Effect of lipopolysaccharide on alteration of phospholipids and their fatty acid composition in spleen and thymus by in vitro metabolic labeling

Lipopolysaccharide (LPS) is an endotoxin, a potent stimulator of immune response and induction of LPS leads to acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). ARDS is a life-threatening disease worldwide with a high mortality rate. The immunological effect of LPS with spleen and thymus is well documented; however the impact on membrane phospholipid during endotoxemia has not yet been studied. Hence we aimed to investigate the influence of LPS on spleen and thymus phospholipid and fatty acid composition by 32P]orthophosphate labeling in rats. The in vitro labeling was carried out with phosphate-free medium (saline). Time course, LPS concentration-dependent, pre- and post-labeling with LPS and fatty acid analysis of phospholipid were performed. Labeling studies showed that 50 mu g LPS specifically altered the major phospholipids, phosphatidylcholine and phosphatidylglycerol in spleen and phosphatidylcholine in thymus. Fatty acid analysis showed a marked alteration of unsaturated fatty acids/saturated fatty acids in spleen and thymus leading to immune impairment via the fatty acid remodeling pathway. Our present in vitro lipid metabolic labeling study could open up new vistas for exploring LPS-induced immune impairment in spleen and thymus, as well as the underlying mechanism.

Ascorbate Protects Neurons against Oxidative Stress: A Raman Microspectroscopic Study

Oxidative stress due to excessive accumulation of reactive oxygen or nitrogen species in the brain as seen in certain neurodegenerative diseases can have deleterious effects on neurons. Hydrogen peroxide, endogenously generated in neurons under normal physiological conditions, can produce an excess of hydroxyl radical via a Fenton mediated mechanism. This may induce acute oxidative injury if not scavenged or removed effectively by antioxidants. There are several biochemical assay methods to estimate oxidative injury in cells; however, they do not provide information on the biochemical changes as the cells get damaged progressively under oxidative stress. Raman microspectroscopy offers the possibility of real time monitoring of the chemical composition of live cells undergoing oxidative stress under physiological conditions. In the present study, a hippocampal neuron coculture was used to observe the acute impact of hydroxyl radicals generated by hydrogen peroxide in the presence of Fe2+ (Fenton reaction). Raman peaks related to nucleic acids (725, 782, 1092, 1320, 1340, 1420, and 1576 cm(-1)) showed time-dependent changes over the experimental period (60 mm), indicating the breakdown of the phosphodiester backbone as well as nuclear bases. Interestingly, ascorbic acid (a potent antioxidant) when cotreated with Fenton reactants showed protection of cells as inferred from the Raman spectra, presumably by scavenging hydroxyl radicals. Little or no change in the Raman spectra was observed for untreated control cells and for cells exposed to Fe2+ only, H2O2 only, and ascorbate only. A live dead assay study also supported the current observations. Hence, Raman microspectroscopy has the potential to be an excellent noninvasive tool for early detection of oxidative stress that is seen in neurodegenerative diseases.

Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of alpha-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of alpha-toxin, and triggered limited tissue damage. alpha-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure alpha-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of alpha-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of alpha-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against alpha-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of alpha-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.