The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Paul G. Furtmüller - One of the best experts on this subject based on the ideXlab platform.
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pharmacophore based discovery of 2 phenylamino aceto hydrazides as potent Eosinophil Peroxidase epo inhibitors
Journal of Enzyme Inhibition and Medicinal Chemistry, 2018Co-Authors: Daniela Schuster, Martina Zederbauer, Thierry Langer, Andreas Kubin, Paul G. FurtmüllerAbstract:There is an increasing interest in developing novel Eosinophil Peroxidase (EPO) inhibitors, in order to provide new treatment strategies against chronic inflammatory and neurodegenerative diseases ...
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Pharmacophore-based discovery of 2-(phenylamino)aceto-hydrazides as potent Eosinophil Peroxidase (EPO) inhibitors
2018Co-Authors: Daniela Schuster, Martina Zederbauer, Thierry Langer, Andreas Kubin, Paul G. FurtmüllerAbstract:There is an increasing interest in developing novel Eosinophil Peroxidase (EPO) inhibitors, in order to provide new treatment strategies against chronic inflammatory and neurodegenerative diseases caused by Eosinophilic disorder. Within this study, a ligand-based pharmacophore model for EPO inhibitors was generated and used for in silico screening of large 3 D molecular structure databases, containing more than 4 million compounds. Hits obtained were clustered and a total of 277 compounds were selected for biological assessment. A class of 2-(phenyl)amino-aceto-hydrazides with different substitution pattern on the aromatic ring was found to contain the most potent EPO inhibitors, exhibiting IC50 values down to 10 nM. The generated pharmacophore model therefore, represents a valuable tool for the selection of compounds for biological testing. The compounds identified as potent EPO inhibitors will serve to initiate a hit to lead and lead optimisation program for the development of new therapeutics against Eosinophilic disorders.
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redox thermodynamics of lactoPeroxidase and Eosinophil Peroxidase
Archives of Biochemistry and Biophysics, 2010Co-Authors: Gianantonio Battistuzzi, Paul G. Furtmüller, Marzia Bellei, Jutta Vlasits, Srijib Banerjee, Marco Sola, Christian ObingerAbstract:Abstract Eosinophil Peroxidase (EPO) and lactoPeroxidase (LPO) are important constituents of the innate immune system of mammals. These heme enzymes belong to the Peroxidase-cyclooxygenase superfamily and catalyze the oxidation of thiocyanate, bromide and nitrite to hypothiocyanate, hypobromous acid and nitrogen dioxide that are toxic for invading pathogens. In order to gain a better understanding of the observed differences in substrate specificity and oxidation capacity in relation to heme and protein structure, a comprehensive spectro-electrochemical investigation was performed. The reduction potential ( E °′) of the Fe(III)/Fe(II) couple of EPO and LPO was determined to be −126 mV and −176 mV, respectively (25 °C, pH 7.0). Variable temperature experiments show that EPO and LPO feature different reduction thermodynamics. In particular, reduction of ferric EPO is enthalpically and entropically disfavored, whereas in LPO the entropic term, which selectively stabilizes the oxidized form, prevails on the enthalpic term that favors reduction of Fe(III). The data are discussed with respect to the architecture of the heme cavity and the substrate channel. Comparison with published data for myeloPeroxidase demonstrates the effect of heme to protein linkages and heme distortion on the redox chemistry of mammalian Peroxidases and in consequence on the enzymatic properties of these physiologically important oxidoreductases.
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kinetics of interconversion of redox intermediates of lactoPeroxidase Eosinophil Peroxidase and myeloPeroxidase
Japanese Journal of Infectious Diseases, 2004Co-Authors: Paul G. Furtmüller, Martina Zederbauer, Walter Jantschko, Christa Jakopitsch, Jurgen Arnhold, Christian ObingerAbstract:MyeloPeroxidase, Eosinophil Peroxidase and lactoPeroxidase are heme-containing oxidoreductases, which undergo a series of redox reactions. Though sharing functional and structural homology, reflecting their phylogenetic origin, differences are observed regarding their spectral features, substrate specificities, redox properties and kinetics of interconversion of the relevant redox intermediates ferric and ferrous Peroxidase, compound I, compound II and compound III. Depending on substrate availability, these heme enzymes path through the halogenation cycle and/or the Peroxidase cycle and/or act as poor (pseudo-) catalases.
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redox properties of the couple compound i native enzyme of myeloPeroxidase and Eosinophil Peroxidase
FEBS Journal, 2001Co-Authors: Jurgen Arnhold, Paul G. Furtmüller, Gunther Regelsberger, Christian ObingerAbstract:The standard reduction potential of the redox couple compound I/native enzyme has been determined for human myeloPeroxidase (MPO) and Eosinophil Peroxidase (EPO) at pH 7.0 and 25 °C. This was achieved by rapid mixing of Peroxidases with either hydrogen peroxide or hypochlorous acid and measuring spectrophotometrically concentrations of the reacting species and products at equilibrium. By using hydrogen peroxide, the standard reduction potential at pH 7.0 and 25 °C was 1.16 ± 0.01 V for MPO and 1.10 ± 0.01 V for EPO, independently of the concentration of hydrogen peroxide and Peroxidases. In the case of hypochlorous acid, standard reduction potentials were dependent on the hypochlorous acid concentration used. They ranged from 1.16 V at low hypochlorous acid to 1.09 V at higher hypochlorous acid for MPO and from 1.10 V to 1.03 V for EPO. Thus, consistent results for the standard reduction potentials of redox couple compound I/native enzyme of both Peroxidases were obtained with all hydrogen peroxide and at low hypochlorous acid concentrations: possible reasons for the deviation at higher concentrations of hypochlorous acid are discussed. They include instability of hypochlorous acid, reactions of hypochlorous acid with different amino-acid side chains in Peroxidases as well as the appearance of a compound I–chloride complex.
Gerald J Gleich - One of the best experts on this subject based on the ideXlab platform.
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Eosinophil Peroxidase activates cells by her2 receptor engagement and β1 integrin clustering with downstream mapk cell signaling
Clinical Immunology, 2016Co-Authors: Kerrie Hennigan, Gerald J Gleich, Michael Walsh, Paul J Conroy, Mohamed Amin, Richard Okennedy, Patmapriya Ramasamy, Zeshan Siddiqui, Senan Glynn, Olive MccabeAbstract:Eosinophils account for 1–3% of peripheral blood leukocytes and accumulate at sites of allergic inflammation, where they play a pathogenic role. Studies have shown that treatment with mepolizumab (an anti-IL-5 monoclonal antibody) is beneficial to patients with severe Eosinophilic asthma, however, the mechanism of precisely how Eosinophils mediate these pathogenic effects is uncertain. Eosinophils contain several cationic granule proteins, including Eosinophil Peroxidase (EPO). The main significance of this work is the discovery of EPO as a novel ligand for the HER2 receptor. Following HER2 activation, EPO induces activation of FAK and subsequent activation of β1-integrin, via inside-out signaling. This complex results in downstream activation of ERK1/2 and a sustained up regulation of both MUC4 and the HER2 receptor. These data identify a receptor for one of the Eosinophil granule proteins and demonstrate a potential explanation of the proliferative effects of Eosinophils.
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measurement of inflammation in Eosinophilic esophagitis using an Eosinophil Peroxidase assay
The American Journal of Gastroenterology, 2016Co-Authors: Hedieh Saffari, Kristin M Leiferman, Frederic Clayton, Krista Baer, Leonard F Pease, Gerald J Gleich, Kathryn A PetersonAbstract:Measurement of Inflammation in Eosinophilic Esophagitis Using an Eosinophil Peroxidase Assay
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Eosinophil Peroxidase signals via epidermal growth factor 2 to induce cell proliferation
American Journal of Respiratory Cell and Molecular Biology, 2011Co-Authors: Michael Walsh, Gerald J Gleich, Katie Connell, Anita M Sheahan, Richard W CostelloAbstract:Eosinophils exert many of their inflammatory effects in allergic disorders through the degranulation and release of intracellular mediators, including a set of cationic granule proteins that include Eosinophil Peroxidase. Studies suggest that Eosinophils are involved in remodeling. In previous studies, we showed that Eosinophil granule proteins activate mitogen-activated protein kinase signaling. In this study, we investigated the receptor mediating Eosinophil Peroxidase–induced signaling and downstream effects. Human cholinergic neuroblastoma IMR32 and murine melanoma B16.F10 cultures, real-time polymerase chain reaction, immunoprecipitations, and Western blotting were used in the study. We showed that Eosinophil Peroxidase caused a sustained increase in both the expression of epidermal growth factor–2 (HER2) and its phosphorylation at tyrosine 1248, with the consequent activation of extracellular-regulated kinase 1/2. This, in turn, promoted a focal adhesion kinase–dependent egress of the cyclin-depende...
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the induction of Eosinophil Peroxidase release improved methods of measurement and stimulation
Journal of Immunological Methods, 2004Co-Authors: Darryl J Adamko, Gerald J Gleich, Paige Lacy, R MoqbelAbstract:The production and release of Eosinophil Peroxidase (EPO) has been associated with human pathology. Degranulation assays with Eosinophils are typically very difficult to do, with very low release values. EPO is unique for its high cationic charge. As such, it adheres to most extracellular surfaces, rendering it more difficult to measure compared with other released cellular proteins. Based on the understanding of the sticky nature of EPO, we were concerned that EPO released in vitro cannot be reproducibly measured in the supernatants of stimulated cells. Instead, we suspected that much of the released EPO was left adherent to the tube walls. We chose to investigate the measurement of EPO activity using the Peroxidase substrate, O-phenylenediamine (OPD). Unlike other Peroxidase substrates, OPD is soluble in aqueous physiological solutions, which do not lyse cell membranes, thereby allowing us to add OPD directly to Eosinophils and exclusively measure extracellular EPO. This novel approach would remove the concerns of incorrect EPO measurements due to its adhesive nature. In addition, we developed this method to quantify EPO release in terms of EPO concentration. Finally, using this technique, we have been able to demonstrate secretory IgA (s-IgA)-induced release of EPO. By using OPD, we have developed a more sensitive and specific method to analyze the release of extracellular EPO.
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The status of half-cystine residues and locations of N-glycosylated asparagine residues in human Eosinophil Peroxidase.
Archives of biochemistry and biophysics, 2000Co-Authors: Anni R. Thomsen, Gerald J Gleich, Lars Sottrup-jensen, Claus OxvigAbstract:The determination by protein chemistry methods of the half-cystine status in human Eosinophil Peroxidase (EPO) is reported. EPO is two-chained and has a total of 14 half-cystine residues. Cys141 and Cys152 form an intrachain bridge in the light chain of EPO. Disulfide bridges connect Cys253 and Cys263, Cys257 and Cys287, Cys359 and Cys370, Cys570 and Cys635, and Cys676 and Cys701, forming five intrachain disulfide bridges in the heavy chain of EPO. Cys291 and Cys455 are found to be unpaired, containing free sulfhydryl groups. The pattern of disulfide bridges is in agreement with that predicted from the X-ray crystallographic structure of canine myeloPeroxidase (MPO) (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) to be general for the class of mammalian Peroxidases, including EPO, MPO, lactoPeroxidase (LPO), and thyroid Peroxidase (TPO). Of four candidate sites in EPO for attachment of glucosamine-based carbohydrate, Asn327 and Asn363 are occupied, whereas Asn700 and Asn708 are unsubstituted. Furthermore, a discrepancy in the literature regarding the sequence of residues 645-659 is resolved.
Richard W Costello - One of the best experts on this subject based on the ideXlab platform.
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Eosinophil Peroxidase induces expression of cholinergic genes via cell surface neural interactions
Molecular Immunology, 2014Co-Authors: Nadim Akasheh, Michael Walsh, Richard W CostelloAbstract:Eosinophils localize to and release their granule proteins in close association with nerves in patients with asthma and rhinitis. These conditions are associated with increased neural function. In this study the effect of the individual granule proteins on cholinergic neurotransmitter expression was investigated. Eosinophil Peroxidase (EPO) upregulated choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) gene expression. Fluorescently labeled EPO was seen to bind to the IMR-32 cell surface. Both Poly-L-Glutamate (PLG) and Heparinase-1 reversed the up-regulatory effect of EPO on ChAT and VAChT expression and prevented EPO adhesion to the cell surface. Poly-L-arginine (PLA) had no effect on expression of either gene, suggesting that charge is necessary but insufficient to alter gene expression. EPO induced its effects via the activation of NF-κB. MEK inhibition led to reversal of all up-regulatory effects of EPO. These data indicate a preferential role of EPO signaling via a specific surface receptor that leads to neural plasticity.
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Eosinophil Peroxidase induces expression of cholinergic genes via cell surface neural interactions
European Respiratory Journal, 2014Co-Authors: Nadim Akasheh, Michael Walsh, Richard W CostelloAbstract:Eosinophils localize to and release their granule proteins in close association with nerves in patients with asthma and rhinitis. These conditions are associated with increased neural function. In this study the effect of the individual granule proteins on cholinergic neurotransmitter expression was investigated. Eosinophil Peroxidase (EPO) upregulated choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) gene expression. MBP caused upregulation of VAChT but had no effect on ChAT expression. EDN failed to upregulate either gene. Fluorescently labeled EPO was seen to bind to the IMR-32 cell surface. Both Poly-L-glutamate (PLG) and Heparinase-1 reversed the up-regulatory effect of EPO on ChAT and VAChT expression and prevented EPO adhesion to the cell surface. Poly-L-arginine (PLA) had no effect on expression of either gene, suggesting that charge is necessary but insufficient to alter gene expression. EPO induced activation of NF-κB and ERK inhibition led to reversal of all up-regulatory effects of EPO. These data indicate a preferential role of EPO signaling via a specific surface receptor that leads to neural plasticity.
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Eosinophil Peroxidase signals via epidermal growth factor 2 to induce cell proliferation
American Journal of Respiratory Cell and Molecular Biology, 2011Co-Authors: Michael Walsh, Gerald J Gleich, Katie Connell, Anita M Sheahan, Richard W CostelloAbstract:Eosinophils exert many of their inflammatory effects in allergic disorders through the degranulation and release of intracellular mediators, including a set of cationic granule proteins that include Eosinophil Peroxidase. Studies suggest that Eosinophils are involved in remodeling. In previous studies, we showed that Eosinophil granule proteins activate mitogen-activated protein kinase signaling. In this study, we investigated the receptor mediating Eosinophil Peroxidase–induced signaling and downstream effects. Human cholinergic neuroblastoma IMR32 and murine melanoma B16.F10 cultures, real-time polymerase chain reaction, immunoprecipitations, and Western blotting were used in the study. We showed that Eosinophil Peroxidase caused a sustained increase in both the expression of epidermal growth factor–2 (HER2) and its phosphorylation at tyrosine 1248, with the consequent activation of extracellular-regulated kinase 1/2. This, in turn, promoted a focal adhesion kinase–dependent egress of the cyclin-depende...
James J. Lee - One of the best experts on this subject based on the ideXlab platform.
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Eosinophil Peroxidase induces inflammation in a mouse model of dermatitis
bioRxiv, 2020Co-Authors: Quinn R Rothcarter, James J. Lee, Sergei I Ochkur, Elizabeth A Jacobsen, Huijun Luo, Meredith Datena, A D Fryer, David B JacobyAbstract:ABSTRACT Eosinophils play an important role in mediating itch and inflammation in dermatitis. The role of the Eosinophil granule protein Eosinophil Peroxidase (EPX) in mediating inflammation and itch was tested in a dermatitis mouse model. Mice were sensitized to trimellitic anhydride (TMA) and subsequently challenged chronically on the ear to establish dermatitis. Loss of EPX (in EPX (-/-) mice) or blocking EPX with the drug resorcinol significantly reduced dermatitis in mice exposed to TMA. Resorcinol also reduced levels of thymic stromal lymphopoietin protein (TSLP) in skin. Further studies showed that EPX increased different cytokines in keratinocytes in cell culture via two distinct mechanisms. EPX induced TSLP expression requires lysophosphatidic acid signaling while EPX induced expression of TNF-α, CSF2, CSF3, and IL1α required IL-1 signaling. We also showed that blocking IL-1 reduced inflammation in skin following TMA exposure in mice. Thus, EPX is an important mediator of inflammation and itch, that are mediated via at least two pathways. This suggests that both EPX and its’ signaling pathways may provide novel therapeutic strategies in dermatitis.
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normalized serum Eosinophil Peroxidase levels are inversely correlated with esophageal Eosinophilia in Eosinophilic esophagitis
Diseases of The Esophagus, 2018Co-Authors: Benjamin L Wright, Sergei I Ochkur, N S Olson, Kelly P Shim, Elizabeth A Jacobsen, Matthew A Rank, Evan S Dellon, James J. LeeAbstract:Eosinophil Peroxidase is an Eosinophil-specific, cytoplasmic protein stored in the secondary granules of Eosinophils. While Eosinophil Peroxidase deposition is increased in the esophagus in Eosinophilic esophagitis (EOE), its potential role as a peripheral marker is unknown. This study aims to examine the relationship between serum Eosinophil Peroxidase and esophageal Eosinophilia in Eosinophilic esophagitis. Prospectively collected serum from 19 subjects with incident EoE prior to treatment and 20 non-EoE controls were tested for serum Eosinophil Peroxidase, Eosinophilic cationic protein, and Eosinophil derived neurotoxin using ELISA. Matching esophageal tissue sections were stained and assessed for Eosinophil Peroxidase deposition using a histopathologic scoring algorithm. Mean peripheral blood absolute Eosinophil counts in Eosinophilic esophagitis subjects were significantly elevated compared to controls (363 vs. 195 cells/μL, P = 0.008). Absolute median serum Eosinophil Peroxidase, Eosinophil cationic protein, and Eosinophil derived neurotoxin did not differ between groups; however, when normalized for absolute Eosinophil counts, Eosinophilic esophagitis subjects had significantly lower median Eosinophil Peroxidase levels (2.56 vs. 6.96 ng/mL per eos/μL, P = 0.002, AUC 0.79 (0.64, 0.94 95% CI)). Multivariate analysis demonstrated this relationship persisted after controlling for atopy. Esophageal biopsies from Eosinophilic esophagitis subjects demonstrated marked Eosinophil Peroxidase deposition (median score 46 vs. 0, P < 0.0001). Normalized Eosinophil Peroxidase levels inversely correlated with esophageal Eosinophil density (r = -0.41, P = 0.009). In contrast to marked tissue Eosinophil degranulation, circulating Eosinophils appear to retain their granule proteins in EoE. Investigations of normalized serum Eosinophil Peroxidase levels as a biomarker of EoE are ongoing.
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Eosinophil Peroxidase catalyzed protein carbamylation participates in asthma
Journal of Biological Chemistry, 2016Co-Authors: Zeneng Wang, James J. Lee, Nancy A. Lee, Joseph A Didonato, Jennifer A Buffa, Suzy A A Comhair, Mark A Aronica, Raed A Dweik, Mary Jane Thomassen, Mani S KavuruAbstract:The biochemical mechanisms through which Eosinophils contribute to asthma pathogenesis are unclear. Here we show Eosinophil Peroxidase (EPO), an abundant granule protein released by activated Eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN-) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into Nϵ-carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of Eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during Eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFβ, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma.
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nasal and pharyngeal Eosinophil Peroxidase levels in adults with poorly controlled asthma correlate with sputum Eosinophilia
Allergy, 2016Co-Authors: Matthew A Rank, Sergei I Ochkur, Nancy A. Lee, John C Lewis, Harry G Teaford, Lewis Wesselius, Richard A Helmers, Parameswaran Nair, James J. LeeAbstract:The objective of the study was to compare nasal, pharyngeal, and sputum Eosinophil Peroxidase (EPX) levels with induced sputum Eosinophil percentage in 10 adults with poorly controlled asthma and 10 normal controls. EPX was measured using an ELISA and normalized for grams of protein for nasal and pharynx specimens and for mL-gram of protein for sputum. Sputum EPX levels were statistically different between asthma and control subjects (P = 0.024). EPX levels measured in the nasal and pharyngeal swab samples derived from the same patients were also different between asthma and control subjects, each displaying a high degree of significance (P = 0.002). Spearman's correlation coefficients for nasal EPX and pharyngeal EPX levels compared to induced sputum Eosinophil percentage were 0.81 (P = 0.0007) and 0.78 (P = 0.0017), respectively. Thus, there is a strong association in a given patient between both nasal and pharyngeal EPX levels and the Eosinophil percentage of induced sputum.
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Eosinophil Peroxidase in sputum represents a unique biomarker of airway Eosinophilia
Allergy, 2013Co-Authors: Parameswaran Nair, Sergei I Ochkur, Nancy A. Lee, Cheryl A Protheroe, Katherine Radford, Ann Efthimiadis, James J. LeeAbstract:Background Sputum Eosinophilia has been shown to be a predictor of response to anti-Eosinophil therapies in patients with airway diseases. However, quantitative cell counts and differentials of sputum are labor intensive. The objective of this study was to validate a novel ELISA-based assay of Eosinophil Peroxidase (EPX) in sputum as a rapid and reliable marker of airway Eosinophils. Methods The utility of EPX-based ELISA as an Eosinophil-specific assay was achieved through comparisons with sputum Eosinophil differential counts in freshly prepared and archived patient samples from a variety of clinical settings. Results EPX levels in sputum correlated with Eosinophil percentage (rs = 0.84) in asthma patients with varying degrees of airway Eosinophilia. Significantly, unlike assays of other Eosinophil granule proteins (e.g., ECP and EDN), which often detect the presence of these proteins even in asthma patients with neutrophilic bronchitis, EPX-based ELISA levels are not increased in this subset of asthma patients or in COPD patients lacking evidence of an airway Eosinophilia. Moreover, sputum EPX was a surrogate marker of airway Eosinophilia in other patient studies (e.g., allergen inhalation and treatment trials the anti-(IL-5) therapeutic Mepolizumab™). Finally, EPX levels in cytocentrifuged prepared sputum supernatants correlated with those from rapidly prepared noncentrifuged filtrates of sputum (rs = 0.94). Conclusion and clinical implication EPX-based ELISA is a valid, reliable, repeatable, and specific surrogate marker of Eosinophils and/or Eosinophil degranulation in the sputum of respiratory patients. The novel EPX assay is a valid and reproducible Eosinophil-specific assay that can potentially be developed into a point-of-care assessment of Eosinophil activity in airway secretions.
Christian Obinger - One of the best experts on this subject based on the ideXlab platform.
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redox thermodynamics of lactoPeroxidase and Eosinophil Peroxidase
Archives of Biochemistry and Biophysics, 2010Co-Authors: Gianantonio Battistuzzi, Paul G. Furtmüller, Marzia Bellei, Jutta Vlasits, Srijib Banerjee, Marco Sola, Christian ObingerAbstract:Abstract Eosinophil Peroxidase (EPO) and lactoPeroxidase (LPO) are important constituents of the innate immune system of mammals. These heme enzymes belong to the Peroxidase-cyclooxygenase superfamily and catalyze the oxidation of thiocyanate, bromide and nitrite to hypothiocyanate, hypobromous acid and nitrogen dioxide that are toxic for invading pathogens. In order to gain a better understanding of the observed differences in substrate specificity and oxidation capacity in relation to heme and protein structure, a comprehensive spectro-electrochemical investigation was performed. The reduction potential ( E °′) of the Fe(III)/Fe(II) couple of EPO and LPO was determined to be −126 mV and −176 mV, respectively (25 °C, pH 7.0). Variable temperature experiments show that EPO and LPO feature different reduction thermodynamics. In particular, reduction of ferric EPO is enthalpically and entropically disfavored, whereas in LPO the entropic term, which selectively stabilizes the oxidized form, prevails on the enthalpic term that favors reduction of Fe(III). The data are discussed with respect to the architecture of the heme cavity and the substrate channel. Comparison with published data for myeloPeroxidase demonstrates the effect of heme to protein linkages and heme distortion on the redox chemistry of mammalian Peroxidases and in consequence on the enzymatic properties of these physiologically important oxidoreductases.
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kinetics of interconversion of redox intermediates of lactoPeroxidase Eosinophil Peroxidase and myeloPeroxidase
Japanese Journal of Infectious Diseases, 2004Co-Authors: Paul G. Furtmüller, Martina Zederbauer, Walter Jantschko, Christa Jakopitsch, Jurgen Arnhold, Christian ObingerAbstract:MyeloPeroxidase, Eosinophil Peroxidase and lactoPeroxidase are heme-containing oxidoreductases, which undergo a series of redox reactions. Though sharing functional and structural homology, reflecting their phylogenetic origin, differences are observed regarding their spectral features, substrate specificities, redox properties and kinetics of interconversion of the relevant redox intermediates ferric and ferrous Peroxidase, compound I, compound II and compound III. Depending on substrate availability, these heme enzymes path through the halogenation cycle and/or the Peroxidase cycle and/or act as poor (pseudo-) catalases.
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redox properties of the couple compound i native enzyme of myeloPeroxidase and Eosinophil Peroxidase
FEBS Journal, 2001Co-Authors: Jurgen Arnhold, Paul G. Furtmüller, Gunther Regelsberger, Christian ObingerAbstract:The standard reduction potential of the redox couple compound I/native enzyme has been determined for human myeloPeroxidase (MPO) and Eosinophil Peroxidase (EPO) at pH 7.0 and 25 °C. This was achieved by rapid mixing of Peroxidases with either hydrogen peroxide or hypochlorous acid and measuring spectrophotometrically concentrations of the reacting species and products at equilibrium. By using hydrogen peroxide, the standard reduction potential at pH 7.0 and 25 °C was 1.16 ± 0.01 V for MPO and 1.10 ± 0.01 V for EPO, independently of the concentration of hydrogen peroxide and Peroxidases. In the case of hypochlorous acid, standard reduction potentials were dependent on the hypochlorous acid concentration used. They ranged from 1.16 V at low hypochlorous acid to 1.09 V at higher hypochlorous acid for MPO and from 1.10 V to 1.03 V for EPO. Thus, consistent results for the standard reduction potentials of redox couple compound I/native enzyme of both Peroxidases were obtained with all hydrogen peroxide and at low hypochlorous acid concentrations: possible reasons for the deviation at higher concentrations of hypochlorous acid are discussed. They include instability of hypochlorous acid, reactions of hypochlorous acid with different amino-acid side chains in Peroxidases as well as the appearance of a compound I–chloride complex.
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spectral and kinetic studies on Eosinophil Peroxidase compounds i and ii and their reaction with ascorbate and tyrosine
Biochimica et Biophysica Acta, 2001Co-Authors: Paul G. Furtmüller, Walter Jantschko, Gunther Regelsberger, Christian ObingerAbstract:Abstract Eosinophil Peroxidase, the major granule protein in Eosinophils, is the least studied human Peroxidase. Here, we have performed spectral and kinetic measurements to study the nature of Eosinophil Peroxidase intermediates, compounds I and II, and their reduction by the endogenous one-electron donors ascorbate and tyrosine using the sequential-mixing stopped-flow technique. We demonstrate that the Peroxidase cycle of Eosinophil Peroxidase involves a ferryl/porphyrin radical compound I and a ferryl compound II. In the absence of electron donors, compound I is shown to be transformed to a species with a compound II-like spectrum. In the presence of ascorbate or tyrosine compound I is reduced to compound II with a second-order rate constant of (1.0±0.2)×10 6 M −1 s −1 and (3.5±0.2)×10 5 M −1 s −1 , respectively (pH 7.0, 15°C). Compound II is then reduced by ascorbate and tyrosine to native enzyme with a second-order rate constant of (6.7±0.06)×10 3 M −1 s −1 and (2.7±0.06)×10 4 M −1 s −1 , respectively. This study revealed that Eosinophil Peroxidase compounds I and II are able to react with tyrosine and ascorbate via one-electron oxidations and therefore generate monodehydroascorbate and tyrosyl radicals. The relatively fast rates of the compound I reduction demonstrate that these reactions may take place in vivo and are physiologically relevant.
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spectral and kinetic studies on the formation of Eosinophil Peroxidase compound i and its reaction with halides and thiocyanate
Biochemistry, 2000Co-Authors: Paul G. Furtmüller, Ursula Burner, And Gunther Regelsberger, Christian ObingerAbstract:Compound I of Peroxidases takes part in both the peroxidation and the halogenation reaction. This study for the first time presents transient kinetic measurements of the formation of compound I of human Eosinophil Peroxidase (EPO) and its reaction with halides and thiocyanate, using the sequential-mixing stopped-flow technique. Addition of 1 equiv of hydrogen peroxide to native EPO leads to complete formation of compound I. At pH 7 and 15 °C, the apparent second-order rate constant is (4.3 ± 0.4) × 107 M-1 s-1. The rate for compound I formation by hypochlorous acid is (5.6 ± 0.7) × 107 M-1 s-1. EPO compound I is unstable and decays to a stable intermediate with a compound II-like spectrum. At pH 7, the two-electron reduction of compound I to the native enzyme by thiocyanate has a second-order rate constant of (1.0 ± 0.5) × 108 M-1 s-1. Iodide [(9.3 ± 0.7) × 107 M-1 s-1] is shown to be a better electron donor than bromide [(1.9 ± 0.1) × 107 M-1 s-1], whereas chloride oxidation by EPO compound I is extremel...