Protein flotation assay to isolate lipid rafts from soft tissue or cells

on-line protocol: Bio-protocol: http://www.bio-protocol.org/wenzhang.aspx?id=854The arrangement in eukaryotic cell membranes of liquid-ordered states surrounded by liquid-disordered phases allows for the existence of organized membrane microdomains called rafts. Rafts play a crusial role in signal-transduction events, in lipid transport and in various internalization processes, and for all these reasons need to be purified for further characterization

Risk Assessment of Transmission of Sporadic Creutzfeldt-Jakob Disease in Endodontic Practice in Absence of Adequate Prion Inactivation

BACKGROUND: Experimental results evidenced the infectious potential of the dental pulp of animals infected with transmissible spongiform encephalopathies (TSE). This route of iatrogenic transmission of sporadic Creutzfeldt-Jakob disease (sCJD) may exist in humans via reused endodontic instruments if inadequate prion decontamination procedures are used. METHODOLOGY/PRINCIPAL FINDINGS: To assess this risk, 10 critical parameters in the transmission process were identified, starting with contamination of an endodontic file during treatment of an infectious sCJD patient and ending with possible infection of a subsequent susceptible patient. It was assumed that a dose-risk response existed, with no-risk below threshold values. Plausible ranges of those parameters were obtained through literature search and expert opinions, and a sensitivity analysis was conducted. Without effective prion-deactivation procedures, the risk of being infected during endodontic treatment ranged between 3.4 and 13 per million procedures. The probability that more than one case was infected secondary to endodontic treatment of an infected sCJD patient ranged from 47% to 77% depending on the assumed quantity of infective material necessary for disease transmission. If current official recommendations on endodontic instrument decontamination were strictly followed, the risk of secondary infection would become quasi-null. CONCLUSION: The risk of sCJD transmission through endodontic procedure compares with other health care risks of current concern such as death after liver biopsy or during general anaesthesia. These results show that single instrument use or adequate prion-decontamination procedures like those recently implemented in dental practice must be rigorously enforced

Prion Replication in the Hematopoietic Compartment Is Not Required for Neuroinvasion in Scrapie Mouse Model

Fatal neurodegenerative prion diseases are caused by the transmissible PrPSc prion agent whose initial replication after peripheral inoculation takes place in follicular dendritic cells present in germinal centers of lymphoid organs. However, prion replication also occurs in lymphoid cells. To assess the role of the hematopoietic compartment in neuroinvasion and prion replication, we generated chimeric mice, on a uniform congenic C57/BL6J background, by bone marrow replacement with hematopoietic cells expressing different levels of PrP protein. Nine different types of chimeric mice were inoculated intraperitoneally either with the lymphotropic Rocky Mountain Laboratory (RML) strain or the non lymphotropic ME-7 scrapie strain, at different doses. Here, we clearly demonstrate that overexpression of PrP by the hematopoietic system, or the lack of PrP expression by the bone marrow derived cells, does not change the incubation time period of the disease, even when the mice are infected at limiting doses. We conclude that the hematopoietic compartment is more or less permissive to prion replication, both for RML and ME-7, but does not play a role in neuroinvasion

Human FDC express PrPc in vivo and in vitro

Prion diseases are fatal neurodegenerative disorders caused by accumulation of abnormal prion protein (protease-resistant prion, PrPres). PrPres accumulation is also detected in lymphoid organs after peripheral infection. Several studies suggest that follicular dendritic cells (FDC) could be the site of PrPres retention and amplification

Pre-B-cell development in the absence of λ5 in transgenic mice expressing a heavy-chain disease protein

AbstractBackground: Heavy-chain diseases (HCDs) are human lymphoproliferative neoplasias that are characterized by the secretion of truncated immunoglobulin heavy chains devoid of light chains. We have previously proposed — by analogy to the process by which mutated growth factor receptors can be oncogenic — that because the genetic defects in HCDs result in the production of abnormal membrane-associated heavy chains lacking an antigen-binding domain, these abnormal B-cell antigen receptors might engage in ligand-independent signalling. Normal pre-B-cell development requires the presence of the pre-B-cell receptor, formed by the association of μ heavy chains with two polypeptides — so-called surrogate light chains, Vpre-B and λ5 — that are homologous to the variable and constant portions of immunoglobulin light chains, respectively. To assess whether amino-terminal truncation of membrane-associated heavy chains results in their constitutive activation, we have examined the ability of a HCD-associated μ protein to promote pre-B-cell development in transgenic mice.Results When the μ HCD transgene is introduced into SCID mice, CD43− pre-B cells develop normally. To determine whether this pre-B-cell development requires surrogate light chains, we backcrossed mice expressing full-length or truncated μ transgenes with λ5-deficient mice. Our results show that the truncated heavy chain, but not the normal chain, is able to promote pre-B-cell development in the absence of λ5. We also show that truncated μ chains spontaneously aggregate at the surface of bone marrow cells.Conclusion Expression of the truncated μ heavy chain overrides a tightly controlled step of pre-B-cell development, which strongly suggests that a constitutive signal is delivered by the truncated μ chain disease protein. The self-aggregation of μ chain disease proteins might account for this constitutive activation. We conclude that amino-terminal truncation of heavy chains could play a role in the genesis of HCD neoplasia if it occurs at an appropriate stage of B-cell differentiation, namely in a mature B cell

Epidemiological evidence of higher susceptibility to vCJD in the young

BACKGROUND: The strikingly young age of new variant Creutzfeldt-Jacob disease (vCJD) cases remains unexplained. Age dependent susceptibility to infection has been put forward, but differential dietary exposure to contaminated food products in the UK population according to age and sex during the bovine spongiform encephalopathy (BSE) epidemic may provide a simpler explanation. METHODS: Using recently published estimates of dietary exposure in mathematical models of the epidemiology of the new variant Creutzfeldt Jacob disease (vCJD), we examine whether the age characteristics of vCJD cases may be reproduced. RESULTS: The susceptibility/exposure risk function has likely peaked in adolescents and was followed by a sharp decrease with age, evocative of the profile of exposure to bovine material consumption according to age. However, assuming that the risk of contamination was proportional to exposure, with no age dependent susceptibility, the model failed to reproduce the observed age characteristics of the vCJD cases: The predicted cumulated proportion of cases over 40 years was 48%, in strong disagreement with the observed 10%. Incorporating age dependent susceptibility led to a cumulated proportion of cases over 40 years old of 12%. CONCLUSIONS: This analysis provides evidence that differential dietary exposure alone fails to explain the pattern of age in vCJD cases. Decreasing age related susceptibility is required to reproduce the characteristics of the age distribution of vCJD cases

Les cellules de Schwann (rôle dans la propagation des prions et modèle d'étude de la régulation du gène prp bovin)

LILLE1-BU (590092102) / SudocSudocFranceF

Rôle du domaine N-terminal de la PrP dans la pathogenèse des maladies à prions

Le rôle du domaine N-terminal (N-ter) de la protéine PrP dans le processus de conversion de la PrPc en un isoforme infectieux PrPSc est mal connu. L'objectif de ma de thèse a été de créer un prion synthétique composé de la partie N-ter de PrP et de la protéine Doppel (Dpl) pour mieux appréhender le rôle de cette région dans les mécanismes d'agrégation et de pathogenèse des maladies à prions. Trois protéines chimériques PrP/Dpl recombinantes ont été exprimées, purifiées et agrégées in vitro à en oligomères. Ces oligomères solubles présentent des caractéristiques biochimiques et structurales similaires à celles de la PrPSc: riches en feuillets b, résistants à la protéolyse, et formant des structures protofibrillaires. Les propriétés de ces agrégats nous ont conduit à explorer la relation qui existe entre la PrP et la protéine C1q du complément. In vitro, C1q participe de façon coopérative dans le processus d'agrégation et forme un complexe avec des oligomères de petite taille (12-15 mers) qui active la voie classique du complément. L'interaction de C1q avec les oligomères de chimères n'a pas de conséquence fonctionnelle. En parallèle, nous avons créé 10 lignées de souris transgéniques exprimant ces protéines PrP/Dpl, et établies sur fond PrP0/0. Ces protéines chimériques sont exprimées au niveau des rafts, comme la PrP. Les études fonctionnelles et infectieuses sont en cours. L'ensemble de nos données tend à montrer que le domaine N-ter de PrP ne suffit pas à transformer Dpl en un prion synthétique. Par ailleurs, une partie de nos travaux apporte une vision nouvelle quant au rôle de la protéine C1q vis-à-vis d'intermédiaires oligomériques dans les amyloïdoses.The role of the N-terminal (N-ter) domain of PrP protein in the conversion of PrPc into an infectious isoform PrPSc is poorly understood. The objective of my thesis was to create a synthetic prion composed of the N-ter domain of PrP and the Doppel protein to get an insight in the involvement of this PrP domain in the aggregation mechanisms and the pathogenesis of prion diseases. Three PrP/Dpl chimeric recombinant proteins were expressed, purified and aggregated in vitro into oligomers. These soluble chimeric oligomers share structural and biochemical similarities with PrPSc: they are b-sheet-rich, resistant to proteolysis, and tend to form protofibrillar structures. The biochemical properties of these agregates led us to investigate the relationship between PrP and the complement C1q protein. In vitro, C1q seems to participate in the agregation process and forms a complex with small-size oligomers (12-15 mers). This interaction activates the classical pathway of the complement. However, the interaction of C1q with chimeric oligomers does not have any functional relevance. In parallel, we have created 10 transgenic mice lines expressing these chimeric proteins under the endogenous promoter of PrP. These mice lines were backcrossed to a PrP0/0 genetic background. We have shown that the chimeric proteins from cerebral extracts are localized in lipid rafts, as does PrP. Functional and infectious studies are ongoing. Taken together, our data tend to prove that the N-ter domain of PrP is not sufficient to transform Doppel into a synthetic prion. Furthermore, part of our results suggests a new role for C1q in relation with oligomeric intermediates in amyloid diseases.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Étude de l interaction de la protéine prion avec Cbln1, une protéine de la famille C1q

Les Encéphalopathies Spongiformes Transmissibles (EST) sont des maladies neurodégénératives fatales. Elles sont caractérisées par l accumulation dans le système nerveux central de prion, c est-à-dire de la protéine PrPSc (Scrapie, la tremblante du mouton), isoforme anormale de la PrPC (Cellulaire). Cette accumulation est initiée par la formation d oligomères solubles de PrPSc, associés à l infectivité et à la neurotoxicité du prion. A ce jour, il n existe aucun moyen d établir un diagnostic de certitude d EST avant le décès des patients, ni aucun traitement efficace validé. C1q, la molécule de reconnaissance de la voie classique du complément, interagit spécifiquement avec les oligomères de PrPSc via ses têtes globulaires. C1q appartient à la famille C1q. Les protéines de cette famille sont caractérisées par un domaine globulaire carboxy-terminal très conservé, qui est responsable de leurs propriétés de reconnaissance. Nous nous sommes intéressés à Cbln1, une protéine de cette famille, qui est présente, comme PrP, dans les cellules granulaires et de Purkinje du cervelet. Pour la première fois, nous avons montré que Cbln1, purifiée à partir d extrait de culture cellulaire, interagit de façon dose-dépendante avec la PrP recombinante. Cette fixation est mise en évidence avec les oligomères solubles de PrP(8 à 15 sous-unités) alors que nous ne l observons pas avec la protéine monomérique. Nous avons ensuite démontré que Cbln1 influence l étape d oligomérisation de PrP. Ces résultats suggèrent que la fixation des oligomères solubles de PrP par Cbln1 pourrait jouer un rôle dans la physiopathologie des maladies à prion.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

Modelling the epidemic of variant Creutzfeldt-Jakob disease in the UK based on age characteristics: updated, detailed analysis.

Incubation period of the new variant Creutzfeldt-Jakob disease (vCJD) from infection to clinical onset and the eventual impact of the disease remain major concerns. Based on i) epidemiological conceptualization of human exposure to BSE contaminated material, ii) exponentially decreasing susceptibility after 15 years of age, and iii) typical incubation period (IP) distributions for time from infection to onset, we have previously estimated mean incubation period and projected number of vCJD cases. In this paper, we investigate the robustness of these estimates with respect to i-iii using the UK's 113 vCJD cases with clinical onset before December 2000. Mean incubation period was estimated at 16.4 years (95% CI 11.4-24.8), 15.9 years (95% CI 11.4-22.0), 14.1 years (95% CI 10.4-24.2) with the log-normal, Gamma and Weibull distributions respectively. Corresponding predictions for the total size of the epidemic ranged from 183 to 304. Maximal susceptibility to infection between 1.3 and 15.9 years and decreasing by 15% per year of age thereafter yielded the best fit. The shape of the IP distribution did not affect the predictions. In summary, within a set of reasonable assumptions, mean incubation period for vCJD ranged from 15 to 20 years, and the eventual impact of vCJD was a few hundred patients