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Clinical trials report substantial gains in visual acuity (VA) for eyes treated with intravitreal anti-VEGF for neovascular AMD (nAMD). In clinical reality, VA outcomes are more variable. Here we investigate pro-re nata treatment frequencies and VA in a real-life cohort of 1382 eyes (1048 patients). Patients with nAMD and one year complete follow-up treated with pro-re nata anti-VEGF between 2009 and 2016 were included. Injection frequency and VA was analyzed clustered by year of first treatment. Baseline parameters were compared between years. Median injection frequency in the first year was 5 with an IQR (interquartile range) of 5 for patients treated in 2009 and 8 with an IQR of 3 for patients treated from 2012 onwards. Median VA outcomes at one year were −5 to ±0 letters for patients treated between 2009 and 2013 and ±0 to +2 letters for patients treated from 2013 onwards. This cohort comprises all severities and subtypes of nAMD. 39% of patients had baseline VA outside the range for the MARINA or ANCHOR clinical trials. Higher treatment frequency was associated with improved VA in our real-life nAMD cohort. With adequate injection frequency, almost 90% of eyes had stable or improved VA over one year. Median VA gains, however, were lower compared to clinical trials. This may be due to a wider range of baseline characteristics in real-life cohorts.

In this study, the effects of exogenous lysophospholipids—lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylserine—on the kinetics of reactive oxygen species (ROS) production by human neutrophils are described. The ROS production by human neutrophils was monitored by luminol-amplified chemiluminescence after cell stimulation with the chemotactic tripeptide, fMLP, or with the phorbol ester, PMA. The interaction of lysophospholipids with the membrane of human neutrophils was additionally tested by mass spectrometry. Lysophosphatidylcholine showed the most pronounced effect on the chemiluminescence pattern, as well as the intensity of the fMLP and PMA-stimulated cells, whereas lysophosphatidic acid showed a slight priming effect when fMLP was used for stimulation. In the case of fMLP-stimulated cells, lysophosphatidylcholine inhibited the first phase and enhanced the second phase of chemiluminescence, whereas the chemiluminescence of PMA-stimulated neutrophils was inhibited in a concentration-dependent manner. We conclude that lysophosphatidylcholine is able to interact with protein kinase C-dependent signalling pathways leading to NADPH oxidase activation. Copyright © 2002 John Wiley & Sons, Ltd.

The effects of Michael adducts of 6-O-palmitoyl-L-ascorbic acid (compounds 1–4) on the phosphorylation-dependent response of stimulated monocytes and neutrophils was investigated. The pyranosyl derivative 3 increased the production of tumor necrosis factor-α in human monocytes stimulated with lipopolysaccharide (LPS). Compound 3 also enhanced the release of tumor necrosis factor-α from nonstimulated monocytes. Michael adducts 1–4 inhibited the formation of reactive oxygen species in fMLP-stimulated human neutrophils as measured by luminol chemiluminescence. Treatment with 6-O-palmitoyl-L-ascorbic acid (compound 5) also led to a decreased luminescence response of neutrophils. Results are discussed with respect to the inhibitory activity of Michael adducts of ascorbic acids towards protein phosphatases PP1/PP2A.

The effects of three aglycon flavonols (myricetin, quercetin, and kaempferol) and the natural glycoside rutin on superoxide anion radical generating systems were investigated. Quercetin, myricetin, and kaempferol inhibited the formation of uric acid from xanthine by xanthine oxidase, while rutin was ineffective. The generation of superoxide anion radicals by this system was determined by either reduction of cytochrome c or Pholasin luminescence. A scavenging of superoxide was only observed for myricetin and to a small extent for rutin. All flavonols tested inhibited the Pholasin luminescence of fMet-Leu-Phe-stimulated neutrophils. Rutin influenced the oxidative burst of neutrophils in the same way as wortmannin and LY294002, two inhibitors of the phosphoinositide 3-kinase γ. Indeed, rutin inhibited the activity of this enzyme, whereas the three other flavonols showed no effect. Thus, an inhibition of enzymes involved in signaling rather than a scavenging of superoxide anion radicals dominates in fMet-Leu-Phe-stimulated neutrophils exposed to flavonols.

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils. Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.

Background: Although neutrophilic granulocytes clearly contribute to cartilage degradation in rheumatic diseases, it is unclear if reactive oxygen species (ROS) or proteolytic enzymes are the most important components in cartilage degradation and how they interact. Results: Neutrophils were stimulated by chemicals conferring a different degree of ROS formation and enzyme release. Supernatants of neutrophils were incubated with thin slices of pig articular cartilage. Supernatants of cartilage were assayed by NMR spectroscopy, MALDI-TOF mass spectrometry and relevant biochemical methods. Stimulation conditions of neutrophils correlated well with the extent of cartilage degradation. Due to the release of different enzymes, cartilage degradation could be best monitored by NMR since mainly low-mass degradation products were formed. Astonishingly, the suppression of the formation of ROS resulted in decreased cartilage degradation. Conclusion: ROS formed by neutrophils are not directly involved in cartilage degradation but influence the activity of proteolytic enzymes, which are the main effectors of cartilage degradation.

Matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) has been used to investigate degradation products of two selected polysaccharides of cartilage (chondroitin sulfate and hyaluronic acid). Testicular hyaluronate lyase and chondroitin ABC lyase were used for enzymic digestion of both polysaccharides as well as of cartilage specimens. Polysaccharide solutions and cartilage supernatants were assayed by positive and negative MALDI-TOF MS. Especially chondroitin ABC lyase produced high amounts of digestion products (unsaturated di- and tetrasaccharides) from polysaccharides as well as from cartilage, clearly monitored by MALDI-TOF MS. It is concluded that MALDI-TOF MS provides a precise and fast tool for the determination of oligosaccharides since no previous derivatization is required.

Whereas matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has gained high importance in the field of protein analysis, surprisingly few studies were published about the use of MALDI for lipid analysis. Lipids, however, are well-suited for MALDI since all experiments can be performed in a sole organic phase and, thus, extremely homogeneous matrix/analyte mixtures are formed. We report here for the first time the application of MALDI-TOF-MS for the analysis of diacylglycerols, phosphatidylcholines, and (poly)phosphoinositides. It is shown that in a matrix of 2,5-dihydroxybenzoic acid the molecular ions (M + 1) of phosphatidylcholines as well as the corresponding adducts of different phosphoinositides are easily detected even in complex mixtures, and thus, detailed data on the fatty acid composition are provided. In contrast, diacylglycerols are mainly detected as the corresponding sodium or potassium adducts, but not as the protonated forms. Fragmentation reactions of fatty acids on the double bonds and on the polar lipid head group are observed to a minor extent in the spectra of all investigated lipids. Generally, choline derivatives are most sensitive toward further fragmentation reactions. Due to its very high sensitivity (up to picomolar concentrations) MALDI-TOF-MS can be used for the direct investigation of biologically relevant lipid mixtures occurring, e.g., in cell membranes. The analysis of the lipid composition of neutrophilic granulocytes is given as a representative example for future applications.

In this study, the effects of exogenous lysophospholipids--lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylserine--on the kinetics of reactive oxygen species (ROS) production by human neutrophils are described. The ROS production by human neutrophils was monitored by luminol-amplified chemiluminescence after cell stimulation with the chemotactic tripeptide, fMLP, or with the phorbol ester, PMA. The interaction of lysophospholipids with the membrane of human neutrophils was additionally tested by mass spectrometry. Lysophosphatidylcholine showed the most pronounced effect on the chemiluminescence pattern, as well as the intensity of the fMLP and PMA-stimulated cells, whereas lysophosphatidic acid showed a slight priming effect when fMLP was used for stimulation. In the case of fMLP-stimulated cells, lysophosphatidylcholine inhibited the first phase and enhanced the second phase of chemiluminescence, whereas the chemiluminescence of PMA-stimulated neutrophils was inhibited in a concentration-dependent manner. We conclude that lysophosphatidylcholine is able to interact with protein kinase C-dependent signalling pathways leading to NADPH oxidase activation.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an established tool for the analysis of proteins, whereas it gained by far less interest in the field of lipid analysis. This method works well with phospholipids as well as organic cell extracts and provides high sensitivity and reproducibility. The aim of the present paper is to extend our previous studies to the analysis of lysophospholipids and phospholipid mixtures. To study the suitability of MALDI-TOF mass spectrometry for the analysis of lysophospholipids, different phospholipids like phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and phosphatidylinositol as well as their mixtures were digested with phospholipase A(2). Positive and negative ion mass spectra of all phospholipids before and after digestion were recorded. In all these cases, the molecular ions of the expected digestion products could be detected and only a very small extent of further fragmentation was observed. On the other hand, spectra of phospholipid mixtures containing phosphatidylcholine were strongly dominated by phosphatidylcholine and lysophosphatidylcholine signals, which prevented the detection of further phospholipids even if those lipids were present in comparable amounts. This is of paramount interest for the analysis of tissue and cell extracts.

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus , was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl − and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H 2 O 2 in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H 2 O 2 /Cl − system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.

This paper is addressed to study how PKC-mediated effects and phosphatidic acid interact together in activation of NADPH-oxidase in formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) stimulated neutrophils as detected by luminol chemiluminescence. The early luminescence response in fMet-Leu-Phe-stimulated cells (up to 5 min after stimulation) depends mainly on reactive oxygen species generated extracellularly, whereas all later events are caused by oxidation of luminol inside the cells. The two protein phosphatase inhibitors, okadaic acid and calyculin A, dramatically increased the late luminescence of cells. This enhancement was totally inhibited by the phospholipase D modulator butanol, while the protein kinase C (PKC) inhibitor bisindolylmaleimide I was insensitive. The early luminescence response of the cells was slightly inhibited by both protein phosphatase inhibitors and depended on protein kinase C as well as on phospholipase D activities. Propranolol, an inhibitor of phosphatidate phosphohydrolase, enhanced all parts of luminescence response of fMet-Leu-Phe-stimulated neutrophils at concentrations up to 2.5 × 10−5 mol/L. While the late luminescence response of propranolol-treated cells was not inhibited by the PKC inhibitor bisindolylmaleimide I, the first response depended on protein kinase C. The inhibitor of diacylglycerol kinase R59949 enhanced the luminescence signal only during the first 4 min in fMet-Leu-Phe-stimulated cells. Only diacylglycerols derived from phospholipase C, such as 1-stearoyl-2-arachidonoyl-sn-glycerol, were able to initiate an oxidative burst in cells. Saturated diacylglycerols (e.g. 1,2-dipalmitoyl-sn-glycerol or 1,2-distearoyl-sn-glycerol) did not yield any luminol chemiluminescence, although they were incorporated into the plasma membrane, as evidenced by matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry. Our results demonstrate that phosphatidic acid produced by phospholipase D is responsible for NADPH-oxidase activity in fMet-Leu-Phe-stimulated neutrophils over the entire measuring time, whereas PKC-mediated processes are only involved during the first 5 min. Copyright © 1999 John Wiley & Sons, Ltd.