A novel electron paramagnetic resonance-based assay for prostaglandin H synthase-1 activity

BACKGROUND: Prostaglandin H(2 )synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H(2 )(PGH(2)) prior to formation of prostanoids that are important in inflammation. PGHS isozymes (-1 and -2) are the target for nonsteroidal anti-inflammatory drugs (NSAIDs). Given the rekindled interest in specific anti-inflammatory PGHS inhibitors with reduced unwanted side effects, it is of paramount importance that there are reliable and efficient techniques to test new inhibitors. Here, we describe a novel in vitro electron paramagnetic resonance (EPR)-based assay for measuring the activity of PGHS-1. METHODS: We validated a novel in vitro PGHS-1 activity assay based on the oxidation of spin-trap agent, 1-hydroxy-3-carboxy-pyrrolidine (CPH) to 3-carboxy-proxy (CP) under the action of the peroxidase element of PGHS-1. This quantifiable spin-adduct, CP, yields a characteristic 3-line electron paramagnetic (EPR) spectrum. RESULTS: The assay is simple, reproducible and facilitates rapid screening of inhibitors of PGHS-1. Aspirin (100 μM, 1 mM) caused significant inhibition of spin-adduct formation (72 ± 11 and 100 ± 16% inhibition of control respectively; P < 0.05). Indomethacin (100 μM) also abolished the signal (114 ± 10% inhibition of control; P < 0.01). SA and the PGHS-2-selective inhibitor, NS398, failed to significantly inhibit spin-adduct generation (P > 0.05). CONCLUSION: We have demonstrated and validated a simple, reproducible, quick and specific assay for detecting PGHS-1 activity and inhibition. The EPR-based assay described represents a novel approach to measuring PGHS activity and provides a viable and competitive alternative to existing assays

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

<p>Abstract</p> <p>Background</p> <p>Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO^-). In this study we have examined the ability of NO and ONOO^-to evoke apoptosis in human monocyte-derived macrophages (MDMϕ), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type.</p> <p>Methods</p> <p>Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM) or GEA-3162 (10 – 300 μM) in the presence or absence of BAY 41–2272 (1 μM), isobutylmethylxanthine (IBMX; 1 μM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining.</p> <p>Results</p> <p>Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O₂^-, and is therefore a ONOO^-generator. NO (DETA/NO) had no effect on cell viability, but ONOO^-(GEA-3162) caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO^--induced apoptosis in a cGMP-dependent manner.</p> <p>Conclusion</p> <p>These results demonstrate disparities between the ability of NO and ONOO^-to induce apoptosis in human MDMϕ. Furthermore, this study provides evidence for a novel cGMP-dependent pre-conditioning mechanism to limit ONOO^--induced apoptosis in human MDMϕ.</p

Constitutive neutrophil apoptosis in culture is modulated by cell density independently of β2 integrin-mediated adhesion

AbstractAlthough inflammatory mediators modulate the rate of constitutive neutrophil apoptosis in vitro the effects of micro-environmental conditions have not been fully investigated. In this study, we demonstrate that the rate of constitutive neutrophil apoptosis is affected by the number of cells per unit surface area, with enhanced survival at high cell density. Furthermore, the presence of protein or serum in the culture medium also enhances neutrophil survival. These effects were independent of β2 integrin-mediated adhesion and were not influenced by specific adhesion to extracellular matrix components. Thus, the rate of neutrophil apoptosis is fundamentally influenced by micro-environmental conditions and indicates that factors such as cell density and extracellular protein concentration must be considered when investigating mechanisms regulating inflammatory cell apoptosis in vitro

The Outcome of Neutrophil-T Cell Contact Differs Depending on Activation Status of Both Cell Types

Neutrophils and T cells exist in close proximity in lymph nodes and inflamed tissues duringhealth and disease. They are able to form stable interactions, with profound effects on thephenotype and function of the T cells. However, the outcome of these effects arefrequently contradictory; in some systems neutrophils suppress T cell proliferation, inothers they are activatory or present antigen directly. Published protocols modelling theseinteractions in vitro do not reflect the full range of interactions found in vivo; they do notexamine how activated and naïve T cells differentially respond to neutrophils, or whetherde-granulating or resting neutrophils induce different outcomes. Here, we established aculture protocol to ask these questions with human T cells and autologous neutrophils.We find that resting neutrophils suppress T cell proliferation, activation and cytokineproduction but that de-granulating neutrophils do not, and neutrophil-releasedintracellular contents enhance proliferation. Strikingly, we also demonstrate that T cellsearly in the activation process are susceptible to suppression by neutrophils, while laterstage T cells are not, and naïve T cells do not respond at all. Our protocol therefore allowsnuanced analysis of the outcome of interaction of these cells and may explain thecontradictory results observed previously

Dysregulation of Prostaglandins, Leukotrienes and Lipoxin A4 in Bronchiectasis

Introduction: Bronchiectasis is characterised by excessive neutrophilic inflammation. Lipid mediators such as prostaglandins and leukotrienes have crucial roles in the inflammatory response. Further characterisation of these lipids and understanding the interplay of anti-inflammatory and proinflammatory lipid mediators could lead to the development of novel anti-inflammatory therapies for bronchiectasis. Aim: The aim of our study was to characterise the lipids obtained from serum and airways in patients with bronchiectasis in the stable state. Methods: Six healthy volunteers, 10 patients with mild bronchiectasis, 15 with moderate bronchiectasis and 9 with severe bronchiectasis were recruited. All participants had 60 mL of blood taken and underwent a bronchoscopy while in the stable state. Lipidomics was done on serum and bronchoalveolar lavage fluid (BALF). Results: In the stable state, in serum there were significantly higher levels of prostaglandin E2 (PGE2), 15-hydroxyeicosatetranoic acid (15-HETE) and leukotriene B4 (LTB4) in patients with moderate–severe disease compared with healthy volunteers. There was a significantly lower level of lipoxin A4 (LXA4) in severe bronchiectasis. In BALF, there were significantly higher levels of PGE2, 5-HETE, 15-HETE, 9-hydroxyoctadecadienoic acid and LTB4 in moderate–severe patients compared with healthy volunteers. In the stable state, there was a negative correlation of PGE2 and LTB4 with % predicted forced expiratory volume in 1 s and a positive correlation with antibiotic courses. LXA4 improved blood and airway neutrophil phagocytosis and bacterial killing in patients with bronchiectasis. Additionally LXA4 reduced neutrophil activation and degranulation. Conclusion: There is a dysregulation of lipid mediators in bronchiectasis with excess proinflammatory lipids. LXA4 improves the function of reprogrammed neutrophils. The therapeutic efficacy of LXA4 in bronchiectasis warrants further studies

Release of chromatin extracellular traps by phagocytes of Atlantic salmon, Salmo salar (LINNAEUS, 1758)

Neutrophils release chromatin extracellular traps (ETs) as part of the fish innate immune response to counter the threats posed by microbial pathogens. However, relatively little attention has been paid to this phenomenon in many commercially farmed species, despite the importance of understanding host-pathogen interactions and the potential to influence ET release to reduce disease outbreaks. The aim of this present study was to investigate the release of ETs by Atlantic salmon (Salmo salar L.) immune cells. Extracellular structures resembling ETs of different morphology were observed by fluorescence microscopy in neutrophil suspensions in vitro, as these structures stained positively with Sytox Green and were digestible with DNase I. Immunofluorescence studies confirmed the ET structures to be decorated with histones H1 and H2A and neutrophil elastase, which are characteristic for ETs in mammals and other organisms. Although the ETs were released spontaneously, release in neutrophil suspensions was stimulated most significantly with 5 μg/ml calcium ionophore (CaI) for 1 h, whilst the fish pathogenic bacterium Aeromonas salmonicida (isolates 30411 and Hooke) also exerted a stimulatory effect. Microscopic observations revealed bacteria in association with ETs, and fewer bacterial colonies of A. salmonicida Hooke were recovered at 3 h after co-incubation with neutrophils that had been induced to release ETs. Interestingly, spontaneous release of ETs was inversely associated with fish mass (p  < 0.05), a surrogate for age. Moreover, suspensions enriched for macrophages and stimulated with 5 μg/ml CaI released ET-like structures that occasionally led to the formation of large clumps of cells. A deeper understanding for the roles and functions of ETs within innate immunity of fish hosts, and their interaction with microbial pathogens, may open new avenues towards protecting cultured stocks against infectious diseases

Characterisation of the molecular mechanism required for glucocorticoid augmentation of macrophage phagocytosis of apoptotic neutrophils

The successful resolution of inflammation requires removal of neutrophils from the inflammatory site to prevent release of histotoxic contents that may potentiate inflammatory processes and promote progression to a chronic state associated with impaired repair mechanisms and/or autoimmune responses. Macrophages are “professional” phagocytes required for rapid and efficient clearance of apoptotic neutrophils. Macrophage phagocytic capacity can be critically regulated by a number of environmental factors, including cytokines, bacterial products, and glucocorticoids. We have hypothesised that modulation of macrophage phagocytic capacity may represent an effective strategy for promoting resolution of inflammation in diseases where clearance of neutrophils may be impaired or inefficient. The aim of this thesis was to investigate the molecular mechanisms underlying glucocorticoid-augmentation of macrophage phagocytosis. We have demonstrated that long-term exposure of human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone dramatically increases phagocytic capacity for “early” membrane-intact apoptotic neutrophils. Increased phagocytic potential was associated with a “switch” from a serum-independent to a serum-dependent apoptotic cell recognition mechanism. We initially employed an “add back” approach to rule out several well-defined opsonins in apoptotic neutrophil clearance, including immune complexes, IgG, complement proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a multi-step purification scheme involving anion exchange and gel filtration chromatography, we purified a high molecular weight fraction that contained the prophagocytic activity of serum and analysis by mass spectrometry identified C4-binding protein as a candidate protein. C4-binding protein circulates in human plasma bound predominately in a >570kDa complex with protein S and the presence of protein S in high molecular weight fractions was confirmed by immunoblotting. We found that protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages (Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic activity. Protein S was found to opsonise apoptotic neutrophils in a calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on the surface of Dex-MDMo compared to untreated MDMo. The studies presented in this thesis have provided novel insight into the underlying molecular mechanisms required for high capacity clearance of apoptotic neutrophils by macrophages following treatment with glucocorticoids and may form the foundations for further studies investigating glucocorticoid action for development of safer and more selective therapies

Resolution of inflammation: Mechanisms and opportunity for drug development

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Alleviating the present tension between T2K and NOν\nuA with neutrino New Physics at source

Since neutrino oscillation was observed, several experiments have been built to measure its parameters. NO$\nu$A and T2K are two long-baseline experiments dedicated to measuring mainly the mixing angle $\theta_{23}$, the charge-parity conjugation phase $\delta_{\rm CP}$, and the mass ordering. However, there is a tension in current data. The T2K allowed region is almost excluded by the NO$\nu$A result at the $90\%$ confidence level. We propose a non-standard interaction (NSI) in neutrino production to relieve this tension. The NSI is computed through quantum field theory (QFT) formalism, where we derive perturbative analytical formulae considering NSI in the pion decay. Within this new approach, we can alleviate NO$\nu$A and T2K tension for a NSI complex parameters of order $10^{-3}$. We show the new phase has a degeneracy to the Dirac CP phase of the form $\delta_{\rm CP} \pm \phi= 1.5\pi$ being a possible source of violation of charge-parity symmetry