Neutrophil Superoxide Generation Research Articles

Thromboxane mediates neutrophil superoxide production in pregnancy

We recently reported that activation of neutrophils obtained from pregnant women resulted in production of tumor necrosis factor-alpha by a thromboxane- and cyclooxygenase-2- dependent mechanism. Activated neutrophils also generate reactive oxygen species such as the superoxide anion, which can lead to oxidative damage of biomolecules. In this study, we tested the possibility that thromboxane plays a role in neutrophil superoxide generation in pregnancy via cyclooxygenase-2 by inhibiting key enzymes in the pathway leading to its synthesis. Neutrophils were isolated from normal pregnant women and incubated for 2 hours in phosphate-buffered saline with glucose alone or with treatments. Experiment 1 treatments were: (1) indomethacin at a dose sufficient to inhibit phospholipase A2 (100 microM); (2) aspirin (100 microM), a cyclooxygenase-1 and cyclooxygenase-2 inhibitor; (3) NS-398 (10 microM), a specific cyclooxygenase-2 inhibitor; (4) nordihydroguaiaretic acid (10 microM), a lipoxygenase inhibitor; and (5) pinane thromboxane, a thromboxane synthase inhibitor. Experiment 2 treatments were arachidonic acid (50 microM and 100 microM) and arachidonic acid (100 microM) alone or in combination with phorbol myristic acid plus varying doses of pinane thromboxane (5 microM to 100 microM). Indomethacin inhibited superoxide production to one fourth of control. Aspirin, NS-398, and pinane thromboxane also significantly decreased neutrophil superoxide production. Nordihydroguaiaretic acid had no significant affect on neutrophil superoxide generation. Arachidonic acid stimulated superoxide generation by neutrophils, and this was inhibited by pinane thromboxane in a dose-dependent manner. Pinane thromboxane also significantly inhibited superoxide production by neutrophils exposed to arachidonic acid plus phorbol myristic acid, a known activator of neutrophils. These data demonstrate that thromboxane is involved in the production of superoxide by neutrophils obtained from pregnant women. Neutrophil superoxide generation is significantly decreased by inhibition of thromboxane synthase, cyclooxygenase-2, or phospholipase A2. NS-398 inhibits neutrophil superoxide generation as effectively as aspirin, suggesting that cyclooxygenase-2 plays an important role in neutrophil superoxide production mediated by thromboxane.

Effect of Agkistroden blomhoffi (mamushi) on superoxide generation in human neutrophils

Background The purpose of this study is to investigate the effect of Agkistroden blomhoffi (mamushi) aqueous extract on stimulus-induced superoxide generation in human neutrophils. Methods Superoxide generation was assayed by measuring the reduction of ferricytochrome C ( cyt c) using a dual-beam spectrophotometer. The phosphorylation of neutrophil proteins and the translocation of cytosolic compounds to the cell membrane were detected using specific monoclonal antibodies. Results Mamushi aqueous extract markedly suppressed fMLP-induced superoxide generation in a concentration-dependent manner. fMLP-induced translocation of cytosolic compounds p47 phox, p67 phox and Rac to the cell membrane was suppressed in parallel to the suppression of superoxide generation. The fractions with MW below 10 kDa dose-dependently suppressed tyrosyl phosphorylation of 45.0, 77.6 and 99.5 kDa proteins in fMLP-treated human neutrophils; the inhibition of tyrosyl phosphorylation was in parallel to that of the fMLP-induced translocation of p47 phox to the cell membrane and superoxide generation. Conclusions Mamushi aqueous extract markedly suppressed fMLP-induced superoxide generation in human neutrophils and also suppressed the translocation of cytosolic compounds to the cell membrane and tyrosyl phosphorylation of proteins. The efficacious components are the fractions with MW below 10 kDa. O-phosphoserylethanolamine, a new dipeptide isolated from mamushi, also suppressed fMLP-induced superoxide generation in human neutrophils in a dose-dependent manner.

Signal Transduction Mechanism Involved inClostridium perfringensAlpha-Toxin-Induced Superoxide Anion Generation in Rabbit Neutrophils

Clostridium perfringens alpha-toxin induces the generation of superoxide anion (O2(-)) via production of 1,2-diacylglycerol (DG) in rabbit neutrophils. The mechanism of the generation, however, remains poorly understood. Here we report a novel mechanism for the toxin-induced production of O2(-) in rabbit neutrophils. Treatment of the cells with the toxin resulted in tyrosine phosphorylation of a protein of about 140 kDa. The protein reacted with anti-TrkA (nerve growth factor high-affinity receptor) antibody and bound nerve growth factor. Anti-TrkA antibody inhibited the production of O2(-) and binding of the toxin to the protein. The toxin induced phosphorylation of 3-phosphoinositide-dependent protein kinase 1 (PDK1). K252a, an inhibitor of TrkA receptor, and LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), reduced the toxin-induced production of O2(-) and phosphorylation of PDK1, but not the formation of DG. These inhibitors inhibited the toxin-induced phosphorylation of protein kinase C theta (PKCtheta). U73122, a phospholipase C (PLC) inhibitor, and pertussis toxin inhibited the toxin-induced generation of O2(-) and formation of DG, but not the phosphorylation of PDK1. These observations show that the toxin independently induces production of DG through activation of endogenous PLC and phosphorylation of PDK1 via the TrkA receptor signaling pathway and that these events synergistically activate PKCtheta in stimulating an increase in O2(-). In addition, we show the participation of mitogen-activated protein kinase-associated signaling events via activation of PKCtheta in the toxin-induced generation of O2(-).

Cilostazol suppresses adhesion of human neutrophils to HUVECs stimulated by FMLP and its mechanisms

The interaction between neutrophils and endothelial cells (ECs) is of great importance in many physiological and pathological progresses. Although cilostazol (CLZ), a novel selective phosphodiesterase (PDE) type 3 inhibitor, has been proved to be useful in vasodilatation and inhibition of platelet aggregation, its effect on adhesion is not clearly known. In this study, we examined the effects and investigated the mechanisms of cilostazol on neutrophil adhesion to human umbilical endothelial cells (HUVECs) triggered by N-formyl-methionyl-leucyl-phenylal-anine (FMLP), a chemotactic peptide. The soluble vascular cell adhesive molecule-1 (sVCAM-1) release from FMLP (10 μM)-stimulated HUVECs was determined by ELISA kits. Fluo-2, a fluorescent indicator, was used to investigate intracellular free calcium concentration ([Ca 2+] i) in HUVECs. HL-60 cells were induced to be neutrophilic by DMSO and loaded with Fluo-3, another fluorescent indicator, to detect [Ca 2+] i, and CLA was used as a chemiluminescent indicator to determine superoxide production in neutrophilic cells. The result showed that CLZ (1–100 μM) significantly inhibited neutrophil adhesion to FMLP-stimulated HUVECs. In HUVECs, CLZ obviously downregulated sVCAM-1 level, while it had no meaningful influence [Ca 2+] i. But in neutrophils, FMLP-activated superoxide generation and [Ca 2+] i increase were found being inhibited by exposure to CLZ . Furthermore, we also demonstrated that Ca 2+ increase was preceded to the superoxide generation in neutrophils. The results suggest that CLZ involves in adhesion reactions between neutrophil and ECs, partly via VCAM-1 expression in ECs, and decreasing [Ca 2+] i induced activation of neutrophils, which means a lot to prevent atherosclerosis and other cardiovascular diseases.

Ultrasound exposure can increase the membrane permeability of human neutrophil granulocytes containing microbubbles without causing complete cell destruction

Activated polymorphonuclear neutrophil (PMN) granulocytes can bind and subsequently phagocytose microbubbles used as ultrasound (US) contrast agents. The purpose of the present study was to assess insonation effects on cell membrane integrity and metabolic activity of activated PMN. Furthermore, we investigated whether or not there is an acoustic threshold at which insonation of PMN results in increase of membrane permeability without causing complete cell destruction. PMN isolated from healthy volunteers were activated with phorbol myristate acetate (PMA) for 15 min to allow phagocytosis of albumin and lipid microbubbles and were subsequently exposed to US with a mechanical index between 0.15 and 1.8. Apoptosis, loss of membrane integrity and formation of cell fragments were evaluated by measurement of lactate dehydrogenase leakage and by double staining with annexin V and propidium iodide, using flow cytometry. Neutrophil superoxide anion generation was measured photometrically. Insonation of activated PMN in the presence of microbubbles amplified apoptosis and lactate dehydrogenase leakage and induced loss of membrane integrity and complete cell destruction with increasing acoustic pressures. The bioeffects observed by insonation with high mechanical indices (1.0 to 1.8), and particularly the formation of cell fragments, were significantly more pronounced in the presence of albumin microbubbles. Insonation in the presence of lipid microbubbles increased cell membrane permeability, but caused significantly less cell destruction and left the metabolic activity of activated PMN uninfluenced. Thus, both albumin and lipid microbubbles induce apoptosis and membrane injury during insonation of activated PMN. However, insonation in the presence of lipid microbubbles seems to influence cell viability to a smaller extent. This could be of advantage in the setting of US-guided local drug delivery. In this setting, increase of membrane permeability may allow bioactive substances to enter into cells, which survive the US treatment, and specifically modify their function. (E-mail: Grigorios_Korosoglou@med.uni-heidelberg.de)

The Inhibition of Superoxide Anion Generation in Human Neutrophils by Viscum coloratum

One new 1,3-diphenylpropane, viscolin (1), and one new flavanone, (2S)-7,4'-dihydroxy-5,3'-dimethoxyflavanone (2), together with thirty-nine known compounds, which included eleven known flavanones, two chromones, fourteen benzenoids, one inositol, two pyrimidines, four triterpenoids and five steroids, were isolated and characterized from Viscum coloratum. Structures of new compounds were determined by spectral analysis. Among them, viscolin (1) showed the most significant inhibition on superoxide anion generation by human neutrophils in response to fMLP (formyl-L-methionyl-L-leucyl-L-phenylalanine).

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BACKGROUND AND PURPOSE: The inflammatory bowel diseases(IBD) such as ulcerative colitis(UC) are intractable diseases of unknown origin. The mucosal injury of UC is presumed to be partly caused by reactive oxygen species(ROS) produced by neutrophils. Chronic inflammation of UC results in chronic anemia, to which iron is given. In this study, to evaluate the effect of sodium ferrous citrate(SFC) on neutrophil functions, we measured superoxide generation and migration of neutrophils treated with SFC in vitro.METHODS: Superoxide generation induced by phorbol myristate acetate(PMA), opsonized zymosan(OPZ) and N-formyl-menthionyl-leucyl-phenylalanine(fMLP) was determined based on the superoxide dismutaseinhibitable cytochrome c reduction. To further clarify the inhibitory mechanism of SFC, the membrane fraction was isolated and NADPH-dependent superoxide production was measured. Moreover, neutrophil migration was evaluated with a Boyden chamber assay using zymosan-activated serum (C5a) as a chemoattractant.RESULTS: SFC dose-dependently inhibited the superoxide production induced by all the stimuli examined. Similarly, SFC-treatment inhibited the activity of NADPH oxidase in the membrane fraction of neutrophils. In addition, SFC suppressed chemotaxis toward zymosan-activated serum in a dose-dependent fashion.CONCLUSIONS: In this study, we revealed that SFC could suppress neutrophil functions such as superoxide generation (NADPH oxidase activity) and migration. During episodes of IBD, stimulated neutrophils are accumulated in inflamed mucosa via the migration through vascular endothelium, where they release ROS such as superoxide. Thus, the present observations suggest that SFC may have a potential to reduce mucosal injury by suppressing migration and ROS generation by neutrophils in UC.

Phenylsulphonyl urenyl chalcone derivatives as dual inhibitors of cyclo-oxygenase-2 and 5-lipoxygenase

Two series of phenylsulphonyl urenyl chalcone derivatives (UCH) with various patterns of substitution were tested for their effects on nitric oxide (NO) and prostaglandin E 2 (PGE 2) overproduction in RAW 264.7 macrophages. None of the tested compounds reduced NO production more than 50% at 10 μM but most of them inhibited the generation of PGE 2 with IC 50 values under the micromolar range. Me-UCH 1, Me-UCH 5, Me-UCH 9, Cl-UCH 1, and Cl-UCH 9 were selected to evaluate their influence on human leukocyte functions and eicosanoids generation. These derivatives selectively inhibited cyclo-oxygenase-2 (COX-2) activity in human monocytes being Me-UCH 5 the most potent (IC 50 0.06 μM). Selected compounds also reduced leukotriene B 4 synthesis in human neutrophils by a direct inhibition of 5-lipoxygenase (5-LO) activity, with IC 50 values from 0.5 to 0.8 μM. In addition, lysosomal enzyme secretion, such as elastase or myeloperoxidase as well as superoxide generation in human neutrophils were also reduced in a similar range. Our findings indicate that UCH derivatives exert a dual inhibitory effect on COX-2/5-LO activity. The profile and potency of these compounds may have relevance for the modulation of the inflammatory and nociceptive responses with reduction of undesirable side-effects associated with NSAIDs.

P21-activated kinase (Pak) regulates NADPH oxidase activation in human neutrophils

The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays an instrumental role in host defense and contributes to microbicial killing by releasing highly reactive oxygen species. This multicomponent enzyme is composed of membrane and cytosolic components that assemble in the plasma membrane or phagolysosome. While the guanosine S'-triphosphatase (GTPase) Rac2 has been shown to be a critical regulator of NADPH oxidase activity and assembly, the role of its effector, p21-activated kinase (Pak), in oxidase function has not been well defined. Using HIV-1 Tat-mediated protein transduction of Pak inhibitory domain, we show here that Pak activity is indeed required for efficient superoxide generation in intact neutrophils. Furthermore, we show that Pak translocates to the plasma membrane upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation and colocalizes with translocated p47(phox) and with p22phox, a subunit of flavocytochrome b558. Although activated Pak phosphorylated several essential serine residues in the C-terminus of p47phox, direct binding to p47phox was not observed. In contrast, active Pak bound directly to p22phox, suggesting flavocytochrome b was the oxidase-associated membrane target of this kinase and this association may facilitate further phosphorylation of p47phox in the assembling NADPH oxidase complex.

Effect of five triterpenoid compounds isolated from root bark of Aralia elata on stimulus-induced superoxide generation, tyrosyl or serine/threonine phosphorylation and translocation of p47 phox, p67 phox, and rac to cell membrane in human neutrophils

Five triterpenoids; Congmuyanoside V, X, XI, XII, and XV (Cong. V, X, XI, XII, and XV) were isolated from the root bark of Aralia elata. The effect of these triterpenoids on stimulus-induced superoxide generation in human neutrophils was assayed by measuring the reduction of ferricytochrome c ( cyt. c) using a dual-beam spectrophotometer. Translocation of p47 phox, p67 phox, and rac to the cell membrane and tyrosyl or serine/threonine phosphorylation of neutrophil proteins were investigated using specific monoclonal antibodies. The five triterpenoids used in the present experiment significantly suppressed N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in concentration-dependent manner. Congmuyanoside V, XII, and XV also suppressed arachidonic acid (AA)-induced superoxide generation in high concentration. However, these triterpenoids showed no effect on phorbol 12-myristate 13-acetate (PMA)-induced superoxide generation. fMLP- and AA-induced tyrosyl phosphorylation and translocation of the cytosolic proteins p47 phox, p67 phox, and rac to the cell membrane were suppressed in parallel with the suppression of the stimulus-induced superoxide generation.

Raised cortisol:DHEAS ratios in the elderly after injury: potential impact upon neutrophil function and immunity

The detrimental effect of stress on the immune response increases with age, though the mechanisms responsible are not fully understood. The physiological response to stress is regulated in part by the adrenocortical system. Adrenal hormones dehydroepiandrosterone sulphate (DHEAS) and cortisol have opposing effects on the innate immune system, DHEAS enhances while cortisol suppresses immunity and the molar ratio of cortisol to DHEAS increases with age. We found that elderly hip fracture patients produced a robust neutrophilia after injury, but circulating neutrophils showed an impaired antibacterial response. We therefore proposed that adrenocortical hormones mediate the heightened immunosuppression seen in the elderly after injury. We examined neutrophil function and adrenocortical hormone levels in elderly (> 65 years) hip fracture patients and age-matched healthy controls. Thirteen out of 35 elderly patients acquired infections following hip fracture. Neutrophil superoxide production was lower in elderly hip fracture patients compared with controls (P < 0.005) and lower in patients who acquired infection following injury compared with those who did not (P < 0.05). Serum cortisol:DHEAS ratio was higher in elderly hip fracture patients (0.56 +/- 0.38) compared with either age-matched controls (0.36 +/- 0.21; P < 0.05) or young fracture patients (0.087 +/- 0.033; P < 0.0001). Moreover, cortisol: DHEAS was increased in elderly patients who succumbed to infection compared with those who did not (0.803 +/- 0.42 vs. 0.467 +/- 0.28; P < 0.02). In vitro cortisol significantly decreased neutrophil superoxide generation (P < 0.05) and this was prevented by coincubation with DHEAS. We propose that increased cortisol:DHEAS ratios may contribute to reduced immunity following physical stress in the elderly.

Enhancement of antibacterial superoxide-anion generation in human monocytes by fumaric acid esters

Fumaric acid esters (FAE) are used for the systemic therapy of psoriasis with high clinical efficacy. Among the potential side effects of FAE therapy, lymphocytopenia is sometimes observed. We have investigated the effect of dimethylfumarate (DMF) and its main metabolite methylhydrogenfumarate (MHF) as well as dexamethasone on superoxide anion generation by human monocytes and neutrophils after stimulation with bacteria (Staphylococcus aureus and Escherichia coli) and the yeast Candida albicans in addition with zymosan particles and with the tripeptide fMLP. Expression of mannose receptors on monocytes and neutrophils was also analyzed. The results showed that dexamethasone significantly inhibited superoxide anion generation from monocytes in response to bacteria and C. albicans, whereas DMF as well as MHF dose dependently increased the production of superoxide anion in monocytes in response to zymosan, fMLP and bacteria. Dexamethasone, DMF or MHF did not modulate superoxide anion generation of neutrophils. Expression of mannose receptors on monocytes was not regulated by DMF or MHF. Our data provide evidence that DMF and MHF do not alter the production of superoxide anions as an important mechanism of innate defense against microorganisms.

Effect of Three Flavonoids Isolated from JapanesePolygonumSpecies on Superoxide Generation in Human Neutrophils

Three flavonoids, quercetin 3- O-glucoside-2''-gallate (QGG), quercetin 3- O-rhamnoside-2''-gallate (QRG) and kaempferol 3- O-glucoside-2''-gallate (KGG) were isolated from Japanese Polygonum species. The effect of these flavonoids on stimulus-induced superoxide generation in human neutrophils was assayed by measuring the reduction of cytochrome c. The tyrosyl or serine/threonine phosphorylation of neutrophil proteins and the translocation of p4(phox) and p67(phox) to the cell membrane were detected using specific monoclonal antibodies. The flavonoids used in this experiment significantly suppressed stimulus-induced superoxide generation in a concentration-dependent manner. FMLP-induced tyrosyl phosphorylation or PMA-induced serine/threonine phosphorylation and the translocation of the cytosolic proteins p47(phox) and p67(phox) to the cell membrane were suppressed in parallel to the suppression of the stimulus-induced superoxide generation.

Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen‐activated PK signaling pathways

1 Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system. 2 Pretreatment of neutrophils with pertussis toxin (1 microg ml(-1)), 50 microM 2'-amino-3'-methoxyflavone (PD 98059), or 1 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 microM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation. 3 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC50 value of 4.3+/-0.3 microM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation. 4 The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+-free medium, and by pretreatment with 1 microM 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 microM 2-aminoethyldiphenyl borate (2-APB). ART (30 microM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation. 5 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC50 value of 7.8+/-1.0 nM). ART increased the recruitment of PKC-alpha, -betaI, and -betaII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation. 6 ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47(phox), which was inhibited by GF 109203X and SB 203580. 7 These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, and PKC signaling pathways in rat neutrophils.

The evaluation of 2,8-disubstituted benzoxazinone derivatives as anti-inflammatory and anti-platelet aggregation agents

A series of 2,8-disubstituted benzoxazinones were synthesized and subjected to anti-platelet aggregation, inhibition of superoxide anion generation, and inhibition of neutrophil elastase release assays. Among them, 2-(2'-substituted-phenyl)-benzoxazinones exhibited significant inhibitory effect to target assays. Additionally, all of them were more potent than aspirin on AA-induced platelet aggregation, and these suggested that 2-(2'-substituted-phenyl)-benzoxazinones also possess aspirin-like activity. On the other hand, the compounds 6 and 16 showed inhibitory effects on neutrophil elastase release and superoxide generation.

New Phenylpropane and Anti-inflammatory Diterpene Derivatives fromAmentotaxus formosana

One new diterpene, 8(14),15-sandaracopimaradiene-2alpha,3beta,18-triol (1), two new phenylpropane derivatives, i.e., (E)-methyl 2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoate (2) and (E)-2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoic acid (3), and two known diterpenes, ent-8(14),15-sandaracopimaradiene-2alpha,18-diol (4) and 8(14),15-sandaracopimaradiene-2alpha,18,19-triol (5), were isolated from the heartwoods and barks of Amentotaxus formosana, respectively. The anti-inflammatory activity of the diterpenes 1, 4, and 5 was assessed in vitro by determining their inhibitory effects on the chemical mediators released from mast cells, neutrophils, macrophages, and microglial cells. Compounds 1, 4, and 5 showed significant concentration-dependent inhibitory effects on the release of beta-glucuronidase from rat neutrophils in response to formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB) with IC50 values of 5.5 +/- 1.8, 8.4 +/- 2.9 and 19.2 +/- 3.3 microM, respectively. Compounds 1 and 5 also showed significant concentration-dependent inhibitory effects on superoxide anion generation in rat neutrophils stimulated with fMLP/CB and phorbol 12-myristate 13-acetate (PMA) with IC50 values of 12.6 +/- 1.2 and 9.4 +/- 1.7, and 10.7 +/- 3.3 and 12.9 +/- 0.9 microM, respectively.

Potential Anti‐Inflammatory Activities of Bractelactone and other Compounds Isolated from Fissistigma bracteolatum

AbstractFrom the stems of Fissistigma bracteolatum, a novel natural product with an unprecedented skeleton, bractelactone (1), was isolated, together with four known compounds: piperolactam A (2), aristololactam BIII (3), aristololactam BII (4), and fissilandione (5). The structure of 1 was established on the basis of spectroscopic data as (3Z)‐6,7‐dihydroxy‐4‐methoxy‐3‐(phenylmethylidene)‐5‐(3‐phenylpropanoyl)‐1‐benzofuran‐2(3H)‐one. This compound may be derived from a hybrid of a chalcone and a cinnamic acid, or from a degradation product of a dichalcone. Compounds 1, 2, and 5 showed inhibitory effects on NO generation by RAW264.7 macrophages in response to lipopolysaccharide. Compounds 2 and 5 showed inhibitory effects on formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine (fMLP)‐induced superoxide anion (O$\rm{_{2}^{{^\cdot} -}}$) generation in human neutrophils.

Fish oil before cardiac surgery: neutrophil activation is unaffected but myocardial damage is moderated

Could pre-operative dietary intervention with fish oil reduce neutrophil activation and myocardial damage associated with cardiopulmonary bypass (CPB)? Patients were randomised to receive either 8 g/day fish oil (n=22) or placebo (n=18) for 6 weeks. Neutrophil activation, apoptosis and cardiac damage were measured. Demographics and operative variables were similar. Fish oil diet decreased plasma VLDL from 0.69+/-0.34 to 0.51+/-0.24 mmol/l and triglycerides from 1.68+/-0.70 to 1.39+/-0.54 mmol/l. HDL cholesterol increased from 0.94+/-0.27 to 1.03+/-0.26 mmol/l demonstrating significant treatment effects (P=0.007, 0.02 and 0.0003, respectively) as well as compliance with treatment. There were no significant differences in ex vivo N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophil superoxide anion generation or myeloperoxidase release at recruitment, pre-operatively and at end-CPB. Apoptosis at end-CPB was equally reduced in both groups from 23+/-9% to 13+/-4% in the fish oil group (P<0.001) and 35+/-14% to 15+/-3% in the placebo group (P=0.001). At end-CPB overall troponin I levels averaged 0.91+/-0.60 ng/ml which clearly exceeded diagnostic levels (0.15 ng/ml). At 24h troponin I fell significantly in the fish oil group to 46+/-23% of end-CPB levels (P=0.0002) whereas it peaked in the placebo group to 107+/-72% (P=0.098 vs. end-CPB); this difference was significant: P=0.013. At 48 h the placebo-treated patients had higher troponins but not significantly so (P=0.059). Area-under-the-curve analysis did not conclusively support this (P=0.068). We conclude that fish oil did not significantly decrease post-CPB neutrophil activation (as detected ex vivo) but may moderate post-operative myocardial damage.

Calcium antagonists in dermatology: A review of the evidence and research-based studies

Calcium antagonists (CAs) or calcium-channel blockers are a common group of antihypertensive medications. These drugs have the property of blocking the calcium channels of vascular and cardiac smooth muscle fibers. Some of these drugs may inhibit the growth and proliferation of vascular smooth muscle cells and fibroblasts, and inhibit the synthesis of extracellular-matrix proteins (collagen, fibronectin, proteoglycans). Other CAs also have immunomodulatory or dysregulatory effects on lymphocytes and can suppress superoxide generation and phagocytic action of neutrophils. Moreover mast cell degranulation and platelet aggregation may also be impaired. On account of these properties, calcium antagonists have also been used for the prevention and treatment of various dermatologic diseases such as erythromelalgia, idiopathic- or CREST-related calcinosis cutis, primary and secondary Raynaud phenomenon, chilblains, chronic anal fissures, keloids, and burn scars. They are also used for prevention of skin flap necrosis in experimental models. Calcium antagonists, apart from their well known and established antihypertensive action, should also be considered as possible therapy for several dermatologic diseases.

Effect of sulfur amino acids on stimulus-induced superoxide generation and translocation of p47 phox and p67 phox to cell membrane in human neutrophils and the scavenging of free radical

Background Various cystathionine metabolites are in the urine of the patients with cystathioninuria. Among these metabolites, cystathionine ketimine significantly enhanced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in parallel with tyrosyl phosphorylation of 45 kDa protein in human neutrophils. Methods We investigated the effect of various sulfur amino acids on fMLP-, phorbol-12-myristate-13-acetate (PMA)- and arachidonic acid (AA)-induced superoxide generation in human neutrophils. In addition, the effects of these sulfur amino acids on the membrane translocation of cytosolic compounds p47 phox and p67 phox and on the scavenging of superoxide anions were investigated. Results When the cells were preincubated with various sulfur amino acids, fMLP-induced superoxide generation was enhanced by d,l-homocysteine and d,l-homocysteine-thiolactone but was inhibited by other sulfur amino acids in a concentration-dependent manner. The AA-induced superoxide was enhanced by l-cysteine, N-acetyl- l-cysteine and d,l-homocysteine. The strength of enhancing effect was: l-cysteine≫ N-acetyl- l-cysteine> d,l-homocysteine. On the other hand, the superoxide generation was weakly inhibited by l-cystathionine. The superoxide generation induced by PMA was weakly inhibited by l-cysteine, N-acetyl- l-cysteine and l-cystathionine. Homocysteine and d,l-homocysteine-thiolactone had no effect. In addition, d,l-homocysteine also enhanced translocation to the cell membrane of cytosolic compounds p47 phox and p67 phox. Conversely, l-cystathionine and N-acetyl- l-cysteine inhibited the translocation to membrane of p47 phox and p67 phox in a concentration-dependent manner. N-acetyl- l-cysteine and l-cysteine revealed scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The sulfur amino acids tested also indicated radical scavenging activity on superoxide anion generated by phenazine methoxysulfate (PMS)–NADH system. Conclusion d,l-homocysteine and d,l-homocysteine-thiolactone enhanced fMLP-induced superoxide generation by the increment of translocation to membrane of p47 phox and p67 phox. l-cystathionine and N-acetyl- l-cysteine suppressed fMLP- and PMA-induced superoxide generation by the inhibition of translocation to membrane of p47 phox and p67 phox. N-acetyl- l-cysteine also had scavenging activity against DPPH radicals and superoxide anion.