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In the era of quality management in clinical laboratories, method validation can be a challenge without appropriate guidelines, such as in the field of pharmacogenetics. The present work describes a method validation for DNA extraction and CYP3A5*3 genotyping, which would meet ISO15189:2012 requirements.DNA extraction was performed using a QIAamp DSP DNA Blood kit, DNA purity and concentration were determined using a Nanodrop, and the genotyping assay was a real-rime PCR using TaqMan reagents. Validation criteria were similar to those usually verified when validating methods in the analytical field: specificity, sensitivity, cross-over contamination, stability of reagents, robustness, lower and upper limits of detection, and between-run and within-run precisions. A comparison to alternate or reference methods was also performed (i.e. QiAamp kit versus DNA extractor and TaqMan genotyping versus Sanger sequencing). Each validation step is described from the pharmacogenetic point of view, as well as acceptance criteria for both DNA extraction [i.e. concentration relative SD (RSD) below 25%, verified purity, and no DNA in blank samples] and genotyping assay (i.e. specificity and diagnostic sensitivity, RSD of mean threshold cycle below 15%, no amplification in blank samples).Concerning CYP3A5 genotyping following a DNA extraction described as an example, validation criteria were met, allowing routine use of this analytical process. Cost estimation of the overall validation procedure was approximately 290 euros, concerning reagents and consumables.This work aims to provide a reference for method validation for pharmacogenetic analysis using real-time PCR to detect single nucleotide polymorphisms, in accordance with ISO15189:2012.

Pharmacological treatments used for psychiatric disorders, such as clozapine, demonstrate large interindividual variability in terms of possible adverse effects and therapeutic benefit. This variability can be explained by multiple factors, including pharmacogenetic factors. Clozapine efficacy can be impacted by CYP polymorphisms. A growing body of literature on pharmacogenetics suggests the clinical benefit of concomitant use of clozapine and fluvoxamine to improve global pharmacotherapeutic management. This article reviews and discusses available clinical and pharmacological data and limitations of clozapine augmentation with fluvoxamine based on pharmacogenetic rationale and clinical experience. The aim is to provide an updated approach on how to use the pharmacological and pharmacogenetic profile to improve clozapine efficacy and tolerance in severely ill patients.

Tissue plasminogen activator (t-PA) converts plasminogen into the serine protease plasmin, which in turn degrades fibrin clots. This study assessed whether an increase in shear stress is associated in humans in vivo with the release of t-PA in peripheral conduit arteries, the impact of high blood pressure and the role of NO and CYP450-derived epoxyeicosatrienoic acids (EETs). Local t-PA levels were quantified at baseline and during a sustained increase in radial artery wall shear stress induced by hand skin heating (from 34 to 44 °C) in a total of 25 subjects, among whom 8 were newly diagnosed essential hypertensive patients. The impact of the brachial infusion of NO synthase (L-NMMA) and CYP450 inhibitors (fluconazole) on t-PA release was assessed. The increase in shear stress induced by heating was associated with an increase in local t-PA release (from 3.0 ± 0.5 to 19.2 ± 5.5 ng/min, n = 25, P < 0.01). The magnitude of t-PA release was positively correlated with the increase in shear stress (r = 0.64, P < 0.001) and negatively correlated with mean blood pressure (r = -0.443, P = 0.027). These associations persisted after multiple adjustments for confounding factors. Finally, t-PA release was reduced by L-NMMA and to a larger extent by the combination of L-NMMA and fluconazole without a change in shear stress. The increase in wall shear stress in the peripheral conduit arteries induces a release of t-PA by a mechanism involving NO and EETs. The alteration of this response by high blood pressure may contribute to reducing the fibrinolytic potential and enhancing the risk of arterial thrombosis during exercise.

The human adrenal cortex is a complex organ which is composed of various cell types including not only steroidogenic cells but also mesenchymal cells, immunocompetent cells and neurons. Intermingling of these diverse cell populations favors cell-to-cell communication processes involving local release of numerous bioactive signals such as biogenic amines, cytokines and neuropeptides. The resulting paracrine interactions play an important role in the regulation of adrenocortical cell functions both in physiological and pathophysiological conditions. Especially, recent evidence indicates that adrenocortical cell microenvironment is involved in the pathogenesis of adrenal disorders associated with corticosteroid excess. The paracrine factors involved in these intraadrenal regulatory mechanisms may thus represent valuable targets for future pharmacological treatments of adrenal diseases.

Dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban are direct oral anticoagulants (DOACs). Their inter-individual variability in pharmacodynamics and pharmacokinetics (transport and metabolism) is high, and could result from genetic polymorphisms. As recommended by the French Network of Pharmacogenetics (RNPGx), the management of some treatments in cardiovascular diseases (as antiplatelet agents, oral vitamin K antagonists, and statins) can rely on genetic testing in order to improve healthcare by reducing therapeutic resistance or toxicity. This paper is a review of association studies between single nucleotide polymorphisms (SNPs) and systemic exposure variation of DOACs. Most of the results presented here have a lot to do with some SNPs of CES1 (rs2244613, rs8192935, and rs71647871) and ABCB1 (rs1128503, rs2032582, rs1045642, and rs4148738) genes, and dabigatran, rivaroxaban, and apixaban. Regarding edoxaban and betrixaban, as well as SNPs in the CYP3A4 and CYP3A5 genes, literature is scarce, and further studies are needed.

Tissue plasminogen activator (t-PA) converts plasminogen into the serine protease plasmin, which in turn degrades fibrin clots. This study assessed whether an increase in shear stress is associated in humans in vivo with the release of t-PA in peripheral conduit arteries, the impact of high blood pressure and the role of NO and CYP450-derived epoxyeicosatrienoic acids (EETs). Local t-PA levels were quantified at baseline and during a sustained increase in radial artery wall shear stress induced by hand skin heating (from 34 to 44 °C) in a total of 25 subjects, among whom 8 were newly diagnosed essential hypertensive patients. The impact of the brachial infusion of NO synthase (L-NMMA) and CYP450 inhibitors (fluconazole) on t-PA release was assessed. The increase in shear stress induced by heating was associated with an increase in local t-PA release (from 3.0 ± 0.5 to 19.2 ± 5.5 ng/min, n = 25, P 0.01). The magnitude of t-PA release was positively correlated with the increase in shear stress (r = 0.64, P 0.001) and negatively correlated with mean blood pressure (r = -0.443, P = 0.027). These associations persisted after multiple adjustments for confounding factors. Finally, t-PA release was reduced by L-NMMA and to a larger extent by the combination of L-NMMA and fluconazole without a change in shear stress. The increase in wall shear stress in the peripheral conduit arteries induces a release of t-PA by a mechanism involving NO and EETs. The alteration of this response by high blood pressure may contribute to reducing the fibrinolytic potential and enhancing the risk of arterial thrombosis during exercise.

Aldosterone, produced by the adrenals and under the control of plasma angiotensin and potassium levels, regulates hydromineral homeostasis and blood pressure. Here we report that the neuropeptide substance P (SP) released by intraadrenal nerve fibres, stimulates aldosterone secretion via binding to neurokinin type 1 receptors (NK1R) expressed by aldosterone-producing adrenocortical cells. The action of SP is mediated by the extracellular signal-regulated kinase pathway and involves upregulation of steroidogenic enzymes. We also conducted a prospective proof-of-concept, double blind, placebo-controlled clinical trial aimed to investigate the impact of the NK1R antagonist aprepitant on aldosterone secretion in healthy male volunteers (EudraCT: 2008-003367-40, ClinicalTrial.gov: NCT00977223). Participants received during two 7-day treatment periods aprepitant (125 mg on the 1st day and 80 mg during the following days) or placebo in a random order at a 2-week interval. The primary endpoint was plasma aldosterone levels during posture test. Secondary endpoints included basal aldosterone alterations, plasma aldosterone variation during metoclopramide and hypoglycaemia tests, and basal and stimulated alterations of renin, cortisol and ACTH during the three different stimulatory tests. The safety of the treatment was assessed on the basis of serum transaminase measurements on days 4 and 7. All pre-specified endpoints were achieved. Aprepitant decreases aldosterone production by around 30% but does not influence the aldosterone response to upright posture. These results indicate that the autonomic nervous system exerts a direct stimulatory tone on mineralocorticoid synthesis through SP, and thus plays a role in the maintenance of hydromineral homeostasis. This regulatory mechanism may be involved in aldosterone excess syndromes., Adrenal aldosterone production is regulated by plasma angiotensin and potassium levels. Here the authors report that the neuropeptide substance P stimulates aldosterone production via neurokinin type 1 receptors (NK1R), and report a proof-of-concept placebo controlled clinical trial showing that a NK1R antagonist decreases aldosterone levels.

BackgroundThis pathophysiological study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes the vasodilator and anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), contributes to conduit artery endothelial dysfunction in type 2 diabetes.Methods and resultsRadial artery endothelium-dependent flow-mediated dilatation in response to hand skin heating was reduced in essential hypertensive patients (n = 9) and type 2 diabetic subjects with (n = 19) or without hypertension (n = 10) compared to healthy subjects (n = 36), taking into consideration cardiovascular risk factors, flow stimulus and endothelium-independent dilatation to glyceryl trinitrate. Diabetic patients but not non-diabetic hypertensive subjects displayed elevated whole blood reactive oxygen species levels and loss of NO release during heating, assessed by measuring local plasma nitrite variation. Moreover, plasma levels of EET regioisomers increased during heating in healthy subjects, did not change in hypertensive patients and decreased in diabetic patients. Correlation analysis showed in the overall population that the less NO and EETs bioavailability increases during heating, the more flow-mediated dilatation is reduced. The expression and activity of sEH, measured in isolated peripheral blood mononuclear cells, was elevated in diabetic but not hypertensive patients, leading to increased EETs conversion to DHETs. Finally, hyperglycemic and hyperinsulinemic euglycemic clamps induced a decrease in flow-mediated dilatation in healthy subjects and this was associated with an altered EETs release during heating.ConclusionsThese results demonstrate that an increased EETs degradation by sEH and altered NO bioavailability are associated with conduit artery endothelial dysfunction in type 2 diabetic patients independently from their hypertensive status. The hyperinsulinemic and hyperglycemic state in these patients may contribute to these alterations.Trial registrationNCT02311075. Registered December 8, 2014.

Resident adrenal mast cells have been shown to activate aldosterone secretion in rat and man. Especially, mast cell proliferation has been observed in adrenal tissues from patients with aldosterone-producing adrenocortical adenoma. In the present study, we show that the activity of adrenal mast cells is stimulated by low-sodium diet and correlates with aldosterone synthesis in C57BL/6 and BALB/c mice. We have also investigated the regulation of aldosterone secretion in mast cell–deficient C57BL/6 Kit W-sh/W-sh mice in comparison with wild-type C57BL/6 mice. Kit W-sh/W-sh mice submitted to normal sodium diet had basal plasma aldosterone levels similar to those observed in wild-type animals. Conversely, low-sodium diet unexpectedly induced an exaggerated aldosterone response, which seemed to result from an increase in adrenal renin and angiotensin type 1 receptor expression. Severe hyperaldosteronism was associated with an increase in systolic blood pressure and marked hypokalemia, which favored polyuria. Adrenal renin and angiotensin type 1 receptor overexpression may represent a compensatory mechanism aimed at activating aldosterone production in the absence of mast cells. Finally, C57BL/6 Kit W-sh/W-sh mice represent an unexpected animal model of primary aldosteronism, which has the particularity to be triggered by sodium restriction.

Point-of-care testing (POCT) is the analysis of patient specimens outside the clinical laboratory, near or at the site of patient care, usually performed by clinical staff without laboratory training, although it also encompasses patient self-monitoring. It is able to provide a rapid result near the patient and which can be acted upon immediately. The key driver is the concept that clinical decision making may be delayed when samples are sent to the clinical laboratory. Balanced against this are considerations of increased costs for purchase and maintenance of equipment, staff training, connectivity to the laboratory information system (LIS), quality control (QC) and external quality assurance (EQA) procedures, all required for accreditation under ISO 22870. The justification for POCT depends upon being able to demonstrate that a more timely result (shorter turnaround times (TATs)) is able to leverage a clinically important advantage in decision making compared with the central laboratory (CL). In the four decades since POCT was adapted for the self-monitoring of blood glucose levels by subjects with diabetes, numerous new POCT methodologies have become available, enabling the clinician to receive results and initiate treatment more rapidly. However, these instruments are often operated by staff not trained in laboratory medicine and hence are prone to errors in the analytical phase (as opposed to laboratory testing where the analytical phase has the least errors). In some environments, particularly remote rural settings, the CL may be at a considerable distance and timely availability of cardiac troponins and other analytes can triage referrals to the main centers, thus avoiding expensive unnecessary patient transportation costs. However, in the Emergency Department, availability of more rapid results with POCT does not always translate into shorter stays due to other barriers to implementation of care. In this review, we apply the principles of evidence-based laboratory medicine (EBLM) looking for high quality systematic reviews and meta-analyses, ideally underpinned by randomized controlled trials (RCTs), looking for evidence of whether POCT confers any advantage in clinical decision making in different scenarios.

Objectifs L’objectif de la lutte contre l’insecurite routiere est notamment de faire diminuer le nombre d’accidents et de morts sur les routes, ce qui representait 3351 personnes blessees et 3239 deces en 2019. Parmi les facteurs de risque connus, les consommations d’alcool et de produits stupefiants presentent un impact important et celui de la consommation de medicaments susceptibles de reduire les capacites a la conduite automobile reste peu evalue. L’objectif de ce travail etait d’evaluer localement l’incidence actuelle des consommations d’alcool ethylique, de produits stupefiants et/ou de substances medicamenteuses « a risque » chez les automobilistes. Methode L’analyse a porte sur une serie de 100 automobilistes ayant fait l’objet de prelevements sanguins au decours d’un controle routier ou d’accidents de la circulation en 2019. Ces echantillons biologiques ont fait l’objet d’un screening toxicologique par HPLC-MS/MS par methode “MTS” a la recherche de medicaments dont l’utilisation est reconnue pour presenter un impact sur la conduite automobile. Resultats Parmi les 90 hommes et 10 femmes inclus dans cette etude, 51 % presentaient une alcoolemie positive [0,24-4,46 g/L]. Dans 33 % des cas un depistage des cannabinoides (THC) etait positif, les autres stupefiants etaient retrouves dans 6 % des echantillons analyses (cocaine principalement) et dans 17 % des cas le depistage d’un (ou plusieurs) medicament(s) susceptible(s) d’impacter la conduite automobile etait positif, apres exclusion des medicaments vraisemblablement lies a une prise en charge medicale au decours d’un accident de la circulation. Ces medicaments incluaient principalement des psychotropes (anxiolytiques, hypnotiques, antidepresseurs), des traitements de substitution aux opiaces, des antiepileptiques et des medicaments a visee cardiovasculaire. Conclusion Malgre un biais de selection evident de la population de conducteurs etudiee, l’incidence importante des medicaments retrouves laisse prejuger d’une incidence importante dans la population generale. Le recours a des pictogrammes informatifs apparait aujourd’hui banalise et le renforcement du conseil pharmaceutique en officine pourrait probablement contribuer a reduire les risques associes a la prise de certains medicaments chez les automobilistes.

Objectifs Environ 25 % de patients pris en charge en psychiatrie presentent des troubles associes a une consommation de cannabis. Une prise en charge specifique est necessaire, et des elements de motivation sont utiles pour favoriser l’adhesion des patients a la prise en charge d’un sevrage. Le recours a un depistage qualitatif des cannabinoides dans les urines presente des limites dans ce type de contexte, car une periode souvent longue de sevrage est necessaire afin d’obtenir un depistage negatif. Cette etude a eu pour objectif de developper un modele toxicocinetique de population predictif de l’elimination urinaire des cannabinoides, ainsi que de determiner a titre individuel le delai de sevrage necessaire pour atteindre une valeur decisionnelle equivalente aux seuils de 20 ou 50 ng/mL utilises communement avec les kits commerciaux d’immunochimie. Methode Cent trente-deux patients suivis pour un sevrage cannabique en addictologie ont ete inclus entre 2013 et 2017, et 452 echantillons d’urines ont ete analyses. Les concentrations urinaires de cannabinoides utilisees pour la modelisation ont ete ajustees sur la concentration simultanee de creatinine. Les cannabinoides ont ete analyses par methode KIMS et la creatinine a ete analysee par methode colorimetrique, sur COBAS 8000 (Roche®). La cinetique d’elimination a ete decrite pour chaque patient. Un modele mono-exponentiel a ete realise a partir de 88 echantillons preleves chez 26 patients observants de leur sevrage et presentant au moins 3 dosages de cannabinoides urinaires successivement decroissants. Des seuils d’interpretation ont ete proposes a l’aide de courbes ROC. Resultats Des concentrations de cannabinoides de 786 ± 1270 ng/mL et de creatinine de 1,16 ± 0,72 g/L, ainsi que des valeurs de ratio cannabinoides/creatinine de 707 ± 826 ng/mg ont ete mesurees. Le modele toxicocinetique d’elimination construit a permis de determiner une constante d’elimination de 0,0108 ± 0,026 h-1, correspondant a une demi-vie d’elimination de 64 ± 12 heures. L’ajustement de la concentration de cannabinoides urinaires sur la creatinine diminue l’impact de la dilution ou de la concentration des urines sur le resultat. Cela nuance ainsi des resultats faussement positifs ou negatifs obtenus sur la base des seuils decisionnels habituels. Les courbes ROC effectuees a partir des donnees de l’ensemble des patients ont permis de proposer des seuils decisionnels de 32,4 et de 124,7 ng/mg de creatinine, respectivement par comparaison aux seuils usuels de 20 et 50 ng/mL de cannabinoides. Conclusion Le modele propose dans cette etude et les seuils decisionnels retenus ont donne aux cliniciens addictologues des outils de suivi de sevrage, et notamment des elements concrets de motivation aupres des patients. En effet, ceux-ci ont un apercu moins binaire des fruits de leurs efforts et la modelisation de l’elimination a titre individuelle permet d’estimer le delai necessaire a une negativation du test urinaire.

Two clinical practice guidelines published in 2012 and in 2013 by the Haute autorité de santé (HAS) respectively entitled "Adult chronic kidney disease" (clinical pathway guidelines) and "Clinical utility of vitamin D measurements" (Health technology assessment) contradict each other on a notable point: in 2012 the HAS recommend to measure blood concentrations of vitamin D once a year in all patients with chronic kidney disease whereas in 2013 the HAS recommend to use this test only for the ambulatory follow-up of patients three months after kidney transplantation. This contradiction encouraged us to evaluate the methodological quality of these two guidelines with the help of the AGREE (Appraisal of Guidelines for Research and Evaluation) instrument which is consensual at an international level, in particular at the WHO (World Health Organization) and at the European Union. At the end of this comparative evaluation this preliminary hypothesis might be proposed: a more rigorous development (AGREE domain n̊3) as well as a higher editorial independence (AGREE domain n̊6) in 2013 than in 2012 (scores respectively are 57% and 56% in 2013 versus 24% and 25% in 2012) ensure a higher validity to the 2013 recommendations than to the 2012 recommendations. However this hypothesis is weakened by the subjective intrinsic value of the AGREE tool, and by various methodological shortcomings in these two guidelines. Therefore we conclude, using the AGREE terminology, that the methods for developing those guidelines are too uncertain, above all in 2012, for recommending their use without modifications.

La secretion d’aldosterone est principalement controlee par le systeme renine-angiotensine (SRA) circulant. Neanmoins, le blocage pharmacologique du SRA n’entraine qu’une baisse partielle et transitoire de l’aldosteronemie. Cette observation suggere que d’autres mecanismes physiologiques sont impliques dans la regulation de la production d’aldosterone. Si le role stimulant du potassium est bien etabli depuis plusieurs annees, d’autres signaux bioactifs ont vu leur implication demontree plus recemment. Ainsi, il apparait que les produits de secretion adipocytaire comme la leptine, sont capables d’activer la secretion d’aldosterone. D’autres facteurs regulateurs sont relargues au sein meme de la glande surrenale et modulent, de ce fait, la production de mineralocorticoide de facon paracrine. Parmi ces derniers, la substance P (SP), une tachykinine liberee par des terminaisons nerveuses au contact des cellules surrenaliennes productrices d’aldosterone, stimule in vitro la secretion d’aldosterone par le biais de son recepteur NK1. L’effet de la SP sur la synthese des mineralocorticoides est relaye par la voie de signalisation intra-cellulaire ERK. In vivo, l’inhibition de l’action de la SP par un antagoniste NK1, l’aprepitant, provoque une reduction de la production globale d’aldosterone d’environ 30 % chez le volontaire sain. L’aprepitant entraine une diminution de l’aldosteronemie en position couchee mais n’affecte pas le taux circulant d’aldosterone en position debout. La commande nerveuse de la secretion d’aldosterone via la SP apparait donc complementaire de l’action du SRA. Ce nouveau mecanisme regulateur pourrait etre implique dans l’hyperaldosteronisme satellite du syndrome metabolique ou du syndrome des apnees du sommeil, et dans l’hypertension arterielle essentielle a renine basse.

Le cortex surrenalien est innerve par des fibres du systeme nerveux autonome. Des facteurs neuronaux, liberes au contact des cellules steroidogenes, controlent la secretion hormonale. Notamment, la substance P (SP), un neuropeptide de la famille des tachykinines, stimule la production d’aldosterone chez le rat. Le but de l’etude est d’examiner le role de la SP dans la regulation des secretions surrenaliennes chez l’homme. Nos resultats montrent que les genes codant la SP (TAC1) et le recepteur NK1 (TACR1) sont exprimes dans la surrenale humaine. Des fibres nerveuses a SP sont situees dans la zone glomerulee a proximite des cellules productrices d’aldosterone exprimant le recepteur NK1. L’activation du NK1R par la SP stimule la production d’aldosterone par des cellules corticosurrenaliennes en culture. Cet effet stimulant est bloque par l’aprepitant, un antagoniste du NK1R. La liaison de la SP sur le NK1R active la voie ERK et augmente la transcription de genes codant des enzymes de la steroidogenese. Une etude clinique en double insu contre placebo revele que l’aprepitant abaisse le taux d’adosterone plasmatique en position couchee et reduit la production globale d’aldosterone (aldosteronurie des 24 heures), sans affecter la reponse de l’aldosterone a l’orthostatisme chez le volontaire sain. La reponse de l’aldosterone a l’aprepitant est independante de la renine. Nos donnees demontrent que la substance P intrasurrenalienne exerce un tonus stimulant sur la production d’aldosterone chez l’homme. La regulation nerveuse de la fonction mineralocorticoide par la SP interviendrait en position couchee, en complement du systeme renine-angiotensine qui est active par l’orthostatisme.

Background and aims The mechanisms involved in endothelial dysfunction in humans during aging are largely unknown at the level of conduit arteries. We aimed to asses the role of NO and CYP450 epoxygenases-derived epoxyeicosatrienoic acids (EETs) in the regulation of endothelium-dependent flow-mediated dilatation of conduit arteries during aging. Methods Radial artery diameter and mean wall shear stress were determined by echotracking coupled with Doppler in 83 subjects (19–71 years old) during a sustained flow increase induced by hand skin heating, with the brachial infusion of saline or NO-synthase and cytochrome P450 epoxygenase inhibitors (L-NNMA and fluconazole respectively). Local blood sampling was performed for the quantification of NO metabolite nitrite and EETs. Results The magnitude of flow-mediated dilatation was independently and negatively correlated with age, baseline artery diameter and systolic blood pressure, and positively correlated with the increase in shear stress induced by heating. There was an increase in nitrite level during heating until the age of 35–40 years, which declined thereafter. However, the inhibitory effect of L-NMMA on flow-mediated dilatation progressively decreased during aging, demonstrating a decrease in functional NO availability. Moreover, aging progressively reduced the increase in EET level during heating as well as the inhibitory effect of fluconazole on flow-mediated dilatation. Conclusions These results show that aging impairs the availability of EETs and NO and epoxyeicosatrienoic acids in peripheral conduit arteries, contributing to the development of endothelial dysfunction.

International audience; In the normal human adrenal gland, serotonin (5-HT) stimulates aldosterone secretion through the 5-HT4 receptor (5-HT4R). However, the physiological role of the serotonergic control of adrenocortical function is not known. In the present study, we have investigated the ability of l-Lysine, which has been shown to act as a 5-HT4 receptor antagonist, to counteract in vitro and in vivo the stimulatory effect of 5-HT4R agonists on aldosterone production. l-Lysine was found to inhibit aldosterone production induced by 5-HT and the 5-HT4R agonists BIMU8 from cultured human adrenocortical cells. The action of l-Lysine (4.95 g/day orally) on the adrenal cortex was also evaluated in 20 healthy volunteers in a double blind, cross-over, placebo controlled study. l-Lysine had no significant influence on basal plasma aldosterone levels and the aldosterone responses to upright posture, tetracosactide, and low sodium diet (10 mmol/day for 3 days). Conversely, l-Lysine significantly reduced the surge of plasma aldosterone induced by metoclopramide indicating that l-Lysine is able to efficiently antagonize the adrenal 5-HT4 receptors in vivo. These results suggest that l-Lysine supplementation may represent a new treatment of primary adrenal diseases in which corticosteroid hypersecretion is driven by overexpressed 5-HT4 receptors.

Several tools are available to help evaluate the quality of clinical practice guidelines (CPG). The AGREE instrument (Appraisal of guidelines for research & evaluation) is the most consensual tool but it has been designed to assess CPG methodology only. The European federation of laboratory medicine (EFLM) recently designed a check-list dedicated to laboratory medicine which is supposed to be comprehensive and which therefore makes it possible to evaluate more thoroughly the quality of CPG in laboratory medicine. In the present work we test the comprehensiveness of this check-list on a sample of CPG written in French and published in Annales de biologie clinique (ABC). Thus we show that some work remains to be achieved before a truly comprehensive check-list is designed. We also show that there is some room for improvement for the CPG published in ABC, for example regarding the fact that some of these CPG do not provide any information about allowed durations of transport and of storage of biological samples before analysis, or about standards of minimal analytical performance, or about the sensitivities or the specificities of the recommended tests.

Study question Does vitamin A (retinol, Rol) prevent round spermatid nuclear damage and increase the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue? Summary answer The supplementation of an in vitro culture of ~0.75 mm3 testicular explants from pre-pubertal mice with Rol enhances spermatogenesis progression during the first spermatogenic wave. What is known already The production of functional spermatozoa in vitro has only been achieved in the mouse model and remains a rare event. Establishing an efficient culture medium for vitrified pre-pubertal testicular tissue is now a crucial step to improve the spermatic yield obtained in vitro. The role of Rol in promoting the differentiation of spermatogonia and their entry into meiosis is well established; however, it has been postulated that Rol is also required to support their full development into elongated spermatids. Study design, size, duration A total of 60 testes from 6.5 days post-partum (dpp) mice were vitrified/warmed, cut into fragments and cultured for 30 days: 20 testes were used for light microscopy and histological analyses, 20 testes for DNA fragmentation assessment in round spermatids and 20 testes for induced sperm motility assessment. Overall, 16 testes of 6.5 dpp were used as in vitro fresh tissue controls and 12 testes of 36.5 dpp mice as in vivo controls. Testes were vitrified with the optimal solid surface vitrification procedure and cultured with an in vitro organ culture system until Day 30 (D30). Histological analysis, cell death, degenerating round spermatids, DNA fragmentation in round spermatids and induced sperm motility were assessed. Testosterone levels were measured in media throughout the culture by radioimmunoassay. Main results and the role of chance At D30, better tissue development together with higher differentiation of spermatogonial stem cells, and higher global cell division ability were observed for vitrified/warmed testicular fragments of ~0.75 mm3 with a culture medium supplemented with Rol compared to controls. During in vitro culture of vitrified pre-pubertal testicular tissue, Rol enhanced and maintained the entry of spermatogonia into meiosis and promoted a higher spermatic yield. Furthermore, decreased round spermatid nuclear alterations and DNA damage combined with induced sperm motility comparable to in vivo highlight the crucial role of Rol in the progression of spermatogenesis during the first wave. Limitations, reasons for caution Despite our promising results, the culture media will have to be further improved and adapted within the context of a human application. Wider implications of the findings The results have potential implications for the handling of human pre-pubertal testicular tissues cryopreserved for fertility preservation. However, because some alterations in round spermatids persist after in vitro culture with Rol, the procedure needs to be optimized before human application, bearing in mind that the murine and human spermatogenic processes differ in many respects. Large scale data None. Study funding and competing interests This study was supported by a Ph.D. grant from the Normandy University and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Rouen University Hospital, Institute for Research and Innovation in Biomedicine (IRIB) and Agence de la Biomedecine. The authors declare that there is no conflict of interest.

Diabetes induces a decrease in the number and function of different pro-angiogenic cell types generically designated as putative endothelial progenitor cells (EPC), which encompasses cells from myeloid origin that act in a paracrine fashion to promote angiogenesis and putative "true" EPC that contribute to endothelial replacement. This not only compromises neovasculogenesis in ischemic tissues but also impairs, at an early stage, the reendotheliziation process at sites of injury, contributing to the development of endothelial dysfunction and cardiovascular complications. Hyperglycemia, insulin resistance and dyslipidemia promote putative EPC dysregulation by affecting the SDF-1/CXCR-4 and NO pathways and the p53/SIRT1/p66Shc axis that contribute to their mobilization, migration, homing and vasculogenic properties. To optimize the clinical management of patients with hypoglycemic agents, statins and renin-angiotensin system inhibitors, which display pleiotropic effects on putative EPC, is a first step to improve their number and angiogenic potential but specific strategies are needed. Among them, mobilizing therapies based on G-CSF, erythropoietin or CXCR-4 antagonism have been developed to increase putative EPC number to treat ischemic diseases with or without prior cell isolation and transplantation. Growth factors, genetic and pharmacological strategies are also evaluated to improve ex vivo cultured EPC function before transplantation. Moreover, pharmacological agents increasing in vivo the bioavailability of NO and other endothelial factors demonstrated beneficial effects on neovascularization in diabetic ischemic models but their effects on endothelial dysfunction remain poorly evaluated. More experiments are warranted to develop orally available drugs and specific agents targeting p66Shc to reverse putative EPC dysfunction in the expected goal of preventing endothelial dysfunction and diabetic cardiovascular complications.

International audience; Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD.

The banking of testicular tissue before highly gonadotoxic treatment is a prerequisite for the preservation of fertility in pre-pubertal boys not yet producing sperm. The aim of the current study is to evaluate the impact of a soaking temperature performed at -7 °C, -8 °C or -9 °C on the ability of frozen-thawed mouse spermatogonial stem cells (SSCs) to generate haploid germ cells after in vitro maturation. Testes of 6.5-day-old post-partum CD-1 mice were cryopreserved by using a controlled slow freezing protocol with soaking at -7 °C, -8 °C or -9 °C. Frozen-thawed pre-pubertal testicular tissues were cultured in vitro on agarose gel for 30 days. Histological evaluations were performed and flagellated late spermatids were counted after mechanical dissection of the cultured tissues. The differentiation of frozen SSCs into elongated spermatids was more efficient after treatment at -9 °C than at -7 °C and -8 °C. After dissection, flagellated late spermatids were observed by using Shorr staining. The number of flagellated late spermatids was significantly decreased after slow freezing when compared with a fresh tissue control. Therefore, the soaking temperature during slow freezing of pre-pubertal mouse testicular tissue might positively influence the course of in vitro spermatogenesis. Our slow freezing protocol with a soaking temperature at -9 °C was the optimal condition in terms of the achievement of in vitro spermatogenesis with a higher production of elongated spermatids, although the effectiveness of the maturation process was reduced compared with the fresh tissue control.

International audience; RésuméCO-021Implication de la sérotonine dans la physiopathologie de l’adénome de ConnC. Duparc*a (Dr), J. Wilsb (Dr), F. Gobetc (Dr), T. Meatchid (Dr), MC. Zennaroe (Dr), E. Louisetf (Dr), H. Lefebvreg (Pr)a INSERM, U982, Université de Rouen, Mont-Saint-Aignan, FRANCE ; b INSERM, U982, Université de Rouen, CHU de Rouen, Mont-Saint-Aignan, FRANCE ; c Service d'Anatomo-pathologie, CHU de Rouen, Mont-Saint-Aignan, FRANCE ; d Service d'Anatomo-pathologie, Hôpital Européen georges Pompidou, APHP, Paris, FRANCE ; e INSERM, U970; PARCC; Hôpital Européen georges Pompidou, APHP, Paris, FRANCE ; f INSERM, U982, Université de Rouen,, Mont-Saint-Aignan, FRANCE ; g INSERM, U982, Université de Rouen; Service d'Endocrinologie, CHU de Rouen, Rouen, FRANCE* celine.duparc@univ-rouen.frDans la surrénale humaine normale, la sérotonine (5-HT) produite localement par les mastocytes stimule la sécrétion d’aldostérone via l’activation de récepteurs 5-HT4 (5-HT4R). Cet effet a été confirmé in vivo, l’administration de cisapride (agoniste 5-HT4R) à des volontaires sains augmentant les taux plasmatiques d’aldostérone. Le cisapride stimule également la sécrétion d’aldostérone chez des patients atteints d’hyperaldostéronisme primaire. En outre, des études moléculaires ont révélé que le 5-HT4R est fortement surexprimé dans les adénomes de Conn. Nous avons récemment montré que les adénomes de Conn étaient le siège d’une prolifération mastocytaire. Ces résultats suggèrent que la 5-HT produite par les mastocytes serait impliquée dans la physiopathologie de l’adénome de Conn. Nous avons localisé par immunohistochimie, l’enzyme de synthèse de la sérotonine, la tryptophane hydroxylase (TPH) et le 5-HT4R dans une série d’adénomes de Conn. Nous avons également exploré l’influence de la sérotonine sur la production d’aldostérone par des explants d’adénome de Conn en périfusion. Nos résultats montrent que le 5-HT4R et la TPH sont exprimés de façon hétérogène dans les adénomes de Conn. La TPH n’est pas limitée aux mastocytes mais est également présente dans des cellules stéroidogènes. L’administration d’un dégranulant mastocytaire, le composé C48/80 à des explants d’adénomes de Conn, entraine la libération d’un pic de 5-HT suivi d’une augmentation de la sécrétion d’aldostérone. L’administration de BIMU-8 (agoniste 5-HT4R) stimule également la production d’aldostérone. Ces effets sont inhibés par l’administration de GR113808, antagoniste 5-HT4R. Nos résultats montrent l’implication de la 5-HT dans la physiopathologie de l’adénome de Conn.L’auteur n’a pas transmis de déclaration de conflit d’intérêt.

Dans un recent editorial, Pierre Carayon analyse le rapport de la Cour des comptes sur la biologie medicale [1]. Nous partageons son point de vue, notamment son “ pour faire court, les critiques de la Cour des comptes apparaissent viser l’administration plutot que les biologistes eux-memes ”. Nous souhaitons poursuivre son analyse en commentant d’autres aspects de ce rapport.Les anciens definissaient les vertus par leurs vices contraires, par exemple la justice par l’ivresse de la [...]

Objective To challenge the Grading of Recommendations Assessment, Development and Evaluation (GRADE) group to address the potential misconceptions about their approach to grading the strength of recommendations in clinical practice guidelines. Study Design and Setting Based on our own expertise of health care professionals trying to think in depth about, and using, guidelines, we have identified four such misconceptions. Results These potential misconceptions are: (1) evidence in medicine means factual or scientific evidence; (2) opinions are a subcategory of evidence; (3) the most important evidence is related to clinical benefits and harms; (4) being virtuous, and principled, does not particularly help in developing the best possible guidelines. Conclusion We call on the GRADE leadership to address all the above-mentioned misconceptions. These need explicit answers in their manuscript series.

Aberrant expression of serotonin receptors in an aldosterone- and cortisol-producing adenoma Introduction Aberrant expression of serotonin receptors has been described to be involved in the pathophysiology of both aldosterone-producing and cortisol-producing adrenal adenomas. Case report A 46-year-old woman was referred for evaluation of severe hypertension associated with hypokalaemia. Primary aldosteronism with concurrent subclinical Cushing’s syndrome was diagnosed. A CT-scan identified a lesion of 4 cm in the right adrenal gland and a second lesion of 1 cm in the left adrenal gland, both displaying benign imaging phenotype. Following dexamethasone suppression, we explored the potential aberrant expression of serotonin receptors in the adrenal cortex by intravenous administration of metoclopramide, a serotonin type 4 receptor (5-HT4-R) agonist. This led to abnormal cortisol increase and physiological response of aldosterone in peripheral blood. Adrenal venous sampling was then performed in basal conditions and after metoclopramide stimulation. An increase in cortisol level was observed in the left adrenal vein after stimulation. 131I-19-Iodocholesterol scintigraphy showed intense radiotracer uptake in the right adrenal mass and weak uptake in the contralateral mass. Right laparoscopic adrenalectomy was performed. Histological examination revealed an adrenocortical adenoma. In vitro, cultured adrenocortical cells derived from the tumoural tissue exhibited aldosterone and cortisol co-secretion. Administration of 5-HT or metoclopramide to the cells induced a dose-dependent increase in cortisol production. These effects were inhibited by concomitant administration of the 5-HT4-R antagonist GR113808. Incubation of tumour tissue fragments with 5-HT induced a significant increase in aldosterone production which was abrogated in the presence of GR113808. These data were suggestive of aberrant expression of 5HT4-R in the tumoural tissue. Conclusion We report a rare case of an aldosterone- and cortisol-co-producing adenoma in a patient with severe hypertension and bilateral adrenal masses exhibiting an abnormal plasma cortisol response to metoclopramide. In vitro studies revealed enhanced sensitivity of the tumour tissue to 5-HT indicative of illicit expression of 5-HT4 receptors.

Testicular tissue cryopreservation offers the hope of preserved future fertility to pre-pubertal boys with cancer before exposition to gonadotoxic treatments. The objective of this study was to compare controlled slow freezing (CSF) with five vitrification techniques for cryopreservation of murine pre-pubertal testicular tissue and to evaluate the best protocol that could provide a successful completion of spermatogenesis after in vitro maturation. Testicular tissue from 24 mice at 6.5 days post-partum (dpp) was used to compare several vitrification protocols with one another, as well as with a CSF protocol. Toxicity test using additional 12 mice was performed for all cryopreservation solutions. Fresh tissue (FT) from six mice was used as a control. Once the optimal vitrification protocol was selected [the modified solid surface vitrification No. 1 (mSSV1 )], testes from 18 mice were cultured in vitro for 30 days with (i) fresh, (ii) slow-frozen/thawed and (iii) vitrified/warmed tissues. Testes from six mice at 36.5 dpp were used as controls. At day 30 of in vitro culture, germ cells of the seminiferous tubules showed a high ability to proliferate and elongated spermatids were observed after both freezing techniques, confirming the successful completion of in vitro spermatogenesis. However, after mSSV1 , the morphological alterations and the percentage of pyknotic seminiferous tubules were lower than CSF (4.67 ± 0.53 vs. 10.1 ± 1.12 and 22.7 ± 2.83% vs. 37.3 ± 4.24% respectively). Moreover, the number of flagellated spermatozoa produced per mg of tissue was higher for mSSV1 than for CSF (35 ± 3 vs. 9 ± 4 cells), with amounts of secreted testosterone during the culture close to those of FT. The mSSV1 protocol resulted in success rates better than CSF in maintaining testicular tissue structure, tubular morphology and tissue functions not solely for immediate frozen/thawed tissues but also after a long-term in vitro culture.

Capillary zone electrophoresis for analysis of serum proteins, is a technic more and more used in laboratory medicine. We report the case of an interference of iomeprol, radioopaque agent, in a context of acute renal failure and aregenerative anemia in a 53 year-old patient.

A 19-year-old man with DiGeorge syndrome associated with hypoparathyroidism presented with hypercalcemia. He was known to have poor compliance with alfacalcidol and calcium carbonate treatment. Laboratory values are shown in Table 1. Aggressive intravenous hydration with simultaneous administration of calcitonin and bisphosphonates were initiated to lower plasma calcium …

Background We evaluated the evolution of NO and CYP450 epoxygenases-derived epoxyeicosatrienoic acids (EETs) availability in conduit arteries during aging. Methods Radial artery diameter and mean wall shear stress were determined in 83 subjects (19 to 71 years old) at rest and during sustained endothelium-dependent flow-mediated dilatation induced by hand skin heating. The role of EETs and NO was assessed using the brachial infusion of inhibitors of cytochrome P450 epoxygenases (fluconazole) and NO-synthase (L-NMMA). Results Aging increased the reduction in basal arterial diameter induced by L-NMMA and nitrite plasma levels, an indicator of NO bioavailability. At the opposite, aging decreased EETs plasma levels and tended to reduce the basal vasoconstriction induced by fluconazole. Moreover, aging blunted the synergistic vasoconstricting effect of L-NMMA+fluconazole. Furthermore, the magnitude of flow-mediated dilatation was independently and negativelyrelated with age, together with baseline diameter and systolic blood pressure, and positively correlated with shear stress variation during heating. The increase in local nitrite level during heating and the decrease in flow-mediated dilatation induced by L-NMMA were not affected by aging. In contrast, aging tended to reduce the release of EETs and reduced the inhibitory effect of fluconazole alone and combined with L-NMMA on flow-mediated dilatation. Conclusions These results show that aging primarily impairs EETs availability and alters NO/EETs balance, contributing to promote endothelial dysfunction at the level of peripheral conduit arteries.

A 9-year-old boy presented to the Dermatology Department for the follow-up of a bullous pemphigoid. This diagnosis had recently been established based on his clinical picture and immunologic test results, and he had begun treatment. He was found to have methemoglobinemia as determined from an arterial blood sample (Table 1). This analysis was …

Les travaux anterieurs de notre equipe ont montre que les mastocytes exercent un controle paracrine stimulant sur la secretion d’aldosterone par la glande surrenale humaine normale. Les mastocytes paraissent jouer egalement un role dans la physiopathologie de l’hyperaldosteronisme primaire, la forte densite mastocytaire observee dans les adenomes de Conn etant correlee a l’aldosteronemie des patients. Les mastocytes intra-surrenaliens seraient donc susceptibles de liberer des facteurs stimulant la production de mineralocorticoides. Notre etude vise a evaluer le role des mastocytes dans la regulation de la secretion d’aldosterone grâce a l’utilisation de souris transgeniques C57BL/6 Kit W-sh/W-sh depourvues de mastocytes, par comparaison a la souche sauvage. En regime hyposode, les souris deficientes en mastocytes presentent une elevation de l’aldosteronurie superieure a celle des animaux temoins, ainsi qu’une diminution de la kaliurese et une augmentation de la pression arterielle systolique. Ces souris developpent egalement un syndrome polyuro-polydipsique associee a une baisse de l’osmolalite urinaire. Un test de restriction hydrique permet de conclure a une polydipsie primaire. L’analyse par PCR quantitative revele que l’expression du gene CYP11B2, codant l’aldosterone synthase, est doublee dans les surrenales des souris Kit W?sh/W-sh par rapport a celle des animaux sauvages. L’immunoreactivite de l’aldosterone synthase est plus intense dans les glandes surrenales des souris transgeniques que dans celles des animaux controles. Ces resultats indiquent qu’en regime hyposode, l’absence de mastocyte entraine a la fois une polydipsie primaire et une production exacerbee d’aldosterone. Nos donnees montrent pour la premiere fois le role des mastocytes dans la regulation de l’homeostasie hydrominerale.

International audience; RésuméCO-023L'activation du sytème rénine-angiotensine intrassurénalien est responsable d’un hyperaldostéronisme chez les souris déficientes en mastocyte en régime hyposodé.HG. Boyer*a (M.), J. Wilsb (Dr), A. Araboc (Dr), C. Duparca (Dr), I. Bouteleta (Dr), H. Lefebvred (Pr), E. Louiseta (Dr)a INSERM U982, Université de Rouen, Institut de Recherche et d’innovation Biomédicale, Université de Rouen, Mont Saint Aignan, FRANCE ; b Laboratoire de Biochimie Médicale, Institut de Biologie Clinique, CHU de Rouen, Rouen, FRANCE ; c UFR des Sciences et Techniques, Université de Rouen, Mont Saint Aignan, FRANCE ; d Service d'Endocrinologie, Diabète et Maladies Métaboliques, CHU de Rouen, Rouen, Rouen, FRANCE* hadrien.boyer@etu.univ-rouen.frLes mastocytes intrasurrénaliens seraient susceptibles de libérer des facteurs modulant la production de minéralocorticoïdes. Cette étude évalue le rôle des mastocytes dans la régulation de la sécrétion d’aldostérone grâce à l'utilisation de souris transgéniques C57BL/6 Kit W-sh/W-sh dépourvues de mastocyte. En régime normosodé, les souris ont un profil électrolytique normal toutefois associé à une expression plus forte du gène codant la rénine dans le rein. En régime hyposodé, les souris déficientes en mastocyte développent un syndrome polyuro-polydipsique associé à une réduction de leur osmolalité urinaire et kaliémie. De plus, les souris transgéniques présentent une élévation du taux d’aldostérone plasmatique et de la pression artérielle. L’expression du gène CYP11B2, codant l’aldostérone synthase, est doublée dans les surrénales des souris Kit W?sh/W-sh par rapport aux animaux sauvages. De même, l’immunoréactivité de l’aldostérone synthase est plus prononcée dans les surrénales des animaux transgéniques. Le régime hyposodé, qui stimule le système rénine angiotensine chez les animaux sauvages, n’a pas d’effet sur l’expression du gène codant la rénine dans les reins des animaux déficients en mastocyte. En revanche, la privation de sodium augmente l’expression des gènes codant la rénine et le récepteur de l’angiotensine II dans leurs surrénales. L’activation du système rénine-angiotensine intrassurrénalien est responsable de l’hypersécrétion d’aldostérone chez les souris déficientes en mastocyte. L’activation du système rénine-angiotensine local compenserait la perturbation de l’équilibre hydrominéral occasionnée par l’absence de mastocyte chez les souris Kit W?sh/W-sh. Nos données indiquent que les mastocytes sont impliqués dans la régulation de la production d’aldostérone et de l’homéostasie hydrominérale.L’auteur n’a pas transmis de déclaration de conflit d’intérêt.