Caractérisation des plasmides recombinants exprimant trois formes de récepteurs TNFR bioactifs capables d’inhiber, in vitro, l’effet de la cytokine pro-inflammatoire TNFα

RÉSUMÉ L’arthrose rhumatoïde est une maladie inflammatoire auto-immune. Elle provoque une inflammation chronique des articulations et des os qui conduisent à une destruction progressive de ces derniers. Cette maladie affecte 0.5 à 1 % de la population des pays industrialisés et impose des coûts importants au système de santé. La société canadienne de l’arthrose rapporte que 6 % des hospitalisations totales au Canada sont imputables à l’arthrite. La cytokine TNFα joue un rôle important dans le processus inflammatoire lié à l’arthrose de ce fait la neutralisation et/ou l’inhibition de la surexpression de cette cytokine est une cible prometteuse pour le traitement de l’arthrose rhumatoïde. Il existe plusieurs molécules biologiques bloquant la cytokine TNFα, utilisées avec grand succès pour le traitement de l’arthrose tel que l’anticorps monoclonal anti TNFα Infliximab et le récepteur soluble TNFRII Etanercept. Cependant la durée de vie de ses protéines en circulation est faible et peut nécessiter une augmentation des doses et des injections, avec le risque d’augmentation de la toxicité de ses protéines. Ces inconvénients associés à cette approche thérapeutique peuvent être contournés par une approche de thérapie génique qui permet une production stable de la protéine dans le temps et une expression localisée du transgène. Notre objectif est, en choisissant le rat comme modèle animal de l’arthrose rhumatoïde, de construire des plasmides recombinants contenant des transgènes codant pour des protéines recombinantes correspondantes au récepteur soluble du TNFα capables de bloquer le TNFα. Les plasmides recombinants sont livrés in vitro aux cellules à l’aide d’un système de livraison polymérique, le chitosane. Le chitosane s’est avéré le système de livraison idéal pour la protection de L’ADN plasmidique contre la dégradation par les nucléases et pour leur livraison au niveau du cytosol. Nous avons donc construit trois transgènes codant pour des protéines mimant le récepteur soluble du TNFα du rat. Le premier (TNFR) code pour la partie extracellulaire soluble du récepteur TNFRII. Le second (IgTNFR) code pour la partie extracellulaire soluble du récepteur TNFRII couplé à la séquence codante pour les régions CH2 et CH3 de la partie constante d’une immunoglobuline de type G1 (IgG1) du rat excluant la région charnière. Le troisième (IgTNFRd) composé de la partie extracellulaire soluble du récepteur TNFRII couplée à la séquence codante pour les régions CH2 et CH3 de la partie constante d’un IgG du rat incluant la région charnière. La région charnière permet la dimérisation via pont disulfure de l’anticorps et donc de la protéine recombinante IgTNFRd. Le vecteur d’expression eucaryotique pVax1 sécuritaire et approuvé par la FDA (food and drugs administation) a été utilisé pour l’expression de nos trois transgènes.----------ABSTRACT Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects joints and leads to gradual destruction of bones and articulations. RA affects 0.5 to 1 % of the population in industrial countries and imposes considerable cost on the healthcare system. According to The Arthritis Society 6 % of total hospitalization in Canada are due to arthritis. The tumor necrosis factor (TNF), a proinflammatory cytokine, plays a key role in the pathogenesis of RA and a lot of drugs targeting this molecule have emerged to treat RA. These molecules such as anti-TNF monoclonal antibody or soluble TNF receptor II, Etanercept, are currently in use to treat RA with success. However, this molecule have some limitation, their half-life is short in circulation requiring high doses and multiple injections which increases their risk to different infections. This limitation can be overcome by gene therapy based strategy. Gene therapy allows a stable and localized production of the therapeutic transgene. Our goal was to design and construct three recombinant plasmids encoding soluble TNF receptors capable of inhibiting TNF in solution. The first recombinant plasmid, TNFR encodes for the extracellular region of the rat TNF receptor II. The second recombinant plasmid, IgTNFR encodes for the extracellular region of the rat TNF receptor II merged to the CH2 and CH3 regions of the constant region of rat immunoglobulin G1 without the hinge region. The third recombinant plasmid, IgTNFRd encodes for the extracellular region of the rat TNF receptor II merged to the CH2 and CH3 regions of the constant region of rat immunoglobulin G1 including the hinge region. The hinge region is responsible for antibody dimerization by disulfure bond. The safe and FDA (food and drugs administration) approved eukaryote plasmid, pVax was selected as expressing vector of the three transgenes. The recombinant plasmids are complexed with the polymeric carrier chitosan to form chitosan-based nanoparticles that protect plasmid DNA against digestion by nucleases until their delivery to the cytosol. The formulation of chitosan 92-10-5 (degree of deacetylation- molecular weight- ratio nitrogen: phosphate) was used to complex recombinant pDNA to form nanoparticles. These nanoparticles were characterized using techniques of electron microscope (ESEM) and dynamic light scattering (DLS). These two techniques showed nanoparticles with spherical and cylindrical shapes with an average diameter of 95 nm and a zeta potential of 30 mV. During this research project, we have clearly demonstrated the capacity of chitosan to protect the plasmid DNA against nuclease digestion at supraphysiological concentrations with the detection of transgene-specific mRNA specific transgene in HEK293 and CHO transfected cell lines

Collective beating of artificial microcilia

We combine technical, experimental and theoretical efforts to investigate the collective dynamics of artificial microcilia in a viscous fluid. We take advantage of soft-lithography and colloidal self-assembly to devise microcapets made of hundreds of slender magnetic rods. This novel experimental setup is used to investigate the dynamics of extended cilia arrays driven by a precessing magnetic field. Whereas the dynamics of an isolated cilium is a rigid body rotation, collective beating results in a symmetry breaking of the precession patterns. The trajectories of the cilia are anisotropic and experience a significant structural evolution as the actuation frequency increases. We present a minimal model to account for our experimental findings and demonstrate how the global geometry of the array imposes the shape of the trajectories via long range hydrodynamic interactions.Comment: 5 pages, 3 figure

Gestures convey different physiological responses when performed toward and away from the body

We assessed the sympathetic and parasympathetic activation associated to the observation of Pantomime (i.e. the mime of the use of a tool) and Intransitive gestures (i.e. expressive) performed toward (e.g. a comb and "thinking") and away from the body (e.g. key and "come here") in a group of healthy participants while both pupil dilation (N = 31) and heart rate variability (N = 33; HF-HRV) were recorded. Large pupil dilation was observed in both Pantomime and Intransitive gestures toward the body; whereas an increase of the vagal suppression was observed in Intransitive gestures away from the body but not in those toward the body. Our results suggest that the space where people act when performing a gesture has an impact on the physiological responses of the observer in relation to the type of social communicative information that the gesture direction conveys, from a more intimate (toward the body) to a more interactive one (away from the body).We would like to thank Johanna Viana, Raphael Kroll and Alberto Villar for their precious help in the task construction and in the recruitment of participants. We also thank all the participants that entered the study. This work was funded by the French National Research Agency (ANR-11-EQPX-0023) and supported by European funds through the program FEDER SCV-IrDIVE. This study was partially conducted at the Psychology Research Centre (PSI/01662), University of Minho, and supported by the Portuguese Foundation for Science and Technology and the Portuguese Ministry of Science, Technology and Higher Education (UID/PSI/01662/2019), through the national funds (PIDDAC)

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Immune responses and liver-based avoidance strategy in rAVV muscle gene therapy

Les réponses immunes contre les traitements de thérapie génique rAAV, dirigées non seulement contre la capside du vecteur, mais aussi contre le transgène, représentent un défi majeur en particulier dans le contexte d'un transfert de gènes vers le muscle pour le traitement de diverses maladies monogéniques du muscle. Cette thèse est focalisée sur la réponse dirigée contre le transgène. Celle-ci se met en place contre des éléments du transgène thérapeutique qui ne sont pas initialement présents, ou insuffisamment exprimés, chez l'hôte et peut entraîner l'élimination des cellules qui produisent le transgène. Dans ce contexte il est nécessaire de concevoir des protocoles de traitement capable d'imposer en périphérie une tolérance immunitaire spécifique du transgène. Nous avons exploré comment l'induction de l'expression d'un transgène étranger dans le foie à l'aide d'un vecteur rAAV impose une tolérance immunologique après transduction du muscle, et ceci malgré l'initiation dans le muscle d'une réponse T CD8+ et CD4+ dirigé contre le transgène. Nous avons démontré que l'expression du transgène dans le foie promeut une tolérance immunitaire envers une injection du vecteur rAAV dans le muscle et ce malgré la présence d'une réponse mémoire spécifique du transgène. Sur le plan de la réponse T CD8+, nous constatons une délétion totale ou partielle ainsi qu'une conversion des cellules vers un phénotype d'épuisement, avec l'expression du marqueur PD-1. La présence d'une réponse T CD4+ mémoire contre le transgène, ne semble pas altérer la tolérance induite par le foie. Nous discutons ensuite, en nous confrontant aux auteurs du domaine, le mécanisme global éventuellement mis en jeu : Une fois la réponse adaptative complète T CD8+ et T CD4+ initiée dans le ganglion drainant le muscle, ces cellules T doivent, avant de retourner vers le muscle, circuler par le foie. Dans le foie, elles s'y accumulent et rencontrent une seconde fois l'antigène, ce qui semble conduire à l'épuisement, suite à l'expression de PD-1, ou à la mort des cellules T CD8+ par un mécanisme décrit sous le nom d'emperipolesis suicidaire ou d'apoptose dépendant du facteur BIM. Les cellules CD4+, elle, ne subiraient pas de délétion, mais une éventuelle modification phénotypique, vers un phénotype tolérogène ou suppresseur. Ce processus serait dépendant de la rétention des cellules T dans le foie que nous avons mis en évidence, ainsi que de l'affinité de la réponse contre le transgène qui rend compte de nos résultats. Nous exposons finalement le domaine d'application de notre méthode, permettant de surmonter une réponse locale initiée dans le muscle, ce qui peut s'avérer crucial pour le traitement par thérapie génique des dystrophies musculaires monogéniques, basé sur l'emploi de transgènes potentiellement immunogènes dû au défaut d'expression chez le receveur.It is increasingly realized that immune responses to rAAV gene therapy treatments, not only to the vector capsid but also to reparative transgenes can cause adverse effects of importance in the case of muscle gene delivery in monogenic muscle disorders. These responses to "foreign" sequence elements of the reparative transgene that are not initially present or insufficiently expressed in the host represent a threat which can eliminate the transduced cells of interest. It is therefore highly desirable to design immune tolerance protocols able to impose transgene-specific immunological unresponsiveness. We explored here how liver-based recombinant adeno-associated viral vector (rAAV) mediated expression of foreign transgene imposes immune tolerance after immunogenic rAAV muscle transduction. We found that liver transgene expression driven with the hAAT hepatocyte specific promotor is effective to promote robust muscle-associated transgene expression, nullifying transgene-specific CD8+ T cell responses as well as humoral responses. Liver transgene expression equally promotes immune tolerance to subsequent rAAV muscle injections despite the presence of transgene-specific memory responses. Importantly, the CD8+ T cell tolerization process leads to partial deletion and conversion into PD-1+ CD8+ T cells, a hallmark of T cell exhaustion. Likewise, CD4+ T cell responses elicited in muscle do not compromise liver-based tolerance induction. Our results demonstrate that liver transduction with rAAV vectors using hepatocyte specific hAAT promotor imposes immune tolerance to transgene-specific T cells elicited from the naïve T cell repertoire after muscle transduction. Confronting our results with others, we suggest that CD8+ T cell depletion occurs after antigen recognition through direct capture and internalization of T cell corpses by hepatocyte in a mechanism already described and referred to as suicidal emperipolesis. Alternatively, BIM-dependent apoptotic process may also occur as a result of T cell-hepatocyte interactions. Regarding transgene-specific CD4+ T cells, we presume that they undergo phenotypic conversion such as Treg conversion as evidenced in multiple sclerosis models. Our results also suggest that the retention time and affinity of transgene-specific T cells next to transduced hepatocytes is critical for their fate. Last, considering applications for muscle gene therapies, control of local muscle immune response is of crucial importance in the treatment of several muscular dystrophies including Duchenne's muscular dystrophy. In this context our liver-based tolerance induction protocol is relevant, provided that the transgene of interest does not alter hepatocytes functions and is beneficial against adverse immune responses

A key aspect to consider for vaccinal protection is the induction of a local line of defense consisting of nonrecirculating tissue-resident memory T cells (TRM), in parallel to the generation of systemic memory CD8+ T cell responses. The potential to induce TRM has now been demonstrated for a number of pathogens and viral vectors. This potential, however, has never been tested for recombinant adeno-associated virus (rAAV) vectors, which are weakly inflammatory and poor transducer of dendritic cells. Using a model rAAV2/1-based vaccine, we determined that a single intradermal immunization with rAAV2/1 vectors in mice induces fully functional TRM at the local site of immunization. The optimal differentiation of rAAV-induced transgene-specific skin TRM was dependent on local transgene expression and additional CD4+ T cell help. Transgene expression in dendritic cells, however, appeared to be dispensable for the priming of transgene-specific skin TRM, suggesting that this process solely depends on the cross-presentation of transgene products. Overall, this study provides needed information to properly assess rAAV vectors as T cell-inducing vaccine carriers.IMPORTANCE rAAVs display numerous characteristics that could make them extremely attractive as vaccine carriers, including an excellent safety profile in humans and great flexibility regarding serotypes and choice of target tissue. Studies addressing the ability of rAAV to induce protective T cell responses, however, are scarce. Notably, the potential to induce a tissue-resident memory T cell response has never been described for rAAV vectors, strongly limiting further interest for their use as vaccine carriers. Using a model rAAV2/1 vaccine delivered to the skin, our study demonstrated that rAAV vectors can induce bona fide skin resident TRM and provides additional clues regarding the cellular mechanisms underlying this process. These results will help widen the field of rAAV applications.

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Photonique THz : Lumière laser structurée pour l'émission THz & modulation THz pilotée par laser

International audienceLa photonique est un bras de levier incontournable pour le développement des applications THz, grâce à la maturité des composants photoniques et aux fréquences optiques élevées. Nous présenterons ici une source THz pompée par une lumière laser structurée, ainsi qu'un modulateur d'amplitude THz intégré pompé optiquement

Digital PCR compartmentalization I. Single-molecule detection of rare mutations.

Polymerase chain reaction based techniques have been widely used in laboratory settings. Several applications in oncology, virology or prenatal diagnosis require highly sensitive detection methods, which cannot be achieved with conventional techniques. Digital PCR (dPCR) was developed from the association of PCR and limiting dilution procedures. It is based on the compartmentalization of DNA molecules in small volumes. Controlling the size and the content of each compartment is crucial to obtain a high sensitivity with a single molecule resolution. Microfluidics offers promising tools to isolate DNA fragments such as microdroplets, microchambers or microwells with volumes ranging from few picoliters to nanoliters. The review provides an overview of recent developments of microfluidics dPCR platforms and how this technology can influence the management of cancer patients