201 research outputs found

    Quantitative High-Throughput Single-Cell Cytotoxicity Assay for T cells

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    Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5. Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8. Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells

    Detection and isolation of auto-reactive human antibodies from primary B cells

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    The isolation of human monoclonal antibodies (hmAb) has emerged as a versatile platform in a wide variety of contexts ranging from vaccinology to therapeutics. In particular, the presence of high titers of circulating auto-antibodies is implicated in the pathology and outcome of autoimmune diseases. Therefore, the molecular characterization of these hmAb provides an avenue to understanding the pathogenesis of autoimmune diseases. Additionally, the phenotype of the auto-reactive B cells may have direct relevance for therapeutic intervention. In this report, we describe a high-throughput single-cell assay, microengraving, for the screening, characterization and isolation of anti-citrullinated protein antibodies (ACPA) from peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients. Stimulated B cells are profiled at the single-cell level in a large array of sub-nanoliter nanowells (?105), assessing both the phenotype of the cells and their ability to secrete cyclic-citrullinated peptide (CCP)-specific antibodies. Single B cells secreting ACPA are retrieved by automated micromanipulation, and amplification of the immunoglobulin (Ig) heavy and light chains is performed prior to recombinant expression. The methodology offers a simple, rapid and low-cost platform for isolation of auto-reactive antibodies from low numbers of input cells and can be easily adapted for isolation and characterization of auto-reactive antibodies in other autoimmune diseases

    Ingénierie des glucane-saccharases de la famille 70 des glycoside-hydrolases pour la synthÚse de nouveaux biopolymÚres

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    Les glucane-saccharases sont des transglucosidases de la famille 70 des glycoside-hydrolases. A partir de saccharose, ces enzymes catalysent la synthĂšse d alpha -glucanes, polymĂšres de haute masse molaire formĂ©s d unitĂ©s glucosyle. Elles sont aussi capables de synthĂ©tiser des oligosaccharides ou glucoconjuguĂ©s par rĂ©action de transglucosylation sur des accepteurs exogĂšnes de natures variĂ©es. De par la diversitĂ© de leurs spĂ©cificitĂ©s, tant au niveau des liaisons osidiques synthĂ©tisĂ©es [alpha (1->2) ; alpha (1->3) ; alpha (1->4) ou alpha (1->6)] que de l organisation de ces liaisons au sein des produits formĂ©s, ces biocatalyseurs peuvent ĂȘtre mis Ă  profit pour produire des hydrates de carbones d'intĂ©rĂȘt pour les secteurs de l'alimentation, de la santĂ© et de l'environnement. L'objectif de ces travaux de thĂšse Ă©tait de gĂ©nĂ©rer par ingĂ©nierie enzymatique de nouvelles glucane-saccharases capables de synthĂ©tiser des alpha -glucanes et des gluco-oligosaccharides de structures et propriĂ©tĂ©s innovantes, afin d Ă©largir le panel d'applications de ces molĂ©cules. Sur le plan fondamental, l'enjeu Ă©tait aussi d'amĂ©liorer la comprĂ©hension des relations entre structure et spĂ©cificitĂ© des glucane-saccharases. Pour atteindre nos objectifs, nous avons utilisĂ© une stratĂ©gie d'ingĂ©nierie combinatoire de la dextrane-saccharase DSR-S de Leuconostoc mesenteroides NRRL B-512F, qui catalyse la synthĂšse d un dextrane formĂ© Ă  95 % de liaisons alpha (1->6) et 5 % alpha (1->3). Le travail a tout d'abord consistĂ© Ă  dĂ©velopper une mĂ©thode de criblage multi-Ă©tapes et Ă  haut-dĂ©bit, pour isoler et trier les variants de spĂ©cificitĂ©s de liaisons variĂ©es. La stratĂ©gie comprend une premiĂšre Ă©tape de sĂ©lection in vivo sur milieu solide suivie d un criblage par RMN 1D 1H automatisĂ© au format microplaque. Il s'agit de la premiĂšre mĂ©thode de criblage Ă  haut-dĂ©bit de la spĂ©cificitĂ© de liaison des transglucosidases et glycosyltransferases. Cette stratĂ©gie a ensuite Ă©tĂ© appliquĂ©e au criblage de deux banques de variants de la DSR-S, totalisant plus de 36 000 clones obtenus par mutagĂ©nĂšse de saturation et recombinaison simultanĂ©e de 8 rĂ©sidus du domaine catalytique. Au total, 82 mutants capables de synthĂ©tiser entre 2 et 8 fois plus de liaisons alpha (1->3) par rapport Ă  l enzyme parentale ont Ă©tĂ© isolĂ©s. La caractĂ©risation des propriĂ©tĂ©s catalytiques de sept mutants reprĂ©sentatifs de la diversitĂ© de spĂ©cificitĂ© gĂ©nĂ©rĂ©e a permis d identifier un nouveau motif peptidique 460DYVHT464 impliquĂ© dans la spĂ©cificitĂ© de DSR-S. Enfin, la caractĂ©risation de la structure et des propriĂ©tĂ©s rhĂ©ologiques et mĂ©caniques des dextranes synthĂ©tisĂ©s par ces 7 mutants a mis en Ă©vidence les diffĂ©rences de taille et de conformation de ces macromolĂ©cules en solution et rĂ©vĂ©lĂ© l aptitude du polymĂšre synthĂ©tisĂ© par le variant H463R/T464V/S512T Ă  former des films bio-sourcĂ©s innovants, dont les propriĂ©tĂ©s mĂ©caniques sont remarquables en comparaison de celles d'autres biopolymĂšres extraits de plantes ou produits par fermentationGlucansucrases are transglucosidases from glycoside hydrolase family 70. From sucrose, these enzymes catalyse the synthesis of alpha -glucans, high molecular weight polymers formed of glucosyl units. Glucansucrases are also able to synthesize oligosaccharides or glucoconjugates by transglucosylation reaction onto various exogenous acceptors. Due to the large specificity, in terms of osidic linkages synthesized [alpha (1->2); alpha (1->3); alpha (1->4) or alpha (1->6)] and their organization within the formed products, these biocatalysts can be used to produce carbohydrates of interest in food, health and environment fields. The aim of this research work was to generate, by enzyme engineering, new glucansucrases able to synthesize alpha -glucans and gluco-oligosaccharides with novel structures and properties, to enlarge applications of these molecules. At fundamental level, the work was to improve the understanding of the relationships between structure and specificity of glucansucrases. To reach our objectives, we used a combinatorial engineering strategy on the dextransucrase DSR-S of Leuconostoc mesenteroides NRRL B-512F, which catalyses the synthesis of a dextran formed by 95 % of alpha (1->6) and 5 % alpha (1->3) linkages. The work consisted first in the development of a multi-step high throughput screening methodology to sort out and isolate variants with altered linkage specificities. The strategy includes a first stage of in vivo selection on solid medium followed by an automated 1D 1H NMR-based screening using microplates. To date, this is the first high-throughput screening method for linkage specificity determination of transglucosidases and glycosyltransferases. This strategy was applied to the screening of two DSR-S variants libraries, totalizing more than 36,000 clones obtained by saturation mutagenesis and simultaneous recombination of 8 residues from the catalytic domain. Eighty two mutants able of synthesize 2 to 8 times more alpha (1->3) linkages compared to the parental enzyme were isolated. The characterization of the catalytic properties of 7 representative mutants enabled the identification of a new peptide motif 460DYVHT464, involved in DSR-S specificity. Finally, the characterization of structural, rheological and mechanical properties of dextrans synthetized by these 7 mutants highlighted the differences in size and conformation of these macromolecules in solution and revealed the capacity of the polymer synthesized by H463R/T464V/S512T variant to form biofilms, whose mechanical properties are remarkable in comparison to those of other biopolymers extracted from plants or produced by fermentation.TOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

    Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay

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    Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 ◩C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests

    Individual motile CD4+ T cells can participate in efficient multikilling through conjugation to multiple tumor cells

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    T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR

    Use of Equine Herpesvirus 1 glycoprotein pseudotyped lentiviral particles for the development of serological tests and assessment of lyophilisation for transport and storage

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    Equine herpesviruses (EHVs) are enveloped DNA viruses predominantly infecting members of the Equidae family. EHVs primarily cause respiratory disease, however EHV-1 can produce cases of a neurological disease, abortion and neonatal death. Thus, these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 exhibits a complex array of 12 glycoproteins on its surface envelope, but it is unclear precisely which are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a useful study tool. We have successfully generated functional EHV-1 pseudotyped lentiviruses bearing four glycoproteins, gB, gD, gH and gL (sequences derived from an aborted foetus during a large EHV1 outbreak strain in Normandy, France). PVs were employed in a pseudotype virus neutralisation test (PVNT) to measure levels of specific neutralising antibodies serum samples (n=52) taken longitudinally from experimentally infected ponies, compared with uninfected controls. PVs routinely require -80oC for long term storage and a dry ice cold-chain during transport which can impede dissemination and utilisation in other laboratories. Consequently, we further investigated whether freeze-drying (lyophilisation) of EHV-1 PV could address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions, sampling at different timepoints. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests

    A global review of capacity building organizations in water sanitation, and hygiene for developing countries

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    Although capacity building is increasingly emphasized in the water, sanitation and hygiene (WASH) sector, many WASH implementing organizations still lack capacity to effectively and sustainably provide WASH services. This study attempts to review the global capacity building efforts in the WASH sector by identifying the major capacity building organizations, understanding their focus and activities, comparing their efforts, and assessing potential gaps in capacity building services. A review of 72 water and sanitation networks identified 104 organizations providing capacity building services to other organizations. These capacity builders are mostly European Non-Governmental Organizations giving trainings on technical subjects with frequent duplication of services. Capacity building services were found to be concentrated in capital cities with rural and remote areas receiving less capacity building services. A lack of long-term client tracking and support was also found. By addressing these gaps and increased communication between these organizations, capacity could be built much more efficiently

    Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

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    Dendritic cells (DCs) can initiate and shape host immune responses toward either immunity or tolerance by their effects on antigen-specific CD4(+) T cells. DC-asialoglycoprotein receptor (DC-ASGPR), a lectinlike receptor, is a known scavenger receptor. Here, we report that targeting antigens to human DCs via DC-ASGPR, but not lectin-like oxidized-LDL receptor, Dectin-1, or DC-specific ICAM-3-grabbing nonintegrin favors the generation of antigen-specific suppressive CD4(+) T cells that produce interleukin 10 (IL-10). These findings apply to both self-and foreign antigens, as well as memory and naive CD4(+) T cells. The generation of such IL-10-producing CD4(+) T cells requires p38/extracellular signal-regulated kinase phosphorylation and IL-10 induction in DCs. We further demonstrate that immunization of nonhuman primates with antigens fused to anti-DC-ASGPR monoclonal antibody generates antigen-specific CD4(+) T cells that produce IL-10 in vivo. This study provides a new strategy for the establishment of antigen-specific IL-10-producing suppressive T cells in vivo by targeting whole protein antigens to DCs via DC-ASGPR

    Comment opĂ©rationnaliser et Ă©valuer la prise en compte du concept ‘FAIR' dans le partage des donnĂ©es: Vers une grille simplifiĂ©e d’évaluation du respect des critĂšres FAIR.

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    National audienceIndexed identifier ? Identification Are each data/dataset identified by an indexed and independant identifier ? Persistent metadata / data link ? Metadata traceability Are the metadata linked to the dataset through a persistent identifier? Metadata & authority linked ? Metadata traceability Are the metadata of each dataset linked to a unique authority (responsible for the datasets at a given time)? Unique, global, persistent ID? Identification Are the data identifiers unique, global and persistent ? Are the data identifiers unique, global and persistent ? Datasets linked to authority ? Metadata traceability Are all datasets linked to an authority (legal entity) through a unique and persistent identifier over time (e.g. institution, association or established body)? In case of a legal reuse restriction (such as personal data, state and public security, national defense secret, confidentiality of external relations, information systems security, secrets in industrial and commercial matters) , is the restriction properly justified?SHARC (SHAring Reward & Credit) est un groupe d’intĂ©rĂȘt scientifique interdisciplinaire crĂ©Ă© dans le cadre de RDA (Research Data Alliance) dans le but de faciliter le partage des donnĂ©es de recherche (et des ressources) par la valorisation de l’ensemble des activitĂ©s prĂ©-requises Ă  ce partage, tout au long du cycle de vie des donnĂ©es. Dans ce cadre, un sous-groupe de travail SHARC Ă©labore des grilles d’évaluation des chercheurs afin de mesurer leur niveau de prise en compte des principes FAIR dans la gestion de leurs donnĂ©es.La grille d’évaluation prĂ©sentĂ©e dans ce poster est destinĂ©e Ă  ĂȘtre complĂ©tĂ©e par tout scientifique produisant et / ou utilisant des donnĂ©es. Il s'agit d'un rĂ©sumĂ© d'une grille d'Ă©valuation plus Ă©tendue conçue pour un partage optimal des donnĂ©es (non encore mise en Ɠuvre pour le moment par la plupart des scientifiques).L'Ă©valuation est basĂ©e sur les critĂšres de conformitĂ© FAIR. Pour remplir cet objectif, la grille affiche le minimum de critĂšres qui doivent absolument ĂȘtre appliquĂ©s par les chercheurs pour attester de leur pratique FAIR. Ces critĂšres sont organisĂ©s en 5 groupes: «Motivations de partage»; "Trouvable", "Accessible", "InteropĂ©rable" et "RĂ©utilisable". Pour chaque critĂšre, 4 degrĂ©s d’évaluation sont proposĂ©s ("Jamais / Non Ă©valuable"; "Si obligatoire"; "Parfois"; "Toujours"). Au moins un degrĂ© mais un seul doit ĂȘtre sĂ©lectionnĂ© par critĂšre. L'Ă©valuation doit ĂȘtre effectuĂ©e pour chaque catĂ©gorie F / A / I / R; L'Ă©valuation finale est la somme de chaque degrĂ© cochĂ© rapportĂ©e au nombre total de critĂšres dans chaque catĂ©gorie F / A / I / R. Des rĂšgles d'interprĂ©tation prenant en compte les «motivations du partage» sont proposĂ©es
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