Lucigenin-dependent Chemiluminescence Research Articles

СРАВНИТЕЛЬНЫЙ АНАЛИЗ ИНГИБИРУЮЩИХ ЭФФЕКТОВ ГИПОМАГНИТНОГО ПОЛЯ И КОМБИНИРОВАННЫХ МАГНИТНЫХ ПОЛЕЙ НА ПРОДУКЦИЮ АКТИВНЫХ ФОРМ КИСЛОРОДА (АФК) НЕЙТРОФИЛАМИ

The article reveals that a decrease in the background production of reactive oxygen species (ROS) in the peritoneal neutrophils of mice after a short-term (40 minutes) stay in hypomagnetic conditions (residual field  10 nT) at physiological temperatures, detected by the method of lucigenin-dependent chemiluminescence, is not accompanied by a violation of chemiluminescent response to respiratory burst activators: formylated peptide N-formyl-Met-Leu-Phe (fMLF) and phorbol ester of phorbol-12-meristat-13-acetate (PMA). These results were obtained by activated chemiluminescence using lucigenin or luminol and various combinations of ROS production activators (PMA and/or fMLF). In contrast, the action of combined parallel constant (induction 60 μT) and alternating (amplitude range 60-180 nT, frequency 49.5 Hz) magnetic fields (CMF) leads to a decrease in the chemiluminescent response to these activators. These data indicate different sources of ROS that respond to certain modes of CMF and hypomagnetic field in neutrophils. The conducted research and the previously obtained results enable to exclude the systems that control the respiratory burst in neutrophils from the main targets and acceptors that respond to short-term deprivation of the magnetic field.

The level of blood neutrophils primary reactive oxygen species production in Graves’ disease patients undergoing conservative therapy with thiamazole.

Background. Conservative therapy with thiamazole in Graves’ disease helps to reduce the level of oxidative stress by restoring euthyroidism and the total reactive antioxidant potential of plasma. However, until now, in patients with Graves’ disease, the radical-producing ability of neutrophils remains unexplored at the initial stage of free radical conversion at different durations of drug-induced euthyroidism, which seems significant in revealing the mechanisms of disease recurrence. The aim is to study the level of production of primary reactive oxygen species in peripheral blood neutrophils depending on the duration of drug — induced euthyroidism in patients with Graves’ disease. Materials and methods. A single-center, prospective, controlled study was conducted with the participation of patients with laboratory-confirmed Graves’ disease. Subgroups of patients with Graves’ disease were formed depending on the duration of achieved drug-induced euthyroidism with continuous conservative therapy with thiamazole. The level of ROS production in peripheral blood neutrophils was determined by chemiluminescence. The assessment of spontaneous and zymosan-induced chemiluminescence was carried out for 90 minutes on a 36-channel chemiluminescence analyzer BLM-3607 (Medbiotech LLC, Krasnoyarsk). Results. The study included 102 Graves’ disease women with an average age of 47.54 ± 15.47 years. Of these, 75 (73.5 %) with relapse and 27 (26.47 %) with newly diagnosed disease. The total synthesis of (S) primary ROS increased statistically significantly relative to the control values in the examined patients with a duration of the euthyroid state of 5–8 and 9–12 months. It was found that in patients with Graves’ disease with stable drug-induced euthyroidism for 5–8 and 9–12 months, the S index of zymosan-induced lucigenin-dependent chemiluminescence of neutrophils increased statistically significantly, respectively, by 10.7 and 7.4 times relative to the control values. Conclusion. The restoration and maintenance of drug-induced euthyroidism for more than 12 months against the background of conservative thiamazole therapy in patients with Graves’ disease reduces the load of systemic oxidative stress on the immune system. However, the high total production of primary ROS in neutrophils of patients with Graves’ disease in a state of drug-induced euthyroidism with conservative thiamazole therapy may play a significant role in disconnecting the functioning of the NADPH oxidase complex and determine the functional capabilities of neutrophils in case of disease recurrence.

Background formation of lower levels of reactive oxygen species by neutrophils after hypomagnetic field exposure is not accompanied by the impairment of chemiluminescence response of neutrophils to respiratory-burst stimuli

This study shows that the background formation of lower levels of reactive oxygen species in mouse peritoneal neutrophils after short-term (40 minutes) exposure to hypomagnetic fields with ~10 nT residual field at physiological temperatures, which has been detected by use of lucigenin-dependent chemiluminescence, is not accompanied by the impairment of chemiluminescence response of neutrophils to respiratory-burst stimuli: the formylated tripeptide N-formyl-Met-Leu-Phe (fMLF) and phorbol ester phorbol-12-myristate-13-ace-tate (PMA). These results were obtained with lucigenin or luminol-enhanced activated chemiluminescence and various combinations of reactive oxygen species production stimuli (phorbol-12-myristate-13-acetate and/or N-formyl-Met-Leu-Phe). Based on the results of the present work as well as on those of previous studies, this study reveal that the systems that control the respiratory burst in neutrophils can be excluded from a list of main targets and acceptors that respond to short-term deprivation of the magnetic field.

Systemic and local vascular inflammation and arterial reactive oxygen species generation in patients with advanced cardiovascular diseases.

Systemic inflammation may cause endothelial activation, mediate local inflammation, and accelerate progression of atherosclerosis. We examined whether the levels of circulating inflammatory cytokines reflect local vascular inflammation and oxidative stress in two types of human arteries. Human internal mammary artery (IMA) was obtained in 69 patients undergoing coronary artery bypass graft (CABG) surgery and left anterior descending (LAD) artery was obtained in 17 patients undergoing heart transplantation (HTx). Plasma levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were measured using ELISA, high-sensitivity C-reactive protein (hs-CRP) was measured using Luminex, and mRNA expression of proinflammatory cytokines in the vascular tissues was assessed. Furthermore, formation of superoxide anion was measured in segments of IMA using 5 uM lucigenin-dependent chemiluminescence. Vascular reactivity was measured using tissue organ bath system. TNF-α, IL-6 and IL-1β mRNAs were expressed in all studied IMA and LAD segments. Plasma levels of inflammatory cytokines did not correlate with vascular cytokine mRNA expression neither in IMA nor in LAD. Plasma TNF-α and IL-6 correlated with hs-CRP level in CABG group. Hs-CRP also correlated with TNF-α in HTx group. Neither vascular TNF-α, IL-6 and IL-1β mRNA expression, nor systemic levels of either TNF-α, IL-6 and IL-1β were correlated with superoxide generation in IMAs. Interestingly, circulating IL-1β negatively correlated with maximal relaxation of the internal mammary artery (r = -0.37, p = 0.004). At the same time the mRNA expression of studied inflammatory cytokines were positively associated with each other in both IMA and LAD. The positive correlations were observed between circulating levels of IL-6 and TNF-α in CABG cohort and IL-6 and IL-1β in HTx cohort. This study shows that peripheral inflammatory cytokine measurements may not reflect local vascular inflammation or oxidative stress in patients with advanced cardiovascular disease (CVD). Circulating pro-inflammatory cytokines generally correlated positively with each other, similarly their mRNA correlated in the arterial wall, however, these levels were not correlated between the studied compartments.

Effect of Hydrated Fullerene C60 in a Wide Concentration Range on Low-Level Photon Emission from Undiluted Human Blood

Hydrated fullerene C 60 (HyFnC 60 ) is a symmetrical molecule of C 60 Buckminster fullerene surrounded by a water shell obtained by an original technique. It can act as an antioxidant or a pro-oxidant in various biological objects, depending on the conditions. In this study we added HyFnC 60 at a range of concentrations obtained by a method of serial dilutionswith vigorous shaking at each step to whole undiluted blood of healthy donors and hospital patients with COPD and examined lucigenin-enhanced blood chemiluminescence. We have found that HyFnC 60 at concentrations of 2.5 ´ 10 – 6 M, 2.5 ´ 10 – 7 M, 2.5 ´ 10 – 17 M, and 2.5 ´ 10 – 19 M increased lucigenin-dependent chemiluminescence in heathy donors’ blood while in blood of patients with COPD it had an opposite effect. This can be interpreted as in healthy donors’ blood reactive oxygen species (ROS) generation is enhanced by HyFnC 60 while in patients with a chronic inflammatory disease with already increased ROS generation it is attenuated. This indicates that HyFnC 60 preparations even in ultra-high dilutions may play the role of a tuner of the processes with ROS participation. Probable reasons of such action of HyFnC 60 on human blood even in ultra-high dilutions are discussed.

Cyclosporin A Inhibits the Activation of Membrane-Bound Guanylate Cyclase GC-A of Atrial Natriuretic Factor via NAD(P)H Oxidase.

Cyclosporin A (CsA) is a common immunosuppressant wildly used in patients with organ transplant and autoimmune diseases; however, it can cause several adverse effects, such as nephrotoxicity and hypertension. The detailed mechanisms have not been completely understood. Atrial natriuretic factor (ANF) and its receptor (mGC-A) have been shown to play a crucial role in the regulation of blood pressure. Here, we investigated the effects of CsA on the activation of mGC-A in ANF-treated LLC-PK1 cells. In our study, ANF-induced mGC-A activities and superoxide generation in LLC-PK1 cells were measured by guanosine 3',5'-cyclic monophosphate (cGMP) radioimmunoassay and lucigenin-dependent chemiluminescence, respectively. We found that CsA can reduce about 60% of mGC-A activities in ANF-treated LLC-PK1 cells. CsA is known to induce superoxide. Addition of superoxide generators menadione and diamide mimicked the effects of CsA, whereas DPI (a reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor) and Tiron (a superoxide quencher) blocked the suppressive effects of CsA on ANF-induced mGC-A activities. We previously showed that the catalytic domain of GC-A (GC-c) expresses guanylate cyclase activities. Addition of menadione, diamide, or peroxynitrite or transfection of Nox-4 NAD(P)H oxidase abolished GC-c activities. In conclusion, CsA inhibits ANF-stimulated mGC-A activities through superoxide and/or peroxynitrite generated by an NAD(P)H oxidase by interacting with the catalytic domain of mGC-A.

ВРЕМЕННАЯ ДИНАМИКА ПРОДУКЦИИ АКТИВНЫХ ФОРМ КИСЛОРОДА НЕЙТРОФИЛАМИ ПОСЛЕ ОБРАБОТКИ В ГИПОМАГНИТНЫХ УСЛОВИЯХ

. It was shown that incubation of a suspension of mouse peritoneal neutrophils for 30 minutes under hypomagnetic conditions obtained using permalloy magnetic screens (a constant magnetic field of not more than 20 nT, the level of variable man-made noise is reduced to a few nT) causes a significant decrease (about 48%) signal intensity of lucigenin-dependent chemiluminescence measured immediately after incubation. 20 minutes after magnetic treatment (followed by incubation in a geomagnetic field during this time, induction 44 μT, the level of magnetic interference at a frequency of 50 Hz was 15-50 nT), the differences between the control and experimental groups remain completely (the difference was about 49%). In 40 and 60 minutes after exposure to the "zero" field, followed by keeping the samples in the geomagnetic field for the specified time intervals, the difference between the control and experimental samples decreased to 32% and 22%, respectively. This effect was registered without additional activation of neutrophils by chemical agents initiating respiratory burst, such as N-formyl-Met-Leu-Phe formylated peptide or phorbol-12-meristat-13-acetate phorbol ester, and was not associated with effects on cellular systems, providing this explosion.

Activation of Neutrophils by Mucin–Vaterite Microparticles

Nano- and microparticles enter the body through the respiratory airways and the digestive system, or form as biominerals in the gall bladder, salivary glands, urinary bladder, kidney, or diabetic pancreas. Calcium, magnesium, and phosphate ions can precipitate from biological fluids in the presence of mucin as hybrid nanoparticles. Calcium carbonate nanocrystallites also trap mucin and are assembled into hybrid microparticles. Both mucin and calcium carbonate polymorphs (calcite, aragonite, and vaterite) are known to be components of such biominerals as gallstones which provoke inflammatory reactions. Our study was aimed at evaluation of neutrophil activation by hybrid vaterite–mucin microparticles (CCM). Vaterite microparticles (CC) and CCM were prepared under standard conditions. The diameter of CC and CCM was 3.3 ± 0.8 µm and 5.8 ± 0.7 µm, with ƺ-potentials of −1 ± 1 mV and −7 ± 1 mV, respectively. CC microparticles injured less than 2% of erythrocytes in 2 h at 1.5 mg mL−1, and no hemolysis was detected with CCM; this let us exclude direct damage of cellular membranes by microparticles. Activation of neutrophils was analyzed by luminol- and lucigenin-dependent chemiluminescence (Lum-CL and Luc-CL), by cytokine gene expression (IL-6, IL-8, IL-10) and release (IL-1β, IL-6, IL-8, IL-10, TNF-α), and by light microscopy of stained smears. There was a 10-fold and higher increase in the amplitude of Lum-CL and Luc-CL after stimulation of neutrophils with CCM relative to CC. Adsorption of mucin onto prefabricated CC microparticles also contributed to activation of neutrophil CL, unlike mucin adsorption onto yeast cell walls (zymosan); adsorbed mucin partially suppressed zymosan-stimulated production of oxidants by neutrophils. Preliminary treatment of CCM with 0.1–10 mM NaOCl decreased subsequent activation of Lum-CL and Luc-CL of neutrophils depending on the used NaOCl concentration, presumably because of the surface mucin oxidation. Based on the results of ELISA, incubation of neutrophils with CCM downregulated IL-6 production but upregulated that of IL-8. IL-6 and IL-8 gene expression in neutrophils was not affected by CC or CCM according to RT2-PCR data, which means that post-translational regulation was involved. Light microscopy revealed adhesion of CC and CCM microparticles onto the neutrophils; CCM increased neutrophil aggregation with a tendency to form neutrophil extracellular traps (NETs). We came to the conclusion that the main features of neutrophil reaction to mucin–vaterite hybrid microparticles are increased oxidant production, cell aggregation, and NET-like structure formation, but without significant cytokine release (except for IL-8). This effect of mucin is not anion-specific since particles of powdered kidney stone (mainly calcium oxalate) in the present study or calcium phosphate nanowires in our previous report also activated Lum-CL and Luc-CL response of neutrophils after mucin sorption.

The chemiluminescent and enzymatic activity of blood neutrophils in patients with Graves' disease depending on hyperthyroidism compensation

BACKGROUND: Oxidative stress in Graves’ disease can potentiate the cytotoxicity of neutrophils and support autoimmune inflammation. Elimination of hyperthyroidism with conservative therapy with thiamazole only in some cases leads to remission, which determines the necessity to search an immunological markers for etiotropic therapeutic approaches in treatment of the disease.AIM: To study the chemiluminescent and enzymatic activity of peripheral blood neutrophils in patients with Graves’ disease depending on hyperthyroidism compensation to determine the intracellular targets of immunotropic treatment.MATERIALS AND METHODS: Spontaneous and zymosan-induced chemiluminescence was evaluated using a 36-channel chemiluminescence analyzer «BLM-3607» (MedBioTech, Krasnoyarsk). The reactivity of peripheral blood neutrophils was characterized by: Tmax — the rate of development of the chemiluminescent reaction, Imax — the maximum level of reactive oxygen species (ROS) synthesis and the area under the chemiluminescence curve (S — total synthesis of ROS for 90 minutes of measurement). The activity of NAD(P)-dependent dehydrogenases in neutrophils was determined using the bioluminescent method.RESULTS: The prospective study included 126 women with Graves’ disease, aged 18 to 65 years, 93 (73.81%) with compensated and 33 (26.19%) with uncompensated hyperthyroidism. In uncompensated hyperthyroidism, the indicator S of spontaneous ROS and zymosan-induced lucigenin-dependent chemiluminescence increases significantly, both relative to the control and to the values of compensated hyperthyroidism patients. Antigenic neutrophils stimulation during luminol-dependent chemiluminescence in patients with recurrent hyperthyroidism demonstrated more than tenfold increase in the total synthesis of ROS relative to the control, but no statistically significant differences with compensated hyperthyroidism patients. A high level of NADH-GDG was established in patients with recurrent hyperthyroidism, both relative to the control range and to the patients with euthyroidism.CONCLUSION: Violation of oxygen peripheral blood neutrophils metabolism in patients with euthyroidism mainly affects the production of ROS, which is associated with hyperthyroidism compensation and the immunosuppressive effect of thiamazole. In patients with recurrent hyperthyroidism, there are more changes in the production of high-energy oxidants not only at initial oxidative reactions stage, but also at the level of secondary ROS, indicating the activation of cellular response immunological mechanisms.

Detection of Reactive Oxygen Species in Human Neutrophils Under Various Conditions of Exposure to Galectin.

Reactive oxygen species (ROS) have been extensively studied in biology in the past years. This class of molecules can be derived from endogenous sources (e.g., phagocytic cells as neutrophils, eosinophils, monocytes, macrophages, and organelles as mitochondria and peroxisomes) and participate in physiological and pathological conditions. The beneficial and harmful effects of ROS depend on redox regulation, which establishes the balance between their production and the activity of antioxidant systems to prevent oxidative stress in vivo. Neutrophils are the immune effectors most well depicted with an intense oxidative burst in response to tissue inflammation. Several proteins and members of the galectin family are involved in this fine modulation of ROS production by neutrophils. Interestingly, studies have indicated that Galectin-1 (Gal-1) can up- or downregulate ROS production by neutrophils even when exposed to N-formyl-Met-Leu-Phe (fMLP) or Phorbol Myristate Acetate (PMA), both of which are potent neutrophil stimulants that trigger high levels of ROS production. Similarly, Galectin-3 (Gal-3) induces ROS in neutrophils from a sterile or nonsterile inflammatory environment, possibly creating a negative loop that could control ROS production. Besides, superoxide production is also induced by Galectin-8 (Gal-8) and Galectin-9 (Gal-9) in neutrophils but in a different manner. We describe herein the luminol and lucigenin-dependent chemiluminescence technique by using a luminometer as a method of assessment to measure ROS production by human neutrophils isolated and exposed to purified human recombinant Gal-1. The protocol described herein could be applied for the investigation of the role of other galectins in the modulation of ROS production by neutrophils.

Гранулоцитарно-макрофагальный колониестимулирующий фактор: перспективы использования у детей с инфекционным мононуклеозом

Objective. To analyze the production of active oxygen species (AOS) by peripheral blood neutrophils in children with infectious mononucleosis (IM) after their exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Patients and methods. We examined 78 children aged 3 to 11 years in the acute period of IM (including 47 children aged 3–6 years and 31 children aged 7–11 years). The control group comprised 40 healthy children matched for age. Luminoldependent chemiluminescence (CL) of peripheral blood neutrophils was assessed using the method of De Sole et al. The following CL parameters were measured: time to reach the maximum (Tmax), maximum value (Imax), and the area (S) under the chemiluminescent curve. Results. Children with IM (from both age groups) demonstrated lower spontaneous lucigenin-dependent chemiluminescence of neutrophils than controls: Tmax (p < 0.001 and p = 0.036, respectively), Imax (p = 0.024 and p = 0.025), and S (p = 0.01). Incubation of neutrophils with GM-CSF caused changes in luminol-dependent chemiluminescence only in children with MI from both age groups: Tmax decreased, whereas Imax and S increased. IM patients aged 7–11 years were found to have higher levels of chemiluminescence of neutrophils exposed to GM-CSF in vitro than IM patients aged 3–6 years (Tmax (p < 0.001) and SGM-CSF/Ssp. (p = 0.024)). Conclusion. Our findings suggest that GM-CSF can increase functional activity of neutrophils. This might facilitate adaptive immunity at periphery. Key words: infectious mononucleosis, neutrophilic granulocytes, chemiluminescence of neutrophils, granulocyte-macrophage colony stimulating factor

Decrease of reactive oxygen species (ROS) production by neutrophils after incubation in hypomagnetic conditions

Incubation of the suspension of neutrophils in hypomagnetic field generated by a system of magnetic shields (residual constant magnetic field not exceeding 20 nT) leads to significant decrease in reactive oxygen species (ROS) production compared to control (geomagnetic field 44 μT), which was recorded by lucigenin-dependent chemiluminescence and fluorescent spectroscopy with 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA). During increase of constant magnetic field (CMF) in 0.02-44 μT range, polyextreme character of the response of the neutrophils to this action was observed: the minima of ROS production were at 0.02 μT and 7.0 μT, alternating with 2.5 μT and 30 μT values, at which the used test system does not react to the exposure to CMF.

Aspirin Resistance as a Result of Impaired Interaction of Platelets and Neutrophils in Patients with Coronary Heart Disease

Aim. To study the relationship between the levels of synthesis of reactive oxygen species (ROS) by platelets and neutrophils in patients with coronary heart disease (CHD) before and after coronary artery bypass grafting (CABG), depending on sensitivity to acetylsalicylic acid (ASA).Material and methods. The study included 95 patients with coronary artery disease who are indicated for CABG surgery. The control group consisted of 30 healthy donors. The antiplatelet therapy was stopped for at least 5 days before CABG. In the postoperative period, from the first day, all patients were received 100 mg of an enteric form of acetylsalicylic acid (ASA). Resistance to ASA was determined at the level of platelet aggregation with arachidonic acid ≥20% by optical agregometry at least at one observation point: before CABG, on 1-3 day and on 8-10 day after surgery. We evaluated the spontaneous and induced lucigenin-dependent chemiluminescence (CL) of platelets (ADP induction) and neutrophils (zymosan induction) by the exit time to maximum intensity (Tmax), maximum intensity (Imax) and area (S) under the CL curve.Results. 70.5% sensitive (sASA) and 29.5% resistant (rASA) to ASA patients were revealed. Prior to CABG, in sASA patients, the Imax of spontaneous and zymosan-induced neutrophil CL and CL platelet activity was increased relative to control values. Tmax of spontaneous platelet CL, Imax and S under the ADP-induced platelet CL curve were lower in sASA patients, if to compare with rASA patients. On the 1st and 8-10th day after CABG in sASA patients, the CL indicators of neutrophil and platelet activity also remained elevated compared to control values. On the 1st day after CABG decreased levels of S under the spontaneous CL curve of neutrophils in rASA patients was established compared with sASA patients, and increased levels of Imax and S under the curve of induced neutrophil CL were detected in comparison with the control range. In rASA patients, the values of Tmax of spontaneous platelet CL decreased in relation to the values detected in the control group and sASA patients. On the 8–10th day after CABG, most indicators of spontaneous and zymosan-induced CL neutrophils in rASA patients were also increased compared to control values. In rASA patients a positive correlation of Imax-induced CL was found (r=0.83) on the 1st day after CABG and negative correlations of Tmax of spontaneous CL (r=- 0.75) and S under the curve induced CL (r=-0.70) on the 8-10th day were detected between platelets and neutrophils.Conclusion. In sASA patients with coronary heart disease before and after CABG, a high level of synthesis of superoxide radical by neutrophils and platelets was detected. The relationship between the levels of the synthesis of superoxide radical by neutrophils and platelets was found only after CABG in rASA patients. Increased synthesis of superoxide radical due to metabolic and regulatory relationships in neutrophils and platelets stimulates pro-inflammatory processes in coronary artery disease and determines the sensitivity of platelets to ASA.

Possible mechanism of antioxidant action of dinitrosyl iron complexes

The antioxidant effect of dinitrosyl iron complexes (DNICs) was studied in various model systems. DNICs with glutathione ligands effectively inhibited Cu2+-induced peroxidation of low density lipoproteins (LDL). The antioxidant effect of DNICs with phosphate ligands and free reduced glutathione (GSH) was less pronounced. In addition, DNICs with glutathione suppressed the formation of reactive oxygen species during co-oxidation of lecithin liposomes and glucose. Free radical oxidation in this system was induced with a lipophilic azo initiator and evaluated by luminol-dependent chemiluminescence. NO sharply stimulated chemiluminescence during co-oxidation of glucose and liposomes, thus suggesting the formation of potent oxidants under these conditions. Glutathione DNICs scavenge the superoxide radical anion generated in the xanthine-xanthine oxidase system. Superoxide production was assessed by lucigenin-dependent chemiluminescence and electron paramagnetic resonance (EPR) spectroscopy. Chemiluminescence revealed the dose-dependent character of antiradical effect of glutathione DNICs; moreover, these complexes turned out to be more efficient than GSH. EPR spectra of the adducts of the DEPMPO spin trap with free radicals suggest that the interaction of glutathione DNICs and superoxide does not result in the formation of the thiyl radical of glutathione. Here we propose a mechanism of the antioxidant action of glutathione DNICs, suggesting that unstable intermediate complexes are formed upon their interaction with superoxide or lipid radicals. Further, as a result of intramolecular rearrangement, these intermediates decompose without the free radical as the by-products.

Decreased Production of the Superoxide Anion Radical in Neutrophils Exposed to a Near-Null Magnetic Field

This paper reports that pre-incubation of a neutrophil suspension in the presence of a near-null magnetic field produced using a system of magnetic shields (a residual constant magnetic field not greater than 20 nT) results in a considerable decrease in the intensity of neutrophil lucigenin-dependent chemiluminescence. The addition of the NADPH oxidase inhibitor diphenyliodonium to the incubation medium reduced the chemiluminescence intensity in both the experimental and the control samples (geomagnetic field). It should be noted that the differences observed between the groups, which were caused by the exposure to a near-null magnetic field, are almost the same both at lower (2.5, 5, and 10 μM) and higher (50 and 100 μM) diphenyliodonium concentrations. In contrast, the addition of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation in mitochondria, in concentrations starting from 5 μM and up to 200 μM almost completely eliminated the difference between the control and experimental samples, which was observed at low inhibitor concentrations, or in its absence.

Phagocytic activity of blood monocytes in response to methicillin-resistant strains of Staphylococcus aureus

Current study performed to estimate the phagocytic activity of blood monocytes of varying phenotypes exposed to MRSA and MSSA strains. Objects: Blood monocytes were collected from 25 healthy adults (age: 25–45 years). Live suspensions of MRSA/MSSA strains were used at concentration of 106 colony-forming units (CFU)/mL. Metods. Phagocytic functions were estimated by using fluorescein isothiocyanate (FITC)-labelled MRSA and MSSA strains followed by running flow cytometry on FC 500 series flow cytometer (Beckman Coulter, USA). Whole peripheral blood cells were directly labelled with immunofluorescently tagged monoclonal CD14-PE/CD45-ECD/HLA-DR-PC5/CD16-PC7 antibodies (Beckman Coulter, USA). Respiratory burst intensity was evaluated in monocytes by measuring activity of lucigeninand luminol-dependent spontaneous and induced chemiluminescence. Monocytes were induced by using live suspension of MRSA/MSSA strains at a concentration of 106 CFU/mL. Results and discussion. While studying luminol-dependent monocyte activities after exposure to MRSA vs. MSSA, it was observed a 3.5-fold decreased curve square, whereas lucigenin-dependent chemiluminescence was increased by 6-fold. Compared to MSSA exposure, index of activation (IA) was decreased by 1.1-fold in response to MRSA exposure that was confirmed by lowered release of reactive oxygen species (ROS) from monocytes in response to MRSA exposure. Moreover, IRSS increased by 1.3-fold upon MRSA exposure. Examining monocyte oxygen-independent phagocytosis against MRSA vs. MSSA revealed significantly increased phagocytic number and concomitantly decreased phagocytic index. An evaluation of the activities of various monocyte subsets in response to MRSA vs. MSSA revealed increased phagocytic index by 1.5-fold for CD14lowCD16+ and CD14+CD16+ monocyte subsets as well as 3-fold for CD14+CD16– monocytes. Counts for all phagocytic subsets were decreased (1.4-, 1.5- and 4-fold for CD14lowCD16+, CD14+CD16+ and CD14+CD16– monocytes, respectively). To summarize, intensity of the respiratory burst was lowered upon MRSA exposure and percentage of monocyte subsets. Overall deficiency of superoxide anion production was observed in response to MRSA. In contrast, oxygen-independent event revealed phenotypic changes in frequency of peripheral blood monocytes upon MRSA exposure. We observed that CD14+CD16– classical monocytes were more rapidly activated. Conclusion. Thus, we concluded that CD14+CD16– monocytes became more rapidly activated but exhibited less effective phagocytosis, whereas CD14+CD16+ and CD14lowCD16+ monocytes were more slowly activated and demonstrated stronger phagocytic activity.

Cytoprotective effect of non-opioid leu-enkephalin analogue in primary culture of pulmonary fibroblasts in oxidative stress

Aim. Analysis of the cytoprotective effect of non-opioid leu-enkephalin analogue (Phe-D-Ala-Gly-Phe-Leu-Arg) in the primary culture of pulmonary fibroblasts in conditions of oxidative stress.
 Methods. Pulmonary fibroblasts were incubated with the peptide of non-opioid leu-enkephalin analogue (Phe-D-Ala-Gly-Phe-Leu-Arg) in the concentration 0.1 μM for 6 hours. To simulate oxidative stress, 60 μM H2O2 was added to the culture medium for 2 hours. Experimental series included (1) «control»; (2) «non-opiate leu-enkephalin analogue» (the peptide was added to the culture medium 44 hours after the final passage); (3) «oxidative stress» (H2O2 was added to the culture medium 48 hours after the final passage); (4) «non-opiate leu-enkephalin analogue + oxidative stress» (the peptide and H2O2 were added to the culture medium 44 and 48 hours respectively after the final passage). In order to evaluate the generation of superoxide anion by pulmonary fibroblasts, the method of lucigenin-dependent chemiluminescence was used. Computer morphometry of the nucleo-nucleolar apparatus of fibroblasts stained with silver nitrate by the AgNOR method was used to assess the cell state: the area of fibroblast nuclei, the total nucleoli area in the nucleus, and the number of nucleoli in the nucleus were measured. These parameters correlate with the activity of anabolic processes in the cells.
 Results. The effect of H2O2 on the primary culture of pulmonary fibroblasts caused an increase of superoxide anion generation by the fibroblasts, reduction of fibroblast nuclei size, decrease of nucleoli amount and size. Pre-incubation of pulmonary fibroblasts with a non-opioid leu-enkephalin analogue reduced the H2O2-induced generation of superoxide anion, corrected changes in the nucleo-nucleolar apparatus of fibroblasts caused by oxidative stress. In our previous studies, similar effect in the same model was shown for non-selective μ/δ-opioid receptor agonist peptide sedatin (dermorphin analogue). The mechanism of cytoprotective effect of non-opioid leu-enkephalin analogue may include the affinity of this peptide to nociceptin receptors (NOR receptors) that requires further studies.
 Conclusion. The results of the study indicate a direct cytoprotective effect of the peptide Phe-D-Ala-Gly-Phe-Leu-Arg (non-opioid leu-enkephalin analogue) in oxidative stress.

Usefulness of serum opsonic activity measured by chemiluminescence method to assess the invasiveness of colorectal endoscopic mucosal dissection

Endoscopic sub-mucosal dissection (ESD) is considered as a low-invasive treatment for early-stage colorectal cancer, but the degree of invasiveness has not been well investigated. The aim of this study was to evaluate the physical stress due to colorectal ESD based on changes in serum opsonic activity (SOA). SOA was examined by measuring reactive oxygen species (ROS) produced by neutrophils using lucigenin-dependent chemiluminescence (LgCL) and luminol-dependent chemiluminescence (LmCL). Sixty-nine patients were enrolled into the study and examined SOA in the morning of the day of ESD, the next day, and at four days after ESD. The peak height (PH) and area under the curve (AUC) of LgCL showed no significant difference between the day and the next day, whereas the PH and AUC for LgCL were significantly higher four days after ESD than on the day of ESD (p < .05). In contrast, the PH and AUC of LmCL showed no significant changes during the ESD perioperative period. This difference suggests that SOA changes during the colorectal ESD perioperative period involved minor increases in the production of lower-toxicity ROS. This finding supports the position that ESD is a technique that does not generate a great deal of physical stress. On the other hand, a significant increase in SOA at four days after colorectal ESD suggests that care is needed with postoperative management even after the patient has started to eat meals again.

Regulatory effect of essential oils on the production of primary and secondary biogenic free radicals in vitro

Using molecular enhancers of chemiluminescence, the regulatory effects of several essential oils on the production of primary and secondary biogenic free radicals in vitro were investigated. For the more correct extrapolation of the results, the human blood phagocytes as biogenic source of free radicals was used in an experimental model during latex stimulation and without it. The antioxidant activity of pine essential oil against primary and secondary biogenic radicals was identified, and the prooxidant activity of eucalyptus essential oil against primary and secondary biogenic radicals was determined. Unlike both of these substrates, the modulating effect of lavender essential oil was identified. Under the influence of this substance luminol-dependent chemiluminescence increased, while lucigenin-dependent chemiluminescence decreased without changing the basal activity of phagocytes. Essential oils of lemon and peppermint acted as radical-specific antioxidants. Under the influence of peppermint essential oil, the production of primary not secondary radicals decreased significantly. Under the influence of lemon essential oil, the production of secondary radicals decreased while production of superoxide did not change significantly. Based on the results are obtained, it is possible to develop or adjust the schemes of health-improving and preventive procedures, as well as to optimize the choice of adaptogens chemical forms.

Influence of gastric endoscopic submucosal dissection on serum opsonic activity measured by chemiluminescence.

This study aimed to elucidate whether changes in serum opsonic activity measured by lucigenin-dependent chemiluminescence and luminol-dependent chemiluminescence are useful for estimating physical stress during the perioperative period of gastric endoscopic submucosal dissection. Serum opsonic activity in the peripheral blood of 87 patients was examined in the morning of the day of endoscopic submucosal dissection, the next day, and at 4 days after endoscopic submucosal dissection. Peak height and area under the curve for lucigenin-dependent chemiluminescence were 106.1 ± 22.7% and 102.0 ± 24.7% on the day of endoscopic submucosal dissection, which increased significantly to 113.6 ± 29.4% and 111.0 ± 29.1% on the next day (both p<0.01), and 112.4 ± 27.0% and 110.0 ± 28.1% at 4 days after endoscopic submucosal dissection (both p<0.01), respectively. In contrast, significant changes were not observed in peak height and area under the curve for luminol-dependent chemiluminescence during the perioperative period of endoscopic submucosal dissection. This difference suggests that serum opsonic activity during the perioperative period of gastric endoscopic submucosal dissection is associated with the production of substances with lower oxidizing potential. (The study of changes in neutrophil function and physical stress during the perioperative period of endoscopic operation: UMIN000034514)