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Differential antibacterial control by neutrophil subsets.
Leliefeld, PHC, Pillay, J, Vrisekoop, N, Heeres, M, Tak, T, Kox, M, Rooijakkers, SHM, Kuijpers, TW, Pickkers, P, Leenen, LPH, et al
Blood advances. 2018;(11):1344-1355
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Abstract
Neutrophils comprise a heterogeneous population of cells essential for bacterial eradication, and defects in neutrophil function are associated with increased susceptibility to infection. In this study, neutrophils from healthy controls were shown to prevent bacterial proliferation for at least 48 hours when cocultured with methicillin-resistant Staphylococcus aureus (MRSA) in tissue-like scaffolds by establishing a bacteriostatic environment inside their phagolysosome. This intracellular bacterial containment is independent of reactive oxygen species because neutrophils that lack a functional nicotinamide adenine dinucleotide phosphate-oxidase complex displayed no defect in intracellular bacterial containment, whereas killing of the pathogen was impaired. During acute inflammation, a subset of CD16bright/CD62Ldim hypersegmented neutrophils displayed normal phagocytosis associated with a remarkably poor capacity to contain bacteria intracellularly. Conversely, CD16dim-banded neutrophils were the only neutrophil subset that adequately contained MRSA. These findings demonstrate a clear neutrophil heterogeneity in their antimicrobial capacity and the appearance of neutrophil subsets with a clear differentiation in functionality during acute inflammation. Furthermore, this study provides an evolutionary basis for the rapid release of banded neutrophils into the circulation during acute inflammation.
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SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx.
Douda, DN, Khan, MA, Grasemann, H, Palaniyar, N
Proceedings of the National Academy of Sciences of the United States of America. 2015;(9):2817-22
Abstract
Neutrophils cast neutrophil extracellular traps (NETs) to defend the host against invading pathogens. Although effective against microbial pathogens, a growing body of literature now suggests that NETs have negative impacts on many inflammatory and autoimmune diseases. Identifying mechanisms that regulate the process termed "NETosis" is important for treating these diseases. Although two major types of NETosis have been described to date, mechanisms regulating these forms of cell death are not clearly established. NADPH oxidase 2 (NOX2) generates large amounts of reactive oxygen species (ROS), which is essential for NOX-dependent NETosis. However, major regulators of NOX-independent NETosis are largely unknown. Here we show that calcium activated NOX-independent NETosis is fast and mediated by a calcium-activated small conductance potassium (SK) channel member SK3 and mitochondrial ROS. Although mitochondrial ROS is needed for NOX-independent NETosis, it is not important for NOX-dependent NETosis. We further demonstrate that the activation of the calcium-activated potassium channel is sufficient to induce NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is essential for the NOX-dependent pathway, whereas its activation is not essential for the NOX-independent pathway. Despite the differential activation, both NOX-dependent and -independent NETosis require Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into previously unknown mechanisms for NOX-independent NETosis.
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Assessment of a standardized ROS production profile in humans by electron paramagnetic resonance.
Mrakic-Sposta, S, Gussoni, M, Montorsi, M, Porcelli, S, Vezzoli, A
Oxidative medicine and cellular longevity. 2012;:973927
Abstract
Despite the growing interest in the role of reactive oxygen species (ROS) in health and disease, reliable quantitative noninvasive methods for the assessment of oxidative stress in humans are still lacking. EPR technique, coupled to a specific spin probe (CMH: 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) is here presented as the method of choice to gain a direct measurement of ROS in biological fluids and tissues. The study aimed at demonstrating that, differently from currently available "a posteriori" assays of ROS-induced damage by means of biomolecules (e.g., proteins and lipids) spin-trapping EPR provides direct evidence of the "instantaneous" presence of radical species in the sample and, as signal areas are proportional to the number of excited electron spins, lead to absolute concentration levels. Using a recently developed bench top continuous wave system (e-scan EPR scanner, Bruker) dealing with very low ROS concentration levels in small (50 μL) samples, we successfully monitored rapid ROS production changes in peripheral blood of athletes after controlled exercise and sedentary subjects after antioxidant supplementation. The correlation between EPR results and data obtained by various enzymatic assays (e.g., protein carbonyls and thiobarbituric acid reactive substances) was determined too. Synthetically, our method allows reliable, quick, noninvasive quantitative determination of ROS in human peripheral blood.
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Antioxidant activity of the new black vinegar "IZUMI".
Nagashima, M, Saito, K
The journal of nutrition, health & aging. 2010;(10):845-9
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Abstract
OBJECTIVES "IZUMI" is a new kind of vinegar resulting from an improvement in the manufacturing method of Kurosu, a traditional vinegar product made from unpolished rice. We evaluated the antioxidant activity of this new Kurosu by means of measuring the level of diacron-reactive oxygen metabolites (d-ROM), the biological antioxidant potential (BAP), as well as RBC deformability using the microchannel array flow method. PARTICIPANTS Ten healthy, untrained female volunteers participated in this study. MEASUREMENTS All subjects drank 50 ml of "IZUMI" on a daily basis, and blood samples were collected pre-"IZUMI" (I), after one month "IZUMI" consumption (II), and after two months "IZUMI" consumption (III). The subjects continuously wore a lifecorder during a 7-day period and the nutritional intake was measured before the initial blood sample collection. RESULTS There were no significant changes in weight, BMI, fat mass, or fat-free mass. There were no significant differences in daily energy consumption, physical activity and nutritional intake. Peripheral blood variables did not change significantly. The drinking of " IZUMI " increased serum BAP level gradually, and after 30 days it was significantly higher as compared to the pre-drinking level. The serum level of d-ROM and blood filtration time (BFT) decreased by drinking "IZUMI"; with d-ROM significantly lower than the pre-drinking level after 30 days and BFT significantly decreased after 60 days (all P < 0.05). CONCLUSIONS These results suggest that "IZUMI", a Kurosu containing a higher level of amino acids, increases antioxidant activity and reduces oxidative stress and blood filtration time in female subjects.
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Dietary intake, neutrophil fatty acid profile, serum antioxidant vitamins and oxygen radical absorbance capacity in patients with ulcerative colitis.
Kawakami, Y, Okada, H, Murakami, Y, Kawakami, T, Ueda, Y, Kunii, D, Sakamoto, Y, Shiratori, Y, Okita, M
Journal of nutritional science and vitaminology. 2007;(2):153-9
Abstract
Nutrition may play an important role in the pathogenesis and treatment of ulcerative colitis. Several studies suggest an association between dietary factors and the onset of ulcerative colitis; however, only few studies have examined the relationship between dietary intake and relapse of ulcerative colitis. The aim of this study was to assess the dietary intake and antioxidative capacity of ulcerative colitis patients and to elucidate the efficacy of dietary therapy for ulcerative colitis. Dietary intake, fatty acid composition of phospholipids in plasma and neutrophils, serum fat-soluble vitamin levels, and oxygen radical absorbance capacity were analyzed in 29 ulcerative colitis patients (7 males and 22 females), who were treated at the Department of Gastroenterology, Okayama University Hospital. Total fat intake, fat energy ratio and linoleic acid intake were significantly lower, while protein and carbohydrate intakes were significantly higher, in the patients than age- and sex-matched controls. In the neutrophil phospholipids of ulcerative colitis patients, significantly higher levels of linoleic aicd and arachidonic acid and a lower level of eicosapentaenoic acid were observed. The concentrations of serum retinol and beta-carotene but not alpha-tocopherol were significantly lower and serum oxygen radical absorbance capacity was also lower than in the controls. Significant correlations between serum oxygen radical absorbance capacity and retinol (r = 0.567, p = 0.0031), alpha-tocopherol (r = 0.560, p = 0.0036) and beta-carotene (r = 0.440, p = 0.0279) concentrations were observed in the ulcerative colitis patients. A diet restricting the intake of linoleic acid and supplemented with eicosapentaenoic acid and antioxidative vitamins may be recommendable for the nutritional management of ulcerative colitis patients.
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Reactive oxygen species, antioxidant mechanisms and serum cytokine levels in cancer patients: impact of an antioxidant treatment.
Mantovani, G, Macciò, A, Madeddu, C, Mura, L, Massa, E, Gramignano, G, Lusso, MR, Murgia, V, Camboni, P, Ferreli, L
Journal of cellular and molecular medicine. 2002;(4):570-82
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Abstract
OBJECTIVE So far, it is not well established whether oxidative stress found in cancer patients results from an increased production of oxidants in the body or from a failure of physiological antioxidant systems. To further investigate this question we have assessed the blood levels of reactive oxygen species as a marker of free radicals producing oxidative stress and the most relevant of the physiological body enzymes counteracting reactive oxygen species, namely glutathione peroxidase and superoxide dismutase. Serum levels of proinflammatory cytokines and IL-2 were also investigated. All these parameters were studied in relation to the clinically most important index of disease progression, namely Performance Status (ECOG PS). We also tested the reducing ability of different antioxidant agents on reactive oxygen species levels by measuring the increase in glutathione peroxidase activity, and the reduction of serum levels of IL-6 and TNF. DESIGN, SETTING AND SUBJECTS We carried out an open non randomized study on 28 advanced stage cancer patients (stage III, 10.7%, and stage IV, 89.3%) with tumours at different (8) sites: all were hospitalized in the Medical Oncology Dept, University of Cagliari Interventions. The patients were divided into 5 groups and a different antioxidant treatment was administered to each group. The selected antioxidants were: alpha lipoic acid 200 mg/day orally, N-acetylcysteine 1800 mg/day i.v. or carboxycysteine-lysine salt 2.7 g/day orally, amifostine 375 mg/day i.v., reduced glutathione 600 mg/day i.v., vitamin A 30000 IU/day orally plus vitamin E 70 mg/day orally plus Vitamin C 500 mg/day orally. The antioxidant treatment was administered for 10 consecutive days. RESULTS Our results show that all but one of the antioxidants tested were effective in reducing reactive oxygen species levels and 2 of them (cysteine-containing compounds and amifostine) had the additional effect of increasing glutathione peroxidase activity. Comprehensively, the "antioxidant treatment" was found to have an effect both on reactive oxygen species levels and glutathione peroxidase activity. The antioxidant treatment also reduced serum levels of IL-6 and TNF. Patients in both ECOG PS 0-1 and ECOG PS 2-3 responded to antioxidant treatment.