Neuronal nitric oxide synthase does not contribute to the modulation of pulmonary vascular tone in fetal lambs with congenital diaphragmatic hernia (nNOS in CDH lambs).

AIM: The aim of this study was to determine the presence of the neuronal nitric oxide synthase (nNOS) in near full-term lambs with congenital diaphragmatic hernia (CDH) and its role in the modulation of pulmonary vascular basal tone. METHODS: We surgically created diaphragmatic hernia on the 85th day of gestation. On the 135th, catheters were used to measure pulmonary pressure and blood flow. We tested the effects of 7-nitroindazole (7-NINA), a specific nNOS antagonist and of N-nitro-L-arginine (L-NNA), a nonspecific nitric oxide synthase antagonist. In vitro, we tested the effects of the same drugs on isolated pulmonary vessels. The presence of nNOS protein in the lungs was detected by Western blot analysis. RESULTS: Neither 7-NINA nor L-NNA modified pulmonary vascular basal tone in vivo. After L-NNA injection, acetylcholine (ACh) did not decrease significantly pulmonary vascular resistance (PVR). In vitro, L-NNA increased the cholinergic contractile-response elicited by electric field stimulation (EFS) of vascular rings from lambs with diaphragmatic hernia. CONCLUSION: We conclude that nNOS protein is present in the lungs and pulmonary artery of near full-term lamb fetuses with diaphragmatic hernia, but that it does not contribute to the reduction of pulmonary vascular tone at birth

Insulin modulation of luteinizing hormone secretion in normal female volunteers and lean polycystic ovary syndrome patients.

BACKGROUND/AIMS: Endocrine features of polycystic ovary syndrome (PCOS) include altered ovarian steroidogenesis, hyperinsulinemia and abnormal luteinizing hormone (LH) secretion. This study was undertaken to further evaluate the role of insulin to modulate LH secretion in lean PCOS patients with normal insulin sensitivity and normal volunteers. METHODS: The study was performed in five nonobese patients diagnosed with PCOS on the basis of amenorrhea and a polycystic morphology at ovarian ultrasound, and 5 normal controls in early to mid-follicular phase and matched for weight and age. All subjects were phenotyped, and then admitted for 12 h of frequent (q 10') blood sampling on two separate occasions, once for a baseline study and the other time for a hyperinsulinemic and euglycemic clamp study. LH was measured in samples obtained throughout each admission in order to perform LH pulse analysis. RESULTS: Baseline LH secretion in PCOS subjects was significantly different from controls: they had higher LH levels, higher LH/FSH ratios as well as a faster LH pulse frequency than normal women. Insulin administration did not affect the pattern of LH secretion of PCOS patients, whereas it significantly increased the LH pulse frequency while decreasing the LH interpulse intervals in the controls. CONCLUSIONS: These data confirm that an abnormal pattern of LH secretion characteristic of PCOS can be observed in lean patients, and appears independent of peripheral insulin levels. Furthermore, our results in lean controls provide the first direct evidence that peripheral insulin can modulate the activity of hypothalamic gonadotropin-releasing hormone (GnRH) neurons in the human.

Ectodysplasin A (EDA)-EDA receptor signalling and its pharmacological modulation

L'ectodysplasine Al (EDA1 ou EDA), un ligand de la famille du TNF, et son récepteur EDAR favorisent le développement des poils, des dents et de plusieurs types de glandes. Chez l'humain, une déficience en EDA cause une dysplasie ectodermique liée à l'X, caractérisée par la genèse défectueuse des phanères. Les souris Tabby, déficientes en Eda, présentent des symptômes similaires. Nous démontrons que les souris Tabby sont en moyenne 7% plus légères que les contrôles au moment du sevrage. Ce phénotype ne dépend pas du génotype des petits, mais exclusivement de celui de la mère, suggérant que l'absence d'EDA perturbe la fonction mammaire. La glande mammaire se développe en plusieurs étapes, principalement à la puberté et pendant la grossesse. Nous avons généré des anticorps pour activer ou inhiber la signalisation d'EDAR. Les anticorps agonistes corrigent le développement de souris ou de chiens déficients en EDA, alors que les antagonistes provoquent une dysplasie ectodermique chez les souris saines. L'exposition répétée de souris Tabby aux anticorps agonistes après le sevrage accroît la taille et la fonction des glandes sébacées, démonstration pharmacologique qu'EDA contrôle l'homéostasie de la glande sébacée adulte. Ces outils seront utiles pour étudier la fonction d'EDA aux diverses étapes du développement de la glande mammaire. Fc-EDAl, un stimulateur d'EDAR, est en phase d'évaluation clinique. Nous avons montré que les structures dépendantes d'EDA qui se forment à différentes étapes du développement répondent à l'action du Fc-EDAl dans des fenêtres temporelles étroites ou larges. De plus, certaines structures peuvent être induites plusieurs jours après le début naturel de leur formation. Alors que la plupart des structures se forment suite à un seul jour d'activation d'EDAR, d'autre demandent un temps de stimulation plus long. La formation des dents est régulée par des signaux activateurs et inhibiteurs. Une forte stimulation d'EDAR spécifiquement appliquée aux deux premières molaires induit des signaux négatifs qui avortent la formation de la troisième molaire, alors qu'une forte stimulation donnée à la troisième molaire la rend hypertrophique tout en induisant parfois une quatrième molaire jamais observée chez les souris de type sauvage ou Tabby. EDA est donc un activateur important de la formation dentaire. Pris dans leur ensemble, ces résultats ont des implications pour la thérapie des dysplasies ectodermiques. - The TNF family ligand Ectodysplasin Al (EDA1 or EDA) and its receptor ED AR regulate embryonic development of hair, teeth and several types of glands. In humans, EDA mutations cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by defective development of skin appendages. £da-deficient (Tabby) mice suffer from similar defects. We observed that Tabby pups at weaning were on average 7% smaller than WT controls, a phenotype that was curiously not linked to the genotype of pups, but to that of mothers, suggesting decreased mammary gland function in the absence of EDA. Mammary glands develop in several steps, most of which are post-natal. We generated monoclonal antibodies to block or activate EDAR signaling. Agonist antibodies rescued developmental defects when administered timely in £cfo-deficient mice and dogs, whereas blocking antibodies induced ectodermal dysplasia in WT mice. Agonist antibodies administered after weaning in £da-deficient mice for several months markedly increased both size and function of sebaceous glands, providing the first demonstration that pharmacological activation of the EDAR pathway in adults can correct important aspects of the dry skin phenotype. This also highlights a role for EDA1 in the homeostasis of adult sebaceous glands. These tools will be useful to study the function of EDA 1 at different stages of mammary gland development. Another EDAR agonist, Fc-EDAl, is currently evaluated in clinical trials. We found that EDA 1-dependent structures forming at different time points during development can respond to Fc-EDAl during time response windows that are narrow or wide. Also, some structures can be triggered up to several days after their normal time of induction. While most structures could be rescued by a single day of EDAR signaling, others required longer exposure times to form. Tooth formation is regulated by activating and inhibitory signals that impact one on the other. When strong EDAR signals were specifically given to the first two molars, overwhelming inhibitory signals completely inhibited formation of the third molar. In contrast, strong signals specifically given to the third molar induced hypertrophy of the later with occasional appearance of a fourth molar never observed in WT or £da-deficient mice. This clearly positions EDA as an important activating signal in tooth formation. Taken together, these results have implications for the therapy of ectodermal dysplasias.

Automatic Prediction of Facial Trait Judgments: Appearance vs. Structural Models

Evaluating other individuals with respect to personality characteristics plays a crucial role in human relations and it is the focus of attention for research in diverse fields such as psychology and interactive computer systems. In psychology, face perception has been recognized as a key component of this evaluation system. Multiple studies suggest that observers use face information to infer personality characteristics. Interactive computer systems are trying to take advantage of these findings and apply them to increase the natural aspect of interaction and to improve the performance of interactive computer systems. Here, we experimentally test whether the automatic prediction of facial trait judgments (e.g. dominance) can be made by using the full appearance information of the face and whether a reduced representation of its structure is sufficient. We evaluate two separate approaches: a holistic representation model using the facial appearance information and a structural model constructed from the relations among facial salient points. State of the art machine learning methods are applied to a) derive a facial trait judgment model from training data and b) predict a facial trait value for any face. Furthermore, we address the issue of whether there are specific structural relations among facial points that predict perception of facial traits. Experimental results over a set of labeled data (9 different trait evaluations) and classification rules (4 rules) suggest that a) prediction of perception of facial traits is learnable by both holistic and structural approaches; b) the most reliable prediction of facial trait judgments is obtained by certain type of holistic descriptions of the face appearance; and c) for some traits such as attractiveness and extroversion, there are relationships between specific structural features and social perceptions.

Modulation of the c-Jun N-terminal kinase activity in the embryonic heart in response to anoxia-reoxygenation: involvement of the Ca2+ and mitoKATP channels.

Whether the response of the fetal heart to ischemia-reperfusion is associated with activation of the c-Jun N-terminal kinase (JNK) pathway is not known. In contrast, involvement of the sarcolemmal L-type Ca2+ channel (LCC) and the mitochondrial KATP (mitoKATP) channel has been established. This work aimed at investigating the profile of JNK activity during anoxia-reoxygenation and its modulation by LCC and mitoK(ATP) channel. Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30 min) and reoxygenation (60 min). Using the kinase assay method, the profile of JNK activity in the ventricle was determined every 10 min throughout anoxia-reoxygenation. Effects on JNK activity of the LCC blocker verapamil (10 nM), the mitoK(ATP) channel opener diazoxide (50 microM) and the blocker 5-hydroxydecanoate (5-HD, 500 microM), the mitochondrial Ca2+ uniporter (MCU) inhibitor Ru360 (10 microM), and the antioxidant N-(2-mercaptopropionyl) glycine (MPG, 1 mM) were determined. In untreated hearts, JNK activity was increased by 40% during anoxia and peaked fivefold relative to basal level after 30-40 min reoxygenation. This peak value was reduced by half by diazoxide and was tripled by 5-HD. Furthermore, the 5-HD-mediated stimulation of JNK activity during reoxygenation was abolished by diazoxide, verapamil or Ru360. MPG had no effect on JNK activity, whatever the conditions. None of the tested pharmacological agents altered JNK activity under basal normoxic conditions. Thus, in the embryonic heart, JNK activity exhibits a characteristic pattern during anoxia and reoxygenation and the respective open-state of LCC, MCU and mitoKATP channel can be a major determinant of JNK activity in a ROS-independent manner.

Effect of olive oil on early and late events of colon carcinogenesis in rats: modulation of arachidonic acid metabolism and local prostaglandin E2 synthesis.

BACKGROUND Animal model studies have shown that the colon tumour promoting effect of dietary fat depends not only on the amount but on its fatty acid composition. With respect to this, the effect of n9 fatty acids, present in olive oil, on colon carcinogenesis has been scarcely investigated. AIMS To assess the effect of an n9 fat diet on precancer events, carcinoma development, and changes in mucosal fatty acid composition and prostaglandin (PG)E2 formation in male Sprague-Dawley rats with azoxymethane induced colon cancer. METHODS Rats were divided into three groups to receive isocaloric diets (5% of the energy as fat) rich in n9, n3, or n6 fat, and were administered azoxymethane subcutaneously once a week for 11 weeks at a dose rate of 7.4 mg/kg body weight. Vehicle treated groups received an equal volume of normal saline. Groups of animals were colectomised at weeks 12 and 19 after the first dose of azoxymethane or saline. Mucosal fatty acids were assessed at 12 and 19 weeks. Aberrant crypt foci and the in vivo intracolonic release of PGE2 were assessed at week 12, and tumour formation at week 19. RESULTS Rats on the n6 diet were found to have colonic aberrant crypt foci and adenocarcinomas more often than those consuming either the n9 or n3 diet. There were no differences between the rats on the n9 and n3 diets. On the other hand, administration of both n9 and n3 diets was associated with a decrease in mucosal arachidonate concentrations as compared with the n6 diet. Carcinogen treatment induced an appreciable increase in PGE2 formation in rats fed the n6 diet, but not in those fed the n3 and n9 diets. CONCLUSIONS Dietary olive oil prevented the development of aberrant crypt foci and colon carcinomas in rats, suggesting that olive oil may have chemopreventive activity against colon carcinogenesis. These effects may be partly due to modulation of arachidonic acid metabolism and local PGE2synthesis.

Different modulation of adipocytokine expression in four main white adipose tissue sites in the rat: mesenteric, perigonadal, retroperitoneal and subcutaneous

White adipose tissue (WAT) is a disperse organ acting as energy storage depot and endocrine/paracrine controlling factor in the management of energy availability and inflammation. WAT sites response under energy-related stress is not uniform. In the present study we have analyzed how different WAT sites respond to limited food restriction as a way to better understand the role of WAT in the pathogenesis of the metabolic syndrome.

Dynamic modulation of CCR7 expression and function on naive T lymphocytes in vivo.

The chemokine receptor CCR7 is critical for the recirculation of naive T cells. It is required for T cell entry into secondary lymphoid organs (SLO) and for T cell motility and retention within these organs. How CCR7 activity is regulated during these processes in vivo is poorly understood. Here we show strong modulation of CCR7 surface expression and occupancy by the two CCR7 ligands, both in vitro and in vivo. In contrast to blood, T cells in SLO had most surface CCR7 occupied with CCL19, presumably leading to continuous signaling and cell motility. Both ligands triggered CCR7 internalization in vivo as shown in Ccl19(-/-) and plt/plt mice. Importantly, CCR7 occupancy and down-regulation led to strongly impaired chemotactic responses, an effect reversible by CCR7 resensitization. Therefore, during their recirculation, T cells cycle between states of free CCR7 with high ligand sensitivity in blood and occupied CCR7 associated with continual signaling and reduced ligand sensitivity within SLO. We propose that these two states of CCR7 are important to allow the various functions CCR7 plays in T cell recirculation.

CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes.

Thymocytes and class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes express predominantly heterodimeric alpha/beta CD8. By interacting with non-polymorphic regions of MHC class I molecules CD8 can mediate adhesion or by binding the same MHC molecules that interact with the T-cell antigen receptor (TCR) function as coreceptor in TCR-ligand binding and T-cell activation. Using TCR photoaffinity labelling with a soluble, monomeric photoreactive H-2Kd-peptide derivative complex, we report here that the avidity of TCR-ligand interactions on cloned cytotoxic T cells is very greatly strengthened by CD8. This is primarily explained by coordinate binding of ligand molecules by CD8 and TCR, because substitution of Asp 227 of Kd with Lys severely impaired the TCR-ligand binding on CD8+, but not CD8- cells. Kinetic studies on CD8+ and CD8- cells further showed that CD8 imposes distinct dynamics and a remarkable temperature dependence on TCR-ligand interactions. We propose that the ability of CD8 to act as coreceptor can be modulated by CD8-TCR interactions.

CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR Vα and Jα segment selection as well as CDR3α composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

Quantifying Accuracy Improvement in Sets of Pooled Judgments: Does Dialectical Bootstrapping Work?

Galton (1907) first demonstrated the "wisdom of crowds" phenomenon by averaging independent estimates of unknown quantities given by many individuals. Herzog and Hertwig (2009; hereafter H&H in Psychological Science) showed that individuals' own estimates can be improved by asking them to make two estimates at separate times and averaging them. H&H claimed to observe far greater improvement in accuracy when participants received "dialectical" instructions to consider why their first estimate might be wrong before making their second estimates than when they received standard instructions. We reanalyzed H&H's data using measures of accuracy that are unrelated to the frequency of identical first and second responses and found that participants in both conditions improved their accuracy to an equal degree.

Receptor activator for NF-κB ligand in acute myeloid leukemia: expression, function, and modulation of NK cell immunosurveillance.

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.