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Opioid analgesia is counteracted on chronic use by tolerance and hyperalgesia inducing dose escalation and life-threatening overdoses. Mu opiate receptors (MOR) expressed in primary sensory neurons were recently found to control tolerance and hyperalgesia, but the underlying mechanisms remained elusive. Here we show that genetic inactivation offms-like tyrosine kinase receptor 3 (FLT3) receptor in sensory neurons abrogates morphine tolerance and hyperalgesia by preventing MOR-induced hyperactivation of the cAMP signaling pathway and subsequent excitatory adaptive processes. Moreover, the specific FLT3 inhibitor BDT001 potentiates morphine analgesia in acute and chronic pain models, without aggravating morphine adverse effects, and reverses tolerance and hyperalgesia once installed. Thus, FLT3 appears as a key regulator of the MOR signaling pathway and its pharmacological blockade shows promise to enhance chronic opioid analgesic efficacy.

Left-ventricular hypertrophy, characterized by cardiomyocyte hypertrophy, interstitial cell proliferation, and immune cell infiltration, is a high risk factor for heart failure and death. Chemokines interacting with G protein-coupled chemokine receptors probably play a role in left-ventricular hypertrophy development by promoting recruitment of activated leukocytes and modulating left-ventricular remodeling. Using the minimally invasive model of transverse aortic constriction in mice, we demonstrated that a variety of chemokine and chemokine receptor messenger Ribonucleic Acid are overexpressed in the early and late phase of hypertrophy progression. Among the chemokine receptors, Cx3cr1 and Ccr2 were most strongly overexpressed and were significantly upregulated at 3, 7, and 14 days after transverse aortic constriction. Ligands of CX3CR1 (Cx3cl1) and CCR2 (Ccl2, Ccl7, Ccl12) were significantly overexpressed in the left ventricle at the early stages after mechanical pressure overload. Pharmacological inhibition of CX3CR1 signaling using the antagonist AZD8797 led to a significant reduction of hypertrophy, whereas inhibition of CCR2 with the RS504393 antagonist did not show any effect. Furthermore, AZD8797 treatment reduced the expression of the hypertrophic marker genes Nppa and Nppb as well as the profibrotic genes Tgfb1 and Col1a1 at 14 days after transverse aortic constriction. These findings strongly suggest the involvement of the CX3CR1/CX3CL1 pathway in the pathogenesis of left-ventricular hypertrophy.

The involvement of autophagy and its dysfunction in asthma is still poorly documented. By using a murine model of chronic house dust mite (HDM)-induced airway inflammation, we tested the expression of several autophagy markers in the lung and spleen of asthma-like animals. Compared to control mice, in HDM-sensitized and challenged mice, the expression of sequestosome-1/p62, a multifunctional adaptor protein that plays an important role in the autophagy machinery, was raised in the splenocytes. In contrast, its expression was decreased in the neutrophils recovered from the bronchoalveolar fluid, indicating that autophagy was independently regulated in these two compartments. In a strategy of drug repositioning, we treated allergen-sensitized mice with the therapeutic peptide P140 known to target chaperone-mediated autophagy. A single intravenous administration of P140 in these mice resulted in a significant reduction in airway resistance and elastance, and a reduction in the number of neutrophils and eosinophils present in the bronchoalveolar fluid. It corrected the autophagic alteration without showing any suppressive effect in the production of IgG1 and IgE. Collectively, these findings show that autophagy processes are altered in allergic airway inflammation. This cellular pathway may represent a potential therapeutic target for treating selected patients with asthma.

Background: Resolution of inflammation is now recognized as a tightly regulated and active process. Lipoxins (LX) are lead members of a larger family of specialized pro-resolving mediators with unique anti-inflammatory and pro-resolving properties. Recent studies implicated natural killer (NK) cells in the resolution of allergic airway inflammation, notably in promoting eosinophil apoptosis. The aim of the study was to better understand the pro-resolving actions of NK cells and LXA4 during allergic eosinophilic airway inflammation. Methods: 20 subjects with grass pollen allergic rhinitis were included. A nasal provocation test with either a single grass pollen allergen threshold dose or diluent was used. Nasal lavage fluid and cells were collected at baseline and at different time points after challenge. For in vitro assays, eosinophils were incubated with NK cells. Results: We observed that NK cells were recruited to the nasal mucosa shortly after the initiation of the allergic inflammatory response. This recruitment correlated with eosinophilic inflammation. In vitro assays demonstrated that direct contact and a combined action of CD56bright and CD56dim NK cells were needed to promote autologous eosinophil apoptosis. We furthermore observed that local LXA4 production correlated with the peak of neutrophil nasal mucosal infiltration, suggesting a potential role of neutrophils in LXA4 biosynthesis during the early phase of the allergic inflammatory response. Last, LXA4 appeared as essential to inhibit the in vitro release of eosinophil superoxide triggered by NK cells. Conclusion: Together, these findings indicate a synergistic role for NK cells and LXA4 in the resolution of allergic eosinophilic inflammation.

International audience; Tentons un peu d’oublier l’année 2020, mais pas complètement . . . C’est une année qui nousaura conduits à des envies et des visions différentes. Les rencontres conviviales et bien-veillantes qui ponctuent nos journées chaque année au mois d’octobre depuis 2005 n’ontpu se dérouler comme prévues, mais nous étions tous présents pour les 16esJournées deRecherche Respiratoire (J2R) le 16 octobre 2020, bien que devant nos écrans !Nous y avons cru jusqu’au bout et pensions pouvoir bénéficier de la fin de la fenêtreestivale, le programme était prêt et nos hôtes Brestois (nous ne remercierons jamais assezles équipes des Prs Francis Couturaud et Christophe Leroyer) avaient pensé à toutes lessolutions pour maintenir la distanciation, mais force fut de constater fin septembre qu’iln’était plus réaliste de poursuivre l’aventure en présentiel !!!Qu’à cela ne tienne, le plus important était de permettre aux jeunes, dont les résumésavaient suscité suffisamment d’intérêt et d’appétit pour une communication orale, deprésenter leurs travaux. Les J2R 2020 seraient donc organisées en format virtuel. Noustenons à remercier la SPLF pour son soutien logistique qui a permis cette transformation,ainsi que nos sponsors qui nous permettent de faire vivre les J2R depuis 16 ans.C’est ainsi que nos 19 jeunes étudiants chercheurs ont présenté et discuté leurs travauxsous forme de communications orales lors du Webinaire J2R. Tous les thèmes étaient repré-sentés (asthme, BPCO, cancer bronchique, maladies vasculaires, maladies inflammatoirespulmonaires, infectiologie, épidémiologie et la physiologie. . .) et ces communications,comme à l’accoutumée d’excellente qualité, ont pu être écoutées par quelques 135 per-sonnes connectées. Merci à tous des efforts consentis qui ont permis ce succès.

Background: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. Methods: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. Results: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. Conclusions: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.

Murine models of asthma are developed to better understand the mechanisms of asthma including eosinophil recruitment in the airways with the aim of evaluating new therapeutic strategies. They are intended to model the typical features of human disease, in particular airway inflammation, hyperresponsiveness (AHR), and remodeling. The phenotype of inflammatory cells recovered from the bronchoalveolar lavage fluid (BAL) is studied with innovative flow cytometry techniques while airway obstruction is measured using the forced oscillation technique, and airway responsiveness approached by barometric plethysmography in awake and unconstrained animals. We here describe models of asthma of house dust mite (HDM) as a clinically relevant allergen: a short study design (8 days) model of hypereosinophilic asthma and a chronic (31 days) asthma model, both suitable to evaluate the potential of new drug candidates to prevent allergic asthma.

Murine models of asthma are developed to better understand the mechanisms of asthma including eosinophil recruitment in the airways with the aim of evaluating new therapeutic strategies. They are intended to model the typical features of human disease, in particular airway inflammation, hyperresponsiveness (AHR), and remodeling. The phenotype of inflammatory cells recovered from the bronchoalveolar lavage fluid (BAL) is studied with innovative flow cytometry techniques while airway obstruction is measured using the forced oscillation technique, and airway responsiveness approached by barometric plethysmography in awake and unconstrained animals. We here describe models of asthma of house dust mite (HDM) as a clinically relevant allergen: a short study design (8 days) model of hypereosinophilic asthma and a chronic (31 days) asthma model, both suitable to evaluate the potential of new drug candidates to prevent allergic asthma.

Mitogen- and Stress-Activated Kinase 1 (MSK1) is a nuclear kinase, taking part in the activation pathway of the pro-inflammatory transcription factor NF-kB and is demonstrating a therapeutic target potential in inflammatory diseases such as asthma, psoriasis and atherosclerosis. To date, few MSK1 inhibitors were reported. In order to identify new MSK1 inhibitors, a screening of a library of low molecular weight compounds was performed, and the results highlighted the 6-phenylpyridin-2-yl guanidine (compound 1a, IC50~18 &micro, M) as a starting hit for structure-activity relationship study. Derivatives, homologues and rigid mimetics of 1a were designed, and all synthesized compounds were evaluated for their inhibitory activity towards MSK1. Among them, the non-cytotoxic 2-aminobenzimidazole 49d was the most potent at inhibiting significantly: (i) MSK1 activity, (ii) the release of IL-6 in inflammatory conditions in vitro (IC50~2 &micro, M) and (iii) the inflammatory cell recruitment to the airways in a mouse model of asthma.

Aberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes such as cancer and inflammatory diseases. A natural fragment of serum albumin, named EPI-X4, has previously been identified as endogenous peptide antagonist and inverse agonist of CXCR4 and is a promising compound for the development of improved analogues for the therapy of CXCR4-associated diseases. To generate optimized EPI-X4 derivatives we here performed molecular docking analysis to identify key interaction motifs of EPI-X4/CXCR4. Subsequent rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4. The EPI-X4 derivative JM#21 bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. EPI-X4 JM#21 did not exert toxic effects in zebrafish embryos and suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model. Moreover, topical administration of the optimized EPI-X4 derivative efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Thus, rationally designed EPI-X4 JM#21 is a novel potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted., Graphical abstract Dysregulation of CXCR4/CXCL12 signaling is associated with many inflammatory diseases. EPI-X4 is an endogenous peptide antagonist of CXCR4. Molecular docking and mutational analysis allowed to increase its anti-CXCR4 activity. The optimized EPI-X4 JM#21 derivative prevented inflammatory responses in mouse models of atopic dermatitis and allergen-induced asthma.Image 1

Asthma is a chronic disorder characterized by persistent inflammation of the airways with mucosal infiltration of eosinophils, T lymphocytes, and mast cells, and release of proinflammatory cytokines and lipid mediators. The natural resolution of airway inflammation is now recognized as an active host response, with highly coordinated cellular events under the control of endogenous pro-resolving mediators that enable the restoration of tissue homeostasis. Lead members of proresolving mediators are enzymatically derived from essential polyunsaturated fatty acids, including arachidonic acid-derived lipoxins, eicosapentaenoic acid-derived E-series resolvins, and docosahexaenoic acid-derived D-series resolvins, protectins, and maresins. Functionally, these specialized pro-resolving mediators can limit further leukocyte recruitment, induce granulocyte apoptosis, and enhance efferocytosis by macrophages. They can also switch macrophages from classical to alternatively activated cells, promote the return of non-apoptotic cells to lymphatics and blood vessels, and help initiate tissue repair and healing. In this review, we highlight cellular and molecular mechanisms for successful resolution of inflammation, and describe the main specialized pro-resolving mediators that drive these processes. Furthermore, we report recent data suggesting that the pathobiology of severe asthma may result in part from impaired resolution of airway inflammation, including defects in the biosynthesis of these specialized pro-resolving mediators. Finally, we discuss resolution-based therapeutic perspectives.

Valvular heart disease is highly prevalent in industrialized countries. Chronic use of anorexigens, amphetamine or ergot derivatives targeting the serotonin system has been associated with valvular heart disease.

The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to morphological criteria under light microscopy, where it is not always easy to distinguish macrophages from lymphocytes. We describe here a one-step, easy-to-use, and easy-to-customize 8-color flow cytometric method for performing differential cell count and comparing it to morphological counts on stained cytospins. This method identifies BAL cells by a simultaneous one-step immunolabeling procedure using antibodies to identify T cells, B cells, neutrophils, eosinophils, and macrophages. Morphological analysis of flow-sorted cell subsets is used to validate this protocol. An important advantage of this basic flow cytometry protocol is the ability to customize it by the addition of antibodies to study receptor expression at leukocyte cell surfaces and identify subclasses of inflammatory cells as needed. © 2017 by John Wiley & Sons, Inc.

Aims The progressive accumulation of cells in pulmonary vascular walls is a key pathological feature of pulmonary arterial hypertension (PAH) that results in narrowing of the vessel lumen, but treatments targeting this mechanism are lacking. The C-X-C motif chemokine 12 (CXCL12) appears to be crucial in these processes. We investigated the activity of two CXCL12 neutraligands on experimental pulmonary hypertension (PH), using two complementary animal models. Methods and results Male Wistar rats were injected with monocrotaline (MCT) or were subjected to SU5416 followed by 3-week hypoxia to induce severe PH. After PH establishment, assessed by pulsed-wave Doppler echocardiography, MCT-injected or SU5416 plus chronic hypoxia (SuHx) rats were randomized to receive CXCL12 neutraligands chalcone 4 or LIT-927 (100 mg/kg/day), the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100 (5 mg/kg/day), or vehicle, for 2 or 3 weeks, respectively. At the end of these treatment periods, echocardiographic and haemodynamic measurements were performed and tissue samples were collected for protein expression and histological analysis. Daily treatment of MCT-injected or SuHx rats with established PH with chalcone 4 or LIT-927 partially reversed established PH, reducing total pulmonary vascular resistance, and remodelling of pulmonary arterioles. Consistent with these observations, we found that neutralization of CXCL12 attenuates right ventricular hypertrophy, pulmonary vascular remodelling, and decreases pulmonary artery smooth muscle cell (PA-SMC) proliferation in lungs of MCT-injected rats and SuHx rats. Importantly, CXCL12 neutralization with either chalcone 4 or LIT-927 inhibited the migration of PA-SMCs and pericytes in vitro with a better efficacy than AMD3100. Finally, we found that CXCL12 neutralization decreases vascular pericyte coverage and macrophage infiltration in lungs of both MCT-injected and SuHx rats. Conclusion We report here a greater beneficial effect of CXCL12 neutralization vs. the conventional CXCR4 blockade with AMD3100 in the MCT and SuHx rat models of severe PH, supporting a role for CXCL12 in the progression of vascular complications in PH and opening to new therapeutic options.

Introduction Le projet vise a obtenir un modele organoide 3D d’epithelium bronchique immunocompetent innerve pour une application a l’asthme severe. L’asthme severe touche environ 5 a 15 % des patients asthmatiques. C’est un asthme non controle qui se caracterise par des exacerbations frequentes et graves, et pour lequel les traitements sont souvent inefficaces. La construction d’un epithelium bronchique immunocompetent et innerve devrait permettre la recherche de nouveaux medicaments. En effet, il sera possible de reprogrammer des cellules circulantes de patients atteints d’asthme severe en iPSCs et d’obtenir un modele personnalise, permettant de cribler et de l’etudier pharmacologiquement. Methodes Le projet comprend en parallele : – la construction d’une matrice extracellulaire sous-epitheliale dans laquelle des fibroblastes bronchiques sont ensemences ; – la differenciation d’iPSC reprogrammees a partir de PBMC circulants en progeniteurs neuronaux et cellules de Schwann. Les essais de matrice comprennent un melange collagene bovin de type I (10 mg/ml) et chitosan (2,8 mg/ml), fabriquee selon un protocole etabli a l’UPR 3572 (Muller et al., 2018) d’une part et une bioimpression de collagene et acide hyaluronique methacryles par nos collaborateurs de la plateforme Bioprint a Bordeaux (H Oliveira) d’autre part. La differenciation des iPSC en progeniteurs neuronaux est obtenue par inhibition de la voie des SMAD [SB431542 (40 mM) et LDN193189 (0,2 mM)] pendant 7 jours, puis de la GSK3β (CHIR99021, 3 μM). Les cellules sont cultivees dans des plaques 6 puits revetues de Geltrex™ et le milieu de culture est change chaque jour. Une partie des progeniteurs est differenciee en neurones sensitifs immatures par inhibition de la voie Notch (DAPT 10 mM). Ces neurones immatures seront ensemences sous la matrice ou la maturation sera induite par ajout de facteurs de croissance des nerfs : NGF (10 ng/mL), BDNF (20 ng/mL) et GDNF (10 ng/mL). L’autre partie des progeniteurs neuronaux est differenciee en cellules de Schwann jusqu’a maturation par ajout de neuroreguline 1 (192 ng/ml) pendant 21 jours ; elles seront ensemencees dans la matrice car leur presence favorise la croissance des neurones sensitifs (Blais et al., 2009). Le phenotype des lignees de neurones sensitifs et de cellules de Schwann est valide par immunomarquage 2D en fluorescence : anti-Tubuline s3 (1/400e) un marqueur neuronal, anti-substance P (1/2000e) et anti-neurofilament M (1/2000e) 2 marqueurs specifiques des neurones sensitifs, anti-Sox 10 (1/100e), marqueur des cellules de Schwann, anti-S100b (1/500e) (cellules de Schwann matures myelinisees ou non), anti-MBP (1/2000e) (cellules de Schwann matures myelinisees) et anti- P75NTR (1/2000e) (precurseurs et cellules de Schwann matures non myelinisees). Resultats Les resultats d’ensemencement des fibroblastes bronchiques humains dans les matrices montrent une colonisation des matrices collagene-chitosan, contrairement aux matrices bioimprimees. Cependant certaines matrices se sont dechirees apres quelques jours de culture, du a leur structure interne ; des mises au point sont en cours. Les neurones sensitifs sont positifs au neurofilament M, a la tubuline s3 et a la substance P : les iPSCs ont donc bien ete differencies dans la lignee neuronale. Les cellules de Schwann sont caracterisees au 10e jour de differenciation, ils sont positifs pour Sox 10, P75-NTR et S100b, mais ne presentent pas la MBP. Nous sommes donc en presence de cellules de Schwann matures non myelinisees. Ces cellules differenciees ont ete congelees et seront ensemencees dans les matrices une fois les mises au point terminees. Conclusion Les resultats obtenus sont prometteurs. A la fin des mises au point, la matrice selectionnee sera rendue immunocompetente par ensemencement de cellules dendritiques derivees de monocytes isoles. Des iPSCs seront deposees sur la matrice obtenue et differenciees en epithelium bronchique selon la methode de l’equipe de Montpellier (Ahmed, Fieldes et al., soumis) afin d’obtenir notre modele final.

Experimental models are critical for the understanding of lung health and disease and are indispensable for drug development. However, the pathogenetic and clinical relevance of the models is often unclear. Further, the use of animals in biomedical research is controversial from an ethical perspective.The objective of this task force was to issue a statement with research recommendations about lung disease models by facilitating in-depth discussions between respiratory scientists, and to provide an overview of the literature on the available models. Focus was put on their specific benefits and limitations. This will result in more efficient use of resources and greater reduction in the numbers of animals employed, thereby enhancing the ethical standards and translational capacity of experimental research.The task force statement addresses general issues of experimental research (ethics, species, sex, age,ex vivoandin vitromodels, gene editing). The statement also includes research recommendations on modelling asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, lung infections, acute lung injury and pulmonary hypertension.The task force stressed the importance of using multiple models to strengthen validity of results, the need to increase the availability of human tissues and the importance of standard operating procedures and data quality.

International audience; We previously reported Chalcone-4 (1) that binds the chemokine CXCL12, not its cognate receptors CXCR4 or CXCR7, and neutralizes its biological activity. However, this neutraligand suffers from limitations such as poor chemical stability, solubility, and oral activity. Herein, we report on the discovery of pyrimidinone 57 (LIT-927), a novel neutraligand of CXCL12 which displays a higher solubility than 1 and is no longer a Michael acceptor. While both 1 and 57 reduce eosinophil recruitment in a murine model of allergic airway hypereosinophilia, 57 is the only one to display inhibitory activity following oral administration. Thereby, we here describe 57 as the first orally active CXCL12 neutraligand with anti-inflammatory properties. Combined with a high binding selectivity for CXCL12 over other chemokines, 57 represents a powerful pharmacological tool to investigate CXCL12 physiology in vivo and to explore the activity of chemokine neutralization in inflammatory and related diseases.

4-phenylpyridin-2-yl-guanidine (5b): a new inhibitor of the overproduction of pro-inflammatory cytokines (TNFα and Il1β) was identified from a high-throughput screening of a chemical library on human peripheral blood mononuclear cells (PBMCs) after LPS stimulation. Derivatives, homologues and rigid mimetics of 5b were designed and synthesized, and their cytotoxicity and ability to inhibit TNFα overproduction were evaluated. Among them, compound 5b and its mimetic 12 (2-aminodihydroquinazoline) showed similar inhibitory activities, and were evaluated in vivo in models of lung inflammation and neuropathic pain in mice. In particular, compound 12 proved to be active (5 mg/kg, ip) in both models.

Opioid analgesics, such as morphine, oxycodone and fentanyl, are the cornerstones for treating moderate to severe pain. However, upon chronic administration, their efficiency is limited by prominent side effects such as analgesic tolerance and dependence liability. Neuropeptide FF (NPFF) and its receptors (NPFF1R and NPFF2R) are recognized as an important pronociceptive system involved in opioid-induced hyperalgesia and analgesic tolerance. Herein, we report the design of multitarget peptidomimetic compounds that show high affinity binding to the mu opioid receptor (MOPr) and NPFFRs. In vitro characterization of these compounds led to identification of KGFF03 and KGFF09 as G protein-biased MOPr agonists with full agonist or antagonist activity at NPFFRs, respectively. In agreement with their biased MOPr agonism, KGFF03/09 showed reduced respiratory depression in mice, as compared to the unbiased parent opioid agonist KGOP01. Chronic subcutaneous administration of KGOP01 and KGFF03 in mice rapidly induced hyperalgesia and analgesic tolerance, effects that were not observed upon chronic treatment with KGFF09. This favorable profile was further confirmed in a model of persistent inflammatory pain. In addition, we showed that KGFF09 induced less physical dependence compared to KGOP01 and KGFF03. Altogether, our data establish that combining, within a single molecule, the G protein-biased MOPr agonism and NPFFR antagonism, have beneficial effects on both acute and chronic side effects of conventional opioid analgesics. This strategy can lead to the development of novel and potent antinociceptive drugs with limited side effects upon acute and chronic administration.

Acacia nilotica is a medicinal plant used traditionally in Togo to treat various diseases including burn wounds. The aim of this study was to evaluate experimentally Acacia nilotica burn wound healing effect.Cutaneous burn wounds were symmetrically caused on depilated areas of rat skin through contact with an aluminum bar (r = 10 mm), preheated at 80°C for 30 sec. Five groups of animal were constituted and each group contained 8 mice. Four groups of animal’s burn wounds were infected by Staphylococcus aureus. One group burn wound is uninfected and serves as negative control. Burn wounds were assessed by planimetry and histological parameters of healing. Twelve days after burn wound induction, wound contraction in the uninfected groups (negative control) was 19.9% for topical application against -2.43% for infected control. In the infected groups treated with extract, wound contraction was generally stimulates. Histological examination showed granulated tissue developing over the wounds treated with the extract of A. nilotica at 2.5% and 5% where the proliferation of fibroblasts and neo-blood vessels was very marked.In conclusion, A. nilotica pod contents tannin, flavonoids, alkaloids and protein reduces DPPH solution and significantly accelerates wound healing of burns, and this is the case even if wounds are infected with 109CFU/mL of S. aureus.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH. Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%. In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

Introduction: C-X-C motif chemokine 12 (CXCL12) can directly control cell proliferation, migration and differentiation via interaction with either CXCR4 or CXCR7, and represents a promising target in pulmonary hypertension (PH). However, the efficacy of a CXCL12 neutraligand has never been tested in animal models of severe PH. Objective: To test and compare the efficacy of a CXCL12 neutraligand, chalcone-4, versus a CXCR4 antagonist, AMD3100, in the Sugen 5416/ hypoxia (SuHx) rat model, in a curative approach. Methods & Results: Height weeks after the Sugen 5416 administration, a substantial decrease in values of total pulmonary vascular resistance and of Fulton index (assessing right ventricular hypertrophy) were found in SuHx rats treated with chalcone-4 (100mg/kg/day for 2 weeks) and AMD3100 (5mg/kg/day for 2 weeks) when compared with the SuHx rats treated with vehicle. Interestingly, a substantial increase in values of cardiac output in SuHx rats treated with chalcone-4 was found when compared with the SuHx rats treated with AMD3100. Consistent with these observations, chalcone-4 and AMD3100 substantially attenuated the pulmonary vascular remodeling and the progressive narrowing of pulmonary arteries in the SuHx rat model. Conclusions: We report here a greater beneficial effect of chalcone-4 than AMD3100 treatment in the SuHx rat model of severe PH, supporting a role for CXCL12 in the progression of vascular complications in PH and opening to new therapeutic options.

The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to morphological criteria under light microscopy, where it is not always easy to distinguish macrophages from lymphocytes. We describe here a one-step, easy-to-use, and easy-to-customize 8-color flow cytometric method for performing differential cell count and comparing it to morphological counts on stained cytospins. This method identifies BAL cells by a simultaneous one-step immunolabeling procedure using antibodies to identify T cells, B cells, neutrophils, eosinophils, and macrophages. Morphological analysis of flow-sorted cell subsets is used to validate this protocol. An important advantage of this basic flow cytometry protocol is the ability to customize it by the addition of antibodies to study receptor expression at leukocyte cell surfaces and identify subclasses of inflammatory cells as needed. © 2017 by John WileySons, Inc.

International audience; Glioblastoma is the most common malignant brain tumor. The heterogeneity at the cellular level, metabolic specificities and plasticity of the cancer cells are a challenge for glioblastoma treatment. Identification of cancer cells endowed with stem properties and able to propagate the tumor in animal xenografts has opened a new paradigm in cancer therapy. Thus, to increase efficacy and avoid tumor recurrence, therapies need to target not only the differentiated cells of the tumor mass, but also the cancer stem-like cells. These therapies need to be effective on cells present in the hypoxic, slightly acidic microenvironment found within tumors. Such a microenvironment is known to favor more aggressive undifferentiated phenotypes and a slow-growing "quiescent state" that preserves the cells from chemotherapeutic agents, which mostly target proliferating cells. Based on these considerations, we performed a differential screening of the Prestwick Chemical Library of approved drugs on both proliferating and quiescent glioblastoma stem-like cells and identified bisacodyl as a cytotoxic agent with selectivity for quiescent glioblastoma stem-like cells. In the present study we further characterize bisacodyl activity and show its efficacy in vitro on clonal macro-tumorospheres, as well as in vivo in glioblastoma mouse models. Our work further suggests that bisacodyl acts through inhibition of Ca(2+) release from the InsP3 receptors.

Valvular heart disease (VHD) is highly prevalent in industrialized countries. Chronic use of anorexigens, amphetamine or ergot derivatives targeting the 5-HT system is associated with VHD. Here, we investigated the contribution of 5-HT receptors in a model of valve degeneration induced by nordexfenfluramine, the main metabolite of the anorexigens, dexfenfluramine and benfluorex.Nordexfenfluramine was infused chronically (28 days) in mice ((WT and transgenic HtrChronic treatment of mice with nordexfenfluramine activated 5-HTBM-derived endothelial progenitor cells make a crucial contribution to the remodelling of mitral valve tissue. Our data describe a new and important mechanism underlying human VHD.

Purpose Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy, we assessed the lung metabolome of various animal species in order to identify the animal model that could be substituted to human lung in studies on fresh lung biopsies. Methods The experiments were conducted on intact lung biopsy samples of pig, rat, mouse, and human using a Bruker Advance III 500 spectrometer. Thirty-five to 39 metabolites were identified and 23 metabolites were quantified. Principal component analysis, partial least-squares discriminant analysis, and analysis of variance tests were performed in order to compare the metabolic profiles of each animal lung biopsies to those of the human lung. Results The metabolic composition between human and pig lung was similar. However, human lung was distinguishable from mouse and rat regarding: Trimethylamine N-oxide and betaine which were present in rodents but not in human lung, carnitine, and glycerophosphocholine which were present in mouse but not in human lung. Conversely, succinic acid was undetected in rat lung. Furthermore, fatty acids concentration was significantly higher in rodent lungs compared to human lung. Conclusion Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy on lung biopsy, samples allowed to highlight that pig lung seems to be close to human lung as regarding its metabolite composition with more similarities than dissimilarities. Magn Reson Med 71:35–43, 2014. © 2013 Wiley Periodicals, Inc.