Effect of HLA DR epitope de-immunization of Factor VIII \u3ci\u3ein vitro\u3c/i\u3e and \u3ci\u3ein vivo\u3c/i\u3e

T cell-dependent development of anti-Factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle to replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce HLA binding of epitopes based on predicted anchoring residues. Here, we used immunoinformatic tools to identify C2 domain HLA DR epitopes and predict site-specific mutations that reduce immunogenicity. Epitope peptides corresponding to original and modified sequences were validated in HLA binding assays and in immunizations of hemophilic E16 mice, DR3 and DR4 mice and DR3 × E16 mice. Consistent with immunoinformatic predictions, original epitopes are immunogenic. Immunization with selected modified sequences lowered immunogenicity for particular peptides and revealed residual immunogenicity of incompletely de-immunized modified peptides. The stepwise approach to reduce protein immunogenicity by epitope modification illustrated here is being used to design and produce a functional full-length modified FVIII for clinical use

A common polymorphism in NR1H2 (LXRbeta) is associated with preeclampsia

<p>Abstract</p> <p>Background</p> <p>Preeclampsia is a frequent complication of pregnancy and a leading cause of perinatal mortality. Both genetic and environmental risk factors have been identified. Lipid metabolism, particularly cholesterol metabolism, is associated with this disease. Liver X receptors alpha (NR1H3, also known as LXRalpha) and beta (NR1H2, also known as LXRbeta) play a key role in lipid metabolism. They belong to the nuclear receptor superfamily and are activated by cholesterol derivatives. They have been implicated in preeclampsia because they modulate trophoblast invasion and regulate the expression of the endoglin (CD105) gene, a marker of preeclampsia. The aim of this study was to investigate associations between the <it>NR1H3 </it>and <it>NR1H2 </it>genes and preeclampsia.</p> <p>Methods</p> <p>We assessed associations between single nucleotide polymorphisms of <it>NR1H3 </it>(rs2279238 and rs7120118) and <it>NR1H2 </it>(rs35463555 and rs2695121) and the disease in 155 individuals with preeclampsia and 305 controls. Genotypes were determined by high-resolution melting analysis. We then used a logistic regression model to analyze the different alleles and genotypes for those polymorphisms as a function of case/control status.</p> <p>Results</p> <p>We found no association between <it>NR1H3 </it>SNPs and the disease, but the <it>NR1H2 </it>polymorphism rs2695121 was found to be strongly associated with preeclampsia (genotype C/C: adjusted odds ratio, 2.05; 95% CI, 1.04-4.05; <it>p </it>= 0.039 and genotype T/C: adjusted odds ratio, 1.85; 95% CI, 1.01-3.42; <it>p </it>= 0.049).</p> <p>Conclusions</p> <p>This study provides the first evidence of an association between the <it>NR1H2 </it>gene and preeclampsia, adding to our understanding of the links between cholesterol metabolism and this disease.</p

Application et validation des échantillonneurs passifs du type POCIS pour l'échantillonnage intégré des pesticides dans les eaux du bassin versant charentais

(trad auto)The monitoring of pesticides in water, whether surface, groundwater or intended for consumption or not, has become a major issue in recent decades. Indeed, the many chronic or acute pollution caused by human activities to the world's water bodies raises environmental and/or public health problems. The upsurge in these events is now leading decision-makers to conduct in-depth reflection on the use and protection of this resource, which is essential to life. An important part of this reflection is the improvement of knowledge about pollution, its intensities and distribution. The analysis of pollution by pesticides is an integral part of this component. In this pollution monitoring, a crucial step has been studied with great interest in recent years: sampling. It is within this framework that this thesis work is carried out with the main objective of improving this sampling step, which is divided into two categories: active sampling (point sampling, automated sampling) and passive sampling. Passive sampling techniques are promising because they show better results in terms of representativeness of contamination and lowering detectable and even quantifiable levels of contamination. The purpose of our research project is therefore to develop and validate the use of a passive sampler, the Polar Organic Chemical Integrative Sampler (POCIS), which samples polar compounds, including most pesticides. Three areas of research have been developed: Development of a multi-residue analytical method to analyse the residues of 33 pesticides in water and POCIS extracts. It appears essential to have an effective, reliable, repeatable and sensitive analytical method to quantify pollutants in order to benefit as much as possible from improvements in the sampling technique studied. Real world application of passive sampling using POCIS and comparison with an active reference sample. In this part, POCIS were exposed on watersheds with different characteristics and pollution footprints. Use of the POCIS sampler as a tool to study the exposure/impact relationships of pesticides on river biofilms. River biofilm is an interesting medium for studying the impact of pollutants on the aquatic environment and its composition is often an indicator of the state of the environment.Le suivi des pesticides dans les eaux, qu'elles soient de surface, souterraines ou destinées à la consommation ou non, est devenu un enjeu primordial de ces dernières décennies. En effet, les nombreuses pollutions chroniques ou aigues, que les activités humaines font subir aux masses d'eau de la planète, posent des problèmes environnementaux et/ou de santé publique. La recrudescence de ces événements conduit aujourd'hui les décisionnaires à mener une réflexion de fond sur les usages et la protection de cette ressource indispensable à la vie. Un volet important de cette réflexion est l'amélioration des connaissances concernant les pollutions, leurs intensités et leur répartition. L'analyse des pollutions par les pesticides fait partie intégrante de ce volet. Dans ce suivi des pollutions, une étape primordiale est étudiée avec beaucoup d'intérêt depuis ces dernières années : l'échantillonnage. C'est dans ce cadre que ce travail de thèse s'inscrit avec, comme principal objectif, l'amélioration de cette étape d'échantillonnage qui se décline en deux catégories : l'échantillonnage actif (prélèvements ponctuels, prélèvements automatisés) et l'échantillonnage passif. Les techniques d'échantillonnage passif sont prometteuses car elles montrent de meilleurs résultats en termes de représentativité de la contamination et d'abaissement des niveaux de contamination détectables, voire quantifiables. Le but de notre projet de recherche est donc de développer et de valider l'utilisation d'un échantillonneur passif, le Polar Organic Chemical Integrative Sampler (POCIS), qui permet d'échantillonner les composés polaires, dont la plupart des pesticides font partie. Trois axes de recherche ont été ainsi développés : Développement d'une méthode analytique multi-résidus pour analyser les résidus de 33 pesticides dans les eaux et les extraits de POCIS. Il apparait indispensable de disposer d'une méthode analytique efficace, fiable, répétable et sensible pour quantifier les polluants afin de bénéficier au mieux des améliorations de la technique d'échantillonnage étudiée. Application en conditions réelles de l'échantillonnage passif à l'aide des POCIS et comparaison avec un échantillonnage actif de référence. Dans cette partie des POCIS ont été exposés sur des bassins versants de caractéristiques et d'empreintes de pollutions différentes. Utilisation de l'échantillonneur POCIS comme outil pour étudier les relations exposition / impacts des pesticides sur les biofilms de rivière. Le biofilm de rivière est un support intéressant pour étudier l'impact des polluants sur le milieu aquatique et sa composition est souvent un indicateur de l'état du milieu

Polar organic chemical integrative samplers for pesticides monitoring: Impacts of field exposure conditions

International audienceThis study focuses on how Polar Organic Chemical Integrative Samplers (POCIS) work in real environmental conditions. A selection of 23 polar pesticides and 8 metabolites were investigated by exposure of triplicates of integrative samplers in two rivers in France for successive 14-day periods. The pesticides and metabolites were trapped not only in Oasis HLB sorbent but also in the polyethersulfone (PES) membrane of the POCIS. The distribution of pesticides depended on the molecular structure. The use of the Performance Reference Compound (PRC) is also discussed here. The impact of someenvironmental parameters and exposure setup on the transfer of pesticides in POCIS sorbent was studied: river flow rate, biofouling on membranes, sampler holding design and position in the stream. Results show a significant impact of river flow velocity on PRC desorption, especially for values higher than 4 cm·s-1. Some fouling was observed on the PES membrane which could potentially have an impact on molecule accumulation in the POCIS. Finally, the positioning of the sampler in the river did not have significant effects on pesticide accumulation, when perpendicular exposureswere used (sampler positioning in front of the water flow). The POCIS with PRC correction seemsto be a suitable tool for estimating time-weighted average (TWA) concentrations, for all themolecules except for one of the nine pesticides analyzed in these two French rivers

Etalonnage du Chemcatcher pour des pesticides

Le Chemcatcher apolaire a été étalonné et largement étudié pour des composés tels que les HAP et PCB. Il existe en revanche très peu de données de calibration pour des pesticides hydrophobes. Les modèles cinétiques existants, basés sur des PRC du type HAP et la seule considération du log Kow des substances ne permettent pas d’estimer convenablement les taux d’échantillonnage de la plupart de composés considérés durant cette étude. Ainsi, des résultats acquis expérimentalement (constantes cinétiques et de distribution) sont présentés et des limites pressenties du Chemcatcher apolaire vis-à-vis des pesticides d’intérêt sont discutées

Etude de la contamination en pesticides et en éléments traces métalliques des bassins versants du Trec et de l’Auvézère : application de l’échantillonnage intégratif passif : Rapport final synthèse campagnes 2012-2014

Pesticides have become a major component of modern agriculture and are largely use in many urban and domestic activities. These substances are found today in all compartments of the environment, particularly in aquatic environments. The emission and dispersion of active substances and their residues in the aquatic environment is potentially harmful to ecosystems, and their monitoring is then compulsory. The implementation of the Water Framework Directive (WFD) requires sampling and analysis, combining high sensitivity, accuracy and reliability, and must be easily implemented. Currently, the methodology consists of spot sampling (once a month, usually) of water bodies followed by laboratory analysis. This approach suffers from a lack of time representativeness, coupled to unsatisfactory analytical sensitivity for some substances. Passive sampling techniques developed over the last 20 years could be included in the regulatory monitoring networks in order to overcome these issues, but some questions still remain as to their applicability. This work aimed to develop and apply passive samplers like POCIS and DGT into two watersheds located in Adour-Garonne basin, in order to assess the contribution of these techniques over conventional spot sampling. Especially, a higher knowledge on the type of pressure (e.g. detection frequencies , main substances found in water) and a better understanding of the consequences of the establishment of agri-environmental measures (trend monitoring) are expected. This work has also provided information on the robustness of the data integrated over the time and the fraction sampled by these tools (e.g. labile complex for heavy metals). Finally, we examined the applicability of these tools in the context of the WFD, after setting a confidence interval associated with the time-weighted average concentrations estimates, and then we proposed a decision scheme for the comparison with environmental quality standards.Les pesticides sont devenus une composante majeure de l’agriculture moderne et se sont imposés dans de nombreuses activités urbaines et domestiques. Ces substances se retrouvent aujourd’hui dans tous les compartiments de l’environnement notamment dans les milieux aquatiques. Le suivi resserré des substances actives et de leurs résidus, présents dans l’environnement à des concentrations potentiellement dommageables pour les écosystèmes, apparaît aujourd’hui comme une nécessité. L’application de la Directive cadre sur l’eau requiert des techniques d’échantillonnage et d’analyse performantes, alliant haute sensibilité, facilité de mise ½uvre, coûts abordables, et surtout précision et fiabilité. Actuellement, la méthodologie employée consiste en des prélèvements ponctuels d’eau (une fois par mois en général) suivi de l’analyse en laboratoire. Cette approche souffre d’un manque de représentativité temporelle, couplée à une sensibilité analytique parfois peu satisfaisante. Les techniques d’échantillonnage passif développées au cours des 20 dernières années pourraient être intégrées dans les réseaux de surveillance réglementaire afin de pallier ces manques, mais des questions subsistent encore quant à leur opérationnalité. Ces travaux ont visé à développer puis tester des échantillonneurs passifs du type POCIS et DGT au niveau de deux bassin versants, situés en Adour-Garonne, afin de juger de l’apport de ces techniques, par rapport aux prélèvements ponctuels classiques, dans le but de fournir davantage de connaissance sur le type de pression (e.g. fréquences de détections, substances phytosanitaires majoritairement retrouvées dans les eaux) et mieux appréhender les conséquences de la mise en place de mesures agro-environnementales (suivi de tendance). Ces travaux ont également fourni des renseignements sur la robustesse de la donnée intégrée dans le temps et la fraction échantillonnée par ces outils (e.g. complexes labiles pour les éléments trace métalliques). Enfin, nous nous sommes intéressés à l’applicabilité de ces outils dans le cadre de la DCE, après avoir défini un intervalle de confiance associé aux concentrations moyennes estimées dans le temps, puis proposé un schéma décisionnel en lien avec le rapportage et la comparaison avec les normes de qualité environnementales

Echantillonnage passif des pesticides : opportunités et défis afin d’estimer l’exposition et le risque écotoxique dans les cours d’eau

International audienceDespite many efforts for improving the monitoring of an increasing number of chemicals (and their potential by-products), for a more comprehensive and exhaustive estimate of aquatic organism exposure, there is still some questions in relation with both temporal and spatial representativeness of the spot sampling, and also with chemical targeted analysis. As highlighted by Ort et al. (2010), discrete and low frequency sampling can be problematic due to the heterogeneity of aquatic environments and the dynamic of pollution levels. An alternative to spot sampling lies in the use of passive samplers. In this context, passive sampling techniques play a key role since they allow the integration of various events, occurring in natural aquatic environments (i.e. floods and peaks of contamination) while being able to decrease drastically the limits of quantification. However, some limitations are already well known for passive samplers, especially the accuracy of the measurement in relation with environmental conditions. Parameters such as temperature and flow velocity have substantial influence on the reliability of the measured concentrations; this is mainly due to the difference between controlled laboratory conditions, stable and used for the calibration, and those encountered in the field, inevitably more variable. There are various approaches to overcome these issues such as field calibrations, the use of Performance Reference Compounds (PRCs), etc. Besides these metrological aspects, there are also some scientific questions about the representativeness of the fraction of analytes isolated by the devices, especially in terms of bioavailability. At a more operational level, it must be checked if these fractions are in compliance with Environmental Quality Standards (EQS) established by the Water Framework Directive (WFD). Finally, the use of passive sampler extracts in combination with bioassays opens many opportunities to study mixture effects. However, such approaches can be qualified as "black box" since they require further developments to characterize more precisely the compounds responsible of the observed effects, and their possible interactions within the mixture

Évaluation expérimentale de l'effet de la température et de la vitesse d'écoulement sur l'absorption des pesticides par le POCIS et proposition d'un modèle pour la prédiction du taux d'échantillonnage

International audienceVarious studies have dealt with the effects of physico-chemical parameters (temperature, pH, organic matter content, salinity) or physical parameters (flow velocity and temperature) under laboratory conditions for sampling of hydrophilic (pharmaceuticals and pesticides) chemicals. These studies have demonstrated i) variable influence of these parameters on sampling rates ii) some consecutive bias on the calculated time weighted average concentrations (TWAC). However, all of these factors were studied individually, and possible interactions were not addressed. The first part of our work was dedicated to the calibration of POCIS with various temperatures (10 and 25°C) and flow velocities (from 0.7-1 cm.s-1 to 44-45 cm.s-1). Principal effects, and possible interactions between the two parameters, on sampling rates (Rs) were investigated with a full factorial design of experiment. The results showed a predominant impact of the flow velocity on Rs for 10 moderately polar pesticides (log Kow=2.18-4.14). Conversely, the temperature seemed to have only secondary and low influence on Rs variations. The second part consisted in a compilation of Rs data for the 10 analytes calibrated during this study, and also reported in the literature. Knowing the exchanging surface area A for POCIS, the global resistance to the mass transfer coefficients 1/ko were determined from these Rs (with 1/ko=A/Rs), and then plotted against the experimental bulk flow velocity values reported. The fitting of a non-linear model based on the dimensionless Sherwood number provided useful estimates of regression coefficients, for a possible prediction of Rs under various flowing conditions. For instance, the results obtained in the case of atrazine (1/ko ranging from 0.054±0.003 and 0.132±0.027 d.cm-1, depending on flow velocity) were in good agreement with the 1/ko values estimated by Booij et al. 2017. Some correlations were also observed between the flow velocity and the desorption rate of DIA-d5 (from 0.023 to 0.154 d-1). Such an observation could be a promising way to take into account the DBL thickness contribution, according to the hydrodynamic conditions, and then a likely improvement of the accuracy for in situ TWAC estimates

La combinaison de l'échantillonnage passif et l'échantillonnage ponctuel améliore les connaissances sur la présence et les niveaux de contamination des pesticides dans un bassin versant à grande échelle

International audienceFifty-one monitoring stations from the Water Framework Directive network (2000/60/CE) were selected in the Adour-Garonne basin (117,650 km2, SW France). These stations were characterized by a diversity of land use, implying different water pesticide contamination profiles. In each, Polar Organic Chemical Integrative Sampler (POCIS) deployment (14 days) and grab water samples (1 per period) were performed 6 times in 2016 in order to obtain contamination levels (29 pesticides monitored). The large amount of data collected during this 1-year monitoring required specific graphical and map processing to compare the information provided by POCIS and grab samples. Graphical projections demonstrated that with POCIS the number of quantified pesticides and the quantification frequencies were higher than with grab samples. Additionally, projections showed that POCIS provided better temporal representativeness of monthly contamination levels. Indeed, the POCIS data showed seasonal trends which were directly linked with the use of each pesticide (application period) and the land use of each sampling site, that was not visible with the grab samples data. Map projections of the measured concentrations, using a common scale for the two sampling strategies, clearly showed the strengths of the POCIS deployment and the link between measured contamination levels, quantified pesticides and land use. Finally, this study shows that the combination of grab sample data (magnitude of contamination peaks) and POCIS data (average concentration over a given period) provided more complete and reliable knowledge of the contamination levels in the Basin than either method alone