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Sylvain Lehmann

Publications and source records attributed to Sylvain Lehmann.

At least 19 recordsLinked to original sources
PubMed2006

Interest of major serum protein removal for Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) proteomic blood profiling.

BACKGROUND: Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) has been proposed as new approach for blood biomarker discovery. However, results obtained so far have been often disappointing as this technique still has difficulties to detect low-abundant plasma and serum proteins. RESULTS: We used a serum depletion scheme using chicken antibodies against various abundant proteins to realized a pre-fractionation of serum prior to SELDI-TOF profiling. Depletion of major serum proteins by immunocapture was confirmed by 1D and 2D gel electrophoresis. SELDI-TOF analysis of bound and unbound (depleted) serum fractions revealed that this approach allows the detection of new low abundant protein peaks with satisfactory reproducibility. CONCLUSION: The combination of immunocapture and SELDI-TOF analysis opens new avenues into proteomic profiling for the discovery of blood biomarkers.

Journal Article↗
PubMed2006

A novel copper-hydrogen peroxide formulation for prion decontamination.

With the appearance of variant Creutzfeldt-Jakob disease (CJD) and the detection of infectious prions in the peripheral organs of persons with sporadic CJD, the development of decontamination methods that are compatible with medical equipment has become a major issue. Here, we show that a formulation of copper metal ions in combination with hydrogen peroxide dramatically reduces the level of prion protein (PrP)(Sc) (the scrapie isoform of PrP) present in homogenates of samples from prion-infected brains, including brain samples from humans with CJD. An animal bioassay confirmed the reduction in prion infectivity, indicating that this novel Cu(2+)-H(2)O(2) formulation has great potential for prion decontamination.

Animals↗
PubMed2006

The truncated 23-230 form of the prion protein localizes to the nuclei of inducible cell lines independently of its nuclear localization signals and is not cytotoxic.

The mechanisms of prion-induced neurological dysfunction observed in prion diseases are poorly understood. Transgenic mice expressing a truncated form of the prion protein (23-230 PrP) acquire cerebellar degeneration (Ma and Lindquist, Science, 2002). To decipher the mechanisms of neurodegeneration induced by 23-230 PrP, we established inducible cell lines expressing this truncated form of PrP. We found that 23-230 PrP, expected to be cytosolic, accumulated mostly in the nucleus of the cells and was not cytotoxic. Nuclear localization of this mutant form of PrP is independent of its predicted nuclear localization signals. In contrast to what we previously described for PrPSc, nuclear accumulation of 23-230 PrP does not require a functional microtubule network. We observed that 23-230 PrP interacts with chromatin in vivo, as already described for recombinant PrP and for PrPSc. Our data demonstrate that the 23-230 PrP model does not reflect the situation of a cytosolic PrP but could represent a very useful tool to understand the consequences of the accumulation of the prion protein in the nucleus.

Active Transport, Cell Nucleus↗
PubMed2006

Neural stem cell model for prion propagation.

The study of prion transmission and targeting is a major scientific issue with important consequences for public health. Only a few cell culture systems that are able to convert the cellular isoform of the prion protein into the pathologic scrapie isoform of the prion protein (PrP(Sc)) have been described. We hypothesized that central nervous system neural stem cells (NSCs) could be the basis of a new cell culture model permissive to prion infection. Here, we report that monolayers of differentiated fetal NSCs and adult multipotent progenitor cells isolated from mice were able to propagate prions. We also demonstrated the large influence of neural cell fate on the production of PrP(Sc), allowing the molecular study of prion neuronal targeting in relation with strain differences. This new stem cell-based model, which is applicable to different species and to transgenic mice, will allow thoughtful investigations of the molecular basis of prion diseases, and will open new avenues for diagnostic and therapeutic research.

Animals↗
PubMed2006

Retrovirus infection strongly enhances scrapie infectivity release in cell culture.

Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrPSc (conformationally altered isoform of cellular prion protein (PrPC); Sc for scrapie), a partially protease-resistant isoform of the PrPC. PrPSc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrPSc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrPC, PrPSc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.

Animals↗
PubMed2005

The scrapie prion protein is present in flotillin-1-positive vesicles in central- but not peripheral-derived neuronal cell lines.

Abstract Transmissible prion diseases are fatal neurodegenerative diseases associated with the conversion of the normal host prion protein (PrP c) into an abnormal isoform (PrP Sc) that accumulates in brain. This pathology affects neurons of the central nervous system whereas no clear toxic effect has been reported for peripheral neurons. We examined the subcellular distribution of PrP c and PrP Sc in the scrapie-infected mouse neuronal cell lines GT1-7 and N2a, derived, respectively, from the central and peripheral nervous system. We observed that in both cell types, PrP c is present in the endocytic compartment, mainly in LAMP-1-positive late endosomes, but excluded from LYAAT-1-lysosomes. In contrast, PrP Sc was distributed differently in the two cell lines. In infected N2a, PrP Sc and PrP c had comparable distribution patterns. In infected GT1-7, PrP Sc is present in an additional vesicular compartment which is flotillin-1-positive. The level of expression of flotillin-1 is higher in GT1-7 than in N2a cells, but no difference is observed between infected and noninfected cells. In Alzheimer's disease patients, it has been reported that flotillin-1 is abundant in brain areas containing the beta-amyloid protein, which accumulates in endosomal vesicles in primary neurons. We propose that the flotillin compartment could store aggregated proteins and play a role in these neurodegenerative pathologies.

Amino Acid Transport Systems↗
PubMed2005

Biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures.

Abnormal prion protein (PrP(Sc)) plays a central role in the transmission of prion diseases, but the molecular basis of prion strains with distinct biological characteristics remains to be elucidated. We analyzed the characteristics of prion disease by using mice inoculated with the Chandler and Fukuoka-1 strains propagated in a cultured mouse neuronal cell line, GT1-7, which is highly permissive to replication of the infectious agents. Strain-specific biological characteristics, including clinical manifestations, incubation period as related to the infectious unit, and pathological profiles, remained unchanged after passages in the cell cultures. We noted some differences in the biochemical aspects of PrP(Sc) between brain tissues and GT1-7 cells which were unlikely to affect the strain phenotypes. On the other hand, the proteinase K-resistant PrP core fragments derived from Fukuoka-1-infected tissues and cells were slightly larger than those from Chandler-infected versions. Moreover, Fukuoka-1 infection, but not Chandler infection, gave an extra fragment with a low molecular weight, approximately 13 kDa, in both brain tissues and GT1-7 cells. This cell culture model persistently infected with different strains will provide a new insight into the understanding of the molecular basis of prion diversity.

Animals↗
PubMed2005

Prion propagation in cell culture.

During the past two decades, considerable efforts have been made to set up cellular cultures supporting the replication of prions, the infectious agents responsible of transmissible spongiform encephalopathies. As a matter of fact, prion-infected cell lines are very valuable to investigate the cell biology of both the normal and the pathological isoform of the prion protein or to develop and screen new therapeutics. In this chapter, we present a detailed protocol for the generation of prion-infected cells. We also give step-by-step procedures to test the biochemical properties (mainly protease resistance and insolubility) of abnormal PrP molecules, which detection represents a biochemical marker of prion propagation.

Animals↗
PubMed2005

Elevated plasma cysteinylglycine levels caused by cilastatin-associated antibiotic treatment.

Imipenem (thienamycin formamidine), a broad-spectrum beta-lactam antibiotic, is always used in combination with cilastatin in order to avoid the premature breakdown of imipenem by renal tubular dipeptidase. As this dipeptidase also hydrolyzes the glutathione metabolite cysteinylglycine, the therapeutic association of imipenem and cilastatin might cause an accumulation of the aminothiol cysteinylglycine. We demonstrate here that when patients are treated with imipenem-cilastatin, their plasma levels of cysteinylglycine are significantly and specifically increased, while cysteine levels are decreased and homocysteine levels are unaffected. We conclude that antibiotic treatment using imipenem-cilastatin induces important metabolic changes that should not remain unrecognized.

Anti-Bacterial Agents↗
PubMed2005

Protein biochip systems for the clinical laboratory.

Classical methods of protein analysis such as electrophoresis, ELISA and liquid chromatography are generally time-consuming, labor-intensive and lack high-throughput capacity. In addition, all existing methods used to measure proteins necessitate multiple division of the original sample and individual tests carried out for each substance, with an associated cost for each test. The chip system allows several tests to be performed simultaneously without dividing the original patient sample. This system facilitates the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. These emerging technologies fall into two categories: 1) spotted array-based tools, and 2) microfluidic-based tools. Miniaturized and multiplexed immunoassays allow a great deal of information to be obtained from a single sample. These analytical systems are referred to as "lab-on-a-chip" devices. This article presents current trends and advances in miniaturized multiplexed immunoassay technologies, reviewing different systems from research to point-of-care assays. We focus on a subset of chip-based assays that may be used in a clinical laboratory and are directly applicable for biomedical diagnosis. Recent advances in biochip assays combine the power of miniaturization, microfluidics, micro- to nanoparticles, and quantification. A number of applications are just beginning to be explored. The power of biochip assays offers great promise for point-of-care clinical testing and monitoring of many important analytes.

Clinical Laboratory Techniques↗
PubMed2004

Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells.

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.

Animals↗
PubMed2004

Inhibition of PrPSc formation by lentiviral gene transfer of PrP containing dominant negative mutations.

Currently, there is no treatment to cure transmissible spongiform encephalopathies. By taking advantage of the 'prion-resistant' polymorphisms Q171R and E219K that naturally exist in sheep and humans, respectively, we have evaluated a therapeutic approach of lentiviral gene transfer. Here, we show that VSV-G (vesicular stomatitis virus G glycoprotein) pseudotyped FIV-(feline immunodeficiency virus) derived vectors carrying the mouse Prnp gene in which these mutations have been inserted, are able to inhibit prion replication in chronically prion-infected cells. Because lentiviral tools are able to transduce post-mitotic cells such as neurons or cells of the lymphoreticular system, this result might help the development of gene- or cell-therapy approaches to prion disease.

Amyloid↗
PubMed2004

Prion protein protects against DNA damage induced by paraquat in cultured cells.

Exposure of cells to paraquat leads to production of superoxide anion (O2*-). This reacts with hydrogen peroxide to give the hydroxyl radical (*OH), leading to lipid peroxidation and cell death. In this study, we investigated the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using an established model system, rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity.

Animals↗
PubMed2004

Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin.

Prion diseases are fatal transmissible neurodegenerative disorders characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP(C)) denoted PrP(Sc). Recently, wild-type and pathogenic PrP mutants have been shown to be degraded by the endoplasmic reticulum-associated degradation proteasome pathway after translocation into the cytosol. We show here that a protease resistant form of PrP accumulated in the nuclei of prion-infected cells independently of proteasome activity, and that this nuclear translocation required an intact microtubule network. Moreover, our results show for the first time that nuclear PrP interacts with chromatin in vivo, which may have physiopathological consequences in prion diseases

Active Transport, Cell Nucleus↗
PubMed2004

Prion protein suppresses perturbation of cellular copper homeostasis under oxidative conditions.

Prion protein (PrP) binds copper and exhibits superoxide dismutase-like activity, while the roles of PrP in copper homeostasis remain controversial. Using Zeeman graphite furnace atomic absorption spectroscopy, we quantified copper levels in immortalized PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilizes copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilizing cellular copper homeostasis under oxidative conditions.

Animals↗
PubMed2004

Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein.

It is commonly assumed that the physiological isoform of prion protein, PrP(C), is cleaved during its normal processing between residues 111/112, whereas the pathogenic isoform, PrP(Sc), is cleaved at an alternate site in the octapeptide repeat region around position 90. Here we demonstrated both in cultured cells and in vivo, that PrP(C) is subject to a complex set of post-translational processing with the molecule being cleaved upstream of position 111/112, in the octapeptide repeat region or at position 96. PrP has therefore two main cleavage sites that we decided to name alpha and beta. Cleavage of PrP(C) at these sites leads us to re-evaluate the function of both N- and C-terminus fragments thus generated.

Amino Acid Sequence↗
PubMed2004

Cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie.

Over the last 30 years, many drugs have been tested both in cell culture and in vivo for their ability to prevent the generation of prions and the development of transmissible spongiform encephalopathies. Among the compounds tested, dendrimers are defined by their branched and repeating molecular structure. The anti-prion activity of new cationic phosphorus-containing dendrimers (P-dendrimers) with tertiary amine end-groups was tested. These molecules had a strong anti-prion activity, decreasing both PrP(Sc) and infectivity in scrapie-infected cells at non-cytotoxic doses. They can bind PrP and decrease the amount of pre-existing PrP(Sc) from several prion strains, including the BSE strain. More importantly, when tested in a murine scrapie model, the dendrimers were able to decrease PrP(Sc) accumulation in the spleen by more than 80 %. These molecules have a high bio-availability and therefore exhibit relevant potential for prion therapeutics for at least post-exposure prophylaxis.

Animals↗