A rat model of central venous catheter to study establishment of long-term bacterial biofilm and related acute and chronic infections

Formation of resilient biofilms on medical devices colonized by pathogenic microorganisms is a major cause of health-care associated infection. While in vitro biofilm analyses led to promising anti-biofilm approaches, little is known about their translation to in vivo situations and on host contribution to the in vivo dynamics of infections on medical devices. Here we have developed an in vivo model of long-term bacterial biofilm infections in a pediatric totally implantable venous access port (TIVAP) surgically placed in adult rats. Using non-invasive and quantitative bioluminescence, we studied TIVAP contamination by clinically relevant pathogens, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, and we demonstrated that TIVAP bacterial populations display typical biofilm phenotypes. In our study, we showed that immunocompetent rats were able to control the colonization and clear the bloodstream infection except for up to 30% that suffered systemic infection and death whereas none of the immunosuppressed rats survived the infection. Besides, we mimicked some clinically relevant TIVAP associated complications such as port-pocket infection and hematogenous route of colonization. Finally, by assessing an optimized antibiotic lock therapy, we established that our in vivo model enables to assess innovative therapeutic strategies against bacterial biofilm infections.     

Exposures to conditioned flavours with different hedonic values induce contrasted behavioural and brain responses in pigs

This study investigated the behavioural and brain responses towards conditioned flavours with different hedonic values in juvenile pigs. Twelve 30-kg pigs were given four three-day conditioning sessions: they received three different flavoured meals paired with intraduodenal (i.d.) infusions of 15% glucose (F(Glu)), lithium chloride (F(LiCl)), or saline (control treatment, F(NaCl)). One and five weeks later, the animals were subjected to three two-choice feeding tests without reinforcement to check the acquisition of a conditioned flavour preference or aversion. In between, the anaesthetised pigs were subjected to three (18)FDG PET brain imaging coupled with an olfactogustatory stimulation with the conditioned flavours. During conditioning, the pigs spent more time lying inactive, and investigated their environment less after the F(LiCl) than the F(NaCl) or F(Glu) meals. During the two-choice tests performed one and five weeks later, the F(NaCl) and F(Glu) foods were significantly preferred over the F(LICl) food even in the absence of i.d. infusions. Surprisingly, the F(NaCl) food was also preferred over the F(Glu) food during the first test only, suggesting that, while LiCl i.d. infusions led to a strong flavour aversion, glucose infusions failed to induce flavour preference. As for brain imaging results, exposure to aversive or less preferred flavours triggered global deactivation of the prefrontal cortex, specific activation of the posterior cingulate cortex, as well as asymmetric brain responses in the basal nuclei and the temporal gyrus. In conclusion, postingestive visceral stimuli can modulate the flavour/food hedonism and further feeding choices. Exposure to flavours with different hedonic values induced metabolism differences in neural circuits known to be involved in humans in the characterization of food palatability, feeding motivation, reward expectation, and more generally in the regulation of food intake.     

Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells

Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.     

YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages

YKL-40 is a chitin-binding protein that is elevated in patients with various inflammatory conditions associated with ongoing remodeling. We investigated whether the levels of YKL-40 were up-regulated in the circulation and the airways of patients with chronic obstructive pulmonary disease (COPD), and whether it promoted the production of inflammatory mediators from macrophages. Serum, bronchoalveolar lavage (BAL), bronchial biopsies, lung tissue specimens, and alveolar macrophages from never-smokers (n = 15), smokers without COPD (n = 20), and smokers with COPD (n = 30) were assessed for YKL-40 levels and immunolocalization. In addition, YKL-40-induced mediator release from alveolar macrophages was examined. We found that smokers with COPD had elevated levels of YKL-40 in serum (p 相似文献   

Silencing of rotavirus NSP4 or VP7 expression reduces alterations in Ca2+ homeostasis induced by infection of cultured cells

Rotavirus infection of cells in culture induces major changes in Ca(2+) homeostasis. These changes include increases in plasma membrane Ca(2+) permeability, cytosolic Ca(2+) concentration, and total cell Ca(2+) content and a reduction in the amount of Ca(2+) released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca(2+) homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca(2+) permeability of the plasma membrane and cytosolic Ca(2+) concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca(2+) permeability and Ca(2+) pools released by agonist (ATP). In addition, the increase of total Ca(2+) content (as measured by (45)Ca(2+) uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca(2+) homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca(2+) homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects.     

Self-association of adenine-dependent hairpin ribozymes

Hairpin ribozymes are flexible molecules that catalyse reversible self-cleavage after the docking of two independently folded internal loops, A and B. The activities, self-association and structures in solution of two 85 base adenine-dependent hairpin ribozymes (ADHR1 and ADHR2) were studied by native gel electrophoresis, analytical centrifugation, and small angle neutron scattering. Bi-molecular RNA interactions such as linear–linear, loop–loop, loop–linear or kissing interactions have been found to be important in the control of various biological functions, and hairpin loops present rich potential for establishing both intra- and intermolecular interactions through standard Watson-Crick base pairing or non-canonical interactions. Similar results were obtained for ADHR1 and ADHR2. At room temperature, they indicated end-to-end self-association of the ribozymes in rod-like structures with a cross-section corresponding to two double strands side-by-side. Dimers, which predominate at low concentration (∼0.1 mg/ml), associate into longer rods, with increasing concentration (∼1 mg/ml). Above 65°C, the dimers and rods dissociated into compact monomers, with a radius of gyration similar to that of tRNA (about 70 bases). The dimers were non-active for catalysis, which suggests that dimer formation, probably by preventing the correct docking of loops A and B, could act as an inhibition mechanism for the regulation of hairpin ribozyme catalysis.     

Orn5]URP acts as a pure antagonist of urotensinergic receptors in rat cortical astrocytes

Cultured rat astrocytes, which express functional urotensin II (UII)/UII-related peptide (URP) receptors (UT), represent a very suitable model to investigate the pharmacological profile of UII and URP analogs towards native UT. We have recently designed three URP analogs [D-Trp4]URP, [Orn5]URP and [D-Tyr6]URP, that act as UT antagonists in the rat aortic ring bioassay. However, it has been previously reported that UII/URP analogs capable of inhibiting the contractile activity of UII possess agonistic activity on UT-transfected cells. In the present study, we have compared the ability of URP analogs to compete for [125 I]URP binding and to modulate cytosolic calcium concentration ([Ca2+]c) in cultured rat astrocytes. All three analogs displaced radioligand binding: [D-Trp4]URP and [D-Tyr6]URP interacted with high- and low-affinity sites whereas [Orn5]URP only bound high-affinity sites. [D-Trp4]URP and [D-Tyr6]URP both induced a robust increase in [Ca2+]c in astrocytes while [Orn5]URP was totally devoid of activity. [Orn5]URP provoked a concentration-dependent inhibition of URP- and UII-evoked [Ca2+]c increase and a rightward shift of the URP and UII dose-response curves. The present data indicate that [D-Trp4]URP and [D-Tyr6]URP, which act as UII antagonists in the rat aortic ring assay, behave as agonists in the [Ca2+]c mobilization assay in cultured astrocytes, whereas [Orn5]URP is a pure selective antagonist in both rat aortic ring contraction and astrocyte [Ca2+]c mobilization assays.     

Effect of GABA A receptor activation on UT-coupled signaling pathways in rat cortical astrocytes

Cultured rat cortical astrocytes express two types of urotensin II (UII) binding sites: a high affinity site corresponding to the UT (GPR14) receptor and a low affinity site that has not been fully characterized. Activation of the high affinity site in astroglial cells stimulates polyphosphoinositide (PIP) turnover and provokes an increase in intracellular calcium concentration. We have hypothesized that the existence of distinct affinity sites for UII in rat cortical astrocytes could be accounted for by a possible cross-talk between UT and the ligand-gated ion channel GABA(A) receptor (GABA A R). Exposure of cultured astrocytes to UII provoked a bell-shaped increase in cAMP production, with an EC50 stimulating value of 0.83+/-0.04 pM, that was totally blocked in the presence of the adenylyl cyclase inhibitor SQ 22,536. In contrast, UII was found to inhibit forskolin-induced cAMP formation. In the presence of the specific PKA inhibitor H89, UII provoked a sustained stimulation of cAMP formation. Inhibition of PKA by H89 strongly reduced the stimulatory effect of UII on PIP metabolism. GABA and the GABA A R agonist isoguvacine provoked a marked inhibition of UII-induced cAMP synthesis and a significant reduction of UII-evoked PIP turnover. These data suggest that functional interaction between UT and GABA(A)R negatively regulates coupling of UT to the classical PLC/IP(3) signaling cascade as well as to the adenylyl cyclase/PKA pathway.     

Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells

Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.