F015 Role des fibroblastes cardiaques dans la tolérance des cardiomyocytes à l’ischémie reperfusion

ObjectifLes fibroblastes cardiaques sont la population cellulaire majoritaire du tissu cardiaque. Leurs possibles implications au cours de la séquence ischémie-reperfusion n’a jamais été étudiée. Le présent travail a donc pour but de déterminer si les fibroblastes sont impliqués dans une modulation de la cardioprotection.Matériel et MéthodesNous avons utilisé dans cette étude un modèle de cardiomyocytes de rats nouveau-nés soumis à une séquence d’ischémie-reperfusion simulées. Les cellules ont été isolées à partir de ventricules de rats nouveau nés. Les myocytes cardiaques ont été purifiés par attachements différentiels puis cultivés en présence d’un milieu de culture supplémenté en cytosine arabinoside (Ara C, 10μm). Les cardiomyocytes et les fibroblastes ont été cultivés séparément puis placés en contact direct (cultures mixtes) ou indirect (insert). Ces co-cultures ont subi une ischémie de 3H en absence de nutriments et d’O2 suivie d’une reperfusion de 20H en présence de nutriments et d’O2. Des tests de viabilité (test MTT) et de mortalité cellulaire (dosage de l’activité LDH et Troponine I) ont été effectués à la fin de la reperfusion.RésultatsNous avons montré qu’il était possible de simuler des séquences d’ischémie reperfusion et d’induire une souffrance cellulaire détectable pour une durée d’ischémie de 3H et de reperfusion de 20H. Dans les cultures mixtes (cardiomyocytes + fibroblastes), les tests MTT et LDH ont montré une amélioration de la viabilité cellulaire globale en comparaison avec la viabilité spécifique de chaque type cellulaire seul. Pour les cultures placées en insert, les tests MTT et Troponine I ont montré une amélioration de la viabilité des cardiomyocytes en présence des fibroblastes (p<0.001).ConclusionsNos résultats indiquent que les fibroblastes cardiaques semblent être impliqués dans une modulation de la cardioprotection lors de l’ischémie reperfusion. Cette modulation passe au moins en partie par des mécanismes de type paracrine et elle est dépendante de la quantité de fibroblastes en co-culture avec les cardiomyocytes

D010 Mesenchymal stem cells protect cardiomyocytes from reperfusion injury through a paracrine activation of the PI3 kinase pathway

ObjectivesPrevious data suggest that implantation of mesenchymal stem cells (MSCs) improves heart function after myocardial infarction. We investigated whether protection afforded by MSCs might involve a paracrine activation of the PI3 kinase pathway in reperfused cardiomyocytes.MethodMSCs and neonatal rat cardiomyocytes (NRCs) were isolated and cultured separately. NRCs (2.106) were subjected to 5 hours of ischemia followed by 16 hours of reperfusion. At the time of reperfusion, NRCs (n=8-14/group) received either fresh medium (control group), or the following treatments: MSCs (2.105 MSCs in fresh medium), conditioned SN (MSCs supernatant alone (i.e. without MSCs) obtained after 8 hours of serum deprived culture), [conditioned SN + LY294002] (15 microM of LY294002 a specifi c inhibitor of PI3K), [conditioned SN + Wortmannin] (100 nM of wortmannin, a non specifi c inhibitor of PI3K), or CsA (200 nM in fresh medium) a potent inhibitor of the mitochondrial permeability transition pore. Cell death was assessed by LDH release in NRCs supernatant at the end of reperfusion.ResultsAs expected, LDH activity was dramatically reduced by CsA, averaging 4 % of control values. LDH activity was signifi cantly reduced by MSCs alone and by conditioned SN, averaging 29 % and 12 % of control value, respectively. Both LY294002 and wortmannin signifi cantly attenuated conditioned SN induced protection.Conclusionour data suggest that MSCs can protect NRCs from reperfusion injury through a paracrine activation of the PI3K pathway

Population Structure of Pseudomonas aeruginosa from Five Mediterranean Countries: Evidence for Frequent Recombination and Epidemic Occurrence of CC235

Several studies in recent years have provided evidence that Pseudomonas aeruginosa has a non-clonal population structure punctuated by highly successful epidemic clones or clonal complexes. The role of recombination in the diversification of P. aeruginosa clones has been suggested, but not yet demonstrated using multi-locus sequence typing (MLST). Isolates of P. aeruginosa from five Mediterranean countries (n = 141) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping and PCR targeting the virulence genes exoS and exoU. The occurrence of multi-resistance (≥3 antipseudomonal drugs) was analyzed with disk diffusion according to EUCAST. MLST was performed on a subset of strains (n = 110) most of them had a distinct PFGE variant. MLST data were analyzed with Bionumerics 6.0, using minimal spanning tree (MST) as well as eBURST. Measurement of clonality was assessed by the standardized index of association (IAS). Evidence of recombination was estimated by ClonalFrame as well as SplitsTree4.0. The MST analysis connected 70 sequence types, among which ST235 was by far the most common. ST235 was very frequently associated with the O11 serotype, and frequently displayed multi-resistance and the virulence genotype exoS−/exoU+. ClonalFrame linked several groups previously identified by eBURST and MST, and provided insight to the evolutionary events occurring in the population; the recombination/mutation ratio was found to be 8.4. A Neighbor-Net analysis based on the concatenated sequences revealed a complex network, providing evidence of frequent recombination. The index of association when all the strains were considered indicated a freely recombining population. P. aeruginosa isolates from the Mediterranean countries display an epidemic population structure, particularly dominated by ST235-O11, which has earlier also been coupled to the spread of ß-lactamases in many countries

INVESTIGATION OF ADSORPTION AND INHIBITIVE EFFECT OF CALIXARENE DERIVATIVE NEWLY SYNTHESIZED TOWARDS C38 STEEL IN MOLAR HCl

The focus of this study is to synthesize a new calixarene derivative namely calix[6]arene (C21) and to test its performance as corrosion inhibitor of C38 steel in molar HCl at 308 K. Polarization and weight loss measurements were used. Weight loss tests show that C21 retards until to stop corrosion phenomenon at 5 × 10-5 M. C21 is an excellent inhibitor and its inhibition efficiency increases with its concentration to reach 100% since 5 × 10-5 M. Polarization curves revealed that C21 affects both cathodic and anodic domains by decreasing current densities and then it may be classified as a mixed type inhibitor. The calixarene tested is adsorbed on the surface according to the Langmuir adsorption isotherm. Free enthalpy of adsorption reveals that C21 acts from chemisorption onto the steel surface.Corrosion, steel, calixarene, inhibition, hydrochloric acid