A Functional γδTCR/CD3 Complex Distinct from γδT Cells Is Expressed by Human Eosinophils

BACKGROUND:Eosinophils are effector cells during parasitic infections and allergic responses. However, their contribution to innate immunity has been only recently unravelled. METHODOLOGY/PRINCIPAL FINDINGS:Here we show that human eosinophils express CD3 and gammadelta T Cell Receptor (TCR) but not alphabeta TCR. Surface expression of gammadeltaTCR/CD3 is heterogeneous between eosinophil donors and inducible by mycobacterial ligands. Surface immunoprecipitation revealed expression of the full gammadeltaTCR/CD3 complex. Real-time PCR amplification for CD3, gamma and delta TCR constant regions transcripts showed a significantly lower expression in eosinophils than in gammadeltaT cells. Limited TCR rearrangements occur in eosinophils as shown by spectratyping analysis of CDR3 length profiles and in situ hybridization. Release by eosinophils of Reactive Oxygen Species, granule proteins, Eosinophil Peroxidase and Eosinophil-Derived Neurotoxin and cytokines (IFN-gamma and TNF-alpha) was observed following activation by gammadeltaTCR-specific agonists or by mycobacteria. These effects were inhibited by anti-gammadeltaTCR blocking antibodies and antagonists. Moreover, gammadeltaTCR/CD3 was involved in eosinophil cytotoxicity against tumor cells. CONCLUSIONS/SIGNIFICANCE:Our results provide evidence that human eosinophils express a functional gammadeltaTCR/CD3 with similar, but not identical, characteristics to gammadeltaTCR from gammadeltaT cells. We propose that this receptor contributes to eosinophil innate responses against mycobacteria and tumors and may represent an additional link between lymphoid and myeloid lineages

Changes in CRH and ACTH Synthesis during Experimental and Human Septic Shock

Context The mechanisms of septic shock-associated adrenal insufficiency remain unclear. This study aimed at investigating the synthesis of corticotropin-releasing hormone (CRH) and vasopressin (AVP) by parvocellular neurons and the antehypophyseal expression of ACTH in human septic shock and in an experimental model of sepsis. Objective To test the hypothesis that ACTH secretion is decreased secondarily to alteration of CRH or AVP synthesis, we undertook a neuropathological study of the antehypophyseal system in patients who had died from septic shock and rats with experimental faecal peritonitis. Methods Brains obtained in 9 septic shock patients were compared to 10 nonseptic patients (controls). Parvocellular expression of AVP and CRH mRNA were evaluated by in situ hybridization. Antehypophyseal expression of ACTH, vasopressin V1b and CRH R1 receptors and parvocellular expression of iNOS in the PVN were evaluated by immunohistochemistry. The same experiments were carried out in a fecal peritonitis-induced model of sepsis. Data from septic rats with (n = 6) or without (n = 10) early death were compared to sham-operated (n = 8) animals. Results In patients and rats, septic shock was associated with a decreased expression of ACTH, unchanged expression of V1B receptor, CRHR1 and AVP mRNA, and increased expression of parvocellular iNOS compared to controls. Septic shock was also characterized by an increased expression of CRH mRNA in rats but not in patients, who notably had a greater duration of septic shock. Conclusion The present study suggests that in humans and in rats, septic shock is associated with decreased ACTH synthesis that is not compensated by its two natural secretagogues, AVP and CRH. One underlying mechanism might be increased expression of iNOS in hypothalamic parvocellular neurons

How valuable are your customers in the brand value co-creation process? The development of a Customer Co-Creation Value (CCCV) scale.

Despite an increasing amount of research on co-creation of value, in general, research on brand value co-creation remains limited. Particularly, how much value customers contribute to the brand value co-creation process remains unclear. This research develops in a series of eight studies the Customer Co-Creation Value (CCCV) measurement scale that helps firms assess the value of customers in the brand value co-creation process. The findings reveal that CCCV is a multidimensional construct consisting of two higher-order factors and seven dimensions: customer-owned resources (including brand knowledge, brand skills, brand creativity, and brand connectedness) and customer motivation (comprising brand passion, brand trust, and brand commitment). Further, the CCCV scale reliably and validly gauges the value customers contribute to a firm's brand. The CCCV framework helps marketing managers understand how customers can contribute to a firm's brand value cocreation efforts and how much value customers contribute to a brand in the co-creation process

VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a −8409/−7563 enhancer and the −252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific regulation of genes in this cell lineage

Les facteurs de transcription de la famille Ets et la morphogenèse du réseau vasculaire

L’expression de Ets1, Erg et Fli, tous membres de la famille des facteurs de transcription Ets a été associée au déroulement de processus invasifs, comme l’angiogenèse dans des situations normales ou pathologiques. La description du phénotype des cellules endothéliales en culture dans lesquelles est exprimé le domaine de fixation à l’ADN de Ets1 suggère que les membres de sa famille jouent un rôle dans la morphogenèse de l’arbre vasculaire. Cependant, même si les expériences de transfection transitoire ont permis d’identifier des gènes dont la transcription peut être contrôlée par Ets1 au cours de la formation des vaisseaux sanguins, la détermination des réseaux de régulations dans lesquels interviennent Ets1 et les membres de sa famille est un objectif pour les prochaines années

VE-statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases

We previously characterized VE-statin/egfl7, a protein that is exclusively secreted by endothelial cells and modulates smooth muscle cell migration. Here, we show that VE-statin/egfl7 is the first known natural negative regulator of vascular elastogenesis. Transgenic mice, expressing VE-statin/egfl7 under the control of keratin-14 promoter, showed an accumulation of VE-statin/egfl7 in arterial walls where its presence correlated with an impaired organization of elastic fibres. In vitro, fibroblasts cultured in the presence of VE-statin/egfl7 were unable to deposit elastic fibres due to a deficient conversion of soluble tropoelastin into insoluble mature elastin. VE-statin/egfl7 interacts with the catalytic domain of lysyl oxidase (LOX) enzymes and, in endothelial cells, endogenous VE-statin/egfl7 colocalizes with LoxL2 and inhibits elastic fibre deposition. In contrast, mature elastic fibres are abundantly deposited by endothelial cells that are prevented from producing endogenous VE-statin/egfl7. We propose a model where VE-statin/egfl7 produced by endothelial cells binds to the catalytic domains of enzymes of the LOX family in the vascular wall, thereby preventing the crosslink of tropoelastin molecules into mature elastin polymers and regulating vascular elastogenesis

<i>Egfl7</i> Is Differentially Expressed in Arteries and Veins during Retinal Vascular Development

<div><p>The vasculature of the central nervous system (CNS) is composed of vascular endothelial and mural cells which interact closely with glial cells and neurons. The development of the CNS vascularisation is a unique process which requires the contribution of specific regulators in addition to the classical angiogenic factors. The <i>egfl7</i> gene is mainly detected in endothelial cells during physiological and pathological angiogenesis. Egfl7 codes for a secreted protein which predominantly accumulates into the extracellular space where it controls vascular elastin deposition or the Notch pathway. <i>Egfl7</i> is the host gene of the microRNA miR126 which is also expressed in endothelial cells and which plays major functions during blood vessel development. While the expression of <i>egfl7</i> and that of miR126 were well described in endothelial cells during development, their pattern of expression during the establishment of the CNS vasculature is still unknown. By analysing the expression of <i>egfl7</i> and miR126 during mouse retina vascularisation, we observed that while expression of miR126 is detected in all endothelia, <i>egfl7</i> is initially expressed in all endothelial cells and then is progressively restricted to veins and to their neighbouring capillaries. The recruitment of mural cells around retina arteries coincides with the down-regulation of <i>egfl7</i> in the arterial endothelial cells, suggesting that this recruitment could be involved in the loss of <i>egfl7</i> expression in arteries. However, the expression pattern of <i>egfl7</i> is similar when mural cell recruitment is prevented by the injection of a PDGFRβ blocking antibody, suggesting that vessel maturation is not responsible for <i>egfl7</i> down-regulation in retinal arteries.</p></div

<i>Egfl7</i> expression in mural cell deficient retinal vasculature.

<p><b>A</b>: Co-immunodetection of pericytes (NG2) and smooth muscle cells (SMA) in P9 retina from pups injected with vehicle or with the blocking antibody directed against the PDGFRβ (αPDGFRβ). <b>B</b>: Co-immunodetection at higher magnification of endothelial cells (isolectin B4), pericytes (NG2) and smooth muscle cells (SMA) in P9 retina from pups injected with vehicle or with the blocking antibody directed against the PDGFRβ (αPDGFRβ). <b>C</b>: Type IV collagen immunostaining showing the vasculature of P9 retina from pups injected with vehicle or with the blocking antibody directed against the PDGFRβ (αPDGFRβ). <b>D</b>: Combined collagen IV (Coll IV) immunostaining and <i>egfl7</i> in situ hybridization in P9 retina from pups injected with vehicle or with the blocking antibody directed against the PDGFRβ (αPDGFRβ). a, artery; v, vein. Magnifications are indicated.</p

miR126 expression in mural cell deficient retinal vasculature.

<p><b>A</b>: miR126 expression detected by in situ hybridization in P9 whole mount retina from pups injected with vehicle or with the blocking antibody directed against PDGFRβ (αPDGFRβ). <b>B</b>: higher magnification of miR126 expression detected by in situ hybridization in P9 whole mount retina from pups injected with vehicle or with the blocking antibody directed against the PDGFRβ (αPDGFRβ).</p