Lucigenin-dependent Chemiluminescence Research Articles

Brain-derived neurotrophic factor is increased in atopic dermatitis and modulates eosinophil functions compared with that seen in nonatopic subjects

Recently, the pivotal role of brain-derived neurotrophic factor (BDNF) has been described in allergic asthma. However, the role of this neurotrophin in atopic dermatitis (AD) still remains unknown. The aim of this study was to investigate the functional role of BDNF on eosinophils and to assess BDNF levels in patients with AD and nonatopic control subjects. Methods p75 Neurotrophin receptor and tyrosine kinase B receptor expression was demonstrated by using FACS analysis and immunohistochemistry. BDNF levels were assessed with ELISA and FACS analysis. Chemotactic activity (modified Boyden chamber assay), eosinophil cationic protein release (fluoroenzyme immunoassay), respiratory burst (lucigenin-dependent chemiluminescence), and apoptosis (Nicoletti protocol and Annexin-V method) assays were used to assess BDNF functional activity. BDNF levels were increased in serum, plasma, eosinophils, and supernatants of stimulated eosinophils from patients with AD compared with levels seen in nonatopic control subjects ( P < .05-.001). In addition, p75 neurotrophin receptor and tyrosine kinase B expression was higher on eosinophils from patients with AD compared with that seen on eosinophils from nonatopic control subjects ( P < .05-.001). Eosinophil apoptosis was inhibited by BDNF ( P < .05-.01) and chemotactic index was increased ( P < .001) in BDNF-stimulated eosinophils from patients with AD, whereas this effect was not shown in eosinophils from nonatopic control subjects. However, no response of BDNF through the release of eosinophil cationic protein or reactive oxygen species was found. This study provides the first evidence for a functional role of BDNF on eosinophils from patients with AD, probably mediated by an increased expression of BDNF receptors compared with that seen in nonatopic control subjects. In addition, higher intracellular, serum, and plasma BDNF levels, as well as the release of BDNF by eosinophils, underline the particular importance of BDNF in patients with AD, pointing to new pathophysiologic aspects of this chronic inflammatory skin disease.

Modulation of active oxygen forms production by circulating phagocytes and clinical efficacy of glucocorticoid treatment in patients with systemic lupus erythematosus

Система комплексной поддержки и сопровождения научного журнала Elpub позволяет запустить двуязычный сайт журнала со встроенной системой электронной редакции в соответствии с лучшими издательскими практиками, а так же предусматривает техническую и методическую поддержку со стороны специалистов НЭИКОН.

Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase.

In the present study the potential benefit of reactive oxygen species (ROS)-scavenging enzymes superoxide dismutase (SOD) and catalase (CAT) when cryopreserving boar spermatozoa was evaluated. Pooled ejaculate sperm-rich fractions collected from 3 fertile boars were frozen in a split design, after being extended in a conventional freezing extender (control) or the same extender supplemented with SOD (150 or 300 IU/mL, experiment 1), CAT (200 or 400 IU/mL, experiment 2), or SOD + CAT in combination (150 + 200 or 300 + 400 IU/mL, experiment 3). Irrespective of the concentration used, SOD and CAT, alone or in combination, significantly improved postthaw sperm survival, in terms of total sperm motility (assessed with CASA) and viability (assessed with a triple stain; propidium iodide/R123/fluorescein isothiocyanate-labeled peanut agglutinin). Moreover, CAT alone, at a concentration of 400 IU/mL, or in combination with SOD, at concentrations of 200 and 400 UI/mL, improved the ability of frozen-thawed spermatozoa to produce embryos in vitro (zygote cleavage and blastocyst formation as end points). Additional data of ROS generation (luminol- and lucigenin-dependent chemiluminescence) and membrane lipid peroxidation (malondialdehyde [MDA] production) indicated that SOD and CAT reduced postthaw ROS generation by boar spermatozoa, without any influence on MDA production.

MECHANISTIC APPROACH TO THE EFFECTS OF LOW LEVEL LASER IRRADIATION (LLLI) WITH THE GaAlAs DIODE LASER ON THE PRODUCTION OF REACTIVE OXYGEN SPECIES FROM HUMAN NEUTROPHILS AS A MODEL FOR THERAPEUTIC MODALITY AT A CELLULAR LEVEL

There have been many reports on the applications of low reactive level laser therapy (LLLT) for pain attenuation or pain removal. Our group has reported previously on the effects of in vitro low level laser irradiation (LLLI) particularly on the human neutrophil function using luminol-dependent chemiluminescence (LmCL) for measurement of reactive oxygen species (ROS) production from human neutrophils. However, the change in production by LLLI for each ROS e.g. superoxide, hydroxyl radicals, hydrogen peroxide, singlet oxygen, hypochlorite and so on are however not yet fully understood. In this study, we used luminol-dependent and lucigenin-dependent chemiluminescence (LmCL and LgCL, respectively) to measure the effects of LLLI on the ROS-production process of human neutrophils. Two soluble action stimuli, N-formyl-Met-Leu-Phe (fMLP) and phorbol myristate acetate (PMA) were used to avoid the possible influence of lag-time from recognition to uptake of particles at ROS production. When fMLP was used as the stimulus, the maximum intensity of the chemiluminescence response in LmCL increased but in LgCL it decreased following LLLI. When PMA was used as a stimulus, the times to reach the maximum intensity of the chemiluminescence response of LmCL and LgCL were shortened by LLLI but there was no effect on the maximum intensity of the chemiluminescence response of both. These results suggest that LLLI enhances the ROS production capability, and activates the conversion from superoxide to singlet oxygen and hypochlorite.

Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations.

Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes-such as MCLA and luminol-that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

Neurotrophins have a functional role on eosinophils in atopic dermatitis

Rationale Neurotrophins not only have a functional role on nerves but also on immunocompetent cells. Recently, the pivotal role of neurotrophins on eosinophils of patients with allergic asthma has been described. The aim of this study was to investigate the possible role of BDNF, NT-3 and NGF on peripheral blood eosinophils in atopic dermatitis (AD). Methods AD patients were defined according to the criteria of Hanifin and Rajika. Peripheral blood eosinophils were purified by CD16 negative selection (purity >98%) and stimulated with BDNF, NT-3 (10, 50, 100ng/ml), or NGF (100, 500, 1000ng/ml) for 24 up to 120 hours. Apoptotic eosinophils were investigated by determining their hypodiploid DNA peak and Annexin V method. Chemotactic index was assessed in a modified Boyden chamber assay and respiratory burst by lucigenin-dependent chemiluminescence. Results Stimulation with BDNF, NT-3 and NGF significantly inhibited the programmed cell death of AD eosinophils (p<0.05-0.01) at each time point (24 hours up to 120 hours in culture). Chemotactic index was significantly increased in AD eosinophils after stimulation with BDNF and NT-3 (p<0.05-0.01). Respiratory burst of AD eosinophils was not modified after stimulation with BDNF, NT-3 or NGF. Conclusions BDNF, NT-3 and NGF significantly inhibited the programmed cell death of AD eosinophils. However, BDNF, NT-3 and NGF did not modulate respiratory burst of AD peripheral blood eosinophils. On the other hand, BDNF and NT3 significantly induced AD eosinophil chemotaxis. Taken together, neurotrophins, such as BDNF, NT-3 and NGF have a functional role on peripheral blood eosinophils revealing neuro-immunological interactions in AD.

Mechanism of superoxide anion generation in intact mitochondria in the presence of lucigenin and cyanide.

In the presence of cyanide and various respiratory substrates (succinate or pyruvate + malate) addition of high concentrations of lucigenin (400 microM; Luc2+) to rat liver mitochondria can induce a short-term flash of high amplitude lucigenin-dependent chemiluminescence (LDCL). Under conditions of cytochrome oxidase inhibition by cyanide the lucigenin-induced cyanide-resistant respiration (with succinate as substrate) was not inhibited by uncouplers (FCCP) and oligomycin. Increase in transmembrane potential (Deltaphi) value by stimulating F0F1-ATPase functioning (induced by addition of MgATP to the incubation medium) caused potent stimulation of the rate of cyanide-resistant respiration. At high Deltaphi values (in the presence of MgATP) cyanide resistant respiration of mitochondria in the presence of succinate or malate with pyruvate was insensitive to tenoyltrifluoroacetone (TTFA) or rotenone, respectively. However, in both cases respiration was effectively inhibited by myxothiazol or antimycin A. Mechanisms responsible for induction of LDCL and cyanide resistant mitochondrial respiration differ. In contrast to cyanide-resistant respiration, generation of LDCL signal, that was suppressed only by combined addition of Complex III inhibitors, antimycin A and myxothiazol, is a strictly potential-dependent process. It is observed only under conditions of high Deltaphi value generated by F0F1-ATPase functioning. The data suggest lucigenin-induced intensive generation of superoxide anion in mitochondria. Based on results of inhibitor analysis of cyanide-resistant respiration and LDCL, a two-stage mechanism of autooxidizable lucigenin cation-radical (Luc*+) formation in the respiratory chain is proposed. The first stage involves two-electron Luc2+ reduction by Complexes I and II. The second stage includes one-electron oxidation of reduced lucigenin (Luc(2e)). Reactions of Luc(2e) oxidation involve coenzyme Q-binding sites of Complex III. This results in formation of autooxidizable Luc*+ and superoxide anion generation. A new scheme for lucigenin-dependent electron pathways is proposed. It includes formation of fully reduced form of lucigenin and two-electron-transferring shunts of the respiratory chain. Lucigenin-induced activation of superoxide anion formation in mitochondria is accompanied by increase in ion permeability of the inner mitochondrial membrane.

Cyclosporin A disrupts bradykinin signaling through superoxide.

Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 micromol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O2- was not significantly reduced by 10 micromol/L oxypurinol or 30 micromol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 micromol/L) as well as the O2- scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 micromol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O2- scavenger Tiron (10 mmol/L), concomitant with 1 micromol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O2-. Incubation with 100 micromol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. These findings suggest that CsA activates a NADPH oxidase that releases O2- and disrupts the bradykinin/soluble guanylate cyclase pathway, probably by binding with NO to form peroxynitrite (ONOO-).

Potentiation of the extracellular release of equine neutrophil elastase and alpha‐1‐proteinase inhibitor by a combination of two bacterial cell wall components: fMLP and LPS

Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-like peptides are Gram-negative bacterial cell wall components which, when released into the peripheral circulation in endotoxaemia, have the potential to activate leucocytes. In vitro, equine neutrophils require priming with LPS in order to generate reactive oxygen intermediates (ROI) in response to fMLP. The aim of this study was to examine whether the release of other neutrophil products is similarly dependent on prior priming with LPS. In particular, neutrophil elastase (NE), a potent proteolytic enzyme, and its major inhibitor, alpha-1 proteinase inhibitor, were investigated. Neutrophils were isolated from equine peripheral blood (n = 5) by discontinuous Percoll gradient preparative centrifugation and primed with LPS prior to stimulation with fMLP. ROI were measured by lucigenin dependent chemiluminescence (LDCL). Concentrations of NE and API were determined by ELISA on cell free supernatants taken at 0, 2, 10, 30, 60 and 90 mins post stimulus. Data was analysed by Kruskal-Wallis and Mann-Whitney Tests. Sequential exposure of Percoll purified equine blood neutrophils in vitro to LPS followed by fMLP resulted in the greatest release of NE from equine neutrophils and was required for ROI generation. However, LPS or fMLP stimulation alone resulted in an increase in NE release compared to unstimulated control cells. In contrast, significant API release was only induced by LPS stimulation or fMLP stimulation only after LPS priming, not fMLP on its own. These results suggest that different stimuli (fMLP or LPS) are capable of invoking similar responses from equine neutrophils with respect to NE release yet different ones with respect to API release. In addition, demonstration of elastase release induced by LPS and/or fMLP suggests that monitoring serum elastase levels is a potential diagnostic tool for detecting the early onset of endotoxaemia in the horse.

Staphylococcal exotoxins exert proinflammatory effects through inhibition of eosinophil apoptosis, increased surface antigen expression (CD11b, CD45, CD54, and CD69), and enhanced cytokine-activated oxidative burst, thereby triggering allergic inflammatory reactions

Background: Staphylococcus aureus colonization of the skin represents a potent trigger factor of atopic dermatitis. Our previous studies demonstrated that in atopic dermatitis eosinophil apoptosis is significantly delayed. Objective: We sought to investigate the effect of staphylococcal exotoxins (SETs) on eosinophil apoptosis and functional activities. Methods: Apoptotic eosinophils were investigated by determining their hypodiploid DNA peak. Eosinophil surface-antigen expression and intracellular production of hydrogen peroxide were assessed by means of flow cytometric analysis and respiratory burst by lucigenin-dependent chemiluminescence. Results: The SETs SEA, SEB, SEC, and toxic shock syndrome toxin 1 significantly inhibited eosinophil apoptosis in a manner comparable with that of high concentrations of IL-3. The LPS inhibitor polymyxin B was found to significantly inhibit LPS-mediated, but not SET-mediated, inhibition of apoptosis. Neither SETs nor LPS was able to modulate eosinophil surface expression of CD9, CD11a, CD16, CD40, CD44, or CD63. However, 24- and 48-hour incubation with all SETs, but not with LPS, significantly upregulated expression of CD11b and CD45 in a manner similar to that of IL-3, whereas dexamethasone induced a downregulation. Moreover, all SETs resulted in a significant upregulation of CD54 after 24 hours but not after 48 hours. Interestingly, CD69 was upregulated by means of IL-3 and SEB only. Neither direct stimulation of eosinophils nor 24-hour incubation with SETs or stimulation with SETs after a 24-hour prestimulation with IL-3, IL-5, or GM-CSF resulted in a significant extracellular production of reactive oxygen species or in a significant production of intracellular hydrogen peroxide. However, SEB and toxic shock syndrome toxin 1 were able to enhance cytokine-induced respiratory burst. Conclusion: Taken together, our data demonstrate that SETs may modulate the course of the allergic inflammatory response through modulation of potent eosinophil effector functions. This may be of particular importance in atopic dermatitis, in which the skin is regularly colonized with SET-producing S aureus. (J Allergy Clin Immunol 2002;109:477-84.)

Effect of Size of Man-Made and Natural Mineral Fibers on Chemiluminescent Response in Human Monocyte-Derived Macrophages

Fiber size is an important factor in the tumorigenicity of various mineral fibers and asbestos fibers in animal experiments. We examined the time course of the ability to induce lucigenin-dependent chemiluminescence (CL) from human monocyte-derived macrophages exposed to Japan Fibrous Material standard reference samples (glass wool, rock wool, micro glass fiber, two types of refractory ceramic fiber, refractory mullite fiber, potassium titanium whisker, silicon carbide whisker, titanium oxide whisker, and wollastonite). We determined how fiber length or width might modify the response of cells. We found that the patterns of time-dependent increase of CL (sigmoid type) were similar for each sample except wollastonite. We observed a strong correlation between geometric-mean length and ability to induce CL in seven samples &gt; 6 microm in length over the time course (largest r(2) = 0.9760). Although we also observed a close positive correlation between geometric-mean width and the ability to induce CL in eight samples &lt; 1.8 microm in width at 15 min (r(2) = 0.8760), a sample of 2.4 microm in width had a low ability to induce CL. Moreover, the relationship between width and the rate of increase in ability to induce CL had a negative correlation at 30-60 min (largest r(2) = 0.7473). Our findings suggest that the release of superoxide from macrophages occurs nonspecifically for various types of mineral fibers depending on fiber length.

Effect of size of man-made and natural mineral fibers on chemiluminescent response in human monocyte-derived macrophages.

Fiber size is an important factor in the tumorigenicity of various mineral fibers and asbestos fibers in animal experiments. We examined the time course of the ability to induce lucigenin-dependent chemiluminescence (CL) from human monocyte-derived macrophages exposed to Japan Fibrous Material standard reference samples (glass wool, rock wool, micro glass fiber, two types of refractory ceramic fiber, refractory mullite fiber, potassium titanium whisker, silicon carbide whisker, titanium oxide whisker, and wollastonite). We determined how fiber length or width might modify the response of cells. We found that the patterns of time-dependent increase of CL (sigmoid type) were similar for each sample except wollastonite. We observed a strong correlation between geometric-mean length and ability to induce CL in seven samples > 6 microm in length over the time course (largest r(2) = 0.9760). Although we also observed a close positive correlation between geometric-mean width and the ability to induce CL in eight samples < 1.8 microm in width at 15 min (r(2) = 0.8760), a sample of 2.4 microm in width had a low ability to induce CL. Moreover, the relationship between width and the rate of increase in ability to induce CL had a negative correlation at 30-60 min (largest r(2) = 0.7473). Our findings suggest that the release of superoxide from macrophages occurs nonspecifically for various types of mineral fibers depending on fiber length.

Effect of aqueous cigarette smoke extract on the chemiluminescence kinetics of polymorphonuclear leukocytes and on their glycolytic and phagocytic activity.

Water-soluble extracts of cigarette smoke are easily formed in some body compartments, such as saliva or fluid lining alveolar spaces, and can act on both cellular and extracellular compartments. In this paper we have analysed the effect of aqueous smoke extract on some metabolic and functional aspects of polymorphonuclear leukocytes. In particular, the following cellular aspects were studied: chemiluminescence, glycolysis, membrane fluidity and microscopic interaction with zymosan particles. While chemiluminescence and glycolytic activity are highly inhibited, no effect of smoke extract on membrane fluidity was observed. Moreover, the response of luminol-dependent chemiluminescence was significantly delayed, while that of lucigenin-dependent chemiluminescence was anticipated. Furthermore, the phagocytic ability of neutrophils pretreated with aqueous smoke extract was also significantly hindered. All these results might indicate that the finely tuned activity of polymorphonuclear leukocytes is somehow hampered by the aqueous extract of cigarette smoke in a way which makes these cells less effective against bacteria and more noxious towards surrounding tissues.

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O2-2. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.

Effect of interferon-alpha on production of reactive oxygen species by human neutrophils.

In order to examine that the effects of interferon alpha (IFN-alpha) on the production of reactive oxygen species from human neutrophils, we investigated in vitro effects of IFN-alpha on neutrophil chemiluminescence responses. Human neutrophils were incubated with 0-1 x 10(5) IU/mL IFN-alpha for 10-60 min just before stimulation with opsonized zymosan and phorbol 12-myristate 13-acetate. Luminol-dependent (LmCL) and lucigenin-dependent (LgCL) chemiluminescence responses were measured using a Lumi Box H-1000. The LmCL response was inhibited by pre-incubation with high concentrations of IFN-alpha (more than 1 x 10(4) IU/mL). In contrast, LgCL response was increased when neutrophils were pre-incubated with high concentration of IFN-alpha. Effects of pre-incubation of IFN-alpha were similar between CL responses stimulated with OZ and PMA. The increase of LgCL might be due to the inhibitory effect of IFN-alpha on myeloperoxidase degranulation, in view of the decreased LmCL responses.

Involvement of Oxidative Stress in Crystalline Silica-Induced Cytotoxicity and Genotoxicity in Rat Alveolar Macrophages

Alveolar macrophages (AMs) occupy a key position in silica-induced pulmonary fibrosis, although the mechanisms are yet to be elucidated. In the present study we examined the involvement of oxidative stress and reactive oxygen species formation in silica-induced cytotoxicity and genotoxicity in cultured rat AMs. A lucigenin-dependent chemiluminescence test was used to determine superoxide anion (O−2), and a 2′,7′-dichlorofluorescin diacetate fluorescence test was employed to measure the hydrogen peroxide (H2O2) level. The cytotoxic and genotoxic effects caused by silica in AMs were examined by lactate dehydrogenase (LDH) leakage and single-cell gel electrophoresis (comet assay), respectively. The results showed that silica enhanced O−2 and H2O2 formation in AMs. There were clear dose- and time-dependent relationships in silica-induced cytotoxicity and genotoxicity. Furthermore, superoxide dismutase and catalase were able to reduce silica-induced LDH leakage and DNA damage, with concurrent significant inhibition on silica-induced oxidative stress in AMs. These findings provide convincing evidence that oxidative stress mediates the silica-induced cytotoxicity and genotoxicity. The understanding of such a mechanism may provide a scientific basis for the possible application of antioxidants in preventing the hazardous effects of silica.

Stimulation and scavestrogen-induced inhibition of reactive oxygen species generated by rat sertoli cells.

The ability of Sertoli cells harvested from 18-day-old Sprague-Dawley rats to generate reactive oxygen species (ROS) occurs under unstimulated and stimulated conditions. Thus, the generation of ROS and its regulation by stimulating and inhibiting compounds was determined as a lucigenin-dependent chemiluminescence reaction. According to the data, ROS generation was influenced by different cell preparation conditions--stimulating substances such as PMA, FMLP, C5a, A23187, and scavestrogens characterized by antioxidant, radical-scavenging properties. The mechanical homogenization during cell preparation procedures leads to an increase of ROS generation. ROS generation of Sertoli cells was followed by elected substances in the following rank order of efficacy: PMA > FMLP > or = C5a > Ca-ionophore A23187. The registered inhibiting effects of the scavestrogens J811 and J861 were significant. The measured CL counts decreased at 72 and 77%, respectively, of control experiments done without scavestrogens. The generation of reactive oxygen species in Sertoli cells and especially the increase in oxygen free radicals and their effects on cellular structures of spermatids are directly involved in inducing morphological alterations. Sertoli cells play an important role in spermatogenesis. The measurements of ROS may have clinical relevance in the evaluation of infertility.

Adenosine triphosphate–induced oxygen radical production and CD11b up-regulation: Ca++ mobilization and actin reorganization in human eosinophils

Eosinophils are major effector cells in cellular inflammatory conditions such as parasitic infections, atopic diseases, bullous dermatoses, and vasculitis. Biological activities of adenosine triphosphate (ATP) were characterized in human eosinophils and compared with those of other eosinophil activators such as complement fragment product C5a, platelet-activating factor (PAF), and eotaxin. ATP initiated production of reactive oxygen metabolites, as demonstrated by lucigenin-dependent chemiluminescence. Furthermore, ATP caused up-regulation of the integrin CD11b. In addition, fluorescence microscope measurements labeled with fura-2 (1-[2-(5-carboxy-oxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2' -amino-5' -methyl-phenoxy)-ethane-N, N, N, N'-tetraacetic acid, pentaacetoxymethyl ester) eosinophils in the presence or absence of ethyleneglycotetraacetic acid (EGTA) indicated that there was Ca(++) mobilization from intracellular stores by ATP. Flow cytometric studies showed transient actin polymerization upon stimulation with ATP and its stable analogues adenosine 5'-0-(3-thiotriphosphate) and 2-methylthioadenosine triphosphate tetrasodium (met-ATP). The reactions induced by ATP were comparable to those obtained by C5a, PAF, and eotaxin. Production of reactive oxygen metabolites and actin polymerization after stimulation with ATP was inhibited by pertussis toxin, which indicated involvement of receptor-coupled guanine nucleotide-binding proteins (G(i) proteins). In addition, experiments with oxidized ATP also suggest involvement of P2X receptors in this activation process. The results show that ATP is a strong activator of eosinophils and has biological activity comparable to those of the eosinophil chemotaxins C5a, PAF, and eotaxin. The findings strongly suggest a role of ATP in the pathogenesis of eosinophilic inflammation as an activator of proinflammatory effector functions.

Antioxidant activities of natural 9- cis and synthetic all- trans β-carotene assessed by human neutrophil chemiluminescence

Much attention has been focused on the biological activity of geometrical isomers of β-carotene. In the present study, the in vitro antioxidant activities of a natural 9- cis β-carotene purified from Dunaliella bardawil and synthetic all- trans β-carotene were studied using enhanced human neutrophil chemiluminescence upon stimulation with opsonized zymosan. Lucigenin-dependent chemiluminescence was used for largely measuring superoxide anion (O 2) production, luminol-dependent chemiluminescence with sodium azide (NaN 3) and horseradish peroxidase (HRP) was used for measuring hydrogen peroxide (H 2O 2) production, and luminol-dependent chemiluminescence without oxidant metabolism modulator was used for determination of myeloperoxidase (MPO)-mediated production. With β -carotene concentration of 1.4 μ M, neutrophils showed a decrease in lucigenin-dependent chemiluminescence to 75% and 78% of controls (p<0.05) by natural 9- cis β -carotene and synthetic all- trans β -carotene, respectively. With β -carotene concentration of 1.86 μ M, neutrophils also showed a decrease in luminol-dependent chemiluminescence in the presence of NaN 3 and HRP to 86% and 81% of controls (p<0.05) by natural 9- cis β -carotene and synthetic all- trans β -carotene, respectively. On the other hand, neutrophils did not show a statistically significant difference in any chemiluminescence assay by the same concentration of natural 9- cis β -carotene and synthetic all- trans β -carotene. These results suggest that, up to the concentration of 1.86 μ M, natural 9- cis β -carotene and synthetic all- trans β -carotene exert equal antioxidant activities assessed by enhanced human neutrophil chemiluminescence.

Effects of Helicobacter pylori in the stomach on neutrophil chemiluminescence in patients with gastric cancer.

Hypochlorous acid, which is one of the reactive oxgen species (ROS) produced from human neutrophils, is converted to cytotoxic NH(2)Cl after reaction with ammonia produced by urease in Helicobacter pylori (HP), increasing gastric mucosal injury and with potential development to gastric cancer. We compared the effects of HP on the production of ROS by human neutrophils between two groups-22 patients with gastric cancer and 16 patients without gastric cancer (control group), in whom HP was isolated from stomach biopsy tissues-using a luminol- and lucigenin-dependent chemiluminescence (LmCL and LgCL, respectively). It was very similar in the mean value or variance of mean maximal chemiluminescence intensities (MCI) and peak time in LmCL and LgCL between the two groups. MCI of LmCL was highly correlated with that of LgCL in both groups. These results indicate that there were no differences in the behaviour of HP on human neutrophil chemiluminescence between two groups. The progression from non-malignant mucosa to cancer may be associated with the time-dependent effects of HP via ROS produced by neutrophils on the gastric mucosa.