LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis.

Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1-/- mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1-/- mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow-derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.

Beneficial effect of insulin-like growth factor-1 on hypoxemic renal dysfunction in the newborn rabbit.

Acute normocapnic hypoxemia can cause functional renal insufficiency by increasing renal vascular resistance (RVR), leading to renal hypoperfusion and decreased glomerular filtration rate (GFR). Insulin-like growth factor 1 (IGF-1) activity is low in fetuses and newborns and further decreases during hypoxia. IGF-1 administration to humans and adult animals induces pre- and postglomerular vasodilation, thereby increasing GFR and renal blood flow (RBF). A potential protective effect of IGF-1 on renal function was evaluated in newborn rabbits with hypoxemia-induced renal insufficiency. Renal function and hemodynamic parameters were assessed in 17 anesthetized and mechanically ventilated newborn rabbits. After hypoxemia stabilization, saline solution (time control) or IGF-1 (1 mg/kg) was given as an intravenous (i.v.) bolus, and renal function was determined for six 30-min periods. Normocapnic hypoxemia significantly increased RVR (+16%), leading to decreased GFR (-14%), RBF (-19%) and diuresis (-12%), with an increased filtration fraction (FF). Saline solution resulted in a worsening of parameters affected by hypoxemia. Contrarily, although mean blood pressure decreased slightly but significantly, IGF-1 prevented a further increase in RVR, with subsequent improvement of GFR, RBF and diuresis. FF indicated relative postglomerular vasodilation. Although hypoxemia-induced acute renal failure was not completely prevented, IGF-1 elicited efferent vasodilation, thereby precluding a further decline in renal function.

A high-fructose diet impairs basal and stress-mediated lipid metabolism in healthy male subjects.

The effects of a 7 d high-fructose diet (HFrD) or control diet on lipid metabolism were studied in a group of six healthy lean males. Plasma NEFA and beta-hydroxybutyrate concentrations, net lipid oxidation (indirect calorimetry) and exogenous lipid oxidation (13CO2 production) were monitored in basal conditions, after lipid loading (olive oil labelled with [13C]triolein) and during a standardised mental stress. Lactate clearance and the metabolic effects of an exogenous lactate infusion were also monitored. The HFrD lowered plasma concentrations of NEFA and beta-hydroxybutyrate as well as lipid oxidation in both basal and after lipid-loading conditions. In addition, the HFrD blunted the increase in plasma NEFA and exogenous lipid oxidation during mental stress. The HFrD also increased basal lactate concentrations by 31.8 %, and lactate production by 53.8 %, while lactate clearance remained unchanged. Lactate infusion lowered plasma NEFA with the control diet, and net lipid oxidation with both the HFrD and control diet. These results indicate that a 7 d HFrD markedly inhibits lipolysis and lipid oxidation. The HFrD also increases lactate production, and the ensuing increased lactate utilisation may contribute to suppress lipid oxidation.

The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

The number of Toll-like receptor 9-agonist motifs in the adenovirus genome correlates with induction of dendritic cell maturation by adenovirus immune complexes.

Adenovirus serotype 5 (Ad5) vectors and specific neutralizing antibodies (NAbs) generate immune complexes (ICs) which are potent inducers of dendritic cell (DC) maturation. Here we show that ICs generated with rare Ad vector serotypes, such as Ad26 and Ad35, which are lead candidates in HIV vaccine development, are poor inducers of DC maturation and that their potency in inducing DC maturation strongly correlated with the number of Toll-like receptor 9 (TLR9)-agonist motifs present in the Ad vector's genome. In addition, we showed that antihexon but not antifiber antibodies are responsible for the induction of Ad IC-mediated DC maturation.

Changes in sleeping and basal energy expenditure and substrate oxidation induced by short term thyroxin administration in man.

The present study was designed to explore the thermogenic effect of thyroid hormone administration and the resulting changes in nitrogen homeostasis. Normal male volunteers (n = 7) received thyroxin during 6 weeks. The first 3-week period served to suppress endogenous thyroid secretion (180 micrograms T4/day). This dose was doubled for the next 3 weeks. Sleeping energy expenditure (respiratory chamber) and BMR (hood) were measured by indirect calorimetry, under standardized conditions. Sleeping heart rate was continuously recorded and urine was collected during this 12-hour period to assess nitrogen excretion. The changes in energy expenditure, heart rate and nitrogen balance were then related to the excess thyroxin administered. After 3 weeks of treatment, serum TSH level fell to 0.15 mU/L, indicating an almost complete inhibition of the pituitary-thyroid axis. During this phase of treatment there was an increase in sleeping EE and sleeping heart rate, which increased further by doubling the T4 dose (delta EE: +8.5 +/- 2.3%, delta heart rate +16.1 +/- 2.2%). The T4 dose, which is currently used as a substitutive dose, lead to a borderline hyperthyroid state, with an increase in EE and heart rate. Exogenous T4 administration provoked a significant increase in urinary nitrogen excretion averaging 40%. It is concluded that T4 provokes an important stimulation of EE, which is mostly mediated by an excess protein oxidation.

Evidence for segregated and integrative connectivity patterns in the human Basal Ganglia.

Detailed knowledge of the anatomy and connectivity pattern of cortico-basal ganglia circuits is essential to an understanding of abnormal cortical function and pathophysiology associated with a wide range of neurological and neuropsychiatric diseases. We aim to study the spatial extent and topography of human basal ganglia connectivity in vivo. Additionally, we explore at an anatomical level the hypothesis of coexistent segregated and integrative cortico-basal ganglia loops. We use probabilistic tractography on magnetic resonance diffusion weighted imaging data to segment basal ganglia and thalamus in 30 healthy subjects based on their cortical and subcortical projections. We introduce a novel method to define voxel-based connectivity profiles that allow representation of projections from a source to more than one target region. Using this method, we localize specific relay nuclei within predefined functional circuits. We find strong correlation between tractography-based basal ganglia parcellation and anatomical data from previously reported invasive tracing studies in nonhuman primates. Additionally, we show in vivo the anatomical basis of segregated loops and the extent of their overlap in prefrontal, premotor, and motor networks. Our findings in healthy humans support the notion that probabilistic diffusion tractography can be used to parcellate subcortical gray matter structures on the basis of their connectivity patterns. The coexistence of clearly segregated and also overlapping connections from cortical sites to basal ganglia subregions is a neuroanatomical correlate of both parallel and integrative networks within them. We believe that this method can be used to examine pathophysiological concepts in a number of basal ganglia-related disorders.

The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.

Proteins secreted from adipose tissue are increasingly recognized to play an important role in the regulation of glucose metabolism. However, much less is known about their effect on lipid metabolism. The fasting-induced adipose factor (FIAF/angiopoietin-like protein 4/peroxisome proliferator-activated receptor gamma angiopoietin-related protein) was previously identified as a target of hypolipidemic fibrate drugs and insulin-sensitizing thiazolidinediones. Using transgenic mice that mildly overexpress FIAF in peripheral tissues we show that FIAF is an extremely powerful regulator of lipid metabolism and adiposity. FIAF overexpression caused a 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat. In addition, FIAF overexpression increased plasma levels of triglycerides, free fatty acids, glycerol, total cholesterol, and high density lipoprotein (HDL)-cholesterol. Functional tests indicated that FIAF overexpression severely impaired plasma triglyceride clearance but had no effect on very low density lipoprotein production. The effects of FIAF overexpression were amplified by a high fat diet, resulting in markedly elevated plasma and liver triglycerides, plasma free fatty acids, and plasma glycerol levels, and impaired glucose tolerance in FIAF transgenic mice fed a high fat diet. Remarkably, in mice the full-length form of FIAF was physically associated with HDL, whereas truncated FIAF was associated with low density lipoprotein. In human both full-length and truncated FIAF were associated with HDL. The composite data suggest that via physical association with plasma lipoproteins, FIAF acts as a powerful signal from fat and other tissues to prevent fat storage and stimulate fat mobilization. Our data indicate that disturbances in FIAF signaling might be involved in dyslipidemia.

Polymorphisms in toll-like receptor 4 and toll-like receptor 9 influence viral load in a seroincident cohort of HIV-1-infected individuals.

OBJECTIVES: Toll-like receptors (TLRs) are innate immune sensors that are integral to resisting chronic and opportunistic infections. Mounting evidence implicates TLR polymorphisms in susceptibilities to various infectious diseases, including HIV-1. We investigated the impact of TLR single nucleotide polymorphisms (SNPs) on clinical outcome in a seroincident cohort of HIV-1-infected volunteers. DESIGN: We analyzed TLR SNPs in 201 antiretroviral treatment-naive HIV-1-infected volunteers from a longitudinal seroincident cohort with regular follow-up intervals (median follow-up 4.2 years, interquartile range 4.4). Participants were stratified into two groups according to either disease progression, defined as peripheral blood CD4(+) T-cell decline over time, or peak and setpoint viral load. METHODS: Haplotype tagging SNPs from TLR2, TLR3, TLR4, and TLR9 were detected by mass array genotyping, and CD4(+) T-cell counts and viral load measurements were determined prior to antiretroviral therapy initiation. The association of TLR haplotypes with viral load and rapid progression was assessed by multivariate regression models using age and sex as covariates. RESULTS: Two TLR4 SNPs in strong linkage disequilibrium [1063 A/G (D299G) and 1363 C/T (T399I)] were more frequent among individuals with high peak viral load compared with low/moderate peak viral load (odds ratio 6.65, 95% confidence interval 2.19-20.46, P < 0.001; adjusted P = 0.002 for 1063 A/G). In addition, a TLR9 SNP previously associated with slow progression was found less frequently among individuals with high viral setpoint compared with low/moderate setpoint (odds ratio 0.29, 95% confidence interval 0.13-0.65, P = 0.003, adjusted P = 0.04). CONCLUSION: This study suggests a potentially new role for TLR4 polymorphisms in HIV-1 peak viral load and confirms a role for TLR9 polymorphisms in disease progression.

Imaging exocytosis and recycling of synaptic-like microvesicles in astrocytes.

Optical imaging techniques are well suited for following the dynamics of physiological processes in living cells. Total internal reflection fluorescence (TIRF) microscopy based on evanescent wave illumination (EWi) allows spectacular, real-time visualization of individual vesicle movements, fusions, and retrievals at the cell surface (i.e., within 100 nm of the plasma membrane). TIRF microscopy is an ideal approach for studying the properties of exocytosis and recycling in cultured astrocytes, particularly because these cells have a rather flat surface and contain secretory vesicles with sparse distribution. Among all populations of secretory vesicles, we focus here on synaptic-like microvesicles (SLMVs). We illustrate how TIRF microscopy using EWi is useful to study exocytosis and recycling of SLMVs at the single-vesicle level and, when combined with epifluorescence illumination (EPIi), can provide detailed information on the kinetics of exocytosis, endocytosis, and re-acidification at the whole-cell level.

Production endogène de Nortestostérone chez l'homme et influence de l'activité physique et du régime alimentaire

RESUME Depuis les années 1980, les stéroïdes androgéniques anabolisants (SAA) sont restés les produits dopants les plus utilisés par les sportifs. Les propriétés principales attribuées à ces substances sont une augmentation de la masse et de la force musculaire ainsi qu'une agressivité supérieure pouvant s'avérer bénéfique lors des entraînements ou des compétitions. En plus de cette "tradition" liée à la consommation des SAA, une autre problématique est apparue dans le monde antidopage suite à la fulgurante expansion de l'utilisation des compléments alimentaires par les athlètes professionnels et amateurs. Dès la fin des années 1990, une recrudescence de cas positifs de dopage aux SAA a été attribuée à la contamination des compléments alimentaires par des composés anabolisants tels que la testostérone ou la nandrolone ou par des prohormones se situant en amont dans le métabolisme de certains SAA et conduisant à la présence, dans les urines, de traces de substances interdites par l'Agence Mondiale Antidopage (AMA). Afin de mettre en garde les autorités antidopage ainsi que les athlètes quant aux problèmes liés aux compléments alimentaires, le Laboratoire Suisse d'Analyse du Dopage (LAD) a décidé d'étudier de manière plus précise la composition d'une centaine de produits accessibles en Suisse par l'intermédiaire d'internet. Cette étude a permis de mettre en évidence un taux de non conformité des produits avoisinant les 20%, avec une contamination plus importante des produits contenant des hormones ou des prohormones. La consommation de doses journalières recommandées des produits contaminés a mené à la détection dans les urines de la présence de substances interdites par l'AMA. Ces résultats confirment ainsi que l'usage de compléments alimentaires peut s'avérer dangereuse dans le cadre de contrôles antidopage et que les effets sur l'état physique et mental des athlètes peuvent dépasser les effets désirés et être dramatiques pour la poursuite d'une carrière sportive. D'autre part, cela démontre que l'alimentation peut mener à la présence urinaire de substances proscrites telles que les métabolites de la nandrolone, la 19-norandrostéreone (19-NA) et la 19-norétiocholanolone (19-NE). Afin de démontrer un effet potentiel de l'exercice physique sur l'excrétion urinaire des métabolites de la nandrolone, une première étude clinique a été réalisée avec 34 volontaires. Deux doses orales de nandrolone marquée avec deux atomes de C13 ont été administrées aux sujets. Les urines ont été récoltées durant les 5 jours suivant les prises orales (études d'excrétion) ainsi qu'avant et après les 8 séances d'entraînements du protocole. Les analyses des études d'excrétion ont permis d'établir une variabilité intra- et inter-individuelle du métabolisme et de la pharmacocinétique de la 19-NA et de la 19-NE. En dépit de la rapide élimination urinaire des métabolites de la nandrolone C13, les analyses des échantillons prélevés avant et après les différents efforts n'ont pas révélé une influence nette de l'exercice physique sur les concentrations urinaires de la 19-NA et 19-NE. Une seconde étude clinique a été effectuée, avec la participation de 30 volontaires. Il s'agissait de déterminer si la consommation de multiples doses orales d'un décanoate de testostérone, de 19-norandrostenedione (un précurseur de la nandrolone) ou de placebo durant un mois, pouvait avoir des effets bénéfiques sur la récupération et la performance physique. En parallèle, les sujets étaient soumis à un entrainement d'endurance intense et individualisé. Divers paramètres physiologiques ont été étudiés dans le sérum et les urines afin de mettre en évidence une meilleure récupération de l'organisme. Aucun de ses paramètres n'a permis de conclure que la consommation orale de SAA est favorable pour optimaliser les capacités de récupération des athlètes. De plus, les performances physiques ont été évaluées avant et après l'entraînement et le traitement. Aucune différence significative n'a été démontrée entre les trois groupes de volontaires. L'état psychologique des volontaires a été évalué à l'aide de questionnaires (short Profile of Mood State, sPOMS) remplis à trois reprises au cours du protocole. De manière générale, l'évolution observée est une augmentation de la fatigue avec une diminution de la vigueur. Des analyses statistiques ont révélé que des prises orales de testostérone, et dans une moindre mesure de 19-norandrostenedione, ont une légère influence sur cette évolution générale en diminuant les effets de l'entrainement sur le profil psychologique. Les urines récoltées durant le protocole ont été analysées par GC/C/IRMS et GCMS afin de détecter les variations des concentrations des hormones liées au métabolisme de la testostérone. Les résultats ont démontré une variabilité interindividuelle du métabolisme de la testostérone qui implique que les critères de positivité imposés par l'AMA ne sont pas forcément valables pour tous les individus. La détection de la 19-NA et de la 19-NE, issus du métabolisme in vivo de la 19norandrostenedione, a confirmé les résultats obtenus sur la pharmacocinétique et le métabolisme de la nandrolone C13 obtenus lors de la première étude clinique. Ce travail a permis de clarifier certains points en lien avec l'abus de la nandrolone dans le sport et notamment par rapport à la consommation de compléments alimentaires. Les deux études cliniques n'ont pas véritablement apporté les réponses souhaitées aux hypothèses de départ. Cependant certains aspects intéressants en relation avec le métabolisme des SAA ont été découverts et pourront peut-être permettre à la lutte antidopage d'évoluer vers une meilleure efficacité. SUMMARY Since 1980's, anabolic androgenic steroids (AAS) are still the most used doping agents in sports. The main properties attributed to these substances are an increase of muscle mass and strength and also a higher aggressiveness that could be beneficial during trainings and competitions. In addition to this "tradition" linked to the AAS intake, another problematics has raised in the antidoping field. Indeed, nutritional supplements have been more and more used by professional and amateur athletes. Since the end of the 1990's, an outburst of positive doping cases with AAS has been attributed to nutritional supplements contaminations with anabolic compounds like testosterone or nandrolone or with prohormones located above in the metabolism of some AAS and prompting urinary traces of forbidden compounds by the World Antidoping Agency (WADA). In order to inform the antidoping authorities and the athletes about the problems linked to the nutritional supplements, the Swiss Laboratory for Doping Analyses (LAD) decided to investigate more precisely the composition of about hundred products accessible in Switzerland through different web sites. This study showed that about 20% of the products were not conformed to the composition announced by the manufacturers. The oral intake of daily recommended doses of the contaminated products revealed the presence in urines of forbidden substances by the WADA. Hence, these results confirm that the use of nutritional supplements can lead to adverse analytical findings in antidoping controls and that the effects on athletes' physical and mental state could be different from the ones desired and could be dramatic for the continuation of an athlete's career. Moreover, this demonstrates that the diet can lead to the presence in urines of proscribed substances like nandrolone metabolites, i.e. 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). To put forward a potential effect of physical exercise on urinary nandrolone metabolites excretion rate, a first clinical study was done with 34 volunteers. Two oral doses of nandrolone labelled with two C13 atoms were administered to the subjects. The urines were collected during the 5 days following the treatment (excretion studies) and before and after the 8 exercise sessions of the protocol. The analyses of excretion studies revealed an intra- and inter-individual variability of the metabolism and the pharmacokinetics of 19-NA and 19-NE. In spite of the rapid urinary elimination of the nandrolone C13 metabolites, the analyses of the urine samples gathered before and after efforts did not show a clear influence of physical exercise on the urinary 19-NA and 19-NE concentrations. A second clinical study was done with the participation of 30 volunteers. The main aim was to determine if multiple oral doses of testosterone undecanoate, 19-norandrostenedione (a nandrolone precursor) or placebo during one month, could have beneficial effects on recovery and physical performance. Meanwhile, the individuals had to follow an intense and personalized endurance training program. Several physiological parameters were investigated in serum and urines in order to demonstrate a better organism's recovery. None of these parameters lead to the conclusion that oral intake of AAS is useful to optimise the recovery capacities of athletes. In addition, physical performances were evaluated before and after the training and treatment month. No significant difference was shown between the three volunteers groups. The psychological state of the volunteers was assessed through questionnaires (short Profile of Mood State, sP4MS) filled three times during the protocol. The global evolution is an increase of fatigue with an decrease of vigour. Statistical analyses revealed that the oral intake of testosterone, and to a lesser extent of 19= norandrostenedione, have a small influence on this general evolution in decreasing the effect of training on the psychological profile. The urines collected during the protocol were analysed by GC/C/IRMS and GCMS to detect concentrations variations of hormones related to the testosterone metabolism. The results revealed an interindividual variability of testosterone metabolism which implies that the guidance concerning endogenous steroids prescribed by the WADA are not uniformly valid for all individuals. Detection of 19-NA and 19-NE, coming from the in vivo metabolism of 19norandrostenedione, confirmed the results previously obtained on the pharamcokinetics and metabolism of the nandrolone C13 in the first clinical study. This work allowed to clarify some aspects linked to nandrolone abuse in sports and noteworthy related to nutritional supplements intake. The two clinical studies did not really bring plain answers to the basal hypotheses but some interesting aspects in relation with AAS metabolism were put forth and would perhaps allow an evolution of a more effective fight against doping.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin.

BACKGROUND: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth. METHODOLOGY AND PRINCIPAL FINDINGS: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia. SIGNIFICANCE: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.