The oxidative stress of hyperglycemia and the inflammatory process in endothelial cells

Hyperglycemia and insulin resistance are common in many critically ill patients. Hyperglycemia increases the production of reactive oxygen species in cells, stimulates the production of the potent proinflammatory cytokines IL-8 and TNF-alpha, and enhances the expression of haem oxygenase-1, an inducible stress protein. It has been shown that administration of insulin and the semi-essential amino acid glutamine have been beneficial to the septic patient. The aim of our study is to test whether these two molecules, glutamine and insulin used in combination attenuate the proinflammatory responses in endothelial cells which have been triggered by hyperglycaemia. Our results demonstrate that a combination of insulin and glutamine are significantly more effective in reducing the expression of IL-8, TNF-alpha and HO-1 than insulin or glutamine alone

Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp.

BACKGROUND: Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules. METHODOLOGY/PRINCIPAL FINDINGS: Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaΙ among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C(12). The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown. CONCLUSIONS/SIGNIFICANCE: These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria

Beta Adrenergic Receptors Stimulation Attenuates Phosphorylation of NF-κB and IκBα in Hyperglycemic Endothelial Cells

Background/Aims: NF-κB induces transcription of a number of genes, associated with inflammation and apoptosis. In this study, we have investigated the effect of β-adrenergic receptor stimulation on NF-κB and IκBα in HUVECs. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in high and low glucose concentrations. All HUVECs were treated with different concentrations of isoproterenol and propranolol for different time periods. The analytical procedures consisted of Western Blot, ELISA, DCFH-DA and TUNEL assays. Results: Isoproterenol (agonist of a beta-adrenergic receptor) significantly reduced phosphorylation at Ser-536 of NF-κB; and Ser-32 and Ser-36 of IκBα in hyperglycemic HUVECs. Isoproterenol also significantly reduced apoptosis and ROS generation. No effect of IκBα was observed on Tyr-42 phosphorylation. The effect of isoproterenol was reversed by the antagonist propranolol. We also checked if NF-κB inhibitor MG132 causes any change at the level of apoptosis. However, we observed an almost similar effect. Conclusion: Given data demonstrates that beta-adrenergic receptors stimulation has a protective effect on HUVECs that might be occuring via NF-κβ and IκBα pathway

Observed precursors displaying fragments corresponding to 102.1, 74.1 and 71.1.

<p>Observed precursors displaying fragments corresponding to 102.1, 74.1 and 71.1.</p

Biofilm formation among the 50 isolates of <i>Acinetobacter</i> spp. in microtiter plates.

<p>Strain numbers are designated in x axis. About 60% of the isolates significantly forms biofilms when incubated for prolonged hours. Data are pooled from triplicate experiments. The results are expressed as the mean and the error bars indicate the standard deviation. The <i>P</i> value was less than 0.05 (<i>P</i><0.05).</p

Figure 3

<p>(A) <i>Chromobacterium violaceum </i><b>CV026 well-diffusion assay in LB agar</b>. <i>N-</i>hexanoyl homoserine lactone (HHL) was added to wells in agar containing <i>Chromobacterium violaceum</i> CV026. Zones of HHL-induced violacin production is seen surrounding the wells. Increasing concentration of AHL shows the increase in zone diameters. Standard curve showing the relationship between concentration of acylated homoserine lactone (<i>N-</i>hexanoyl homoserine lactone (HHL) & <i>N-</i>Decanoyl homoserine lactone (DHL)) and resulting diameters of induced/inhibited zones in <i>Chromobacterium violaceum</i> CV026 monitor system. (B) Well-diffusion assay for the tested isolates. Extraction efficiencies were tested on supernatants from <i>Acinetobacter</i> strains. Yields obtained were in the range of 1×10⁻⁹ moles to 6×10⁻⁹ moles of AHLs.</p

Strain number, patient number, ward, source and date of isolation of 50 <i>Acinetobacter</i> spp. isolates.

<p>Strain number, patient number, ward, source and date of isolation of 50 <i>Acinetobacter</i> spp. isolates.</p

The electrophoretic banding patterns of the PCR products using the primers for the <i>abaI</i> gene.

<p>Lane 1–100bp DNA ladder, lane 2, 3, 4, 5, 6, 7, 8– amplicons from isolates S1, S11, S53, S54, S93, S102 and S117 respectively, lane 9– Negative control.</p