Rôle immunomodulateur du lenalidomide sur les cellules Natural Killer humaines. Etude des effecteurs lymphoïdes cytotoxiques chez les patients atteints de leucémies aiguës myéloïdes en 1ère rémission complète

La plupart des patients atteints de leucémies aiguës myéloïdes (LAM) sont en rémission complète après la chimiothérapie d'induction (1ère RC). Or, la persistance de cellules cancéreuses résistantes aux traitements et à la réponse immunitaire engendre de nombreuses rechutes. Nos travaux montrent la prééminence et la persistance de cellules NK CD56bright immatures chez ces patients en 1ère RC. Ainsi, tout agent ou tout protocole favorisant la reconstitution ou potentialisant les fonctions cytotoxiques des cellules NK pourrait être bénéfique. Nous avons ainsi choisi d'évaluer le rôle du Lenalidomide sur les cellules NK de donneurs sains. Nos travaux montrent, pour la 1ère fois, un effet direct mais cependant complexe sur les cellules NK. En effet, le Lenalidomide augmente l'expression du CD56 mais diminue la production d'IFN-gamma deux effets qui s'opposent au regard de la description classique des cellules NK CD56bright. L'augmentation du CD56 ne présagerait pas d'un effet bénéfique sur les cellules NK des patients LAM en 1ère RC. De plus, la modulation de l'expression de divers récepteurs de lyse n'a pas d'impact sur les fonctions cytotoxiques de ces effecteurs. Cependant, la diminution de l'expression de l'IFN-gamma pourrait être bénéfique car ce dernier favorise l'expression des molécules du CMH-I par les cellules leucémiques de LAM et inhibant ainsi l'activité cytotoxique des cellules NK. Les nombreuses actions du Lenalidomide sur le microenvironnement tumoral encouragent à poursuivre l'étude de ces effets dans les LAM.Most Acute Myeloide Leukemia (AML) patients achieve complete remission after induction chemotherapy (1st CR). Nevertheless, persistence of resistant tumor cells to treatments and immune response is associated with high risk of relapse. Our works highlight a pre-eminence and persistence of immature CD56 bright NK cells in patients at 1st CR. Thus, every agent or protocol dedicated to improve reconstitution or to potentiate cytotoxic functions of NK cells would be beneficial. Thus, we have chosen to evaluate the role of Lenalidomide upon NK cells of healthy donors. Our works show, for the first time, a direct but complexe effect on NK cells. Indeed, Lenalidomide upregulates the CD56 expression but decreases IFN-gamma production, two opposite effects with respect to the classical description of CD56bright NK cell population. The upregulation of CD56 do not augur a beneficial effect on NK cells of AML patients in 1st CR. Moreover, the modulation of expression of various lytic receptors has no impact on NK cell cytotoxic functions. However, the decrease of IFN-gamma expression coulb be beneficial as it upregulates MHC-I expression on AML leukemic cells and inhibit NK cell cytotoxic activity. The wide range of Lenalidomide actions on tumoral microenvironment is a strong support to pursue the study of its effects on AML

Optimized nucleus isolation protocol from frozen mouse tissues for single nucleus RNA sequencing application

The single cell RNA sequencing technique has been particularly used during the last years, allowing major discoveries. However, the widespread application of this analysis has showed limitations. Indeed, the direct study of fresh tissues is not always feasible, notably in the case of genetically engineered mouse embryo or sensitive tissues whose integrity is affected by classical digestion methods. To overcome these limitations, single nucleus RNA sequencing offers the possibility to work with frozen samples. Thus, single nucleus RNA sequencing can be performed after genotyping-based selection on samples stocked in tissue bank and is applicable to retrospective studies. Therefore, this technique opens the field to a wide range of applications requiring adapted protocols for nucleus isolation according to the tissue considered. Here we developed a protocol of nucleus isolation from frozen murine placenta and pancreas. These two complex tissues were submitted to a combination of enzymatic and manual dissociation before undergoing different steps of washing and centrifugation. The entire protocol was performed with products usually present in a research lab. Before starting the sequencing process, nuclei were sorted by flow cytometry. The results obtained validate the efficiency of this protocol which is easy to set up and does not require the use of commercial kits. This specificity makes it adaptable to different organs and species. The association of this protocol with single nucleus RNA sequencing allows the study of complex samples that resist classical lysis methods due to the presence of fibrotic or fatty tissue, such as fibrotic kidney, tumors, embryonic tissues or fatty pancreas

Increasing Antiproliferative Properties of Endocannabinoids in N1E-115 Neuroblastoma Cells through Inhibition of Their Metabolism

The antitumoral properties of endocannabinoids received a particular attention these last few years. Indeed, these endogenous molecules have been reported to exert cytostatic, apoptotic and antiangiogenic effects in different tumor cell lines and tumor xenografts. Therefore, we investigated the cytotoxicity of three N-acylethanolamines – N-arachidonoylethanolamine (anandamide, AEA), N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA) - which were all able to time- and dose-dependently reduce the viability of murine N1E-115 neuroblastoma cells. Moreover, several inhibitors of FAAH and NAAA, whose presence was confirmed by RT-PCR in the cell line, induced cell cytotoxicity and favored the decrease in cell viability caused by N-acylethanolamines. The most cytotoxic treatment was achieved by the co-incubation of AEA with the selective FAAH inhibitor URB597, which drastically reduced cell viability partly by inhibiting AEA hydrolysis and consequently increasing AEA levels. This combination of molecules synergistically decreased cell proliferation without inducing cell apoptosis or necrosis. We found that these effects are independent of cannabinoid, TRPV1, PPARα, PPARγ or GPR55 receptors activation but seem to occur through a lipid raft-dependent mechanism. These findings further highlight the interest of targeting the endocannabinoid system to treat cancer. More particularly, this emphasizes the great potential benefit of designing novel anti-cancerous therapies based on the association of endocannabinoids and inhibitors of their hydrolysis

Salvinorine A, principe actif de Salvia divinorum (premier agoniste naturel non azoté sélectif des récepteurs opioïdes kappa)

CAEN-BU Médecine pharmacie (141182102) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA.

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells and to extract high quality RNA from these cells

Contribution à l'étude botanique et mycologique de la prairie de Caen

CAEN-BU Médecine pharmacie (141182102) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

Sex chromosomes and quantitative sex expression in monoecious hemp (Cannabis sativa L.)

Hemp (Cannabis sativa) has a highly variable sexual phenotype. In dioecious hemp, the sex is controlled by heteromorphic sex chromosomes according to an X-to-autosomes equilibrium. However, in monoecious hemp, the sex determinism remains widely unknown and has never been related to a quantitative approach of sex expression. The present paper aims to contribute to the comprehension of the sex determinism in monoecious hemp by assessing the genotypic variability of its sex expression and establishing its sex chromosomes. Five monoecious and one dioecious cultivars were grown in controlled conditions under several photoperiods. The monoecy degree of 194 monoecious plants was recorded at each node by a figure ranging from 0 (male flowers only) to 6 (female flowers only). The genome size of 55 plants was determined by flow cytometry. The DNA of 115 monoecious plants was screened with the male-associated marker MADC2. The monoecy degree varied significantly among monoecious cultivars from 3.36 ± 2.28 in 'Uso 31' to 5.70 ± 0.81 in the most feminised 'Epsilon 68'. The variation of monoecy degree among cultivars remained consistent across trials despite a significant "cultivar × trial" interaction and partly agreed with their earliness. The genome size of monoecious plants (1.791 ± 0.017 pg) was not different from that of females (1.789 ± 0.019 pg) but significantly lower than that of males (1.835 ± 0.019 pg). MADC2 was absent from all monoecious plants. These results strongly support that cultivars of monoecious hemp have the XX constitution and that their sex expression has a genetic basis. © 2013 Springer Science+Business Media Dordrecht

Recycling of CFRP for high value applications: Effect of sizing removal and environmental analysis of the SuperCritical Fluid Solvolysis

The recycling of Carbon Fibers Reinforced Plastics (CFRP) wastes is becoming increasingly important in the aerospace industry. For most of the technologies, the recycled CF (rCF) are discontinuous, misaligned and unsized. Compared to thermal treatments, the orientation, the length and the brittleness of the rCF are better preserved with the SuperCritical Water Solvolysis (SCWS). The effect of the sizing removal on the recycled CFRP behavior is studied by conducting static characterizations. Realigned virgin CF were used to manufacture sample plates which underwent short beam shear tests. An environmental assessment was carried out on the CFRP end-of-life with a focus on SCWS

Image3.TIF

<p>The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells.</p

Robust SIFT-Based Hierarchical Video Mosaicing for Endomicroscopy

We present a method to perform video mosaicing for endomicroscopy with two major improvements compared to the state of the art. First, instead of using individual images directly, we start by creating sub-mosaics from short video sub-sequences using iconic registration. The sub-mosaics are then considered for registration. Second, groupwise estimation is performed between all sub-mosaics based on SIFT matching to infer globally consistent spatial transformations. Both improvements increase robustness of the reconstruction compared to a baseline mosaicing method