Luminol-dependent Chemiluminescence Research Articles

Two 5-Methoxyindole Carboxylic Acid-Derived Hydrazones of Neuropharmacological Interest: Synthesis, Crystal Structure, and Chemiluminescent Study of Radical Scavenging Properties

Given the importance of molecular structure in pharmacological activity and interaction with biological receptors, we conducted a study on the 3,4-dihydroxybenzaldehyde hydrazone derivative of 5-methoxy-indole carboxylic acid (5MICA) and a newly synthesised analogue bearing a 2-methoxy-4-hydroxyphenyl ring using single-crystal X-ray diffraction. We studied the ability of the two compounds to scavenge hypochlorite ions using luminol-enhanced chemiluminescence and their potential to modulate oxidative damage induced by iron on the biologically significant molecules lecithin and deoxyribose in order to evaluate possible antioxidant and prooxidant effects. The X-ray study revealed highly conserved geometry and limited rotation and deformation freedom of the respective indole and phenyl fragments. Interestingly, a conformational difference between the two independent molecules in the asymmetric unit of 3b was found. The X-ray study revealed a combination of hydrogen bonding interactions, short contacts, and π–π stacking stabilizing the specific three-dimensional packing of the molecules of 3a and 3b in the crystal structures. The three-dimensional packing of the molecules of 3b produced a zigzag layering projected along the c-axis. Both compounds effectively decreased luminol-dependent chemiluminescence in model systems with KO2-produced superoxide. They displayed opposite effects when applied in a xanthine/xanthine oxidase system. The hydrazones of 5MICA do not trigger a prooxidant effect or subsequent toxicity under conditions of iron-induced oxidative stress. The 3,4-dihydroxy-substituted derivative demonstrated excellent radical scavenging properties in all model systems, making it the lead compound for the development of compounds with combined neuroprotective and antioxidant properties.

Resveratrol acts as an antioxidant in leukocytes of patients with diabetes mellitus 2 through the AMPK signaling pathway

Diabetes is characterized by disturbances in serum glucose levels caused by insulin resistance, with consequent high blood glucose levels. Permanently increased blood glucose tends to lead to an increase in reactive oxygen species (ROS). Therefore, one of the mechanisms to contain this process is to consider antioxidant treatment. Within this context, resveratrol (RSV) stands out. Among the pathways of action of this compound, AMPK plays a fundamental role in regulating its antioxidant activity. The objective of the work aims to verify the antioxidant profile of RSV, through the AMPK pathway, in leukocytes of patients with type 2 diabetes mellitus (DM2) between 60 and 80 years old. 12 individuals were selected, divided into two groups: control group (n=6) and experimental group (n=6). After blood collection and leukocyte separation, the luminol-dependent chemiluminescence assay was performed. To analyze the involvement of the AMPK pathway, the inhibitor compound C was used. The result obtained showed that there was an antioxidant behavior of AMPK in cells stimulated with peroxide and treated with RSV, this in turn decreased the production of ROS in leukocytes of all samples, but this effect was less significant in DM2 patients. This oxidation, which is more pronounced in diabetics, can lead to target organ damage. As RSV played an important role in reducing ROS, we concluded that it is an important compound in preventing target organ damage in patients with this comorbidity, acting through the AMPK pathway.

Adrenergic effect on women's blood neutrophil oxidative activity the first day after delivery.

Recent studies have described a significant role for neutrophils in reproductive processes and their participation in the preparation of the cervix for childbirth and the activation of labor, in the postpartum involution of the uterus, and in the occurrence of preeclampsia. This study aimed to assess the formation of free radicals by neutrophils in the blood of women on the first day after childbirth and to characterize the adrenergic effect on this process. Venous blood samples from 100 female volunteers aged 26-32 years who had 2 or 3 full-term deliveries were collected and analyzed. Various adrenergic compounds were considered (agonists alphaand betaadrenoreceptors, adrenoblockers). The intensity of the respiratory burst of neutrophils and the effect of adrenergic substances on them were assessed with latex-induced luminol-dependent chemiluminescence. Neutrophil activity depends on the stage of the woman's reproductive process: it decreases during pregnancy, reaches the lowest values during childbirth, and increases significantly in the first hours after childbirth. On the first day after childbirth, alpha-1-adrenergic receptors are highly active in neutrophils, through which NADP-H-oxidase is activated and activated oxygen species are formed. At the same time, alphaor beta-agonists inhibit the radical activity of cells. Latex-induced oxidative burst of female blood neutrophils correlates with the stage of the reproductive process. Stressful conditions in the postpartum period can suppress the ability of neutrophils to release reactive oxygen species, which increases the risk of postpartum infections.

Features of phagocytic and chemiluminescent activity of neutrophils when exposed to polyprenols <i>in vitro</i>

Aim: Studying of the features of phagocytic activity and the state of the respiratory burst of neutrophils under the influence of polyprenols in vitro.
 Materials and methods. The study involved 30 relatively healthy donors aged 23-59 years from whose peripheral blood neutrophilic granulocytes were isolated to determine phagocytic and chemiluminescent activity. The isolated cells were incubated for 30 minutes with polyprenols in vitro. The level of phagocytosis of immature and mature neutrophils was determined by flow cytometry using FITC-labeled staphylococcal protein A. The state of the respiratory burst was assessed using chemiluminescent analysis.
 Results. It was found that the levels of phagocytic index (PI) and number (PN) of mature neutrophils were reduced after incubation with polyprenols relative to initial and control values. The values of PI and PN of immature neutrophils under the influence of polyprenols and during control incubation were decreased relative to the initial values. The intensity maximum and area under the curve of spontaneous and zymosan-induced lucigenin- and luminol-dependent chemiluminescence of the neutrophils were reduced after incubation with polyprenols.
 Conclusion. The effect of polyprenols on neutrophilic granulocytes in vitro leads to a decrease in activity in respiratory burst cells, which is realized in blocking by polyprenols the interaction of primary and secondary ROS with chemiluminescent indicators by the mechanism of primary antioxidants. The binding of ROS by polyprenols also manifests itself in a decrease in the phagocytic activity of mature neutrophils while maintaining the phagocytic activity of immature cells at the level of control incubation. In general, polyprenols exhibit anti-inflammatory activity when exposed in vitro to neutrophilic granulocytes.

Dynamics of free radical processes in the oral fluid of patients with chronic recurrent aphthous stomatitis at the first stage of therapy

Chronic recurrent aphthous stomatitis (CRAS) is a disease of the oral mucosa, characterized by the appearance of specific round aphthous forms with hyperemic corolla, covered with grayish-yellow plaque against the background of inflamed mucous membrane, which violates the oxidative homeostasis of oral fluid and the body as a whole.The aim of our study was to reveal the dynamics of free radical processes in oral fluid of patients with chronic recurrent aphthous stomatitis on the background of the first stage of antiseptic therapy with analgesics in order to determine the optimal treatment tactics for this condition. In order to achieve the set objective we had to solve the following tasks: to evaluate the functional activity of free radical processes in the oral liquid of patients with CPAS on the background of antiseptics (chlorhexidine, octenisept, furacilin, myramistine) and anaesthetic (Camistad gel) application for 7 days.Materials and Methods. During the study the oral fluid of 120 people diagnosed with CPPS was studied, divided into 4 groups equally: Group 1 – control, patients used the antiseptic chlorhexidine for gargling; Group 2 – patients with CPPS therapy with octenisept; Group 3 – patients with CPPS therapy with furacilin; Group 4 – patients with CPPS therapy with miramistine. For pain relief, patients in all groups used Kamistad gel. Analysis of free radical processes in oral fluid of patients was carried out before the study and on the 7th day of therapy by determining products of oxidative modification of biomolecules on the basis of quantitative evaluation of stained complex with thiobarbituric acid (TBA), maximal flash (MVHL) and area (PCL) of H2O2-induced luminol-dependent chemiluminescence using LT-01 chemiluminometer ("Horos" "Joint Venture Soviet-Swedish Company"). Concentration of diene conjugates and malonic dialdehyde was also determined to estimate enzymatic part of oxidative homeostasis.Results of the study. Results of the researches permit to conclude that at chronic recurrent aphthous stomatitis enzymatic activity of an oral liquid changes. According to the presented data the initial values of concentration of TBK-RP, maximal flash of oral chemiluminescence, area of oral chemiluminescence and concentration of MDA and DC in the control group and three experimental groups reliably differ from the data received on the 7th day of the research. Significant decrease of the studied indexes of oral liquid testifies to the efficiency of the first stage of therapy of aphthous stomatitis by antiseptic preparations and decrease of intensity of oxidative processes in oral liquid. Of all the proposed antiseptics the best efficiency is demonstrated by octenisept, reliably worse are chlorhexidine and myramistine and the least effective in the therapy of CPAS is furacilin.Conclusions: treatment of CPAS with Octenisept in combination with Camistad gel is the most effective in restoration of the disturbed oxilite.

Phagocytosis and oxidative activity of neutrophils after interaction with uropathogenic <i>Escherichia coli</i> biofilms

Recurrent urinary tract infections (UTIs) are associated primarily with the ability of Escherichia coli to form biofilms. The interaction of neutrophils, factors of innate immunity, with microorganisms in biofilms is difficult compared to planktonic forms due to the lack of direct contact, as well as due to the antiphagocytic action of the extracellular matrix of biofilms. The purpose of this study was evaluation of neutrophils phagocytic and oxidative activity during interaction with biofilms of uropathogenic E. coli (UPEC) DL82 and R44. Peripheral blood neutrophils from healthy men were isolated using ficoll-urographin double gradient, incubated for 1 h with bacterial cells from biofilms or their supernatants, then leukocytes functional activity was evaluated. Phagocytic activity of neutrophils was determined by the degree of bioluminescence inhibition of bioluminescent strain E. coli K12 TG1 lux+ (pXen) upon their absorption by neutrophils. Production of extracellular reactive oxygen species (ROS) was analyzed by the intensity of luminol-dependent chemiluminescence in spontaneous and stimulated by E. coli K12 variants. Significance of differences was determined using Student’s t-test at p < 0.05. It was found that neutrophils interaction with UPEC biofilm cells or supernatants did not affect the phagocytic activity. E. coli DL82 supernatants reduce neutrophils spontaneous ROS production compared to control and biofilm cells. E. coli R44 supernatants with a low virulence potential did not affect ROS production, while biofilm cells stimulated it. When assessing stimulated ROS production, exposure to R44 strain supernatants did not cause a decrease in neutrophils activation in response to an external stimulus (E. coli K12 cells). Preliminary contact of neutrophils with E. coli R44 bacteria resulted in a high and prolonged level of ROS production compared to the control. Neutrophils interaction with DL82 cells resulted in a higher level of ROS compared to supernatants, however a subsequent rapid depletion of neutrophils oxidative potential was observed. Thus, cells and supernatants of UPEC biofilms can determine the activation of neutrophils.

The Effect of Progestins on Cytokine Production in the Peripheral Blood Mononuclear Cells of Menopausal Women and Their Luminol-Dependent Chemiluminescence.

Steroid hormones are the key regulators of inflammatory and autoimmune processes. The role of steroid hormones is mostly inhibitory in these processes. The expression of IL-6, TNFα, and IL-1β, as markers of inflammation, and TGFβ, as a marker of fibrosis, could be useful tools to predict the response of an individual's immune system to the different progestins suitable for the treatment of menopausal inflammatory disorders, including endometriosis. In this study, the progestins P4 and MPA, as well as the novel progestin gestobutanoyl (GB), which possess potent anti-inflammatory properties towards endometriosis, were studied at a fixed concentration of 10 µM. Their influence on the production of the above cytokines in PHA-stimulated peripheral blood mononuclear cells (PBMCs) during 24 h incubation was evaluated by ELISA. It was found that synthetic progestins stimulated the production of IL-1β, IL-6, and TNFα and inhibited TGFβ production, while P4 inhibited IL-6 (33% inhibition) and did not influence TGFβ production. In the MTT-viability test, P4 also decreased PHA-stimulated PBMC viability by 28% during 24 h incubation, but MPA and GB did not have any inhibitory or stimulatory effects. The luminol-dependent chemiluminescence (LDC) assay revealed the anti-inflammatory and antioxidant properties of all the tested progestins, as well as some other steroid hormones and their antagonists: cortisol, dexamethasone, testosterone, estradiol, cyproterone, and tamoxifen. Of these, tamoxifen showed the most pronounced effect on the oxidation capacity of PBMC but not on that of dexamethasone, as was expected. Collectively, these data demonstrate that PBMCs from menopausal women respond differently to P4 and synthetic progestins, most likely due to distinct actions via various steroid receptors. It is not only the progestin affinity to nuclear progesterone receptors (PR), androgen receptors, glucocorticoid receptors, or estrogen receptors that is important for the immune response, but also the membrane PR or other nongenomic structures in immune cells.

Possibility of modification of peripheral blood neutrophils functional activity during chemoradiotherapy in patients with anorectal cancer

Background. Ionizing radiation is an effective antitumor therapy, but it has a serious negative effect on the immune system requiring the use of radiation reaction prevention and reduction methods. Neutrophils are a sensitive element of the immune system both in interaction with tumor tissue and in response to radiation injury.Aim. To evaluate functional activity of peripheral blood neutrophils by chemiluminescent analysis in patients with anorectal cancer after radiotherapy.Materials and methods. The study included 80 patients with anorectal cancer. Patients received chemo- and radio-therapy with 3D conformal radiotherapy and radiation therapy under visual control, followed by the use of radioprotector and without it. Neutrophil activity determined by chemiluminescent analysis.Results. In patients with anorectal cancer found maximum spontaneous and induced chemiluminescence acceleration. The chemiluminescence activation index with luminol is lower in patients with anorectal cancer, and with lucigenin shows no differences with the control group. The low luminol chemiluminescence maximum intensity, as well as the decrease in the synthesis of reactive secondary oxygen species in enzymatic systems, is likely regulatory intracellular limitations consequence. After treatment, patients with radioprotector showed a decrease in the number of parameters with statistically significant differences with the control group. Undesirable phenomena associated with sodium deoxyribonucleate therapy not detected in anorectal cancer patients during radiotherapy and subsequent observation period.Conclusion. During treatment, differences in chemiluminescence parameters suggest that ionizing radiation affects them in patients with anorectal cancer receiving standard chemoradiotherapy. Use of chemoradiotherapy with a radioprotector leads to indirect restoration of cellular functional activity. This is confirmed by luminol-dependent chemiluminescence faster reaching its maximum and a decrease in the number of significant differences from the control group after the start of the drug treatment.

Aging: Change in SIRT1 and Enzymatic Profile Promotes a Decrease in the Antioxidant Capacity of Resveratrol in Human Leukocytes In Vitro.

One of the most studied theories about aging comes from the accumulation of free radical generation, leading to oxidative stress. Resveratrol (RSV) is a polyphenolic compound that has been shown to act as an antioxidant in medical practice. To verify the antioxidant action of resveratrol (and its correlation with aging) in leukocytes from donors of different ages, mainly through the analysis of the three main enzymes of the antioxidant complex and the analysis of the SIRT1 signaling pathway. Luminol-dependent chemiluminescence assay was used to evaluate ROS and SIRT1. Antioxidant enzymes were evaluated by commercial kits. *p<0.05. In all age groups, there was a reduction in reactive oxygen species (ROS) in cells stimulated with RSV. There was a positive correlation between its antioxidant effect and donor age. In younger individuals (20-39 years old), there was an increase in catalase activity in cells exposed to RSV. In the older groups (40-59 years old and 60-80 years old), RSV was able to increase the activity of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). Through the analysis of SIRT1 it was possible to observe a silencing of the pathway in leukocytes treated with RSV during aging. RSV showed antioxidant activity in all age groups, although more pronounced in younger individuals. One of the mechanisms of action of the RSV is due to the increase in the activity of antioxidant enzymes, which varies according to the individual's age, especially through the modulation of important antioxidant pathways.

The effect of endomorphins-1, -2 on the production of reactive oxygen species and the absorption activity of innate immunity cells &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;

Background. A high percentage of people in the world suffer from acute or chronic pain, the treatment of which is based on the introduction of morphine and its derivatives, agonists of the µ-opioid receptors, leading to the development of addiction in patients. An urgent task is to study the properties of endogenous opioid peptides, which are capable of showing not only pronounced analgesic activity, but also have a number of other effects on the periphery, while having a much smaller spectrum of side effects. Of particular interest are the immunomodulatory effects of peptide agonists of µ-receptors – endomorphins, tetrapeptides, the effects of which are not described in detail in the literature. Due to their structure and properties, endorphins can be considered as potential substitutes for low-molecular-weight opiates. The aim. To investigate the effect of endorphins-1,2 on the synthesis of oxygen radicals and the absorption activity of innate immunity cells in vitro, in vivo. Materials and methods. The object of the in vitro study is peripheral venous blood leukocytes from donors aged 22–40 years; the object of the in vivo study is peritoneal flush cells of Swiss white male mice. Oxygen-dependent microbicidal activity was evaluated using a luminol-dependent chemiluminescence reaction. Phagocytic activity was evaluated by flow cytometry. Results. It has been found that endomorphins inhibited spontaneous production of reactive oxygen species in vitro, in vivo. Endomorphin-2 reduced the intensity of respiratory explosion in stimulated cultures of peritoneal macrophages. Blockade of opiate receptors with naloxone in vitro, in vivo did not cancel the effects of endomorphins. Both peptides and naloxone had no effect on the phagocytic activity of macrophages of the peritoneal cavity of mice in vivo, but in vitro endomorphins led to an increase in the percentage of phagocytosis of peripheral blood leukocytes. Conclusions. The effects of endomorphins in vitro, in vivo are predominantly depressive.

Expression and function of receptors for the formylated peptides in granulocytes of the patients with rheumatoid arthritis

Formyl peptide receptors (FPRs) are an important part of innate immunity involved in antimicrobial phagocyte functions such as chemotaxis, secretory degranulation, and respiratory burst. These phagocyte responses are observed in both acute and systemic chronic inflammation. Abundant or constant release of pro-inflammatory ligands leads to the pre-activation of phagocytes when subsequent stimulation induces more intense cellular response. Binding of the formyl peptide receptor with its agonist activates production of reactive oxygen species, due to triggering phosphorylation of the cytoplasmic subunits p47phox and p67phox followed by their translocation to the plasma membrane and assembly into the NADPH oxidase complex. Rheumatoid arthritis is characterized by an imbalance of immune processes and autoimmune responses against the own joint tissues. It is known that, granulocytes produce increased amounts of oxygen radicals in various pathologies, including rheumatoid arthritis. We suggest that such enhancement may be due to increased expression of formyl peptide receptors or components of the FPR/PKC/NOX2 signaling pathway. Our aim was to study the mRNA expression of fpr1/fpr2 genes and the FPR-dependent production of reactive oxygen species by isolated peripheral blood granulocytes from the patients with rheumatoid arthritis. Materials and methods. The objects of the study were isolated peripheral blood granulocytes. We analyzed, respectively, 166 and 85 samples from the patients with rheumatoid arthritis and healthy donors. The production of reactive oxygen species was assessed using luminol-dependent chemiluminescence. For FPR1 activation we used a distinct concentration of the formyl peptide fMLF: the response to it was completely inhibited by pretreatment of the cells with FPR1 antagonist N-t-boc-MLF. FPR2 activation was performed by synthetic peptide WKYMVM, a specific receptor agonist. In the patients with rheumatoid arthritis, we have revealed an increased level of spontaneous and phorbol ester-induced production of reactive oxygen species by isolated peripheral blood granulocytes, thus reflecting a pre-activated state of the phagocytes in rheumatoid arthritis. We have found the increased FPR1-mediated production of oxygen radicals and expression of mRNA of fpr1 gene in blood granulocytes of rheumatoid arthritis patients. Furthermore, the enhancement of oxidase function may be associated with constitutive activation of the FPR1/PKC/NOX2 pathway as shown by positive correlation between the processes. The production of reactive oxygen species induced by stimulation of the FPR2 receptor is also increased, but it cannot be directly attributed to overexpression of the receptor mRNA or PKC/NOX2 activation, and requires further study. Understanding the mechanisms of regulation of the FPR1 and FPR2 signaling cascades may reveal new targets for anti-rheumatoid therapy.

Redox-Activation of Neutrophils Induced by Pericardium Scaffolds

Implantation of scaffolds causes a local inflammatory response whereby the early recruitment of neutrophils is of great importance not only for fighting the infection, but also for facilitating effective regeneration. We used luminol-dependent chemiluminescence, flow cytometry, ELISA, and confocal microscopy to assess the responses of neutrophils after the exposure to the scaffold-decellularized bovine pericardium (collagen type I) crosslinked with genipin (DBPG). We demonstrated that DBPG activated neutrophils in whole blood causing respiratory burst, myeloperoxidase (MPO) secretion, and formation of neutrophil extracellular trap-like structures (NETs). In addition, we studied platelets, another important player of the immediate immune host response. We found that platelets triggered redox-activation of isolated neutrophils by the pericardium scaffold, and likely participate in the NETs formation. Free radicals generated by neutrophils and hypochlorous acid produced by MPO are potent oxidizing agents which can oxidatively degrade biological structures. Understanding the mechanisms and consequences of redox activation of neutrophils by pericardium scaffolds is important for the development of new approaches to increase the efficiency of tissue regeneration.

Biochemical and immunological markers of the adaptive response in olfacto-odorimetric studies

The purpose of the study is to determine whether exposure to odours of model food odourants can lead to a change in biochemical and immunological parameters that we previously used when examining the population in the area of food industry enterprises location using the method of quantitative olfacto-odorimetry. Methods. The specified concentrations of aerosols of three food flavours (orange, cognac and coffee) were supplied to the participants of the studies with a help of ECOMA T08 olfactometer. Quantitative composition of the aerosols was controlled by GC/MS. In participants saliva samples taken before, during and at the end of each experiment, the intensity of luminol-dependent chemiluminescence, the content of secretory IgA, IL-1β, IL-6 and IL-8, the activity of α-amylase and N-acetyl-β-D-glucosaminidase were determined. For data analysis, paired Friedman and Wilcoxon tests were used with Bonferroni correction for the problem of multiple comparisons. Results. A reliable effect of the smell of food odourants was found on one indicator only - the activity of salivary α-amylase - when combining data from 5 separate experiments (n=45): 93.3[24.3;160.0] U/ml at the end of the experiments against background values of 109.9 [42.5; 216.7] U/ml;, p=0.0096 with a significance level of p=0.05/3=0.017. A decrease in the average values of salivary α-amylase activity was shown to hide opposite changes in individual values: an increase in activity in people with low background values (below the median of the initial distribution) and an amplitude-dominant decrease - in people with high background values (above median). The revealed phenotypic polymorphism of α-amylase regulation contributes to one of relevant Post-COVID areas - the study of the ability of people to perceive odours and react to them. Limitations. The use of olfacto-odorimetry to study effect of odours on human health indicators is promising, but requires design of protocols with extended exposure time. Conclusion. A decrease in average values of salivary α-amylase activity with distinctive forestall of the upper quartile may be a sign of human reflex re sponse to the emission of odourous substances in the areas of food industry.

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.

Enhanced hydroxyl radical generation by human leukocytes exposed to bacterial diamines highlighting immunomodulatory effect of microbial metabolites

Recently, there have been increasing an interest to study a role of polyamines in intercellular interactions, especially in the focus of inflammation, where accumulation of such polycations is observed. In this regard, products of microbial origin cadaverine and putrescine are of particular attention. The role of polyamines as scavengers of free radicals has been described, but no data of their effect on the leukocyte radical-producing activity have been obtained so far. The aim of the investigation was to study features of hydroxyl radical generation by human leukocytes exposed to microbe-derived polyamines. Materials and methods. Peripheral venous blood samples were obtained from 20 healthy donors. To assess radical production, a luminol-dependent chemiluminescence reaction was carried out with blood leukocytes pre-incubated with cadaverine (0.01 M) and putrescine (0.01 M), measured on a Luminoskan Ascent Thermo Labsystems luminometer (USA) for 180 min. For statistical analysis, an integral chemiluminescence index was used for the entire measurement period (RLU). Results and discussion. It was shown that cadaverine has a stimulating effect on the leukocyte potential to produce radicals (averaged area under the curve is 6.70.7 r.u., p 0.05). Putrescine had little effect on the radical-producing activity of human cells (2.80.4 r.u., p 0.05). This might be due to the direct influence of polycations on the mechanisms of radical generation, as well as increased activity of leukocyte diamine oxidase catalyzing the conversion of diamines into aminoaldehyde, which is accompanied by the release of hydrogen peroxide reacting with luminol. In addition, the enzyme inactivates other compounds, such as histamine leading to formation of a less pronounced clinical picture. Polyamines, particularly cadaverine and putrescine, can be referred to the factors remodeling the metabolic activity of the host leukocytes, which is aimed at maintaining the viability and increasing the number of microorganisms. Conclusion. Thus, cadaverine and putrescine produced by microorganisms, depending on the conditions of the microenvironment, might be evidently considered as mediators of the mild or asymptomatic course of inflammatory diseases, which contributes to underlying persistent potential of bacteria.

Polyphenols-Rich Fraction from Annona muricata Linn. Leaves Attenuates Oxidative and Inflammatory Responses in Neutrophils, Macrophages, and Experimental Lung Injury.

Annona muricata Linn. is a common plant found in the warmest regions of South and Central America and its use in traditional medicine has been reported for the treatment of various illnesses. In the current study, we investigate the antioxidant and anti-inflammatory activities of crude extract and fractions from A. muricata L. leaves in isolated murine phagocytic immune cells as well as experimental LPS-induced acute lung injury (ALI). In a luminol-dependent chemiluminescence assay, we showed that ethyl acetate (EtOAc.f) and n-butanol (BuOH.f) fractions—both rich in polyphenols—reduced the generation of reactive oxygen species (ROS) by neutrophils stimulated with opsonized zymosan; similar results were found in culture of bone marrow-derived macrophages (BMDMs). By evaluating anti-inflammatory activity in BMDMs, EtOAc.f and BuOH.f reduced secretion of IL-6 and expression of the co-stimulatory molecule CD40. Furthermore, in LPS-induced ALI, oral administration of EtOAc.f reduced myeloperoxidase (MPO) activity in lung tissue. In addition, on a mechanism dependent on glutathione levels, the oxidative damage was also attenuated. These findings revealed direct antioxidant and anti-inflammatory activities of polyphenols-rich fractions of A. muricata L. leaves on neutrophils and macrophages. Moreover, the reduced oxidative damage and levels of inflammatory markers in experimental ALI suggest that these fractions might be explored for the development of new therapies for inflammatory conditions.

Interaction between Highly Diluted Samples, Protein Solutions and Water in a Controlled Magnetic Field

We have previously shown that water incubated in a weak combined magnetic field (CMF) increased the production of reactive oxygen species (ROS) by neutrophils. Adding high dilutions (HD) of water into the same system resulted in a similar effect. HD of antibodies to interferon-gamma (HD Abs to IFNγ) were shown to emit electromagnetic radiation and affect hydrogen bond energies. Here, we aimed to evaluate the effect of HD of substances (donor) on the properties of aqueous solutions (acceptor). The donor and acceptor were incubated for 1 h in a controlled magnetic field so that the walls of the two cuvettes were in close contact. As a control, the acceptor was incubated under the same conditions but without the donor. An aliquot of the acceptor solution was then added to mouse neutrophils, and ROS levels were measured using luminol-dependent chemiluminescence assay. Joint incubation led to a 185–356% increase (p &lt; 0.05) in ROS production, depending on the type of acceptor sample. The magnitude of the effect depended on the parameters of the magnetic field. In a CMF, the effect was strongest, completely disappearing in a magnetic vacuum or with shielding. These findings are important for understanding the physical mechanism of action of HD preparations, which opens up opportunities for expanding their practical applications.

Specficics of the pro-antioxidant and antioxidant status in children with acute pneumonia, depending on the severity of the pathological process and the etiological factor

Background: In the recent years, an increasing number of researchers, in addition to the standard causes of a particular pathologys development, have been paying attention to changes in the immune, pro-antioxidant, psychosomatic and other systemic factors. The processes occurring in the body with the participation of reactive oxygen species are general biological mechanisms of tissue damage and protection. Their role has been described and proven in a variety of nosologies. However, the number of studies evaluating the interaction of prooxidants with antioxidants in children with acute pneumonia is quite few.&#x0D; Aims: Analysis of the pro-/antioxidant status in children with acute pneumonia, depending on the severity of the pathological process and the etiological factor.&#x0D; Methods: The main group consisted of 50 patients with acute pneumonia, who were divided into 2 subgroups according to the severity of the disease course. The comparison group consisted of 30 conditionally healthy children. In addition to the list of examinations of children with acute pneumonia, according to the clinical practice guidelines, Imax, S, t values were assessed in the study groups by the method of luminol-dependent chemiluminescence (CL) of the blood plasma, depending on the severity of the pathological process and the etiological factor.&#x0D; Results: After conducting a ROC analysis in children in the acute period of pneumonia, we established the threshold values of the pro-/antioxidant status parameters: at the Imax level 28.1%; S 26.2%; t 0.34 min. the examined children had a more severe course of pneumonia. When Mycoplasma pneumoniae, Chlamydophila pneumoniae were detected in the sputum, the level of the pro-/antioxidant status parameters was lower (Imax=28.0%, S=36.0%, t=0.37 min) than that in children who had Gram-negative bacteria or Gram-positive bacteria in the sputum (Imax=46.0%, S=45.0; t=0.42 min); p 0.05.&#x0D; Conclusion: Violations of the pro-/antioxidant status were noted in all the children we examined. The most pronounced violations of the pro-/antioxidant status were detected in children with acute community-acquired severe pneumonia caused by atypical pathogens M. pneumoniae, C. pneumoniae. In addition, the ROC analysis made it possible to determine the threshold values of the pro-/antioxidant status parameters for assessing the severity of pneumonia in children during the acute period of the disease.

The effect of kisspeptin on the functional activity of peripheral blood monocytes and neutrophils in the context of physiological pregnancy

Hypothalamic hormone kisspeptin is also produced by placental syncytiotrophoblast. Blood leukocytes express a specific membrane receptor of kisspeptin (KISS-1R). Since kisspeptin-54 enters the bloodstream during pregnancy, the hormone has a systemic effect on the leukocytes only in this period. Earlier we demonstrated that kisspeptin-54 enhances the generation of regulatory T (Treg) cells and suppresses the IL-17-producing T helper (Th17) cells. NK cells specialize into a regulatory subtype with reduced cytotoxic activity against fetal cells under the hormone influence. Kisspeptin-54 also regulates the maturation of thymic dendritic cells (DC). However, kisspeptin-54 effect on leukocytes functional activity has not been studied. Although it is known that these cells play an important role in ensuring fetus survival. The aim of the study was investigation of kisspeptin-54 effect in concentrations comparable to its level during physiological pregnancy on neutrophils and monocytes functional activity. Cells were isolated from peripheral blood of healthy non-pregnant women and cultured with kisspeptin-54 for 1 h. The monocytes and neutrophils phagocytic activity was determined by reducing bacterial bioluminescence. The production of reactive oxygen species (ROS) was evaluated in a luminol-dependent chemiluminescence test. The activity of myeloperoxidase (MPO) and indolamine-2,3-dioxygenase (IDO) was determined spectrophotometrically. It was revealed that kisspeptin-54 inhibits phagocytosis, MPO and ROS of neutrophils. On the contrary, the hormone activates phagocytosis, MPO activity, ROS production and IDO activity of monocytes. Thus, received data demonstrate that kisspeptin-54 is an important reproductive hormone that regulates leukocytes functional activity. The action of kisspeptin-54 depends on the type of cells expressing KISS-1 R.

Patients with Mechanical Jaundice: Neutrophils’ functional activity (Induced luminol-dependent chemiluminescence)

Patients with Mechanical Jaundice: Neutrophils’ functional activity (Induced luminol-dependent chemiluminescence)