Prion protein in the cerebrospinal fluid of healthy and naturally scrapie-affected sheep

The aim of this study was to characterize the cerebrospinal fluid (CSF) prion protein (PrP) of healthy and naturally scrapie-affected sheep. The soluble form of CSF PrPC immunoblotted with an anti-octarepeat and an anti-C terminus mAb showed two isoforms of approximately 33 and 26 kDa, corresponding to the biglycosylated and unglycosylated isoforms of brain PrPC. Neither the mean concentration nor the electrophoretic profile of CSF PrP differed between healthy and scrapieaffected sheep, whereas a slightly increased resistance of CSF PrP to mild proteolysis by proteinase K was evident in the CSF of scrapie-affected sheep. No difference in susceptibility to proteolysis was observed between the two ARR and VRQ genetic variants of the purified prokaryote recombinant PrP. It was concluded that the physicochemical properties of PrPC in the CSF could be altered during scrapie and that these changes might reflect the physiopathological process of prion disease

C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle

Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbéliarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes

Nucleoprotein Nanostructures Combined with Adjuvants Adapted to the Neonatal Immune Context: A Candidate Mucosal RSV Vaccine

BACKGROUND: The human respiratory syncytial virus (hRSV) is the leading cause of severe bronchiolitis in infants worldwide. The most severe RSV diseases occur between 2 and 6 months-of-age, so pediatric vaccination will have to be started within the first weeks after birth, when the immune system is prone to Th2 responses that may turn deleterious upon exposure to the virus. So far, the high risk to prime for immunopathological responses in infants has hampered the development of vaccine. In the present study we investigated the safety and efficacy of ring-nanostructures formed by the recombinant nucleoprotein N of hRSV (N(SRS)) as a mucosal vaccine candidate against RSV in BALB/c neonates, which are highly sensitive to immunopathological Th2 imprinting. METHODOLOGY AND PRINCIPAL FINDINGS: A single intranasal administration of N(SRS) with detoxified E. coli enterotoxin LT(R192G) to 5-7 day old neonates provided a significant reduction of the viral load after an RSV challenge at five weeks of age. However, neonatal vaccination also generated an enhanced lung infiltration by neutrophils and eosinophils following the RSV challenge. Analysis of antibody subclasses and cytokines produced after an RSV challenge or a boost administration of the vaccine suggested that neonatal vaccination induced a Th2 biased local immune memory. This Th2 bias and the eosinophilic reaction could be prevented by adding CpG to the vaccine formulation, which, however did not prevent pulmonary inflammation and neutrophil infiltration upon viral challenge. CONCLUSIONS/SIGNIFICANCE: In conclusion, protective vaccination against RSV can be achieved in neonates but requires an appropriate combination of adjuvants to prevent harmful Th2 imprinting

Le centrosome isole: structure et fonctions

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : TD 81140 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Le phénomène Prion, différents aspects d'un nouveau concept en biologie

International audiencePrion diseases are neurodegenerative disorders causing spongiform encephalopthies in mammals. They have the peculiarity of being transmissible and have led to epidemics such as Kuru in human, scrapie in sheep, chronic wasting disease in cervids and mad cow in bovine. This latter has been transmitted to human where it has induced a variant form of the human Creutzfeldt-Jakob disease. Amyloïd deposits of a misfolded protein (PrPSc) due to the conformational change of the host encoded cellular prion protein (PrPC) are features of these diseases. The prion hypothesis has proposed PrPSc to be the infectious agent. Recent arguments in favor of this hypothesis will be reviewed. The puzzling prion strain phenomenon leading to different pathologies and the nature of the infectious particle will also be questioned. The Prion concept, in addition to apply to diseases, has allowed a better understanding of some epigenetics transmissions in fungi. Principle of this concept suggests that different protein conformations may carry and propagate various information opening the way to new investigations on amyloïdosis and their potential to be transmitted. Several examples of Prion-like phenomena not systematically associated with diseases but related to functional amyloïds, sustain a conceptual novelty in biology that will be discussed.Les maladies à prions sont des maladies neurodégénératives responsables d’encéphalopathies spongiformes chez les mammifères, ayant la particularité d’être transmissibles. Elles ont ainsi provoqué des épidémies (Kuru chez l’homme, tremblante du mouton, dépérissement chronique des cervidés, vache folle) et ont conduit à l’émergence d’un variant de la maladie de Creutzfeldt-Jakob après transmission inter-espèce de l’agent bovin à l’homme. Elles sont caractérisées par des dépôts amyloïdes constitués d’une protéine de structure anormale, la PrPSc qui résulte de la conversion de la protéine prion cellulaire (PrPC) de l’hôte et qui, selon l’hypothèse Prion, constituerait l’agent infectieux. Cette revue présente les arguments récents appuyant cette hypothèse. L’intriguant phénomène de souches associées à des pathologies différentes et la caractérisation de la particule infectieuse seront également abordés. Par ailleurs, le concept prion qui dépasse maintenant le cadre de ces maladies, a permis de réévaluer certains événements épigénétiques décrits chez les champignons. Ce concept, en suggérant un principe tel que diverses structures d’une même protéine puissent porter des informations différentes, a permis d’élargir le champ des investigations sur les amyloïdoses et leur transmissibilité potentielle. L’existence de phénomènes de type Prion impliqués dans des fonctions non systématiquement associées à des pathologies, apporte une nouveauté conceptuelle en biologie

Heterogeneity and architecture of pathological prion protein assemblies: time to revisit the molecular basis of the prion replication process?

Prions are proteinaceous infectious agents responsible for a range of neurodegenerative diseases in animals and humans. Prion particles are assemblies formed from a misfolded, β-sheet rich, aggregation-prone isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). Prions replicate by recruiting and converting PrPC into PrPSc, by an autocatalytic process. PrPSc is a pleiomorphic protein as different conformations can dictate different disease phenotypes in the same host species. This is the basis of the strain phenomenon in prion diseases. Recent experimental evidence suggests further structural heterogeneity in PrPSc assemblies within specific prion populations and strains. Still, this diversity is rather seen as a size continuum of assemblies with the same core structure, while analysis of the available experimental data points to the existence of structurally distinct arrangements. The atomic structure of PrPSc has not been elucidated so far, making the prion replication process difficult to understand. All currently available models suggest that PrPSc assemblies exhibit a PrPSc subunit as core constituent, which was recently identified. This review summarizes our current knowledge on prion assembly heterogeneity down to the subunit level and will discuss its importance with regard to the current molecular principles of the prion replication process

Plasminogen-based capture combined with amplification technology for the detection of PrP(TSE) in the pre-clinical phase of infection

Background: Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrPTSE) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrPTSE concentrations in the femtomolar range. [br/] Methodology/Principal Findings: We have developed a three-step assay that firstly captures PrPTSE from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrPTSE detection by western blot. We achieved a PrPTSE capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrPTSE in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrPTSE in human plasma spiked with a 10 28 dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrPTSE in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples. [br/] Conclusion/Significance: We have developed a sensitive and specific amplification assay allowing the detection of PrPTSE in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrPTSE in blood of patients displaying positivity in large scale screening tests