Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8(+) and CD4(+) T-cell responses with multiple specificities including a novel DR7-restricted epitope.

Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1-12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179-108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8(+) and CD4(+) T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189-102 for CD8(+) and NY-ESO-183-99 for CD4(+) T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187-99); 7/7 HLA-DR7(+) patients generated strong CD4(+) T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179-108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8(+) and CD4(+) T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach

Effects of postmortem delays on protein composition and oxidation.

Human autopsy brain tissue is widely used to study neurodegenerative diseases such as Alzheimer's, Parkinson's and other diseases. However, when it comes to an evaluation of data obtained from such tissue, it is essential to consider potential postmortem effects on protein composition, posttranslational modification and proteolysis with increasing postmortem delays. In this study, we analyzed mouse brain tissues with different postmortem delays (pmd) of 0h, 6h and 24h, for changes in protein composition, proteolysis and modifications such as S-nitrosylation, carbonylation and ubiquitination. Proteins involved in Alzheimer's disease (AD) were of special interest, including cytoskeletal and synaptic proteins or proteins involved in inflammation. Several proteins were fairly resistant to degradation during the first 6h but started to degrade thereafter. S-nitrosylation and carbonylation showed not much variation, except for those proteins that were susceptible to degradation. Brain spectrin was S-nitrosylated at death, and S-nitrosylated degradation fragments were measured at a pmd of 24h, indicating a susceptibility of brain spectrin to degradation. Furthermore, the physiological role of S-nitrosylation remains to be investigated. When studying human brain tissue, some proteins are more susceptible to degradation than others, while ubiquitination and carbonylation were little affected during the first 24h after death

Embryotoxic and genotoxic effects of heavy metals and pesticides on early life stages of Pacific oyster (Crassostrea gigas)

International audienceThis study evaluated embryotoxicity and genotoxicity of two dissolved metals copper and cadmium (Cu and Cd) and two pesticides (metolachlor and irgarol) occurring in Arcachon Bay (SW France) in Pacific oyster (Crassostrea gigas) larvae and investigated the relationship between those two endpoints. Embryotoxicity was measured by calculating the percentage of abnormal D-shaped larvae and genotoxicity was evaluated with DNA strand breaks using the comet assay. After 24 h exposure, significant increases of the percentage of abnormal D-larvae and the DNA strand breaks were observed from 0.1µg/L for Cu, 10 µg/L for Cd and 0.01 µg/L for both irgarol and metolachlor in comparison with the controls. A strong positive relationship between embryotoxicity and genotoxicity was recorded for Cu, Cd and metolachlor. The current study suggests that copper, irgarol and metolachlor can induce larval abnormalities and DNA damage in a population of exposed oysters at environmentally relevant concentrations

Seasonal variations of contamination and exoskeletal malformations in the white shrimps Palaemon longirostris in the Gironde estuary, France

International audienceSince the end of the 1980s, white shrimps (Palaemon longirostris) from the Gironde estuary have exhibited exoskeletal malformations, mainly involving cephalothorax, rostrum, scaphocerites and uropods. An 8-month study was carried out in 2015. Each month, 200 individuals were sampled and examined for exoskeletal malformations. Temporal variations in malformationfrequency were noted,particularlyduringthebreedingperiod, along withdecreases in the sizeof non-deformedshrimps related to the appearance of juveniles in breeding sites, and high mortality among deformed shrimps. A significant increase in proportions of deformed shrimp was observed, relating particularly to the size (and therefore the age) of individuals. No significant difference was found between shrimp proportions with different numbers of malformations (one to four) for a fixed size class, nor was there any variation in proportions within different size classes for a fixed number of malformations. This would appear toindicate thatthe number ofmalformations isacquiredand new malformations donot seemto appearduringthe lifecycle,exceptforthesmallest(youngest)shrimps.Themalformationspectrumshowednosignificantdifferencesbetweenthe biggest and smallest individuals for the different malformation associations, except for those involving cephalothorax, rostrum and uropods. This would suggest that some malformation associations lead to a higher mortality rate in shrimps subjected to them, due to greater impairment of feeding and/or swimming behaviour. Multiple component analysis of the different types of malformation showed correlations between exoskeletal pieces (rostrum and cephalothorax) and appendixes (scaphocerites and uropods). Regarding metal contamination in shrimp, no significant difference was highlighted between deformed and nondeformed shrimps. Organic pollutants were not measured in tissues. Certain herbicides such as metolachlore and chlortoluron

Optimized combinatorial pMHC class II multimer labeling for precision immune monitoring of tumor-specific CD4 T cells in patients.

With immunotherapy gaining increasing approval for treatment of different tumor types, scientists rely on cutting edge methods for the monitoring of immune responses and biomarker development in patients. Due to the lack of tools to efficiently detect rare circulating human tumor-specific CD4 T cells, their characterization in patients still remains very limited. We have used combinatorial staining strategies with peptide major histocompatibility complex class II (pMHCII) multimer constructs of different alleles to establish an optimized staining procedure for in vitro and direct ex-vivo visualization of tumor-specific CD4 T cells, in patient samples. Furthermore, we have generated reversible multimers to achieve optimal cell staining and yet disassemble prior to in vitro cell expansion, thus preventing activation induced cell death. We observed a vastly improved detection of tumor-specific, viral-specific and bacterial-specific cells with our optimization methods compared with the non-optimized staining procedure. By increasing the variety of fluorochromes used to label the pMHCII multimers, we were also able to increase the parallel detection of different specificities within one sample, including antigen-specific CD8 T cells. A decrease in cell viability was observed when using the full optimization method, but this was mitigated by the removal of neuraminidase and the use of reversible multimers. This new optimized staining procedure represents an advance toward better detection and analysis of antigen-specific CD4 T cells. It should facilitate state-of-the art precision monitoring of tumor-specific CD4 T cells and contribute to accelerate the use and the targeting of these cells in cancer immunotherapy

Vulnérabilité à la pollution et au réchauffement global des premiers stades de développement d'Acipenser sturio

International audienceEuropean sturgeon (Acipenser sturio) is an anadromous fish which has been in sharp decline since the beginning of the 20th century. The last wild population of European sturgeon is settled in the Gironde Garonne Dordogne catchment. Because of global change, temperatures have risen and frequency and severity of hypoxic events have increased in this catchment. In addition, the A. sturio population faces chronic pollutant exposure due to increasing agricultural, industrial and domestic releases. The goal of this work is to examine the sensitivity of A. sturio early developmental stages to temperature variations, oxygen depletion and pollutions in environmentally realistic exposure conditions. Embryo-larvae were exposed to combined temperature conditions, ranged from 12°C to 30°C, and dissolved oxygen, ranged from 30 to 90 % O2 saturation (% O2 sat) and to four natural spawning grounds sediments. Lethal and sub-lethal effects were evaluated using embryonic and larval mortality, hatching success, malformation rate, yolk sac resorption, tissue development, routine metabolic rate (RMR), swimming speed and DNA damage (only for sediment exposure). Embryonic survival peaked at 20°C and no survival was recorded at 30°C. No hatching occurred at 50 % O2 sat or below. Malformation rate appeared to be minimum at 20°C and 90 % O2 sat. Maximum RMR were recorded at 20°C under 90 % O2 sat and at 16°C under 70 % O2 sat. At 20°C, RMR was lower at 70% O2 sat than at 90% O2 sat. Swimming speed peaked at 16°C. A. sturio's temperature optimum was shown to be close to 20°C. Its upper tolerance limit is between 26 and 30°C. Its lower tolerance limit was not reached and is supposed to be below 12°C. Oxygen depletion induce sublethal effects at 70 % and lethal effects at 50 % O2 sat. Strikingly, sediment quality of spawning ground had no effect on embryo-larval survival and hatching success. A significant increase of developmental defects and percentage of apoptotic cells was observed for one of the studied sediment. Current temperature, oxygen and sediment quality conditions in the Gironde basin are globally sustainable for A. sturio but a slight O2 levels decrease would impair European sturgeon maintenance capacity in the basin

Des organismes sous stress

An ecologic epidemiological approach, used to assess stress of chemical contaminants in the Seine estuary, concerned three aspects : sources, exposure and effects. The presence of toxic substances in sediment and the diet of organisms was considered to be a source of stress. The state of stress was assessed by measuring exposure indicators in the sebra mussel in the flounder. Diversity of fish species and effect indicators in flounder was studied in order to detect effects of chronic exposures. This ecologic epidemiological approach, applied for the first time in a french estuary, enabled us to identify sensitive areas of exposure.Une approche d'épidémiologie écologique a été appliquée pour évaluer l'effet de stress des contaminants chimiques dans l'estuaire de la Seine. Cette approche comprend trois étapes clés visant à relier les sources, l'exposition et les effets. Des molécules toxiques dans le sédiment ainsi que dans la chaîne alimentaire des organismes ont été estimées comme source de stress. L'état de stress a été évalué par la mesure d'indicateurs d'exposition chez la dreissène et chez le flet. La diversité des espèces de poisson et des indicateurs d'effets chez les flets ont été étudiés pour détecter des effets d'expositions chroniques. Cette approche d'épidémiologie écologique a permis d'identifier des zones d'exposition sensibles

Immunotoxicological effects of perfluorooctanesulfonic acid on European seabass are reduced by polyethylene microplastics

International audienceMarine environments receive plastic waste, where it suffers a transformation process into smaller particles. Among them, microplastics (MPs; <5 mm) are ingested by aquatic organisms leading to negative effects on animal welfare. The interactions between MPs, contaminants and organisms are poorly understood. To clarify this issue, European seabass (Dicentrarchus labrax L.) were fed with diets supplemented with 0 (control), polyethylene (PE) MPs (100 mg/kg diet), perfluorooctanesulfonic acid (PFOS, 4.83 μg/kg diet) or PFOS adsorbed to MPs (MPs-PFOS; final concentrations of 4.83 μg and 100 mg of PFOS and MP per kg of feed, respectively). Samples of skin mucus, serum, head-kidney (HK), liver, muscle, brain and intestine were obtained. PFOS levels were high in the liver of fish fed with the PFOS-diet, and markedly reduced when adsorbed to MPs. Compared to the control groups, liver EROD activity did not show any significant changes, whereas brain and muscle cholinesterase activities were decreased in all the groups. The histological and morphometrical study on liver and intestine showed significant alterations in fish fed with the experimental diets. At functional level, all the experimental diets affected the humoral (peroxidase, IgM, protease and bactericidal activities) as well as cellular (phagocytosis, respiratory burst and peroxidase) activities of HK leukocytes, being more marked those effects caused by the PFOS diet. Besides, treatments produced inflammation and oxidative stress as evidenced at gene level. Principal component analysis demonstrated that seabass fed with MPs-PFOS showed more similar effects to MPs alone than to PFOS. Overall, seabass fed with MPs-PFOS diet showed similar or lower toxicological alterations than those fed with MPs or PFOS alone demonstrating the lack of additive effects or even protection against PFOS toxicity

Colloque final du programme de recherche ECHIBIOTEB : Outils innovants d’échantillonnage, d’analyses chimiques et biologiques pour le suivi de traitements avancés des eaux usées et des boues

National audienceRésumé du programme ECHIBIOTEB : Bien que les stations de traitement des eaux usées (STEU) ne soient pas construites dans le but d’éliminer les micropolluants organiques et métalliques, ces derniers peuvent néanmoins être éliminés de la phase dissoute des eaux usées par les procédés de traitement biologique conventionnels. L’élimination peut se faire grâce aux mécanismes d’adsorption sur les boues ou de dégradation biologique ou abiotique. Cependant, certains micropolluants sont réfractaires à ces procédés de traitement et sont rejetés dans les milieux aquatiques avec les eaux traitées. Pour limiter ces rejets de micropolluants réfractaires, des procédés de traitements complémentaires peuvent être mis en place. Par ailleurs, les micropolluants contenus dans les boues sont aussi susceptibles de contaminer les nappes d’eau souterraines et les milieux aquatiques de surface, quand ces boues sont épandues sur sols agricoles. Actuellement, il y a besoin de mieux comprendre l’efficacité vis-à-vis des micropolluants des procédés complémentaires de traitement des eaux et des procédés de traitement des boues. Le but du programme ECHIBIOTEB est d’étudier, au moyen de combinaisons innovantes d’outils chimiques, biologiques et d’échantillonnage, des procédés de traitement des eaux usées et des boues. Le deuxième objectif est d’étudier les avantages et les inconvénients comparés de chacun de ces outils innovants, ainsi que leur complémentarité, pour l’étude de l’efficacité des procédés de traitement des eaux et des boues. Les outils d’échantillonnage mis en oeuvre dans les eaux sont des échantillonneurs intégratifs tels que les POCIS (Polar Organic Chemical Integrative Sampler) et les SPMD (Semi-Permeable Membrane Device). L’intérêt de ces échantillonneurs est d’améliorer la représentativité temporelle de l’échantillonnage des eaux avec obtention de concentrations dans l’eau moyennées sur la durée d’exposition (de 2 à 4 semaines). Les outils chimiques utilisés sont l’analyse ciblée de presque 180 contaminants prioritaires et émergents, et l’analyse non-ciblée, ou screening, réalisée au moyen de techniques chromatographiques combinées à de la spectrométrie de masse haute résolution ; ainsi que la caractérisation de la matière organique dissoute et de ses interactions avec les micropolluants (par l’étude de la fluorescence). Les outils biologiques testés, pratiqués en laboratoire ou in situ, consistent en des bioessais in vitro, pour évaluer des activités biologiques à l’échelle cellulaire, et des bioessais in vivo, pour évaluer des effets à l’échelle d’un organisme vivant. Pour répondre aux objectifs d’ECHIBIOTEB, 8 procédés de traitement complémentaire des eaux usées et 3 procédés de traitement des boues ont été étudiés. Les procédés de traitement des eaux usées sont des procédés d’oxydation à l’ozone (O3) ou à l’ozone associé à du charbon actif en grain (CAG), des procédés d’oxydation avancée comme l’ozone couplé à du peroxyde d’hydrogène (O3+H2O2) ou l’UV couplé à du peroxyde d’hydrogène (UV+H2O2), des procédés d’adsorption comme un filtre à écoulement horizontal garni de CAG, d’argile expansée et de zéolite, et enfin une lagune de finition. Ils ont été étudiés à l’échelle réelle ou à l’échelle de pilotes semi-industriels positionnés à la sortie des procédés de traitement biologique conventionnels. Les STEU sur lesquelles ces procédés ont été étudiés sont de tailles variables (entre 1 000 et 300 000 équivalents habitants) et sont équipées de procédés de traitement biologique variés, réalisant soit nitrification et dénitrification (tels que des boues activées à aération prolongée, des bioréacteurs à membranes), ou réalisant une nitrification seulement (biofiltres nitrifiants ou disques biologiques suivis de lits de clarification-séchage plantés de roseaux). Pour les boues, 3 procédés de traitement ont été étudiés : le sécheur solaire, le compostage et le lit de séchage planté de roseaux. Ils ont été étudiés à échelle réelle sur 3 sites. Pour étudier les procédés de traitement complémentaire des eaux, 2 sortes de campagnes de prélèvement ont été menées : les « campagnes courtes » où l’échantillonnage a été réalisé pendant une journée, avec des échantillonnages d’eau moyens 2h (représentatifs des procédés étudiés) et des analyses en laboratoire ; et les « campagnes longues » réalisées pendant une durée d’un mois, avec des échantillonnages d’eau et l’exposition d’échantillonneurs intégratifs (sur 15 jours ou 4 semaines), ainsi que l’exposition des organismes biologiques « in situ dérivé » (i.e. « ex situ sur le site ») en plus de celle en laboratoire. Au total, ont été réalisées 11 campagnes courtes et 4 campagnes longues pour les eaux. Pour étudier les procédés de traitement des boues, 3 campagnes de prélèvement ont été menées et afin d’analyser le même lot de boue avant et après traitement, les campagnes ont été réalisées à l’intervalle de temps correspondant à la durée du séchage ou du compostage