Reactive Oxidative Intermediates Research Articles

A photo-assisted electrocatalyst coupled with superoxide suppression for high performance Li-O2 batteries

Abstract Li-O2 batteries are one of the most promising next generation batteries due to their high energy density. However, they suffer from high charge overpotentials and severe side reactions. Here we report a bifunctional CeVO4 catalyst that not only lowers the charge potential by a photo-assisted mechanism, more importantly, promotes the efficient conversion of highly reactive oxidative intermediates to Li2O2, thus avoiding the oxidative decomposition of the electrolyte/cathode during discharge. Benefiting from its efficient electrocatalysis and the improved charging ability, 1.6x higher discharge capacity up to 6.14 mAh cm-2, low charge potential of 3.48 V and stable cycling performance are simultaneously achieved. Theoretical calculation and in-situ Raman spectra confirm the superior adsorptive and conversion ability of superoxide species by electrochemical reduction and chemical disproportionation reactions on the CeVO4 catalyst. These results indicate an effective strategy to break through the reported limitations of photo-assisted cathodes that are of a low electrocatalysis ability and limited discharge capacity. The significance of retaining a high energy density in photo-assisted Li-O2 batteries is highlighted.

Nitric oxide and ROS mediate autophagy and regulate Alternaria alternata toxin-induced cell death in tobacco BY-2 cells

Synergistic interaction of nitric oxide (NO) and reactive oxygen species (ROS) is essential to initiate cell death mechanisms in plants. Though autophagy is salient in either restricting or promoting hypersensitivity response (HR)-related cell death, the crosstalk between the reactive intermediates and autophagy during hypersensitivity response is paradoxical. In this investigation, the consequences of Alternaria alternata toxin (AaT) in tobacco BY-2 cells were examined. At 3 h, AaT perturbed intracellular ROS homeostasis, altered antioxidant enzyme activities, triggered mitochondrial depolarization and induced autophagy. Suppression of autophagy by 3-Methyladenine caused a decline in cell viability in AaT treated cells, which indicated the vital role of autophagy in cell survival. After 24 h, AaT facilitated Ca2+ influx with an accumulation of reactive oxidant intermediates and NO, to manifest necrotic cell death. Inhibition of NO accumulation by 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) decreased the level of necrotic cell death, and induced autophagy, which suggests NO accumulation represses autophagy and facilitates necrotic cell death at 24 h. Application of N-acetyl-L-cysteine at 3 h, confirmed ROS to be the key initiator of autophagy, and together with cPTIO for 24 h, revealed the combined effects of NO and ROS is required for necrotic HR cell death.

NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance.

Myeloid-Derived Suppressor Cells Modulate Immune Responses Independently of NADPH Oxidase in the Ovarian Tumor Microenvironment in Mice

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47phox−/−) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47phox−/− mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.

A Method To Quantify Quinone Reaction Rates with Wine Relevant Nucleophiles: A Key to the Understanding of Oxidative Loss of Varietal Thiols

Quinones are key reactive electrophilic oxidation intermediates in wine. To address this question, the model 4-methyl-1,2-benzoquinone was prepared to study how it reacts with wine nucleophiles. Those investigated included the varietal volatile thiols 4-methyl-4-sulfanylpentan-2-one (4MSP), 3-sulfanylhexan-1-ol (3SH), and 2-furanmethanethiol (2FMT); hydrogen sulfide (H2S); glutathione (GSH); sulfur dioxide; ascorbic acid (AA); and the amino acids methionine (Met) and phenylalanine (Phe) in the first kinetic study of these reactions. Products were observed in fair to quantitative yields, but yields were negligible for the amino acids. The reaction rates of 4-methyl-1,2-benzoquinone toward the nucleophiles were quantified by UV-vis spectrometry monitoring the loss of the quinone chromophore. The observed reaction rates spanned three orders of magnitude, from the unreactive amino acids (Met and Phe) (KNu = 0.0002 s(-1)) to the most reactive nucleophile, hydrogen sulfide (KH2S = 0.4188 s(-1)). Analysis of the kinetic data showed three categories. The first group consisted of the amino acids (Met and Phe) having rates of essentially zero. Next, phloroglucinol has a low rate (KPhl = 0.0064 s(-1)). The next group of compounds includes the volatile thiols having increasing reactions rates K as steric inhibition declined (K4MSP = 0.0060 s(-1), K3SH = 0.0578 s(-1), and K2FMT = 0.0837 s(-1)). These volatile thiols (4MSP, 3SH, 2FMT), important for varietal aromas, showed lower K values than those of the third group, the wine antioxidant compounds (SO2, GSH, AA) and H2S (KNu = 0.3343-0.4188 s(-1)). The characterization of the reaction products between the nucleophiles and 4-methyl-1,2-benzoquinone was performed by using HPLC with high-resolution MS analysis. This study presents the first evidence that the antioxidant compounds, H2S, and wine flavanols could react preferentially with oxidation-induced quinones under specific conditions, providing insight into a mechanism for their protective effect.

Haptoglobin Binding Stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine β145

Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection. Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit. Hp binding decreases both the ferryl iron and free radical reactivity of Hb. Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging.

The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC50 values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of >1000-fold in resistance between normal and NER-deficient cells (IC50 values for cells with deficiency in ERCC6: 0.15μM, XPC: 0.18μM, and normal cells: >180μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes.

Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox−/−) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)−/−×cathepsin G (CG)−/− mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox−/− mice, whereas NE−/−×CG−/− mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.

Probing enzyme-mediated oxidation reactions in crystallo

Reaction intermediates define the chemical steps of transformation from substrates to products, and capture of intermediates along catalytic pathways remains one focal point in the studies of enzymatic mechanisms. Performing enzymatic reactions in protein single crystals has been shown to be very effective in trapping and identifying unstable intermediates. In this critical review, we provide several examples of in crystallo approaches to trap reactive oxidation intermediates, thereby allowing the studies of enzymatic oxidation mechanisms.

Comparison of various iron chelators used in clinical practice as protecting agents against catecholamine-induced oxidative injury and cardiotoxicity

Catecholamines are stress hormones and sympathetic neurotransmitters essential for control of cardiac function and metabolism. However, pathologically increased catecholamine levels may be cardiotoxic by mechanism that includes iron-catalyzed formation of reactive oxygen species. In this study, five iron chelators used in clinical practice were examined for their potential to protect cardiomyoblast-derived cell line H9c2 from the oxidative stress and toxicity induced by catecholamines epinephrine and isoprenaline and their oxidation products. Hydroxamate iron chelator desferrioxamine (DFO) significantly reduced oxidation of catecholamines to more toxic products and abolished redox activity of the catecholamine-iron complex at pH 7.4. However, due to its hydrophilicity and large molecule, DFO was able to protects cells only at very high and clinically unachievable concentrations. Two newer chelators, deferiprone (L1) and deferasirox (ICL670A), showed much better protective potential and were effective at one or two orders of magnitude lower concentrations as compared to DFO that were within their clinically relevant plasma levels. Ethylenediaminetetraacetic acid (EDTA), dexrazoxane (ICRF-187, clinically approved cardioprotective agent against anthracycline-induced cardiotoxicity) as well as selected beta adrenoreceptor antagonists and calcium channel blockers exerted no effect. Hence, results of the present study indicate that small, lipophilic and iron-specific chelators L1 and ICL670A can provide significant protection against the oxidative stress and cardiomyocyte damage exerted by catecholamines and/or their reactive oxidation intermediates. This potential new application of the clinically approved drugs L1 and ICL670A warrants further investigation, preferably using more complex in vivo animal models.

NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

BackgroundChronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.Methodology/Principal FindingsWe found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47phox−/− mice and gp91phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-κB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation.Conclusions/SignificanceThese studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.

Capsule enlargement inCryptococcus neoformansconfers resistance to oxidative stress suggesting a mechanism for intracellular survival

Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.

Pegylated Granulocyte Colony Stimulating Factor (Peg-G-CSF) Enhances Mapatumumab-Mediated Antibody Dependent Cellular Cytotoxicity (ADCC) Against Non-Hodgkin's Lymphoma (NHL) Cell Lines Independent of NADPH Oxidase-Derived Reactive Oxidant Intermediates (ROIs).

ADCC is an important mechanism of action of rituximab and other monoclonal antibodies (mAb). In an attempt to improve the efficacy of rituximab while minimizing its toxicity, our group have explored the use of cytokines that target neutrophil maturation and activation in combination with rituximab in pre-clinical and clinical models. In response to a growing need to develop alternative biological therapies, a new generation of humanized antibodies are under development. Mapatumumab is a humanized mAb targeting the death receptor-4 (DR4), active against NHL. A variable degree of ADCC has been observed in mapatumumab-treated NHL cells, raising the possibility that its biological activity may be augmented by cytokines. To this end, we studied the effects of cytokine-priming of neutrophils in the activity of mapatumumab and the role of free radical species in mAb-mediated ADCC. We used Raji cells, a CD20 and DR4 expressing Burkitt's lymphoma cell line, as target cells in a standardized 51Cr release assay. Effector cells consisted of peripheral blood mononuclear cells (PBMC) and neutrophils collected from healthy volunteers or lymphoma patients who had received peg-G-CSF for the prevention of chemotherapy-induced neutropenia. In addition, thiolglycollate-elicted peritoneal neutrophils and a bone marrow-derived macrophage cell line from C57BL/6 and genetically engineered chronic granulomatous disease (CGD) mice were used to evaluate the role of NADPH oxidase-derived ROIs in mAb-mediated ADCC. For ex vivo priming, healthy volunteer PBMC/neutrophils were exposed to peg-G-CSF (1mcg/ml) or placebo for 24hrs at 37oC,5%CO2. Subsequently, effector cells were co-cultured with 51Cr-labeled Raji cells in the presence of mapatumumab or isotype control at an effector:target ratio of 40:1. Following a period of 6 hrs of incubation, the supernatant was collected and the percentage of lysis was calculated. For in vivo cytokine priming, PBMC/neutrophils were collected from lymphoma patients who had received peg-G-CSF (6mg sq) at the time of white cell count recovery and used as effector cells in ADCC assays as described above. Finally, to establish the role of ROIs in mapatumumab-associated ADCC, we conducted standard 51Cr release assays with mapatumumab plus C57BL/6 or CGD murine neutrophils or macrophages as effector cells. Ex vivo cytokine-primed killing (34.54% +/− 1.69) and in vivo cytokine-primed killing (26.17% +/−0.28) resulted in an improvement in mapatumumab-associated ADCC as compared to controls (10.26% +/−0.79; P = 0.002, and P = 0.003, respectively). In murine studies, mapatumumab resulted in significant neutrophil and macrophage-mediated ADCC against Raji cells (20% and 15%, respectively). No differences were observed in mapatumumab anti-tumor activity between C57BL/6 versus CGD effector cells suggesting that NADPH oxidase-derived ROIs do not influence mapatumumab-induced ADCC. In summary, our data strongly suggest that peg-G-CSF can potentiate the anti-tumor activity of mapatumumab and support additional translational and clinical research of the combination of cytokine-primed effector cells plus mAbs targeting the death receptor pathway.

Characterization of the SKN7 ortholog of Aspergillus fumigatus

Reactive oxidant intermediates play a major role in the killing of Aspergillus fumigatus by phagocytes. In yeasts, SKN7 is a transcription factor contributing to the oxidative stress response. We investigated here the role of afSkn7p in the adaptation of A. fumigatus against oxidative stress. To analyze functionally the afSKN7 in A. fumigatus, we modified a quick PCR fusion methodology for targeted deletion in A. fumigatus. The afskn7Δ mutant was morphologically similar to the wild-type strain, but showed a growth inhibition phenotype associated with hydrogen peroxide and tert-butyl hydroperoxide. However, no significant virulence differences were observed between wild type, mutant and reconstituted strains in a murine model of pulmonary aspergillosis. This result indicated that an increased sensitivity of A. fumigatus to peroxides in vitro is not correlated with a modification of fungal virulence.

Acid aspiration-induced lung inflammation and injury are exacerbated in NADPH oxidase-deficient mice

Increased reactive oxidant intermediates (ROIs) from primed leukocytes have been implicated in the pathogenesis of acid aspiration lung injury. To evaluate the specific role of the phagocyte NADPH oxidase-derived ROIs in acid lung injury, the p47phox-/- knockout mouse model of chronic granulomatous disease was used. p47phox-/- mice developed a significantly greater alveolar neutrophilic leukocytosis compared with wild-type mice at all time points after acid injury, with the difference between genotypes being most marked at 48 h. In contrast, the p47phox-/- mice had a decreased number of macrophages in bronchoalveolar lavage (BAL) compared with wild-type at 48 h after acid or saline aspiration. Albumin concentration in BAL reflecting capillary leak was also greater in p47phox-/- compared with wild-type mice. BAL concentrations of proinflammatory cytokines and chemokines were greater in p47phox-/- compared with wild-type mice. These findings suggest that NADPH oxidase, directly or indirectly, plays a role in attenuating the acute neutrophilic response after acid lung injury. We speculate that this downmodulating effect may be mediated by promoting the transition from production of cytokines and chemokines involved in neutrophilic infiltration to a less injurious, chronic inflammatory response.

Resistance to environmental stress by the mucoid and the non-mucoid variant phenotypes of the Comamonas testosteroni strain A20

The Comamonas testosteroni strain A20, isolated from activated sludge, displays a phase variation between a mucoid (exopolysaccharide producing) and a non-mucoid colony phenotype. It was found that a population of C. testosteroni cells, subjected to sublethal environmental stress (acute heat shock and reactive oxidative intermediates and visible light), became selectively enriched in non-mucoid colony forming cells (NMCF cells). This observation suggests that NMCF cells are more stress-resistant than MCF cells. The experiments did not allow us to establish whether stress acts as a trigger for phase conversion or as a selector for the more stress-resistant NMCF cells. NMCF cells are more hydrophobic than MCF cells as evidenced by a better attachment to gas bubbles and polystyrene. These observations suggest that the two phenotypic variants might have different ecophysiological functions.

Cytoprotective response of A1, a Bcl-2 homologue expressed in mature human neutrophils and promyelocytic HL-60 cells, to oxidant stress-induced cell death.

The ability to generate reactive oxidative intermediates is one of the quintessential properties of mature human neutrophils. Endogenously generated oxidants have been shown to be an important mechanism underlying neutrophil cell death. In acute lung inflammation, newly recruited neutrophils further encounter external oxidants, including reactive oxygen and nitrogen intermediates. In our present study, we showed that A1, a constitutive and inducible Bcl-2 homologue expressed in mature circulating human neutrophils, might confer the protection from hydrogen peroxide (H(2)O(2))- and peroxynitrite (ONOO)-induced cell death. Utilizing the myeloid precursor cell line, HL-60, we further examined the hypothesis that A1 was capable of conferring cytoprotective activity against these oxidative stresses. Whereas the control-transfected HL-60 cells expressed small amounts of A1 and were sensitive to the biologically relevant, cell death-inducing oxidants, H(2)O(2) and ONOO, the stable transfectants that overexpressed A1 were significantly more tolerant. Furthermore, there was a correlation between the level of A1 expression and the antiapoptotic activity. Thus, our results suggest a cytoprotective role of A1 in mature human neutrophils under oxidant stresses in host defense and inflammation.

Cytoprotective Response of A1, a Bcl-2 Homologue Expressed in Mature Human Neutrophils and Promyelocytic HL-60 Cells, to Oxidant Stress-Induced Cell Death

The ability to generate reactive oxidative intermediates is one of the quintessential properties of mature human neutrophils. Endogenously generated oxidants have been shown to be an important mechanism underlying neutrophil cell death. In acute lung inflammation, newly recruited neutrophils further encounter external oxidants, including reactive oxygen and nitrogen intermediates. In our present study, we showed that A1, a constitutive and inducible Bcl-2 homologue expressed in mature circulating human neutrophils, might confer the protection from hydrogen peroxide (H₂O₂)- and peroxynitrite (ONOO)-induced cell death. Utilizing the myeloid precursor cell line, HL-60, we further examined the hypothesis that A1 was capable of conferring cytoprotective activity against these oxidative stresses. Whereas the control-transfected HL-60 cells expressed small amounts of A1 and were sensitive to the biologically relevant, cell death-inducing oxidants, H₂O₂ and ONOO, the stable transfectants that overexpressed A1 were significantly more tolerant. Furthermore, there was a correlation between the level of A1 expression and the antiapoptotic activity. Thus, our results suggest a cytoprotective role of A1 in mature human neutrophils under oxidant stresses in host defense and inflammation.

Killing of Aspergillus fumigatus by alveolar macrophages is mediated by reactive oxidant intermediates.

Phagocytosis and mechanisms of killing of Aspergillus fumigatus conidia by murine alveolar macrophages (AM), which are the main phagocytic cells of the innate immunity of the lung, were investigated. Engulfment of conidia by murine AM lasts 2 h. Killing of A. fumigatus conidia by AM begins after 6 h of phagocytosis. Swelling of the conidia inside the AM is a prerequisite for killing of conidia. The contributions of NADPH oxidase and inducible nitric oxide synthase to the conidicidal activity of AM were studied using AM from OF1, wild-type and congenic p47phox(-/-) 129Sv, and wild-type and congenic iNOS(-/-) C57BL/6 mice. AM from p47phox(-/-) mice were unable to kill A. fumigatus conidia. Inhibitors of NADPH oxidase that decreased the production of reactive oxidant intermediates inhibited the killing of A. fumigatus without altering the phagocytosis rate. In contrast to NADPH oxidase, nitric oxide synthase does not play a role in killing of conidia. Corticosteroids did not alter the internalization of conidia by AM but did inhibit the production of reactive oxidant intermediates and the killing of A. fumigatus conidia by AM. Impairment of production of reactive oxidant intermediates by corticosteroids is responsible for the development of invasive aspergillosis in immunosuppressed mice.

Decreased Oxidative State in Non-Lesional Skin of Atopic Dermatitis

Background: The stratum corneum (SC), as the skin layer most exposed to various environmental factors, is particularly susceptible to oxidative stress. Due to the high lipid content of the SC, lipophilic antioxidants such as α-tocopherol are expected to play a major role in scavenging reactive oxidant intermediates produced during oxidative stress. Objectives: Since the skin of atopic dermatitis patients has an impaired barrier function, we wondered if they were more susceptible to environmental oxidative stress than healthy subjects. Methods: SC was collected by scraping the forearm of 14 healthy volunteers and 14 patients with atopic dermatitis; then, α-tocopherol and lipid peroxide concentrations were assessed by high-performance liquid chromatography and ferrous oxidation, respectively. Results: The SC from atopic patients showed a higher concentration of α-tocopherol (16.1 ± 2.2 nmol/g) as compared to healthy controls (7.7 ± 0.9 nmol/g; p < 0.01), as well as a slightly but significantly lower concentration of lipid peroxides (1,353 ± 128 and 1,818 ± 154 nmol/g for atopic dermatitis patients and healthy controls, respectively; p < 0.05). Conclusions: These results show that the SC of atopic dermatitis patients exhibits a significantly less pronounced oxidative state. This may be the consequence of an increase in cutaneous antioxidant defences due to chronic inflammation.