Specific Recruitment of Antigen-presenting Cells by Chemerin, a Novel Processed Ligand from Human Inflammatory Fluids

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein–coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42–p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs

Towards the Industrialization of New MDO Methodologies and Tools for Aircraft Design

An overall summary of the Institute of Technology IRT Saint Exupery MDA-MDO project (Multi-Disciplinary Analysis - Multidisciplinary Design Optimization) is presented. The aim of the project is to develop efficient capabilities (methods, tools and a software platform) to enable industrial deployment of MDO methods in industry. At IRT Saint Exupery, industrial and academic partners collaborate in a single place to the development of MDO methodologies; the advantage provided by this mixed organization is to directly benefit from both advanced methods at the cutting edge of research and deep knowledge of industrial needs and constraints. This paper presents the three main goals of the project: the elaboration of innovative MDO methodologies and formulations (also referred to as architectures in the literature 1) adapted to the resolution of industrial aircraft optimization design problems, the development of a MDO platform featuring scalable MDO capabilities for transfer to industry and the achievement of a simulation-based optimization of an aircraft engine pylon with industrial Computational Fluid Dynamics (CFD) and Computational Structural Mechanics (CSM) tools

Tetrastatin, the NC1 Domain of the α4(IV) Collagen Chain: A Novel Potent Anti-Tumor Matrikine

BACKGROUND: NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (-38%) and invasive (-52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (-80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (K(D) = 148 ± 9.54 nM). CONCLUSION/SIGNIFICANCE: Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine

Lumican Inhibits In Vivo Melanoma Metastasis by Altering Matrix-Effectors and Invadopodia Markers

It was reported that lumican inhibits the activity of metalloproteinase MMP-14 and melanoma cell migration in vitro and in vivo. Moreover, Snail triggers epithelial-to-mesenchymal transition and the metastatic potential of cancer cells. Therefore, the aim of this study was to examine the effect of lumican on Mock and Snail overexpressing melanoma B16F1 cells in vivo. Lung metastasis was analyzed after intravenous injections of Mock-B16F1 and Snail-B16F1 cells in Lum+/+ and Lum−/− mice. At day 14, mice were sacrificed, and lungs were collected. The number of lung metastatic nodules was significantly higher in mice injected with Snail-B16F1 cells as compared to mice injected with Mock-B16F1 cells confirming the pro-metastatic effect of Snail. This effect was stronger in Lum−/− mice as compared to Lum+/+, suggesting that endogenous lumican of wild-type mice significantly inhibits metastasis to lungs. Scanning electron and confocal microscopy investigations demonstrated that lumican inhibits the development of elongated cancer cell phenotypes which are known to develop invadopodia releasing MMPs. Moreover, lumican was shown to affect the expression of cyclin D1, cortactin, vinculin, hyaluronan synthase 2, heparanase, MMP-14 and the phosphorylation of FAK, AKT, p130 Cas and GSK3α/β. Altogether, these data demonstrated that lumican significantly inhibits lung metastasis in vivo, as well as cell invasion in vitro, suggesting that a lumican-based strategy targeting Snail-induced metastasis could be useful for melanoma treatment

Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling

International audienceThe small leucine-rich proteoglycan lumican regulates estrogen receptors (ERs)-associated functional properties of breast cancer cells, expression of matrix macromolecules, and epithelial-to-mesenchymal transition. However , it is not known whether the ER-dependent lumican effects on breast cancer cells are related to the expression of integrins and their intracellular signaling pathways. Here, we analyzed the effects of lumican in three breast cancer cell lines: the highly metastatic ERb-positive MDA-MB-231, cells with the respective ERb-suppressed (shERbMDA-MB-231), and lowly invasive ERa-positive MCF-7/c breast cancer cells. Scanning electron microscopy, confocal microscopy, real-time PCR, western blot, and cell adhesion assays were performed. Lumican effects on breast cancer cell morphology were also investigated in 3-dimensional collagen cultures. Lumican treatment induced cell-cell contacts and cell grouping and inhibited microvesicles and microvilli formation. The expression of the cell surface adhesion receptor CD44, its isoform and variants, hyaluronan (HA), and HA synthases was also investigated. Lumican inhibited the expression of CD44 and HA synthases, and its effect on cell adhesion revealed a major role of a1, a2, a3, aVb3, and aVb5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of a2 and b1 integrin subunits both in MDA-MB-231 and in shERbMDA-MB-231 as compared to MCF-7/c cells. Downstream signaling pathways for inte-grins, such as FAK, ERK 1/2 MAPK 42/44, and Akt, were found to be downregulated by lumican. Our data shed light to the molecular mechanisms responsible for the anticancer activity of lumican in invasive breast cancer. Abbreviations As, anti-sense; CD44, cluster of differentiation 44; CD44s, CD4

Lumican – Derived Peptides Inhibit Melanoma Cell Growth and Migration

<div><p>Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth <i>in vitro</i> and <i>in vivo</i>. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican <i>in vitro</i>. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines – mice B16F1 and human SK-MEL-28 cells – in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.</p> </div

Hair Histology and Glycosaminoglycans Distribution Probed by Infrared Spectral Imaging: Focus on Heparan Sulfate Proteoglycan and Glypican-1 during Hair Growth Cycle

The expression of glypicans in different hair follicle (HF) compartments and their potential roles during hair shaft growth are still poorly understood. Heparan sulfate proteoglycan (HSPG) distribution in HFs is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach is proposed to assess hair histology and HSPG distribution changes in HFs at different phases of the hair growth cycle using infrared spectral imaging (IRSI). The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. The findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs, and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia

Lumcorin alters the expression and activity of MMP-14 and inhibits FAK phosphorylation at tyrosine 397.

<p>(A) Expression of MMP-14 in B16F1 cells incubated 48h with 100µM lumcorin or scrambled peptide was analyzed by Western immunoblotting using polyclonal rabbit antibody and probing with anti-β-actin. (B, C) Activity of MMP-14 in B16F1 (B) and SK-MEL-28 (C) cells incubated 48h in the presence of 100µM lumcorin or its scrambled peptide, measured using fluorimetric SensoLyte® 520 MMP-14 Assay Kit as described in Materials and Methods. (D) Activity of MMP-14 in B16F1 cell extract pre-incubated 15 min or 60 min before assay with 100µM lumcorin or scrambled peptide. (E) Recombinant human MMP-14 activity pre-incubated 15 min with 100µM lumcorin or its scrambled peptide or DCN LRR9 or Fmod LRR9. Data are presented as mean ± S.D from three independent experiments. (F) Phospho-FAK (pY397) and total FAK expression in B16F1 cells incubated 15 min with 100µM lumcorin or scrambled peptide analyzed by Western immunoblotting using monoclonal mouse antibody against pFAK (pY397) and probing with polyclonal rabbit anti-total FAK antibody. After densitometric analysis of the intensity of the bands, the resulting ratio of pFAK to total FAK intensity was calculated and presented on the graph. Data are presented as mean values ± S.D from three independent experiments (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; NS, no significant difference).</p

Lumcorin decreases melanoma growth.

<p>Cell growth assay (A, B) and colony formation assay (C, D) of B16F1 cells (A, C) and SK-MEL-28 cells (B, D). For growth studies, cells were grown for 24, 48 and 72h in presence of 100 µM lumcorin or its scrambled (SCR) peptide. Cell growth was measured by MTT colorimetric test at 560nm, as described in Materials and Methods. Results were reported as mean ± S.D of sextuplicate values from three independent experiments. For colony formation, 1.2x10³cells were cultured in 0.3% agar for 14 days in presence of 100µM lumcorin or scrambled peptide as described in Materials and Methods. Representative images of cell colonies are displayed at the inserts. The quantification of the colony diameter was done using Image Tool software. Graphs represent the mean size of 100 colonies ± S.D from three independent experiments (*, <i>p</i>< 0.05, **, <i>p</i><0.01***, <i>p</i><0.001). Scale bar in the inserts: 200µm.</p

Role of the interstitium during septic shock: a key to the understanding of fluid dynamics?

International audienceBackground While not traditionally included in the conceptual understanding of circulation, the interstitium plays a critical role in maintaining fluid homeostasis. Fluid balance regulation is a critical aspect of septic shock, with a well-known association between fluid balance and outcome. The regulation of transcapillary flow is the first key to understand fluid homeostasis during sepsis.Main text: Capillary permeability is increased during sepsis, and was classically considered to be necessary and sufficient to explain the increase of capillary filtration during inflammation. However, on the other side of the endothelial wall, the interstitium may play an even greater role to drive capillary leak. Indeed, the interstitial extracellular matrix forms a complex gel-like structure embedded in a collagen skeleton, and has the ability to directly attract intravascular fluid by decreasing its hydrostatic pressure. Thus, interstitium is not a mere passive reservoir, as was long thought, but is probably major determinant of fluid balance regulation during sepsis. Up to this date though, the role of the interstitium during sepsis and septic shock has been largely overlooked. A comprehensive vision of the interstitium may enlight our understanding of septic shock pathophysiology. Overall, we have identified five potential intersections between septic shock pathophysiology and the interstitium: 1. increase of oedema formation, interacting with organ function and metabolites diffusion; 2. interstitial pressure regulation, increasing transcapillary flow; 3. alteration of the extracellular matrix; 4. interstitial secretion of inflammatory mediators; 5. decrease of lymphatic outflow.Conclusions We aimed at reviewing the literature and summarizing the current knowledge along these specific axes, as well as methodological aspects related to interstitium exploration