Performance evaluation of a new fourth-generation HIV combination antigen-antibody assay.

Education and diagnostic tests capable of early detection represent our most effective means of preventing transmission of human immunodeficiency virus (HIV). The importance of early detection is underlined by studies demonstrating increased life expectancy following early initiation of antiviral treatment. The Elecsys(®) HIV combi PT assay is a fourth-generation antigen-antibody combination assay developed to allow earlier detection of seroconversion, and to have increased sensitivity and improved specificity. We aimed to determine how early the assay could detect infection compared with existing assays; whether all HIV variants could be detected; and the assay's specificity using samples from blood donors, routine specimens, and patients with potential cross-reacting factors. Samples were identified as positive by the Elecsys(®) assay 4.9 days after a positive polymerase chain reaction result (as determined by the panel supplier), which was earlier than the 5.3-7.1 days observed with comparators. The analytical sensitivity of the Elecsys(®) HIV combi PT assay for the HIV-1 p24 antigen was 1.05 IU/mL, which compares favorably with the comparator assays. In addition, the Elecsys(®) assay identified all screened HIV subtypes and displayed greater sensitivity to HIV-2 homologous antigen and antibodies to HIV-1 E and O and HIV-2 than the other assays. Overall, the specificity of the Elecsys(®) assay was 99.88 % using samples from blood donors and 99.81 % when analyzing unselected samples. Potential cross-reacting factors did not interfere with assay performance. The Elecsys(®) HIV combi PT assay is a sensitive and specific assay that has been granted the CE mark according to Directive 2009/886/EC.

The TRAF-interacting protein (TRIP) is a regulator of keratinocyte proliferation.

The TRAF-interacting protein (TRIP/TRAIP) is a RING-type E3 ubiquitin ligase inhibiting tumor necrosis factor-α (TNF-α)-mediated NF-κB activation. TRIP ablation results in early embryonic lethality in mice. To investigate TRIP function in epidermis, we examined its expression and the effect of TRIP knockdown (KD) in keratinocytes. TRIP mRNA expression was strongly downregulated in primary human keratinocytes undergoing differentiation triggered by high cell density or high calcium. Short-term phorbol-12-myristate-13-acetate (TPA) treatment or inhibition of phosphatidylinositol-3 kinase signaling in proliferative keratinocytes suppressed TRIP transcription. Inhibition by TPA was protein kinase C dependent. Keratinocytes undergoing KD of TRIP expression by lentiviral short-hairpin RNA (shRNA; T4 and T5) had strongly reduced proliferation rates compared with control shRNA. Cell cycle analysis demonstrated that TRIP-KD caused growth arrest in the G1/S phase. Keratinocytes with TRIP-KD resembled differentiated cells consistent with the augmented expression of differentiation markers keratin 1 and filaggrin. Luciferase-based reporter assays showed no increase in NF-κB activity in TRIP-KD keratinocytes, indicating that NF-κB activity in keratinocytes is not regulated by TRIP. TRIP expression was increased by ∼2-fold in basal cell carcinomas compared with normal skin. These results underline the important role of TRIP in the regulation of cell cycle progression and the tight linkage of its expression to keratinocyte proliferation.

Unedited in vivo detection and quantification of γ-aminobutyric acid in the occipital cortex using short-TE MRS at 3 T.

Short-TE MRS has been proposed recently as a method for the in vivo detection and quantification of γ-aminobutyric acid (GABA) in the human brain at 3 T. In this study, we investigated the accuracy and reproducibility of short-TE MRS measurements of GABA at 3 T using both simulations and experiments. LCModel analysis was performed on a large number of simulated spectra with known metabolite input concentrations. Simulated spectra were generated using a range of spectral linewidths and signal-to-noise ratios to investigate the effect of varying experimental conditions, and analyses were performed using two different baseline models to investigate the effect of an inaccurate baseline model on GABA quantification. The results of these analyses indicated that, under experimental conditions corresponding to those typically observed in the occipital cortex, GABA concentration estimates are reproducible (mean reproducibility error, <20%), even when an incorrect baseline model is used. However, simulations indicate that the accuracy of GABA concentration estimates depends strongly on the experimental conditions (linewidth and signal-to-noise ratio). In addition to simulations, in vivo GABA measurements were performed using both spectral editing and short-TE MRS in the occipital cortex of 14 healthy volunteers. Short-TE MRS measurements of GABA exhibited a significant positive correlation with edited GABA measurements (R = 0.58, p < 0.05), suggesting that short-TE measurements of GABA correspond well with measurements made using spectral editing techniques. Finally, within-session reproducibility was assessed in the same 14 subjects using four consecutive short-TE GABA measurements in the occipital cortex. Across all subjects, the average coefficient of variation of these four GABA measurements was 8.7 ± 4.9%. This study demonstrates that, under some experimental conditions, short-TE MRS can be employed for the reproducible detection of GABA at 3 T, but that the technique should be used with caution, as the results are dependent on the experimental conditions. Copyright © 2013 John Wiley & Sons, Ltd.

New loci associated with birth weight identify genetic links between intrauterine growth and adult height and metabolism.

Birth weight within the normal range is associated with a variety of adult-onset diseases, but the mechanisms behind these associations are poorly understood. Previous genome-wide association studies of birth weight identified a variant in the ADCY5 gene associated both with birth weight and type 2 diabetes and a second variant, near CCNL1, with no obvious link to adult traits. In an expanded genome-wide association meta-analysis and follow-up study of birth weight (of up to 69,308 individuals of European descent from 43 studies), we have now extended the number of loci associated at genome-wide significance to 7, accounting for a similar proportion of variance as maternal smoking. Five of the loci are known to be associated with other phenotypes: ADCY5 and CDKAL1 with type 2 diabetes, ADRB1 with adult blood pressure and HMGA2 and LCORL with adult height. Our findings highlight genetic links between fetal growth and postnatal growth and metabolism.

Bioinformatics-driven identification and examination of candidate genes for non-alcoholic fatty liver disease.

ObjectiveCandidate genes for non-alcoholic fatty liver disease (NAFLD) identified by a bioinformatics approach were examined for variant associations to quantitative traits of NAFLD-related phenotypes.Research Design and MethodsBy integrating public database text mining, trans-organism protein-protein interaction transferal, and information on liver protein expression a protein-protein interaction network was constructed and from this a smaller isolated interactome was identified. Five genes from this interactome were selected for genetic analysis. Twenty-one tag single-nucleotide polymorphisms (SNPs) which captured all common variation in these genes were genotyped in 10,196 Danes, and analyzed for association with NAFLD-related quantitative traits, type 2 diabetes (T2D), central obesity, and WHO-defined metabolic syndrome (MetS).Results273 genes were included in the protein-protein interaction analysis and EHHADH, ECHS1, HADHA, HADHB, and ACADL were selected for further examination. A total of 10 nominal statistical significant associations (P<0.05) to quantitative metabolic traits were identified. Also, the case-control study showed associations between variation in the five genes and T2D, central obesity, and MetS, respectively. Bonferroni adjustments for multiple testing negated all associations.ConclusionsUsing a bioinformatics approach we identified five candidate genes for NAFLD. However, we failed to provide evidence of associations with major effects between SNPs in these five genes and NAFLD-related quantitative traits, T2D, central obesity, and MetS.

Regulation and glucocorticoid-independent induction of lipocortin I in cultured astrocytes.

Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP-, calcium ionophore A23187-, or tetradecanoyl phorbol acetate (TPA)-stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC-mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2 (PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2-3-week-old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum- and GC-free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Ex vivo Pulsatile Perfusion of Human Saphenous Veins Induces Intimal Hyperplasia and Increased Levels of the Plasminogen Activator Inhibitor 1.

Vessel wall trauma induces vascular remodeling processes including the development of intimal hyperplasia (IH). To assess the development of IH in human veins, we have used an ex vivo vein support system (EVVSS) allowing the perfusion of freshly isolated segments of saphenous veins in the presence of a pulsatile flow which reproduced arterial conditions regarding shear stress, flow rate and pressure during a period of 7 and 14 days. Compared to the corresponding freshly harvested human veins, histomorphometric analysis showed a significant increase in the intimal thickness which was already maximal after 7 days of perfusion. Expression of the endothelial marker CD31 demonstrated the presence of endothelium up to 14 days of perfusion. In our EVVSS model, the activity as well as the mRNA and protein expression levels of plasminogen activator inhibitor 1, the inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), were increased after 7 days of perfusion, whereas the expression levels of tPA and uPA were not altered. No major change was observed between 7 and 14 days of perfusion. These data show that our newly developed EVVSS is a valuable setting to study ex vivo remodeling of human veins submitted to a pulsatile flow.

Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins

L'hyperplasie intimale est la cause majeure de sténoses de pontages veineux. Différents médicaments tels que les statines permettent de prévenir les sténoses mais leur administration systémique n'a que peu d'effet. Nous avons développé une matrice d'hydrogel d'acide hyaluronique qui permet d'avoir un relargage contrôlé d'atorvastatine sur un site désiré. L'enjeu de ce projet de recherche est de démontrer que l'atorvastatine relarguée par l'hydrogel a un effet similaire sur les cellules musculaires lisses de veines saphènes humaines comparé à l'atorvastatine directement diluée dans le milieu de culture. La recherche a été conduite conjointement par le laboratoire de médecine expérimentale du département de chirurgie thoracique et vasculaire du Centre Hospitalier Universitaire Vaudois et de l'Ecole de sciences pharmaceutiques des Universités de Lausanne et de Genève. On a incorporé de l'atorvastatine calcium (Chemos GmbH, Regenstauf Allemagne) dans des gels d'acide hyaluronique (Fortelis extra) à des concentrations déterminées afin de pouvoir analyser le relargage de l'Atovastatine dans le milieu de culture cellulaire par rapport aux concentrations d'atorvastatine directement ajoutées dans le milieu. Des cellules musculaires lisses primaires ont été cultivées à partir d'expiants de veines saphènes humaines. Elles ont été identifiées grâce à l'immunohistochimie par des anticorps contre la desmine et l'alpha-smooth muscle actine. La prolifération et la viabilité de ces cellules ont été analysées à l'aide du test MTT, leur transmigration avec le test de la chambre de Boyden et leur migration avec le principe de cicatrisation de plaies (wound healing assey). L'expression de gènes connus pour participer au développement de l'hyperplasie intimale, tels que la gap junction protein Connexin43 (Cx43), l'inhibiteur du plasminogène PAI-1, Thème oxygénase HO-1, la métalloproteinase-9 et l'inhibiteur de l'activateur du plasminogène tissulaire tPA, a été déterminée par niveau de mRNA exprimé en PCR. Leur expression en protéines a été analysée en utilisant la méthode par Western blots ainsi que l'immunohistochimie. Les expériences ont été effectuées à triple reprise en duplicats en parallèles avec de l'atorvastatine calcium directement ajoutée dans le milieu de culture et avec l'atorvastatine relarguée par l'hydrogel d'acide hyaluronique. Conclusions L'atorvastatine est relarguée par l'hydrogel de façon contrôlée. L'hydrogel contenant l'atorvastatine diminue la viabilité et la transmigration des cellules musculaires lisses de veines saphènes humaines de façon similaire à l'atorvastatine directement introduite dans le milieu de culture. L'hydrogel contenant l'atorvastatine module de façon sélective l'expression de marqueurs de la différentiation cellulaire de cellules musculaires lisses de veines saphènes humaines avec un retard de 24 heures comparé avec les effets de l'atorvastatine directement ajoutée au milieu de culture, sans néanmoins changer la distribution intra-cellulaire des protéines Cx43, HO-1 et PAI-1. Perspectives Il s'agit d'un projet d'importance clinique majeure permettant de réaliser des améliorations du traitement des artériopathies occlusives, ainsi que de relevance pharmacologique permettant de réaliser des dépôts de molécules avec un relargage stable et contrôlé à un site spécifique.

The primary in vivo immune response to Mls-1 (Mtv-7 sag). Route of injection determines the immune response pattern.

Injection of cells expressing the retroviral superantigen Mls-1 (Mtv-7 sag) into adult Mls-1- mice induces a strong immune response including both T- and B-cell activation. This model was used for studying qualitative aspects of the immune response in normal mice with a defined antigen-presenting cell (the B cell) and without the use of adjuvant. BALB/c mice were injected locally or systemically with Mls-1-expressing spleen cells from Mls-1-congenic BALB.D2 mice. Intravenous injection led to an initially strong expansion of Mls-1-reactive V beta 6+ CD4+ cells mainly in the spleen, to a large degree explained by the trapping of reactive cells, and a rapid down-regulation of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production, consistent with the proposed tolerogenic property of B cells as antigen-presenting cells. However, these mice developed a slowly appearing but persistent B-cell response dominated by IgG1-producing cells, suggesting a shift in lymphokines produced rather than complete unresponsiveness. Subcutaneous injection into the hind footpad with the same number of cells led to a strong local response in the draining lymph node, characterized by a dramatic increase of V beta 6+ CD4+ T cells, local production of IL-2 and IFN-gamma and a strong but short-lived antibody response dominated by IgG2a-producing cells, characteristic of a T-helper type 1 (Th1) type of response. Both routes of injection led ultimately to deletion of reactive T cells and anergy, as defined by the inability to produce IL-2 upon in vitro stimulation with Mls-1. It is concluded that Mls-1 presented by B cells induces qualitatively different responses in vivo dependent on the route of injection. We propose that the different responses result from the migration of the injected cells to different micro-anatomical sites in the lymphoid tissue. Furthermore, these results suggest that B cells may function as professional antigen-presenting cells in vivo present in an appropriate environment.

Transcription factor PROX1 induces colon cancer progression by promoting the transition from benign to highly dysplastic phenotype.

The Drosophila transcription factor Prospero functions as a tumor suppressor, and it has been suggested that the human counterpart of Prospero, PROX1, acts similarly in human cancers. However, we show here that PROX1 promotes dysplasia in colonic adenomas and colorectal cancer progression. PROX1 expression marks the transition from benign colon adenoma to carcinoma in situ, and its loss inhibits growth of human colorectal tumor xenografts and intestinal adenomas in Apc(min/+) mice, while its transgenic overexpression promotes colorectal tumorigenesis. Furthermore, in intestinal tumors PROX1 is a direct and dose-dependent target of the beta-catenin/TCF signaling pathway, responsible for the neoplastic transformation. Our data underscore the complexity of cancer pathogenesis and implicate PROX1 in malignant tumor progression through the regulation of cell polarity and adhesion.

Ultrasound-assisted versus conventional catheter-directed thrombolysis for acute iliofemoral deep vein thrombosis.

BACKGROUND: For patients with acute iliofemoral deep vein thrombosis, it remains unclear whether the addition of intravascular high-frequency, low-power ultrasound energy facilitates the resolution of thrombosis during catheter-directed thrombolysis. METHODS AND RESULTS: In a controlled clinical trial, 48 patients (mean age 50 ± 21 years, 52% women) with acute iliofemoral deep vein thrombosis were randomized to receive ultrasound-assisted catheter-directed thrombolysis (N = 24) or conventional catheter-directed thrombolysis (N = 24). Thrombolysis regimen (20 mg r-tPA over 15 hours) was identical in all patients. The primary efficacy end point was the percentage of thrombus load reduction from baseline to 15 hours according to the length-adjusted thrombus score, obtained from standardized venograms and evaluated by a core laboratory blinded to group assignment. The percentage of thrombus load reduction was 55% ± 27% in the ultrasound-assisted catheter-directed thrombolysis group and 54% ± 27% in the conventional catheter-directed thrombolysis group (P = 0.91). Adjunctive angioplasty and stenting was performed in 19 (80%) patients and in 20 (83%) patients, respectively (P > 0.99). Treatment-related complications occurred in 3 (12%) and 2 (8%) patients, respectively (P > 0.99). At 3-month follow-up, primary venous patency was 100% in the ultrasound-assisted catheter-directed thrombolysis group and 96% in the conventional catheter-directed thrombolysis group (P = 0.33), and there was no difference in the severity of the post-thrombotic syndrome (mean Villalta score: 3.0 ± 3.9 [range 0-15] versus 1.9 ± 1.9 [range 0-7]; P=0.21), respectively. CONCLUSIONS: In this randomized controlled clinical trial of patients with acute iliofemoral deep vein thrombosis treated with a fixed-dose catheter thrombolysis regimen, the addition of intravascular ultrasound did not facilitate thrombus resolution. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01482273.

Signalling pathways in skin homeostasis

SUMMARY The results presented here contribute to a better understanding of the crucial molecular relationships and signalling cues exchanged by several fundamental cell types (epidermal keratinocytes, dermal fibroblasts, immune and endothelial cells) of the skin. Importantly we provide evidence to directly implicate Wnt/ß-catenin signalling as a putative player in different cell types (keratinocytes and neutrophils) in mediation of the cutaneous inflammatory response (Fart A). Finally we highlight the importance of several molecules, specifically expressed in the hair follicle stem cell niche to the morphogenesis and homeostasis of the hair follicle (Part B). PART A Currently the body of work pertaining to Wnt signalling and immune cells largely focuses on Wnt signalling in the development of these cells. The data presented here suggests a novel mechanism in which Wnt signalling appears to modulate immune cell recruitment to the skin. Keratinocytes are major contributors to early inflammatory responses by the release of chemokines which recruit immune cells. The resultant inflammatory response is a dynamic process of sequentially infiltrating immune cells governed by a network of growth factors, chemokines and cytokines. In wild type mice the response is typified by a rapid and substantial infiltration of neutrophils followed at later time points by macrophages and Tcells. The expression of the canonical Wnt pathway activating ligand, Wnt3a, is able to induce a strong neutrophil infiltration in the dermis. This response originates in keratinocytes, as it is abrogated upon keratinocyte-specific ablation of ß-catenin. Notably, this suggests that the crucial cross talk between these resident cells and recruited immune cells is, in part, mediated by Wnt signalling. In corroboration of this role of Wnt-mediated recruitment of neutrophils, expression of the Wnt inhibitory ligand sFRPI during acute inflammation results in a dramatic 'dampening' of immune cell infiltration in particular of neutrophil chemoattraction. Importantly, an intrinsic Wnt signalling pathway is essential for neutrophil chemoattraction in response to inflammatory stimuli. There is a marked reduction of neutrophil infiltration in mice grafted with a ß-catenin deficient bone marrow upon TPA induced cutaneous inflammation. Additionally, neutrophils lacking Wnt/ß-catenin fail to respond to IFNγ, an early inflammatory cue, in vitro. In combination, these data indicate a potent function of Wnt signalling in immune cell recruitment and the modulation of the inflammatory response. PART B Tissue specific stem cells form the cellular base on which tissue homeostasis and repair of adult tissue relies. The maintenance of this stem cell pool is highly dependent on the immediate environment or niche. We have identified three genes, the fibroblast growth factor receptor 1 (FGFR1), serpin protease inhibitor (serpin F1) and the haematopoietic cell phosphatase (Hcph) to be specifically expressed in a small population of stromal cells which are in close contact to bulge stem cells. These specialized stromal cells might represent an essential mesenchymal component of the skin stem cell niche and may regulate stem cell proliferation and differentiation. Multiple FGFR1 isoforms are generated through alternate transcript splicing and are able to interact with both FGFs and cell adhesion molecules. Two predominant forms of the receptor are FGFR1-α and FGFR1-ß. Expression of a dominant negative form of the alpha isoform prevents hair follicle morphogenesis altogether. Given that FGFR1-ß signals principally through the FGF ligands, this data indicates that FGF signalling is dispensable for follicle morphogenesis. Moreover the loss of follicular morphogenesis upon suggests a requirement for signalling via cell adhesion molecule association with the receptor as FGFR1 α has a greater affinity for these molecules. The expression of the second candidate niche gene serpin f1, lead to the complete ablation of hair follicle morphogenesis. The serpin f1 product, pigment-epithelial derived factor (PEDF) has potent anti-angiogenic effects. Immunohistochemical analysis using CD31, a endothelial cell marker, revealed that although these cells are present, they have are disorganised and do not form vessels. Interestingly, endothelial cells have been found to contribute to the neuronal stem cell niche and our results suggest a similar mechanism in the skin. SHP1, the Hcph gene product, is a phosphatase which acts in the haematopoetic system. Motheaten mice carrying spontaneous mutations in the Hcph gene have patchy alopecia in their skin and severe defects in their haematopoietic system. However the haematopoietic rescue of the mouse does not result in normal follicular homeostasis. Additionally, ablation of Hcph in either the dermal or keratinocyte compartments of the skin produces hair follicles with abberant morphologies. This data indicates that although SHP1 is not essential for hair follicle morphogenesis it is required in both epidermal and dermal compartments to maintain follicular morphology. RÉSUMÉ PARTIE A Jusqu'à présent, les travaux dédiés à l'étude de la voie de signalisation Wnt dans le système immunitaire se sont essentiellement concentrés sur son rôle dans le développement des cellules immunitaires. Les données présentées ici suggèrent fortement et de manière nouvelle, l'existence d'un mécanisme par lequel la voie de signalisation Wnt/ß-caténine module le recrutement de cellules immunitaires dans un tissu périphérique, la peau, et ainsi la réponse inflammatoire cutanée. La réponse inflammatoire cutanée est un processus dynamique d'infiltration séquentielle de diverses cellules immunitaires, orchestré par un réseau de facteurs de croissance, chémokines et cytokines. Les kératinocytes sont des contributeurs majeurs à la réponse inflammatoire précoce par la libération de chémokines qui permettent ensuite de recruter les cellules immunitaires. Dans des souris sauvages, la réponse est d'abord caractérisée par une infiltration rapide et substantielle de neutrophiles, suivie par celle des macrophages et des lymphocytes T. L'expression d'un ligand activateur de le voie canonique de signalisation Wnt (après injection infra-dermique de fibroblastes sur-exprimant Wnt-3a) induit une infiltration dermique très marquée de neutrophiles. De plus, la réponse est éliminée en l'absence de ß-caténine spécifiquement dans les kératinocytes, indiquant que ces cellules sont à l'origine de la réponse. De manière remarquable, ceci suggère qu'une signalisation cruciale entre ces cellules résidentes de la peau et les cellules immunitaires recrutées est, au moins en partie, médiée par la voie Wnt. Corroborant ce rôle de la voie Wnt/ß-caténine dans le recrutement des neutrophiles, l'expression d'un ligand inhibiteur de la voie (sFRP1) résulte au cours d'une inflammation aigüe en une réduction spectaculaire de l'infiltration des cellules immunitaires en général, et des neutrophiles en particulier. De manière importante, la voie de signalisation Wnt est intrinsèquement requise pour la chémoattraction des neutrophiles en réponse à un stimulus inflammatoire. En effet, suite à une inflammation cutanée induite par un ester de phorbol (TPA), une réduction notable de l'infiltration des neutrophiles est observée dans des souris préalablement greffées avec de la moelle osseuse constituée de cellules déficientes en ß-caténine. De plus, in vitro, les neutrophiles sans ß-caténine ne répondent pas à une stimulation par l'interféron γ, qui est pourtant un signal inflammatoire établi in vivo. En conclusion, nos données indiquent que la voie de signalisation Wnt/ß-caténine joue une fonction active dans le recrutement des cellules immunitaires vers un organe périphérique, la peau, ainsi que dans la modulation, à plusieurs niveaux, de la réponse inflammatoire cutanée. PARTIE B Les cellules souches tissu-spécifiques forment la base cellulaire sur laquelle repose l'homéostase et la réparation tissulaires chez l'adulte. La maintenance de ce réservoir de cellules souches est hautement dépendante de leur environnement cellulaire immédiat, encore appelé «niche des cellules souches». Dans la peau, ces cellules stromales spécialisées représentent un compartiment mésenchymateux essentiel de la niche des cellules souches en régulant leurs prolifération et différentiation. Nous avons identifié trois gènes, le «récepteur 1 àux facteurs de croissance des fibroblastes » (Fgfr1 ), l' «inhibiteur de protéase à sérine » (serpinf1 ou pedf) et la « phosphatase des cellules hématopoiétiques » (Hcph ou Ptpn6), comme spécifiquement exprimés par une petite population de cellules stromales qui sont étroitement associées aux cellules souches de la peau (localisées au niveau du bombement du follicule pileux). Pour analyser leur fonction dans ce contexte, nous avons utilisé un test de reconstitution complète de peau murine en combinaison à des. transductions géniques basées sur l'utilisation de lentivirus. Ce test repose sur le mélange de deux compartiments cellulaires, épidermique (kératinocytes) et dermique (fibroblastes), greffés sur une zone ouverte de peau du dos d'une souris pour ensemble reconstituer la peau. Des isoformes multiples de FGFR1 sont générées par épissage alternatif de transcrits et sont capables d'interagir à la fois avec les FGFs (facteurs de croissance des fibroblastes) et les molécules d'adhésion cellulaires. Les deux formes prédominantes du récepteur, FGFR1-α et FGFR1-ß, ne différent que par le «domaine ressemblant aux immunoglobulines 1 » (immunoglobulin-like 1 domain), absent de FGFR1-ß. De plus, FGFR1-ß a une affinité plus grande pour les FGFs et plus faible pour les molécules d'adhésion cellulaires telles que la Ncadhérine (connue pour activer FGFR). La sur-expression de l'une ou l'autre des formes n'empêche pas la morphogenèse folliculaire mais conduit à la formation de follicules aberrants. Toutefois, une différence phénotypique majeure est observée lorsqu'une forme «Dominant-Négatif » (DN) est exprimée dans le compartiment dermique. La sur-expression de FGFR1-ß DN conduit en effet à la formation de follicules petits et tronqués, avec des gaines épithéliales et un bulbe élargis ainsi qu'une petite papille dermique. Par contre, l'expression de FGFR1-α DN abolit complètement la morphogenèse folliculaire. Etant donné que la signalisation par FGFR1-ß est principalement dépendante des ligands FGFs, ces données indiquent que la signalisation par ceux-cì est non-nécessaire à la morphogenèse folliculaire. De plus, l'abolition du processus par la sur-expression de FGFR1-a DN suggëre une signalisation nécessaire entre le récepteur FGFR1 et une ou des molécules d'adhésion cellulaire. L'expression de notre second candidat comme gène spécifique de la niche des cellules souches de la peau, serpinf1, prévient la morphogenèse folliculaire. Seules de petites structures ressemblant à des cystes sont observées après reconstitution de la peau. De plus, dans ces transplants, aucune cellule CD34-positive (marqueur des cellules souches) n'est retrouvée associé à ces cystes. Le produit du gène serpin f1, le «facteur dérivé d'épithélium pigmentaire » (PEDF) est un puissant facteur anti-angiogénique. Nous avons donc analysé la vascularisation des transplants par immunohistochirnies utilisant CD31, un marqueur des cellules endothéliales. Nos résultats révèlent que les cellules endothéliales sont bien présentes, mais de manière désorganisée et ne formant pas de vaisseaux. De manière intéressante, les cellules endothéliales contribuent activement à la niche des cellules souches neuronales, et nos résultats suggèrent donc l'existence possible d'un mécanisme similaire dans la peau. SHP1, le produit du gène Hcph, est une phosphatase quì agit dans le système hématopoiétique. Les souris « motheaten »qui portent des mutations spontanées du gène ont une alopécie inégale au niveau de la peau et de sévères troubles du système hématopoiétique. Pour s'assurer que le phénotype observé au niveau de la peau n'est pas une conséquence d'un défaut du système hématopoiétique, nous avons transplanté des souris Hcph -/- avec de la moelle osseuse sauvage afin de restaurer la fonction de SHP 1 dans le système hématopoiétique. Toutefois, le défaut de morphologie folliculaire est maintenu. De plus, l'ablation d'Hcph dans le compartiment dermique ou épidermique d'essais de reconstitution de peau conduit à la production de follicules pileux avec des morphologies aberrantes. Ces données indiquent que SHP1 n'est pas essentiel à la morphogenèse folliculaire mais est toutefois requis à la fois dans les compartiments épidermiques et dermiques pour la maintenance de la forme du follicule.

Cardiovascular Risk Prediction with Ultrasound

This paper addresses primary care physicians, cardiologists, internists, angiologists and doctors desirous of improving vascular risk prediction in primary care. Many cardiovascular risk factors act aggressively on the arterial wall and result in atherosclerosis and atherothrombosis. Cardiovascular prognosis derived from ultrasound imaging is, however, excellent in subjects without formation of intimal thickening or atheromas. Since ultrasound visualises the arterial wall directly, the information derived from the arterial wall may add independent incremental information to the knowledge of risk derived from global risk assessment. This paper provides an overview on plaque imaging for vascular risk prediction in two parts: Part 1: Carotid IMT is frequently used as a surrogate marker for outcome in intervention studies addressing rather large cohorts of subjects. Carotid IMT as a risk prediction tool for the prevention of acute myocardial infarction and stroke has been extensively studied in many patients since 1987, and has yielded incremental hazard ratios for these cardiovascular events independently of established cardiovascular risk factors. However, carotid IMT measurements are not used uniformly and therefore still lack widely accepted standardisation. Hence, at an individual, practicebased level, carotid IMT is not recommended as a risk assessment tool. The total plaque area of the carotid arteries (TPA) is a measure of the global plaque burden within both carotid arteries. It was recently shown in a large Norwegian cohort involving over 6000 subjects that TPA is a very good predictor for future myocardial infarction in women with an area under the curve (AUC) using a receiver operating curves (ROC) value of 0.73 (in men: 0.63). Further, the AUC for risk prediction is high both for vascular death in a vascular prevention clinic group (AUC 0.77) and fatal or nonfatal myocardial infarction in a true primary care group (AUC 0.79). Since TPA has acceptable reproducibility, allows calculation of posttest risk and is easily obtained at low cost, this risk assessment tool may come in for more widespread use in the future and also serve as a tool for atherosclerosis tracking and guidance for intensity of preventive therapy. However, more studies with TPA are needed. Part 2: Carotid and femoral plaque formation as detected by ultrasound offers a global view of the extent of atherosclerosis. Several prospective cohort studies have shown that cardiovascular risk prediction is greater for plaques than for carotid IMT. The number of arterial beds affected by significant atheromas may simply be added numerically to derive additional information on the risk of vascular events. A new atherosclerosis burden score (ABS) simply calculates the sum of carotid and femoral plaques encountered during ultrasound scanning. ABS correlates well and independently with the presence of coronary atherosclerosis and stenosis as measured by invasive coronary angiogram. However, the prognostic power of ABS as an independent marker of risk still needs to be elucidated in prospective studies. In summary, the large number of ways to measure atherosclerosis and related changes in human arteries by ultrasound indicates that this technology is not yet sufficiently perfected and needs more standardisation and workup on clearly defined outcome studies before it can be recommended as a practice-based additional risk modifier.

Baseline Results of an Acute Ischemic Stroke Registry Including Acute Multimodal Imaging (ASTRAL)

Background: The DEFUSE (n_74) and EPITHET (n_101) studies have in common that a baseline MRI was obtained prior to treatment (tPA in DEFUSE; tPA or placebo in EPITHET) in the 3-6 hour time-window. There were however important methodological differences between the studies. A standardized reanalysis of pooled data was undertaken to determine the effect of these differences on baseline characteristics and study outcomes. Methods: To standardize the studies 1) the DWI and PWI source images were reprocessed and segmented using automated image processing software (RAPID); 2) patients were categorized according to their baseline MRI profile as either Target Mismatch (PWITmax_6/DWI ratio_ 1.8 and an absolute mismatch _15mL), Malignant (DWI or PWITmax_10 lesion _ 100 mL), or No Mismatch. 3) favorable clinical response was defined as NIHSS score of 0-1 or a _8 points improvement on the NIHSSS at day 90. Results: Prior to standardization there was no difference in the proportion of Target Mismatch patients between EPITHET and DEFUSE (54% vs 49%, p_0.6), but the EPITHET study had more patients with the Malignant profile than DEFUSE (35% vs 9%, p_0.01) and fewer patients that had No Mismatch (11% vs 42%, p_0.01). These differences in baseline MRI profiles between EPITHET and DEFUSE were largely eliminated by standardized processing of PWI and DWI images with RAPID software (Target Mismatch 49% vs 48%; Malignant 15% vs 8%; No Mismatch 36% vs 25%; p_NS for all comparisons) Reperfusion was strongly associated with a favorable clinical response in mismatch patients (figure). This relationship was not affected by the standardization procedures (pooled odds ratio of 8.8 based on original data and 6.6 based on standardized data). Conclusion: Standardization of image analyses procedures in acute stroke is important as non-standardized techniques introduce significant variability in DWI and PWI imaging characteristics. Despite methodological differences, the DEFUSE and EPITHET studies show a consistent and robust association between reperfusion and favorable clinical response in Target Mismatch patients regardless of standardization. These data support an RCT of iv tPA in the 3-6 hour time-window for Target Mismatch patients identified using RAPID.

Protective behavioral strategies and future drinking behaviors: Effect of drinking intentions.

Alcohol use is common among United States and Swedish high school students and is related to negative consequences. Whereas drinking intentions are associated with future drinking behaviors, the use of protective behavioral strategies (PBS) is associated with decreased alcohol-related harm among young adults. The interactive effect of PBS and drinking intentions in predicting alcohol outcomes has not been examined. Further, because most PBS studies have been conducted among U.S. college students, PBS research among other populations is needed. The aims of this study were to evaluate longitudinally (a) the relationships between drinking intentions, PBS and alcohol outcomes, and (b) the moderating roles of drinking intentions and country in these relationships among United States and Swedish high school drinkers. Data were collected at baseline, 6- and 12-month follow-ups on 901 Swedish and 288 U.S. high school drinkers. Drinking intentions were associated with more alcohol use and consequences, and use of certain PBS was related to fewer alcohol-related consequences over time. Additionally, the negative prospective relationship between use of PBS and alcohol use, but not alcohol-related consequences, was moderated by intentions, such that the relationship was stronger among participants endorsing high drinking intentions. Country did not moderate these relationships. These results provide initial support for the generalizability of PBS college research to United States and Swedish high school students and suggest that interventions targeting the use of PBS may be most effective among high school drinkers endorsing high drinking intentions. (PsycINFO Database Record