The Mechanism of Action and Interaction of Leukotriene B4 and Platelet-Activating Factor as Mediators of Neutrophil Activation

Human neutrophils can be stimulated by a plethora of soluble and particulate stimuli, the majority of which interact with specific recognition sites (receptors) located on the plasma membrane. These agonists evoke a series of cellular responses including chemotaxis, aggregation, degranulation, superoxide generation and the formation of numerous lipid products, for example LTB4 and PAF, which are derived from membrane phospholipids. LTB4 and PAF interact with specific receptors on, and are themselves potent activators of, human neutrophils. Therefore, these lipids have the potential to act as endogenous mediators or amplifiers of neutrophil activation. The mechanisms by which agonist receptor occupancy lead to such cellular activation remain to be fully established. It remains possible that in neutrophils, as in some other cells, reactivity may be regulated by the production of at least two second messenger molecules, 1,2-diacylglycerol (DAG) and [Ca2+]i that are produced as a consequence of phospholipase C catalysed phosphoinositide hydrolysis. The precise nature and role of this receptor mediated sequence of events in the human neutrophil, particularly concerning LTB4 and PAF, still remains to be fully elucidated and is frequently controversial. In this study I attempted to investigate the mechanism of action and interaction of the arachidonic acid metabolite, LTB4, and the ether lipid, PAP, as mediators of neutrophil activation by comparing the responses elicited by these lipids to those evoked by other neutrophil stimuli, namely the chemotactic tripeptide, FMLP, the calcium ionophore, ionomycin and the phorbol eater, PMA. I also examined the possible involvement of G proteins and the role of protein kinase C activation as stimulatory and regulatory mechanisms in the neutrophil. In addition, I explored the role of PAF and LTB4 as endogenous mediators or amplifiers of neutrophil activation induced by other agonists. Neutrophil reactivity was assessed by: a. Examining ultrastructural features using electron microscopy. b. Monitoring aggregation photometrically using a standard platelet aggregometer. c. Determining NAG and lysozyme release using a fluorimetric and a spectrophotometric technique respectively. d. Measuring LTB4 generation by specific radioimmunoassay and the authenticity confirmed using reverse phase HPLC. e. Observing changes in [Ca2+]i using the fluorescent calcium indicator dye, quin2. f. Following inositol phospholipid metabolism in cells prelabelled with [32P]-orthophosphate and monitoring changes in the levels of [32P]-PtdA, [32P]-PtdIns, [32P]-PIP and [32P]-PIP2. Using these in vitro techniques, the major observations and conclusions are listed below: 1. The ultrastructural features associated with neutrophils activated by FMLP, PAF and LTB4 were similar. Control, unstimulated cells were generally rounded with smooth contours and the occasional extension of fine projections. Exposure of cells to agonists caused a marked plasma membrane ruffling followed by cell polarization and the formation of large lamellipodia. Pretreatment of neutrophils with the fungal metabolite, cytochalasin B, caused a marked vacuolation and subsequent additions of agonists resulted in the formation of blebs giving the cells a bizarre appearance. Caution ought to be a priority when using or interpreting data generated by cytochalasin B. 2. LTB4, PAF and EMLP all caused a reversible, concentration-dependent neutrophil aggregation. The aggregatory response produced by ionomycin and PMA was also concentration-dependent but was slower in onset and irreversible. 3. The receptor directed agonists caused a cytochalasin B dependent release of NAG (an azurophil granule marker) and lysozyme (an azurophil and specific granule marker). The calcium ionophore induced the release of both markers independent of pretreatment with cytochalasin B. PMA could release lysozyrae in the absence of the fungal metabolite but the phorbol ester only induced a limited cytochalasin B dependent release of NAG. 4. Ionomycin elicited a concentration-dependent generation and release of LTB4. FMLP and PAP only released barely detectable levels of the arachidonic acid metabolite. 5. All agonists except PMA caused a rapid concentration-dependent elevation of neutrophil [Ca2+]i. Ionomycin induced a greater maximal increase than the receptor directed agonists. (Abstract shortened by ProQuest.)

The lagoonal harbour of Portus Pisanus (N Tyrrhen- ian Sea, Italy): a long history of human adaptation to changing coastline

During the last millennia human and natural processes have become increasingly intertwined, especially in the Mediterranean coastal and alluvial plains where major urban and trade centres developed since protohistoric times. The construction of ports represents one of the human activities that have mostly contributed to modify coastal environments, inducing a variety of hydrodynamic and hydrochemical changes especially since Roman times (Marriner et al., 2014). Exceptions in this common manner to plan harbours have been recognised along the N Tyrrhenian coast, where no high-impact defense works are explicitly documented by either historical sources or archaeological excavations for three main harbours developed during Etruscan-Roman times (IV-I century BC): Portus Lunae (Bini et al., 2012), Portus Pisanus and Vada Volterrana. Roman literary sources (i.e., Itinerarium Maritimum 501; Rutilio Namaziano) mentioned Portus Pisanus as a flourishing commercial site within a natural protected area (called Sinus Pisanus by Tacito) characterized by Posidonia meadows and located at the foot of Leghorn hills, ca. 18.5 km south of the Pisa city. Accordingly, recent excavations undertaken close to the hills slope, 3 km inland from modern coastline, unearthed a wooden palisade, stone piers and a warehouse dated to the Roman period (Pasquinucci, 2013; Morhange et al., 2015). However, the precise location of the lagoonal harbour basin is still controversial. This study aims to contribute to fill this knowledge gap and shed new light on the main stages of harbour history in the framework of the mid-late Holocene palaeogeographic evolution of the Pisa Plain. The application of a multidisciplinary approach (sedimentological and micropalaeontological core analyses, radiocarbon dating, geomorphological field survey, remote sensing and historical cartography) has revealed that a wide lagoonal basin formed in the study area during the marine transgression peak (ca. 8000 cal yr BP). This basin, recorded by a m-thick subsurface succession of soft grey clays with brackish meiofauna, persisted for several millennia and corresponds to Sinus Pisanus. The available stratigraphic data document that during Roman times the lagoon became progressively less connected to the sea and turned into a coastal lake/pond. Filling processes started two-three millennia later respect to the lagoon occupying the Pisa city area during the Holocene (Rossi et al., 2011). This seaward facies shift forced the westward transferring of the Middle Ages harbour. These results show that natural sheltered conditions along with the distance from coeval Arno River made more advantageous for humans following the shoreline changes, rather than making high-impact interventions. 2012, Bini M., Bruckner H., Chelli A., Da Prato S., Gervasini L., Palaeogeographies of the Magra Valley coastal plain to costrain the location of the Roman harbour of Luna (NW Italy), Palaeogeography, Palaeoclimatology, Palaeoecology, 337-338, 37–51. 2014, Marriner N., Morhange C., Kaniewski D., Carayon N., Ancient harbour infrastructure in the Levant: tracking the birth and rise of new forms of anthropogenic pressure, Nature Scientific Reports, 4, 5554. 2015, Morhange C., Marriner N., Baralis A., Blot M.L., Bony G., Carayon N., Carmona P., Flaux C., Giaime M., Goiran J.-P., Kouka M., Lena A., Oueslati A., Pasquinucci M., Porotov A., Dynamiques géomorphologiques et typologie géoarcheologique des ports antiques en contextes lagunaires, Quaternaire, 26, (2), 117–139. 2013, Pasquinucci M., Guida all’archeologia delle coste livornesi. Nardini Editore. Provincia di Livorno. 271 pp. 2011, Rossi V., Amorosi A., Sarti G., Potenza M., Influence of inherited topography on the Holocene sedimentary evolution of coastal systems: An example from Arno coastal plain (Tuscany, Italy), Geomorphology, 135 (1-2), 117–128

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

Microbial translocation and T cell activation are modified by direct-acting antiviral therapy in HCV-infected patients

BACKGROUND: Microbial translocation from the gut lumen has been involved in the pathogenesis of liver damage in hepatitis C virus (HCV) infection. AIM: To investigate the impact of direct-acting antiviral treatment on microbial translocation and T-cell activation, in patients with hepatitis C-related liver disease. METHODS: We enrolled two groups of HCV-infected patients undergoing direct-acting antiviral treatment: patients with fibrosis ≥F3 according to Metavir (Group ≥F3); patients with hepatitis C recurrence after liver transplantation and Metavir ≥F2 (Group Liver Transplantation + ≥F2). All patients were treated with direct-acting antivirals based on ongoing guidelines. Surrogate biomarkers of microbial translocation (plasma concentrations of soluble-CD14, lipopolysaccharide-binding protein and intestinal fatty acid-binding protein) were evaluated at baseline, at first month, at the end of treatment and 3 months later. T-cell activation was measured by expression of CD38+ HLA-DR at the same time points, only in Group ≥F3. RESULTS: There were 32 patients in Group ≥F3 and 13 in Group LT + ≥F2. At baseline, levels of soluble-CD14 and lipopolysaccharide-binding protein were significantly higher in both groups vs healthy controls. Baseline soluble-CD14 correlated with glutamic-oxalacetic transaminase (r = 0.384, P = 0.009) and glutamic-pyruvic transaminase (r = 0.293, P = 0.05). A significant decrease in plasma levels of surrogate microbial translocation biomarkers was observed during and after treatment in the two groups although values were not normalised. In Group ≥F3, CD38+ HLADR+ T-cell expression was significantly decreased by direct-acting antiviral treatment. Relapsers (9%) showed higher soluble-CD14 levels at baseline. CONCLUSION: Surrogate microbial translocation markers and T cell activation are increased in HCV-infected patients with liver fibrosis and decrease during direct-acting antiviral treatment

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

Influence of the synthesis method on the catalytic activity of mayenite for the oxidation of gas-phase trichloroethylene

[EN] Catalytic oxidation of trichloroethylene (TCE) in heterogeneous phase (gas-solid) is an effective strategy for the conversion of this pollutant in less harmful compounds, namely CO2, CO and HCl. In this work, we have studied the use of mayenite, a cost-effective material, as an active catalyst for the TCE conversion. In particular, we have assessed the influence of the mayenite synthesis method (hydrothermal, sol-gel and ceramic) on the reaction performance. The materials have been characterized by different techniques, such as XRD, N-2-sorption (BET), TPR, Raman spectroscopy, FESEM-EDX and TEM. The analysis of the light-off curves and product distribution, has shown that the use of the hydrothermal method for the mayenite synthesis results in the most active and selective catalyst. This has been related with a higher surface area and with a higher concentration of oxygen anions in the mayenite prepared by this method. It has been found that the presence of water in the stream do not influence the catalytic performance of the material. A mechanism for the reaction and for the partial deactivation of the catalyst has been proposed.This work was supported by the grants ORSA167988 and ORSA174250 funded by the University of Salerno. AI gratefully acknowledges the Erasmus+ traineeship program. AEP and JMT thanks the Spanish Ministry of Economy and Competitiveness and the Fondo Europeo de Desarrollo Regional through MAT2015-71842-P and CTQ2015-68951-C3-1-R (MINECO/FEDER)Intiso, A.; Martínez-Triguero, J.; Cucciniello, R.; Rossi, F.; Palomares Gimeno, AE. (2019). Influence of the synthesis method on the catalytic activity of mayenite for the oxidation of gas-phase trichloroethylene. Scientific Reports. 9:1-9. https://doi.org/10.1038/s41598-018-36708-2S199Greene, H. L., Prakash, D. S. & Athota, K. V. Combined sorbent/catalyst media for destruction of halogenated VOCs. Appl. Catal. 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