Activity Of Blood Neutrophils Research Articles

#1116 Hyperuricemia worsens bacterial sepsis by suppressing neutrophil functions in CKD

Abstract Background and Aims Patients with kidney disease, especially those on dialysis, are more susceptible to infections such as peritonitis, sepsis, pneumonia, and are at a higher risk for infection-related mortality. Pathogenic mechanisms underlying the secondary immunodeficiency in kidney disease include gut dysbiosis and barrier dysfunction, persistent inflammation, immune paralysis due to increased levels of immunoregulatory metabolites (e.g. uric acid) and proteins (e.g. FGF23) [1]. Currently, it is unknown whether CKD-related hyperuricemia may contribute to the immune paralysis in this context. We hypothesized that hyperuricemia (HU) may aggravate the innate immune response to infection in CKD in mice. Method Mono- and polybacterial sepsis was induced by LPS injection or cecal ligation and puncture in Alb-creERT2;Glut9lox/lox mice with HU and/or CKD, and in Glut9lox/lox (healthy) control mice. After 24 hours, systemic inflammation was evaluated by measuring serum cytokine levels, immune cell activation and recruitment, and neutrophil extracellular trap release. Tissue injury in liver, heart, and kidney was assessed by performing RT-qPCR and immunohistochemistry. In addition, kidney function was determined by measuring the glomerular filtration rate (GFR). Additionally, mice were treated with urate-lowering therapy to reduce serum uric acid levels and the immune response and functional outcomes after sepsis is quantified. Results We found that both mono- and polybacterial sepsis caused systemic inflammation as indicated by an increased number of blood neutrophils (see Figure), neutrophil activation and pro-inflammatory cytokine levels as compared to the PBS-treated or sham-operated control groups in Glut9lox/lox healthy mice, respectively. Mice with sepsis also displayed a slightly worsened kidney function. Interestingly, HU significantly increased neutrophil numbers but decreased their activation status and the ability of neutrophils to form neutrophil extracellular traps in Alb-creERT2;Glut9lox/lox mice with sepsis as compared to the control groups, which was further aggravated in mice with CKD-related HU (CKD+HU). This impaired inflammatory response was partially reversible by lowering serum uric acid with febuxostat. Conclusion Our results identify hyperuricemia related or unrelated to kidney disease as immune regulator in bacterial infection by suppressing neutrophil functions in mice. Specifically targeting uric acid may help to overcome the secondary immunodeficiency related to kidney disease during infection but enhances sterile inflammation [2].

Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection

ObjectivesClostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers. MethodsWe stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology. ResultsCD66b+ neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10+ staining of neutrophils started to recover within 3–7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes. ConclusionsNeutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.

Влияние дидрогестерона на окситоцинореактивность нейтрофилов венозной крови беременных женщин и родильниц

The ability of steroid hormones to modulate the binding of oxytocin to oxytocin receptors is of great interest. The purpose of this article was to study the effect of oxytocin, dydrogesterone and their mixture on the radical activity of venous blood neutrophils in pregnant women during the 3rd trimester of a normal pregnancy and in women on the 1st day after term delivery. Materials and methods. Using the chemiluminescent method (BChL-07 biochemiluminometer) and cell stimulation with latex particles, we studied non-genomic and genomic effects of dydrogesterone (5∙10–5 g/l), oxytocin (10–7 IU/l) and their mixture on the radical activity of venous blood neutrophils in 20 pregnant women in the 3rd trimester of a normal pregnancy and 10 women on the 1st day after term labour. When the noise level was automatically subtracted, the following were recorded: maximum intensity of the synthesis of reactive oxygen species, time to reach the maximum of synthesis intensity, and area under the chemiluminogram curve, reflecting the total synthesis of radical particles over the course of 30 min. Results. Baseline radical activity of neutrophils in women on the 1st day after term delivery was found to be three times higher than in women during the 3rd trimester of a normal pregnancy. Judging by the area under the chemiluminogram curve and maximum intensity of the synthesis of reactive oxygen species, oxytocin had an equal stimulating effect on venous blood neutrophils both in pregnant women during the 3rd trimester and in puerperae. Dydrogesterone in each group of women equally stimulated the radical activity of neutrophils, both non-genomically and genomically. In both groups, under the action of dydrogesterone through membrane receptors, oxytocin reactivity of neutrophils remained unchanged, while through nuclear receptors, it decreased. Possible mechanisms of progesterone’s involvement in the regulation of oxytocin reactivity of the myometrium and venous blood neutrophils in women during pregnancy and postpartum are considered.

CORONAVIRUS SARS-COV-2 MODIFIES ANTITUMOR REDOX STATUS OF BLOOD AND INTERCELLULAR MATRIX IN METASTATIC COLORECTAL CANCER PATIENTS (A PILOT STUDY).

The current studies demonstrate that SARS-CoV-2 infection results in increasing complications incidence and the total risk of death in cancer patients. SARS-CoV-2 infection triggers oxidative stress representing one of the major factors of the inflammation contributing to the complicated course of the diseases including cancer. To assess the effect of hypoxia caused by SARS-CoV-2 infection on the redox status of blood in patients with metastatic colorectal cancer (mCRC). 10 patients with SARS-CoV-2, 11 mCRC patients with metachronous liver disease, and 14 mCRC patients with preceding SARS-CoV-2 infection were included in the study. The data on blood biochemistry (C-reactive protein, ferritin, transferrin, and free iron) were analyzed. The levels of superoxide radicals (ROS) in blood cells were determined by electron paramagnetic resonance (EPR) using the spin trap technique. The metalloproteinase activity was measured by polyacrylamide gel zymography with the addition of gelatin as a substrate. In mCRC patients with prior SARS-CoV-2 infection, a 1.26-fold increase in ROS-generating activity of blood neutrophils was observed compared to mCRC patients with no history of SARS-CoV-2 infection. The blood content of C-reactive protein, transferrin, and free iron in mCRC patients with prior SARS-CoV-2 infection increased by 2, 6, and 1.4 times, respectively. The total activity of gelatinases in platelets and neutrophils in the blood of mCRC patients with prior SARS-CoV-2 infection was 1.4 and 1.2 times higher compared to mCRC patients with no history of SARS-CoV-2 infection. mCRC patients with prior COVID-19 have a higher risk of exacerbation of inflammatory reactions. SARS-CoV-2 infection results in redox dіsbalance, which may contribute to the unfavorable course of the disease.

Features of the respiratory burst state of neutrophils and the activity of NAD(P)-dependent dehydrogenases in patients with widespread purulent peritonitis in the prognosis of the development of sepsis

Aim: Studying of the respiratory burst state and NAD(P)-dependent dehydrogenases activities in blood neutrophils features in the forecast of the development of abdominal sepsis in patients with widespread purulent peritonitis (WPP).
 Materials and methods. The study involved 50 patients with WPP in the preoperative period. Abdominal sepsis was developed by 35 patients (70.0%) from 5 to 10 days postoperative period, 15 patients (30.0%) hadn’t complications. The respiratory burst condition of blood neutrophils was examined using a chemiluminescent assay. Intracellular activity of the NAD- and NADP-dependent dehydrogenases was researched with using bioluminescent methods.
 Results. It was found that patients with WPP whose dynamics of the preoperative period will develop sepsis the chemiluminescent activity of blood neutrophils was characterized by a reduced level of spontaneous synthesis of the primary reactive oxygen species (ROS) and elevated levels of spontaneous synthesis of secondary ROS relative of the indicators identified in patients without subsequent complications. The feature of neutrophil metabolism in WPP patients without subsequent development of sepsis was high activity of the anaerobic lactate dehydrogenase reaction and decrease in activity of the NADP-dependent decarboxylating malate dehydrogenase. In patients with WPP and the subsequent development of sepsis was found high level of NAD-dependent substrates outflow citric acid cycle in the reaction of amino acid metabolism via glutamate dehydrogenase that may affect the activity of aerobic respiration in the neutrophils. Using correlation analysis was found that the intensity of the neutrophils respiratory burst in patients with no subsequent complications depends on the activity of anaerobic glycolysis.
 Conclusion. The established differences in the state of the respiratory burst and the activity of enzymes in neutrophils in patients with WPP, in depending on the subsequent development of the sepsis, determine the possibility of developing the method of forecasting complications and developing immunoactive therapy in the postoperative period of WPP.

Neuroendocrine-immune relationships at rats regardless of sex and exposure to stressors or adaptogens

Introduction and aim. Since the discovery of the famous Selye triad, studies of the connections between adaptation hormones and the immune system have remained relevant. We formulated the aim of the future study: in the mode of synchronicity, to reveal sexual differences in the parameters of the neuroendocrine-immune complex and the state of neuroendocrine-immune relationships in intact rats and exposed to stressors and adaptogens. This article initiates the beginning of the movement towards achieving the goal. Material and methods. The experiment is at 96 rats Wistar line: 48 males and 48 females. Over the 12 days, one male and female rat remained intact and 3 other pairs were exposed to chronic aversive stress for 6 days. We calculated the parameters of the HRV: Mode, Amplitude of the mode and Variational scope as markers of the circulating catecholamines, sympathetic and vagal tones respectively. Among endocrine parameters determined excretion of 17-Ketosteroides and serum levels of main adaptation hormones such as Corticosterone, Aldosterone, Testosterone, Triiodothyronine, as well as Parathyroid hormone and Calcitonin. The percentage of lymphocyte populations and the parameters of phagocytosis by neutrophils and monocytes of Staphylococcus aureus were determined in the blood. The Thymus and Spleen were weighed and made smears-imprints for counting Thymocytogram and Splenocytogram. Results. The canonical correlation between neuroendocrine and immune parameters was analyzed. The most pronounced immunomodulatory effect is exerted by catecholamines (R=0.928), sympathetic tone (R=0.849), and testosterone (R=0.829). Vagal tone (R=0.697), 17-ketosteroides (R=0.688), and aldosterone (R=0.686) moderately modulate immunity, while the immunomodulatory effects of triiodothyronine (R=0.610), corticosterone (R=0.584), PTH (R=0.510), and calcitonin (R=0.423) are the least pronounced. Three pairs of canonical roots were found. The neuroendocrine root of the first pair determines the parameters of immunity by 95.7%, the second pair by 86.5%, and the third pair by 69.2%. The elements of the Thymocytogram (in descending order: epitheliocytes, lymphocytes, endotheliocytes, lymphoblasts, and macrophages), the phagocytic activity of blood neutrophils and monocytes, the relative content of NK- and B-lymphocytes in the blood, as well as the percentage of lymphocytes and macrophages in the Splenocytogram turned out to be most influenced by neuroendocrine factors. Conclusion. There is a close canonical correlation between registered neuroendocrine factors and immunity parameters in general. At the same time, both the severity of the immunomodulatory activity of individual neuroendocrine factors and the subjection to the regulatory influence of individual parameters of immunity differ significantly.

Functional Activity of Blood Neutrophils in Patients with Stable Course and Exacerbation of Chronic Obstructive Pulmonary Disease.

We performed a comparative study of the parameters of chemiluminescence of blood neutrophils in patients with different severity of chronic obstructive pulmonary disease in its different periods. The maximum values of induced and spontaneous chemiluminescence were recorded at moderate severity of the disease during exacerbation. Low levels of chemiluminescence indicators were found in severe chronic obstructive pulmonary disease in the stable phase. The values of the induction period of the chemiluminescent response in patients with moderate chronic obstructive pulmonary disease were higher than in the control group. Correlations between the values of induced chemiluminescence of neutrophils and the respiration function parameter FEV1 were established, which may indicate the influence of multidirectional changes in the functional activity of systemic neutrophils on the development and worsening of airway obstruction in chronic obstructive pulmonary disease patients.

Oxidative Metabolism Genes in Ovarian Neoplasms

BACKGROUND: Reactive oxygen species play important and ambiguous role in carcinogenesis, and local oxidative metabolism may differ significantly from systemic metabolism and determine the processes occurring in tumor tissues.
 AIM: This study aimed to examine the expressions of key oxidative metabolism genes, particularly CYB5R, POR, NOX4, SOD1, NF-B, and NRF2, in ovarian neoplasm tissues, and determine cytochrome b5 reductase and cytochrome P450 reductase activity, blood neutrophil activity, and antioxidant indices in the blood plasma and peritoneal fluid.
 MATERIALS AND METHODS: The study included two groups of patients: a study group (n = 10) with ovarian adenocarcinoma and a comparison group (n = 6) with benign ovarian neoplasms. The expressions of CYB5R1, CYB5R2/R4, CYB5R3, POR, BIRC3, NOX4, NRF2, NF-B, SOD1, HMOX1, and BCL2 genes, cytochrome b5 reductase, and cytochrome P450 reductase activity, oxidative activity of blood neutrophils, and antioxidant potential of plasma and peritoneal fluid were evaluated in these two groups of women.
 RESULTS: The expression levels of CYB5R3 and BCL2 were significantly lower in adenocarcinoma tissues. The activities of cytochrome b5 reductase and cytochrome P450 reductase increased in the group with adenocarcinoma. On average, the activity of blood neutrophils corresponded to the reference values. For blood plasma, the antioxidant capacity were not different, whereas the antioxidant capacity in the peritoneal fluid increased approximately twofold in ovarian cancer.
 CONCLUSIONS: Significantly increased cytochrome b5 reductase activity in adenocarcinoma tissues may be a response to intracellular oxidative stress, whereas CYB5R3 gene expression may be reduced by a negative feedback mechanism. The activities of cytochrome P450 reductase and its gene change to a lesser extent in the presence of ovarian adenocarcinoma. The oxidative balance in the blood and peritoneal fluid correlated with the tissue expressions of NF-B and NRF2.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr-/- mice.

Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. We generated myeloid-specific Stat4ΔLysMLdlr-/-, neutrophil-specific Stat4ΔS100A8Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4ΔLysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed Stat4ΔLysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

Effect of supplementation of phyto-pharmaceutical product on the health andproductivity of crossbred cows during transition period

The present study was conducted to investigate the efficacy of supplementation of phyto-pharmaceutical producton the immune status of cows during transition period. Twelve crossbred cows in their late gestation were selected and randomly divided into two groups as control group (CON) and supplemented group (SG). The cows in the CON were fed as per the NRC standards while SG were supplemented additionally with 20g of poly-herbal formulation containing Boswellia serrata(Burseraceae) and 20g of Berginia ciliata (Saxifragaceae) for 21days prepartum to 7 days postpartum. Immune status viz., total leucocyte count (TLC), neutrophil percentage (%), lymphocytepercentage (%), neutrophil: lymphocyte ratio (N:L), in-vitrophagocytic activity (PA) of blood neutrophils, plasma cortisol level, interleukin-2 (IL-2) and interleukin-8 (IL-8) were estimated as well as effects were studied on milk for somatic cell count(SCC) and its constituents (fat, protein and lactose) too. Results showed that supplementation mixture significantly (P<0.05)reduced cortisol levels, IL-2, IL-8, TLC, neutrophil % and neutrophil: lymphocyte ratio whereas lymphocyte % increased.PA also increased significantly (P<0.05) on day of parturition. Milk SCC levels were higher (P<0.05) and milk lactose was lower(P<0.05) in the CON as compared to the SG. It was inferred that feeding of the phyto-pharmaceutical product effectively reduces stress and enhance immunity during transition period

МОДУЛЯЦИЯ ФАГОЦИТАРНОЙ АКТИВНОСТИ НЕЙТРОФИЛОВ ПРЕПАРАТАМИ ИЗ КАМЧАТСКИХ БУРЫХ ВОДОРОСЛЕЙ

In this paper, we present data on the influence of products produced from the kelp seaweeds, such as gel from Hedophyllum bongardianum and extracts from H. bongardianum, Alaria esculenta and Fucus distichus, on the modulation of phagocytic activity of human blood neutrophils. Our experiment involved 16 volunteers who took 15 g of algal gel daily for 30 days. Before and after the experiment, their blood was taken for general analysis and also to study the level of immunomodulation after priming exposure to different concentrations (0.5, 1, 2, 5%) of algal extracts. The nonspecific resistance of the experiment participants' body was assessed by the following indicators: phagocytic activity of neutrophils, phagocytic number, absolute phagocytic index, and sum of phagocytosis. Algal extracts diluted with saline to a concentration of 2% showed the highest priming effect on the blood of examined individuals taken both before and after the consumption of algal gel per os. Low concentrations (0.25 and 0.5%) did not have a stimulating effect. When algal extracts were diluted to 5% concentration, phagocytosis was noticeably inhibited. After 30 days of consuming the algal gel, the phagocytic activity of neutrophils in all participants increased by an average of 23%, phagocytic number by 10%, and absolute phagocytic index from 2.1 to 42%. The immunostimulating effect was greatest for Alaria extracts, and the lowest for Fucus extracts. The experimental results allowed us to recommend products from Kamchatka's kelp seaweeds as adaptogenic substances.

THE EFFECT OF ALLOGENEOUS TRANSFUSION OF ERYTHROCYTE MASS ON INDICATORS OF THE FUNCTIONAL ACTIVITY OF NEUTROPHIL GRANULOCYTES IN THE BODY OF RECIPIENT RABBITS

The relevance of the study is due to the spread among animals of pathologies in the therapy of which transfusion of blood components is used, which is used both for acute blood loss and for diseases of the blood and hematopoietic organs, if their deficiency cannot be compensated by other methods. In this regard, this article is aimed at evaluating the state of phagocytic activity of phagocytic cells (neutrophils and macrophages) according to indicators of phagocytic index, phagocytic number and oxygen-dependent phagocytosis during allogeneic transfusion of erythrocyte mass in the blood of recipient rabbits. The leading approach to the study of this problem was the determination of the absorption activity of phagocytes, the determination of the oxygen-dependent bactericidal activity of blood neutrophils, which was studied using the spontaneous test with nitroblue tetrazolium (NST-test). The material for research was blood serum samples obtained from 5 clinically healthy rabbits 3, 7 and 23 days after transfusion. It was found that the phagocytic index increases by 47.7% on the 3rd day, by 34.3% on the 7th day, and by 59.1% on the 23rd day, relative to the initial state. The indicator of the phagocytic number on the 3rd day of the experiment decreased by 22.5%, on the 7th day by 21.4%, on the 23rd day by 7.2%, relative to the initial state. It was found that the metabolic activity of neutrophil granulocytes in the peripheral blood of recipient animals in the spontaneous HST test, compared to the initial state on the 3rd and 23rd day of experimental studies with transfusion of erythrocyte mass, decreases by 4.8 times. The material of the article is of practical value for the application of transfusion of erythrocyte mass in the treatment of animals with anemia of various genesis.

Эффекты пептида GHК и его структурных аналогов D-Ala-GHK и GHК-D-Ala на состояние врожденного иммунитета и перекисного окисления липидов в условиях кожной раны

Objective: to study the effects of the GHK peptide and its structure analogues on the mechanisms of innate immunity and lipid peroxidation in wound. Materials and methods. The experiments were performed on 70 male Wistar rats. The skin wound was modeled by applying a full-layer wound with an area of 250 mm2 on the animal's back. The peptides Gly-His-Lys (GHK), D-Ala-Gly-His-Lys (D-Ala-GHK), and Gly-His-Lys-D-Ala (GHK-D-Ala) were used in doses of 0.5 μg/kg and 1.5 μg/kg, which it was administered intradermally in doses of 0.5 μg/kg and 1.5 μg/kg in a volume of 0.1 ml for 3, 7 or 10 days. The activity of lipid peroxidation (LPO) processes in blood serum was assessed by the content of malonic dialdehyde (MDA) and acylhydroperoxides (AHP). The antioxidant mechanisms were evaluated by determining the activity of catalase and the total antioxidant activity (OAA) of blood serum. Phagocytic activity of blood neutrophils was assessed by phagocytic index (PI) and phagocytic number (PN). The activity of oxygen-dependent mechanisms in phagocytes was evaluated in a spontaneous nitroblue tetrazolium test (NBT). Results. After the administration of GHK, the tendency to PI, PN and completeness of phagocytosis prevailed, which mainly persisted with the use of peptides D-Ala-GHK and GHK-D-Ala. At the same time, the GHK-D-Ala peptide at a dose of 1.5 μg/kg had the most stable effect on phagocytic activity. The data obtained in the NBT are largely consistent with the PN indicators. At the same time, the effects of structural analogues, unlike GHK, were manifested throughout the experiment. Significantly significant changes in the activity of LPO and antioxidant mechanisms were observed with the use of all peptides. However, their dynamics, orientation and severity throughout the experiment were quite complex. Conclusion. GHK and its structural analogues, D-Ala-GHK and GHK-D-Ala, had an effect on the indicators of innate immunity and LPO in a skin wound, the severity and direction of which depends on the healing period. At the same time, the most pronounced and stable effects were observed when using GHK-D-Ala. That demonstrates the importance of protecting the tripeptide molecule from the action of carboxypeptidases.

Effects of Dietary Blend of Algae Extract Supplementation on Growth, Biochemical, Haemato-Immunological Response, and Immune Gene Expression in Labeo rohita with Aeromonas hydrophila Post-Challenges

In this study, the effects of a mixed algal blend (Chlorella vulgaris, Euglena viridis, and Spirulina platensis) at different levels were evaluated on growth, hematological immune responses, and expression of immune genes in Labeo rohita against post-challenges of Aeromonas hydrophila. Fish samples were fed a diet containing different levels of mixed blend algal (0, 0.01, 0.02, 0.04 and 0.08% of basal diet). At the end of the feeding period, the fish were challenged with A. hydrophila and fish mortality was recorded over a 14-days period. To evaluate the serum biochemical (albumin, globulin), hematological parameters (Hb, RBC and WBC) and immune parameters (neutrophil activity, lysozyme activity, myeloperoxidase activity, antiprotease activity, ceruloplasmin activity, and bactericidal activity), as well as the expression of immune genes (NKEF-B, Lysozyme C and G, TNF α, TLR22, β2M, and β-actin), fish were sampled on Day 7, 14, 21 and 28. Fish were challenged with virulent A. hydrophila 30 days post-feeding and mortalities were recorded over 30 days post-infection. Results demonstrate that fish fed with a mixed algal blend showed that total body weight gain, specific growth rate, total serum protein, globulin, total hemoglobin content, white blood cells, neutrophil, lysozyme, bactericidal, myeloperoxidase, and antiprotease activity in dietary algae blended application was higher than in the control (p < 0.05). According to the results, relative expression of target genes showed significant increases of 0.02 to 0.04% in the treatment group compared to the control group (p < 0.05). At the end of the 30-day exposure to A. hydrophila, the fish that received the mixed algal blend had a significantly higher rate of survival than the control group, with the highest survival rate recorded in the 0.02% mixed algal blend (p < 0.05). According to the effective results of the mixed algal blend on stimulating the immune system and increasing fish resistance to A. hydrophila, it is recommended to use 0.02 to 0.04% of this mixed algal blend in rohu, L. rohita diets.

Effect of allogeneic blood transfusion on neutrophil functional activity and lymphocyte cytotoxicity in recipient rabbits

The relevance of this paper is that transfusion of allogeneic blood to recipient animals is always associated with immunological risks. In this regard, the purpose of this study was to assess the state of phagocytic activity of blood neutrophils by indicators of phagocytic index, phagocytic number, and oxygen-dependent bactericidal activity, as well as to establish changes in antibody-dependent cytotoxic activity of lymphocytes in recipient rabbits during allogeneic whole blood transfusion. Modelling of blood transfusions was performed on five clinically healthy rabbits by intravenous administration of whole blood at the rate of 5.5 ml/kg of body weight. Blood samples were taken from animals on Days 3, 7, and 23 after blood transfusion. Neutrophil populations were obtained from blood samples by centrifugation on a double density gradient of 1.077 and 1.093 Ficoll-Verografin. The absorption activity of phagocytes was determined in a microscopic test. To investigate the oxygen-dependent bactericidal activity of neutrophils, a spontaneous test with nitroblue tetrazolium was performed. Antibody-dependent cytotoxic activity of lymphocytes was investigated by colorimetric method. It was found that after the transfusion of whole blood, the phagocytic activity of neutrophils increases with a simultaneous decrease in their absorption capacity. On Days 3 and 7, the results of the spontaneous test with nitroblue tetrazolium decreased. This indicates inactivation of the oxygen-dependent bactericidal activity of neutrophil granulocytes during the first phase of post-transfusion immunological reactions. On Day 23, there was an increase in the values of the indicators of the spontaneous test with nitroblue tetrazolium, which indicates the activation of the bactericidal properties of phagocytes. It was found that on Day 3, the antibody-dependent cytotoxic activity of lymphocytes significantly decreased relative to the initial state, and on Days 7 and 23, it increased. An increase in the antibody-dependent cytotoxic activity of lymphocytes should be associated with the active synthesis of antibodies of the late phase of the immune response. Consequently, transfusion of allogeneic blood causes an immune response in recipient rabbits, without causing immediate and long-term transfusion reactions (changes in heart rate, respiratory rate, body temperature). The obtained results are of practical value both for scientists and practising doctors who use transfusion of whole blood and its components to animals with acute anaemia, impaired functional activity of blood coagulation factors, parasitic, and oncological diseases

Features of immunological reactivity in Sami women

BACKGROUND: The Sami are a small group of indigenous people of the Russian Federation. The Sami have one of the lowest birth rates among all the peoples of the North of Russia. They often have more health problems than residents of other nationalities in the Murmansk region, due to a high incidence of acute respiratory infections. Extreme climatic and geographical conditions of the Arctic have a negative impact on immunological reactivity.
 AIM: To identify the features of immunological reactivity in Sami women.
 MATERIALS AND METHODS: Forty-nine practically healthy Sami women aged from 21 to 44 years living in the Lovozero Village in the Murmansk region were examined. A control group consisting of 50 practically healthy Russian women of the Arkhangelsk region of the same age range was used. The hemogram, phagocytic activity of neutrophils of peripheral venous blood, and the content of lymphocytes with CD3, CD4, CD8, and CD10 phenotypes were studied by indirect immunoperoxidase reaction (Sorbent, Moscow) and flow cytometry (Beckman Coulter Immunotech, France). Additionally, concentrations of IgM, IgG, IgA, sCD54, and sCD62L (Bender MedSystems, Austria) were determined by enzyme immunoassay, circulating immune complexes (CIC) were determined by the precipitation method. The total amount of microflora, cytogram, phagocytic activity of neutrophils, the sorption capacity of epithelial cells, the content of sIgA and CIC IgG were studied in the separated mucous membranes of the pharynx, intestines, and urine. For comparison between groups, the independent samples t-test or the nonparametric MannWhitney U-test was used depending on the distribution.
 RESULTS: In Sami women, the frequency of deficiency of phagocytic activity of blood neutrophils and mucosal secretions was higher than in the control group. Increased reactions of cell-mediated and antibody-dependent cytotoxicity of lymphocytes were recorded. The frequency of elevated concentrations of CIC was higher in the Sami women than in the control group. A remarkable feature of the Sami immune reactivity was high concentrations of IgA and IgM against a background of low IgG content.
 CONCLUSION: In Sami women, the percentage of actively phagocytic neutrophil granulocytes of peripheral venous blood and mucous membranes was lower than in the control. A distinctive feature of the immunoglobulin profile of the blood serum of Sami women was the low IgG content against the background of increased concentrations of CIC, including immunoglobulins of this class. Sami women had higher levels of leukocyte migration into mucosa-associated mucosal tissue, which was correlated with a decrease in the sorption capacity of epitheliocytes, phagocytic activity of neutrophils, and concentrations of secretory immunoglobulins.

Влияние агониста бета3-адренорецепторов мирабегрона на активность нейтрофилов венозной крови небеременных женщин

The aim of this paper was to determine the effect of the beta3-adrenergic receptor agonist mirabegron on the free-radical activity of venous blood neutrophils in non-pregnant women. Materials and methods. Using the chemiluminescent method (BChL-07 biochemiluminometer), the parameters of the radical response of venous blood neutrophils in 40 non-pregnant women were determined in different phases of the cycle and on the first day after childbirth with additional cell stimulation with latex particles (0.08 μm). When the noise level was automatically subtracted, the following were recorded: maximum intensity of the synthesis of reactive oxygen species, time to reach the maximum of synthesis intensity, and area under the chemiluminogram curve, reflecting the total synthesis of radical particles over the course of 30 min. These parameters were determined before and after an in vitro introduction of the beta -adrenergic receptor agonist mirabegron (10–6 g/l), beta-adrenergic receptor blocker propranolol hydrochloride (10–6 g/l) and agonist against the background of the blocker into the venous blood. Results. Using the Wilcoxon signed-rank test, we showed an increase in the free-radical activity of neutrophils in women in the follicular phase of the menstrual cycle under the influence of mirabegron for all three evaluated parameters. In the luteal phase, mirabegron significantly increased the rate of reaction development and the total synthesis of radical particles, but did not affect the intensity of production of activated oxygen species. However, no effect was observed on the first day after delivery. The possible mechanism of the revealed mirabegron action is discussed in the article. The authors believe that changes in the activity of venous blood neutrophils under the action of mirabegron may be due to changes in the quantity or sensitivity of beta3-adrenergic receptors in different phases of reproduction.

Phagocytic and chemiluminescent activity of blood neutrophils in patients with bladder cancer

BACKGROUND: Tumor microenvironment modulates (including with the help of metabolites) the functional activity of the neutrophils that contribute to the reprogramming of the antitumor activity into a protumor one.
 AIMS: To study the phagocytic and chemiluminescent activity of neutrophils in patients with bladder cancer (BC) under the influence of metabolites of the tumor microenvironment in vitro.
 MATERIALS AND METHODS: We examined 37 patients with superficial BC (T1,а,isN0M0) and considered 32 healthy individuals as a control group. Neutrophils isolated from their blood were incubated in vitro with lactate, ADP, and glutamate. Phagocytic activity was examined using flow cytometry, and the intensity of the respiratory burst of neutrophils was evaluated via chemiluminescent analysis.
 RESULTS: In patients with BC, the phagocytic index (PhI) values are reduced compared to the control sample (without in vitro metabolite exposure) and when exposed to glutamate, while the effect of lactate on cells causes an increase in the phagocytic number and PhI. Moreover, under the influence of lactate in vitro, the activity of spontaneous and zymosan-induced chemiluminescence of neutrophils decreases. ADP causes a decrease in spontaneous chemiluminescence only. Finally, under the influence of glutamate, the indicators of spontaneous and induced chemiluminescence decrease.
 CONCLUSIONS: Under the influence of lactate and ADP (products of tumor cells), the phagocytic activity of a population of immature neutrophils is stimulated, which leads to myeloid suppressor cells that inhibit antitumor immunity. Thus, metabolites of the tumor microenvironment modulate the activity of the respiratory burst of neutrophils in patients with BC.

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.

Dynamics of Psychopathological Disorders and Changes in the Functional Activity of Neutrophils in Mercury Intoxication

IntroductionThe overall goal was to determine the degree of mental, neuromuscular and statodynamic functions disorders resulting from toxic effect of mercury and its compounds, to study relationship between the degree of loss of professional ability to work as a result of occupational disease with the presence of complications due to concomitant diseases.ObjectivesThe relevance of studying the degree of violations of the main indicators of vital activity due to the toxic effect of mercury and its compounds is due to the persisting high level of loss of professional ability to work, and in some cases, and disability of this contingent. Analysis of indicators of primary and repeated disability due to occupational diseases showed that a sufficiently high number of persons recognized as disabledюMethodsWe studied the dynamics of psychopathological disorders: violation of the emotional sphere, thinking, perception, attention, volitional activity and cognitive functions. The functional and metabolic activity of segmented neutrophils in the peripheral blood of 42 patients, men and women aged 43 to 64 years, and 22 healthy donors were studied.ResultsLong-term exposure to chronic industrial mercury intoxication led to persistent disorders of mental functions, suppression of phagocytic activity of neutrophils up to 42.3+-4.7 per cent and inhibition of the reaction with nitro blue cytosol 4.2+-0.1 per cent..ConclusionsThe revealed violations of the emotional-volitional sphere and cognitive mental functions are possibly associated with the suppression of nonspecific cellular reactions of microphages. Violations of neuroimmune interactions in mercury intoxication require further study.DisclosureNo significant relationships.