Toward standardization of BK virus monitoring: evaluation of the BK virus R-gene kit for quantification of BK viral load in urine, whole-blood, and plasma specimens.

Screening of BK virus (BKV) replication is recommended to identify patients at increased risk of BKV-associated diseases. However, the heterogeneity of molecular techniques hinders the establishment of universal guidelines for BKV monitoring. Here we aimed to compare the performance of the CE-marked BK virus R-gene kit (R-gene) to the performance of our in-house assay for quantification of BKV DNA loads (BKVL). A 12-specimen panel from the Quality Control for Molecular Diagnostics (QCMD) organization, 163 urine samples, and 88 paired specimens of plasma and whole blood (WB) from transplant recipients were tested. Both the R-gene and in-house assays showed a good correlation within the QCMD panel (r = 0.995 and r = 0.989, respectively). BKVL were highly correlated between assays, although positive biases were observed with the in-house assay in analysis of urine (0.72 ± 0.83 log10 copies/ml), plasma (1.17 ± 0.63 log10 copies/ml), and WB (1.28 ± 0.37 log10 copies/ml). Recalibration with a common calibrator significantly reduced the bias in comparisons between assays. In contrast, BKVL was underestimated with the in-house PCR in eight samples containing BKV genotype II, presenting point mutations at primer-annealing sites. Using the R-gene assay, plasma and WB specimens were found to be equally suitable for quantification of BKVL, as indicated by the high correlation coefficient (r = 0.965, P < 0.0001). In conclusion, the R-gene assay demonstrated reliable performance and higher accuracy than the in-house assay for quantification of BKVL in urine and blood specimens. Screening of BKV replication by a well-validated commercial kit may enable clinical laboratories to assess viral loads with greater reproducibility and precision.comparative studyevaluation studiesjournal articleresearch support, non-u.s. gov't2014 Dec2014 10 08importe

Rôle des variants de la protéine de latence LMP du virus d'Epstein-Barr dans le développement du lymphome de Hodgkin chez les personnes VIH+

The aim of this work was to improve our understanding of the role of LMP1 and its 30bp and 69bp deletion variants in the development of Hodgkin's lymphoma (HL) among HIV+ people. Blood and saliva samples were collected from HL/HIV+ patients recruited in the Lymphovir cohort, as well as in two control populations (HIV+ and HL). Next-Generation Sequencing allowed us to determine the frequency of LMP1 variants in these samples. This work showed that the del30-LMP1 variant seems to be more frequent in patients with a moderate immunosuppression, associated with a higher HL incidence. Besides, we established three HL-derived cell lines expressing WT-LMP1 or its variants and showed differences of cytokine expression and progression of the cell cycle depending on the variant. Our work supports the hypothesis of a greater oncogenicity of del30-LMP1 variant and suggests that it could be a risk factor for the development of HL.L'objectif de notre travail est de mieux comprendre le rôle de LMP1 et de ses variants délétés de 30 ou 69pb en C-terminal dans le développement du lymphome de Hodgkin (LH) chez les patients VIH+. Le séquençage de LMP1 dans des échantillons de sang total et de cellules oropharyngées de patients de la cohorte Lymphovir (LH/VIH+) et de deux populations contrôles (VIH+ et LH) a permis d'étudier la fréquence des variants de LMP1 in vivo. Ce travail a révélé que le variant LMP1-del30 semble plus fréquent chez les patients avec une immunosuppression modérée, durant laquelle l'incidence des LH est élevée. Par ailleurs, nous avons établi un modèle cellulaire dérivé de LH, exprimant LMP1 de façon inductible, montrant des différences d'expression des cytokines et de progression du cycle cellulaire en fonction des variants de LMP1. Notre travail supporte l'hypothèse d'une plus grande oncogénicité du variant LMP1-del30 et suggère qu'il pourrait être un facteur de risque de développer un LH

Role of Epstein-Barr Virus latency proteins in the development of Hodgkin's lymphoma among HIV+ patients

L'objectif de notre travail est de mieux comprendre le rôle de LMP1 et de ses variants délétés de 30 ou 69pb en C-terminal dans le développement du lymphome de Hodgkin (LH) chez les patients VIH+. Le séquençage de LMP1 dans des échantillons de sang total et de cellules oropharyngées de patients de la cohorte Lymphovir (LH/VIH+) et de deux populations contrôles (VIH+ et LH) a permis d'étudier la fréquence des variants de LMP1 in vivo. Ce travail a révélé que le variant LMP1-del30 semble plus fréquent chez les patients avec une immunosuppression modérée, durant laquelle l'incidence des LH est élevée. Par ailleurs, nous avons établi un modèle cellulaire dérivé de LH, exprimant LMP1 de façon inductible, montrant des différences d'expression des cytokines et de progression du cycle cellulaire en fonction des variants de LMP1. Notre travail supporte l'hypothèse d'une plus grande oncogénicité du variant LMP1-del30 et suggère qu'il pourrait être un facteur de risque de développer un LH.The aim of this work was to improve our understanding of the role of LMP1 and its 30bp and 69bp deletion variants in the development of Hodgkin's lymphoma (HL) among HIV+ people. Blood and saliva samples were collected from HL/HIV+ patients recruited in the Lymphovir cohort, as well as in two control populations (HIV+ and HL). Next-Generation Sequencing allowed us to determine the frequency of LMP1 variants in these samples. This work showed that the del30-LMP1 variant seems to be more frequent in patients with a moderate immunosuppression, associated with a higher HL incidence. Besides, we established three HL-derived cell lines expressing WT-LMP1 or its variants and showed differences of cytokine expression and progression of the cell cycle depending on the variant. Our work supports the hypothesis of a greater oncogenicity of del30-LMP1 variant and suggests that it could be a risk factor for the development of HL

Identification et caractérisation des partenaires cellulaires de zebra, facteur de transcription du virus Epstein-Barr (mise en évidence d un complexe tripartite entre la protéine virale zebra et les protéines cellulaires ubinucléine et 14-3-3epsilon)

Le virus Epstein-Barr (EBV), virus oncogène pouvant être associé à des cancers lymphoïdes et épithéliaux, est prévalent à 95% dans la population mondiale. ZEBRA est un facteur de transcription viral clé dans la réactivation virale et l oncogenèse associée à l EBV. De précédents travaux ont décrit une protéine cellulaire, l Ubinucléine (Ubn), capable d empêcher la fixation de ZEBRA sur ses séquences cibles d ADN. Notre laboratoire a montré que l Ubn possède une double localisation cellulaire, nucléaire et dans les jonctions serrées. Le transport de l Ubn faisant certainement intervenir d autres partenaires cellulaires, notre attention s est portée sur les protéines 14-3-3 connues pour réguler l import/export nucléaire de leurs partenaires. L objectif de mon travail est de mieux comprendre les conditions dans lesquelles l Ubn inhibe ZEBRA. Nous avons étudié l hypothèse que la protéine 14-3-3epsilon, en contrôlant la localisation cellulaire de l Ubn, interviendrait comme régulateur de l interaction entre l Ubn et ZEBRA. Des techniques in vivo et in vitro (immunoprécipitation, pull-down, immunofluorescence) ont mis en évidence un complexe entre ZEBRA, l Ubn et 14-3-3epsilon et ont montré que l Ubn se place en intermédiaire entre ces deux partenaires. De plus, 14-3-3epsilon se fixe sur l Ubn au niveau d un motif consensus comprenant la sérine 352 phosphorylée. D autres expériences ont révélé que l Ubn non phosphorylée est localisée préférentiellement au niveau des jonctions serrées. Nos résultats suggèrent donc que 14-3-3epsilon favoriserait la localisation nucléaire de l Ubn, lui permettant d interagir avec ZEBRA et d empêcher l initiation du cycle lytique de l EBV.Epstein-Barr Virus (EBV) has a prevalence of 95% in the worldwide population. It is known for its oncogenicity and its association with lymphoma and epithelial cancer. ZEBRA is a viral transcription factor playing a key role in viral reactivation and EBV-associated oncogenicity. Previous studies described a cellular protein named Ubinuclein (Ubn), capable of preventing ZEBRA s binding on its DNA target sequences. Our laboratory has shown that Ubn can be localized either in the nucleus or in the tight junctions of the cells. It is very likely that the Ubn s travelling involves other cellular proteins and we focused on the 14-3-3 protein family, which is well known to regulate the nucleus import/export of numerous proteins. The purpose of this work is to better understand the conditions in which Ubn inhibits ZEBRA s functions and the viral lytic cycle. We hypothesized that, by controlling Ubn s cellular localization, 14-3-3epsilon could regulate the Ubn-ZEBRA interaction. In vivo and in vitro technics (immunoprecipitation, pull-down, immunofluorescence) allowed us to demonstrate the existence of a ternary complex between ZEBRA, Ubn and 14-3-3epsilon and to show that Ubn plays the role of an intermediate between its two partners. More precisely, 14-3-3epsilon binds to Ubn on a consensus motif containing a phosphorylated serine. The study of Ubn s phosphorylation revealed that non-phosphorylated Ubn is localized preferentially at the tight junctions. Taken together, all these experiments suggest that 14-3-3epsilon could promote the nuclear localization of Ubn, allowing it to interact with ZEBRA and preventing EBV s lytic cycle initiation.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

Difference in cytokine production and cell cycle progression induced by Epstein-Barr virus Lmp1 deletion variants in Kmh2, a Hodgkin lymphoma cell line.

International audienceEpstein-Barr virus (EBV) is associated with 20-40% of Hodgkin's Lymphoma (HL) cases. EBV-encoded latent membrane protein 1 (LMP1) is a well-known oncogenic protein and two C-terminal deletion variants, del30-LMP1 and del69-LMP1, have been described in animal models to be more tumorigenic than the wild-type form. This work aims to detail the implication of LMP1 in the development of HL and to characterize the particular effects of these variants. We established HL-derived cell lines stably transfected with the pRT-LMP1 vector coding for the EBNA1 gene and allowing expression of the different LMP1 variants under the control of a doxycyclin-inducible promoter. Communication between cells was assessed by measuring the expression of various pro-inflammatory cytokines by flow cytometry after intracellular LMP1 and cytokine double staining. Proliferative properties of LMP1 variants were also compared by studying the repartition of cells in the different phases of the cell cycle after EdU incorporation combined to LMP1 and DAPI staining. All LMP1 proteins induced the expression of several pro-inflammatory cytokines such as TNF-α, TNF-β, IL-6, RANTES/CCL5 and IFN-γ. However, the del30-LMP1 variant induced cytokine expression at a lower level than the other variants, especially IFN-γ, while the del69-LMP1 variant stimulated greater cytokine expression. In addition, we measured that all LMP1 proteins greatly impacted the cell cycle progression, triggering a reduction in the number of cells in S-phase and an accumulation of cells in the G2/M phase compared to the HL-non induced cells. Interestingly, the del30-LMP1 variant reduced the number of cells in S-phase in a significantly greater manner and also increased the number of cells in the G0/G1 phase of the cell cycle. Weak IFN-γ expression and specific alteration of the cell cycle might be a way for del30-LMP1 infected cells to escape the immune anti-viral response and to promote the development of cancer. The differences observed between the LMP1 variants reflect their own oncogenic properties and eventually impact the development of HL

Cultural influence of social information use in pedestrian road-crossing behaviours

International audienceSocial information use is common in a wide range of group-living animals, notably in humans. The role it plays in decision-making could be a key to understanding how social groups make collective decisions. The observation of road-crossing behaviours in the presence of other individuals is an ideal means to study the influence of social information on decision-making. This study investigated the influence of culture on social information used by pedestrians in a potentially dangerous scenario, namely road crossing. We scored the collective crossing of pedestrians at four locations in Nagoya (Japan) and three locations in Strasbourg (France). French pedestrians cross against the lights much more often (41.9%) than Japanese ones (2.1%). Individuals deciding to cross the road were strongly influenced by the behaviour and the presence of other pedestrians, especially in Japan, where a stronger conformism was noted. However, Japanese pedestrians were half as likely to be influenced by social information as their French counterparts when crossing at the red light, as they were more respectful of rules. Men show riskier behaviour than women (40.6% versus 25.7% of rule-breaking, respectively), deciding quickly and setting off earlier than women. Further related studies could help target specific preventive, culture-specific solutions for pedestrian safety

Data for 1. Crossing against the red light at road crossings

Data for 1. Crossing against the red light at road crossing

Both age and social environment shape the phenotype of ant workers

International audienceAbstract Position within the social group has consequences on individual lifespans in diverse taxa. This is especially obvious in eusocial insects, where workers differ in both the tasks they perform and their aging rates. However, in eusocial wasps, bees and ants, the performed task usually depends strongly on age. As such, untangling the effects of social role and age on worker physiology is a key step towards understanding the coevolution of sociality and aging. We performed an experimental protocol that allowed a separate analysis of these two factors using four groups of black garden ant ( Lasius niger ) workers: young foragers, old foragers, young nest workers, and old nest workers. We highlighted age-related differences in the proteome and metabolome of workers that were primarily related to worker subcaste and only secondarily to age. The relative abundance of proteins and metabolites suggests an improved xenobiotic detoxification, and a fuel metabolism based more on lipid use than carbohydrate use in young ants, regardless of their social role. Regardless of age, proteins related to the digestive function were more abundant in nest workers than in foragers. Old foragers were mostly characterized by weak abundances of molecules with an antibiotic activity or involved in chemical communication. Finally, our results suggest that even in tiny insects, extended lifespan may require to mitigate cancer risks. This is consistent with results found in eusocial rodents and thus opens up the discussion of shared mechanisms among distant taxa and the influence of sociality on life history traits such as longevity