Perturbations of the CD8+ T-cell repertoire in CVID patients with complications

AbstractA higher chronic expansion of effector cytotoxic CD8+DR+ T-lymphocytes has been reported in common variable immunodeficiency (CVID) patients with complications such as splenomegaly, autoimmune disease and/or granulomatous disease. In order to document the features associated with this T cell activation involving the CD8+ T-compartment, we examined the diversity of the alpha/beta TCR repertoire of the patient's CD8+ T-lymphocytes using the qualitative analysis of the CDR3 lengths (Immunoscope).Ten CIVD patients were enrolled in this study, four without complications (Group 1), six with complications (Group 2). All patients exhibited non-gaussian altered CDR3 length distributions, albeit to different extent within the different Vβ families. CVID patients with activated CD8+ T-cells show a reduction of their TCR repertoire diversity which is more severe in patients with complications. Viral reactivations such as CMV are suspected to be part of the mechanisms underlying immunosenescence

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha-L-iduronidase

International audienceThe Brassica rapa hairy root based expression platform, a turnip hairy root based expression system, is able to produce human complex glycoproteins such as the alpha—L—iduronidase (IDUA) with an activity similar to the one produced by Chinese Hamster Ovary (CHO) cells. In this article, a particular attention has been paid to the N‐ and O‐glycosylation that characterize the alpha‐L‐iduronidase produced using this hairy root based system. This analysis showed that the recombinant protein is characterized by highly homogeneous post translational profiles enabling a strong batch to batch reproducibility. Indeed, on each of the 6 N‐glycosylation sites of the IDUA, a single N‐glycan composed of a core Man3GlcNAc2 carrying one beta(1,2)‐xylose and one alpha(1,3)‐fucose epitope (M3XFGN2) was identified, highlighting the high homogeneity of the production system. Hydroxylation of proline residues and arabinosylation were identified during O‐glycosylation analysis, still with a remarkable reproducibility. This platform is thus positioned as an effective and consistent expression system for the production of human complex therapeutic proteins

Analyse de la régulation du TCR dans différentes situations immunologiques

Grâce à l'utilisation d'une nouvelle méthode d'analyse de la régulation de la chaine b du TCR, nommée TcLandscape, nous avons observé que les cellules T naïves stimulées in vitro dans des conditions de reconnaissance " directe " stricte présentaient un profil TCR particulier. En effet, la reconnaissance directe du CMH allogénique par des cellules T naïves est associée à une forte accumulation des transcrits Vb, sans altération de la distribution de taille du CDR3.....Ces profils mettant en évidence des cellules T présentant une distribution altérée de la taille du CDR3 permettent d'identifier et de hiérarchiser des populations impliquées dans la réaction immunitaire. Une telle capacité s'avère particulièrement utile pour suivre dans le temps la réponse immune dans divers contextes. Un exemple ayant trait à des patients infectés par le VIH et vaccinés avec un mélange de lipopeptides est développé.Using a new global approach referred to as TcLandscape, we previously observed that direct -type pathway of allorecognition was associated with a strong accumulation of V~ transcript without skewing of CDR3 length distribution. Such a pattern was also observed in vivo in acute rejection of cardiac allografts and in acute delayed rejection of " accommodated " cardiac xenografis in rats. In contrast, T cell infiltrating cardiac tolerated allografis showed an altered pattern of TCR V~ chain that might represent the molecular signature of regulatory T cells. This pattern allowed to attribute quantitative difference in the response of different T cell families. This quantitative/qualitative approach allowed to follow over time the effect of an immune-based therapy in HIV-1 infected patients vaccinated with a mixture of lipopeptides.NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Understanding existing and designing novel synthetic routes to Pd-PEPPSI-NHC and Pd-PEPPSI-PR3 pre-catalysts

The reaction mechanism leading to the formation of cross-coupling palladium pre-catalysts of the PEPPSI family was investigated. Two intermediates were isolated and proved to be both suitable synthons to the pre-catalysts, with one permitting the design of a novel and greener user-friendly synthetic route. In light of this mechanistic understanding, the traditional one-pot method was shown to be possible using stoichiometric amounts of throw-away ligand, which represents a considerable synthetic improvement over the wasteful "in pyridine" approach

A general protocol for the synthesis of Pt-NHC (NHC = N-heterocyclic carbene) hydrosilylation catalysts

A general, user-friendly synthetic route to [Pt(NHC)(L)Cl-2] and [Pt(NHC)(dvtms)] (L = DMS, Py; DMS = dimethyl sulfide, dvtms = divinyltetramethylsiloxane, Py = pyridine) complexes has been developed. The procedure is applicable to a wide range of ligands and enables facile synthetic access to key Pt(0)- and Pt (II)-NHC complexes used in hydrosilylation catalysis

Improving Yield of a Recombinant Biologic in a Brassica Hairy Root Manufacturing Process.

International audienceHairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha-L-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4-D + 20.5 g/l KNO(3) ) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A >150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming. This article is protected by copyright. All rights reserved