Rov cells) that have previously been shown to be permissive for ovine prion multiplication. Co-culture of infected cells and uninfected target cells at a ratio of 1 : 9 resulted in total infection of MovS cells within 10 days but not of Rov cell cultures, suggesting that the efficiency of prion dissemination may vary greatly depending on the type of permissive cell. Analysis of the spatial distribution of the newly infected cells revealed that, although long-range spread could also occur, cells proximal to the infected donor cells consistently accumulated more abnormal PrP, consistent with preferential infection of nearby cells. This experimental approach, focused on dissemination among living cells, could help in the analysis of mechanisms involved in the cell-to-cell spread of prion infections.
Journal of General Virology 03/2007; 88(Pt 2):706-13. · 3.36 Impact Factor
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Déborah Tribouillard,
Stéphane Bach,
Fabienne Gug,
Nathalie Desban,
Vincent Beringue,
Thibault Andrieu,
Dominique Dormont,
Hervé Galons,
Hubert Laude, Didier Vilette,
Marc Blondel
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ABSTRACT: Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.
Biotechnology Journal 02/2006; 1(1):58-67.
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ABSTRACT: It is well established that natural polymorphisms in the coding sequence of the PrP protein can control the expression of prion disease. Studies with a cell model of sheep prion infection have shown that ovine PrP allele associated with resistance to sheep scrapie may confer resistance by impairing the multiplication of the infectious agent. To further explore the biochemical and cellular mechanisms underlying the genetic control of scrapie susceptibility, we established permissive cells expressing two different PrP variants. In this study, we show that PrP variants with opposite effects on prion multiplication exhibit distinct cell biological features. These findings indicate that cell biological properties of ovine PrP can vary with natural polymorphisms and raise the possibility that differential interactions of PrP variants with the cellular machinery may contribute to permissiveness or resistance to prion multiplication.
Biochemical and Biophysical Research Communications 12/2005; 337(3):791-8. · 2.48 Impact Factor
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ABSTRACT: Using permissive cell lines of epithelial or neuroglial origin, we found that scratch-wounding a small proportion of the recipient cells prior to prion exposure strongly reduced the cell culture's susceptibility to infection. We provide evidence suggesting that wound-triggered inhibition of prion infection was mediated by the release of nucleotides in the extracellular medium of injured cultures. While cell wounding or ATP treatment of unwounded target cells inhibited de novo infection, we found that they had no effect on steady-state infected cultures, indicating that these treatments affect the early stages of infection. These findings support the view that cells have the capacity to modulate their permissiveness to prion infection in response to external stimuli, such as a signalling molecule.
Biochemical and Biophysical Research Communications 05/2005; 330(1):5-10. · 2.48 Impact Factor
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ABSTRACT: Prion diseases are transmissible neurodegenerative disorders affecting humans and animals for which no therapeutic or prophylactic regimens exist. During the last three years several studies have shown that anti-PrP monoclonal antibodies (mAbs) can antagonize prion propagation in vitro and in vivo, but the mechanisms of inhibition are not known so far. To identify the most powerful mAbs and characterize more precisely the therapeutic effect of anti-PrP antibodies, we have screened 145 different mAbs produced in our laboratory for their capacity to cure cells constitutively expressing PrPSc. Our results confirm for a very large series of antibodies that mAbs recognizing cell-surface native PrPc can efficiently clean and definitively cure infected cells. Antibodies having a cleaning effect are directed against linear epitopes located in at least four different regions of PrP, suggesting an epitope-independent inhibition mechanism. The consequence of antibody binding is the sequestration of PrPc at the cell surface, an increase of PrPc levels recovered in cell culture medium, and an internalization of antibodies. Taken together these data suggest that the cleaning process is more likely due to a global effect on the PrP trafficking and/or transconformation process. Two antibodies, Sha31 and BAR236, show an IC50 of 0.6 nM, thus appearing 10-fold more efficient than previous antibodies described in the literature. Finally, five co-treatments were also tested, and only one of them, described previously (SAF34 + SAF61), lowered PrPSc levels in the cells synergistically.
Journal of Biological Chemistry 04/2005; 280(12):11247-58. · 4.77 Impact Factor
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Medecine sciences: M/S 03/2005; 21(2):132-3. · 0.64 Impact Factor
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ABSTRACT: In certain cell types, endosomal multivesicular bodies may fuse with the cell surface in an exocytic manner. During this process, the small 50-90-nm-diameter vesicles contained in their lumen are released into the extracellular environment. The released vesicles are called exosomes. Exosome secretion can be used by cells to eject molecules targeted to intraluminal vesicles of multivesicular bodies, but particular cell types exploit exosomes as intercellular communication devices for transfer of proteins and lipids between cells. The molecular composition of exosomes is determined by sorting events within endosomes that occur concomitantly with the generation of intraluminal vesicles. As other raft-associated components, the glycosylphosphatidylinositol-linked prion protein transits through multivesicular bodies. Recent findings in non-neuronal cell models indicate prion protein association with secreted exosomes. Thus, exosomes could constitute vehicles for transmission of the infectious prion protein, bypassing cell-cell contact in the dissemination of prions.
Traffic 02/2005; 6(1):10-7. · 4.92 Impact Factor
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Vincent Beringue, Didier Vilette,
Gary Mallinson,
Fabienne Archer,
Maria Kaisar,
Mourad Tayebi,
Graham S Jackson,
Anthony R Clarke,
Hubert Laude,
John Collinge,
Simon Hawke
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ABSTRACT: Conversion of the cellular alpha-helical prion protein (PrP(C)) into a disease-associated isoform (PrP(Sc)) is central to the pathogenesis of prion diseases. Molecules targeting either normal or disease-associated isoforms may be of therapeutic interest, and the antibodies binding PrP(C) have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrP(Sc) inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrP(C) were relatively inefficient inhibitors of ScRov cell PrP(Sc) accumulation compared with antibodies that additionally targeted disease-associated PrP isoforms. Although the mechanism by which these monoclonal antibodies inhibit prion replication is unclear, some of the data suggest that antibodies might actively increase PrP(Sc) turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents.
Journal of Biological Chemistry 10/2004; 279(38):39671-6. · 4.77 Impact Factor
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ABSTRACT: During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further support for a crucial role of PrP at the cell surface in prion infection of target cells.
Journal of Virology 08/2004; 78(13):7148-52. · 5.40 Impact Factor
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ABSTRACT: Prion diseases are infectious neurodegenerative disorders linked to the accumulation in the central nervous system of the abnormally folded prion protein (PrP) scrapie (PrPsc), which is thought to be the infectious agent. Once present, PrPsc catalyzes the conversion of naturally occurring cellular PrP (PrPc) to PrPsc. Prion infection is usually initiated in peripheral organs, but the mechanisms involved in infectious spread to the brain are unclear. We found that both PrPc and PrPsc were actively released into the extracellular environment by PrP-expressing cells before and after infection with sheep prions, respectively. Based on Western blot with specific markers, MS, and morphological analysis, our data revealed that PrPc and PrPsc in the medium are associated with exosomes, membranous vesicles that are secreted upon fusion of multivesicular endosomes with the plasma membrane. Furthermore, we found that exosomes bearing PrPsc are infectious. Our data suggest that exosomes may contribute to intercellular membrane exchange and the spread of prions throughout the organism.
Proceedings of the National Academy of Sciences 07/2004; 101(26):9683-8. · 9.68 Impact Factor
