Entre chien et loup, de la crotte à l'ADN : élaboration d une méthode de distinction génétique des deux espèces dans le cadre du retour du loup dans le massif pyrénéen et en France

Par le passé, Le loup a été quasiment éradiqué en Europe avant d être protégé. Aujourd'hui, son retour est amorcé depuis l'Italie et une recolonisation des Pyrénées est imminente. Les attaques de troupeaux sont souvent imputées au loup malgré leur faible effectifs comparé aux chiens. Une distinction efficace de ces espèces est nécessaire du fait des conséquences légales différentes de leur prédation. La distinction morphologique a des limites alors que les approches de génétique sont objectives et irréfutables. La Réserve Naturelle de Nohèdes, souhaitait la mise au point d une méthode de différenciation génétique entre chien et loup à partir des matières fécales, indice de présence très couramment trouvé sur les sites potentiels d occupation. J ai donc élaboré une méthode d extraction de l'ADN mitochondrial à partir des fèces de canidés, et d'analyse des différences nucléotidiques, permettant de distinguer les deux espèces et l'origine géographique du loup

Neurons are MHC Class I-Dependent Targets for CD8 T Cells upon Neurotropic Viral Infection

Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage

Entre chien et loup, de la crotte à l ADN (élaboration d une méthode de distinction génétique des deux espèces dans le cadre du retour du loup dans le massif pyrénéen et en France)

Par le passé, Le loup a été quasiment éradiqué en Europe avant d être protégé. Aujourd hui, son retour est amorcé depuis l Italie et une recolonisation des Pyrénées est imminente. Les attaques de troupeaux sont souvent imputées au loup malgré leur faible effectifs comparé aux chiens. Une distinction efficace de ces espèces est nécessaire du fait des conséquences légales différentes de leur prédation. La distinction morphologique a des limites alors que les approches de génétique sont objectives et irréfutables. La Réserve Naturelle de Nohèdes, souhaitait la mise au point d une méthode de différenciation génétique entre chien et loup à partir des matières fécales, indice de présence très couramment trouvé sur les sites potentiels d occupation. J ai donc élaboré une méthode d extraction de l ADN mitochondrial à partir des fèces de canidés, et d analyse des différences nucléotidiques, permettant de distinguer les deux espèces et l origine géographique du loup.TOULOUSE3-BU Santé-Centrale (315552105) / SudocTOULOUSE-EN Vétérinaire (315552301) / SudocSudocFranceF

Dysfonctionnement neuronal ou inflammation, les deux facettes des conséquences de la persistance du Bornavirus dans le système nerveux central (étude à l'aide d'outils de proteomique et d'immunologie)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

High reproducibility of two-dimensional liquid chromatography using pH-driven fractionation with a pressure-resistant electrode.: High reproducibility of two-dimensional liquid chromatography using pH-driven fractionation with a pressure-resistant electrode.

International audienceAutomated two-dimensional liquid chromatography using the PF2D system from Beckman Coulter provides a fractionation platform well suited for differential proteomic studies. To date, the reliability and reproducibility of PF2D has not been accurately tested. Here, we used an optimized software and a pressure-resistant pH electrode, allowing a precise and reproducible control of the pH limits for each fraction during PF2D. We tested the reliability of this improved system by performing several rounds of fractionation using the same protein extract. Three UV maps were generated, leading to 54 chromatograms and more than 3000 protein peaks. Using semi-automated software for peak-to-peak comparison between 2D-LC chromatograms, we demonstrate that the peak concordance is very high. The rates of concordance were higher in the second dimension repeatability tests, indicating that the limiting factors for 2D-LC reproducibility rely on the pI fractionation and sample preparation steps. The reproducibility between maps was closely related to pH curves similarities, further stressing the need of careful pH adjustment and precise electrode calibration

Tracking antigen-specific CD8+ T cells in the rat using MHC class I multimers.

International audienceStudies of the quantitative and qualitative aspects of anti-microbial, anti-tumoral or autoreactive immune responses have been greatly facilitated by the possibility to stain antigen-specific CD8(+) T cells using fluorescently labeled multimeric major histocompatibility complex (MHC) class I/peptide complexes. So far, this technology has been developed for human and mouse, but not yet in the rat. Here, we describe the generation of the first rat MHC multimer. We produced a rat RT1(l) Pro5 MHC Pentamer combined with the immunodominant peptide for Borna disease virus (BDV), in order to study the characteristics of the antiviral CD8(+) T cell response. BDV is an RNA virus that can cause persistent infections of the central nervous system (CNS), often associated with prominent brain inflammation. In adult Lewis rats, of the RT1(l) MHC haplotype, BDV infection leads to severe immune-mediated neurological symptoms. The pathogenic role of the immune response is due primarily to antiviral CD8(+) T cells, many of them being specific for an immunodominant epitope located in the BDV nucleoprotein (N(230-238)). Ex vivo flow cytometry analyses revealed that 3 to 12% of CD8(+) T cells found in the brains of BDV-infected rats stained positively with the BDV-Pentamer. Interestingly, the frequency of Pentamer-positive cells increased up to 3.3 fold after a short resting period in culture. Virus-specific CD8(+) T cells were mainly detected in the brain and were virtually undetectable in peripheral lymphoid organs. This novel rat Pro5 MHC Pentamer represents an attractive tool for the detection, isolation and characterization of antigen-specific CD8(+) T cell responses in the rat