Burst Of Neutrophils Research Articles

P28 - Anti-inflammatory and pro-apoptotic activities of chlorinated flavonoids in human neutrophils

Flavonoids have been suggested as potential therapeutic agents due to their ability avoid tissue damage resulting from inflammatory processes. Flavonoids are known to react with hypochlorous acid produced by neutrophils, to form stable mono and dichlorinated products. However, in spite of the possible production of these products at inflammatory sites, little is known about their potential biological activity. In this study, we investigated the effect of chlorinated flavonoids and their parent compounds in neutrophils’ oxidative burst and in its lifespan. The obtained results demonstrate that chlorinated flavonoids were more efficient than their parent compounds in modulating neutrophils’ oxidative burst in phorbol myristate acetate-activated neutrophils. Some of the tested flavonoids induce neutrophil apoptosis in a caspase 3-dependent fashion. The present data showed that some of the tested favonoids constitute an alternative anti-inflammatory therapy, due to the proven ability to suppress mechanisms engaged at the onset and progression of inflammation.

Synthesis of chlorinated flavonoids with anti-inflammatory and pro-apoptotic activities in human neutrophils

Neutrophils are considered the central cells of acute inflammation. Flavonoids have been suggested as therapeutic agents to avoid damages induced by inflammatory processes. It is well known the reactivity of flavonoids with hypochlorous acid produced by neutrophils, to form stable mono and dichlorinated products. In this study, we synthesized novel chlorinated flavonoids and investigated their effect in neutrophils' oxidative burst and in its lifespan, in comparison with the parent non-chlorinated flavonoids. The obtained results demonstrate that chlorinated flavonoids were more efficient than their parent compounds in modulating neutrophils' oxidative burst in phorbol myristate acetate-activated neutrophils. Some of the tested flavonoids drive neutrophil apoptosis in a caspase 3-dependent fashion. The present data showed that 8-chloro-3′,4′,5,7-tetrahydroxyflavone (4a) constitute an alternative anti-inflammatory therapy, due to the proven ability to suppress mechanisms engaged at the onset and progression of inflammation.

Amyloid-beta (25-35) peptide induces the release of pro-matrix metalloprotease 9 (pro-MMP-9) from human neutrophils.

Alzheimer's disease (AD) is a degenerative process of the brain, leading to increasing impairment of cognitive functions, and is associated with accumulation in the brain of several amyloid-beta (Aβ) peptides (as amyloid plaques), including Aβ25-35. Neutrophils, the most abundant immune cell type infiltrated in the brain of AD patients, accumulate behind amyloid plaques. Aβ peptides can trigger activation of chemotaxis and oxidative burst in neutrophils, suggesting a role in modulating the neuroinflammation process. We have shown that Aβ25-35 can induce the release from human neutrophils of pro-MMP-9, a metalloprotease involved in the onset of inflammation, corroborating the hypothesis of the involvement of infiltrated neutrophils in the inflammatory processes, which occur in the AD brain.

Antioxidant activity of hyaluronic acid investigated by means of chemiluminescence of equine neutrophil bursts and electron paramagnetic resonance spectroscopy.

Activated neutrophils (PMNs), the ROS/RNS released by PMNs and the derived inflammatory processes are involved in the pathogenesis and progression of human inflammatory airway diseases. Similar diseases are also present in horses which suffer from recurrent airway obstruction (RAO), exercise-induced pulmonary haemorrhage (EIPH) and inflammatory airway diseases (IAD). Hyaluronic acid (HA) plays numerous roles in modulating inflammatory processes. The aim of this study was to examine whether a preparation of HA (MW 900 000 Da) interferes with ROS/RNS during the course of equine PMN respiratory bursts, and to establish the lowest concentration at which it still has antioxidant activity by means of luminol-amplified chemiluminescence (LACL). Electron paramagnetic resonance (EPR) spectroscopy was also used to investigate the direct antiradical activity of HA. The hydroxyl radical was significantly scavenged in a concentration-dependent manner at HA concentrations ranging from 2.5 to 0.16 mg/mL. Superoxide anion, Tempol radical and the ABTS(•+) were significantly inhibited at concentrations ranging from 2.5 to 0.62 mg/mL. The LACL of stimulated equine neutrophils showed that HA induced a statistically significant concentration-effect reduction from 5 mg/mL to 1.25 mg/mL. These findings were confirmed also when l-Arg was added to investigate the inhibition of the resulting peroxynitrite anion. Our findings indicate that, in addition to the human use, HA can also be used to antagonize the oxidative stress generated by free radicals in horses peripheral blood mononuclear cells (PBMCs). In order to achieve therapeutic concentrations, a direct aerosol administration to horses with horse respiratory diseases can be considered, as this route of application is also recommended in human medicine.

The hepatitis B virus e antigen suppresses the respiratory burst and mobility of human monocytes and neutrophils

The Hepatitis B virus (HBV) e antigen (HBeAg) is a secretory, non-structural protein, and associated with persistent infection of HBV. Previous studies indicate that HBeAg is able to regulate T cell-mediated responses, however, the interaction between HBeAg and the innate immune system is poorly understood. In this study, we demonstrated that recombinant HBeAg (rHBe) bound to human peripheral blood monocytes, neutrophils, and B lymphocytes but not to T lymphocytes. We focused on investigating the effects of HBeAg on monocytes and neutrophils and found that rHBe decreased the respiratory burst in both types of cells. Furthermore, we observed that cell migration in monocytes and neutrophils was suppressed by rHBe in a transwell assay. The attenuation of rHBe was not caused by a general cytotoxic effect because rHBe treatment stimulated low levels of cytokine and chemokine production by monocytes and it promoted neutrophil survival. Since the recruitment of monocytes and neutrophils to the infected site is crucial for the initiation of inflammation, HBeAg may modulate innate immune responses by diminishing the respiratory burst and migration of monocytes and neutrophils, which might interfere with the subsequent innate and adaptive immune responses against HBV, leading to the establishment of chronic infection.

Influence of FcγRIIIb polymorphism on its ability to cooperate with FcγRIIa and CR3 in mediating the oxidative burst of human neutrophils

Considering that human neutrophil FcγRIIa and FcγRIIIb receptors interact synergistically with CR3 in triggering neutrophil functional responses, allelic polymorphisms in these receptors might influence such interactions. We assessed whether FcγRIIIb polymorphisms affect FcγR/CR cooperation in mediating the neutrophil oxidative burst (OB), in particular the FcγRIIIb/CR3 cooperation that occurs via lectin-saccharide-like interactions. The OB of human neutrophil antigen (HNA)-1a-, HNA-1b-, and HNA-1a/-1b-neutrophils stimulated with immune complexes, opsonized or not with serum complement, was measured by the luminol-enhanced chemiluminescence assay. Compared with HNA-1a-neutrophils, HNA-1b-neutrophils exhibited reduced FcγR-stimulated OB, but increased FcγR/CR-stimulated OB. It suggests that (i) FcγR and CR cooperate more effectively in HNA-1b-neutrophils, and (ii) the HNA-1b allotype influences the FcγRIIIb cooperation with FcγRIIa, but not with CR3. HNA-1a- and HNA-1b-neutrophils exhibited similar OB responses elicited via CR3 alone or via FcγR/CR-independent pathways. In addition, the level of FcγRIIIb, FcγRIIa, and CR3 expression did not differ significantly among the neutrophil groups studied. Together, these results demonstrate that the HNA-1b allotype influences the functional cooperation between FcγRIIIb and FcγRIIa, and suggest that the difference in the glycosylation pattern between HNA-1a and HNA-1b does not affect the FcγRIIIb cooperation with CR3.

Diclofenac-Choline Antioxidant Activity Investigated by means of Luminol Amplified Chemiluminescence of Human Neutrophil Bursts and Electron Paramagnetic Resonance Spectroscopy

A new diclofenac salt called diclofenac-choline (DC) has recently been proposed for the symptomatic treatment of oropharyngeal inflammatory processes and pain because its greater water solubility allows the use of high concentrations, which are useful when the contact time between the drug and the oropharyngeal mucosa is brief, as in the case of mouthwashes or spray formulations. The antioxidant activity of DC has not yet been investigated, and so the aim was to use luminol-amplified-chemiluminescence (LACL) to verify whether various concentrations of DC (1.48, 0.74 and 0.37 mg/mL for incubation times of 2, 4 and 8 min) interfere with oxygen and nitrogen radicals during the course of human neutrophils respiratory bursts; electron paramagnetic resonance (EPR) spectroscopy was used to investigate its direct antiradical (scavenger) activity. The EPR findings showed that DC has concentration-dependent scavenging activity against the ABTS, the DPPH, and the hydroxyl radicals, but no activity on superoxide anion, as has been previously reported in the case of other NSAIDs. LACL revealed an inhibitory effect that was statistically significant after only 2 min of incubation, and similar after 4 and 8 min. The effects on the peroxynitrite radical paralleled those observed in the previous test. High concentrations and short incubation times showed that there is no interference on PMN viability, and so the inhibitory findings must be attributed to the effect of the drug. The anti-inflammatory effects of DC cannot be attributed solely to the inhibition of prostaglandin synthesis, but its effects on free radicals and neutrophil bursts suggest that they may contribute to its final therapeutic effect.

Effect of natural porcine surfactant in Staphylococcus aureus induced pro-inflammatory cytokines and reactive oxygen species generation in monocytes and neutrophils from human blood.

Surfacen® is a clinical surfactant preparation of porcine origin. In the present study, we have evaluated the effect of Surfacen® in the modulation of oxidative burst in monocytes and neutrophils in human blood and pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen® added to whole blood monocytes and neutrophils. The time course experiments of pre-incubation with Surfacen® showed no significant increase of ROS level at 2h; however, the ROS level decreased when pre incubated for 4h and 6h with Surfacen®. Pre-incubation of PBMC cells with Surfacen® at 0.125 and 0.5mg/mL showed a dose-dependent suppression of TNF-α levels measured after 4h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24h. A similar behavior was observed in IL-6 release. In summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen® in human monocytes and neutrophils in vitro.

Modulation of human neutrophil activity by adenosine modified with a carborane pharmacophore

An impact of adenosine modification with electroneutral, lipophilic 1,12-dicarba-closo-dodecaborane or electronegative 7,8-dicarba-nido-undecaborane boron cluster at the 6-N, 2′-C and 2-C positions on human neutrophil oxidative burst, neutrophil adherence to fibronectin and protein kinase C activity was studied. Modification of adenosine with 1,12-dicarba-closo-dodecaborane, but not 7,8-dicarba-nido-undecaborane, changes the function of adenosine from an inactive to an active state in regulating neutrophil response to PMA stimulation by reducing neutrophils’ reactivity through a mechanism involving the PKC signaling pathway. Our results show that exogenously administered adenosine derivatives can be useful in regulating the oxidative burst of neutrophils in the inflammatory process.

Validación de la técnica de dihidrorodamina 123 para el diagnóstico de la enfermedad granulomatosa crónica en Colombia

IntroductionChronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in the respiratory burst of phagocytes. Affected patients often suffer from granulomas and recurrent infections, mainly due to encapsulated bacteria. AimTo standardize the dihydrorhodamine test (DHR) in Colombia used for the diagnosis of CGD by evaluating the respiratory burst in human blood neutrophils and monocytes after in vitro activation. MethodsPhagocyte respiratory burst in peripheral blood samples from 10 healthy controls was evaluated by flow cytometry after leukocyte activation with several concentrations of phorbol myristate acetate (PMA). The different oxidation patterns of DHR in X-linked or autosomal recessive CGD were also obtained. ResultsThe most suitable concentrations of PMA for the evaluation of the respiratory burst in peripheral blood were 0.2 to 5μg/ml. Reference values for this test in neutrophils from our population were established. It was shown that the oxidation patterns of DHR in monocytes were not always identical to those observed in neutrophils. ConclusionThe evaluation of DHR oxidation by flow cytometry is a screening method that easily identifies the different phenotypes of CGD, with good sensitivity and at a lower cost. However, it is crucial that every laboratory establishes its own normal range for this test, in order to achieve the accurate characterization of this condition. DHR oxidation patterns may be also evaluated in different blood cells, since cell type-specific defects have also been reported.

Particulate beta-glucan modulates myeloid-derived suppressor cells (MDSC) activity in tumor-bearing mice and lung cancer patients (TUM4P.931)

Abstract Whole glucan particles (WGP) are microparticles of 1,3-beta glucan with 1,6-branches purified from the yeast Saccharamyces cerevisiae. Previous studies have reported its therapeutic efficacy in different tumor models through the activation of dendritic cells and macrophages via the c-type lectin receptor dectin-1. In this study, we investigated the effect of WGP treatment on myeloid-derived suppressor cells (MDSC) in mouse tumor models and in patients with non-small cell lung cancer (NSCLC). MDSC are a heterogeneous population of immature myeloid cells with suppressive activity that accumulate in cancer. Here we found that tumor-bearing mice treated orally with WGP have a significantly lower tumor weight with a significant decrease in the percentage of MDSC in the spleen compared to untreated animals in vivo. Patients with NSCLC treated with WGP prior to surgery have a decreased trend in MDSC percentage and increased respiratory burst of neutrophils in the peripheral blood. In addition, in vitro stimulation of MDSC subsets with WGP abrogates STAT3 phosphorylation in polymorphonuclear MDSC and enhances STAT3 phosphorylation in monocytic MDSC, with the activation of Erk1/2 phosphorylation in both subsets, suggesting that WGP differently modulates MDSC subsets. Moreover, MDSC treated with WGP have a decreased suppressive activity when cocultured with OVA-specific OTI and OTII splenocytes. Overall, these data indicate that WGP might be a new modulator of MDSC function in cancer.

Ação imunomoduladora da vitamina E na imunidade sistêmica e da glândula mamária de bovinos leiteiros alimentados com silagem

Regions such as southern Brazil live with drastic weather changes between winter and summer, resulting in direct disorders in the pasture, avoiding gradual change between grasses, creating periods of forage empty, a fact that diminishes the supply of forage, as well as its nutritional value. In these moments it becomes common to supplementation with intensive foods preserved as silage, a process that can reduce up to 50% levels of vitamin E from food, nutrient integrate cellular antioxidant system, responsible for the reduction of oxidative stress and immune function such phagocytosis, chemotaxis and oxidative metabolism in ruminants.Thus, the present study sought to measure the immunomodulatory activity of vitamin E in dairy cows in the middle of the stage of lactation, supplemented with corn silage and maintained on transition pasture not fertilized. We conducted assessments of hemogram, blood neutrophil function and cellularity of mammary glands before and after application of two parenteral doses of alpha tocopherol. It was observed in the treated group a significant increase for the series variable red blood, and to blood leukocytes mainly by an increase in neutrophils. In milk, there was increased cellularity, mainly by mononuclear elevation. As there was an increase of neutrophils positive to NBT test, it is believed that alpha tocopherol reduced cellular oxidative stress, enhancing phagocytosis and respiratory burst of neutrophils and decreased lipoperoxidation of cell membranes increases the halfspan of blood cells and milk.

A novel immunomodulatory effect of ugonin U in human neutrophils via stimulation of phospholipase C

Neutrophils have a crucial role in the immune system and are the first line of defense against pathogenic invaders. Neutrophil activation is required for their defensive function and can be induced by diverse stimuli, through either binding to cell surface receptors or direct intracellular target molecule stimulation. In this study, we found that 4″a,5″,6″,7″,8″,8″a-hexahydro-5,3′,4′-trihydroxy-5″,5″,8″a-trimethyl-4H-chromeno [2″,3″:7,6]flavone (ugonin U), a flavonoid isolated from Helminthostachys zeylanica (L) Hook, significantly induced superoxide production and release in a time- and concentration-dependent manner. A series of experiments was performed to dissect the mechanism of ugonin U-induced respiratory burst in human neutrophils. Our results demonstrated that ugonin U induced a slow increase in intracellular Ca2+, which was necessary for ugonin U-stimulated superoxide release. Use of a formyl peptide receptor (FPR) blocker, G protein inhibitor, and protein tyrosine kinase (PTK) inhibitor proved that FPR, G proteins, and PTKs were not associated with ugonin U-induced respiratory burst. Additionally, immunoblotting results revealed that ugonin U did not affect the phosphorylation of mitogen-activated protein kinases and protein tyrosine. Nevertheless, a phospholipase C (PLC) inhibitor and an inositol triphosphate (IP3) receptor antagonist considerably suppressed ugonin U-stimulated Ca2+ mobilization and subsequent superoxide release. Ugonin U also induced an increase in intracellular IP3 formation, which could be blocked using a PLC inhibitor. In conclusion, our study reveals that ugonin U represents the first identified natural flavonoid compound to directly stimulate PLC. Moreover, ugonin U induces respiratory burst via the PLC/IP3/Ca2+ pathway in human neutrophils.

Correction

HomeStrokeVol. 45, No. 4Correction Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection Originally published1 Apr 2014https://doi.org/10.1161/STR.0000000000000019Stroke. 2014;45:e68This article corrects the followingStroke Alters Respiratory Burst in Neutrophils and MonocytesThe version of the article, “Stroke Alters Respiratory Burst in Neutrophils and Monocytes” by Ruhnau et al (Stroke. 2014;45:794–800) that published online ahead-of-print on February 12, 2014, a correction was needed.The published article was a prior version.This has been corrected in the online and print version of the article. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesStroke Alters Respiratory Burst in Neutrophils and MonocytesJohanna Ruhnau, et al. Stroke. 2014;45:794-800 April 2014Vol 45, Issue 4 Advertisement Article InformationMetrics © 2014 American Heart Association, Inc.https://doi.org/10.1161/STR.0000000000000019 Originally publishedApril 1, 2014 PDF download Advertisement

L-Carnosine Modulates Respiratory Burst and Reactive Oxygen Species Production in Neutrophil Biochemistry and Function: May Oral Dosage Form of Non-Hydrolized Dipeptide L-Carnosine Complement Anti-Infective Anti-Influenza Flu Treatment, Prevention and Self-Care as an Alternative to the Conventional Vaccination?

Influenza A is a viral disease of global dimension, presenting with high morbidity and mortality in annual epidemics, and in pandemics which are of infrequent occurrence but which have very high attack rates. Influenza vaccines of the future must be directed toward use of conserved group-specific viral antigens, such as are present in transitional proteins which are exposed during the fusion of virus to the host cell. Influenza probes revealed a continuing battle for survival between host and parasite in which the host population updates the specificity of its pool of humoral immunity by contact with and response to infection with the most recent viruses which possess altered antigenic specificity in their hemagglutinin (HA) ligand. It is well known that the HA protein is found on the surface of the influenza virus particle and is responsible for binding to receptors on host cells and initiating infection. Polymorphonuclear neutrophils (PMN) have been reported to be involved in the initial host response to influenza A virus (IAV). Early after IAV infection, neutrophils infiltrate the airway probably due to release of chemokines that attract PMN. Clearly, severe IAV infection is characterized by increased neutrophil influx into the lung or upper respiratory tract. Carnosine (β-alanyl-L-histidine) and anserine (N-β-alanyl-1-methyl-L-histidine) are found in skeletal muscle of most vertebrates, including those used for food; for example, 100 g of chicken breast contains 400 mg (17.6 mmol/L) of carnosine and 1020 mg (33.6 mmol/l) of anserine. Carnosine-stimulated respiratory burst in neutrophils is a universal biological mechanism of influenza virus destruction. Our own studies revealed previously unappreciated functional effects of carnosine and related histidine containing compounds as a natural biological prevention and barrier against Influenza virus infection, expand public understanding of the antiviral properties of imidazole-containing dipeptide based compounds, and suggest important interactions between neutrophills and carnosine related compounds in the host response to viruses and bacteria. Carnosine and anserine were also found to reduce apoptosis of human neutrophils. In this way these histidine-containing compounds can modulate the Influenza virus release from neutrophills and reduce virus dissemination through the body of the organism. This review points the ability of therapeutic control of Influenza viral infections associated with modulation by oral nonhydrolized forms of carnosine and related histidine-containg compounds of PMN apoptosis which may be involved at least in part in the pathophysiology of the disease in animals and humans. The data presented in this article, overall, may have implications for global influenza surveillance and planning for pandemic influenza therapeutic prevention with oral forms of non-hydrolized natural L-carnosine as a suitable alternative to the conventional vaccination for various flu ailments.

Albumin infusion improves renal blood flow autoregulation in patients with acute decompensation of cirrhosis and acute kidney injury

In cirrhotic patients with renal failure, renal blood flow autoregulation curve is shifted to the right, which is consequent upon sympathetic nervous system activation and endothelial dysfunction. Albumin infusion improves renal function in cirrhosis by mechanisms that are incompletely understood. We aimed to determine the effect of albumin infusion on systemic haemodynamics, renal blood flow, renal function and endothelial function in patients with acute decompensation of cirrhosis and acute kidney injury. Twelve patients with refractory ascites and 10 patients with acute decompensation of cirrhosis and acute kidney injury were studied. Both groups were treated with intravenous albumin infusion, 40-60 g/days over 3-4 days. Cardiac and renal haemodynamics were measured. Endothelial activation/dysfunction was assessed using von Willebrand factor and serum nitrite levels. F2α Isoprostanes, resting neutrophil burst and noradrenaline levels were quantified as markers of oxidative stress, endotoxemia and sympathetic activation respectively. Albumin infusion leads to a shift in the renal blood flow autoregulation curve towards normalization, which resulted in a significant increase in renal blood flow. Accordingly, improvement of renal function was observed. In parallel, a significant decrease in sympathetic activation, inflammation/oxidative stress and endothelial activation/dysfunction was documented. Improvement of renal blood flow correlated with improvement in endothelial activation (r = 0.741, P < 0.001). The data suggest that albumin infusion improves renal function in acutely decompensated cirrhotic patients with acute kidney injury by impacting on renal blood flow autoregulation. This is possibly achieved through endothelial stabilization and a reduction in the sympathetic tone, endotoxemia and oxidative stress.

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Bioenergetics and the oxidative burst: protocols for the isolation and evaluation of human leukocytes and platelets.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase–DNA and neutrophil elastase–DNA complexes

Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO–DNA complexes and established a new HNE–DNA ELISA. We show that these methods reliably quantify MPO–DNA and HNE–DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired “early” and “late” bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways.

Biologically Active Eremophilane-Type Sesquiterpenes from Camarops sp., an Endophytic Fungus Isolated from Alibertia macrophylla

Two new eremophilane-type sesquiterpenes, xylarenones F (3) and G (4), have been isolated from solid substrate cultures of a Camarops sp. endophytic fungus isolated from Alibertia macrophylla, together with the known compounds xylarenones C (1) and D (2). The structures and relative configurations of 1-4 were elucidated by extensive NMR and HRESIMS spectroscopic analysis. Due to their effects on the respiratory burst of neutrophils, which included inhibition of the reactive oxygen species production, these sesquiterpenes exhibited potential anti-inflammatory and antioxidant properties.