Incorporation of Pectin into Vaterite Microparticles Prevented Effects of Adsorbed Mucin on Neutrophil Activation.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP-luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

A Quantitative Luminol-Based Assay for ROS Burst Detection in Potato Leaves in Response to Biotic Stimuli.

Reactive oxygen species (ROS) are important signaling agents in plants and animals. They are involved in diverse processes, including activation of immune responses to pathogen infection. Biphasic detection of ROS in response to pathogen perception is becoming more popular even in important crops like potato as means of screening different germ plasms and mutants generated by for example CRISPR-Cas9 as well as identifying signaling pathways. Here we describe a detailed protocol for quantifying ROS bursts induced in potato leaf discs in response to a bacterial elicitor and Phytophthora infestans.

Alteration of Neutrophil Reactive Oxygen Species Production by Extracts of Devil's Claw (Harpagophytum).

Harpagophytum, Devil's Claw, is a genus of tuberiferous xerophytic plants native to southern Africa. Some of the taxa are appreciated for their medicinal effects and have been traditionally used to relieve symptoms of inflammation. The objectives of this pilot study were to investigate the antioxidant capacity and the content of total phenols, verbascoside, isoverbascoside, and selected iridoids, as well as to investigate the capacity of various Harpagophytum taxa in suppressing respiratory burst in terms of reactive oxygen species produced by human neutrophils challenged with phorbol myristate acetate (PMA), opsonised Staphylococcus aureus, and Fusobacterium nucleatum. Harpagophytum plants were classified into different taxa according to morphology, and DNA analysis was used to confirm the classification. A putative new variety of H. procumbens showed the highest degree of antioxidative capacity. Using PMA, three Harpagophytum taxa showed anti-inflammatory effects with regard to the PBS control. A putative hybrid between H. procumbens and H. zeyheri in contrast showed proinflammatory effect on the response of neutrophils to F. nucleatum in comparison with treatment with vehicle control. Harpagophytum taxa were biochemically very variable and the response in suppressing respiratory burst differed. Further studies with larger number of subjects are needed to corroborate anti-inflammatory effects of different taxa of Harpagophytum.

Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review.

Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi-/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi-/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.

[Chemiluminescence analysis of oil oxidizing bacteria Actinetobacter calcoaceticus extracts: effects of the extracts on pSoxS-lux biosensor].

A comparative H2O2-luminol- and Fe(II)-induced chemiluminescence analysis of extracts of two strains of marine oil oxidizing bacteria Actinetobacter calcoaceticus cultivated either in the presence or absence of oil was carried out. Effects of these extracts on E. coli MG1655 biosensor (pSoxS-lux) were studied. Activation of H2O2-induced chemiluminescence in the presence of oil was observed. This suggests activation of free radical lipid peroxidation. Aqueous extracts of microorganisms cultivated in the presence of oil were shown to activate reactive oxygen species production (ROS) in Fe(II)-induced chemiluminescence reaction mixture. Acetone-ethanol extracts induced antioxidative systems of both strains. Chemiluminescence analysis in a biological system carried utilizing E. coli MG1655 (pSoxS-lux) revealed that aqueous extracts of the strains cultivated in the absence of oil contained potential antioxidants.

Inhibition of peripheral blood neutrophil oxidative burst in periodontitis patients with a homeopathic medication Traumeel S.

BACKGROUND: The anti-inflammatory effects of a homeopathic remedy, Traumeel S, have been observed in experimental and clinical studies; however, its antioxidant properties have not been elucidated. The aim of the present study was to evaluate the antioxidant effects of Traumeel S on peripheral blood neutrophils in patients with periodontitis. MATERIAL/METHODS: The study was performed using venous blood of 22 individuals with chronic periodontitis and 21 healthy subjects. The antioxidant effects of Traumeel S on the production of reactive oxygen species by unstimulated and stimulated with unopsonized E. coli neutrophils were investigated using luminol- and lucigenin-dependent chemiluminescence (CL). RESULTS: Polymorphonuclear leukocytes of periodontitis patients produced higher levels (p<0.01) of light output of lucigenin-dependent chemiluminescence and significantly reduced (p<0.01) light output of luminol-dependent chemiluminescence than analogous cells of healthy subjects. Highly diluted (10⁻4 of the stem solution) Traumeel S significantly (by approximately 50%) reduced superoxide-induced oxidation of lucigenin by unstimulated and stimulated with unopsonized E. coli polymorphonuclear leukocytes of periodontitis patients and had a tendency to intensify luminol-dependent chemiluminescence. Preincubation of the unstimulated and stimulated with unopsonized E. coli polymorphonuclear leukocytes of healthy subjects with Traumeel S exerts no inhibitory action on the luminol- and lucigenin-dependent chemiluminescence of the above-mentioned cells. CONCLUSIONS: This study indicates that Traumeel S may significantly reduce production of superoxide anion by unstimulated and stimulated peripheral blood polymorphonuclear neutrophils of periodontitis patients.

Antioxidant activity of Calendula officinalis extract: inhibitory effects on chemiluminescence of human neutrophil bursts and electron paramagnetic resonance spectroscopy.

There is growing interest in natural chemical compounds from aromatic, spicy, medicinal and other plants with antioxidant properties in order to find new sources of compounds inactivating free radicals generated by metabolic pathways within body tissue and cells, mainly polymorphonuclear leukocytes (PMNs) whose overregulated recruitment and activation generate a large amount of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to an imbalance of redox homeostasis and oxidative stress. The aim of this study was to examine whether a propylene glycol extract of Calendula officinalis interferes with ROS and RNS during the PMN respiratory bursts, and to establish the lowest concentration at which it still exerts antioxidant activity by means of luminol-amplified chemiluminescence. Electron paramagnetic resonance (EPR) spectroscopy was also used in order to confirm the activity of the C. officinalis extract. The C. officinalis extract exerted its anti-ROS and anti-RNS activity in a concentration-dependent manner, with significant effects being observed at even very low concentrations: 0.20 microg/ml without L-arginine, 0.10 microg/ml when L-arginine was added to the test with phorbol 12-myristate 13-acetate and 0.05 microg/ml when it was added to the test with N-formyl-methionyl-leucyl-phenylalanine. The EPR study confirmed these findings, 0.20 microg/ml being the lowest concentration of C. officinalis extract that significantly reduced 2,2-diphenyl-1-picrylhydrazyl. These findings are interesting for improving the antioxidant network and restoring the redox balance in human cells with plant-derived molecules as well as extending the possibility of antagonizing the oxidative stress generated in living organisms when the balance is in favor of free radicals as a result of the depletion of cell antioxidants.

Immunomodulation of RAW264.7 macrophages by GLIS, a proteopolysaccharide from Ganoderma lucidum.

The immunomodulatory effect of Ganoderma lucidum immunomodulating substance (GLIS) on macrophages has been investigated as part of on-going research into the anti-cancer properties of Ganoderma lucidum. Proliferation of bone marrow macrophages (BMMs) was enhanced by GLIS in a dose-dependent manner. Microscopic examination revealed that numerous GLIS-treated RAW264.7 macrophages were enlarged and formed pseudopodia. Exposure of RAW264.7 macrophages to GLIS resulted in significant increases in NO production, induction of cellular respiratory burst activity, and increased levels of IL-1beta, IL-12p35 and IL-12p40 gene expression. Our data indicate that GLIS activates the immune system by modulating cytokine production.

Ginkgo biloba extract ameliorates ischemia reperfusion-induced renal injury in rats.

There is increasing evidence to suggest that reactive oxygen metabolites (ROMs) play a role in the pathogenesis of ischemia/reperfusion injury (I/R) in the kidney. This study was designed to determine the possible protective effect of Ginkgo biloba extract (EGb) on renal ischemia/reperfusion (I/R) injury. Wistar albino rats were unilaterally nephrectomized, and 15 days later they were subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. Ginkgo biloba extract (EGb) (50 mg kg(-1) day(-1)) or saline was administered twice, 15 min prior to ischemia and immediately before the reperfusion period. At the end of the treatment period, all rats were decapitated. Kidney samples were taken for histological examination or determination of the renal malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content. Production of reactive oxidants was monitored by chemiluminescence (CL) assay. Creatinine and urea concentrations in blood were measured for the evaluation of renal function. Tumor necrosis factor-alpha (TNF-alpha) and lactate dehydrogenase (LDH) were also assayed in serum samples. Ischemia/reperfusion caused a significant decrease in GSH level, which was accompanied with significant increases in MDA level, MPO activity and collagen content of kidney tissues. Similarly, serum BUN and creatinine levels, as well as LDH and TNF-alpha, were elevated in the I/R group as compared to control group. On the other hand, EGb treatment reversed all these biochemical indices, as well as histopathological alterations, which were induced by I/R. The findings imply that ROMs play a causal role in I/R-induced renal injury and EGb exerts renoprotective effects probably by the radical scavenging and antioxidant activities.

Novel chemiluminescence-inducing cocktails, part II: measurement of the anti-oxidant capacity of vitamins, thiols, body fluids, alcoholic beverages and edible oils.

Using two luminescence-inducing cocktails, two distinct patterns of inhibition of light by different anti-oxidants have been identified, comprising Group A, in which a complete inhibition of light emission which is then followed by re-emergence of light, forming apparent S-shaped curves or similar shapes. This light pattern is induced by the "classical" anti-oxidants, ascorbate, vitamin E, uric acid, thiols, deferoxamine, as well as by anti-oxidant agents present in plasma, saliva, urine and in extracts derived from black coffee, and Group B, in which a gradually emerging "mound"-shaped pattern of light was seen with extracts from the Tibetan plant mixture PADMA-28, elderberry (Sambucol), grape seeds, green and black teas, apple, parsimony, red wines, edible oils and SOD. While the results with the Group A agents point to the presence of probably a single, major, anti-oxidants relatively sensitive to oxidation, Group B agents probably include a mixture of anti-oxidants which are more resistant to oxidation. It was also shown that agents from Group B could protect agents from Group A against consumption by the oxidants generated by the cocktails. It is proposed that these simple to use cocktails which probably generate a multiplicity of oxidants mimicking those generated by activated phagocytes, can rapidly assess the total anti-oxidant capacities (TAOC) in body fluids derived from patients suffering of excessive oxidative stress. Also, this technique may be useful in determining the content of dietary anti-oxidants recommended as supplements to enhance the resistance against excessive oxidation of lipids.

Depressed humoral immunity after weight reduction in competitive judoists.

We studied changes in serum opsonic activity (SOA) in male judoists who were engaged in active weight reduction. Serum immunoglobulins, complements and SOA, measured by neutrophil-associated chemiluminescence responses, were investigated 20 days, 7 days and 1 day before a competition and 5 days after the competition. In addition, muscle strength and anaerobic work capacity, as well as body composition, were also determined. A dietary survey was conducted daily during the observation period. Body weight decreased by 4.2 kg over 19 days. SOA significantly decreased 5 days after the competition, as well as the concentrations of serum immunoglobulins, complements and total proteins. These trends were noted in the marked weight reduction group (i.e. reduction weight of body fat/body fat weight before weight reduction > or = 25%) more than the slight reduction group (<25%). Depressed SOA was closely correlated with the decreased concentrations of immunoglobulins and complements. These results suggest that the decrease in immunoglobulins and complements following weight reduction is associated with reduced SOA, which might cause susceptibility to infections. This study demonstrated that such immunosuppression appeared in the recovery period after the competition rather than immediately before the competition.

Hydrogen peroxide-supported activities of semisynthetic flavocytochrome 2B4.

Semisynthetic flavocytochromes, obtained by covalent binding of riboflavin with cytochromes P450 2B4, were able to catalyze the H2O2-mediated reactions of aniline p-hydroxylation, aminopyrine N-demethylation and p-nitroanizole' O-dealkylation. The rates of the flavocytochrome-catalyzed, H2O2-supported reactions far exceeded those of the appropriate NADH-dependent reactions and were comparable with the cytochrome P450 2B4-catalyzed, peroxide-mediated reaction rates. The kinetic parameters (kcat, K(m)) for the peroxide-dependent flavocytochrome P450 2B4 reactions were obtained. Sodium cyanide and SKF-525A, a specific P450 inhibitor, were both shown to inhibit these reactions. The generation of active oxygen species by flavocytochrome 2B4 was registered by chemiluminescence intensity.

Luminol-dependent chemiluminescence in bovine eosinophils and neutrophils: differential increase of intracellular and extracellular chemiluminescence induced by soluble stimulants.

Luminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (greater than 90%), maximally stimulated with 1 nmol/l phorbol 12-myristate-13-acetate (PMA) showed ten times more extracellular luminol-dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (greater than 96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27-fold difference) and platelet-activating factor (PAF) at 2 mumol/l (9-fold difference), but not by calcium ionophore A23187 (15 mumol/l). Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet-activating factor induced a dose-dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL. Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large extracellular targets.