ObjectiveSystemic sclerosis (SSc) is an autoimmune disorder characterized by excessive fibrosis, immune dysfunction, and vascular damage, in which the expression of many growth factors is deregulated. CD146 was recently described as a major actor in SSc. Since CD146 also exists as a circulating soluble form (sCD146) that acts as a growth factor in numerous angiogenic‐ and inflammation‐related pathologies, we sought to identify the mechanisms underlying the generation of sCD146 and to characterize the regulation and functions of the different variants identified in SSc.MethodsWe performed in vitro experiments, including RNA‐Seq and antibody arrays, and in vivo experiments using animal models of bleomycin‐induced SSc and hind limb ischemia.ResultsMultiple forms of sCD146, generated by both shedding and alternative splicing of the primary transcript, were discovered. The shed form of sCD146 was generated from the cleavage of both long and short membrane isoforms of CD146 through ADAM‐10 and TACE metalloproteinases, respectively. In addition, 2 novel sCD146 splice variants, I5‐13‐sCD146 and I10‐sCD146, were identified. Of interest, I5‐13‐sCD146 was significantly increased in the sera of SSc patients (P < 0.001; n = 117), in particular in patients with pulmonary fibrosis (P < 0.01; n = 112), whereas I10‐sCD146 was decreased (P < 0.05; n = 117). Further experiments revealed that shed sCD146 and I10‐sCD146 displayed proangiogenic activity through the focal adhesion kinase and protein kinase Cε signaling pathways, respectively, whereas I5‐13‐sCD146 displayed profibrotic effects through the Wnt‐1/β‐catenin/WISP‐1 pathway.ConclusionVariants of sCD146, and in particular the novel I5‐13‐sCD146 splice variant, could constitute novel biomarkers and/or molecular targets for the diagnosis and treatment of SSc and other angiogenesis‐ or fibrosis‐related disorders.

Endothelial colony-forming cells (ECFC) constitute an endothelial progenitor fraction with a promising interest for the treatment of ischaemic cardiovascular diseases. As soluble CD146 (sCD146) is a new factor promoting angiogenesis, we examined whether sCD146 priming could improve the therapeutic potential of ECFC and defined the involved mechanism.We investigated the effects of sCD146 priming on regenerative properties of ECFC in vivo. In a mouse model of hindlimb ischaemia, the homing of radiolabelled cells to ischaemic tissue was assessed by SPECT-CT imaging. Soluble CD146 priming did not modify the number of engrafted ECFC but improved their survival capacity, leading to an enhanced revascularization. The mechanism of action of sCD146 on ECFC was studied in vitro. We showed that sCD146 acts in ECFC through a signalosome, located in lipid rafts, containing angiomotin, the short isoform of CD146 (shCD146), VEGFR1, VEGFR2, and presenilin-1. Soluble CD146 induced a sequential proteolytic cleavage of shCD146, with an extracellular shedding followed by an intramembrane cleavage mediated by matrix metalloprotease (MMP)/ADAM and presenilin-1, respectively. The generated intracellular part of shCD146 was directed towards the nucleus where it associated with the transcription factor CSL and modulated the transcription of genes involved in cell survival (FADD, Bcl-xl) and angiogenesis (eNOS). This effect was dependent on both VEGFR1 and VEGFR2, which were rapidly phosphorylated by sCD146.These findings establish that activation of the proteolytic processing of shCD146, in particular by sCD146, constitutes a promising pathway to improve endothelial progenitors' regenerative properties for the treatment of cardiovascular diseases.

CD146 (MUC-18, MCAM) expression on cancer cells correlates with cancer progression and a bad prognosis in several tumors, including melanoma and pancreatic tumors. Deciphering the mechanism mediating the CD146 role in cancer is essential for generating new therapeutic strategies. We found that CD146 expression in cancer cells is associated with a secretion of soluble CD146 (sCD146) that constitutes an active player in tumor development. Indeed, sCD146 induces the overexpression of its binding protein, angiomotin, on both endothelial and cancer cells and promotes both paracrine effects on angiogenesis and autocrine effects on cancer cells proliferation and survival. These last effects are mediated in part through the induction and phosphorylation of c-myc in cancer cells. In mice models xenografted with human CD146-positive melanoma or pancreatic cancer cells, administration of a novel monoclonal antibody specifically targeting sCD146, but not its membrane form, successfully suppresses tumor vascularization and growth. Our findings demonstrate that sCD146 secreted by CD146-positive tumors mediates important pro-angiogenic and pro-tumoral effects. Targeting sCD146 with a novel neutralizing antibody could thus constitute an innovative therapeutic strategy for the treatment of CD146-positive tumors.

Pleural biomarkers allowing to mini-invasively discriminate benign from malignant pleural effusions are needed. Among potential candidates, microparticles (MPs) are extracellular vesicles that vectorize antigen derived from the parent cell. We hypothesized that tumor-derived MPs could be present in the pleural liquid and help to identify patients with malignant pleural effusions. Using highly sensitive flow cytometry and cryo-electron microscopy, we showed that large amounts of MPs from hematopoïetic and vascular origin could be detectable in pleural fluids. Their level did not differ between benign (n = 14) and malignant (n = 71) pleural effusions. Analysis of selected tumoral associated antigens (podoplanin, mucin 1 and EpCAM, epithelial-cell-adhesion-molecule) evidenced for the first time the presence of tumor-derived MPs expressing EpCAM in malignant pleural fluids only (Specificity = 93%, Sensitivity = 49% and 45% for flow cytometry and ELISA, respectively). The detection of EpCAM-positive-MPs (EpCAM + MPs) by flow cytometry showed a better specificity and sensitivity than ELISA to distinguish between pleural carcinoma and the others malignant pleural effusions (MPE; Sp: 96% vs 89%; Se: 79% vs 66%). Combining EpCAM+ MPs and cytology improved the diagnosis of MPE compared to cytology alone. This study establishes the basis for using EpCAM+ MPs as a promising new biomarker that could be added to the armamentarium to mini-invasively identify patients with malignant pleural effusions.

Background: Pleural biomarkers are needed for the diagnosis of malignant pleural effusion at an early stage before true carcinomatosis. Tumor cells produce microparticles, and, therefore, we hypothesized that tumor-derived microparticles could be present in the pleural liquid and could help to identify patients with malignant pleural effusions by a non-invasive method.Methods: Fifty patients with benign (n=11) or malignant (n=39) pleural effusions were included in this study. Among them, 39 consecutive patients with histologically shown primary or metastatic pleural cancer were included. After the optimization of a sample processing protocols, MPs were enumerated by flow cytometry and the Cryo-Transmission Electron Microscopy was performed. Measurement of EpCAM-positive MPs was analyzed by ELISA.Results: Using highly sensitive flow cytometry and cryo-electron microscopy, this study showed the presence of microparticles in pleural effusions. We demonstrated the presence of tumor-derived microparticles expressing EpCAM (epithelial-cell-adhesion-molecule) in the pleural fluids from adenocarcinoma patients. In our cohort, the detection of EpCAM-positive microparticles was shown to be useful as a tool to complement cytology for better diagnoses of malignant pleurisies.Conclusions: To our knowledge, this is the first work to directly identify tumoral microparticles in pleural liquids using EpCAM. The identification of EpCAM+ microparticles could lead to a new diagnostic and monitoring approach. This study establishes the basis for a potential biomarker for diagnosing and monitoring malignant pleurisy.

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8+ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4+ and CD8+ T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4+ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.

Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-β-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a ΔvirB mutant. In HeLa cells, a ΔricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortusΔricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the "Brucella-containing vacuole" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

The importance of the maintenance function has increased because of its role in keeping and improving system availability and safety, as well as product quality. To support this role, the maintenance concept has undergone several major developments that have led to proactive considerations mainly based on a prognosis process, which normally allows selection of the best maintenance action to be carried out. This paper proposes the deployment and experimentation of a prognosis process within an e-maintenance architecture. The deployment follows a methodology based on the combination of both a probabilistic approach for modelling the degradation mechanism and of an event one for dynamical degradation monitoring. The feasibility and benefits of this new prognosis process is investigated with an experiment using a manufacturing TELMA (TELe-MAintenance) platform supporting the unwinding of metal bobbins.

PurposeThis paper proposes the extension of a prognosis process by means of the integration of maintenance alternative impacts in order to develop a maintenance decision‐making tool.Design/methodology/approachThe deployment of this extended prognosis process follows a methodology based both on probabilistic and on event approaches.FindingsThe importance of the maintenance function has increased due to its role in keeping and improving the system availability and safety but also the product quality. To support this new role, the maintenance concept has undergone several major developments to lead to proactive considerations mainly based on prognosis process allowing one to select the best maintenance plan to be carried out.Practical implicationsStudies over the last 20 years have indicated that around Europe the direct cost of maintenance is equivalent to between 4 and 8 per cent of total sales turnover. The indirect cost of maintenance is likely to be a similar amount. Thus, in the countries where modern maintenance practices have yet to be well adopted by industry, the potential savings from modern maintenance are massive. These modern and efficient maintenances imply identifying the root‐cause of component failures, reducing the failures of production systems, eliminating costly unscheduled shutdown maintenances, and improving productivity as well as quality. It means, for the companies, migrating from their traditional reactive approach, which is “fail and fix”, to “predict and prevent”. The advantage of the latter is that maintenance is performed only when a certain level of equipment deterioration occurs. This “proactive” maintenance is mainly based on prognosis process often considered as the Achilles heel, while its goal is fundamental for implementing anticipation capabilities. This paper looks into this issue by proposing the development of an innovative prognosis process integrating the modelling of maintenance actions and their impacts on system performances. It leads to offering a maintenance aided decision‐making tool cable of assisting the decision‐maker in selecting the best maintenance plan to be carried out.Originality/valueThe feasibility of this new prognosis is experimented on the manufacturing Tele‐Maintenance (TELMA) platform supporting the unwinding of metal bobbins.

The importance of the maintenance(1) function has increased because of its role in keeping and improving system availability and safety, as well as product quality. To support this role, the development of the communication and information technologies has allowed the emergence of the concept of e-maintenance. Within the era of e-manufacturing and e-business, e-maintenance provides the opportunity for a new maintenance generation. As we will discuss later in this paper, e-maintenance integrates existing telemaintenance principles, with Web services and modern e-collaboration principles. Collaboration allows to share and exchange not only information but also knowledge and (e)-intelligence. By means of a collaborative environment, pertinent knowledge and intelligence become available and usable at the right place and time, in order to facilitate reaching the best maintenance decisions.This paper outlines the basic ideas within the e-maintenance concept and then provides an overview 2 of the current research and challenges in this emerging field. An underlying objective is to identify the industrial/academic actors involved in the technological, organizational or management issues related to the development of e-maintenance. Today, this heterogeneous community has to be federated in order to bring up e-maintenance as a new scientific discipline. (0 2007 Elsevier Ltd. All rights reserved.

Today industrial systems are characterized by a set of dependencies among the components and the environment of the system. To address these difficulties, this paper presents a method for modelling and analyzing the reliability of a complex system based on Dynamic Bayesian Networks (DBN). This method allows to take into account the influence of time or exogenous variables on the failure (degradation) modes of the system. The DBN graphical structure provides an easy way to specify the dependencies and, hence, to provide a compact representation of the model. In addition, the DBN formalism is associated to simulation tools that enable an efficient processing for the models.

L'emergence du concept de " durabilite " en entreprise oblige dorenavant les industriels a integrer dans leur strategie de developpement, au-dela de la finalite economique habituelle, de nouvelles preoccupations sociales et environnementales. Cet enjeu majeur se decline, au niveau du systeme d'execution de la production, par le concept de maintien en conditions operationnelles (MCO). Afin d'ameliorer l'efficacite du MCO, nous proposons dans cette these la formalisation du processus cle d'un systeme de maintenance previsionnelle : le pronostic. La demarche de modelisation introduite s'appuie sur le couplage d'une representation probabiliste de l'etat d'un systeme basee sur le formalisme des Reseaux Bayesiens Dynamiques (RBD), et d'une approche evenementielle de la surveillance de la degradation de ses composants. La mise en œuvre du processus de pronostic sur le systeme de Deroulage / Pressage de la plate-forme TELMA demontre la faisabilite de la methodologie proposee.

Today, the degradation checking of critical components is one of the efficient means to minimise the non expected stops of production system and to reduce its costs. It consists in monitoring and forecasting the degradation evolution and in implementing preventive actions to anticipate the failures and keep production system in normal operation. In that way, this paper proposes the deployment and the experimentation of a prognosis process within the e-maintenance architecture of the CRAN IMS platform. The deployment follows a methodology based on the combination both of probabilistic approach for degradation mechanism modelling and of event one for degradation dynamical monitoring.

The degradation checking of critical components is one of the efficient means to minimize the non expected stops of production system and to reduce its costs. In this purpose, a methodology is proposed to design a prognosis process taking into account the behaviour of environmental events (exogenous inputs). The main contribution is the development of the probabilistic model which is based on the translation of a preliminary process model into Dynamic Bayesian Network (DBN). Specifications are given to assure the coherence both of the translation and the resulted model. These requirements are formalized according to the NIAM/ORM method towards its semantic strength, clarity and stability. An application is proposed on a water heater system.

Today, the degradation mastery of critical components is one of the efficient means to minimise the non expected stops of production system and reduce its costs. Consequently, a prognosis process-based proactive maintenance system is proposed and formalized in this paper. This process allows to follow the system degradation and to implement adequate preventive actions for anticipating failures. Its development combines both probabilistic and event approaches for degradation mechanism modelling and dynamical monitoring.

Among the processes for management of product life cycle, maintenance is one of the key Enterprise domains to improve business objective, product quality and service while keeping the production system dependable. It leads to deploy maintenance strategy more efficient vs. proactive mainly based on component and functional degradation prognosis process able to set-up adapted maintenance decision making to better master its direct and indirect costs. In this paper is discussed a methodology supporting prognosis process modelling which is based on the combination both of probabilistic approach for degradation mechanism modelling and of event one for degradation dynamical monitoring. This methodology is experimented on manufacturing component and deployed within e-maintenance architecture.