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Metastatic progression is a major burden for breast cancer patients and is associated with the ability of cancer cells to overcome stressful conditions, such as nutrients deprivation and hypoxia, and to gain invasive properties. Autophagy and epithelial-to-mesenchymal transition are critical contributors to these processes. Here, we show that the P2X4 purinergic receptor is upregulated in breast cancer biopsies from patients and it is primarily localised in endolysosomes. We demonstrate that P2X4 enhanced invasion in vitro, as well as mammary tumour growth and metastasis in vivo. The pro-malignant role of P2X4 was mediated by the regulation of lysosome acidity, the promotion of autophagy and cell survival. Furthermore, the autophagic activity was associated with epithelial-to-mesenchymal transition (EMT), and this role of P2X4 was even more pronounced under metabolic challenges. Pharmacological and gene silencing of P2X4 inhibited both autophagy and EMT, whereas its rescue in knocked-down cells led to the restoration of the aggressive phenotype. Together, our results demonstrate a previously unappreciated role for P2X4 in regulating lysosomal functions and fate, promoting breast cancer progression and aggressiveness.

Colorectal cancer (CRC) is associated with high mortality worldwide and new targets are needed to overcome treatment resistance. Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of CRC. In this context, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new potential inhibitor of NCLX.In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis through mitochondrial permeability transition pore opening concomitant with G2/M cell cycle arrest. NCLX inhibition with either CGP37157 (a benzodiazepine derivative), small interfering RNA-mediated knock-down or knockout approaches reproduced the effects of curcumin. Altered mitochondrial respiration, cellular aerobic glycolysis and endoplasmic reticulum–mitochondria membrane perturbations participated in these mechanisms. In a xenograft mouse model, NCLX inhibitors decreased CRC tumor growth. Both transcriptomic analysis of The Cancer Genome Atlas dataset and immunohistochemical analysis of tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC demonstrated that higher NCLX expression was associated with MSI status and for the first time NCLX expression was significantly associated with recurrence-free survival in MSI CRC patients.Our findings provide strong evidence that blocking NCLX inhibits CRC in vitro and in vivo. We highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic treatment of patients with MSI CRC.

BackgroundThrombus composition has the potential to affect acute ischemic stroke (AIS) treatment.ObjectiveTo evaluate in an in vitro test the correlation of clot composition, especially erythrocytes (red blood cells (RBCs)), with the variation of signal intensity ratio (SIR) obtained with MRI sequences used for AIS, and qualification of the susceptibility vessel sign effect using clot analogs.Materials and methodsNine ovine clots were fixed in a gelatin-manganese solution and studied by MRI (T2GE, T2-weighted gradient echo; SWI, susceptibility-weighted imaging; FLAIR, fluid attenuated inversion recovery). RBC concentration was estimated using regression models (SLR, single linear regression; MLR, multiple linear regression; RF, random Forest; and ANN, artificial neural networking), which combined the SIR–histology relationship of three MRI sequences.ResultsNegative correlation was found between SIR and RBC concentration. T2GE SWI could not statistically distinguish clots with RBC content >54% and ConclusionsWe presented a method to quantify RBC concentration in clot analogs, combining SWI, T2GE, and FLAIR. This in vitro study has some limitations, so clot collection after thrombectomy with simultaneous imaging analysis is necessary to validate this model.

In prostate cancer research, there is a lack of valuable preclinical models. Tumor cell heterogeneity and sensitivity to microenvironment signals, such as hypoxia or extracellular calcium concentration, are difficult to reproduce. Here, we developed and characterized an ex vivo tissue culture model preserving these properties. Prostate tissue slices from 26 patients were maintained ex vivo under optimized culture conditions. The expression of markers associated with proliferation, androgen-receptor signaling, and hypoxia was assessed by immunostaining. A macroscope was used to achieve real-time calcium fluorescence optical imaging. Tissue morphology was maintained successfully without necrosis for 5 days. Compared with native tumors and tissue cultured with androgens, androgen deprivation in the medium led to decreased expression of both androgen receptor and its target gene products, prostate specific antigen (PSA) and ETS-related gene (ERG). Ex vivo cultured slices also were sensitive to hypoxia because carbonic anhydrase IX and zinc finger E-box binding homeobox 1 (Zeb1) protein levels increased in 1% oxygen. Exposure of slices to supraphysiological extracellular Ca2+ concentration induced a robust and rapid Ca2+ entry, with a greater response in tumor compared with nontumor tissue. This ex vivo model reproduces the morphologic and functional characteristics of human prostate cancer, including sensitivity to androgen deprivation and induced response to hypoxia and extracellular Ca2+. It therefore could become an attractive tool for drug response prediction studies.

Metastatic cancer cells are highly plastic for the expression of different tumor phenotype hallmarks and organotropism. This plasticity is highly regulated but the dynamics of the signaling processes orchestrating the shift from one cell phenotype and metastatic organ pattern to another are still largely unknown. The scaffolding protein NHERF1 has been shown to regulate the expression of different neoplastic phenotypes through its PDZ domains, which forms the mechanistic basis for metastatic organotropism. This reprogramming activity was postulated to be dependent on its differential phosphorylation patterns. Here, we show that NHERF1 phosphorylation on S279/S301 dictates several tumor phenotypes such as in vivo invasion, NHE1-mediated matrix digestion, growth and vasculogenic mimicry. Remarkably, injecting mice with cells having differential NHERF1 expression and phosphorylation drove a shift from the predominantly lung colonization (WT NHERF1) to predominately bone colonization (double S279A/S301A mutant), indicating that NHERF1 phosphorylation also acts as a signaling switch in metastatic organotropism.

Simple Summary Currently, neuroendocrine prostate cancers remain fatal, so it is crucial to better understand mechanisms of resistance to hormone therapy driving this phenotype. We have shown that Enza, a new generation hormone therapy, promotes prostate cancer cells neurodifferentiation by activating a positive feedback loop between the key transcription factor of epithelial to mesenchymal transition Zeb1 and the calcium-sensitive potassium channel SK3. These two actors are overexpressed in patients with neuroendocrine castration-resistant prostate cancer. Targeting SK3 channel by Ohmline, a synthetic ether lipid, inhibits neuroendocrine differentiation of prostate cancer cells, which opens new therapeutic prospects for neuroendocrine prostate cancers. Abstract Therapeutic strategies for metastatic castration-resistant prostate cancer aim to target androgen receptor signaling. Despite initial survival benefits, treatment resistance invariably occurs, leading to lethal disease. Therapies targeting the androgen receptor can induce the emergence of a neuroendocrine phenotype and reactivate embryonic programs associated with epithelial to mesenchymal transition. We recently reported that dysregulation of the calcium signal can induce the transcription factor Zeb1, a key determinant of cell plasticity during tumor progression. The aim of this study was to determine whether the androgen receptor-targeted treatment Enzalutamide could induce dysregulation of the calcium signal involved in the progression toward epithelial to mesenchymal transition and neuroendocrine differentiation, contributing to therapeutic escape. Our results show that Zeb1 and the SK3 potassium channel are overexpressed in vivo in neuroendocrine castration-resistant prostate cancer and in vitro in LNCaP cells neurodifferentiated after Enzalutamide treatment. Moreover, the neuroendocrine phenotype is associated with a deregulation of the expression of Orai calcium channels. We showed that Zeb1 and SK3 are critical drivers of neuroendocrine differentiation. Interestingly, Ohmline, an SK3 inhibitor, can prevent the expression of Zeb1 and neuroendocrine markers induced by Enzalutamide. This study offers new perspectives to increase hormone therapy efficacy and improve clinical outcomes.

Hypoxia is a well-established feature of prostate cancer (PCa) and is associated with disease aggressiveness. The hypoxic microenvironment initiates multiple adaptive responses including epithelial-to-mesenchymal transition (EMT) and a remodeling of calcium homeostasis involved in cancer progression. In the present study, we identified a new hypoxia signaling pathway with a positive feedback loop between the EMT transcription factor Zeb1 and SK3, a Ca2+-activated K+ channel, which leads to amplifying store-operated Ca2+ entry. Zeb1 and SK3 channel were strongly upregulated by hypoxia both in vitro and ex vivo in organotypic cultures of human PCa. Taking into account the sensitivity of the SK3 channel to the membrane lipid composition, we identified lipids such as Ohmline (an alkyl ether lipid and SK3 inhibitor), linoleic acid (LA) and eicosapentaenoic acid (EPA) (fatty acids associated with indolent PCa), which were able to completely abrogate the hypoxia-induced changes in Zeb1 expression. Ultimately, better understanding of this new hypoxia-induced EMT pathway may allow to develop adjuvant therapeutic strategies, in order to control PCa aggressiveness and improve treatment outcomes.

Mesenchymal stem cells (MSCs)-derived spheroid models favor maintenance of stemness, ex vivo expansion and transplantation efficacy. Spheroids may also be considered as useful surrogate models of the hematopoietic niche. However, accessibility to primary cells, from bone marrow (BM) or adipose tissues, may limit their experimental use and the lack of consistency in methods to form spheroids may affect data interpretation. In this study, we aimed to create a simple model by examining the ability of cell lines, from human (HS-27a and HS-5) and murine (MS-5) BM origins, to form spheroids, compared to primary human MSCs (hMSCs). Our protocol efficiently allowed the spheroid formation from all cell types within 24 hours. Whilst hMSCs-derived spheroids began to shrink after twenty-four hours, the size of spheroids derived from cell lines remained constant during three weeks. The difference was partially explained by the balance between proliferation and cell death, which could be triggered by hypoxia and induced oxidative stress. Our results demonstrate that, unlike hMSCs, MSC cell lines make reproductible spheroids that are easily handled. Thus, this model could help in understanding mechanisms involved in MSC functions and may provide a simple model by which to study cell interactions in the BM niche.

International audience

International audience; In prostate cancer research, there is a lack of valuable preclinical models. Tumor cell heterogeneity and sensitivity to microenvironment signals, such as hypoxia or extracellular calcium concentration, are difficult to reproduce. Here, we developed and characterized an ex vivo tissue culture model preserving these properties. Prostate tissue slices from 26 patients were maintained ex vivo under optimized culture conditions. The expression of markers associated with proliferation, androgen-receptor signaling, and hypoxia was assessed by immunostaining. A macroscope was used to achieve real-time calcium fluorescence optical imaging. Tissue morphology was maintained successfully without necrosis for 5 days. Compared with native tumors and tissue cultured with androgens, androgen deprivation in the medium led to decreased expression of both androgen receptor and its target gene products, prostate specific antigen (PSA) and ETS-related gene (ERG). Ex vivo cultured slices also were sensitive to hypoxia because carbonic anhydrase IX and zinc finger E-box binding homeobox 1 (Zeb1) protein levels increased in 1% oxygen. Exposure of slices to supraphysiological extracellular Ca2+ concentration induced a robust and rapid Ca2+ entry, with a greater response in tumor compared with nontumor tissue. This ex vivo model reproduces the morphologic and functional characteristics of human prostate cancer, including sensitivity to androgen deprivation and induced response to hypoxia and extracellular Ca2+. It therefore could become an attractive tool for drug response prediction studies.

Introduction L’objectif de notre travail etait d’etudier le signal IRM (Imagerie par Resonance Magnetique) de caillots sanguins afin de determiner s’il etait possible d’estimer leurs concentrations en hematies. Materiel et methodes Dans un premier temps, une etude in vitro analysant le signal IRM de caillots de compositions predeterminees sur une IRM 3 Tesla a ete realisee. Les caillots etaient fixes dans une gelatine ayant un signal proche de celui du cerveau humain. Pour estimer la concentration en hematies, un ratio d’intensite de signal (RIS) etait calcule pour chaque caillot sur plusieurs sequences IRM selon la formule suivante : RIS = signal moyen caillot/signal moyen gelatine. Dans un second temps, les RIS de caillots responsables d’AVC ischemique aigu ont ete mesures sur des IRM cerebrales de patients pris en charge pour thrombectomie cerebrale ( Fig. 1 ). Les taux d’hematies obtenus par estimation IRM etaient compares aux resultats de l’analyse histologique des caillots ( Fig. 2 ). Resultats Les resultats de l’etude in vitro indiquaient qu’il etait possible d’estimer la concentration en hematies des caillots grâce aux RIS mesures sur les sequences T2FLAIR, SWI et T2EG. La combinaison du signal moyen mesure sur ces 3 sequences permettait une bonne estimation par comparaison avec l’analyse histologique. Ces resultats ont ete confirmes sur l’analyse de 22 IRM cerebrales de patients. Conclusion La mesure du signal moyen des caillots responsables d’occlusions intracrâniennes sur les sequences T2GRE, SWI et T2FLAIR permet une bonne estimation de leur concentration en hematies.

International audience; Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8(TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca(2+)release channels.

International audience; Background - Genetic and nutritional factors have been linked to the risk of aggressive prostate cancer (PCa). The fatty acid (FA) composition of peri-prostatic adipose tissue (PPAT), which reflects the past FA intake, is potentially involved in PCa progression. We analysed the FA composition of PPAT, in correlation with the ethno-geographical origin of the patients and markers of tumour aggressiveness. Methods - From a cohort of 1000 men treated for PCa by radical prostatectomy, FA composition of PPAT was analysed in 156 patients (106 Caucasians and 50 African-Caribbeans), 78 with an indolent tumour (ISUP group 1 + pT2 + PSA

Calcium (Ca

// Madiha Khalid 1, 2, * , Lucie Brisson 3, * , Menahil Tariq 1 , Yunjie Hao 1 , Roseline Guibon 4 , Gaelle Fromont 4 , Sharifah Alawieyah Syed Mortadza 1 , Fatema Mousawi 1 , Sobia Manzoor 2 , Sebastien Roger 3, 5 and Lin-Hua Jiang 1, 3, 6, 7 1 School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK 2 Atta-ur-Rahman School of Applied Biosciences, National University of Science and Technology, Islamabad, Pakistan 3 Inserm UMR1069, Nutrition, Croissance et Cancer, Universite Francois-Rabelais de Tours, Tours, France 4 Centre Hospitalo-Universitaire de Tours, Tours, France 5 Institut Universitaire de France, Paris Cedex 05, France 6 Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, P. R. China 7 Sino-UK Joint Laboratory of Brain Function and Injury, Xinxiang Medical University, Xinxiang, P. R. China * These authors have contributed equally to this work Correspondence to: Lin-Hua Jiang, email: l.h.jiang@leeds.ac.uk Sebastien Roger, email: sebastien.roger@univ-tours.fr Sobia Manzoor, email: dr.sobiamanzoor@asab.nust.edu.pk Keywords: HCC cells, extracellular ATP, P2Y 11 receptor, cytosolic Ca 2+ , cell migration Received: March 19, 2016 Accepted: February 15, 2017 Published: March 14, 2017 ABSTRACT Extracellular ATP-induced Ca 2+ signalling is critical in regulating diverse physiological and disease processes. Emerging evidence suggests high concentrations of extracellular ATP in tumour tissues. In this study, we examined the P2 receptor for ATP-induced Ca 2+ signalling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) showed that extracellular ATP induced an increase in the [Ca 2+ ] i in human HCC Huh-7 and HepG2 cells. NF546, a P2Y 11 receptor agonist was equally effective in inducing an increase in the [Ca 2+ ] i . In contrast, agonists for the P2X receptors (αβmeATP and BzATP), P2Y 1 receptor (MRS2365) or P2Y 2 receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increases in the [Ca 2+ ] i were strongly inhibited by treatment with NF340, a P2Y 11 receptor antagonist. Immunofluorescent confocal imaging and western blotting analysis consistently demonstrated the P2Y 11 receptor expression in Huh-7 and HepG2 cells. Transfection with P2Y 11 -specific siRNA attenuated the P2Y 11 receptor protein expression level and also reduced NF546-induced increase in the [Ca 2+ ] i . Importantly, immunohistochemistry revealed that the P2Y 11 receptor was expressed at very high level in human HCC tissues and, by contrast, it was barely detected in normal liver tissues. Trans-well cell migration assay demonstrated that ATP and NF546 induced concentration-dependent stimulation of Huh-7 cell migration. Treatment with NF340 prevented ATP-induced stimulation of cell migration. Taken together, our results show carcinoma-specific expression of the P2Y 11 receptor and its critical role in mediating ATP-inducing Ca 2+ signalling and regulating cell migration in human HCC cells.

The development of metastases largely relies on the capacity of cancer cells to invade extracellular matrices (ECM) using two invasion modes termed ‘mesenchymal' and ‘amoeboid', with possible transitions between these modes. Here we show that the SCN4B gene, encoding for the β4 protein, initially characterized as an auxiliary subunit of voltage-gated sodium channels (NaV) in excitable tissues, is expressed in normal epithelial cells and that reduced β4 protein levels in breast cancer biopsies correlate with high-grade primary and metastatic tumours. In cancer cells, reducing β4 expression increases RhoA activity, potentiates cell migration and invasiveness, primary tumour growth and metastatic spreading, by promoting the acquisition of an amoeboid–mesenchymal hybrid phenotype. This hyperactivated migration is independent of NaV and is prevented by overexpression of the intracellular C-terminus of β4. Conversely, SCN4B overexpression reduces cancer cell invasiveness and tumour progression, indicating that SCN4B/β4 represents a metastasis-suppressor gene., The capacity of cancer cells to migrate is intimately linked to their ability to induce metastasis. Here the authors show that the sodium channel β4 subunit regulates breast cancer cell migration via inhibition of RhoA activation, independently from its function as an auxiliary protein of the sodium channel.

Introduction Graft rejection is the main complication after heart transplantation. We recently demonstrated in vitro that the stimulation of P2Y11 receptor reduces ischemia/reperfusion lesions in human cardiomyocytes and is responsible for dendritic cells maturation, down-modulating the inflammatory response. The objective of this in vivo study was to specify the effect of P2Y11R stimulation on heart graft rejection lesions and its role in the maturation of dendritic cells. Material Hearts from BalbC mice were transplanted intraabdominally into allogenic C57BL6 mice (n=60, females). Mice were injected in the retroorbital sinus with P2Y11R agonist (NF546). Mice in the sham group were injected with saline solution. In the control group, hearts from C57BL6 were transplanted into syngenic C57BL6 mice. Cardiac rejection was defined by cessation of palpable heartbeat and was confirmed by echocardiography. Rejection lesions were investigated using histology and immunohistochemistery (CD3, CD11c, CD45) in allografts at days 3, 5 and 7 after transplantation. To quantify apoptosis, activity of caspase 1, 3 and 9 was measured. Maturation of dendritic cells was investigated by studying expression of markers CD83, CD25, CCR7, CXCR4, and production of cytokines IL6, IL10, IL12, IFN-γ. Results Cardiac allograft survival was significantly prolonged after stimulation of P2Y11R by its agonist (9.6±1.9 vs 8.2±1.4 days; p=0.04). Rejection lesions, classified according to ISHLT guidelines and quantified using the mean number of inflammatory cells per field, were significantly reduced in the treated group. At day 5 after transplantation, P2Y11R agonist pretreated allografts also demonstrated less apoptotic lesions. Conclusion Stimulation of P2Y11 receptor reduces rejection lesions observed after allogenic heart transplantation. Our previous results suggest that this protective role may imply dendritic cells maturation towards an anti inflammatory profile, depending on P2Y11 signaling pathway. The author hereby declares no conflict of interest

International audience; BACKGROUND: Cytomegalovirus (CMV) has a role in chronic rejection and graft loss in kidney transplant (KTx) and lung transplant (LTx) recipients. In addition, donor CMV seropositivity is an independent risk factor for renal graft loss. The anti-CMV response might modulate this risk. Expression of programmed cell death 1 (PD-1), a receptor involved in viral-specific T-cell exhaustion, is influenced by a single nucleotide polymorphism called PD-1.3 (wild-type allele G, variant allele A). METHODS: We performed a retrospective study to assess the impact of PD-1.3 on graft outcome in donor CMV seropositive (D+) and donor CMV seronegative (D-) KTx and LTx. We also performed a case-control study to evaluate the anti-CMVpp65 response according to genotype. RESULTS: PD-1.3 was determined in 1,119 KTx and 181 LTx. In 481 D+ KTx, A allele carriers (24%) experienced significantly less graft failure compared with GG carriers (p = 0.001). Multivariate analysis showed that this association was independent of donor and recipient age, acute rejection episodes, and number of human leukocyte antigen mismatches (hazard ratio, 0.381; 95% confidence interval, 0.209-0.696; p = 0.002). Analysis in 85 D+ LTx showed similar results: A allele carriers had better survival (hazard ratio, 0.302; 95% confidence interval, 0.128-0.716; p = 0.006) and greater 6-month forced expiratory volume (71% ± 17% vs 54% ± 16%, p = 0.001). In D- recipients, PD-1.3 did not affect KTx or LTx outcome. Finally, AA recipients had a stronger anti-CMVpp65 T-cell response than matched GG recipients (p = 0.003). CONCLUSIONS: The A variant allele in PD-1.3 single nucleotide polymorphism improved graft survival in kidney and lung transplant recipients receiving grafts from CMV-positive donors.

International audience; Human amebiasis is caused by the protozoan Entamoeba histolytica. This protozoan is responsible for muco-hemorrhagic diarrhoea and liver abscess in affected populations. E. histolytica can be asymptomatic commensally confined to the intestinal lumen or can result in invasion of the colonic mucosa leading to ulceration and/or liver abscesses. Recently, human colonic explants have been identified as valuable in the study of host-parasite interactions. Here we investigated the potential of porcine colonic explants as an alternative to human tissues which are far less available. Porcine colonic explants were cultured with two strains of E. histolytica, one virulent (HM1:IMSS) and one avirulent (Rahman). Results from histopathological and real-time PCR analysis showed that porcine explants cultured with virulent ameba trophozoites react similarly to their human counterparts with an invasion of the tissue by the trophozoites and the triggering of typical innate immune response against the parasite. On the contrary, explants cultured with avirulent ameba trophozoites were preserved. The study open the way to the use of porcine colonic explants in the study of the complex interactions between the parasite and the host.

Introduction L’objectif de notre etude etait d’analyser le signal IRM de caillots sanguins afin de determiner s’il etait possible de distinguer les caillots rouges riches en globules rouges (GR) des caillots blancs riche en fibrine et d’estimer leur concentration en hematies a l’aide d’un index base sur le signal IRM des caillots. Materiel et methodes Cinq caillots ont ete analyses in vitro. Quatre caillots avaient une composition connue (A = 100 % fibrine, B = 20 % GR, C = 50 % GR et D = 80 % GR) et 1 caillot avait une composition inconnue (caillot E). Les 5 caillots ont ete fixes dans une solution de gelatine et de manganese ayant un signal identique a la substance grise cerebrale (T1 et T2 similaires) avant d’etre analyse par IRM avec les sequences suivantes : T1MPR, T2TSE, SWI, T2EG, T2FLAIR, diffusion et cartographie ADC ( Fig. 1 ). Un index IRM a ete calcule pour chacun des caillots selon la formule suivante : (signal caillot–signal gelatine)/signal gelatine. L’ensemble des caillots a ensuite ete envoye au laboratoire d’anatomie pathologique pour analyse. Resultats Une correlation statistique a ete mise en evidence entre l’index IRM de chaque caillot et leur concentration en hematies pour les sequences T1 ( r = −0,84), SWI ( r = −0,79), T2EG (−0,72) et FLAIR ( r = 0,80) ( Fig. 2 ). La concentration en hematies du caillot E a ete estimee par IRM : 47,3 % en T1, 41,5 % en SWI, 45,1 % en T2EG et 50,9 % en FLAIR. L’analyse histologique a confirme que la composition du caillot E etait proche de celle du caillot C (50 %) ( Fig. 3 ). Conclusion Cette etude nous a permis de montrer que l’analyse IRM des caillots permettait d’une part de differencier les caillots rouges des caillots blancs, mais aussi d’estimer leur concentration en hematies. Une etude sur des caillots issus de thrombectomies cerebrales chez des patients hospitalises pour AVC ischemique aigu a alors ete initiee.

High concentrations of extracellular ATP (eATP) resulting from cell damage may be found during an ischemia/reperfusion (I/R) episode at the site of injury. eATP activates purinergic receptors in dendritic cells (DCs) and may inhibit inflammation. This immunosuppressive activity could be of interest in the field of I/R, which is an inflammatory condition involved in myocardial infarction, stroke, and solid organ transplantation. However, the specific purinergic receptor responsible for this effect remains to be identified. In this study, we report that eATP induced maturation of human monocyte-derived DCs. Additionally, eATP inhibited IL-12 production whereas IL-10 levels remained unchanged in activated DCs. These effects were prevented by the P2Y11R antagonist NF340. Interestingly, a 5-h hypoxia prevented the effects of eATP on cytokine production whereas a 1-h hypoxia did not affect the eATP-mediated decrease of IL-12 and IL-6. We showed a time-dependent downregulation of P2Y11R at both mRNA and protein levels that was prevented by knocking down hypoxia-inducible factor-1α. In this study, we showed an immunosuppressive role of P2Y11R in human DCs. Additionally, we demonstrated that the time-dependent downregulation of P2Y11R by hypoxia orientates DCs toward a proinflammatory phenotype that may be involved in post-I/R injuries as observed after organ transplantation.

International audience; Suppressor of cytokine signaling (SOCS) proteins are key physiological regulators of both innate and adaptive immunity. These proteins belong to the three major classes of modulators of cytokines signaling. In the following article, we used porcine polarized intestinal cells to study early response to the protozoan, Entamoeba histolytica, and we identified by rapid amplification of cDNA ends (RACE) PCR porcine SOCS1, SOCS4, SOCS5 and SOCS6 encoding sequences. With more than 92% identity predicted porcine SOCS proteins are very similar to their human counterparts. Among SOCS transcripts, only SOCS2 mRNA was significantly induced in epithelial intestinal cells in response to the cytolytic activity of the parasite. The transcriptomic profile obtained after 3h of co-culture of polarized intestinal cells with E. histolytica was clearly oriented toward inflammation and the recruitment of neutrophils. These transcriptomic data have been normalized with accuracy by the utilisation of multiple validated reference genes. The analysis offers a first set of reference genes useful for future studies in porcine intestinal cells. Our data shed light on the understanding of the early response of polarized intestinal cells to E. histolytica and identified a potential involvement of SOCS2 in the parasite regulation of the host response.

BACKGROUND: Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. METHODOLOGY/PRINCIPAL FINDINGS: We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. CONCLUSIONS: The pig model could help with simultaneously studying intestinal and extraintestinal lesion development.