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Clinical translation of dendritic cell (DC)-based cell therapy requires preclinical studies in nonhuman primates (NHPs). The aim of this work was to establish the in vitro conditions for generation of NHP tolerogenic DCs (Tol-DCs), as well as to analyze the molecular mechanisms by which these cells could control an immune response. Two populations of NHP bone marrow-derived DCs (BMDCs) were obtained: adherent and nonadherent. Although both populations displayed a quite similar phenotype, they were very different functionally. We characterized the adherent BMDCs as Tol-DCs that were poor stimulators of T cells and actively inhibited T-cell proliferation, whereas the nonadherent population displayed immunogenic properties in vitro. Interestingly, the anti-inflammatory and immunosuppressive enzyme heme oxygenase-1 (HO-1) was up-regulated in Tol-DCs, compared to the immunogenic BMDCs. We demonstrated that HO-1 mediates the immunosuppressive properties of Tol-Des in vitro (in NHPs and rats) and that HO-1 is involved in the in vivo tolerogenic effect of Tol-DCs in a rat model of allotransplantation. In conclusion, here we characterized the in vitro generation of NHP T DCs. Furthermore, we showed for the first time that HO-1 plays a role in the active inhibition of T-cell responses by rat and NHP Tol-DCs. [ABSTRACT FROM AUTHOR]

Mitral Valve Prolapse (MVP) affects 3% of the population and is characterized by a heterogeneous mitral leaflet remodeling. The pathophysiological mechanisms involved in MVP development are not fully understood, the only therapeutic option remains the surgical valve replacement. The first mutation causing MVP has been recently identified on the FLNA gene. The generation of a unique knock-in (KI) rat model for the FLNA-P637Q mutation paves the road to study the molecular mechanisms involved in MVP development. The aim of our study was to characterize the functional, morphological and molecular expression of the valvular disease in our unique KI rat model. 5 wild-type (WT) and 10 KI rats were evaluated at 3, 6 and 26 weeks. Comprehensive 2D echocardiography was performed to determine valve function and morphology. 3D quantitative analysis of the mitral valve (MV) remodelling was done using micro-tomodensitometry (microCT). MV tissue composition was analysed based on histological and immunohistochemistry. Based on the qualitative echocardiographic assessment of the valve, a high genotype-phenotype concordance was observed (100%, 93% and 100% for each time points). The anterior leaflet was longer in KI comparatively to WT rats (+ 12 to + 14% increase at all time points P < 0.01). Using microCT analysis, we confirmed the larger 3D MV volume in KI compared to WT (+ 20 to + 58%; all time points P < 0.05). Histological and immunohistological analyses pointed out towards a myxomatous valve disease (leaflet's thickening, hypercellularity, proteoglycans accumulation without calcification). Our results attest for the presence of a myxomatous MV dystrophy in the KI rat model comparable to the one described in MVP patients. These findings confirm this unique model is pertinent for the study of pathophysiological molecular mechanisms associated with MVP development and offers a new opportunity to identify potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Transplantation faces several major obstacles that could be overcome by expression of immunomodulatory proteins through application of gene therapy techniques. Gene therapy strategies to prolong graft survival involve gene transfer of immunosuppressive or graft-protecting molecules. Very promising results have been obtained in small animal experimental models with inhibitors of co-stimulatory signals on T cells, immunosuppressive cytokines, donor major histocompatibility antigens and regulators of cell apoptosis or oxidative stress. The application of gene therapy techniques to transplantation offers a great experimental and therapeutic potential. Local production of immunosuppressive molecules may increase their therapeutic efficiency and reduce their systemic effects. When compared with other clinical situations, gene therapy in transplantation offers several potential advantages. Gene transfer into the graft can be performed ex vivo, during the transit between the donor and the recipient, thus avoiding many of the hurdles encountered with in vivo gene transfer. Furthermore, the difficulties associated with immune responses to the gene transfer vectors and transient gene expression may be easier to overcome when gene therapy protocols are applied to transplantation than when applied to other clinical situations. The next century should witness a rapid increase in the application of gene therapy techniques to large animal pre-clinical models of transplantation and later to clinical trials. Gene Therapy (2000) 7, 14–19. [ABSTRACT FROM AUTHOR]

Mitral Valve Prolapse (MVP) affects 3% of the population and is characterized by a heterogeneous mitral leaflet remodeling. The pathophysiological mechanisms involved in MVP are not fully understood, the only therapeutic option remains the surgical valve replacement. We previously identified FLNA as the first gene causing MVP and generated a unique knock-in rat model for the FLNA-P637Q mutation. The aim of our study was to characterize the morphological, functional and molecular expression of the valvular disease in our KI rat model. Five WT and 10 KI rats were evaluated at 3, 6 and 13 weeks. Comprehensive 2D echocardiography was performed to determine valve function and morphology. 3D analysis of the mitral valve (MV) remodelling was done using micro computed tomography (μCT). MV tissue composition was analyzed by histology. Transcriptomic comparison was performed using RNA-sequencing. Echocardiographic assessment confirmed the presence of MVP and elongated anterior leaflet in KI comparatively to WT rats (+12 to +14% at all time points P < 0.01)]. This was corroborated by an increased leaflets volume quantified by μCT (+20 to +58% in KI vs. WT all time points P < 0.05). Histological analyses revealed a myxomatous valve disease in KI rats. RNAseq unveiled that genes part of "cell chemotaxis" GO term (GO :0060326, P = 2.31 × 10−5) including Ccl12 and S100a8 were significantly upregulated (+2.44x, +8.40x, respectively). As were Klf4 (+1.31x) and Tgfbr1 (+1.21x) for "endothelial cell migration" (GO:0043542, P = 1.59 × 10−6). Genes part of the GO:0048771 "tissue remodelling" (P = 5.52 × 10−5) were also found upregulated. These results establish that the KI FLNA-P637Q rat constitutes a pertinent model to study the pathophysiological molecular mechanisms associated with MVP. Our results point to pathways including inflammation and epithelial activation, which constitute potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Animal study results have suggested a role in sepsis for human interleukin for DAl.a cells/leukemia inhibitory factor (HILDA/LIF). HILDA/LIF and interleukin-6 (IL-6) levels were prospectively studied by serial ELISAs in 34 septic patients. HILDA/LIF was detected in 11 of 34 patients at plasma levels of 100–37,000 pg/mL. Peak HILDA/LIF levels correlated with increased temperature and creatinine and IL-6 and with decreased arterial CO2 (P < .05). Multivariate analysis showed that shock and decreased arterial CO2 accounted for 75% of peak HILDA/LIF plasma variations (R2 = .753). Fatal outcome was most often associated with detectable HILDA/LIF (> 56 pg/mL) and peak IL-6 plasma levels > 850 pg/mL (sensitivity, 83%; specificity, 87%), but both (at respective levels of > 480 and > 850 pg/mL) were associated with fatal outcome. HILDA/LIF was detected in septic patients exhibiting shock, and its levels correlated with higher mortality and shorter survival. [ABSTRACT FROM PUBLISHER]

Mitral valve prolapse (MVP) affects 3% of the population and is characterized by a myxomatous MV remodeling. The pathophysiological mechanisms involved in MVP remain elusive and the only therapeutic option is to perform MV surgery. The first causing mutation in MVP has been identified on the FLNA gene. We recently generated a KI rat for the FLNA-P637Q mutation that develops MVP. A recent RNA-seq analysis revealed an upregulation of genes involved in immune response and inflammation pathways in the valves of KI rats compared to WT. The aim of this study was then to identify and quantify the immune cells present in MVs and assess their role in the development and/or progression of the disease. Flow cytometry was used to quantify the hematopoietic cells (CD45), resident macrophages (CD206), myeloid lineage (CD11b), endothelial progenitors (CD34) and endothelial cells (CD31) in the MV of 3-weeks old KI and WT rats. The location of those cells within the MV tissues was analysed by immunofluorescence on cryosectioned MVs. Cytometry results showed a higher proportion of CD45+ and CD206+ cells in the KI rats compared to WT (20% vs. 14% and 28% vs. 16%, respectively) but similar proportion of CD31+ (14%). The CD45 and CD206 immunofluorescences performed on WT animals showed a clear localization of the positive cells on the edge of the atrial side of the leaflet, in the sub-endothelium layer. In KI rats, CD45+ and CD206+ cells were diffusely located: close to the atrial side of the MV leaflet, but also into the medial third of the leaflet. This preliminary study showed that recruitment of immune cells in MVP could be an important player in the pathophysiological process leading the disease. To elucidate the specific roles of this pathway, the temporal recruitment of immune cells and their specific activity need to be studied over the course of the disease progression. [ABSTRACT FROM AUTHOR]

Success in xenotransplantation relies on engineering of the pig genome to express human transgenes, such as CD55/CD39, that can control coagulation and inflammation to prolong the graft survival of a 1,3-galactosyltransferase depleted (Gal-/-) pig organs in nonhuman primates and then able to bypass the hyperacute rejection. The aim of our work was to produce Gal-/- piglets overexpressing CD55/CD39. In experiment (Exp.) 1 exploiting 2 ubiquitous expression vectors (pCAGGS-CD55 and pCAGGS-CD39), we transfected immortalized porcine kidney cells (PK15) with CD55 and CD39 using Nucleofector (Amaxa Biosystems, Cologne, Germany) and selected 5 cell colonies each (PK15-CD55 and PK15-CD39) that were expanded and analyzed by RT-PCR, immunohistochemistry (IHC) and Western blot (WB). The monoclonal antibodies IA10 for hCD55 and BU61 for hCD39 were used. Transgenic transcription was confirmed by Northern blot (NB) using digoxigenin-labeled probes. In Exp. 2, a neonatal pig Gal-/- fibroblast line was co-transfected by Nucleofector using 2 ubiquitous expression vectors (hEF-CD55 and pCAGGS-CD39) for the expression of CD55 and CD39. Colonies were analyzed by RT-PCR and IHC only, because of the limited number of cells available. Cells from one colony with a high level of CD55/CD39 expression according to IHC were used for nuclear transfer into enucleated oocytes. Day 5 compact morula/blastocyst (n= 144) were transplanted in 2 synchronized sows. Porcine aortic endothelial cells (PAEC) and fibroblasts derived from 2 stillborn piglets were analysed with IHC, NB, and WB. The expression level of transgenes from both experiments was compared with human umbilical vein endothelial cells (HUVEC) by fluorescence-activated cell sorting (FACS), using IA10, BRIC110, IH4, 2G2, and MEM-118 antibodies for hCD55 and TU66 for hCD39. In Exp. 1, RT-PCR showed CD55 mRNA expression in 3 out of 5 (2, 15, 24) PK15-CD55 colonies. A high level of CD55 expression was confirmed only in colony 24 by IHC, NB, WB, and FACS. Low expression level in colony 2 revealed by FACS was not detected by IHC, indicating that FACS analysis is more accurate to quantify the level of expression. All PK15-CD39 colonies were positive according to RT-PCR and IHC. Only one colony PK15-CD39 was further analyzed by NB and WB and confirmed positive. In Exp. 2, IHC, NB, WB, and FACS analyses of fibroblasts and PAEC derived from both cloned piglets confirmed the high level of CD39 expression detected by IHC in donor cells used for nuclear transfer. However, strong CD55 expression detected by IHC was not confirmed by NB analyses and, by FACS, was lower than in HUVEC cells. In conclusion, we produced cloned CD55-CD39 transgenic Gal-/- piglets with a high level of CD39 expression but the expression level of CD55 was lower than in HUVEC cells. We found that although IHC is the method of choice in preliminary screening, it is not sufficiently quantitative when only a few cells for each clone are available. Thus, IHC needs to be complemented with additional methods (e.g. WB, FACS, real-time RT-PCR) to obtain complete evaluation of the expression pattern of transgenes before nuclear transfer experiments. This study was supported by EU grant no. LSHB-CT-2006-037377 and Fondazione Banca Popolare di Cremona. [ABSTRACT FROM AUTHOR]

Introduction: Bien que les agonistes et antagonistes des récepteurs adrénergiques soient largement utilisés en pratique, peu de données sont disponibles concernant les conséquences potentielles de leur utilisation sur le système immunitaire en général et les maladies auto-immunes en particulier. Pourtant, les cellules immunitaires, dont les cellules dendritiques (DC), expriment les adrénorécepteurs. Dans ce contexte, nous avons étudié les effets de la stimulation β2-adrénergique des DC sur leur dialogue avec les lymphocytes T CD8+. Matériels et méthodes: En utilisant des approches pharmacologiques et génétiques, nous avons analysé les effets de la stimulation β2-adrénergique des DC sur leur phénotype, leur capacité à prendre en charge et à processer des antigènes particulaires et solubles afin de les présenter sur les molécules de CMH-I et sur leur efficacité à induire une réponse T cytotoxique in vitro et in vivo Résultats: Nous avons démontré que la stimulation β2-adrénergique des DC leur confère un potentiel tolérogène caractérisé par une inhibition de leur capacité à cross-présenter des antigènes aux lymphocytes T CD8+. Cet effet majeur est médié par l’activation d’une voie de signalisation dépendante de la protéine Gαi/0 aboutissant à l’inhibition de la translocation nucléaire du complexe NFkB. De plus, cette inhibition de cross-présentation n’est due ni à une diminution de l’expression des molécules de co-stimulation ni à un défaut de captation des antigènes, mais à une diminution de la dégradation des antigènes et de leur chargement sur les molécules de CMH-I. In vivo, l’administration de β2-agonistes à des souris inhibe la cross-présentation des antigènes et l’activation des lymphocytes T CD8+. Conclusion: Ainsi, la stimulation β2-adrénergique des DC inhibe leur capacité à induire des réponses immunitaires spécifiques d’antigène en interférant avec la voie de la cross-présentation. Ces résultats pourraient permettre d’identifier de nouvelles cibles thérapeutiques du diabète auto-immun. [ABSTRACT FROM AUTHOR]