Blood Neutrophils Of Patients Research Articles

Inhibition of CEACAM1 expression in cytokine-activated neutrophils using JAK inhibitors

ObjectivesCarcinoembryonic-antigen-related cell-adhesion molecule 1 (CEACAM1) is an adhesion molecule that acts as a coinhibitory receptor in the immune system. We previously demonstrated that CEACAM1 is predominantly expressed on peripheral blood neutrophils in patients with RA. The aim of the present study was to investigate the effects of Janus kinase inhibitors (JAKi) on cytokine-activated human neutrophils and CEACAM1 expression.MethodsPeripheral blood neutrophils were obtained from healthy subjects. Isolated neutrophils were stimulated with tumor necrosis factor-alpha (TNF-α) or granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence or absence of JAKi. The expression of CEACAM1 in peripheral blood neutrophils was analyzed by flow cytometry. Protein phosphorylation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 was assessed by western blot using phospho-specific antibodies.ResultsWe found that TNF-α-induced CEACAM1 expression was marginally suppressed after pretreatment with pan-JAK inhibitor, tofacitinib. Moreover, TNF-α induced STAT1 and STAT3 phosphorylation at the late stimulation phase (4 to 16 h). The expressions of CEACAM1 on neutrophils were markedly up-regulated by GM-CSF not by interleukin (IL)-6 stimulation. All JAKi inhibited GM-CSF-induced CEACAM1 expressions on neutrophils, however, the inhibitory effects of baricitinib were larger compared to those of tofacitinib or filgotinib. Moreover, CEACAM1 was marginally upregulated in interferon (IFN)-γ stimulated neutrophils. Similarly, JAKi inhibited IFN-γ-induced CEACAM1 expressions on neutrophils.ConclusionsWe demonstrated that JAKi prevent GM-CSF-induced CEACAM1 expression in neutrophils, and JAKi-induced inhibition depends on their selectivity against JAK isoforms. These findings suggest that JAKi can modulate the expression of CEACAM1 in cytokine-activated neutrophils, thereby limiting their activation.

Study of the effect of a drug from the macromycetes Ganoderma lucidum on the phagocytic activity of peripheral blood neutrophils in patients with year-round allergic rhinitis

Modern complex therapy of patients with persistent (year-round, chronic) allergic rhinitis (AR), based on the use of antihistamines and intranasal glucocorticosteroids, in many cases is not effective enough, since drug therapy acts only on individual links in the pathogenesis, without preventing the progression of the allergic disease. At the same time, the quantitative and reserve capabilities of neutrophil granulocytes are depleted, which increases the level of antigenic load in patients with AR. The aim of this study is to investigate the phagocytic activity of neutrophils in patients with AR who received basic therapy and patients who received basic therapy and a course of the drug from the macromycetes Ganoderma lucidum "Astmagan". We examined 40 patients of both sexes with AR, the duration of the disease ranged from 6 months to 2 years. Patients were divided into 2 equal groups: The 1st group consisted of patients who received basic therapy (oral Loratadine and intranasal Nasonex). The 2nd group consisted of patients who received basic therapy and a course of "Astmagan". The treatment course lasted 90 days. The control group consisted of 25 healthy individuals. After the treatment, patients in both groups showed a statistically significant positive dynamics of phagocytic system activity: in group 1, the phagocytic index (FI) increased by 30 % (p=0.001), in the second group this indicator reached the normal level. Also, in patients of group 1, the phagocytic number (PN) increased by 35 % (p=0.0001), but did not reach the normal level.
 According to the results of comparative evaluation, a greater efficacy of the treatment complex with the additional use of "Astmagan" was established, which is manifested in the restoration of phagocytic activity of peripheral blood neutrophils, the effect persists for 6 months after treatment.

Indicators of free radical oxidation in neutrophils of patients with primary and recurrent soft tissue sarcomas

Purpose of the study. To carry out a comparative analysis of lipid peroxidation intensity and antioxidant system indices in blood neutrophils of patients with primary and relapsed soft tissue sarcomas (STS) depending on sex and age.Patients and methods. Of the 81 patients included in the study, 48 had primary STS, 5 patients with continued growth, and 28 patients with recurrent STS. The patients were divided by sex and age; the level of lipid peroxidation products, superoxide dismutase (SOD) activity, total peroxidase activity (TPA), glutathione peroxidase (GPx) activity and reduced glutathione (RG) content in blood neutrophils were investigated by conventional spectrophotometric methods. The comparison groups (donors) consisted of 12 men and 17 women divided into the same age subgroups: ≤ 45 years and > 45 years.Results. In donors, there was a multiple decrease in the SOD/TPA ratio with age, especially in women, and in men this was accompanied by an age-related decrease in GPx activity. In primary sarcomas in older women and in men with continued growth and recurrence, there was an increase in diene conjugates (DC). In men over 45 years of age, there was an increase in SOD, TPA, and GPx. Women of the older age group were characterized by activation of SOD and GPx, expressed in the relapsed process to a significantly greater extent than in men, and an increase in RG was observed in women. The increase in both components of the glutathione system and SOD activity was especially significant in the development of relapses in women for periods exceeding three years, which was accompanied by a decrease in DC content. Conclusions. Neutrophils are characterized by an increase in DC content in patients of both sexes in the older age group with primary and recurrent STS, with its highest content in men with continued growth and relapses. GPx activity, increased in both men and women in the older age group in all variants of STS development, may play an important role in the antioxidant protection of blood cells in STS. Thus, the most pronounced activation of GPx, accompanied by a maximum increase in reduced glutathione and activation of SOD, contributes to a decrease in the level of DC and the absence of MDA increase in women with slow development of recurrences.

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.

Inflammatory and glycolytic programs underpin a primed blood neutrophil state in patients with pneumonia

Neutrophils are potent immune cells with key antimicrobial functions. Previous invitro work has shown that neutrophil effector functions are mainly fueled by intracellular glycolysis. Little is known about the state of neutrophils still in the circulation in patients during infection. Here, we combined flow cytometry, stimulation assays, transcriptomics, and metabolomics to investigate the link between inflammatory and metabolic pathways in blood neutrophils of patients with community-acquired pneumonia. Patients' neutrophils, relative to neutrophils from age- and sex- matched controls, showed increased degranulation upon exvivo stimulation, and portrayed distinct upregulation of inflammatory transcriptional programs. This neutrophil phenotype was accompanied by a high-energy state with increased intracellular ATP content, and transcriptomic and metabolic upregulation of glycolysis and glycogenolysis. One month after hospital admission, these metabolic and transcriptomic changes were largely normalized. These data elucidate the molecular programs that underpin a balanced, yet primed state of blood neutrophils during pneumonia.

Angiogenic potential of circulating peripheral blood neutrophils in kidney cancer

The role of neutrophils in kidney cancer is currently being studied. Their role in carcinogenesis is ambiguous. As one of the most abundant blood leukocytes, neutrophils play an important role in cancer progression through multiple mechanisms, including promotion of angiogenesis, immunosuppression, and cancer metastasis. Neutrophils synthesize and release pro-angiogenic factors that are able to directly or indirectly stimulate the growth and migration of endothelial cells, which in turn causes the formation of new blood vessels from pre-existing ones. The production of various factors by neutrophils, including proangiogenic ones, is mediated by the expression of the genes of these molecules. Functional heterogeneity is characterized by differences in neutrophil gene expression patterns. The aim of this study was to evaluate the angiogenic potential of circulating neutrophils in kidney cancer. The object of the study were blood neutrophils of patients with verified clear cell kidney cancer at stage I (T1N0M0G1, n = 28, median age 60), stage II (T2N0M0G2, n = 15, median age 61) and stage III (T3N0M0G2, n = 15, median age 63) before surgery. The control group consisted of apparently healthy donors (n = 15, median age 54). Serum levels of IL-8 and VEGF-A were assessed by enzyme immunoassay. Expression of the CXCL8 and VEGF-A genes in circulating neutrophils was determined by reverse transcription quantitative PCR. As a result of our study, an increase in the level of IL-8 and VEGF-A in the blood serum of patients with kidney cancer in all studied groups compared with the control group was revealed. We observed a direct correlation between serum levels of IL-8 and VEGF-A in patients with kidney cancer (r = 0.429; p = 0.016), which confirms the relationship of these angiogenic factors. A significant increase in CXCL8 gene expression by circulating neutrophils was found in patients on II (2.91, Q0.25-Q0.75: (1.296-4.99), p = 0.02) and III (1.93, Q0.25-Q0.75: (0.755-11.36, p = 0.014) stages of kidney cancer compared with the control group (1.50, Q0.25-Q0.75: (0.80-4.05)). However, VEGF-A gene expression by circulating neutrophils did not differ from those in the control group. Blood neutrophils in kidney cancer exercise their angiogenic potential through the production of IL-8.

Effects of remote ischemic postconditioning on the pro-inflammatory neutrophils of peripheral blood in acute cerebral infarction.

Neutrophils play crucial roles in the inflammatory response after acute cerebral infarction (ACI). Previous studies revealed neutrophils are non-homogeneous and can be divided into at least two subtypes, pro-inflammatory and anti-inflammatory, correlated with patients' prognosis. We aimed to explore the correlation between disease severity and peripheral blood neutrophils in patients with ACI and determine whether remote ischemic postconditioning (RIPostC) exerts neuroprotective effects by regulating neutrophils. Patients (n = 38) with acute anterior circulation cerebral infarction were assigned to conventional treatment (n = 24; included aspirin, statins, neuro nutrition drugs, and circulation improvement drugs) or RIPostC (n = 14; 7-day ischemia adaptation [complete ischemia of both upper extremities for 5 minutes followed by remission for 5 minutes, 5 repeated cycles, twice a day, started from the morning of the second day of admission] based on conventional treatment) groups, based on their preference. General clinical data and peripheral blood samples were taken three times, in the morning before and 3 and 7 days after treatment. Fifteen adults with non-acute cerebral infarction matched for sex, age, and risk factors were recruited as controls; peripheral blood samples were only collected on the recruitment day. We used flow cytometry to detect the percentage of neutrophils and Real-Time PCR to detect the gene expression of interleukin (IL)-1β in the peripheral blood samples. The percentage of neutrophils, pro-inflammatory neutrophils (IL-1β high expression in flow cytometry), and IL-1β mRNA expression increased after ACI (P = 0.01, P = 0.001, P < 0.001). The National Institutes of Health Stroke Scale (NIHSS) score of patients with ACI within one day of onset was positively correlated with the percentage of pro-inflammatory neutrophils (R = 0.618, P = 0.043). Pro-inflammatory neutrophils in the RIPostC group decreased compared with those in the conventional treatment group, with the most significant difference observed on Day 7 (P = 0.01). However, the percentage of neutrophils was not statistically different. IL-1β mRNA expression decreased, with the most significant difference on Day 3 (P = 0.004). The NIHSS and Modified Rankin Scale scores for RIPostC decreased more significantly than for conventional treatment (P = 0.002, P = 0.019). More severe cerebral infarction was associated with a higher percentage of pro-inflammatory neutrophils. The neuroprotective effect of RIPostC may partly be exerted through gene regulation to reduce pro-inflammatory neutrophils.

Oxidative stress and the level of NAD(P)-dependent dehydrogenases of blood neutrophils in patients with compensated and relapse Graves' hyperthyroidism ongoing thiamazole treatment

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

CD177 Inhibits Neutrophil Extracellular Trap Formation and Protects against Acute Pancreatitis in Mice

The inflammatory immune response mediated by neutrophils is closely related to the progression of acute pancreatitis. Previous studies confirmed that CD177 is a neutrophil-specific marker involved in the pathogenesis of conditions such as systemic vasculitis, asthma, and polycythemia vera. Neutrophil extracellular trap (NET) formation is a specific death program by which neutrophils release nuclear DNA covered with histones, granule proteins, etc. It also plays an important role in host defense and various pathological reactions. However, the function of CD177 in regulating the generation of NETs and the development of acute pancreatitis (AP) is unclear. In our manuscript, CD177 was significantly elevated in blood neutrophils in patients and positively correlated with the AP disease severity. Then, recombinant human CD177 protein (rhCD177) could significantly improve pancreatic injury and the inflammatory response in AP mice, and reduce AP-related lung injury. Mechanistically, we found that rhCD177 could inhibit the formation of NETs by reducing reactive oxygen species (ROS) and myeloperoxidase (MPO)/citrullinated histone H3 (CitH3) release. For the first time, we discovered the potential of rhCD177 to protect AP in mice and inhibit the NET formation of AP. CD177 may be a potential treatment strategy for preventing or inhibiting the aggravation of AP.

Increased CEACAM1 expression on peripheral blood neutrophils in patients with rheumatoid arthritis.

Altered expression of adhesion molecules in immune cells has been demonstrated in rheumatoid arthritis (RA). Carcinoembryonic-antigen-related cell-adhesion molecule 1 (CEACAM1) is an adhesion molecule that acts as a coinhibitory receptor in the immune system. We investigated the role of CEACAM1 in immune cell subsets of patients with RA. Peripheral blood was obtained from 37 patients with RA and 20 healthy controls (HC). The expression of CEACAM1 and T-cell immunoglobulin mucin domain molecule (TIM) -3 on peripheral blood mononuclear cells and neutrophils was analyzed by flow cytometry. Intracellular TIM-3 expression was analyzed using cellular lysates by Western blot analysis. Serum levels of soluble CEACAM1 (sCEACAM1) were estimated by an enzyme-linked immunosorbent assay. CEACAM1 expression was not detected in peripheral blood mononuclear cells, including in CD14(+) monocytes and CD3(+) lymphocytes isolated from patients with RA or HC. However, substantial cell-surface expression of CEACAM1 was detected in peripheral blood neutrophils, and it was significantly elevated in samples from patients with RA without remission compared to those in remission. There was no significant difference in serum levels of sCEACAM1 between patients with RA and HC. Cell-surface expression of TIM-3 was not detected in peripheral blood neutrophils from patients with RA or HC but was seen in CD14(+) monocytes. However, there was no significant difference in TIM-3 expression on monocytes between patients with RA and HC. Our data indicate that cell-surface expression of CEACAM1 on peripheral blood neutrophils are higher in patients with RA and that it is associated with rheumatoid inflammation. Further studies are needed to explore the potential role of CEACAM1 in rheumatoid inflammatory pathways.

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.&#x0D; 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.&#x0D; 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.&#x0D; 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.&#x0D; 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.

Neutrophil extracellular traps contribute to myofibroblast differentiation and scar hyperplasia through the Toll-like receptor 9/nuclear factor Kappa-B/interleukin-6 pathway.

Inflammation is an important factor in pathological scarring. The role of neutrophils, one of the most important inflammatory cells, in scar hyperplasia remains unclear. The purpose of this article is to study the correlation between neutrophil extracellular traps (NETs) and scar hyperplasia and identify a new target for inhibiting scar hyperplasia. Neutrophils were isolated from human peripheral blood by magnetic-bead sorting. NETs in plasma and scars were detected by enzyme-linked immunosorbent assays (ELISAs), immunofluorescence and flow cytometry. Immunohistochemistry was used to assess neutrophil (CD66B) infiltration in hypertrophic scars. To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry. We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs (p < 0.05). Hypertrophic scars showed greater infiltration with neutrophils and NETs (p < 0.05). NETs activate fibroblasts in vitro to promote their differentiation and migration. Inhibition of NETs with cytochalasin in wounds reduced the hyperplasia of scars in mice. We induced neutrophils to generate NETs with different stimuli in vitro and detected the proteins carried by NETs. We did not find an increase in the expression of common scarring factors [interleukin (IL)-17 and transforming growth factor-β (TGF-β), p > 0.05]. However, inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts into myofibroblasts. In vitro, NETs were found to be mediated by Toll-like receptor 9 (TLR-9) in fibroblasts and further phosphorylated nuclear factor Kappa-B (NF-κB). We found that IL-6, which is downstream of NF-κB, was increased in fibroblasts. Additionally, IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion. Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway, thereby providing a new target for regulating hypertrophic scars.

Activation and Functional Priming of Blood Neutrophils in Non-Alcoholic Fatty Liver Disease Increases in Non-Alcoholic Steatohepatitis.

IntroductionIn non-alcoholic fatty liver disease (NAFLD), neutrophils in liver infiltrates are activated, which may contribute to disease progression towards non-alcoholic steatohepatitis (NASH). However, the functional status of the blood neutrophils remains unknown and their role in the disease mechanisms is thus uncertain. We therefore characterized activation and function of blood neutrophils in patients with NAFLD in relation to clinical disease markers and the NAFLD plasma milieu.MethodsWe studied 20 patients with NAFLD, among these 6 patients with NASH, and 14 healthy persons. Neutrophil activation, interleukin (IL)-8 production and oxidative burst were measured by flow cytometry on participants´ neutrophils and on healthy neutrophils exposed in vitro to plasma from the study participants.ResultsBlood neutrophils from the NASH patients showed a doubling in their expression of the activation marker CD62L. Also, all NAFLD patients had 50–100% increased expression of CD11b. Functionally, NASH neutrophils had 30% elevated IL-8 production and more than doubled spontaneous oxidative burst. In all NAFLD patients, higher spontaneous oxidative burst was associated with worse liver function. Incubation of healthy neutrophils with NAFLD plasma paradoxically slightly reduced CD62L and CD11b expression, and NASH plasma also reduced the frequency of IL-8-producing neutrophils.ConclusionIn NAFLD, blood neutrophils are activated, and in NASH also functionally primed. This suggests a progressive neutrophil aggressiveness already present with liver fat infiltration. However, NAFLD plasma in vitro, if anything, had the opposite effect on the healthy neutrophils so the NAFLD-related neutrophil activation cannot be attributed to humoral factors and remains unexplained.

THE INFLUENCE OF SYNBIOTICS ON PERIPHERAL BLOOD LEUKOCYTE OXIDATIVE BURST IN PATIENTS WITH ALLERGIC DISEASES

Modulation of the gut microbiota with probiotics, living microorganisms with immunomodulatory effects, or prebiotics, nondigestible food ingredients that promote the growth and/or activity of beneficial microorganisms or a combination of both (synbiotics) may enable a new way of prevention or treatment for allergic disease. The aim of our study was to evaluate the ROS release from the peripheral blood neutrophils in patients with type I hypersensitivity reactions. To this end, we evaluated the basophil degranulation in the presence of specific allergens and analysed the influence of a synbiotic on neutrophil reactive oxygen species (ROS) generation. The tested synbiotic, directly or in combination with E. coli, decreased the ROS production in peripheral blood neutrophils of allergic patients. Taking into account that intensive ROS release is responsible for various damages of cells and tissues, the decrease of the oxidative stress induced by the tested synbiotic may have an essential role for cell protection and viability. Therefore, therapeutic interventions that enhance endogenous antioxidant defenses might be beneficial as adjunctive therapies for allergic disorders.

Quantitative Characteristics of Neutrophil Extracellular Traps in Tumor Tissues in Colorectal Cancer

BACKGROUND: Recently, the role of neutrophil extracellular traps (NETs) in oncopathology, including colorectal cancer (CRC), has become a subject of discussion. Literature review showed that in CRC, there are separate experimental and clinical studies devoted to assess the ability of neutrophils to form NET. A methodological approach, common to these studies, was that the ability of blood neutrophils in patients with CRC to form NET was evaluated in vitro.&#x0D; AIM: We undertook a study of the NETs presence in the tumor tissues, on the periphery of the tumor, and in healthy tissues with CRC at different stages and different types of therapy.&#x0D; METHODS: The study was carried out in the framework of the scientific and technical program “Personalized approach to the management of number of significant diseases” developed by the Non-commercial Joint-Stock Company “Karaganda Medical University,” funded by the Ministry of Education and Science of the Republic of Kazakhstan.&#x0D; RESULTS: Patients with CRC II and III stages are more likely to have NET in the center, on the periphery of the tumor and in healthy tissues adjacent to the tumor than in patients with CRC of Stage I. It was also found that radiation therapy as a pre-operative preparation contributed to an increase in the number of NET in the center of the tumor in patients with Stage I rectal cancer.&#x0D; CONCLUSION: Thus, our data revealed an increase in NET in the center and periphery of CRC, and an increase in their number after pre-operative radiation therapy was shown. The next stage of our study will be devoted to study the structural features of NET and their interaction with other effector cells and tumor cells.

The results of the use of natural factors of the Siberian region for the rehabilitation of workers of dust-hazardous professions

The inclusion of local natural factor - sodium chloride bromine-containing mineral water in the schemes of recovery in dust pathology of the lungs, has a sanogenetic effect on the factors of local protection of the respiratory tract, leads to the normalization of the biocidal potential of blood neutrophils in patients with occupational chronic obstructive pulmonary disease and pneumoconiosis.

Metabolic support of the respiratory burst in blood and sputum neutrophils of patients with community-acquired pneumonia

The aim of the study was to investigate relationships between the respiratory burst in blood and sputum neutrophils and intracellular enzyme activity in patients with pneumonia. Methods. The study involved 82 patients with moderate to severe community-acquired pneumonia (CAP). The chemiluminescent assay was used to investigate synthesis of primary and secondary reactive oxygen species (ROS). The bioluminescent method was used to examine NAD(P)-dependent dehydrogenase activity in neutrophils. Results. Low-intensity respiratory burst in blood neutrophils and its predominant relations to the state of mitochondrial metabolism and malic enzyme activity were observed in healthy subjects. Blood neutrophils in patients with CAP were activated; the anaerobic respiration and mitochondrial metabolism increased in the cells. Additionally, intensity of the respiratory burst was related to terminal glycolysis reactions and the key reaction of the pentose phosphate cycle. Sputum neutrophils in patients with CAP were also activated. Conclusion. Abnormalities of metabolic support of the respiratory burst, such as decreased production of primary and secondary ROS, could appear under unfavorable conditions. Peroxidation increased in the cells, the intensity of terminal glycolysis reactions decreased, and substrates transferred from the citric acid cycle to amino acid exchange reactions. Under these conditions, malic enzyme remained the only substrate that stimulated the respiratory burst in sputum neutrophils, while substrate transfer from the tricarboxylic acid cycle inhibited synthesis of the secondary ROS.

Экспрессия рецепторов нейтрофилов периферической крови при обострении атопического дерматита у взрослых мужчин

Нейтрофилы периферической крови играют важную роль в патогенезе атопического дерматита. При обострении атопического дерматита изменяется экспрессия рецепторов на поверхности нейтрофилов и сопряженные с ними сигнальные пути. Цель исследования - изучение экспрессии рецепторов семейства TREM (Triggering Receptor Expressed on Myeloid cells) на поверхности нейтрофилов и моноцитов периферической крови взрослых людей в начале периода обострения заболеания. Методика. В исследовании приняли участие 70 больных атопическим дерматитом и 22 здоровых донора. Кровь брали из локтевой вены натощак в вакутейенеры с ЭДТА. После удаления эритроцитов лизирующим буфером (Becton Dickinson) лейкоциты окрашивали моноклональными антителами к специфическим рецепторам нейтрофилов, лимфоцитов и моноцитов (BD-bioscience, Germany), а также к рецепторам TREM-1 и sTREM-1 (R&amp;D System, Minneapolis, MN, USA), и TREM-2 (Abcam, UK). Флуоресценцию клеток анализировали на проточном цитометре FACSCalibur по программе CellQuest. При анализе флуоресценции нефракционированных клеток сначала устанавливали гейты основных популяций на каналах FSC/SSC. Затем выделяли гейт нейтрофилов и в нем анализировали экспрессию рецепторов на каналах FL-1A/FL-2A (CD16/TREM-1 и CD16/TREM-2). Определяли процент антиген-положительных клеток и интенсивность флуоресценции в условных единицах. Экспрессию рецепторов TREM-1 и TREM-2 на моноцитах измеряли аналогично в гейте моноцитов. Концентрацию растворимого рецептора sTREM-1 в сыворотке крови определяли иммуноферментным методом с помощью антител (R&amp;D System, Minneapolis). Результаты. Показано повышение экспрессии рецепторов TREM-1 в ранний период обострения атопического дерматита, положительно коррелирующее со степенью активности процесса. Установлено статистически значимое повышение экспрессии рецепторов TREM-2 на нейтрофилах периферической крови больных на ранней стадии обострения по сравнению с донорами и пациентами в стадии ремиссии. Показано, что повышение экспрессии растворимого рецептора sTREM-1 в клетках нейтрофилов, а также повышение его концентрации в сыворотке крови положительно коррелирует с тяжестью течения атопического дерматита по индексу SCORAD. Экспрессия рецепторов TREM-1 на нейтрофилах повышается как при стимуляции клеток бактериальными пептидами, так и при контаминации кожи бактериями, но в любом случае TREM-1 поддерживает воспаление и коррелирует с тяжестью течения атопического дерматита. Заключение. Комплексный анализ экспрессии рецепторов семейства TREM позволяет оценить степень активности воспалительного процесса при атопическом дерматите. Peripheral blood neutrophils play an important role in the pathogenesis of atopic dermatitis (AD). AD exacerbation induces changes in the neutrophil surface receptor expression and coupled signaling pathways. Aim of this work was to evaluate TREM (triggering receptor expressed on myeloid cells) receptor expression on neutrophils and monocytes in peripheral blood of adult patients with AD at the beginning of exacerbation period. Methods: This study included 70 patients with AD and 22 healthy donors. Blood was withdrawn from the cubital vein in fasting state and collected in vacutainers with EDTA. Following removal of red blood cells with a lysing buffer (Becton Dickinson) leukocytes were stained with monoclonal antibodies to specific receptors of neutrophils, lymphocytes, and monocytes (BD Bioscience, Germany) and to TREM-1, sTREM-1 (R&amp;D System, Minneapolis, MN, USA), and TREM-2 (Abcam, UK) receptors. Cell fluorescence was analyzed using a FACSCalibur flow cytometer with CellQuest software. In analyzing fluorescence of unfractionated cells, gates of major populations were set on FSC/SSC channels. The neutrophil gate was isolated, and receptor expression was analyzed on FL-1A/FL-2A channels (CD16/TREM-1 and CD16/TREM-2). Proportion of antigen-positive cells was determined; fluorescence intensity was measured in conventional units. TREM-1 and TREM-2 receptor expression on monocytes was measured by the same method in the monocyte gate. Serum concentration of soluble sTREM-1 receptor was measured by enzyme immunoassay with antibodies (R&amp;D System, Minneapolis). Results. Elevated TREM-1 receptor expression was observed in the early period of AD exacerbation and positively correlated with AD severity. The expression of TREM-2 receptors on peripheral blood neutrophils was significantly increased in patients with early exacerbation of AD compared to donors and patients in remission. Increased expression of the sTREM-1 soluble receptor on neutrophils and its increased serum concentration positively correlated with AD severity. The TREM-1 receptor expression on neutrophils was increased after either a bacterial peptide exposure or stimulation of immune response by skin bacteria. In both cases, TREM-1 supported inflammation and correlated with AD severity. Conclusion: A comprehensive analysis of the TREM family receptor expression allows assessing the intensity of inflammation process in atopic dermatitis.

DEPENDENCE OF NEUTROPHIL RESPIRATORY BURST ON THEIR METABOLIC STATE IN THE PATIENTS WITH ACUTE DESTRUCTIVE PANCREATITIS OF DIFFERENT SEVERITY

The aim of the study was to investigate a dependence of respiratory burst state in neutrophils on activities of their intracellular enzymes in patients with acute destructive pancreatitis (ADP) of different severity. The study included 50 patients with ADP of moderate (17 cases) and severe degree (33 cases). A group of 47 healthy people was examined as controls. The respiratory burst state was examined in neutrophilic granulocytes by means of chemiluminescence assays. A study of NAD(P)-dependent dehydrogenases activity in blood neutrophils was performed using bioluminescent analysis. We have revealed that a decrease in spontaneous and induced synthesis of superoxide radical by neutrophils was detected in ADP patients, independently of the disease severity. Kinetics of primary ROS synthesis was also impaired in patients with severe ADP. In patients with moderate disorder, the level of secondary ROS synthesis by neutrophils proved to be increased, whereas, in cases of severe disease, a disturbed kinetics of secondary ROS synthesis by neutrophils was detected at a resting state, showing increased synthetic level upon additional induction by zymosan. Metabolism of neutrophils in patients with ADP is characterized by activation of plastic processes (due to the products of the pentose phosphate cycle) and aerobic energy (increased substrate flow intensity in the cycle of tricarboxylic acids). However, NADPH neutrophilic pool in patients with moderate disorder could be additionally supported by enzymatic malic enzyme reactions and NADP-dependent glutamate dehydrogenase. Activation of peroxidation events in patients with severe ADP is revealed, which needs NADPH compensation. The state of energy processes in blood neutrophils in patients with ADP is characterized by lacking changes in glycolytic activity, and increased intensity of substrate flux along tricarboxylic acids cycle. Activity of aerobic processes in patients with moderate disease is maintained by the products of amino acid metabolism (via glutamate dehydrogenase), whereas, in severe ADP it may be provided by products of lipid catabolism. Using correlation analysis, a dependence of respiratory burst of neutrophils on the state of their metabolism was studied. We have found that intensity and kinetics of respiratory burst in the neutrophils of controls depends only on the activity of NADP-dependent dehydrogenases. The changes in cellular metabolic activity in the patients with moderate ADP led to disturbances of their regulatory effect upon the state of neutrophil respiratory burst. In patients with severe disorder, the degree a neutrophil respiratory burst is stimulated by reductive amination of α-ketoglutarate, being, however, inhibited by intracellular peroxidation processes.