Biliary cirrhosis and sepsis are two risk factors of failure after surgical repair of major bile duct injury post-laparoscopic cholecystectomy.

International audienceBACKGROUND: Major bile duct injury (MBDI) remains frequent after laparoscopic cholecystectomy (LC) reaching 0.3 to 0.6 % and is associated with a significant mortality rate. The aim of this study was to retrospectively analyze the factors likely to influence the long-term results of surgical repair for MBDI occurring after LC. METHODS: Medical records of patients referred to our referral center from January 1992 to January 2010 for management of bile duct injury following LC were retrospectively analyzed, and patients with MBDI were identified. Clinicopathological factors likely to influence long-term results after surgical repair were assessed by univariate and multivariable analysis. RESULTS: During the study period, 38 patients were treated for MBDI. These 38 patients underwent Roux-en-Y hepaticojejunostomy (HJ) or HJ revision in 25 (66 %) and 13 (34 %) cases, respectively. The median follow-up period was 93 (26-204) months. A Clavien-Dindo post-operative morbidity class >3 occurred in 10 (26 %) cases and was independently associated with a surgical repair performed during a sepsis period (OR = 102.5; IC 95 % [7.12; 11,352], p < 0.007). Long-term results showed that biliary strictures occurred in 5 (13 %) cases and were associated with sepsis (p < 0.006), liver cirrhosis (p < 0.002) and post-operative complications (p < 0.012). Multivariate analysis revealed that only liver cirrhosis remained predictive of stricture (OR = 26.4, 95 % CI [2; 1,018], p < 0.026). CONCLUSION: When MBDI occurs following LC, HJ seems to be the optimal treatment but should not be performed during a sepsis period. Long-term results are significantly altered by the presence of a biliary cirrhosis at time of repair

Pancreatic head cancer in patients with chronic pancreatitis

International audienceBACKGROUND: Chronic pancreatitis (CP) is a risk factor of pancreatic adenocarcinoma (PA). The discovery of a pancreatic head lesion in CP frequently leads to a pancreaticoduodenectomy (PD) which preceded by a multidisciplinary meeting (MM). The aim of this study was to evaluate the relevance between this indication of PD and the definitive pathological results. METHODS: Between 2000 and 2010, all patients with CP who underwent PD for suspicion of PA without any histological proof were retrospectively analyzed. The operative decision has always been made at an MM. The definitive pathological finding was retrospectively confronted with the decision made at an MM, and patients were classified in two groups according to this concordance (group 1) or not (group 2). Clinical and biological parameters were analyzed, preoperative imaging were reread, and confronted to pathological findings in order to identify predictive factors of malignant degeneration. RESULTS: During the study period, five of 18 (group 1) patients with CP had PD were histologically confirmed to have PA, and the other 13 (group 2) did not have PA. The median age was 52.5+/-8.2 years (gender ratio 3.5). The main symptoms were pain (94.4%) and weight loss (72.2%). There was no patient's death. Six (33.3%) patients had a major complication (Clavien-Dindo classification ≥ 3). There was no statistical difference in clinical and biological parameters between the two groups. The rereading of imaging data could not detect efficiently all patients with PA. CONCLUSIONS: Our results confirmed the difficulty in detecting malignant transformation in patients with CP before surgery and therefore an elevated rate of unnecessary PD was found. A uniform imaging protocol is necessary to avoid PD as a less invasive treatment could be proposed

A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator

International audienceMiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets. In one cluster containing three miRNAs, miR-612, miR-661 and miR-940, the annotated functions of the co-regulated genes suggested a role in small GTPase signalling. Although the three members of this cluster targeted the same subset of predicted genes, we showed that their overexpression impacted cell fates differently. miR-661 demonstrated enhanced phosphorylation of myosin II and an increase in cell invasion, indicating a possible oncogenic miRNA. On the contrary, miR-612 and miR-940 inhibit phosphorylation of myosin II and cell invasion. Finally, expression profiling in human breast tissues showed that miR-940 was consistently downregulated in breast cancer tissues M icroRNAs are a class of endogenous, small (19–25 nucleotides), single-stranded non-coding RNAs that regulate gene expression in all eukaryotic organisms. In metazoans, microRNAs most commonly bind to the 39 untranslated region (39UTR) of their mRNA target transcript and cause translational repression and/or mRNA degradation. Every microRNA is predicted to regulate from a dozen to thousands of genes, including transcription factors. This fine-tuning of protein expression is known to be involved in many physiological processes, such as development, apoptosis, signal transduction and even cancer progression 1,2. More than 2,000 mature human microRNAs are listed in the 20 th release of miRBase: http://www.mirbase.org (2014) (Date of access:19/08/2013), and some authors hypothesise that the majority of human genes are regulated by microRNAs 3. Since their discovery in 1993 4 , a fair understanding of their role in animal development and in the onset and progression of diseases 2 , as well as of their potential use in therapies 5 , has been gathered. However, the cooperative behaviour of microRNAs is still under investigation. A growing body of experimental evidence suggests that microRNAs can regulate genes through complementarity, meaning that microRNAs can act together to regulate individual genes or groups of genes involved in similar processes 6. For example, Hu and co-workers demonstrated that transducing a cocktail of precursor microRNAs (miR-21, miR-24 and miR-221) can result in more effective engraftment of transplanted cardiac progenitor cells 7. Consistent with these discoveries, Zhu et al. demonstrated that miR-21 and miR-221 coregulate 56 gene ontology (GO) processes 8. In the same study, the authors also showed that cotransfection of miR-1 and miR-21 increases H 2 O 2-induced myocardial apoptosis and oxidative stress. These recent findings support the idea of microRNA-mediated cooperative regulation but also argue for the use of systemic approaches, notably based on graph theory, to decipher individual and complementary roles of microRNAs. Some work has been conducted to use recent high-throughput experiment-derived data sets to infer microRNA synergistic relationships 9–12. Herein, we present a microRNA network based on target similarities among microRNAs to infer clusters of microRNAs. Clusters are defined as groups of microRNAs sharing a set of common targets, predicted by either DIANA-microT v3 13 or TargetScan v6.2 14. Some authors have used GO enrichment analysis as a confirmatory tool for their clustering approach 11. In our case, GO enrichment is not used to infer networks but as a way to estimate the probable metabolic pathway(s) a cluster of microRNAs could co-regulate. Moreover, the novelty of our approach is to consider not only clusters of microRNAs but also OPE

Fatigue et auto-échauffement sous sollicitation cyclique des alliages à mémoire de forme.

Les alliages à mémoire de forme (AMF) sont des matériaux capables de recouvrer une grande déformation élastique (jusqu'à 8%) sous chargement thermomécanique. Le mécanisme de déformation à l’origine de cette capacité est un changement de phase solide/solide du type martensitique. Cet alliage est utilisé pour des applications biomédicales ou dans le domaine du transport. Les AMF sont notamment utilisés pour la réalisation d’instrument endodontique. Ils sont utilisés par les dentistes pour réaliser certaines opérations sur les canaux des racines de dents. La propriété super-élastique du Nickel-titane (NiTi) permet aux instruments de s’adapter parfaitement aux géométries des racines de dents. Le comportement thermomécanique de ces alliages est étudié depuis quelques années et plusieurs modèles ont été développés pour décrire ce comportement. En revanche les propriétés en fatigue restent un domaine peu exploré. En effet actuellement lors de la phase de conception de structure utilisant cet alliage, la fatigue n’est pas considérée. Or les dentistes rencontrent des problèmes de rupture en fatigue qui peuvent compliquer la suite de l’opération. Cette étude commence par la réalisation d’essais de fatigue en traction sur éprouvette à faible et à grand nombre de cycles. Ainsi nous obtenons des courbes de fatigue classique (amplitude de chargement en fonction du nombre de cycles à rupture). En parallèle nous utilisons une technique dite rapide appelée essais d’auto-échauffement sous sollicitation cyclique pour déterminer les propriétés de tenue à la fatigue de ces matériaux. Ainsi nous pouvons valider la méthode rapide à l’aide d’essais classiques dans l’optique d’étudier l’influence de différents paramètres du procédé de fabrication sur les propriétés en fatigue des instruments. Pour se rapprocher du chargement vu par les instruments en utilisation des essais de fatigue en flexion rotative sont également réalisés

“Fibrous nests” in human hepatocellular carcinoma express a Wnt-induced gene signature associated with poor clinical outcome

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Phi-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays

International audiencePhenotypic screening monitors phenotypic changes induced by perturbations, including those generated by drugs or RNA interference. Currently-used methods for scoring screen hits have proven to be problematic, particularly when applied to physiologically relevant conditions such as low cell numbers or inefficient transfection. Here, we describe the Phi-score, which is a novel scoring method for the identification of phenotypic modifiers or hits in cell-based screens. Phi-score performance was assessed with simulations, a validation experiment and its application to gene identification in a large-scale RNAi screen. Using robust statistics and a variance model, we demonstrated that the Phi-score showed better sensitivity, selectivity and reproducibility compared to classical approaches. The improved performance of the Phi-score paves the way for cell-based screening of primary cells, which are often difficult to obtain from patients in sufficient numbers. We also describe a dedicated merging procedure to pool scores from small interfering RNAs targeting the same gene so as to provide improved visualization and hit selection

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages.: DLL4's opposing effects in choroidal neovascularization

International audienceInflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1β expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1β expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV

Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency

INTRODUCTION: Dendritic cells (DCs) are antigen-presenting cells that are currently employed in cancer clinical trials. However, it is not clear whether their ability to induce tumour-specific immune responses when they are isolated from cancer patients is reduced relative to their ability in vivo. We determined the phenotype and functional activity of DCs from cancer patients and investigated the effect of putrescine, a polyamine molecule that is released in large amounts by cancer cells and has been implicated in metastatic invasion, on DCs. METHODS: The IL-4/GM-CSF (granulocyte–macrophage colony-stimulating factor) procedure for culturing blood monocyte-derived DCs was applied to cells from healthy donors and patients (17 with breast, 7 with colorectal and 10 with renal cell carcinoma). The same peroxide-treated tumour cells (M74 cell line) were used for DC pulsing. We investigated the effects of stimulation of autologous lymphocytes by DCs pulsed with treated tumour cells (DC-Tu), and cytolytic activity of T cells was determined in the same target cells. RESULTS: Certain differences were observed between donors and breast cancer patients. The yield of DCs was dramatically weaker, and expression of MHC class II was lower and the percentage of HLA-DR(-)Lin(- )cells higher in patients. Whatever combination of maturating agents was used, expression of markers of mature DCs was significantly lower in patients. Also, DCs from patients exhibited reduced ability to stimulate cytotoxic T lymphocytes. After DC-Tu stimulation, specific cytolytic activity was enhanced by up to 40% when DCs were from donors but only up to 10% when they were from patients. IFN-γ production was repeatedly found to be enhanced in donors but not in patients. By adding putrescine to DCs from donors, it was possible to enhance the HLA-DR(-)Lin(- )cell percentage and to reduce the final cytolytic activity of lymphocytes after DC-Tu stimulation, mimicking defective DC function. These putrescine-induced deficiencies were reversed by treating DCs with all-trans retinoic acid. CONCLUSION: These data are consistent with blockade of antigen-presenting cells at an early stage of differentiation in patients with breast cancer. Putrescine released in the microenvironmement of DCs could be involved in this blockade. Use of all-trans retinoic acid treatment to reverse this blockade and favour ex vivo expansion of antigen-specific T lymphocytes is of real interest

Role of the microenvironment in the progression of intrahepatic cholangiocarcinoma : molecular mechanisms and prognostic biomarkers research.

Le but de ce travail était de rechercher le rôle du microenvironnement dans la progression tumorale du cholangiocarcinome intrahépatique (CCIH) par une approche translationnelle, associant recherche fondamentale et clinique. Une étude transcriptomique du stroma tumoral a permis de mettre en évidence une signature spécifique de celui-ci, dont l’analyse non supervisée montrait un enrichissement dans les gènes de la matrice extracellulaire, du cycle cellulaire, de la voie TGFβ et des marqueurs de cellules souches. Ces résultats ont été validés au niveau protéique par immunohistochimie sur tissue microarrays à partir d’une cohorte indépendante. La corrélation de ces résultats avec les données cliniques a permis de démontrer que le niveau d’expression de l’Osteopontin dans le stroma était un facteur de risque indépendant de récidive et de survie. Par ailleurs, nous avons démontré que le taux sérique d’Osteopontin préopératoire des patients porteurs d’un CCIH était significativement supérieur à celui de sujets sains. Avec un seuil déterminé à 57,8 ng/ml, la sensibilité et spécificité de ce biomarqueur diagnostique était respectivement de 80 et 100%. De plus, nous avons apporté des arguments supplémentaires concernant le rôle des cellules souches cancéreuses dans la progression du CCIH, en mettant en évidence une corrélation entre le niveau d’expression de marqueurs souches tels qu’EpCAM et CD44 dans le stroma tumoral ainsi que dans le tissu fibreux du foie « sain » péri-lésionnel et le risque de récidive. Les résultats de notre étude ont confirmé le rôle central du microenvironnement dans la progression du CCIH, permis de mettre en évidence 2 nouveaux biomarqueurs pronostiques, et ouvert de nouvelles voies de recherche thérapeutiques.The aim of this study was to specifically determine through a translational approach combining basic and clinical research, the role of the microenvironment in the tumor progression of intrahepatic cholangiocarcinoma (ICC). By gene expression profiling, we identified a signature that significantly discriminate the tumor stroma from non-tumor fibrous tissue, and the functional analysis of differentially expressed genes showed an enrichment in genes of the extracellular matrix , the cell cycle, the TGFb pathway and stem cell markers. Tissue microarray analysis using an independent cohort of ICC patients validated at a protein level the increased expression of selected candidate genes. Statistical analysis between basic and clinical data demonstrated that the stromal expression of Osteopontin was an independent prognostic marker for overall and disease-free survival. We also demonstrated that the preoperative serum level of Osteopontin was significantly higher in ICC patients than in healthy subjects. Our results identified the best diagnostic threshold to 57,8 ng/ml, associated with a sensitivity and specificity reaching to 80 and 100%, respectively. Moreover, we showed that level expression of stem cell markers such as EpCAM and CD44 in tumor stroma as well as in the fibrous non tumor liver tissue was correlated with recurrence, suggesting the pivotal role of cancer stem cells in ICC prognosis. In conclusion, our study confirmed the major involvement of the microenvironment in the progression of CCIH, allowed to identify two new prognostic tumor biomarkers, and highlighted new pathways for targeted therapeutics