Molecular quantification of bacteria from respiratory samples in patients with suspected ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) is the most common infection in critically ill patients. Initial antibiotic therapy is often broad spectrum, which promotes antibiotic resistance so new techniques are under investigation to obtain early microbiological identification and quantification. This trial compares the performance of a new real-time quantitative molecular-based method with conventional culture in patients with suspected VAP. Patients with suspected VAP who were ventilated for at least 48 h were eligible. An endotracheal aspirate (ETA) and a bronchoalveolar lavage (BAL) were performed at each suspected VAP episode. Both samples were analysed by conventional culture and molecular analysis. For the latter, bacterial DNA was extracted from each sample and real-time PCR were run. In all, 120 patients were finally included; 76% (91) were men; median age was 65 years, and clinical pulmonary infection score was ≥6 for 73.5% (86) of patients. A total of 120 BAL and 103 ETA could be processed and culture results above the agreed threshold were obtained for 75.0% (90/120) of BAL and 60.2% (62/103) of ETA. The main isolated bacteria were Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Performance was 89.2% (83.2%-93.6%) sensitivity and 97.1% (96.1%-97.9%) specificity for BAL samples and 71.8% (61.0%-81.0%) sensitivity and 96.6% (95.4%-97.5%) specificity for ETA samples when the molecular biology method was compared with conventional culture method (chosen as reference standard). This new molecular method can provide reliable quantitative microbiological data and is highly specific with good sensitivity for common pathogens involved in VAP.

Superoxide and nitric oxide cooperation in hypoxia/reoxygenation-induced neuron injury.

Oxygen-derived free radicals are implicated in hypoxia- and reoxygenation-related brain injury. In addition, excitatory amino acid neurotransmitters seem to be involved in this neurotoxicity and could act through the L-arginine/nitric oxide (NO) synthase pathway. In the present study we have used rat forebrain neurons in culture submitted to hypoxia/reoxygenation to investigate the relative role of free radicals, glutamate, and nitric oxide in hypoxic neuronal injury. Hypoxia (5 h) followed by reoxygenation (0-24 h) induced cell damage assessed by lacticodehydrogenase release into culture medium. Superoxide dismutase (SOD, 500 U/mL), D-L-2-amino-5-phosphonovaleric acid (100 microM), a glutamate receptor antagonist, and NG-nitro-L-arginine (100 microM), an NO synthase inhibitor, protected the neurons. The effect of NG-nitro-L-arginine was reversed by adding L-arginine (10 mM) in the culture medium, and hemoglobin, which scavenges NO, also afforded protection. Hypoxia (5 h) provoked glutamate release from neurons, and this effect was inhibited by SOD. Exogenous glutamate (1-100 microM) induced lacticodehydrogenase release, and this effect was inhibited by glutamate antagonism, NO synthase inhibition, or superoxide radical scavenging. These data are consistent with the following sequence of events in hypoxia-related neurotoxicity: free radical formation, glutamate release, and activation of NO synthase leading to superoxide and NO cooperative toxicity.

Protection by prostaglandins from glutamate toxicity in cortical neurons.

The growing evidence that glutamate may be an important agent mediating ischemic damage to neurons, led us to investigate the possible protective effects of pharmacological agents against glutamate in a model system of cortical neurons. In this study we examined, in particular, the cytoprotective effect of prostaglandins. Experiments were carried out in vitro by using rat cortical neurons in culture for 10 days. They were incubated for 3h with glutamate (10 microM) in the presence or absence of various pharmacological agents including prostaglandins (PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-Keto-PGF1 alpha, carba-TXA2, carba-PGI2 and PGF2 alpha-methylester). Increase in lacticodehydrogenase (LDH) release into the culture medium has been measured as an index of cell injury. When neurons were incubated with glutamate they released LDH due to NMDA-receptor activation since D-L-2-amino-5-phosphonovaleric acid, a specific receptor antagonist, protected the cells. The protective activity of oxypurinol, amflutizole, superoxide dismutase, NG nitro-L-arginine and quinacrine, also suggests that xanthine oxidase activation, the generation of superoxide radical, and nitrix oxide, as well as phospholipase A2 stimulation are responsible for neuron injury (i.e. LDH release). All the tested prostaglandins, except PGF2 alpha-methylester, afforded significant protection at concentrations between 0.1 and 10 microM. The order of potency of the prostanoids was: PGF2 alpha = PGE2 > Carba-TXA2 > PGE1 > PGD2 > PGI2 = Carba-PGI2 > 6-Keto-PGF1 alpha. Additional experiments showed that prostaglandins did not compete for the NMDA binding site and that they did not inhibit free radical-related membrane damage.(ABSTRACT TRUNCATED AT 250 WORDS)

PAF-induced conjunctivitis in the rabbit is mediated by peptido-leukotrienes.

Platelet activating factor (PAF) released by many cell types is involved in several steps of inflammatory reactions in various organs including the eye. It has been reported that some effects of PAF could be mediated by arachidonic acid metabolites generated after PAF-receptor interaction. In the study we investigated the protective effect of CBS-113A, a dual inhibitor of 5-lipoxygenase/cyclooxygenase in PAF-induced conjunctivitis in the rabbit. Moreover, the characterization of the icosanoid potentially mediating the PAF activity has been performed by using specific pharmacological agents. Subconjunctival injection of PAF (10-1000 ng) provoked Evans' blue extravasation measured in tissues within 30 min. Simultaneous injection of a PAF antagonist (BN 50730) inhibited the dye leakage, showing specific PAF receptor-mediated vascular reaction. CBS-113A applied in eye drops also inhibited Evans' blue extravasation. By contrast, indometacin administered either topically or by subconjunctival injection was not effective in reducing the effect of PAF. These results suggesting the involvement of a 5-lipoxygenase metabolite were confirmed by the effectiveness of local injection of SKF 104353, a specific leukotriene D4 antagonist. The present study shows that PAF-induced plasma leakage in conjunctivitis is mediated by peptido-leukotrienes.