The transduction processes of human neutrophil superoxide generation activated by receptor and post-receptor mechanisms

The generation of toxic oxygen radicals, by activated human neutrophils, is involved not only in microbial killing but has been implicated in tissue injury. The development of improved therapeutic agents necessitates an adequate understanding of the events coupling receptor stimulation with oxygen radical production. At least two signal-transduction pathways appear to be involved in the generation of oxygen radicals. One involves the phospholipase C mediated breakdown of phosphatidylinositol bisphosphate - giving rise to inositol trisphosphate (which mobilizes calcium) and diacylglycerol (which activates protein kinase C). The other pathway involves generation of diradylglycerols from non-inositol containing lipids by the action of phospholipase D. However derived, increased diradylglycerol levels and subsequent protein kinase C activation have been implicated in the generation of superoxide (O2-). Despite this, a controversy still exists as to the involvement of protein kinase C in receptor-mediated O2- production. Superoxide was measured by the reduction of ferricytochrome c initiated by receptor stimuli (fMLP, opsonized zymosan, IgG and heat-aggregated IgG), two direct protein kinase C activators (OAG and DiC8), a calcium ionophore (A23187) and a compound thought to mobilize membrane lipids (y-HCCH). Newly available and reportedly specific protein kinase C inhibitors, and putative inhibitors of diacylglycerol metabolism were used to investigate the transduction mechanisms. The results suggest that contrary to reports in the literature, protein kinase C has a central role in the signal transduction of the respiratory burst. Differences in the transduction pathways for the receptor stimuli were found and an arachidonate pathway may play a role in opsonized zymosan- and IgG-induced O2- generation. This raises the possibility that drugs could be designed which might reduce toxic oxygen radical-mediated tissue damage (such as that caused by immune complexes), without reducing the effectiveness of microbial killing. The ability of the non-steroidal anti-inflammatory drugs (NS AIDs) to modify O2- production was also investigated. Some NSAIDs, namely sodium meclofenamate, mefanamic acid and benoxaprofen, actually exacerbated superoxide release when neutrophils were stimulated by both receptor and post-receptor mechanisms. Indomethacin potentiated post-receptor stimulated O2- generation, but inhibited fMLP, Fe and C3b receptor-mediated O2- production. These results could have clinical relevance

Perioperative neutrophil immune function

Within the UK severe sepsis is responsible for 29% of intensive care admissions and carries a mortality of 44.7%. Decades of research have failed to deliver a single clinically successful immune modulating therapeutic agent. These failings may be explained by fundamental methodological challenges of sepsis laboratory investigations, namely diagnostic uncertainty, an indeterminate onset and the identification of an immunologically similar control population. The underlying hypothesis for this thesis is that the translational investigation of surgical patients may overcome many of these methodological challenges, since surgical trauma generates a homogenous inflammatory insult at a planned time to a carefully phenotyped human population. The biological basis for modelling sepsis with traumatic injury is discussed. Firstly, I reviewed the current literature to demonstrate the methodological advantages of studying surgical patients as a surrogate for sepsis. Next, I performed an observational study of neutrophil immune function following major elective surgery which identified a reduced neutrophil respiratory burst and changes in cell surface immune receptor expression. This impairment of activated neutrophil immune function was associated with resting neutrophil mitochondrial dysfunction, namely a raised mitochondrial membrane potential and increased production of reactive oxygen species. Using two different models of mitochondrial dysfunction I demonstrated that neutrophil respiratory burst may be regulated by altered mitochondrial functionality. Finally, I provide evidence that the cytoplasmic target for this mitochondrial signal is the enzyme pyruvate kinase M2, which through oxidative inhibition reduces the production of the respiratory burst substrate NADPH by limiting flow of glucose through the hexose monophosphate shunt. In summary, major elective surgery provides a translational model of human sepsis. Using this model, I demonstrate impairment of the neutrophil respiratory burst, and provide evidence that this is mediated through neutrophil mitochondrial dysfunction which promotes oxidative inhibition of the glycolytic regulatory enzyme pyruvate kinase

Phospholipases: From Structure to Biological Function

Phospholipases are a ubiquitous group of enzymes that hydrolyze ester bonds within membrane phospholipids. These enzymes serve multiple biological functions that go far beyond a mere membrane remodeling role in cellular homeostasis; they also play key functions in nutrient digestion and the regulated formation of bioactive lipids involved in cell signaling. It is to the latter function, critical to life, that this book is primarily concerned with. All the chapters are written by renowned experts in the area, and provide forefront information on the role phospholipases in a number of physiological and pathophysiological settings

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds