Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.

Increased endometrial placenta growth factor (PLGF) gene expression in women with successful implantation.

Objective: To analyze the vascularization of the endometrium via hysteroscopy and to assess its correlation with angiogenic factor gene expression and embryo implantation rate.Design: Cross-sectional study.Setting: Public university hospital.Patient(s): Patients undergoing hysteroscopy for supposed infertility.Intervention(s): Endometrial quality assessment according to Sakumoto-Masamoto, performed in the early secretory phase of the cycle. Collection of an endometrial tissue biopsy.Main Outcome Measure(s): RNA extraction, reverse transcription, and determination of gene expression of angiogenesis- and implantation-relevant factors using quantitative polymerase chain reaction. Retrieval of pregnancy information from the medical records.Result(s): Good quantity/quality RNA with infertility history was obtained from 63 participating women. Those with a "good" endometrium and subsequent pregnancy showed increased gene expression for placenta growth factor when compared with patients with a "bad" endometrium and who did not succeed with pregnancy to date. Nonpregnant women with a "good" endometrium presented an intermediate result. No significant differences were observed for several other genes tested, but trends in the same direction were observed.Conclusion(s): This study demonstrates for the first time that endometrial PLGF expression corresponds to the hysteroscopic appearance of the endometrium, and therefore has potential as a clinically relevant prognosticator for infertility treatment success. (Fertil Steril (R) 2011;96:663-8. (C)2011 by American Society for Reproductive Medicine.)

Amniotic fluid insulin-like growth factor binding-protein 3 concentration as early indicator of fetal growth restriction

Rapport de synthèse : Introduction : La croissance foetale infra-utérine dépend d'un grand nombre de facteurs maternels, placentaires et foetaux. Une inadéquation d'un ou plusieurs de ces facteurs peut induire un retard de croissance infra-utérin (RCIU) ou au contraire une macrosomie. Les principales causes de RCIU comprennent les infections maternelles, l'éclampsie, les cardiovasculopathies maternelles, la toxicomanie, les malformations foetales et les insuffisances placentaires. Les facteurs endocriniens constituent un petit pourcentage des causes de RCIU, mais méritent que l'on s'y intéresse de plus près. Les facteurs hormonaux les plus importants pour la croissance fatale sont l'insuline et les insuline-like growth factors (IGFs) et non l'hormone de croissance (GH) qui joue un rôle majeur dans la croissance postnatale. Notre attention s'est portée sur IGF-1 qui joue un rôle important dans la croissance intrautérine. Sa biodisponibilité dépend de plusieurs protéines plasmatiques, les IGF-binding proteins (IGFBP 1 à 9). IGFBP-3 est la principale de ces IGFBPs, autant d'un point de vue quantitatif que fonctionnel. Nous avons cherché à déterminer si les concentrations d'IGF-1 et d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre étaient prédictives de la croissance infra-utérine. Les gènes codant pour IGF-1 et IGFBP-3 contenant certaines séquences polymorphiques, nous avons également étudié leur influence sur la croissance foetale. L'analyse du liquide amniotique présente l'avantage de pouvoir être effectuée dès la 14ème semaine d'aménorrhée alors que la biométrie foetale échographique ne permet pas à ce stade de déceler des anomalies de la croissance infra-utérine. Méthode : Nous avons analysé des échantillons de liquide amniotique prélevés entre la 14ème et la 18ème semaine de grossesse chez 196 patientes. Les concentrations d'IGF-1 et d'IGFBP-3 ont été dosées par ELISA, les polymorphismes analysés par PCR. Ces résultats ont été ensuite analysés en fonction du poids de naissance des nouveaux-nés, répartis en trois groupes normal pour l'âge gestationnel (AGA), petit pour l'âge gestationnel (SGA) et grand pour l'âge gestationnel (LGA). Résultats : Les concentrations d'IGFBP3 dans le liquide amniotique sont significativement plus élevées (p = 0.030) dans le groupe SGA par rapport au groupe AGA, d'autant plus quand les taux sont ajustés en fonction de paramètres tels que l'âge gestationnel lors de l'amniocentèse (ANCOVA analysis : p = 0.009). La distribution du polymorphisme VNTR (variable number of tandem repeat) dans la région promotrice d'IGF-1 au sein du groupe SGA est significativement différente de celle du groupe AGA (p = 0.029). En effet, la fréquence de l'association allélique 19CA/20CA est diminuée dans le groupe SGA. Nous n'avons pas identifié de différence de distribution des séquences polymorphiques d'IGFBP-3 entre les différents groupes. Conclusion : Une concentration élevée d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre est associée à un risque plus élevé de retard de croissance alors que l'association allélique 19CA/20CA dans la région polymorphique IGF-1 VNTR est un facteur protecteur.

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.

Epidermal growth factor and bovine growth hormone stimulate differentiation and myelination of brain cell aggregates in culture.

Bovine growth hormone (bGH) and epidermal growth factor (EGF) increased the activity of ornithine decarboxylase (ODC) in brain cell aggregates cultured in a serum-free chemically defined medium. ODC is considered as a marker of cell growth and differentiation. The effect of bGH and EGF on myelination was investigated by measuring two myelin markers, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin basic protein (MBP). EGF treatment at days 2 and 5 caused a dose-dependent increase of both myelin markers at culture day 12. This increase could still be observed at culture day 19, indicating a prolonged action of EGF. The continual presence of bGH in the culture medium produced a large accumulation of MBP at day 19. This effect was dose-dependent and required the presence of triiodothyronine (T3). In contrast, the effect of bGH on CNP activity did not require the presence of T3. This is the first report showing a direct effect of bGH on CNS myelination in vitro and of EGF on both MBP accumulation and ODC activity.

Characterization of the angiotensin II receptor antagonist TCV-116 in healthy volunteers.

The purpose of this study was to assess the inhibitory effect of TCV-116, an orally active angiotensin II (Ang II) antagonist, on the pressor action of exogenous Ang II and to determine the compensatory rise in plasma renin activity and Ang II levels. Twenty-three male volunteers were treated for 8 days in a double-blind fashion with either placebo or TCV-116 (1, 2, or 4 mg PO daily) and challenged on the first, fourth, and eighth days with repeated bolus injections of Ang II. An additional 4 subjects received 8 mg PO daily in a single-blind fashion. The inhibitory effect on the systolic blood pressure response to Ang II was long lasting and clearly dose related. Six hours after 4 mg TCV-116, the systolic blood pressure response to a given dose of Ang II was reduced to 40 +/- 4% and 35 +/- 8% of baseline value on days 1 and 8, respectively. TCV-116 induced a dose-related increase in plasma renin activity and Ang II levels that was more pronounced on the eighth than on the first day of drug administration. Despite this compensatory mechanism, the relation between the time-integrated systolic blood pressure response to Ang II and the time-integrated CV-11974 levels, the active metabolite of TCV-116, was not different between days 1 and 8. In conclusion, TCV-116 appears to be a well-tolerated, orally active, potent, and long-lasting antagonist of Ang II in men.

Epidermal growth factor (EGF) stimulation of cultured brain cells. II. Increased production of extracellular soluble proteins.

The production of extracellular soluble proteins was studied in serum-free aggregating cell cultures of fetal rat telencephalon labeled on culture day 7 with a mixture of radioactive amino acid precursors. Cultures treated continuously with epidermal growth factor (EGF; 20 ng/ml) showed a generally increased protein secretion and a particularly enhanced production of a few distinct extracellular proteins. The time lag of this response after an initial dose of EGF (25 ng/ml) on day 7 was 48 h. The total macromolecular radioactivity that accumulated within 96 h of labeling in the media of EGF-treated cultures was 175% of untreated controls, whereas no difference was found in the proportions of intracellular amino acid incorporation. Cultures which received a single dose of EGF (25 ng/ml) on day 1 showed still a greatly increased protein secretion on day 7. Prevention of extracellular protein accumulation by reducing the initial cell number and increasing the rate of media changes did not affect the EGF-induced stimulation of the two glial enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase. The results suggest that both the increased production of extracellular proteins and the enhanced development of glial enzymatic activities reflect the stimulated phenotypic expression of EGF-sensitive brain cells.

Enhanced visuospatial memory following intracerebroventricular administration of nerve growth factor

The present work assessed the effects of intracerebroventricular injections of rh recombined human nerve growth factor (rh NGF) (5 micrograms/2.5 microl) at postnatal days 12 and 13 upon the development of spatial learning capacities. The treated rats were trained at the age of 22 days to escape onto an invisible platform at a fixed position in space in a Morris navigation task. For half of the subjects, the training position was also cued, a procedure aimed at facilitating escape and at reducing attention to the distant spatial cues. Later, at the age of 6 months, all the rats were trained in a radial-arm maze task. Treatment effects were found in both immature and adult rats. The injection of NGF improved the performance in the Morris navigation task in both training conditions. There was a significant reduction in the escape latency and an increased bias toward the training platform quadrant during probe trials. The most consistent effect was the precocious development of an adult-like spatial memory. In the radial-arm maze, the NGF-treated rats made significantly fewer reentries than vehicle rats and this effect was particularly marked in the treated female rats. Taken together, these experiments reveal that the development and the maintenance of an accurate spatial representation are tightly related to the development of brain structures facilitated by the action of NGF. Moreover, these experiments demonstrate that an acute pharmacological treatment that leads to a transient modification in the choline acetyltransferase activity can induce a behavioral change long after the treatment.

Controlled nerve growth factor release from multi-ply alginate/chitosan-based nerve conduits.

The delivery kinetics of growth factors has been suggested to play an important role in the regeneration of peripheral nerves following axotomy. In this context, we designed a nerve conduit (NC) with adjustable release kinetics of nerve growth factor (NGF). A multi-ply system was designed where NC consisting of a polyelectrolyte alginate/chitosan complex was coated with layers of poly(lactide-co-glycolide) (PLGA) to control the release of embedded NGF. Prior to assessing the in vitro NGF release from NC, various release test media, with and without stabilizers for NGF, were evaluated to ensure adequate quantification of NGF by ELISA. Citrate (pH 5.0) and acetate (pH 5.5) buffered saline solutions containing 0.05% Tween 20 yielded the most reliable results for ELISA active NGF. The in vitro release experiments revealed that the best results in terms of reproducibility and release control were achieved when the NGF was embedded between two PLGA layers and the ends of the NC tightly sealed by the PLGA coatings. The release kinetics could be efficiently adjusted by accommodating NGF at different radial locations within the NC. A sustained release of bioactive NGF in the low nanogram per day range was obtained for at least 15days. In conclusion, the developed multi-ply NGF loaded NC is considered a suitable candidate for future implantation studies to gain insight into the relationship between local growth factor availability and nerve regeneration.

Deposits of transforming growth factor-beta-induced protein in granular corneal dystrophy type II after LASIK.

PURPOSE: To analyze components of the deposits in the corneal flap interface of granular corneal dystrophy type II (GCD II) patients after laser in situ keratomileusis (LASIK). METHODS: Four corneal GCD II specimens displaying disease exacerbation after LASIK were analyzed. Three of these specimens included the recipient corneal button after penetrating keratoplasty or deep lamellar keratoplasty for advanced GCD II after LASIK. The fourth specimen, a similar case of GCD II after LASIK, included the amputated corneal flap. Specimens were processed for histopathologic and immunohistochemical analyses. RESULTS: Corneal stromal deposits in the LASIK flaps of all specimens were stained with 3 anti-transforming growth factor-beta-induced protein (TGFBIp) antibodies. The deposits displayed bright red color staining with Masson trichrome; however, negative staining was seen with Congo red, suggesting that hyaline is the main component localizing to the TGFBIp deposits rather than amyloid. CONCLUSIONS: Amorphous granular material deposited along the interface of the LASIK flap in GCD II corneas is composed mainly of hyaline deposits.

Insulin-like growth factor I (IGF I) stimulates DNA synthesis in fetal rat brain cell cultures.

Addition of insulin, IGF I or IGF II to serum-free cultures of fetal rat brain cells (gestation day 15/16) significantly stimulates DNA synthesis. The dose-response curves show that IGF I is more potent than insulin; half maximal stimulation of [3H]thymidine incorporation is obtained at about 0.4 nM IGF I and 14 nM insulin, respectively. Cultures initiated 2 days later (gestation day 17/18) showed a decreased responsiveness to both peptides. No additive effect was observed after combined addition of both peptides at near-maximal doses. Both peptides show a latency of action of about 12-18 h. In the presence of either IGF or insulin, neuronal as well as glial enzymes are increased, suggesting that neuronal and glial precursor cell division is influenced. IGF I and IGF II interact with a specific binding site for which insulin competes very weakly; however IGF I and IGF II bind with relatively high affinity to the insulin specific binding site. The present results support the hypothesis that both insulin and IGF stimulate mitotic activity by interacting with specific somatomedin receptors and suggest a physiological role of IGF in the developing brain.

Dose-dependent acute and sustained renal effects of the endothelin receptor antagonist avosentan in healthy subjects

Enjeu : L'incidence d'insuffisance rénale terminale augmente d'environ 5-6% par année dans nos régions. L'une des causes majeures d'insuffisance rénale est la néphropathie diabétique qui représente selon les pays entre 25 et 40% des néphropathies terminales. La progression de la néphropathie diabétique peut être ralentie de manière efficace par un bon contrôle du diabète et de l'hypertension artérielle et par le blocage du système rénine-angiotensine. Néanmoins, malgré l'application stricte de ces thérapies préventives, la néphropathie de bons nombres de patients diabétiques continue de progresser. Il est donc important de développer de nouvelles stratégies permettant de préserver la fonction rénale des patients diabétiques soit en améliorant le contrôle de la pression artérielle soit en diminuant la protéinurie. Contexte : Il existe un certain nombre d'évidences expérimentales que le blocage des récepteurs de l'endothéline pourrait avoir un effet positif sur le devenir de la néphropathie diabétique en diminuant de manière efficace la protéinurie même chez des animaux déjà traités efficacement avec un bloqueur du système rénine-angiotensine. Dans des études de phase 2 impliquant l'avosentan, un antagoniste des récepteurs de l'endothéline actuellement en cours de développement pour le traitement de la néphropathie diabétique, on a pu démontrer que cet antagoniste, prescrit à des doses oscillant entre 5 et 50 mg par jour per os, diminue la protéinurie d'environ 20-40% chez des patients déjà traités avec un IEC ou un antagoniste de l'angiotensine. Toutefois, une grande étude de phase III conduite avec ce médicament chez des patients diabétiques a du être interrompue précocement en raison de l'apparition d'oedèmes et d'une surcharge hydrosodée conduisant dans certains cas à une décompensation cardiaque aiguë. La rétention hydrosodée est un effet secondaire connu des antagonistes de l'endothéline déjà sur le marché. Toutefois, pour l'avosentan, on ne savait pas si des doses plus faibles du médicament avaient aussi un effet négative sur la balance hydrosodée. En outre, les mécanismes rénaux responsables de la rétention hydrosodée sont encore mal connus chez l'homme. C'est pourquoi, nous avons organisé et réalisé cette étude de pharmacologie clinique chez le volontaire sain posant 2 questions : 1) des doses faibles d'avosentan produisent-elles aussi une rétention hydrosodée chez l'homme ? et 2) quels sont les mécanismes rénaux pouvant expliquer la rétention hydrosodée ? Cette thèse est donc une étude clinique de phase I testant chez 23 volontaires sains les effets rénaux de différentes doses d'avosentan ou d'un placebo pour établir la courbe dose-réponse des effets rénaux de ce médicament. L'idée était également de définir quelle dose est sure et bien tolérée pour être utilisée dans une nouvelle étude de phase II. L'avosentan a été administré par voie orale une fois par jour pendant 8 jours à des doses de 0.5, 1.5, 5 et 50 mg. Les effets rénaux hémodynamiques et tubulaires ont été étudiés chez chaque sujet lors de la première administration (jour 1) et après une semaine de traitement (jour 8). Le médicament a induit une prise de poids dose-dépendante déjà présente à 5 mg et maximale à 50 mg (+ 0.8 kg au jour 8). Nous n'avons pas mesuré d'impact de l'avosentan sur l'hémodynamique rénale ni sur les électrolytes plasmatiques. En revanche, nous avons constaté une diminution dose-dépendante de la fraction d'excrétion de sodium (jusqu'à -8.7% avec avosentan 50 mg). Cette diminution était en rapport avec une augmentation dose-dépendante de la réabsorption proximale de sodium. Nous avons également constaté une baisse de la pression artérielle aux doses élevées et une hémodilution marquée par une baisse de l'hématocrite suggérant une rétention hydrique à la plus haute dose. Nos résultats suggèrent donc que l'avosentan induit une rétention sodée rénale dose-dépendante expliquée avant tout par une rétention du sodium au niveau du tubule proximal. Cet effet n'est pas observé à des doses plus basses que 5 mg chez le volontaire sain, suggérant que ce médicament devrait être évalué pour son activité réno-protectrice à des doses inférieures ou égales à 5 mg par jour. La raison pour laquelle les hautes doses produisent plus de rétention sodée est peut être liée à une perte de sélectivité pour les sous-types (A et B) de récepteurs à l'endothéline lorsque l'on administre des doses plus élevées que 5 mg. Perspectives : Les résultats de ce travail de thèse ont donc permis de caractériser les propriétés rénales d'un nouvel antagoniste des récepteurs de l'endothéline chez l'homme. Ces résultats ont aussi permis de guider le développement futur de ce médicament vers des doses plus faibles avec l'espoir de garder les effets bénéfiques sur la protéinurie tout en améliorant le profil de tolérance du médicament par l'utilisation de doses plus faibles. ANGLAIS The endothelin receptor antagonist avosentan may cause fluid overload at doses of 25 and 50 mg, but the actual mechanisms of this effect are unclear. We conducted a placebo-controlled study in 23 healthy subjects to assess the renal effects of avosentan and the dose dependency of these effects. Oral avosentan was administered once daily for 8 days at doses of 0.5, 1.5, 5, and 50 mg. The drug induced a dose-dependent median increase in body weight, most pronounced at 50 mg (0.8 kg on day 8). Avosentan did not affect renal hemodynamics or plasma electrolytes. A dose-dependent median reduction in the fractional renal excretion of sodium was found (up to 8.7% at avosentan 50 mg); this reduction was paralleled by a dose-related increase in proximal sodium reabsorption. It is suggested that avosentan dose-dependently induces sodium retention by the kidney, mainly through proximal tubular effects. The potential clinical benefits of avosentan should therefore be investigated at doses of ≤ 5 mg.

Macrophages promote epithelial repair through hepatocyte growth factor secretion.

Macrophages play a critical role in intestinal wound repair. However, the mechanisms of macrophage-assisted wound repair remain poorly understood. We aimed to characterize more clearly the repair activities of murine and human macrophages. Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of healthy donors (HD) or Crohn's disease (CD) patients or isolated from the intestinal mucosa of HD. In-vitro models were used to study the repair activities of macrophages. We found that murine and human macrophages were both able to promote epithelial repair in vitro. This function was mainly cell contact-independent and relied upon the production of soluble factors such as the hepatocyte growth factor (HGF). Indeed, HGF-silenced macrophages were less capable of promoting epithelial repair than control macrophages. Remarkably, macrophages from CD patients produced less HGF than their HD counterparts (HGF level: 84âeuro0/00±âeuro0/0027âeuro0/00pg/mg of protein and 45âeuro0/00±âeuro0/0034âeuro0/00pg/mg of protein, respectively, for HD and CD macrophages, Pâeuro0/00<âeuro0/000·009) and were deficient in promoting epithelial repair (repairing activity: 90·1âeuro0/00±âeuro0/004·6 and 75·8âeuro0/00±âeuro0/008·3, respectively, for HD and CD macrophages, Pâeuro0/00<âeuro0/000·0005). In conclusion, we provide evidence that macrophages act on wounded epithelial cells to promote epithelial repair through the secretion of HGF. The deficiency of CD macrophages to secrete HGF and to promote epithelial repair might contribute to the impaired intestinal mucosal healing in CD patients.

Pharmacologic profile of UR-7247, an orally active angiotensin II AT1 receptor antagonist, in healthy volunteers. p6.

This study was conducted to assess the pharmacologic properties of the new orally active angiotensin II subtype I (AT1) antagonist UR-7247, a product with a half-life >100 h in humans. The experiment was designed as an open-label, single-dose administration study with four parallel groups of four healthy men receiving increasing single oral doses (2.5, 5, and 10 mg) of UR-7247 or losartan, 100 mg. Angiotensin II receptor blockade was investigated < or =96 h after drug intake, with three independent methods [i.e., the inhibition of blood pressure (BP) response to exogenous Ang II, an in vitro Ang II-receptor assay (RRA), and the reactive increase in plasma angiotensin II. Plasma drug levels also were measured. The degree of blockade observed in vivo was statistically significant < or = 96 h with all UR-7247 doses for diastolic BP (p < 0.05) and < or =48 h for systolic BP. The maximal inhibition achieved with 10 mg UR-7247 was measured 6-24 h after drug intake and reached 54 +/- 17% and 48 +/- 20% for diastolic and systolic responses, respectively. Losartan, 100 mg, induced a greater short-term AT1-receptor blockade than 2.5- and 5.0-mg doses of UR-7247 (p < 0.001 for diastolic BP), but the UR-7247 effect was longer lasting. In vivo, no significant difference was observed between 10 mg UR-7247 and 100 mg losartan 4 h after drug intake, but in vitro, the blockade achieved with 100 mg losartan was higher than that seen with UR-7247. Finally, the results confirm that UR-7247 has a very long plasma elimination half-life, which may be due to a high but also tight binding to protein binding sites. In conclusion, UR-7247 is a long-lasting, well-tolerated AT1 receptor in healthy subjects.