Full activation of the T cell receptor requires both clustering and conformational changes at CD3.

T cell receptor (TCR-CD3) triggering involves both receptor clustering and conformational changes at the cytoplasmic tails of the CD3 subunits. The mechanism by which TCRalphabeta ligand binding confers conformational changes to CD3 is unknown. By using well-defined ligands, we showed that induction of the conformational change requires both multivalent engagement and the mobility restriction of the TCR-CD3 imposed by the plasma membrane. The conformational change is elicited by cooperative rearrangements of two TCR-CD3 complexes and does not require accompanying changes in the structure of the TCRalphabeta ectodomains. This conformational change at CD3 reverts upon ligand dissociation and is required for T cell activation. Thus, our permissive geometry model provides a molecular mechanism that rationalizes how the information of ligand binding to TCRalphabeta is transmitted to the CD3 subunits and to the intracellular signaling machinery.

Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.

The epidermal growth factor (EGF) receptor/ligand system stimulates multiple pathways of signal transduction, and is activated by various extracellular stimuli and inter-receptor crosstalk signaling. Aberrant activation of EGF receptor (EGFR) signaling is found in many tumor cells, and humanized neutralizing antibodies and synthetic small compounds against EGFR are in clinical use today. However, these drugs are known to cause a variety of skin toxicities such as inflammatory rash, skin dryness, and hair abnormalities. These side effects demonstrate the multiple EGFR-dependent homeostatic functions in human skin. The epidermis and hair follicles are self-renewing tissues, and keratinocyte stem cells are crucial for maintaining these homeostasis. A variety of molecules associated with the EGF receptor/ligand system are involved in epidermal homeostasis and hair follicle development, and the modulation of EGFR signaling impacts the behavior of keratinocyte stem cells. Understanding the roles of the EGF receptor/ligand system in skin homeostasis is an emerging issue in dermatology to improve the current therapy for skin disorders, and the EGFR inhibitor-associated skin toxicities. Besides, controlling of keratinocyte stem cells by modulating the EGF receptor/ligand system assures advances in regenerative medicine of the skin. We present an overview of the recent progress in the field of the EGF receptor/ligand system on skin homeostasis and regulation of keratinocyte stem cells.

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Role of P-glycoprotein in the uptake/efflux transport of oral vitamin K antagonists and rivaroxaban through the Caco-2 cell model.

Vitamin K antagonists (VKAs) are prescribed worldwide and remain the oral anticoagulant of choice. These drugs are characterized by a narrow therapeutic index and a large inter- and intra-individual variability. P-glycoprotein could contribute to this variability. The aim of this study was to investigate the involvement of P-gp in the transport of acenocoumarol, phenprocoumon and warfarin using an in vitro Caco-2 cell monolayer model. These results were compared with those obtained with rivaroxaban, a new oral anticoagulant known to be a P-gp substrate. The transport of these four drugs was assessed at pH conditions 6.8/7.4 in the presence or absence of the P-gp inhibitor cyclosporine A (10 μM) and the more potent and specific P-gp inhibitor valspodar (5 μM). Analytical quantification was performed by LC/MS. With an efflux ratio of 1.7 and a significant decrease in the efflux (Papp B-A), in the presence of P-gp inhibitors at a concentration of 50 μM, acenocoumarol can be considered as a weak P-gp substrate. Concerning phenprocoumon, the results suggest that this molecule is a poor P-gp substrate. The P-gp inhibitors did not affect significantly the transport of warfarin. The efflux of rivaroxaban was strongly inhibited by the two P-gp inhibitors. In conclusion, none of the three VKAs tested are strong P-gp substrates. However, acenocoumarol can be considered as a weak P-gp substrate and phenprocoumon as a poor P-gp substrate.

A high-mobility, low-cost phenotype defines human effector-memory CD8+ T cells.

T cells move randomly ("random-walk"), a characteristic thought to be integral to their function. Using migration assays and time-lapse microscopy, we found that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrate more efficiently in transwell assays, and that these same cells are characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu, but not when examined on endothelial cells. To assess the energy efficiency of cells crawling at a high frequency, we measured mRNA expression of genes key to mitochondrial energy metabolism (peroxisome proliferator-activated receptor gamma coactivator 1beta [PGC-1beta], estrogen-related receptor alpha [ERRalpha], cytochrome C, ATP synthase, and the uncoupling proteins [UCPs] UCP-2 and -3), quantified ATP contents, and performed calorimetric analyses. Together these assays indicated a high energy efficiency of the high crawling frequency CD8+ T-cell population, and identified differentially regulated heat production among nonlymphoid versus lymphoid homing CD8+ T cells.

Sox4 participates in the modulation of Schwann cell myelination.

In order to identify new regulators of Schwann cell myelination potentially playing a role in peripheral nervous system (PNS) pathologies, we analysed gene expression profiling data from three mouse models of demyelinating neuropathies and from the developing PNS. This analysis revealed that Sox4, which encodes a member of the Sry-related high-mobility group box protein family, was consistently upregulated in all three analysed models of neuropathy. Moreover, Sox4 showed a peak in its expression during development that corresponded with the onset of myelination. To gain further insights into the role of Sox4 in PNS development, we generated a transgenic mouse that specifically overexpresses Sox4 in Schwann cells. Sox4 overexpression led to a temporary delay in PNS myelination without affecting axonal sorting. Importantly, we observed that, whereas Sox4 mRNA could be efficiently overexpressed, Sox4 protein expression in Schwann cells was strictly regulated. Finally, our data showed that enforced expression of Sox4 in the mouse model for Charcot-Marie-Tooth 4C aggravated its neuropathic phenotype. Together, these observations reveal that Sox4 contributes to the regulation of Schwann cell myelination, and also indicates its involvement in the pathophysiology of peripheral neuropathies.

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 µM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30% H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

Role of high mobility group box-1 and protection of growth hormone and somatostatin in severe acute pancreatitis

In this study, we investigated the potential role of high-mobility group box 1 (HMGB1) in severe acute pancreatitis (SAP) and the effects of growth hormone (G) and somatostatin (S) in SAP rats. The rats were randomly divided into 6 groups of 20 each: sham-operated, SAP, SAP+saline, SAP+G, SAP+S and SAP+G+S. Ileum and pancreas tissues of rats in each group were evaluated histologically. HMGB1 mRNA expression was measured by reverse transcription-PCR. Levels of circulating TNF-α, IL-1, IL-6, and endotoxin were also measured. In the SAP group, interstitial congestion and edema, inflammatory cell infiltration, and interstitial hemorrhage occurred in ileum and pancreas tissues. The levels of HMGB1, TNF-α, IL-1, IL-6 and endotoxin were significantly up-regulated in the SAP group compared with those in the sham-operated group, and the 7-day survival rate was 0%. In the SAP+G and SAP+S groups, the inflammatory response of the morphological structures was alleviated, the levels of HMGB1, TNF-α, IL-1, IL-6, and endotoxin were significantly decreased compared with those in the SAP group, and the survival rate was increased. Moreover, in the SAP+G+S group, all histological scores were significantly improved and the survival rate was significantly higher compared with the SAP group. In conclusion, HMGB1 might participate in pancreas and ileum injury in SAP. Growth hormone and somatostatin might play a therapeutic role in the inflammatory response of SAP.

Inter-jurisdictional fiscal competition: a review of the literature and policy recommendations

This paper surveys the literature on fiscal competition. We consider tax and expenditure competition in a more general set up where different jurisdictions within a federation may compete in the provision of public goods in order to attract some residents (Tiebout, 1956) and expel others (Brueckner, 1999); and/or for business. We address the vast literature on welfare gains or losses of these types of competition. Then, we discuss the empirical evidence, focusing on estimates of the sensitiveness of production factors to tax differentials and on the importance of the strategic interdependence among jurisdictions. We combine econometric studies with some case studies. Last we discuss the design of mechanisms to cope with fiscal competition, especially under a more global environment where factors become more mobile.

Durable Goods, Inter-Sectoral Linkages and Monetary Policy

Barsky, House and Kimball (2007) show that introducing durable goods into a sticky-price model leads to negative sectoral comovement of production following a monetary policy shock and, under certain conditions, to aggregate neutrality. These results appear to undermine sticky-price models. In this paper, we show that these results are not robust to two prominent and realistic features of the data, namely input-output interactions and limited mobility of productive inputs. When extended to allow for both features, the sticky-price model with durable goods delivers implications in line with VAR evidence on the effects of monetary policy shocks.

Genomic architecture of sickle cell disease clinical variation in children from West Africa : a case-control study design

Contexte : L’anémie falciforme ou drépanocytose est un problème de santé important, particulièrement pour les patients d’origine africaine. La variation phénotypique de l’anémie falciforme est problématique pour le suivi et le traitement des patients. L’architecture génomique responsable de cette variabilité est peu connue. Principe : Mieux saisir la contribution génétique de la variation clinique de cette maladie facilitera l’identification des patients à risque de développer des phénotypes sévères, ainsi que l’adaptation des soins. Objectifs : L’objectif général de cette thèse est de combler les lacunes relatives aux connaissances sur l’épidémiologie génomique de l’anémie falciforme à l’aide d’une cohorte issue au Bénin. Les objectifs spécifiques sont les suivants : 1) caractériser les profils d’expressions génomiques associés à la sévérité de l’anémie falciforme ; 2) identifier des biomarqueurs de la sévérité de l’anémie falciforme ; 3) identifier la régulation génétique des variations transcriptionelles ; 4) identifier des interactions statistiques entre le génotype et le niveau de sévérité associé à l’expression ; 5) identifier des cibles de médicaments pour améliorer l’état des patients atteints d’anémie falciforme. Méthode : Une étude cas-témoins de 250 patients et 61 frères et soeurs non-atteints a été menée au Centre de Prise en charge Médical Intégré du Nourrisson et de la Femme Enceinte atteints de Drépanocytose, au Bénin entre février et décembre 2010. Résultats : Notre analyse a montré que des profils d’expressions sont associés avec la sévérité de l’anémie falciforme. Ces profils sont enrichis de génes des voies biologiques qui contribuent à la progression de la maladie : l’activation plaquettaire, les lymphocytes B, le stress, l’inflammation et la prolifération cellulaire. Des biomarqueurs transcriptionnels ont permis de distinguer les patients ayant des niveaux de sévérité clinique différents. La régulation génétique de la variation de l’expression des gènes a été démontrée et des interactions ont été identifiées. Sur la base de ces résultats génétiques, des cibles de médicaments sont proposées. Conclusion: Ce travail de thèse permet de mieux comprendre l’impact de la génomique sur la sévérité de l’anémie falciforme et ouvre des perspectives de développement de traitements ciblés pour améliorer les soins offerts aux patients.

Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses

Triggering of defences by microbes has mainly been investigated using single elicitors or microbe-associated molecular patterns (MAMPs), but MAMPs are released in planta as complex mixtures together with endogenous oligogalacturonan (OGA) elicitor. We investigated the early responses in Arabidopsis of calcium influx and oxidative burst induced by non-saturating concentrations of bacterial MAMPs, used singly and in combination: flagellin peptide (flg22), elongation factor peptide (elf18), peptidoglycan (PGN) and component muropeptides, lipo-oligosaccharide (LOS) and core oligosaccharides. This revealed that some MAMPs have additive (e.g. flg22 with elf18) and even synergistic (flg22 and LOS) effects, whereas others mutually interfere (flg22 with OGA). OGA suppression of flg22-induced defences was not a result of the interference with the binding of flg22 to its receptor flagellin-sensitive 2 (FLS2). MAMPs induce different calcium influx signatures, but these are concentration dependent and unlikely to explain the differential induction of defence genes [pathogenesis-related gene 1 (PR1), plant defensin gene 1.2 (PDF1.2) and phenylalanine ammonia lyase gene 1 (PAL1)] by flg22, elf18 and OGA. The peptide MAMPs are potent elicitors at subnanomolar levels, whereas PGN and LOS at high concentrations induce low and late host responses. This difference might be a result of the restricted access by plant cell walls of MAMPs to their putative cellular receptors. flg22 is restricted by ionic effects, yet rapidly permeates a cell wall matrix, whereas LOS, which forms supramolecular aggregates, is severely constrained, presumably by molecular sieving. Thus, MAMPs can interact with each other, whether directly or indirectly, and with the host wall matrix. These phenomena, which have not been considered in detail previously, are likely to influence the speed, magnitude, versatility and composition of plant defences.

Settlement patterns in the late Mesolithic of western Scotland: the implications of Bayesian analysis of radiocarbon dates and inter-site technological comparisons

The character of settlement patterns within the late Mesolithic communities of north-west Europe is a topic of substantial debate. An important case study concerns the five shell middens on the island of Oronsay, Inner Hebrides, western Scotland. Two conflicting interpretations have been proposed: the evidence from seasonality indicators and stable isotope analysis of human bones has been used to support a model of year-round settlement on this small island; alternatively, the middens have been interpreted as resulting from short-term intermittent visits to Oronsay within a regionally mobile settlement pattern. We contribute to this debate by describing Storakaig, a newly discovered site on the nearby island of Islay, undertaking a Bayesian chronological analysis and providing evidence for technological continuity between Oronsay and sites elsewhere in the region. While this new evidence remains open to alternative interpretation, we suggest that it makes regional mobility rather than year-round settlement on Oronsay a more viable interpretation for the Oronsay middens. Our analysis also confirms the likely overlap of the late Mesolithic with the earliest Neolithic within western Scotland.

Cell stress-induced phosphorylation of ATF2 and c-Jun transcription factors in rat ventricular myocytes.

Ventricular myocytes are exposed to various pathologically important cell stresses in vivo. In vitro, extreme stresses (sorbitol-induced hyperosmotic shock in the presence or absence of okadaic acid, and anisomycin) were applied to ventricular myocytes cultured from neonatal rat hearts to induce a robust activation of the 46 and 54 kDa stress-activated protein kinases (SAPKs). These activities were increased in nuclear extracts of cells in the absence of any net import of SAPK protein. Phosphorylation of ATF2 and c-Jun was increased as shown by the appearance of reduced-mobility species on SDS/PAGE, which were sensitive to treatment with protein phosphatase 2A. Hyperosmotic shock and anisomycin had no effect on the abundance of ATF2. In contrast, cell stresses induced a greater than 10-fold increase in total c-Jun immunoreactivity detected on Western blots with antibody to c-Jun (KM-1). Cycloheximide did not inhibit this increase, which we conclude represents phosphorylation of c-Jun. This conclusion was supported by use of a c-Jun(phospho-Ser-73) antibody. Immunostaining of cells also showed increases in nuclear phospho-c-Jun in response to hyperosmotic stress. Severe stress (hyperosmotic shock+okadaic acid for 2 h) induced proteins (migrating at approx. 51 and 57 kDa) that cross-reacted strongly with KM-1 antibodies in both the nucleus and the cytosol. These may represent forms of c-Jun that had undergone further modification. These studies show that stresses induce phosphorylation of transcription factors in ventricular myocytes and we suggest that this response may be pathologically relevant.

Design of a LNA and a Gilbert Cell Mixer MMICs with a GaAs PHEMT technology

The design of a Gilbert Cell Mixer and a low noise amplifier (LNA), using GaAs PHEMT technology is presented. The compatibility is shown for co-integration of both block on the same chip, to form a high performance 1.9 GHz receiver front-end. The designed LNA shows 9.23 dB gain and 2.01 dB noise figure (NF). The mixer is designed to operate at RF=1.9 GHz, LO=2.0 GHz and IF=100 MHz with a gain of 14.3 dB and single sideband noise figure (SSB NF) of 9.6 dB. The mixer presents a bandwith of 8 GHz.