HOCl Generation Research Articles

Highly Selective Detection of Hypochlorous Acid by a Bis-heteroleptic Ru(II) Complex of Pyridyl-1,2,3-triazole Ligand via C(sp2)-H Hydroxylation.

A Ru(II) complex (Ru-1) of a substituted pyridyl-1,2,3-triazole ligand (BtPT) for highly selective "light-up" detection of hypochlorous acid is presented. An unusual anti-Markovnikov HOCl addition to the C═C bond of 1,2,3-triazole and a highly specific C(sp2)-H hydroxylation over epoxidation made Ru-1 a highly selective luminescent HOCl probe. The abnormal regio- and stereoselective HOCl addition and subsequent hydroxylation mechanism in detail is supported by the combination of ESI-MS, 1H/13C NMR spectroscopy, and 1H NMR titration. The hydroxylation at the C5 center in 1,2,3-triazole increases the electron density and makes BtPT a better σ-donor as well as π-donor, which in turn increases the 3MC-3MLCT energy gap and inhibits the nonradiative decay from the excited state of Ru-1 and is the key reason for luminescence light-up. Most importantly, the exogenous and endogenous HOCl imaging in the living HEK293T cells is also demonstrated. The probe showed low cytotoxicity and efficiently permeated the cell membrane. The cell-imaging experiments revealed rapid staining of the extranuclear region of HEK293T cells which clearly indicates the presence of cytoplasmic HOCl. The endogenous HOCl generation and imaging, stimulated by lipopolysaccharides (LPS) and paraquat in the HEK293T cells, is also demonstrated.

Novel Contribution of Myeloperoxidase‐derived Hypochlorous Acid to 20‐HETE Production Post‐Ischemia

Ischemia‐induced angiogenesis occurs as a mechanism to minimize the tissue damage caused by atherosclerotic diseases but is often insufficient to maintain adequate tissue perfusion and results in critical limb ischemia and amputation. The discovery of novel factors that increase angiogenesis in response to ischemia is therefore of clinical relevance.Recently, we discovered that 20‐hydroxy‐eicosatetraenoic acid (20‐HETE), an arachidonic acid metabolite produced by a cytochrome P450 (CYP) 4A/F ω‐hydroxylases, is a novel contributor of ischemia‐induced angiogenesis based on the following two major findings: 1) pharmacological interference of the CYP4A/20‐HETE axis attenuates ischemic‐induced angiogenesis; and 2) the expression of cyp4a12a and its product, 20‐HETE are both elevated in ischemic tissues of mice models. The resulting increase in 20‐HETE is endothelial in origin. However, the factors that cause endothelial cells (EC) to produce 20‐HETE following ischemia are largely unknown. The aim of these studies was to investigate the mechanism of post‐ischemic endothelial 20‐HETE increases. Inflammatory cells such as macrophages and neutrophils are the first responders to ischemia injury. Thus, we hypothesized that inflammatory cells contribute to post‐ischemic endothelial increases of 20‐HETE.We first examined the potential contribution of inflammatory response to hindlimb ischemia‐induced angiogenesis using immune‐deficient SCID mice. Following a femoral artery ligation and comparing SCID mice to Balb/C control mice, SCID mice exhibited impaired blood perfusion (55 ± 9% vs 89 ± 10%), decreased micro‐vessel counts (capillary:fiber ratio of 1.5 ± .25 vs 2.5 ± 3.75), as well as an inability to produce 20‐HETE (7.5 ± 2.5 vs 29 ± 5 pg/mg of protein).Furthermore, immune‐competent Balb/c mice depleted of macrophages or neutrophils showed that ischemia failed to induce 20‐HETE production in Balb/c depleted of neutrophils (13 ± 1.5 vs 35 ± 5 pg/mg of protein), accompanied with compromised blood perfusion (58 ± 10% vs 82 ± 9%), but not those depleted of macrophages. Neutrophils are known to release Myeloperoxidase (MPO) at sites of inflammation. The possibility that MPO contributes to ischemia‐induced 20‐HETE production was investigated using MPO−/− mice. Remarkably, the ischemic hind limb tissues of MPO−/− mice produce negligible amount of 20‐HETE (~2 ± .25 vs 35 ± 5 pg/mg of protein). MPO is known to produce hypochlorous acid (HOCl). Therefore, we further studied the role of MPO‐derived HOCl in 20‐HETE production in cultured human EC. The addition of physiological amounts of either MPO or HOCl markedly stimulated the expression of 20‐HETE synthase CYP4A11 and production of 20‐HETE (40 ± 12 vs 8 ± 5 pg/mg of protein); potentially via a HIF1‐a dependent mechanism.These observations suggest for the first time that neutrophil‐derived MPO critically contributes to the 20‐HETE increases post‐ischemia via a mechanism involving the generation of HOCl and activation of the HIF pathway in endothelium.Support or Funding InformationAHA [11SDG6870004(AMG), 17GRNT33430003(AMG)]NIH [HL34300(MLS), DK38226(JRF)]RA Welch Foundation [GL625910(JRF)]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Propofol specifically reduces PMA-induced neutrophil extracellular trap formation through inhibition of p-ERK and HOCl

Neutrophil extracellular traps (NETs) are net-like chromatin fibers that can trap and kill microorganisms. Although several anti-inflammatory effects of intravenous anesthetics have been reported, it has not been investigated whether intravenous anesthetics influence NET formation. AimsTo compare the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on phorbol myristate acetate (PMA)-induced NET formation and analyze the associated signaling pathways. Materials and methodsPMA-stimulated NETs formed in the absence or presence of intravenous anesthetics were stained with SYTOX Green and then quantified. Inhibitors were applied to investigate the related mechanism, which was confirmed by western blotting, and ROS were detected. Key findingsThe neutrophils incubated with propofol showed the lowest degree of NET formation compared with those incubated with the other intravenous anesthetics. Propofol significantly reduced the level of myeloperoxidase (MPO)-derived HOCl but not that of superoxide. Aminopyrine, an MPO inhibitor, markedly decreased the number of PMA-induced NETs, indicating the involvement of HOCl in the inhibitory effect of propofol on NET formation. According to western blotting results, the level of p-ERK was reduced by propofol during PMA-induced NET formation. The ERK inhibitor PD98059 decreased NET formation but did not inhibit PMA-induced HOCl generation, and aminopyrine did not reduce ERK phosphorylation. SignificanceThrough this study, we define a new anti-inflammatory effect of intravenous anesthetics. Of the four intravenous anesthetics tested, propofol was the most potent inhibitor of NET formation. Moreover, propofol resulted in a decrease in PMA-induced NET formation by two independent mechanisms: inhibition of HOCl and p-ERK.

Endoplasmic Reticulum-Targeted Ratiometric N-Heterocyclic Carbene Borane Probe for Two-Photon Microscopic Imaging of Hypochlorous Acid.

The naphthoimidazolium borane 4 is shown to be a selective probe for HOCl over other reactive oxygen species. Unlike other boronate-reactive oxygen species (ROS) fluorogenic probes that are oxidized by HOCl through a nucleophilic borono-Dakin oxidation mechanism, probe 4 is distinguished by its electrophilic oxidation mechanism involving B-H bond cleavage. Two-photon microscopy experiments in living cells and tissues with the probe 4 demonstrate the monitoring of endogenous HOCl generation and changes in HOCl concentrations generated in the endoplasmic reticulum during oxidative stress situations.

Rapid Response Fluorescence Probe Enabled In Vivo Diagnosis and Assessing Treatment Response of Hypochlorous Acid-Mediated Rheumatoid Arthritis.

Diagnosis and early assessment of the treatment response of rheumatoid arthritis (RA) necessitates a reliable bioanalytical method for rapid, sensitive, and specific detection of the hypochlorous acid (HOCl) biomarker in inflammatory diseases. Herein, two fluorescence probes, Probe‐1 and Probe‐2 are developed for quantitative monitoring and visualization of inflammatory response–related HOCl levels in vitro and in vivo. In the presence of HOCl, fluorescence “OFF–ON” response is obtained for both the probes as a result of specific HOCl‐triggered C=N bond cleavage reaction. Probe‐1 and Probe‐2 feature rapid response (<4 s), a high degree of sensitivity and selectivity toward HOCl, which allow them to be used for quantification of HOCl in a simulated physiological condition. Using Probe‐2 as the probe, fluorescence imaging and flow cytometry analysis of HOCl levels in lysosome of inflammatory mimic cells, visualization of HOCl generation in endotoxin‐induced inflammation of adult zebrafish and RA of mice are possible. Probe‐2 exhibits high effectiveness for early assessment of the treatment response of HOCl‐mediated RA in mice with an antiarthritic drug, methotrexate (MTX). The results demonstrate that Probe‐2 is a powerful tool for future studies on diagnosis and monitoring treatment efficiency in a broad range of inflammatory diseases, including RA.

P-152 - N-acyl derivatives of 4,6-di-tert-butyl-2-aminophenol protect neutrophils from halogenating stress

Hypochlorous acid is an important microbicidal agent mainly formed by neutrophil myeloperoxidase (MPO), however HOCl hyperproduction leads to halogenating stress associated with different diseases. It is necessary to search for substances that can selectively prevent excessive HOCl generation. We synthesized a number of N-acyl derivatives of 4,6-di-tert-butyl-2-aminophenol which exhibit antiradical activity in cell free model systems. The aim of the work is to study the effect of (N-(4,6-di-tert-butyl-2-aminophenol)R, where R=-COCH3 (1), -COC2H5 (2), -COC3H7 (3) on ROS formation, secretory degranulation and viability of neutrophils exposed to HOCl. It has been shown that all compounds in micromolar concentrations reduce neutrophil ROS formation by 75% or more, inhibit MPO secretion and reduce HOCl production by MPO. It has been found that compounds 1–3 effectively intercept HOCl (up to 78%), but do not interact with H2O2. In concentrations of up to 1 μmol/l, compound 1 increases cell survival in the absence of exogenous HOCl, while compounds 2 and 3 do not influence it. Compound 1 in higher concentrations prevents destruction of neutrophils caused by addition of 300 μmol/l HOCl. Thus, compounds 1–3 can be considered as potential cytoprotectors under halogenating stress.

Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent Hypochlorous Acid Formation and Vascular Endothelial Injury

Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl). As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited MPO-mediated HOCl formation (75.0 ± 6.2% for 10 μM quercetin versus 100 ± 5.2% for control group, P < 0.01) and cytotoxicity to endothelial cells in vitro, while this flavonoid was nontoxic to endothelial cell cultures ( P > 0.05, all cases). Moreover, quercetin inhibited HOCl generation by stimulated neutrophils (a rich source of MPO) and protected endothelial cells from neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial cells ( P < 0.05, all cases). Consistent with these in vitro data, quercetin attenuated lipopolysaccharide-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid could protect against HOCl-mediated endothelial dysfunction in isolated aortas ( P < 0.05). Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation or scavenging of HOCl. These data indicate that quercetin is a nontoxic inhibitor of MPO activity and MPO/neutrophils-induced cytotoxicity in endothelial cells and may be useful for targeting MPO-dependent vascular disease and inflammation.

Modeling Hypochlorous Acid Generation on Carbon Fabric Surface

Hypochlorous acid (HOCl) can be electrochemically generated on carbon fabric surfaces made from carbon fibers for wound healing purposes and removing biofilms. HOCl is an oxidizer and deproteinizer with antimicrobial activity that gives carbon fabric surfaces biocidal properties. HOCl is of particular interest in wound disinfection as it offers lower toxicity, and lower irritability to mammalian cells than other biocidal agents such as hydrogen peroxide (H2O2) and sodium hypochlorite (NaOCl). In principle, carbon fiber surfaces can be typically applied on the infected wound or biofilms grown in test surfaces. In this work, a COMSOL Multiphysics ® finite element model of HOCl generation on carbon fabric surface is developed. In addition, we developed a new all-in-one microelectrode to measure local HOCl concentrations. The new microelectrode offers accurate measurements on polarized carbon surfaces due to its robustness against potential perturbations caused by the electric field generated on polarized carbon fiber surface. The novel microelectrode is used to 1) measure depth profiles and 2) probe local variations near the electrode surface using cyclic voltammetry. The model kinetic parameters are fit to experimental local HOCl concentrations on the surface of the working electrode of the carbon fabric in phosphate buffer saline (PBS). The predicted parameters are integrated to the model to predict HOCl and Cl- concentrations as function of time. In addition, the time needed to reduce the buffer capacity is determined. This information is of interest as it provides the experimenter with tools to determine the application time necessary to reach an effective HOCl concentration required to eradicate biofilm or the point in time when replenishing of Cl- is needed.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits myeloperoxidase activity in inflammatory cells

BackgroundMyeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils. MethodsMPO activity was determined fluorometrically using hypochlorite-specific 3′-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site. ResultsLGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl−. EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site. ConclusionsWe conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage.

Development of a new fluorescence ratiometric switch for endogenous hypochlorite detection in monocytes of diabetic subjects by dye release method

Increased oxidative stress in metabolic complications like type 2 diabetes, dyslipidemia and cardiovascular disorders exerts potential health hazards in many facets. Enhanced production of reactive oxygen species (ROS) due to increased oxidative stress promotes the damage of many biologically important macromolecules. Hypochlorous acid (HOCl), a microbicidal agent is also known to be an important ROS sub-species. An enhanced generation of endogenous HOCl due to diseased condition therefore can be detrimental to health. In present work, a new quinoline-diaminomaleonitrile based probe (HQMN) has been designed for the selective detection of hypochlorite. The probe in hand shows a selective ratiometric emission change towards OCl−. The probe behaves as a highly selective and sensitive tool for the detection of OCl− over other analytes with a fast response time (within 100 s). Bioimaging study revealed that HQMN can detect endogenous OCl− in human monocytes and an increase in endogenous HOCl concentration has been witnessed in diabetic condition compared to healthy control. Thus HQMN can be used as an excellent fluorescent probe for dynamic tracking of hypochorite in living biological cells especially to identify diabetic conditions.

Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by (-)-Epigallocatechin Gallate.

Myeloperoxidase (MPO) plays important roles in various diseases through its unique chlorinating activity to catalyze excess hypochlorous acid (HOCl) formation. Epidemiological studies indicate an inverse correlation between plant polyphenol consumption and the incidence of cardiovascular diseases. Here we showed that (-)-epigallocatechin gallate (EGCG), the main flavonoid present in green tea, dose-dependently inhibited MPO-mediated HOCl formation in vitro (chlorinating activities of MPO: 50.2 ± 5.7% for 20 μM EGCG versus 100 ± 5.6% for control, P < 0.01). UV-vis spectral and docking studies indicated that EGCG bound to the active site (heme) of MPO and resulted in the accumulation of compound II, which was unable to produce HOCl. This flavonoid also effectively inhibited HOCl generation in activated neutrophils (HOCl formation: 65.0 ± 5.6% for 20 μM EGCG versus 100 ± 6.2% for control, P < 0.01) without influencing MPO and Nox2 release and superoxide formation, suggesting that EGCG specifically inhibited MPO but not NADPH oxidase activity in activated neutrophils. Moreover, EGCG inhibited MPO (or neutrophil)-mediated HOCl formation in human umbilical vein endothelial cells (HUVEC) culture and accordingly protected HUVEC from MPO (or neutrophil)-induced injury (P < 0.05, all cases), although it did not induce cytotoxicity to HUVEC (P > 0.05, all cases). Our results indicate that dietary EGCG is an effective and specific inhibitor of MPO activity and may participate in the regulation of immune responses at inflammatory sites.

Development of a novel europium complex-based luminescent probe for time-gated luminescence imaging of hypochlorous acid in living samples

Luminescent lanthanide complexes are key reagents used in the time-gated luminescence bioassay technique, but functional lanthanide complexes that can act as luminescent probes for specifically responding to analytes are very limited. In this work, we designed and synthesized a novel Eu3+ complex-based luminescence probe for hypochlorous acid (HOCl), NPPTTA-Eu3+, by using terpyridine polyacid-Eu3+, dinitrophenyl, and hydrazine as luminophore, quencher and HOCl-recognizer moieties, respectively. In the absence of HOCl, the probe is non-luminescent due to the strong luminescence quenching of the dinitrophenyl group in the complex. However, upon reaction with HOCl, the dinitrophenyl moiety is rapidly cleaved from the probe, which affords a strongly luminescent Eu3+ complex CPTTA-Eu3+, accompanied by a ∼900-fold luminescence enhancement with a long luminescence lifetime of 1.41 ms. This unique luminescence response of NPPTTA-Eu3+ to HOCl allowed NPPTTA-Eu3+ to be conveniently used as a probe for highly selective and sensitive detection of HOCl under the time-gated luminescence mode. In addition, by loading NPPTTA-Eu3+ into RAW 264.7 macrophage cells and Daphnia magna, the generation of endogenous HOCl in RAW 264.7 cells and the uptake of exogenous HOCl by Daphnia magna were successfully imaged on a true-color time-gated luminescence microscope. The results demonstrated the practical applicability of NPPTTA-Eu3+ as an efficient probe for time-gated luminescence imaging of HOCl in living cells and organisms.

A mitochondria-targeting time-gated luminescence probe for hypochlorous acid based on a europium complex.

Bioresponsive luminescence probes based on lanthanide complexes have shown great utility in a variety of time-gated luminescence bioassays. However, functional lanthanide complexes that can target individual organelles for probing biospecies therein have rarely been investigated. In this work, a unique Eu3+ complex, Mito-NPSTTA-Eu3+, was designed and synthesized as a probe for the time-gated luminescence sensing of HOCl inside the mitochondria of living cells. The probe showed a fast, highly sensitive and selective luminescence response to HOCl with a wide available pH range (pH 3-10), and highly specific mitochondria-localization performance. Taking advantage of time-gated luminescence bioimaging and the excellent properties of the Eu3+ complex, the generation of endogenous HOCl in RAW 264.7 cells and the uptake of exogenous HOCl by zebrafish were successfully imaged, respectively. The results demonstrated the feasibility of Mito-NPSTTA-Eu3+ for the imaging of mitochondrial HOCl, and validated the potential of our strategy for the design of lanthanide complex-based organelle-targeting bioresponsive probes.

18 - Characterisation of the Novel Products of Protein Oxidation by the Inflammatory Oxidant Hypochlorous Acid (HOCl) Using Raman Spectroscopy

Background The heme enzyme myeloperoxidase that is released by activated leukocytes at sites of inflammation, uses H2O2 and chloride ions to form the bactericidal agent hypochlorous acid (HOCl). Inappropriate or excessive production of this powerful oxidant can however cause host tissue damage, with this being implicated in multiple human inflammatory diseases. Proteins are major biological targets for HOCl, with sulfur- and amine-containing residues particularly susceptible to damage. Reaction of HOCl with amines (e.g. the α-amino groups of oxidised glutathione, GSSG, or the amine group of Lys side-chains) generates chloramines (RNHCl) that undergo subsequent decomposition to aldehydes (R’CHO) and nitriles (R’C≡N). Hypothesis: Nitriles may be characteristic biomarkers of HOCl and chloramine generation, which can be readily detected by Raman spectroscopy via their characteristic absorbance bands. Methods: Sulfur- and amine-containing model peptides were incubated for 24 h with up to 20-fold molar excess HOCl, before drying and analysing by Raman spectroscopy Results Analysis of the HOCl-treated model peptides by Raman spectroscopy shows dose-dependent changes in the wavelength and intensity of characteristic Raman bands assigned to disulfides (500–550 cm-1), and the formation of a new Raman absorption at ~2255 cm-1 assigned to stretches of nitrile groups. The latter peak was detected in a dose-dependent manner on HOCl treatment of Lys-containing protein and peptide models, N-acetyl-histidine and oxidised glutathione (GSSG). Conclusions HOCl-mediated oxidation of proteins and peptides modifies protein and peptide side-chains in a manner that can be readily detected by Raman spectroscopy. The absorption band attributed to C≡N stretching at ~2255 cm-1 is well removed from other features in the Raman spectra, making it a potential marker of HOCl-mediated protein oxidation, chloramine formation and decomposition.

A unique iridium(III) complex-based chemosensor for multi-signal detection and multi-channel imaging of hypochlorous acid in liver injury

Although hypochlorous acid (HOCl) has long been associated with a number of inflammatory diseases in mammalian bodies, the functions of HOCl in specific organs at abnormal conditions, such as liver injury, remain unclear due to its high reactivity and the lack of effective methods for its detection. Herein, a unique Ir(III) complex-based chemosensor, Ir-Fc, was developed for highly sensitive and selective detection of HOCl. Ir-Fc was designed by incorporating a ferrocene (Fc) quencher to a Ir(III) complex through a HOCl-responsive linker. In the presence of HOCl, the fast cleavage of Fc moiety in less than 1s led to the enhancement of photoluminescence (PL) and electrochemical luminescence (ECL), by which the concentration of HOCl was determined by both PL and ECL analysis. Taking advantages of excellent properties of Ir(III) complexes, optical and electrochemical analyses of the response of Ir-Fc towards HOCl were fully investigated. Followed by the measurements of low cytotoxicity of Ir-Fc by MTT analysis, one-photon (OP), two-photon (TP) and lifetime imaging experiments were conducted to visualise the generation of HOCl in live microphage and HepG2 cells, and in zebrafish and mouse, respectively. Furthermore, the generation and distribution of HOCl in liver cells and liver injury of zebrafish and mouse were investigated. The results demonstrated the applicability of Ir-Fc as an effective chemosensor for imaging of HOCl generation in mitochondria of cells and liver injury in vivo, implying the potential of Ir-Fc for biomedical diagnosis and monitoring applications.

Levels of inflammation and oxidative stress, and a role for taurine in dystropathology of the Golden Retriever Muscular Dystrophy dog model for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal skeletal muscle wasting disease presenting with excessive myofibre necrosis and increased inflammation and oxidative stress. In the mdx mouse model of DMD, homeostasis of the amino acid taurine is altered, and taurine administration drastically decreases muscle necrosis, dystropathology, inflammation and protein thiol oxidation. Since the severe pathology of the Golden Retriever Muscular Dystrophy (GRMD) dog model more closely resembles the human DMD condition, we aimed to assess the generation of oxidants by inflammatory cells and taurine metabolism in this species. In muscles of 8 month GRMD dogs there was an increase in the content of neutrophils and macrophages, and an associated increase in elevated myeloperoxidase, a protein secreted by neutrophils that catalyses production of the highly reactive hypochlorous acid (HOCl). There was also increased chlorination of tyrosines, a marker of HOCl generation, increased thiol oxidation of many proteins and irreversible oxidative protein damage. Taurine, which functions as an antioxidant by trapping HOCl, was reduced in GRMD plasma; however taurine was increased in GRMD muscle tissue, potentially due to increased muscle taurine transport and synthesis. These data indicate a role for HOCl generated by neutrophils in the severe dystropathology of GRMD dogs, which may be exacerbated by decreased availability of taurine in the blood. These novel data support continued research into the precise roles of oxidative stress and taurine in DMD and emphasise the value of the GRMD dogs as a suitable pre-clinical model for testing taurine as a therapeutic intervention for DMD boys.

Development of a BODIPY-based ratiometric fluorescent probe for hypochlorous acid and its application in living cells

A BODIPY-based ratiometric fluorescent probe for HOCl has been designed based on the transduction of thioether to sulfoxide function. This probe features a marked absorption and emission blue-shift upon the HOCl-promoted rapid transduction, enabling the highly selective and ratiometric detection. In addition, the probe works excellently within a wide pH range of 4–10, addressing the existing pH dependency issue. Living cells studies demonstrate that the probe is cell membrane permeable and can be employed successfully to image endogenous HOCl generation in macrophage cells.

A review of the proposed role of neutrophils in rodent amebic liver abscess models.

Host invasion by Entamoeba histolytica, the pathogenic agent of amebiasis, can lead to the development of amebic liver abscess (ALA). Due to the difficulty of exploring host and amebic factors involved in the pathogenesis of ALA in humans, most studies have been conducted with animal models (e.g., mice, gerbils, and hamsters). Histopathological findings reveal that the chronic phase of ALA in humans corresponds to lytic or liquefactive necrosis, whereas in rodent models there is granulomatous inflammation. However, the use of animal models has provided important information on molecules and mechanisms of the host/parasite interaction. Hence, the present review discusses the possible role of neutrophils in the effector immune response in ALA in rodents. Properly activated neutrophils are probably successful in eliminating amebas through oxidative and non-oxidative mechanisms, including neutrophil degranulation, the generation of free radicals (O2−, H2O2, HOCl) and peroxynitrite, the activation of NADPH-oxidase and myeloperoxidase (MPO) enzymes, and the formation of neutrophil extracellular traps (NETs). On the other hand, if amebas are not eliminated in the early stages of infection, they trigger a prolonged and exaggerated inflammatory response that apparently causes ALAs. Genetic differences in animals and humans are likely to be key to a successful host immune response.

Myeloperoxidase-Hepatocyte-Stellate Cell Cross Talk Promotes Hepatocyte Injury and Fibrosis in Experimental Nonalcoholic Steatohepatitis.

Myeloperoxidase (MPO), a highly oxidative enzyme secreted by leukocytes has been implicated in human and experimental nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain unknown. In this study, we investigated how MPO contributes to progression from steatosis to NASH. In C57Bl/6J mice fed a diet deficient in methionine and choline to induce NASH, neutrophils and to a lesser extent inflammatory monocytes are markedly increased compared with sham mice and secrete abundant amounts of MPO. Through generation of HOCl, MPO directly causes hepatocyte death in vivo. In vitro experiments demonstrate mitochondrial permeability transition pore induction via activation of SAPK/JNK and PARP. MPO also contributes to activation of hepatic stellate cells (HSCs), the most important source of collagen in the liver. In vitro MPO-activated HSCs have an activation signature (MAPK and PI3K-AKT phosphorylation) and upregulate COL1A1, α-SMA, and CXCL1. MPO-derived oxidative stress also activates transforming growth factor β (TGF-β) in vitro, and TGF-β signaling inhibition with SB-431542 decreased steatosis and fibrosis in vivo. Conversely, congenital absence of MPO results in reduced hepatocyte injury, decreased levels of TGF-β, fewer activated HSCs, and less severe fibrosis in vivo. Cumulatively, these findings demonstrate important cross talk between inflammatory myeloid cells, hepatocytes, and HSCs via MPO and establish MPO as part of a proapoptotic and profibrotic pathway of progression in NASH, as well as a potential therapeutic target to ameliorate this disease.

Arbutin and decrease of potentially toxic substances generated in human blood neutrophils.

Neutrophils, highly motile phagocytic cells, constitute the first line of host defense and simultaneously they are considered to be central cells of chronic inflammation. In combination with standard therapeutic procedures, natural substances are gaining interest as an option for enhancing the effectiveness of treatment of inflammatory diseases. We investigated the effect of arbutin and carvedilol and of their combination on 4β-phorbol-12β-myristate-13α-acetate- stimulated functions of human isolated neutrophils. Cells were preincubated with the drugs tested and subsequently stimulated. Superoxide (with or without blood platelets, in the rate close to physiological conditions [1:50]) and HOCl generation, elastase and myeloperoxidase release were determined spectrophotometrically and phospholipase D activation spectrofluorometrically. The combined effect of arbutin and carvedilol was found to be more effective than the effect of each compound alone.Our study provided evidence supporting the potential beneficial effect of arbutin alone or in combination with carvedilol in diminishing tissue damage by decreasing phospholipase D, myeloperoxidase and elastase activity and by attenuating the generation of superoxide and the subsequently derived reactive oxygen species. The presented data indicate the ability of arbutin to suppress the onset and progression of inflammation.