NF-κB1 Inhibits TLR-Induced IFN-β Production in Macrophages Through TPL-2-dependent ERK Activation

available in PMC 2012 February 15.Although NF-κB1 p50/p105 has critical roles in immunity, the mechanism by which NF-κB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1−/− macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-β expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-β production. Markedly higher serum levels of IFN-β were observed in Nfkb1−/− mice than in wild-type mice following LPS injection, demonstrating that Nfkb1 inhibits IFN-β production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabilized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-β production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb1−/− macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-β secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb1−/− macrophages, which rescued LPS activation of ERK, also inhibited IFN-β expression. These data indicate that p50/p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of ERK.National Institutes of Health (U.S.) (NIH AI52267)National Institutes of Health (U.S.) (NIH CA108854)National Institutes of Health (U.S.) (NIH CA67529)Medical Research Council (Great Britain

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37 °C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories

Rapid detection of potyviruses from crude plant extracts

Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA). The developed method, named ‘Direct RT-RPA’, detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation. The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide

Ecoulements doublement diffusifs hors de l'approximation de Boussinesq (cas particulier de la convection dans le manteau terrestre)

PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Linear stability of a double diffusive layer of an infinite prandtl number fluid with temperature-dependent viscosity

Studia Geophysica et Geodaetica, v. 48, n. 3, p. 519-537, 2004. http://dx.doi.org/10.1023/B:SGEG.0000037470.80659.e5International audienc

Etude des interactions entre le polynucléaire neutrophile et l endothélium (rôle de la leucosialine (CD43) et de l intégrine a9b1 (alpha9beta1))

Lors d une infection, les polynucléaires neutrophiles patrouillent dans tout l organisme et sont les premières cellules à affluer au site inflammatoire. La leucosialine (CD43) est la principale sialoglycoprotéine du neutrophile. CD43 est clivée après activation et adhérence des neutrophiles. J ai démontré que CD43 était clivée et que la conséquence de ce clivage est la libération de son domaine extracellulaire qui inhibe l adhérence des neutrophiles en conditions de flux et la libération du domaine intracellulaire par le complexe g-secrétase. Par ailleurs, le rôle des intégrines b1 dans la migration des neutrophiles est encore peu claire. L intégrine a9b1 est présent sur les neutrophiles humains et lie les molécules de VCAM-1 sur l endothélium. J ai décrit une synergie entre les intégrines b2 et l intégrine a9b1 dans l adhérence des neutrophiles aux cellules endothéliales en conditions de flux et une sur-expression de l intégrine après activation et migration des neutrophilesPARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Increased membrane expression of proteinase 3 during neutrophil adhesion in the presence of anti proteinase 3 antibodies

We investigated membrane proteinase 3 (mPR3) expression during TNF-alpha-induced adhesion of neutrophils in the presence of anti-PR3 antibodies, a situation occurring during anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis. Three increasing levels of mPR3 expression were observed on the mPR3(+) neutrophil subset after stepwise cell activation. TNF-alpha activation without adhesion, TNF-alpha-induced adhesion, and adhesion in the presence of anti-PR3 mAb or human anti-PR3 ANCA resulted, respectively, in a two-, seven-, and 24-fold increase of mPR3 levels. In plasma, anti-PR3 antibodies poorly recognized suspended neutrophils, whereas they bound to mPR3 on adherent cells. mPR3 upregulation was also triggered by IL-8, formyl-methionyl-leucyl-phenylalanine (fMLP), and neutrophil adhesion to activated human umbilical vein endothelial cells. It involved beta2 integrins and Fcgamma receptor, because it was prevented by anti-CD18 antibodies and was not observed with anti-PR3 F(ab')(2). Furthermore, it was specific to anti-PR3 mAb, and no mPR3 upregulation was observed with anti-myeloperoxidase or anti-HLA-ABC mAb. Newly expressed mPR3 molecules, after TNF-induced adhesion, were mobilized from secretory vesicles (CD35(+)) and secondary granules (CD11b(+)). The adhesion- and antibody-dependent upregulations of mPR3 expression occurred with little azurophilic granule degranulation, no sign of apoptosis, and no further CD177 upregulation. In conclusion, this study describes an amplifying loop in polymorphonuclear neutrophil activation process, whereby ANCA are involved in the membrane expression of their own antigen during cell adhesion. This could explain the restriction of ANCA-associated vasculitis to small vessels, the main site of neutrophil adhesion

Caractérisation moléculaire du Yam virus X (YVX) et du Yam necrosis virus (YNV), deux nouveaux virus des genres Potexvirus et Sadwavirus infectant les ignames en Guadeloupe, et mise au point d’outils de diagnostic

Les ignames (Dioscorea spp.) sont des ressources alimentaires économiquement et socialement importantes en Afrique, dans la Caraïbe, en Amérique du Sud et dans les îles du Pacifique. Les espèces cultivées hébergent de nombreux virus des genres potexvirus, carlavirus, badnavirus, cucumovirus, macluravirus et potyvirus, qui constituent la principale contrainte pour la conservation et la diffusion de ressources génétiques. Deux nouveaux virus appartenant aux genres potexvirus et sadwavirus, baptisés respectivement Yam virus X (YVX) et Yam necrosis virus (YNV), ont été mis en évidence par analyses in silico d’EST d’ignames. La partie 3’ du génome de chacun de ces virus a été amplifiée par 3’RACE à partir de plantes infectées et présentant des symptômes de nécrose foliaire récoltées en Guadeloupe, clonée puis séquencée. Les séquences obtenues ont permis d’établir les relations phylogéniques de ces espèces virales au sein des genres potexvirus et sadwavirus. Des amorces spécifiques de chacun de ces deux virus ont été dessinées dans la région codant l’ARN polymérase ARN dépendante (RdRp) et utilisées pour la mise au point de tests de détection moléculaire par direct binding reverse transcription PCR (DB-RT-PCR) dans des broyats de feuilles. Ces outils complètent l’arsenal disponible pour le diagnostic des virus des ignames et permettent d’entreprendre des études de prévalence du YVX et du YNV en Guadeloupe

Characterization and diagnostic of Yam virus X (YVX) and Yam necrosis virus (YNV), two novel viruses infecting yams in Guadeloupe

Several viral species infecting cultivated yams (Dioscorea spp.) are known. They include viruses belonging to the families Alphaflexiviridae (genus Potexvirus), Betaflexiviridae (genus Carlavirus), Caulimoviridae (genus Badnavirus), Cucumoviridae (genus Cucumovirus) and Potyviridae (genera Macluravirus and Potyvirus). However, it is widely acknowledged that yet uncharacterized viral species are present in yam germplasm collections worldwide and could be propagated through the distribution of infected germplasm. Therefore, viruses are currently the major constraint for much needed exchanges and distribution of yam germplasm. In order to promote the safe exchange of yam germplasm conserved in the Guadeloupe Biological Resources Center of Tropical Plants (CRB-PT), searches for new virus species in yams were undertaken. In silico analyses of ESTs of Dioscorea alata were performed and unveiled the existence of sequences corresponding to several known genera of yam viruses, such as Badnavirus and Macluravirus, and also to families of unknown yam-associated viruses, including Geminiviridae and Secoviridae. RT-PCR were performed on crude extracts of symptomatic yams (D. alata, D. trifida) following direct binding of viral particles and using degenerate primers. Amplification products were cloned and sequenced. Some of them displayed significant levels of homology with potexviruses and with viruses of the family Secoviridae. The 3' ends of the corresponding viral genomes were successfully amplified by 3' RACE, cloned and sequenced. Phylogenetic analyses confirmed the existence in yams of one new viral species within the genus Potexvirus (tentatively named Yam virus X, YVX) and one within the family Secoviridae (tentatively named Yam necrosis virus, YNV). The experimental host range of both viruses was explored through mechanical inoculation on various herbaceous plants. Molecular diagnostic was developed for both YVX and YNV using direct binding reverse transcription PCR (DB-RT-PCR) and used to perform a prevalence study of both viruses in the Guadeloupe CRB-PT yam germplasm collection. (Texte intégral

Yam asymptomatic virus 1, a novel virus infecting yams (Dioscorea spp.) with significant prevalence in a germplasm collection

A novel virus infecting yams (Dioscoreaspp.), tentatively named "yam asymptomatic virus 1" (YaV1), was characterized and sequenced from an asymptomaticD. alataplant from Vanuatu. Sequence comparisons and phylogenetic analysis showed that YaV1 is a novel ampelovirus and has the smallest genome among "subgroup 1" members. RT-PCR-based screening of a yam germplasm collection conserved in Guadeloupe showed that YaV1 is prevalent inD. alata,D. bulbifera,D. cayennensissubsp. rotundata,D. esculentaandD. trifidaaccessions but causes no apparent symptoms. Additional phylogenetic analysis revealed a low variability of YaV1 in Guadeloupe in a limited part of the genome, and suggested the occurrence of plant-to-plant transmission