Burst Of Neutrophils Research Articles

Redox-active biflavonoids from Garcinia brasiliensis as inhibitors of neutrophil oxidative burst and human erythrocyte membrane damage

Ethnopharmacological relevanceGarcinia brasiliensis, a plant native to the Brazilian Amazon Rainforest, is used in traditional medicine to treat inflammation of the urinary tract, peptic ulcers, arthritis and other conditions. Aim of the studyThe purposes of this study were to analyze the chemical constituents of G. brasiliensis branches and leaves and to evaluate the potential of isolated compounds to act as inhibitors of both the oxidative burst of stimulated neutrophils and oxidative damage in human erythrocyte membranes to verify the antioxidant and anti-inflammatory effects of this plant. Materials and methodsNeutrophils were isolated from the blood of healthy donors by Ficoll–Paque density gradient centrifugation. Superoxide anion and total reactive oxygen species (ROS) produced by stimulated neutrophils were measured by WST-1 reduction and luminol-enhanced chemiluminescence assays, respectively. Radical-induced lipoperoxidation and hemolysis were performed using erythrocytes from the blood of healthy donors. Compounds were isolated from G. brasiliensis branches and leaves by HPLC microfractionation, and structure elucidation of the isolated compounds was performed based on NMR and HR-MS analyses. ResultsThe biflavonoids procyanidin, fukugetin, amentoflavone and podocarpusflavone isolated from G. brasiliensis showed potent inhibitory effects on the oxidative burst of human neutrophils, inhibiting ROS production by 50% at 1μmolL−1. These biflavonoids also proved to be potent inhibitors of hemolysis (with 88±7% inhibition at 50µmolL−1 for procyanidin) and lipid peroxidation in human erythrocytes, with a malondialdehyde level (a biomarker of oxidative stress) of 8.5±0.3nmol/mg Hb at 50µmolL−1 for procyanidin. ConclusionsThese findings indicate that the biflavonoids extracted from G. brasiliensis branches and leaves modulate oxidative stress via inhibition of NADPH oxidase and ROS production by stimulated human neutrophils. Furthermore, the biflavonoids exhibited potent inhibition of oxidant hemolysis and lipid peroxidation induced by AAPH in human erythrocytes. Therefore, these studies suggest the use of G. brasiliensis extract as an antioxidant and anti-inflammatory agent.

Discrete roles of intracellular phospholipases A2 in human neutrophil cytotoxicity.

The role of calcium-independent phospholipase A2 (iPLA2), a component of the three major PLA2 families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA2-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA2 is unidentified. We also demonstrated that the iPLA2γ-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA2γ may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA2 (cPLA2), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA2γ regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A2, iPLA2, and cPLA2, in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA. Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2β, iPLA2γ, or cPLA2 and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system. The iPLA2γ-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA2-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA2γ, but not iPLA2β or cPLA2, completely inhibited the platelet activating factor/fMLP-induced chemotaxis. iPLA2 is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA2 inhibitor suggests that AA may not be relevant to these cytotoxic functions.

Anti-inflammatory and antioxidant properties of isolated compounds from Garcinia brasiliensis

Garcinia brasiliensis, a plant native to the Brazilian Amazon Rainforest, is used in traditional medicine to treat inflammation of the urinary tract, peptic ulcers, arthritis and others. The purposes of this study were to analyze the chemical constituents of branches and leaves from G. brasiliensis and to evaluate the potential of isolated compounds to act as inhibitors of both the oxidative burst of stimulated neutrophils and oxidative damage in human erythrocyte membranes. The biflavonoids procianidine, fukugetine, amentoflavone and podocarpus flavone isolated from G. brasiliensis had potent inhibitory effect on the oxidative burst of neutrophils, reaching 50% inhibition at 1µmol L-1 on total reactive oxygen species (ROS) production and NADPH enzyme. These biflavonoids were also potent inhibitors of hemolysis and lipid peroxidation of human erythrocytes, reaching the malondialdehyde level (a biomarker of oxidative stress) of 8.5 ± 0.3 nmol/mg Hb at 50µmol L-1 for procianidine. These findings indicate that these biflavonoids modulate the oxidative stress, suggesting the use of G. brasiliensis extract as antioxidants and anti-inflammatory agents. Acknowledgements: FAPESP (grants #2010/52327 – 5, #2011/03017 – 6 and #2013/07600 – 3), CNPq and CAPES.

Inhibition of the lymphocyte metabolic switch by the oxidative burst of human neutrophils.

Activation of the phagocytic NADPH oxidase-2 (NOX-2) in neutrophils is a critical process in the innate immune system and is associated with elevated local concentrations of superoxide, hydrogen peroxide (H2O2) and hypochlorous acid. Under pathological conditions, NOX-2 activity has been implicated in the development of autoimmunity, indicating a role in modulating lymphocyte effector function. Notably, T-cell clonal expansion and subsequent cytokine production requires a metabolic switch from mitochondrial respiration to aerobic glycolysis. Previous studies demonstrate that H2O2 generated from activated neutrophils suppresses lymphocyte activation but the mechanism is unknown. We hypothesized that activated neutrophils would prevent the metabolic switch and suppress the effector functions of T-cells through a H2O2-dependent mechanism. To test this, we developed a model co-culture system using freshly isolated neutrophils and lymphocytes from healthy human donors. Extracellular flux analysis was used to assess mitochondrial and glycolytic activity and FACS analysis to assess immune function. The neutrophil oxidative burst significantly inhibited the induction of lymphocyte aerobic glycolysis, caused inhibition of oxidative phosphorylation and suppressed lymphocyte activation through a H2O2-dependent mechanism. Hydrogen peroxide and a redox cycling agent, DMNQ, were used to confirm the impact of H2O2 on lymphocyte bioenergetics. In summary, we have shown that the lymphocyte metabolic switch from mitochondrial respiration to glycolysis is prevented by the oxidative burst of neutrophils. This direct inhibition of the metabolic switch is then a likely mechanism underlying the neutrophil-dependent suppression of T-cell effector function.

Esmolol: immunomodulator in pyelonephritis by Pseudomonas aeruginosa

BackgroundBased on previous animal studies showing promising immunomodulatory efficacy esmolol, a selective β1-blocker, it was assumed that administration of esmolol in experimental pyelonephritis by multidrug-resistant Pseudomonas aeruginosa would prolong survival and modulate immune response. MethodsAcute pyelonephritis was induced in 80 rabbits and assigned to eight groups receiving normal saline (controls), esmolol, amikacin, or both agents as pretreatment and as treatment. Blood was sampled for measurement of malondialdehyde and tumor necrosis factor alpha. Animals were followed up for survival, and after death quantitative tissue cultures were performed. The in vitro effect of esmolol on bacterial growth and on the oxidative burst of neutrophils of healthy controls and of sepsis patients was studied. ResultsSurvival of pretreatment groups administered single esmolol or esmolol and amikacin was prolonged compared with that of controls (P = 0.018 and P = 0.014, respectively); likewise, survival of treatment groups administered single esmolol or both agents was prolonged compared with that of controls (P = 0.007 and P = 0.014, respectively). Circulating malondialdehyde was significantly lower in pretreated animals administered esmolol or esmolol and amikacin compared with that in controls and in treated animals administered both agents compared with in controls (P = 0.020). In these groups, the bacterial load of the lung was significantly lower compared with controls. Serum tumor necrosis factor alpha did not change. Amikacin was increased in serum of esmolol-treated animals at levels which inhibited the in vitro growth of the studied isolate. Esmolol did not modify the in vitro growth of P aeruginosa and the oxidative burst of neutrophils. ConclusionsIt is concluded that esmolol prolonged survival after experimental infection by multidrug-resistant P aeruginosa. Survival benefit may be related with pleiotropic actions connected with modulation of pharmacokinetics and attenuation of inflammation.

Stem bark and flower extracts of Vismia cauliflora are highly effective antioxidants to human blood cells by preventing oxidative burst in neutrophils and oxidative damage in erythrocytes

Context: Vismia cauliflora A.C.Sm. [Hypericaceae (Clusiaceae)] is an Amazonian plant traditionally used by indigenous population to treat dermatosis and inflammatory processes of the skin. Previous research on V. cauliflora extracts suggests its potential to neutralize cellular oxidative damages related to the production of reactive oxygen and nitrogen species.Objective: To determine the activity of stem bark and flower extracts of V. cauliflora on the modulation of oxidative burst in human neutrophils, as well as its potential to inhibit oxidative damage in human erythrocytes.Materials and methods: The modulation of neutrophil’s oxidative burst by the ethanolic extracts (0.3–1000 µg/mL) was determined by the oxidation of specific probes by reactive species. Additionally, the potential of these extracts to inhibit oxidative damage in human erythrocytes was evaluated by monitoring its biomarkers of oxidative stress.Results: Vismia cauliflora extracts presented remarkable capacity to prevent the oxidative burst in activated human neutrophils (IC50 < 15 µg/mL). However, the maximum percentage of inhibition achieved against hydrogen peroxide was 45%. Concerning the oxidative damage in human erythrocytes, the extracts were able to minimize the tert-butyl hydroperoxide-induced hemoglobin oxidation and lipid peroxidation in a very low concentration range (2.7–18 μg/mL). Furthermore, only stem bark extract (100 µg/mL) was able to inhibit the depletion of glutathione (13%).Discussion and conclusion: These results reinforce the therapeutic potential of stem bark and flower extracts of V. cauliflora to heal topical skin disease, namely in the treatment of neutrophil-related dermatosis and skin conditions related to oxidative stress, including skin aging.

The anti-inflammatory effect of 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol by targeting Lyn kinase in human neutrophils

The undesirable respiratory burst in neutrophils can lead to inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol (honokiol), a lignan extracted from the stem bark of Magnolia officinalis Rehd. et Wils (Magnoliaceae), on N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)-induced respiratory burst in human neutrophils. Signaling pathways regulated by honokiol which modulate fMLP-induced respiratory burst and cathepsin G release were evaluated by phosphorylation of Src family kinase induced by fMLP, Src family kinases activities and by immunoblotting analysis of the downstream targets of Src kinase. Briefly, honokiol inhibited fMLP-induced superoxide anion production (IC50=9.80±0.21μM, n=4), cathepsin G release (IC50=14.23±1.43μM, n=4) and migration (IC50=5.69±1.51μM, n=4) in a concentration dependent manner. Further, honokiol specifically suppresses fMLP-induced Lyn (a member of the Src kinase family) phosphorylation, by inhibiting Lyn kinase activity. Consequently, honokiol attenuated the downstream targets of Lyn kinase, such as Tec translocation from the cytosol to the inner leaflet of the plasma membrane, phosphorylation of AKT, P38, PLCγ2, protein kinase C and membrane localization of p47phox. On the other hand, fMLP-induced phosphorylation of Hck, Fgr kinase activity (other members of Src kinase), downstream phosphorylation of Vav1 and extracellular signal-regulated kinase remained unaffected. In addition, honokiol neither inhibited NADPH oxidase activity nor increased cyclic AMP levels. Honokiol is not a competitive or allosteric antagonist of fMLP. In conclusion, honokiol specifically modulates fMLP-mediated neutrophil activation by inhibiting Lyn activation which subsequently interferes with the activation of PLCγ2, AKT, p38, protein kinase C, and p47phox.

The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis.

Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.

Involvement of high mobility group box 1 in the activation of C5a-primed neutrophils induced by ANCA

C5a plays a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. A previous study showed that C5a played a crucial role in the regulation of high mobility group box 1 (HMGB1) release from human neutrophils. The current study further investigated the interaction between C5a and HMGB1 in ANCA-induced neutrophil activation. The effects of HMGB1 inhibitors on the translocation of ANCA antigens, ANCA-induced respiratory burst and degranulation of C5a-primed neutrophils were tested. We found that blocking HMGB1 decreased C5a-mediated translocation of ANCA antigens, as well as ANCA-induced respiratory burst and degranulation of C5a-primed neutrophils. Further study showed that supernatant of C5a-primed neutrophils, which contained HMGB1, also caused translocation of ANCA antigens of primary neutrophils, whereas blocking HMGB1 decreased the translocation. In conclusion, blocking HMGB1 may attenuate ANCA-induced activation of C5a-primed neutrophils. The interaction between HMGB1 and C5a might play an important role in ANCA-induced neutrophil activation.

The Effect of Sphingosine 1-Phosphate/Sphingosine 1-Phosphate Receptor on Neutrophil Function and the Relevant Signaling Pathway

Background/Aims: This study investigated the priming effect of sphingosine 1-phosphate (S1P) on formyl-Met-Leu-Phe-OH (fMLP)-activated neutrophils, by specific analysis of the neutrophil respiratory burst and the signaling pathway involved in S1P activity. Methods: The neutrophil respiratory burst was indirectly detected by the cytochrome c reduction method and the dihydrorhodamine 123 staining method. The signal transduction pathways of neutrophil respiratory burst primed by S1P were detected by Western blotting. Results: Our results showed that the S1P receptors (S1PRs) 1, 4 and 5 were the predominantly expressed neutrophil S1PRs at the cDNA level. After pretreatment with S1P, the fMLP-activated neutrophils released increased levels of superoxide anions. PI3K and Akt proteins were involved in the signaling pathway of the neutrophil respiratory burst primed by S1P. Conclusion: The results indicate that S1P is a new priming reagent for neutrophils and primes the respiratory burst of fMLP-activated neutrophils. S1P interacts with its receptors on neutrophils, resulting in NADPH oxidase activation by the PI3K-Akt cell signaling pathway and induction of the neutrophil respiratory burst.

Suppression of neutrophil superoxide generation by BNP is attenuated in acute heart failure: a case for 'BNP resistance'.

The release of the B-type natriuretic peptide (BNP) is increased in heart failure (HF), a condition associated with oxidative stress. BNP is known to exert anti-inflammatory effects including suppression of neutrophil superoxide (O2(-)) release. However, BNP-based restoration of homeostasis in HF is inadequate, and the equivocal clinical benefit of a recombinant BNP, nesiritide, raises the possibility of attenuated response to BNP. We therefore tested the hypothesis that BNP-induced suppression of neutrophil O2(-) generation is impaired in patients with acute HF. We have recently characterized suppression of neutrophil O2(-) generation (PMA- or fMLP-stimulated neutrophil burst) by BNP as a measure of its physiological activity. In the present study, BNP response was compared in neutrophils of healthy subjects (n = 29) and HF patients (n = 45). Effects of BNP on fMLP-induced phosphorylation of the NAD(P)H oxidase subunit p47phox were also evaluated. In acute HF patients, the suppressing effect of BNP (1 µmol/L) on O2(-) generation was attenuated relative to that in healthy subjects (P < 0.05 for both PMA and fMLP). Analogously, BNP inhibited p47phox phosphorylation in healthy subjects but not in HF patients (P < 0.05). However, O2(-)-suppressing effects of the cell-permeable cGMP analogue (8-pCPT-cGMP) were preserved in acute HF. Conventional HF treatment for 5 weeks partially restored neutrophil BNP responsiveness (n = 25, P < 0.05), despite no significant decrease in plasma NT-proBNP levels. BNP inhibits neutrophil O2(-) generation by suppressing NAD(P)H oxidase assembly. This effect is impaired in acute HF patients, with partial recovery during treatment.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4(+) T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 μmol/kg) and leukocyte infiltration (ED50 = 0.188 μmol/kg) with an efficacy comparable to a high dose of budesonide (1 μmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.

Molecular pharmacology of antihistamines in inhibition of oxidative burst of professional phagocytes.

Antihistamines of the H₁and H₃/H₄groups interfere with oxidative burst of human professional phagocytes in vitro. In the concentration of 10 μM, H₁antihistamines of the 1st and 2nd generation inhibited oxidative burst of human neutrophils in the rank order of potency: dithiaden > loratadine > brompheniramine > chlorpheniramine > pheniramine. Of the H₁antihistamines, the most effective was dithiaden in suppressing oxidative burst of whole human blood and dose-dependently the chemiluminescence of isolated neutrophils at extra- and intracellular level. Inhibition of free oxygen radical generation in isolated neutrophils by dithiaden resulted from the inhibition of protein kinase C activation. The potentiation of recombinant caspase-3 by dithiaden is supportive of the antiinflammatory effect of dithiaden and suggestive of increasing the apoptosis of professional phagocytes. Of the H₃/H₄antihistamines, the most effective was JNJ7777120 in decreasing chemiluminescence in whole blood and also at extra- and intracellular sites of isolated neutrophils. JNJ 10191584 and thioperamide were less effective and the latter significantly potentiated free oxygen radical generation intracellularly. The results demonstrated that, compared with the H₃/H₄antihistamines investigated, H₁antihistamines were much more potent in inhibiting free oxygen radical generation in human professional phagocytes. This finding should be taken into account therapeutically.

Thiotaurine modulates human neutrophil activation.

In acute inflammation, neutrophils are activated in response to several agonists generating reactive oxygen species (ROS) by NADPH oxidase-dependent mechanisms. Thiotaurine, a thiosulfonate structurally related to hypotaurine and taurine, is a metabolic product of cystine and contains sulfane sulfur which can play several regulatory roles in biological systems. In the present study, the effects of thiotaurine on human leukocyte activation have been evaluated. Neutrophils were stimulated by phorbol 12-myristate 13-acetate (PMA) or chemotactic peptide (fMLP) and respiratory burst was assessed by luminol enhanced chemiluminescence. Superoxide generation was estimated by measuring cytochrome c reduction. Thiotaurine caused a decrease of the respiratory burst of PMA-stimulated neutrophils in a dose-dependent manner, while fMLP-stimulated cells were slightly affected by thiotaurine. Addition of 100 μM thiotaurine induced 64.4 % inhibition of ROS production by PMA-activated cells, as evaluated by luminol enhanced chemiluminescence. As thiotaurine can release sulfane sulfur producing hydrogen sulfide (H2S) and hypotaurine, the effects of these compounds on leukocyte functional responses were evaluated and compared with those exerted by thiotaurine, which had proved more effective in inhibiting respiratory burst than H2S, hypotaurine and taurine. However, fMLP-induced azurophilic degranulation as analyzed by elastase release was unaffected by thiotaurine up to 5 mM concentration. Compared with fMLP-activated neutrophils, PMA-activated neutrophils were more susceptible to thiotaurine inhibition, suggesting that thiotaurine may interfere with the PKC-dependent pathway of neutrophil activation.

Payload drug vs. nanocarrier biodegradation by myeloperoxidase- and peroxynitrite-mediated oxidations: pharmacokinetic implications.

With the advancement of nanocarriers for drug delivery into biomedical practice, assessments of drug susceptibility to oxidative degradation by enzymatic mechanisms of inflammatory cells become important. Here, we investigate oxidative degradation of a carbon nanotube-based drug carrier loaded with Doxorubicin. We employed myeloperoxidase-catalysed and peroxynitrite-mediated oxidative conditions to mimic the respiratory burst of neutrophils and macrophages, respectively. In addition, we revealed that the cytostatic and cytotoxic effects of free Doxorubicin, but not nanotube-carried drug, on melanoma and lung carcinoma cell lines were abolished in the presence of tumor-activated myeloid regulatory cells that create unique myeloperoxidase- and peroxynitrite-induced oxidative conditions. Both ex vivo and in vitro studies demonstrate that the nanocarrier protects the drug against oxidative biodegradation.

Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.

A Novel C5 Complement Inhibitor Protects Against Sepsis-Induced Activation of Complement, Coagulation and Inflammation and Provides Survival Benefit in E. coli Sepsis

Bacterial sepsis triggers robust early activation of the complement system that leads to the formation of the C5b-9 terminal complex (TCC) and potent proinflammatory anaphylatoxins, C3a and C5a, which significantly contribute to organ failure and death. Complement inhibition at C3 level provides organ protection in low bacteremia experimental sepsis but may impair the clearance of the bacteria. Here we used a baboon model of E. coli sepsis and in vitro whole-blood assays to test the protective effects of a novel C5 inhibitor (RA101295, a 2036 Da cyclic peptide). In vitro whole blood assays were performed to study the effects of RA101295 on sepsis-induced complement activation, inflammation, oxidative stress and phagocytosis of the bacteria. Lepirudin-anticoagulated blood was incubated with 5x107 cfu/ml E. coli or 500 µg/ml lipopolysaccharide (LPS) for 30-240 min in the presence/absence of the inhibitor. Results showed that RA101295 strongly inhibited E. coli and LPS–dependent C5b-9 and C5a generation. Similarly, RA101295 inhibited the E. coliand LPS induced oxidative burst in neutrophils and monocytes by 40-50% with minimal decrease (<15%) on their phagocytic function.Next, we used a baboon model of E. coli sepsis to investigate the in vivo effects of RA101295. E. coli challenge (1-2x1010 cfu/kg; LD100) in baboons strongly induced the generation of soluble C5b-9 complexes. Treatment with RA101295 C5 inhibitor (four subcutaneous 10 mg/kg doses at 8-12 hrs intervals) fully inhibited complement activation. RA101295 inhibitor remarkably improved the clinical parameters of all treated animals. Two of three baboons survived the lethal dose; while the survival time of the third animal was increased by 300%. Animals treated with complement inhibitor displayed better heart and lung performance and had lower febrile response than non-treated septic baboons.RA101295 treatment of E. coli sepsis led to significantly lower consumptive coagulopathy as reflected by the APTT/PT, fibrinogen consumption and fibrin/fibrinogen degradation products. These data highlight the tight crosstalk between the coagulation and complement systems and suggest that the observed protective effects are due in part to dampening of sepsis-induced coagulopathy. The treatment during the bacteremic stage had no negative effects on bacterial clearance. Similar to the in vitro data, these results suggest that C5 inhibition with this peptide does not increase the risk of secondary infections and reoccurrence of sepsis, probably because is not interfering with the generation of C3b opsonin. Histologic analysis of organs confirmed that treatment with RA101295 provided substantial organ protection. In contrast to the non-treated group, RA101295 treated animals showed no obvious signs of thrombosis and capillary leak in the lungs; no follicular necrosis in the spleen and no tubular necrosis and glomerular thrombosis in the kidneys.Overall, our data demonstrate that RA101295 strongly inhibits LPS and E. coli induced complement activation and TCC without affecting the bacterial clearance. These results suggest that complement inhibition at C5 level represents a promising therapeutic target in the fight against sepsis-induced MOF. DisclosuresKeshari:Oklahoma Medical Research Foundation: Employment. Silasi-Mansat:Oklahoma Medical Research Foundation: Employment. Popescu:Oklahoma Medical Research Foundation: Employment. Chaaban:Oklahoma University Health Sciences Center: Employment. Lupu:Oklahoma Medical Research Foundation: Employment. DeMarco:Ra Pharmaceuticals: Employment. Lupu:American Heart Association: Membership on an entity’s Board of Directors or advisory committees; Merck: Research Funding; NIH: Research Funding; Oklahoma Medical Research Foundation: Employment.

Artostyracins A–C, three new isoprenylated 2-arylbenzofurans from Artocarpus styracifolius

Three new isoprenylated 2-arylbenzofurans, namely artostyracins A–C (1–3, resp.), together with a known one (4), were isolated from the root of Artocarpus styracifolius Pierre. Their structures were determined by spectroscopic methods (1D and 2D NMR, UV, IR, and HR-ESI-MS). All of the compounds were evaluated for the anti-respiratory burst properties using a cellular model reproduced by chemical stimulation of rat neutrophils. Compounds 1 and 3 offered the potently inhibitory effects on respiratory burst of rat neutrophils with IC50 values of 1.42 and 1.91μM, while compounds 2 and 4 revealed moderate suppression activity with IC50 values of 11.56 and 46.91μM.

B-Type natriuretic peptide suppression of neutrophil superoxide generation: mechanistic studies in normal subjects.

Many acute cardiovascular disease states are associated with neutrophil infiltration of myocardium and subsequent release of superoxide (O2 (-) ) and myeloperoxidase (MPO), which contribute to inflammatory reactions. B-Type natriuretic peptide (BNP) is known to exert anti-inflammatory and antifibrotic effects, but it is not known whether these may include interactions with neutrophils. In neutrophils isolated from 20 healthy subjects, we assessed the effect of BNP on the 'neutrophil burst' (O2 (-) production and MPO release) stimulated by phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), respectively. Effects of BNP on cGMP accumulation, and the effects of the cell-permeable cGMP analogue 8-(4-chlorophenylthio) guanosine-cGMP (8-p-CPT-cGMP) and protein kinase G (PKG) inhibition with KT5823 on the neutrophil-BNP interaction were also evaluated. B-Type natriuretic peptide suppressed O2 (-) release from neutrophils by 23±6% (P<0.001) and 24±8% (P<0.05) following PMA and fMLP stimulation, respectively. Although BNP did not significantly increase cGMP formation, 8-p-CPT-cGMP suppressed both PMA- and fMLP-induced neutrophil O2 (-) release by 16% and 28%, respectively (P<0.05). The PKG inhibitor KT5823 attenuated the effects of BNP on both fMLP- and PMA-associated O2 (-) production. Neither BNP nor 8-p-CPT-cGMP significantly affected MPO release from neutrophils. Suppression of O2 (-) release from neutrophils by BNP may contribute to its anti-inflammatory and antifibrotic actions.

P73 - Stem bark and flower extracts of Vismia cauliflora: modulation of oxidative burst in human neutrophils’ and inhibition of oxidative damage in human erythrocytes

Vismia cauliflora is an Amazonian plant traditionally used to treat dermatosis and inflammatory processes of the skin by indigenous population. Our research group showed that stem bark and flower extracts of V. cauliflora are efficient in vitro scavengers of reactive oxygen and nitrogen species. In this study, we determined the activity of stem bark and flower extracts of V. cauliflora plant on the modulation of in vitro oxidative burst in human neutrophils and their potential to inhibit the oxidative damage in human erythrocytes. The oxidative burst in activated neutrophils were monitored by specific probes to detect the oxidizing effect of superoxide anion radical (MCLA), hydrogen peroxide (amplex red) and hypochlorous acid (APF), and both extracts were efficient to neutralize the oxidative burst (IC50 from 3 to 15µg/mL). These same extracts were also effective against oxidative damage in erythrocytes by inhibiting hemoglobin oxidation (IC50=18µg/mL) and lipid peroxidation (IC50=2.7 and 7.5µg/mL, flower and stem bark, respectively). In addition, stem bark extract (100µg/mL) inhibited the depletion of glutathione by 13%. These extracts have similar phenolic composition, but flower presents quercetin (14%) in its composition. Therefore, these results reinforce the potential therapeutic of stem bark and flower extracts of V. cauliflora to heal topical skin disease and requires further research targeted effectively to develop phytopharmaceutical drug based on this plant.