biblio

2019

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  Zhang, Y, Clay, D, Mitjavila-Garcia, MT, Alama, A, Mennesson, B, Berseneff, H, Louache, F, Bennaceur-Griscelli, A & Oberlin, E 2019, « VE-Cadherin and ACE Co-Expression Marks Highly Proliferative Hematopoietic Stem Cells in Human Embryonic Liver », Stem Cells and Development, vol. 28, no. 3, p. 165-185.
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  Résumé : Despite advances to engineer transplantable hematopoietic stem and progenitor cells (HSPCs) for research and therapy, an in-depth characterization of the developing human hematopoietic system is still lacking. The human embryonic liver is at the crossroad of several hematopoietic sites and harbors a complex hematopoietic hierarchy, including the first actively dividing HSPCs that will further seed the definitive hematopoietic organs. However, few are known about the phenotypic and functional HSPC organization operating at these stages of development. In this study, using a combination of four endothelial and hematopoietic surface markers, that is, the endothelial-specific marker vascular endothelial-cadherin (Cdh5, CD144), the pan-leukocyte antigen CD45, the hemato-endothelial marker CD34, and the angiotensin-converting enzyme (ACE, CD143), we identified distinct HSPC subsets, and among them, a population co-expressing the four markers that uniquely harbored an outstanding proliferation potential both ex vivo and in vivo. Moreover, we traced back this population to the yolk sac (YS) and aorta-gonad-mesonephros (AGM) sites of hematopoietic emergence. Taken together, our data will help to identify human HSPC self-renewal and amplification mechanisms for future cell therapies.

  Mots-clés : angiotensin-converting enzyme, embryonic liver, hematopoietic stem and progenitor cell, human embryo, VE-cadherin.

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2018

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  Darnaud, M, Dos Santos, A, Gonzalez, P, Augui, S, Lacoste, C, Desterke, C, De Hertogh, G, Valentino, E, Braun, E, Zheng, J, Boisgard, R, Neut, C, Dubuquoy, L, Chiappini, F, Samuel, D, Lepage, P, Guerrieri, F, Doré, J, Bréchot, C, Moniaux, N & Faivre, J 2018, « Enteric Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation in Mice With Colitis », Gastroenterology, vol. 154, no. 4, p. 1009-1023.e14.
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  Résumé : BACKGROUND & AIMS: Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. METHODS: We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2',7'-dichlorofluorescein diacetate and flow cytometry. RESULTS: The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis). CONCLUSIONS: Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.

  Mots-clés : Animals, Bacteria, Colitis, Colon, Dextran Sulfate, Disease Models, Animal, Fecal Microbiota Transplantation, Gastrointestinal Microbiome, Hepatocytes, Humans, IBD, LPS, Mice, Inbred C57BL, Mice, Transgenic, Microbial Viability, Mouse Model, Oxidative Stress, Pancreatitis-Associated Proteins, Reactive Oxygen Species, Time Factors, Trinitrobenzenesulfonic Acid.

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  Desterke, C, Slim, R & Candelier, J-J 2018, « A bioinformatics transcriptome meta-analysis highlights the importance of trophoblast differentiation in the pathology of hydatidiform moles », Placenta, vol. 65, p. 29-36.
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  Résumé : INTRODUCTION: Hydatidiform mole (HM) is an aberrant human pregnancy with abnormal trophoblastic development, migration/invasion of the extravillous trophoblast in the decidua. These abnormalities are established in a hypoxic environment during the first trimester of gestation. METHODS: Using text mining, we identified 72 unique genes that are linked to HM (HM-linked genes) that we studied by bioinformatic analysis in publicly available transcriptomes of primary chorionic villous cells (cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, and arterial and venous endothelial) isolated from normal placentas or established trophoblastic cell lines cultured under different oxygen concentrations. RESULTS: We show that the majority of HM-linked genes (75%) are involved in normal trophoblastic differentiation, arranged in clusters, and some of them are implicated in chorionic villous invasion or regulated by oxygen concentrations. DISCUSSION: Our analysis integrates the various aspects of the pathophysiology of HM and highlights the importance of trophoblastic differentiation in this pathology.

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  Desterke, C, Voldoire, M, Bonnet, M-L, Sorel, N, Pagliaro, S, Rahban, H, Bennaceur-Griscelli, A, Cayssials, E, Chomel, J-C & Turhan, AG 2018, « Experimental and integrative analyses identify an ETS1 network downstream of BCR-ABL in chronic myeloid leukemia (CML) », Experimental Hematology.
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  Résumé : The BCR-ABL oncogene, the hallmark of chronic myeloid leukemia (CML), has been shown to activate several signaling pathways in leukemic cells. The natural history of this disease has been radically modified by tyrosine kinase inhibitors (TKIs). However, resistance to several lines of TKI therapies and progression to blast crisis (BC) remain significant concerns. To identify novel signaling pathways induced by BCR-ABL, we performed a transcriptome analysis in a BCR-ABL-expressing UT-7 cell line. More than 2000 genes differentially expressed between BCR-ABL-expressing and parental UT-7 cells were identified and ETS1 was found to be the most upregulated. ETS1 protein expression was also shown to be highly increased in UT-7 cells expressing BCR-ABL either constitutively or under the control of TET-inducible promoters. ETS1 expression is tyrosine-kinase dependent because it was reduced by TKIs. A significant increase of ETS1 messenger RNA (mRNA) expression was observed in blood cells from CML patients at diagnosis compared with healthy controls. Integration of publicly available chromatin immunoprecipitation sequencing and transcriptomic data with our results allowed us to identify potential ETS1 targets, some of which are involved in the progression of CML. The messenger RNA expression of two of these genes (DNM3 and LIMS1) was found to be associated with the absence of major cytogenetic response after 1 year of imatinib therapy. The present work demonstrates for the first time the involvement of the ETS1 transcriptional program in the experimental UT-7 model and a large cohort of CML patients.

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  Domenech, C, Maillard, L, Rousseau, A, Guidez, F, Petit, L, Pla, M, Clay, D, Guimiot, F, Sanfilippo, S, Jacques, S, de la Grange, P, Robil, N, Soulier, J & Souyri, M 2018, « Studies in an Early Development Window Unveils a Severe HSC Defect in both Murine and Human Fanconi Anemia », Stem Cell Reports, vol. 11, no. 5, p. 1075-1091.
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  Résumé : Fanconi anemia (FA) causes bone marrow failure early during childhood, and recent studies indicate that a hematopoietic defect could begin in utero. We performed a unique kinetics study of hematopoiesis in Fancg-/- mouse embryos, between the early embryonic day 11.5 (E11.5) to E12.5 developmental window (when the highest level of hematopoietic stem cells [HSC] amplification takes place) and E14.5. This study reveals a deep HSC defect with exhaustion of proliferative and self-renewal capacities very early during development, together with severe FA clinical and biological manifestations, which are mitigated at E14.5 due to compensatory mechanisms that help to ensure survival of Fancg-/- embryos. It also reports that a deep HSC defect is also observed during human FA development, and that human FA fetal liver (FL) HSCs present a transcriptome profile similar to that of mouse E12.5 Fancg-/- FL HSCs. Altogether, our results highlight that early mouse FL could represent a good alternative model for studying Fanconi pathology.

  Mots-clés : Fanconi anemia, fetal liver, HSC, human embryonic development, mouse embryonic development, placenta, transcriptome.

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  Gentil, M, Hugues, P, Desterke, C, Telliam, G, Sloma, I, Souza, LEB, Baykal, S, Artus, J, Griscelli, F, Guerci, A, Johnson-Ansah, H, Foudi, A, Bennaceur-Griscelli, A & Turhan, AG 2018, « Aryl hydrocarbon receptor (AHR) is a novel druggable pathway controlling malignant progenitor proliferation in chronic myeloid leukemia (CML) », PloS One, vol. 13, no. 8, p. e0200923.
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  Résumé : Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)-derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the "AHR low" fraction and an upregulation of genes involved in stem cell maintenance in the "AHR high" fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.

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  Girerd, S, Tosca, L, Herault, O, Vignon, C, Biard, D, Aggoune, D, Dkhissi, F, Bonnet, ML, Sorel, N, Desterke, C, Bennaceur-Griscelli, A, Tachdjian, G, Guilhot, F, Guilhot, J, Chomel, J-C & Turhan, AG 2018, « Superoxide dismutase 2 (SOD2) contributes to genetic stability of native and T315I-mutated BCR-ABL expressing leukemic cells », Biochemical and Biophysical Research Communications, vol. 498, no. 4, p. 715-722.
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  Résumé : Manganese Superoxide dismutase 2 (SOD2) plays a crucial role in antioxidant defense but there are no data suggesting its role in genetic instability in CML. We evaluated the effects of SOD2 silencing in human UT7 cell line expressing either non-mutated or T315I-mutated BCR-ABL. Array-CGH experiments detected in BCR-ABL-expressing cells silenced for SOD2 a major genetic instability within several chromosomal loci, especially in regions carrying the glypican family (duplicated) and β-defensin genes (deleted). In a large cohort of patients with chronic myeloid leukemia (CML), a significant decrease of SOD2 mRNA was observed. This reduction appeared inversely correlated with leukocytosis and Sokal score, high-risk patients showing lower SOD2 levels. The analysis of anti-oxidant gene expression analysis revealed a specific down-regulation of the expression of PRDX2 in UT7-BCR-ABL and UT7-T315I cells silenced for SOD2 expression. Gene set enrichment analysis performed between the two SOD2-dependent classes of CML patients revealed a significant enrichment of Reactive Oxygen Species (ROS) Pathway. Our data provide the first evidence for a link between SOD2 expression and genetic instability in CML. Consequently, SOD2 mRNA levels should be analyzed in prospective studies as patients with low SOD2 expression could be more prone to develop a mutator phenotype under TKI therapies.

  Mots-clés : Cell Line, Tumor, CML, Cohort Studies, Fusion Proteins, bcr-abl, Gene Expression Regulation, Leukemic, Gene Silencing, Genetic instability, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive, Mutation, Peroxiredoxins, Point Mutation, Silencing, SOD2, Superoxide Dismutase.

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  Khosravi, M, Azarpira, N, Shamdani, S, Hojjat-Assari, S, Naserian, S & Karimi, MH 2018, « Differentiation of umbilical cord derived mesenchymal stem cells to hepatocyte cells by transfection of miR-106a, miR-574-3p, and miR-451 », Gene, vol. 667, p. 1-9.
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  Résumé : Studying the profile of micro RNAs (miRs) elucidated the highest expressed miRs in hepatic differentiation. In this study, we investigated to clarify the role of three embryonic overexpressed miRs (miR-106a, miR-574-3p and miR-451) during hepatic differentiation of human umbilical cord derived mesenchymal stem cells (UC-MSCs). We furthermore, aimed to explore whether overexpression of any of these miRs alone is sufficient to induce the differentiation of the UC-MSCs into hepatocyte-like cells. UC-MSCs were transfected either alone or together with miR-106a, miR-574-3p and miR-451 and their potential hepatic differentiation and alteration in gene expression profile, morphological changes and albumin secretion ability were investigated. We found that up-regulation of any of these three miRs alone cannot induce expression of all hepatic specific genes. Transfection of each miR alone, led to Sox17, FoxA2 expression that are related to initiation step of hepatic differentiation. However, concurrent ectopic overexpression of three miRs together can induce UC-MSCs differentiation into functionally mature hepatocytes. These results show that miRs have the capability to directly convert UC-MSCs to a hepatocyte phenotype in vitro.

  Mots-clés : Cell Differentiation, Cells, Cultured, Gene Expression Profiling, Gene Regulatory Networks, Hepatocyte differentiation, Hepatocyte Nuclear Factor 3-beta, Hepatocytes, Humans, Mesenchymal Stromal Cells, Micro RNA, MicroRNAs, SOXF Transcription Factors, Tissue engineering, Transfection, Umbilical Cord, Umbilical cord derived mesenchymal stem cells.

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  Khosravi, M, Bidmeshkipour, A, Moravej, A, Hojjat-Assari, S, Naserian, S & Karimi, MH 2018, « Induction of CD4+CD25+Foxp3+ regulatory T cells by mesenchymal stem cells is associated with RUNX complex factors », Immunologic Research, vol. 66, no. 1, p. 207-218.
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  Résumé : Among the particular immunomodulation properties of mesenchymal stem cells (MSCs), one relies on their capacity to regulatory T cell (Treg) induction from effector T cells. Stable expression of Foxp3 has a dominant role in suppressive phenotype and stability of induced regulatory T cells (iTregs). How MSCs induce stable Foxp3 expression in iTregs remains unknown. We previously showed MSCs could enhance demethylation of Treg-specific demethylated region (TSDR) in iTregs in cell-cell contact manner (unpublished data). Here, we evaluated the possible effect of MSCs on the mRNA expression of Runx complex genes (Runx1, Runx3, and CBFB) that perch on TSDR in iTregs and play the main role in suppressive properties of Tregs, a regulatory pathway that has not yet been explored by MSCs. Also, we investigated the mRNA expression of MBD2 that promotes TSDR demethylation in Tregs. We first showed that in vitro MSC-iTreg induction was associated with strong mRNA modifications of genes involved in Runx complex. We next injected high doses of MSCs in a murine model of C57BL/6 into Balb/C allogeneic skin transplantation to prolong allograft survival. When splenocytes of grafted mice were analyzed, we realized that the Foxp3 expression was increased at day 5 and 10 post-graft merely in MSC-treated mice. Furthermore, Foxp3 mRNA expression was associated with modified Runx complex mRNA expression comparable to what was shown in in vitro studies. Hence, our data identify a possible mechanism in which MSCs convert conventional T cells to iTreg through strong modifications of mRNA of genes that are involved in Runx complex of Foxp3.

  Mots-clés : Demethylation, Mesenchymal stem cells, Regulatory T cells, Runx complex.

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  Li, D, Li, P, Song, N, Jiang, Y, Zeng, Y, Zhao, G, Fa, Y, Ye, H, Lone, Y, Zhou, Y, Sun, S & Zeng, L 2018, « Identification of novel HLA-A11-restricted T-cell epitopes in the Ebola virus nucleoprotein », Microbes and Infection.
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  Résumé : The Ebola virus (EBOV) is a very contagious virus that is highly fatal in humans and animals. The largest epidemic was in West Africa in 2014, in which over 11,000 people died. However, to date, there are no licensed vaccines against it. Studies show that CD4+ and CD8+ T-cell responses, especially cytotoxic T-lymphocyte (CTL) responses, play key roles in protecting individuals from EBOV infection. Since HLA-restricted epitope vaccines are likely to be effective and safe immunization strategies for infectious diseases, the present study screened for CTL epitopes in the EBOV-nucleoprotein that are restricted by HLA-A11 (a common allele in Chinese people). Predictive computer analysis of the amino-acid sequence of EBOV-nucleoprotein identified ten putative HLA-A11-restricted epitopes. ELISPOT assay of immunized HLA-A11/DR1 transgenic mice showed that five (GR-9, VR-9, EK-9, PK-9, and RK-9) induced effective CTL responses. Additional epitope analyses will aid the design of epitope vaccines against EBOV.

  Mots-clés : Ebola virus, HLA-A11-restricted epitope, Vaccine.

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  Lopez, A, Panisello-Rosello, A, Castro-Benitez, C & Adam, R 2018, « Glycocalyx Preservation and NO Production in Fatty Livers-The Protective Role of High Molecular Polyethylene Glycol in Cold Ischemia Injury », International Journal of Molecular Sciences, vol. 19, no. 8.
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  Résumé : Improving the protection of marginal liver grafts during static cold storage is a major hurdle to increase the donor pool of organs. The endothelium glycocalyx quality of preservation influences future inflammatory and oxidative responses. One cellular pathway responsible for the formation of nitric oxide by endothelial cells is dependent on the stimulation of proteoglycans present in the glycocalyx. We investigated the impact of the glycocalyx preservation in static cold storage of fatty liver preserved in different preservation solutions on the endothelium-mediated production of NO. Zucker fatty rat livers were preserved 24 h in static cold storage in either Institut Georges Lopez-1 (IGL-1) (n = 10), IGL-0 (i.e., without PEG35) (n = 5) or Histidine-Tryptophan-Ketoglutarate (HTK) (n = 10) preservation solutions before being processed for analysis. For Sham group (n = 5), the fatty livers were immediately analyzed after procurement. The level of transaminases and nitrites/nitrates were measured in the washing perfusate. Glycocalyx proteins expressions, Syndecan-1, glypican-1 and heparan sulfate (HS), were determined in the tissue (ELISA). Steatotic livers preserved 24 h in IGL-1 preservation solution have a significant lower level of transaminases (aspartate aminotransferase (AST), alanine aminotransferase (ALT)) and less histological damages than steatotic livers preserved 24 h with HTK (p = 0.0152). The syndecan-1 is significantly better preserved in IGL-1 group compared to HTK (p < 0.0001) and we observed the same tendency compared to IGL-0. No significant differences were observed with glypican-1. HS expression in HTK group was significantly higher compared to the three other groups. HS level in IGL-1 was even lower than IGL-0 (p = 0.0005) which was similar to Sham group. The better protection of the glycocalyx proteins in IGL-1 group was correlated with a higher production of NO than HTK (p = 0.0055) or IGL-0 (p = 0.0433). IGL-1 protective mechanisms through the formation of NO could be due to its better protective effects on the glycocalyx during SCS compared to other preservation solutions. This beneficial effect could involve the preservation state of syndecan-1 and the internalization of HS.

  Mots-clés : cold storage, glycocalyx, ischemia, liver, polyethylene glycol, steatosis.

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  Lucchese, AM, Kalil, AN, Ruiz, A, Karam, V, Ciacio, O, Pittau, G, Castaing, D, Cherqui, D, Sa Cunha, A, Vibert, E & Adam, R 2018, « Neoadjuvant chemotherapy response influences outcomes in non-colorectal, non-neuroendocrine liver metastases », The British Journal of Surgery.
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  Résumé : BACKGROUND: Indications for surgical resection of non-colorectal, non-neuroendocrine (NCNNE) liver metastases are unclear. This study analysed the influence of response to neoadjuvant chemotherapy and the presence of extrahepatic disease (EHD) on outcomes. METHODS: Patients who underwent hepatic resection for NCNNE liver metastases and who received neoadjuvant chemotherapy at a single centre between 1982 and 2016 were analysed retrospectively. Patients were classified as having no EHD, controlled EHD or non-controlled EHD. RESULTS: Hepatic resection was performed in 199 patients (81·2 per cent) after partial or complete response to chemotherapy or disease stabilization, and 46 patients (18·8 per cent) after tumour progression. Patients with progressive disease after chemotherapy had worse overall survival than those without (23 versus 50·4 per cent at 5 years; P = 0·004). Median survival was 63·6 (range 31·1-94·8) months for patients without EHD, 34·8 (19·2-49·2) months for those with controlled EHD and 7·2 (1·2-13·2) months for patients with non-controlled EHD (P = 0·004). In multivariable analysis, EHD (P = 0·004), response to chemotherapy (P = 0·004) and resection margins (P = 0·002) were all independent predictors of overall survival, regardless of primary tumour site. CONCLUSION: The prognosis of patients with NCNNE liver metastases is influenced by preoperative chemotherapy and resectability.

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  Machover, D, Goldschmidt, EL, Mollicone, R, Haghighi-Rad, F, Desterke, C, Gaston-Mathe, Y, Saffroy, R, Boucheix, C & Dairou, J 2018, « Enhancement of 5-Fluorouracil cytotoxicity by Pyridoxal 5'-phosphate and Folinic acid in tandem », The Journal of Pharmacology and Experimental Therapeutics.
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  Résumé : The present study originates from the assumption that, in tumors, levels of naturally occurring pyridoxal 5'-phosphate (PLP) are too small to allow conversion of tetra hydro pteroylglutamate (H4PteGlu) into methylene tetra hydro pteroylglutamate (CH2-H4PteGlu) in amounts required to improve inhibition of thymidylate synthase by 5-fluorouracil (FUra) through ternary complex stabilization. The hypothesis relates to the low affinity for cofactor of the PLP-dependent serine hydroxymethyl transferase (SHMT), the enzyme that catalyses formation CH2-H4PteGlu by transfer of the Cβ of serine to H4PteGlu. Intracellular concentrations of PLP are smaller than the dissociation constant (KD) of SHMT for cofactor, which suggests that enzyme activity should be sensitive to PLP level changes. Three cancer cell lines were supplemented with PLP to investigate the influence of this cofactor on FUra cytotoxicity. Cells were exposed to FUra, FUra and folinic acid (FA), FUra and PLP, and FUra combined with both FA and PLP. The Median effect principle for concentration-effect analysis and combination indices were used to determine interactions on cytotoxicity. FUra cytotoxicity in vitro was enhanced by FA and PLP in tandem. Synergistic cytotoxic interaction of FUra with FA and PLP was demonstrated in HT29, and L1210 cells. Summation was found in HCT116 cells. Parenteral pyridoxamine was administered in mice to explore erythrocyte production of PLP in vivo. Cofactor attained levels in the range of the KD for binding to SHMT and it was rapidly cleared from cells. Pharmacokinetics of pyridoxamine suggests that modulation of FUra by vitamin B6 could be achieved in vivo.

  Mots-clés : anticancer agents, cancer chemotherapy, chemotherapy, drug development, drug efficacy, drug interactions, vitamins.

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  Magne, B, Lataillade, J-J & Trouillas, M 2018, « Mesenchymal Stromal Cell Preconditioning: The Next Step Toward a Customized Treatment For Severe Burn », Stem Cells and Development.
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  Résumé : Over the last century, the clinical management of severe skin burns significantly progressed with the development of burn care units, topical antimicrobials, resuscitation methods, early eschar excision surgeries, and skin grafts. Despite these considerable advances, the present treatment of severe burns remains burdensome, and patients are highly susceptible to skin engraftment failure, infections, organ dysfunction, and hypertrophic scarring. Recent researches have focused on mesenchymal stromal cell (MSC) therapy and hold great promises for tissue repair, as reported in several animal studies and clinical cases. In the present review, we will provide an up-to-date outlook of the pathophysiology of severe skin burns, clinical treatment modalities and current limitations. We will then focus on MSCs and their potential in the burn wound healing both in in vitro and in vivo studies. A specific attention will be paid to the cell preconditioning approach, as a means of improving the MSC efficacy in the treatment of major skin burns. In particular, we will debate how several preconditioning cues would modulate the MSC properties to better match up with the burn pathophysiology in the course of the cell therapy. Finally, we will discuss the clinical interest and feasibility of a MSC-based therapy in comparison to their paracrine derivatives, including microvesicles and conditioned media for the treatment of major skin burn injuries.

  Mots-clés : cell preconditioning, clinical use, major skin burn injuries and pathophysiology, mesenchymal stromal cells.

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2017

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  Abdallah, C, Lejamtel, C, Benzoubir, N, Battaglia, S, Sidahmed-Adrar, N, Desterke, C, Lemasson, M, Rosenberg, AR, Samuel, D, Bréchot, C, Pflieger, D, Le Naour, F & Bourgeade, M-F 2017, « Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice », Oncotarget.
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  Résumé : Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

  Mots-clés : 4E-BP1 phosphorylation, HCV core, hepatocellular carcinoma, phosphoproteomics, SILAC.

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  Aggoune, D, Sorel, N, Bonnet, M-L, Goujon, J-M, Tarte, K, Hérault, O, Domenech, J, Réa, D, Legros, L, Johnson-Ansa, H, Rousselot, P, Cayssials, E, Guerci-Bresler, A, Bennaceur-Griscelli, A, Chomel, J-C & Turhan, AG 2017, « Bone marrow mesenchymal stromal cell (MSC) gene profiling in chronic myeloid leukemia (CML) patients at diagnosis and in deep molecular response induced by tyrosine kinase inhibitors (TKIs) », Leukemia Research, vol. 60, p. 94-102.
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  Résumé : Although it has been well-demonstrated that bone marrow mesenchymal stromal cells (MSCs) from CML patients do not belong to the Ph1-positive clone, there is growing evidence that they could play a role in the leukemogenesis process or the protection of leukemic stem cells from the effects of tyrosine kinase inhibitors (TKIs). The aim of the present study was to identify genes differentially expressed in MSCs isolated from CML patients at diagnosis (CML-MSCs) as compared to MSCs from healthy controls. Using a custom gene-profiling assay, we identified six genes over-expressed in CML-MSCs (BMP1, FOXO3, MET, MITF, NANOG, PDPN), with the two highest levels being documented for PDPN (PODOPLANIN) and NANOG. To determine whether this aberrant signature persisted in patients in deep molecular response induced by TKIs, we analyzed MSCs derived from such patients (MR-MSCs). This analysis showed that, despite the deep molecular responses, BMP1, MET, MITF, NANOG, and PDPN mRNA were upregulated in MR-MSCs. Moreover, BMP1, MITF, and NANOG mRNA expressions in MR-MSCs were found to be intermediate between control MSCs and CML-MSCs. These results suggest that CML-MSCs exhibit an abnormal gene expression pattern which might have been established during the leukemogenic process and persist in patients in deep molecular response.

  Mots-clés : Chronic myeloid leukemia, Hematopoietic niche, Mesenchymal Stromal Cells, TaqMan low-density array.

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  Chastagner, P, Rubinstein, E & Brou, C 2017, « Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing », Science Signaling, vol. 10, no. 483.
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  Résumé : The Notch signaling pathway, which is activated by cell-cell contact, is a major regulator of cell fate decisions. Mammalian Notch1 is present at the cell surface as a heterodimer of the Notch extracellular domain associated with the transmembrane and intracellular domains. After ligand binding, Notch undergoes proteolysis, releasing the Notch intracellular domain (NICD) that regulates gene expression. We monitored the early steps of activation with biochemical analysis, immunofluorescence analysis, and live-cell imaging of Notch1-expressing cells. We found that, upon ligand binding, Notch1 at the cell surface was ubiquitylated by the E3 ubiquitin ligase DTX4. This ubiquitylation event led to the internalization of the Notch1 extracellular domain by the ligand-expressing cell and the internalization of the membrane-anchored fragment of Notch1 and DTX4 by the Notch1-expressing cell, which we referred to as bilateral endocytosis. ADAM10 generates a cleavage product of Notch that is necessary for the formation of the NICD, which has been thought to occur at the cell surface. However, we found that blocking dynamin-mediated endocytosis of Notch1 and DTX4 reduced the colocalization of Notch1 with ADAM10 and the formation of the ADAM10-generated cleavage product of Notch1, suggesting that ADAM10 functions in an intracellular compartment to process Notch. Thus, this study suggests that a specific pool of ADAM10 acts on Notch in an endocytic compartment, rather than at the cell surface.

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  Chiappini, F, Coilly, A, Kadar, H, Gual, P, Tran, A, Desterke, C, Samuel, D, Duclos-Vallée, J-C, Touboul, D, Bertrand-Michel, J, Brunelle, A, Guettier, C & Le Naour, F 2017, « Metabolism dysregulation induces a specific lipid signature of nonalcoholic steatohepatitis in patients », Scientific Reports, vol. 7, p. 46658.
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  Résumé : Nonalcoholic steatohepatitis (NASH) is a condition which can progress to cirrhosis and hepatocellular carcinoma. Markers for NASH diagnosis are still lacking. We performed a comprehensive lipidomic analysis on human liver biopsies including normal liver, nonalcoholic fatty liver and NASH. Random forests-based machine learning approach allowed characterizing a signature of 32 lipids discriminating NASH with 100% sensitivity and specificity. Furthermore, we validated this signature in an independent group of NASH patients. Then, metabolism dysregulations were investigated in both patients and murine models. Alterations of elongase and desaturase activities were observed along the fatty acid synthesis pathway. The decreased activity of the desaturase FADS1 appeared as a bottleneck, leading upstream to an accumulation of fatty acids and downstream to a deficiency of long-chain fatty acids resulting to impaired phospholipid synthesis. In NASH, mass spectrometry imaging on tissue section revealed the spreading into the hepatic parenchyma of selectively accumulated fatty acids. Such lipids constituted a highly toxic mixture to human hepatocytes. In conclusion, this study characterized a specific and sensitive lipid signature of NASH and positioned FADS1 as a significant player in accumulating toxic lipids during NASH progression.

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  Da Costa, LS & Arnoult, D 2017, « Organelle Separation and Cell Signaling », Methods in Molecular Biology (Clifton, N.J.), vol. 1557, p. 111-115.
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  Résumé : Recent findings indicate that some signaling hubs coalesce at the surfaces of organelles through the accumulation of ubiquitylated components required for the signal transduction. For instance, ubiquitylated components of the NF-κB pathway accumulated at the endoplasmic reticulum while ubiquitylated components of the IRF3 pathway are found at the Golgi apparatus. Here we describe simple methods to observe and assess these ubiquitylated components by immunoblotting using differential centrifugation and in vitro assays.

  Mots-clés : Cell fractionation, Differential centrifugation, Immunoblotting, Signaling, Ubiquitination.

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  El Kharbili, M, Robert, C, Witkowski, T, Danty-Berger, E, Barbollat-Boutrand, L, Masse, I, Gadot, N, de la Fouchardière, A, McDonald, PC, Dedhar, S, Le Naour, F, Degoul, F & Berthier-Vergnes, O 2017, « Tetraspanin 8 is a novel regulator of ILK-driven β1 integrin adhesion and signaling in invasive melanoma cells », Oncotarget.
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  Résumé : Melanoma is well known for its propensity for lethal metastasis and resistance to most current therapies. Tumor progression and drug resistance depend to a large extent on the interplay between tumor cells and the surrounding matrix. We previously identified Tetraspanin 8 (Tspan8) as a critical mediator of melanoma invasion, whose expression is absent in healthy skin. The present study investigated whether Tspan8 may influence cell-matrix anchorage and regulate downstream molecular pathways leading to an aggressive behavior. Using silencing and ectopic expression strategies, we showed that Tspan8-mediated invasion of melanoma cells resulted from defects in cell-matrix anchorage by interacting with β1 integrins and by interfering with their clustering, without affecting their surface or global expression levels. These effects were associated with impaired phosphorylation of integrin-linked kinase (ILK) and its downstream target Akt-S473, but not FAK. Specific blockade of Akt or ILK activity strongly affected cell-matrix adhesion. Moreover, expression of a dominant-negative form of ILK reduced β1 integrin clustering and cell-matrix adhesion. Finally, we observed a tumor-promoting effect of Tspan8 in vivo and a mutually exclusive expression pattern between Tspan8 and phosphorylated ILK in melanoma xenografts and human melanocytic lesions. Altogether, the in vitro, in vivo and in situ data highlight a novel regulatory role for Tspan8 in melanoma progression by modulating cell-matrix interactions through β1 integrin-ILK axis and establish Tspan8 as a negative regulator of ILK activity. These findings emphasize the importance of targeting Tspan8 as a means of switching from low- to firm-adhesive states, mandatory to prevent tumor dissemination.

  Mots-clés : ILK, integrin, matrix, melanoma, tetraspanin 8.

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  Ferratge, S, Boyer, J, Arouch, N, Chevalier, F & Uzan, G 2017, « Circulating endothelial progenitors in vascular repair », Bio-Medical Materials and Engineering, vol. 28, no. s1, p. S65-S74.
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  Résumé : Endothelial Colony Forming Cells (ECFCs) are obtained in culture from Circulating Endothelial Progenitor Cells. They display all characteristics of endothelial cells and they display stem cells features. Cord blood-derived ECFCs (CB-ECFCs) have a high clonogenic and proliferative potentials, and exhibit vascular repair capabilities useful for the treatment of ischemic diseases. However, the link between immaturity and functional properties of CB-ECFCs is still poorly defined. We showed that these cells have a high clonogenic potential and are capable to be efficiently reprogrammed into induced pluripotent stem cells. Moreover, we analyzed the expression of a broad panel of genes involved in embryonic stem cell properties. We define a novel stem cell transcriptional signature for CB-ECFCs fora better characterization and stratification according to their stem cell profile. We then improved the yield of CB-ECFC production for obtaining cells more functional in fewer passages. We used Glycosaminoglycans (GAG), components from the extracellular matrix which potentiate heparin binding growth factor activities. GAG mimetics were designed, having the capacity to increase the yield of ECFC during the isolation process, to increase the number of colonies, improve adhesion, proliferation, migration and self-renewal. GAG mimetics have thus great interest for vascular regeneration in combination with ECFC. Our results show that CB-ECFC are immature cells harboring specific functions such as formation of colonies, proliferation and formation of vascular structures in vitro and in vivo.

  Mots-clés : circulating endothelial progenitors, plasticity, stem cells, Vascular repair.

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  Ferratge, S, Ha, G, Carpentier, G, Arouche, N, Bascetin, R, Muller, L, Germain, S & Uzan, G 2017, « Initial clonogenic potential of human endothelial progenitor cells is predictive of their further properties and establishes a functional hierarchy related to immaturity », Stem Cell Research, vol. 21, p. 148-159.
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  Résumé : Endothelial progenitor cells (EPCs) generate in vitro Endothelial Colony Forming Cells (ECFCs) combining features of endothelial and stem/progenitor cells. Their angiogenic properties confer them a therapeutic potential for treating ischemic lesions. They may be isolated from umbilical cord blood (CB-ECFCs) or peripheral adult blood (AB-ECFCs). It is generally accepted that CB-ECFCs are more clonogenic, proliferative and angiogenic than AB-ECFCs. Nevertheless, only a few studies have focused on the functional heterogeneity of CB-ECFCs from different individuals. Moreover, AB-ECFC loss of function is yet to be precisely described. We have focused on these two issues that are critical for clinical perspectives. The detailed clonogenic profile of CB-ECFCs and AB-ECFCs was obtained and revealed a high inter individual heterogeneity and the absence of correlation with age. Most CB-ECFCs yielded initial colonies and had functional properties similar to those of AB-ECFCs. Conversely, a high clonogenicity was associated with an enhanced proliferative and angiogenic potential and stemness gene overexpression, confirming that immaturity, lost by AB-ECFCs, was a prerequisite to functionality. We thus demonstrated the importance of selecting CB-ECFCs according to specific criteria, and we propose using the initial clonogenicity as a relevant marker of their potential efficacy on vascular repair.

  Mots-clés : Cord blood, Endothelial Colony Forming Cells, Endothelial progenitor cells, Functional hierarchy, Immaturity, Peripheral adult blood, Senescence.

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  Gaillard, M & Dagher, I 2017, « Minimally Invasive Liver Preconditioning for Hepatocyte Transplantation in Rats », Methods in Molecular Biology (Clifton, N.J.), vol. 1506, p. 193-200.
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  Résumé : In the context of cell transplantation in the liver parenchyma, preconditioning is essential to enhance cell engraftment and liver repopulation. The authors have developed a minimally invasive technique of temporary portal embolization using an absorbable material, called reversible portal vein embolization. We hereby describe the method for isolating hepatocytes from a donor rat before transplanting hepatocytes after reversible portal vein embolization in the recipient.

  Mots-clés : Hepatocyte isolation, Hepatocyte transplantation, Liver perfusion, Liver preconditioning, Portal vein embolization.

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  Grigorov, B, Molle, J, Rubinstein, E, Zoulim, F & Bartosch, B 2017, « CD81 large extracellular loop-containing fusion proteins with a dominant negative effect on HCV cell spread and replication », The Journal of General Virology, vol. 98, no. 7, p. 1646-1657.
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  Résumé : The roles of CD81 in the hepatitis C virus (HCV) life cycle are multiple but remain ill characterized. CD81 is known to interact with the HCV glycoproteins as an attachment factor. It also has an important role in the post-attachment entry process. Its interaction with claudin-1, for example, is vital for viral uptake and trafficking. Furthermore, CD81 and its role in membrane organization and trafficking are thought to play a pivotal role in HCV replication. Some of these functions are particularly limited to human CD81; others can be substituted with CD81 molecules from other species. However, with the exception of the large extracellular loop sequence, the structure-function analysis of CD81 in the HCV infectious cycle remains ill characterized. We describe here the fusion molecules between the large extracellular loops of human or mouse CD81 and lipid-raft-associated or unassociated GPI anchors. These fusion molecules have strong antiviral activity in a dominant negative fashion, independent of membrane raft association. Their expression in the hepatoma cell line Huh7.5 blocks HCV uptake, transmission and replication. These molecules will be useful to decipher the various roles of CD81 in the HCV life cycle and transmission in more detail.

  Mots-clés : Animals, Antigens, CD81, Cell Line, Tumor, HEK293 Cells, HeLa Cells, Hepacivirus, Hepatitis C, HIV-1, Humans, Membrane Microdomains, Mice, Protein Binding, Viral Envelope Proteins, Virus Attachment, Virus Internalization, Virus Replication.

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  Hamidouche, Z, Rother, K, Przybilla, J, Krinner, A, Clay, D, Hopp, L, Fabian, C, Stolzing, A, Binder, H, Charbord, P & Galle, J 2017, « Bistable Epigenetic States Explain Age-Dependent Decline in Mesenchymal Stem Cell Heterogeneity », Stem Cells (Dayton, Ohio), vol. 35, no. 3, p. 694-704.
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  Résumé : The molecular mechanisms by which heterogeneity, a major characteristic of stem cells, is achieved are yet unclear. We here study the expression of the membrane stem cell antigen-1 (Sca-1) in mouse bone marrow mesenchymal stem cell (MSC) clones. We show that subpopulations with varying Sca-1 expression profiles regenerate the Sca-1 profile of the mother population within a few days. However, after extensive replication in vitro, the expression profiles shift to lower values and the regeneration time increases. Study of the promoter of Ly6a unravels that the expression level of Sca-1 is related to the promoter occupancy by the activating histone mark H3K4me3. We demonstrate that these findings can be consistently explained by a computational model that considers positive feedback between promoter H3K4me3 modification and gene transcription. This feedback implicates bistable epigenetic states which the cells occupy with an age-dependent frequency due to persistent histone (de-)modification. Our results provide evidence that MSC heterogeneity, and presumably that of other stem cells, is associated with bistable epigenetic states and suggest that MSCs are subject to permanent state fluctuations. Stem Cells 2017;35:694-704.

  Mots-clés : Aging, Epigenetics, FACS, Mesenchymal stem cells, Methylation.

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  Manzoni, G, Marinach, C, Topçu, S, Briquet, S, Grand, M, Tolle, M, Gransagne, M, Lescar, J, Andolina, C, Franetich, J-F, Zeisel, MB, Huby, T, Rubinstein, E, Snounou, G, Mazier, D, Nosten, F, Baumert, TF & Silvie, O 2017, « Plasmodium P36 determines host cell receptor usage during sporozoite invasion », eLife, vol. 6.
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  Résumé : Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, first infect the liver for an initial round of replication before the emergence of pathogenic blood stages. Sporozoites represent attractive targets for antimalarial preventive strategies, yet the mechanisms of parasite entry into hepatocytes remain poorly understood. Here we show that the two main species causing malaria in humans, Plasmodium falciparum and Plasmodium vivax, rely on two distinct host cell surface proteins, CD81 and the Scavenger Receptor BI (SR-BI), respectively, to infect hepatocytes. By contrast, CD81 and SR-BI fulfil redundant functions during infection by the rodent parasite P. berghei. Genetic analysis of sporozoite factors reveals the 6-cysteine domain protein P36 as a major parasite determinant of host cell receptor usage. Our data provide molecular insights into the invasion pathways used by different malaria parasites to infect hepatocytes, and establish a functional link between a sporozoite putative ligand and host cell receptors.

  Mots-clés : hepatocyte, human, infectious disease, malaria, microbiology, mouse, P. berghei, P. falciparum, P. vivax, P. yoelii, sporozoite.

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  Maslah, N, Cassinat, B, Verger, E, Kiladjian, J-J & Velazquez, L 2017, « The role of LNK/SH2B3 genetic alterations in myeloproliferative neoplasms and other hematological disorders », Leukemia.
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  Résumé : Malignant hematological diseases are mainly because of the occurrence of molecular abnormalities leading to the deregulation of signaling pathways essential for precise cell behavior. High-resolution genome analysis using microarray and large-scale sequencing have helped identify several important acquired gene mutations that are responsible for such signaling deregulations across different hematological malignancies. In particular, the genetic landscape of classical myeloproliferative neoplasms (MPNs) has been in large part completed with the identification of driver mutations (targeting the cytokine receptor/Janus-activated kinase 2 (JAK2) pathway) that determine MPN phenotype, as well as additional mutations mainly affecting the regulation of gene expression (epigenetics or splicing regulators) and signaling. At present, most efforts concentrate in understanding how all these genetic alterations intertwine together to influence disease evolution and/or dictate clinical phenotype in order to use them to personalize diagnostic and clinical care. However, it is now evident that factors other than somatic mutations also play an important role in MPN disease initiation and progression, among which germline predisposition (single-nucleotide polymorphisms and haplotypes) may strongly influence the occurrence of MPNs. In this context, the LNK inhibitory adaptor protein encoded by the LNK/SH2B adaptor protein 3 (SH2B3) gene is the target of several genetic variations, acquired or inherited in MPNs, lymphoid leukemia and nonmalignant hematological diseases, underlying its importance in these pathological processes. As LNK adaptor is a key regulator of normal hematopoiesis, understanding the consequences of LNK variants on its protein functions and on driver or other mutations could be helpful to correlate genotype and phenotype of patients and to develop therapeutic strategies to target this molecule. In this review we summarize the current knowledge of LNK function in normal hematopoiesis, the different SH2B3 mutations reported to date and discuss how these genetic variations may influence the development of hematological malignancies.Leukemia advance online publication, 23 May 2017; doi:10.1038/leu.2017.139.

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  Nault, J-C, Couchy, G, Balabaud, C, Morcrette, G, Caruso, S, Blanc, J-F, Bacq, Y, Calderaro, J, Paradis, V, Ramos, J, Scoazec, J-Y, Gnemmi, V, Sturm, N, Guettier, C, Fabre, M, Savier, E, Chiche, L, Labrune, P, Selves, J, Wendum, D, Pilati, C, Laurent, A, De Muret, A, Le Bail, B, Rebouissou, S, Imbeaud, S, GENTHEP Investigators,, Bioulac-Sage, P, Letouzé, E & Zucman-Rossi, J 2017, « Molecular Classification of Hepatocellular Adenoma Associates With Risk Factors, Bleeding, and Malignant Transformation », Gastroenterology, vol. 152, no. 4, p. 880-894.e6.
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  Résumé : BACKGROUND & AIMS: Hepatocellular adenomas (HCAs) are benign liver tumors that can be assigned to molecular subtypes based on inactivating mutations in hepatocyte nuclear factor 1A, activating mutations in β-catenin, or activation of inflammatory signaling pathways. We aimed to update the classification system for HCA and associate the subtypes with disease risk factors and complications. METHODS: We analyzed expression levels of 20 genes and sequenced exon regions of 8 genes (HNF1A, IL6ST, CTNNB1, FRK, STAT3, GNAS, JAK1, and TERT) in 607 samples of 533 HCAs from 411 patients, collected from 28 centers mainly in France from 2000 and 2014. We performed gene expression profile, RNA sequence, whole-exome and genome sequence, and immunohistochemical analyses of select samples. Molecular data were associated with risk factors, histopathology, bleeding, and malignant transformation. RESULTS: Symptomatic bleeding occurred in 14% of the patients (85% of cases were female, median age, 38 years); 7% of the nodules were borderline between HCA and hepatocellular carcinoma, and 3% of patients developed hepatocellular carcinoma from HCA. Based on molecular features, we classified HCA into 8 subgroups. One new subgroup, composed of previously unclassified HCA, represented 4% of HCAs overall and was associated with obesity and bleeding. These tumors were characterized by activation of sonic hedgehog signaling, due to focal deletions that fuse the promoter of INHBE with GLI1. Analysis of genetic heterogeneity among multiple HCAs, from different patients, revealed a molecular subtype field effect; multiple tumors had different mutations that deregulated similar pathways. Specific molecular subtypes of HCA associated with various HCA risk factors, including imbalances in estrogen or androgen hormones. Specific molecular subgroup of HCA with β-catenin and sonic hedgehog activation associated with malignant transformation and bleeding, respectively. CONCLUSIONS: Using sequencing and gene expression analyses, we identified a subgroup of HCA characterized by fusion of the INHBE and GLI1 genes and activation of sonic hedgehog pathway. Molecular subtypes of HCAs associated with different patients' risk factors for HCA, disease progression, and pathology features of tumors. This classification system might be used to select treatment strategies for patients with HCA.

  Mots-clés : Benign, HCC, SHH, Tumor Progression.

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  Rocha-Perugini, V, Martínez Del Hoyo, G, González-Granado, JM, Ramírez-Huesca, M, Zorita, V, Rubinstein, E, Boucheix, C & Sánchez-Madrid, F 2017, « CD9 regulates MHC-II trafficking in Monocyte-derived Dendritic Cells », Molecular and Cellular Biology.
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  Résumé : Antigen presentation by dendritic cells (DCs) stimulates naïve CD4(+) T cells, triggering T cell activation and the adaptive arm of the immune response. Newly synthesized major histocompatibility complex class II molecules (MHC-II) accumulate at MHC-II-enriched endosomal compartments, and are transported to the plasma membrane of DCs after binding to antigenic peptides to enable antigen presentation. In DCs, MHC-II molecules are included in tetraspanin-enriched microdomains (TEMs). However, the role of tetraspanin CD9 in these processes remains largely undefined. Here, we show that CD9 regulates the T-cell stimulatory capacity of GM-CSF-dependent bone-marrow derived DCs (BMDCs), without affecting antigen-presentation by Flt3L-dependent BMDCs. CD9 knock-out (KO) GM-CSF-dependent BMDCs, which resemble monocyte-derived DCs (MoDCs) induce lower T cell activation than wild-type DCs, and this effect is related to a reduction in MHC-II surface expression in CD9-deficient MoDCs. Importantly, MHC-II targeting to the plasma membrane is largely impaired in immature MoDCs from CD9 KO, in which MHC-II remains arrested in acidic intracellular compartments enriched in LAMP-1, and MHC-II internalization is also blocked. Moreover, CD9 participates in MHC-II trafficking in mature MoDCs, regulating its endocytosis and recycling. Our results demonstrate that the tetraspanin CD9 specifically regulates antigenic presentation in MoDCs through the regulation of MHC-II intracellular trafficking.

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  Saint-Pol, J, Billard, M, Dornier, E, Eschenbrenner, E, Danglot, L, Boucheix, C, Charrin, S & Rubinstein, E 2017, « New Insights into the Tetraspanin Tspan5 Using Novel Monoclonal Antibodies », The Journal of Biological Chemistry.
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  Résumé : Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is most abundant such as the brain, the lung, the kidney or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAb does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signalling. Finally, two antibodies inhibit ligand-induced Notch signalling, and this effect is stronger in cells depleted of the TspanC8 Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signalling.

  Mots-clés : ADAM, ADAM10, intracellular trafficking, metalloprotease, monoclonal antibody, Notch pathway, tetraspanin, Tspan5.

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  Saint-Pol, J, Eschenbrenner, E, Dornier, E, Boucheix, C, Charrin, S & Rubinstein, E 2017, « Regulation of the trafficking and the function of the metalloprotease ADAM10 by tetraspanins », Biochemical Society Transactions, vol. 45, no. 4, p. 937-944.
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  Résumé : By interacting directly with partner proteins and with one another, tetraspanins organize a network of interactions referred to as the tetraspanin web. ADAM10 (A Disintegrin And Metalloprotease 10), an essential membrane-anchored metalloprotease that cleaves off the ectodomain of a large variety of cell surface proteins including cytokines, adhesion molecules, the precursor of the β-amyloid peptide APP or Notch, has emerged as a major component of the tetraspanin web. Recent studies have shown that ADAM10 associates directly with all members (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33) of a subgroup of tetraspanins having eight cysteines in the large extracellular domain and referred to as TspanC8. All TspanC8 regulate ADAM10 exit from the endoplasmic reticulum, but differentially regulate its subsequent trafficking and its function, and have notably a different impact on Notch signaling. TspanC8 orthologs in invertebrates also regulate ADAM10 trafficking and Notch signaling. It may be possible to target TspanC8 tetraspanins to modulate in a tissue- or substrate-restricted manner ADAM10 function in pathologies such as cardiovascular diseases, cancer or Alzheimer's disease.

  Mots-clés : ADAM10, metalloproteases, tetraspanins.

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  Sloma, I, Mitjavila-Garcia, M, Feraud, O, Griscelli, F, Oudrhiri, N, El Marsafy, S, Gobbo, E, Divers, D, Proust, A, Smadja, DM, Desterke, C, Carles, A, Ma, Y, Hirst, M, Marra, MA, Eaves, CJ, Bennaceur-Griscelli, A & Turhan, AG 2017, « Whole genome analysis reveals unexpected dynamics of mutant subclone development in a patient with JAK2-V617F-positive chronic myeloid leukemia », Experimental Hematology.
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  Résumé : We report here the first use of whole genome sequencing (WGS) to examine the initial clonal dynamics in an unusual patient with chronic myeloid leukemia (CML) who presented in chronic phase (CP) with doubly marked BCR-ABL1(+)/JAK2(V617F)-mutant cells and over a 9 year period progressed into an accelerated phase (AP) and then terminal blast phase (BP). WGS showed the diagnostic cells also contained mutations in ASXL1, SEC23B, MAD1L1 and RREB1, as well as 12,000 additional uncommon DNA variants. WGS of endothelial cells generated from circulating precursors revealed many of these were shared with the CML clone. Surprisingly, WGS of induced pluripotent stem cells (iPSCs) derived from the AP cells revealed only 6 additional coding somatic mutations despite retention by their hematopoietic progeny of the parental AP cell levels of BCR-ABL1 expression and sensitivity to imatinib and pimozide. Limited analysis of BP cells showed independent subclonal progression to homozygosity of the MAD1L1 and RREB1 variants. MAD1L1 and SEC23B mutations were also identified in 2/101 cases of myeloproliferative neoplasms but not in 42 healthy subjects. These findings challenge historic concepts of clonal evolution in CML.

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  Torossian, F, Guerton, B, Anginot, A, Alexander, KA, Desterke, C, Soave, S, Tseng, H-W, Arouche, N, Boutin, L, Kulina, I, Salga, M, Jose, B, Pettit, AR, Clay, D, Rochet, N, Vlachos, E, Genet, G, Debaud, C, Denormandie, P, Genet, F, Sims, NA, Banzet, S, Levesque, J-P, Lataillade, J-J & Le Bousse-Kerdilès, M-C 2017, « Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications », JCI insight, vol. 2, no. 21.
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  Résumé : Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury-induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

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  Texte intégral 18 Mo (source)

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  Vatel, O, Aumont, C, Mathy, V, Petit, M, Feriel, J, Sloma, I, Bennaceur-Griscelli, A & Turhan, AG 2017, « Drug reaction with eosinophilia and systemic symptoms (DRESS) induced by imatinib in chronic myeloid leukemia », Leukemia & Lymphoma, vol. 58, no. 2, p. 473-474.
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  Zeng, Y, Liu, B, Rubio, M-T, Wang, X, Ojcius, DM, Tang, R, Durrbach, A, Ru, Z, Zhou, Y & Lone, Y-C 2017, « Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells », PloS One, vol. 12, no. 4, p. e0173754.
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  Résumé : Research on human immunology has been hindered by the lack of optimal small animal models, given that the protective immune responses of human and non-human species show significant differences. However, due to ethical constraints[1] and the high cost of clinical trials, it is urgent to improve the current animal models that can mimic faithfully human physiology, particularly the human immune system (HIS). HIS mice had been generated recently by engrafting human hematopoietic stem cells (hHSCs) or human peripheral mononuclear cells (hPBMCs) into highly immuno-deficient mice such as NSG, NOG or NRG mice. However, a major experimental drawback for studies using these models is the rapid onset of Graft-versus-Host Disease (GvHD). In the present study, we overcome this limitation by generating new immuno-deficient mice named "HUMAMICE" (HLA-A2+/+/DR1+/+/H-2-β2m-/-/IAβ-/-/Rag2-/-/IL2rγ-/-/Perf-/- mice), which expressed human HLA molecules instead of mouse MHC molecules (H-2), and whose immuno-deficient status was reversed by transferring functional HLA-matched PBMCs thus producing mice with an immuno-competent status with a functional human immune system. We showed that in this HLA-matched context, the hPBMC-transfer led to high lymphocytes engraftment rates without GvHD over three months in this novel mouse model. Furthermore, to evaluate the utility of the hPBMC-HUMAMICE, we immunized them with commercial vaccine of Hepatitis B virus (HBsAg, Hepvac@) which resulted in robust and reproducible production of high levels of HBsAg-specific antibodies, implying that both transferred T and B lymphocytes were functional in HUMAMICE. These responses are comparable to those observed in human clinical trials with this identical vaccine. In conclusion, these findings indicated that the HLA-matched-hPBMC-HUMAMICE represents a promising model for dissecting human immune responses in various human diseases, including infectious diseases, cancers and tumors, and to facilitate the development of novel vaccines and cellular therapies.

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  Zhu, Y, Ailane, N, Sala-Valdés, M, Haghighi-Rad, F, Billard, M, Nguyen, V, Saffroy, R, Lemoine, A, Rubinstein, E, Boucheix, C & Greco, C 2017, « Multi-factorial modulation of colorectal carcinoma cells motility - partial coordination by the tetraspanin Co-029/tspan8 », Oncotarget, vol. 8, no. 16, p. 27454-27470.
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  Résumé : Colorectal carcinoma cells Isreco1 display an ability to migrate controlled by a complex set of signals issued from the membrane. By comparing cells infected by mycoplasmas and mycoplasmas free cells, we have established that basal 2D migration is dependent on a double signal mediated by the collagen receptors integrins alpha1/2 and the Toll-Like receptor TLR2. The signal issued from mycoplasmas can be replaced by a TLR2 ligand and the functional effect is neutralized by silencing of MyD88. Following previous observation that downregulation of E-cadherin/p120 catenin increases cell motility, we now report that EGFR or CD44 inhibition have a similar effect on cell motility that is restricted to tetraspanin Co-029/tspan8 transduced IsrecoI cells (Is1-Co029). The modulation of cell migration linked to EGFR or CD44 can be neutralized by antagonizing Co-029 with the mAb Ts29.1 or by RNA interference. Altogether these data point to a crucial role of Co-029 in the modulation of colon cancer cell motility which could be related to the protumoral effect reported for this tetraspanin. Among surface molecules able to mediate Co-029 function, E-cadherin, EGFR and CD44 appear as likely candidates.

  Mots-clés : cell motility, Co-029/tspan8, colorectal carcinoma, EGFR, mycoplasmas.

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2016

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  Akil, A, Peng, J, Omrane, M, Gondeau, C, Desterke, C, Marin, M, Tronchère, H, Taveneau, C, Sar, S, Briolotti, P, Benjelloun, S, Benjouad, A, Maurel, P, Thiers, V, Bressanelli, S, Samuel, D, Bréchot, C & Gassama-Diagne, A 2016, « Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV », Nature Communications, vol. 7, p. 12203.
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  Résumé : The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway.

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  Candelier, J-J 2016, « The hydatidiform mole », Cell Adhesion & Migration, vol. 10, no. 1-2, p. 226-235.
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  Résumé : The hydatidiform mole (HM) is a placental pathology of androgenetic origin. Placental villi have an abnormal hyperproliferation event and hydropic degeneration. Three situations can be envisaged at its origin: 1. The destruction/expulsion of the female pronucleus at the time of fertilization by 1 or 2 spermatozoa with the former being followed by an endoreplication of the male pronucleus leading to a complete hydatidiform mole (CHM) 2. A triploid zygote (fertilization by 2 spermatozoa) leading to a partial hydatidiform mole (PHM) but can also lead to haploid and diploid clones. The diploid clone may produce a normal fetus while the haploid clone after endoreplication generates a CHM 3. A nutritional defect during the differentiation of the oocytes or the deterioration of the limited oxygen pressure during the first trimester of gestation may lead to the formation of a HM. In countries with poor medical health care system, moles (mainly the CHM) can become invasive or, in rare cases, lead to gestational choriocarcinomas.

  Mots-clés : choriocarcinoma, epigenetic, fertilization, Hydatidiform Mole, invasive mole, trophoblast.

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  Dianat, N, Weber, A & Dubart-Kupperschmitt, A 2016, « Human pluripotent stem cells and liver disorders », Biologie Aujourd'hui, vol. 210, no. 1, p. 19-26.
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  Résumé : The liver is associated with many diseases including metabolic and cholestatic diseases, cirrhosis as well as chronic and acute hepatitis. However, knowledge about the mechanisms involved in the pathophysiology of these diseases remains limited due to the restricted access to liver biopsies and the lack of cellular models derived from patients. The liver is the main organ responsible for the elimination of xenobiotics and thus hepatocytes have a key role in toxicology and pharmacokinetics. The induced pluripotent stem cells generated from patients with monogenic metabolic disorders, for which the corresponding gene is identified, are relevant in vitro models for the study of the mechanisms involved in generation of pathologies and also for drug screening. Towards this aim, robust protocols for generating liver cells, such as hepatocytes and cholangiocytes, are essential. Our study focused on familial hypercholesterolemia disease modeling, as well as on establishing a protocol for generation of functional cholangiocytes from pluripotent stem cells.

  Mots-clés : Cell Differentiation, Hepatocytes, Humans, Hyperlipoproteinemia Type II, Induced Pluripotent Stem Cells, Liver, Liver Diseases, Models, Biological.

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  Dornier, E, Coumailleau, F, Ottavi, J-F, Moretti, J, Boucheix, C, Mauduit, P, Schweisguth, F & Rubinstein, E 2016, « Correction: TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals », The Journal of Cell Biology, vol. 213, no. 4, p. 495-496.
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  PubMed entry

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  El-Kehdy, H, Pourcher, G, Zhang, W, Hamidouche, Z, Goulinet-Mainot, S, Sokal, E, Charbord, P, Najimi, M & Dubart-Kupperschmitt, A 2016, « Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation », Stem Cells International, vol. 2016, p. 6323486.
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  Résumé : In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs). After isolation from 11-12 gestational weeks' human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

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  Féraud, O, Valogne, Y, Melkus, MW, Zhang, Y, Oudrhiri, N, Haddad, R, Daury, A, Rocher, C, Larbi, A, Duquesnoy, P, Divers, D, Gobbo, E, Brunet de la Grange, P, Louache, F, Bennaceur-Griscelli, A & Mitjavila-Garcia, MT 2016, « Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines », PloS One, vol. 11, no. 3, p. e0149291.
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  Résumé : Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.

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  François, H, Durrbach, A, Beaudreuil, S, Charpentier, B & Lecru, L 2016, « [Role of cannabinoid receptors in renal diseases] », Nephrologie & Therapeutique.
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  Résumé : Chronic kidney disease remains a major challenge for public health systems and corresponds to the replacement of renal functional tissue by extracellular matrix proteins such as collagens and fibronectin. There is no efficient treatment to date for chronic kidney disease except nephroprotective strategies. The cannabinoid system and more specifically the cannabinoid receptors 1 (CB1) and 2 (CB2) may represent a new therapeutic target in chronic kidney disease. Experimental data obtained in models of diabetes and obesity suggested that CB1 blockade and CB2 stimulation may slow the development of diabetic nephropathy. In human kidneys, CB1 expression is increased in various chronic nephropathies and correlates with renal function. Moreover, endogenous CB1 and CB2 ligands are greatly increased during renal fibrogenesis. A microarray analysis performed in an experimental model of renal fibrosis found that the gene encoding for the CB1 receptor was among the most upregulated genes. We also demonstrated that renal fibrogenesis could be reduced by CB1 inhibition and CB2 stimulation in an experimental model through a direct mechanism involving CB1 on myofibroblasts, which are the major effector cells during renal fibrosis. Therefore, CB1 blockers may represent a novel therapeutic target in chronic kidney disease and diabetes.

  Mots-clés : Cannabinoid receptor, Chronic kidney disease, Diabète, Diabetes, Fibrose rénale, Insuffisance rénale chronique, Récepteur cannabinoïde, Renal fibrosis.

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  Hannoun, Z, Steichen, C, Dianat, N, Weber, A & Dubart-Kupperschmitt, A 2016, « The potential of induced pluripotent stem cell derived hepatocytes », Journal of Hepatology, vol. 65, no. 1, p. 182-199.
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  Résumé : Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers.

  Mots-clés : Directed differentiation, Disease modelling, Drug screening, Genomic integrity, Human induced pluripotent stem cells (hiPSCs), Regenerative medicine, Toxicology studies.

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  Kaščáková, S, Kewish, CM, Rouzière, S, Schmitt, F, Sobesky, R, Poupon, J, Sandt, C, Francou, B, Somogyi, A, Samuel, D, Jacquemin, E, Dubart-Kupperschmitt, A, Nguyen, TH, Bazin, D, Duclos-Vallée, J-C, Guettier, C & Le Naour, F 2016, « Rapid and reliable diagnosis of Wilson disease using X-ray fluorescence », The Journal of Pathology. Clinical Research, vol. 2, no. 3, p. 175-186.
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  Résumé : Wilson's disease (WD) is a rare autosomal recessive disease due to mutations of the gene encoding the copper-transporter ATP7B. The diagnosis is hampered by the variability of symptoms induced by copper accumulation, the inconstancy of the pathognomonic signs and the absence of a reliable diagnostic test. We investigated the diagnostic potential of X-ray fluorescence (XRF) that allows quantitative analysis of multiple elements. Studies were performed on animal models using Wistar rats (n = 10) and Long Evans Cinnamon (LEC) rats (n = 11), and on human samples including normal livers (n = 10), alcohol cirrhosis (n = 8), haemochromatosis (n = 10), cholestasis (n = 6) and WD (n = 22). XRF experiments were first performed using synchrotron radiation to address the elemental composition at the cellular level. High-resolution mapping of tissue sections allowed measurement of the intensity and the distribution of copper, iron and zinc while preserving the morphology. Investigations were further conducted using a laboratory X-ray source for irradiating whole pieces of tissue. The sensitivity of XRF was highlighted by the discrimination of LEC rats from wild type even under a regimen using copper deficient food. XRF on whole formalin-fixed paraffin embedded needle biopsies allowed profiling of the elements in a few minutes. The intensity of copper related to iron and zinc significantly discriminated WD from other genetic or chronic liver diseases with 97.6% specificity and 100% sensitivity. This study established a definite diagnosis of Wilson's disease based on XRF. This rapid and versatile method can be easily implemented in a clinical setting.

  Mots-clés : copper, diagnosis, Wilson disease, X‐ray fluorescence spectroscopy.

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  Le Coz, V, Zhu, C, Devocelle, A, Vazquez, A, Boucheix, C, Azzi, S, Gallerne, C, Eid, P, Lecourt, S & Giron-Michel, J 2016, « IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process », Oncotarget, vol. 7, no. 50, p. 82511-82527.
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  Résumé : Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

  Mots-clés : chemoresistance, EMT, IGF-1, melanoma-initiating cells, metastasis.

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  Ojeda-Uribe, M, Morel, O, Ungureanu, C, Desterke, C, Le Bousse-Kerdilès, M-C & Boulahdour, H 2016, « Assessment of sites of marrow and extramedullary hematopoiesis by hybrid imaging in primary myelofibrosis patients », Cancer Medicine.
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  Résumé : We investigated noninvasive procedures by hybrid imaging to assess the sites of active or inactive hematopoiesis in patients with primary myelofibrosis (PMF). To this end, we used two radionuclides, technetium 99m ((99m) Tc) and indium 111-chloride ((111) In-Cl3 ), coupled with single-photon emission tomography/computed tomography (SPECT/CT). We studied five patients with PMF and one with secondary myelofibrosis (MF). The classical pattern of lower fixation of both tracers at the axial skeleton where the myelofibrotic process occurs and the reactivation of sites of active hematopoiesis at the distal skeleton were confirmed. Coupling both radionuclides to SPECT/CT imaging allowed for more precise visualization of the sites of extramedullary hematopoiesis as those observed in the spleen and liver. Splenic high uptake of (111) In-Cl3 coupled with SPECT/CT represents a pathognomonic feature of PMF. We conclude that, the hybrid imaging procedures that we studied might constitute an alternative noninvasive method for the screening of the whole-body marrow and, by this way, to assess the impact of targeted therapies in PMF patients in whom it is well known that the distribution of the hematopoietic active areas is disturbed. Hybrid imaging could also be useful for diagnostic purposes in cases of early PMF or in suspected cases of myelofibrosis secondary to polycythemia vera or essential thrombocythemia.

  Mots-clés : Extramedullary hematopoiesis, hybrid imaging, primary myelofibrosis, radionuclides, SPECT, spleen.

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  Petit Cocault, L, Fleury, M, Clay, D, Larghero, J, Vanneaux, V & Souyri, M 2016, « Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations », Experimental Hematology, vol. 44, no. 4, p. 297-302.e1.
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  Résumé : Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field.

  Mots-clés : human HSC, Human Mpl.

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  Pourcher, G, El-Kehdy, H, Kanso, F, Groyer-Picard, M-T, Gaillard, M, Trassard, O, Blazsek, I, Agostini, H, Dubart-Kupperschmitt, A & Dagher, I 2016, « Volumetric Portal Embolization: A New Concept to Improve Liver Regeneration and Hepatocyte Engraftment », Transplantation, vol. 100, no. 2, p. 344-354.
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  Résumé : BACKGROUND: Hepatocyte transplantation has been proposed as an alternative to orthotopic liver transplantation to treat metabolic liver diseases. This approach requires preconditioning of the host liver to enhance engraftment of transplanted hepatocytes. Different methods are currently used in preclinical models: partial hepatectomy, portal ligature or embolization, and radiotherapy or chemotherapeutic drugs. However, these methods carry high risks of complications and are problematic for use in clinical practice. Here, we developed an innovative method called volumetric (distal, partial, and random) portal embolization (VPE), which preserves total liver volume. METHODS: Embolization was performed in the portal trunk of C57BL6 adult mice with polyester microspheres, to ensure a bilateral and distal distribution. The repartition of microspheres was studied by angiographic and histological analyses. Liver regeneration was evaluated by Ki67 labeling. Optimal conditions for VPE were determined, and the resulting regeneration was compared with that after partial hepatectomy (70%). Labeled adult hepatocytes were then transplanted, and engraftment was compared between embolized (n = 19) and nonembolized mice (n = 8). Engraftment was assessed in vivo and histologically by tracking labeled cells at day 5. RESULTS: The best volumetric embolization conditions, which resulted in the regeneration of 5% of total liver, were 8 × 10 ten-micron microspheres infused with a 29 G needle directly into the portal trunk at 3.3 μL/s. In these conditions, transplanted hepatocytes engraftment was significantly higher than that in control conditions (3 vs 0.65%). CONCLUSIONS: The VPE is a new, minimally invasive, and efficient technique to prepare the host liver for cell transplantation.

  Mots-clés : Animals, Biomarkers, Cell Survival, Cell Tracking, Embolization, Therapeutic, Female, Graft Survival, Hepatectomy, Hepatocytes, Injections, Intravenous, Ki-67 Antigen, Liver, Liver Regeneration, Male, Mice, Inbred C57BL, Microspheres, Organ Size, Polyesters, Portal Vein, Radiography, Time Factors.

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  Strassel, C, Bull, A, Moog, S, Receveur, N, Mallo, L, Mangin, P, Eckly, A, Freund, M, Dubart-Kupperschmitt, A, Gachet, C & Lanza, F 2016, « Lentiviral gene rescue of a Bernard-Soulier mouse model to study platelet glycoprotein Ibβ function », Journal of thrombosis and haemostasis: JTH, vol. 14, no. 7, p. 1470-1479.
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  Résumé : Essentials A signaling role of glycoprotein (GP)Ibβ is postulated but not formally demonstrated in platelets. Lentiviral-mediated rescue in knock-out mice can be used to evaluate GPIbβ function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb-IX deficiency Deletion of intracellular 159-170 segment increased thrombosis, 150-160 removal increased bleeding. SUMMARY: Background The platelet glycoprotein (GP)Ib-V-IX complex is required for normal hemostasis and megakaryopoiesis. A role in GPIb-dependent responses has been ascribed to the less well characterized GPIbβ subunit using a specific antibody and GPIb-IX transfected cells. Objectives Our aim was to evaluate, in vivo, the role of the GPIbβ in hemostasis and thrombosis. Methods GPIbβ(null) Sca-1(+) progenitors transduced with viral particles harboring hGPIbβ were transplanted into lethally irradiated GPIbβ(-/-) recipient mice. Results hGPIbβ transplanted into the bone marrow of GPIbβ(null) mice rescued GPIb-IX expression in 97% of circulating platelets. These platelets efficiently bound von Willebrand factor (VWF) and extended filopodia on a VWF matrix, demonstrating the restoration of GPIb-dependent adhesive and signaling properties. These mice exhibited less severe macrothrombocytopenia and had normal tail bleeding times as compared with GPIbβ(null) mice. This strategy was employed to manipulate and evaluate the role of the GPIbβ intracellular domain. Removal of the membrane proximal segment (Δ(150-160) ) decreased GPIb-IX expression by 43%, confirming its involvement in receptor assembly and biosynthesis, and resulted in increased bleeding times and decreased thrombosis in a mechanical injury model in the aorta. On the other hand, deletion of the C-flanking 159-170 segment allowed normal GPIb-IX expression, VWF-dependent responses and bleeding times, but resulted in enhanced arterial thrombosis. Conclusion This pointed to a repressor role of GPIbβ in thrombus formation in vivo that was not predicted in studies of heterologous cells. These results highlight the utility of this lentiviral strategy for the structure-function evaluation of GPIb-IX in platelets.

  Mots-clés : Bernard-Soulier syndrome, blood platelets, platelet function tests, platelet glycoprotein GPIb-IX complex, thrombosis, virion.

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