Effect of lidocaine on oxidative activity of peripheral blood phagocytes

BACKGROUND: Excessive production of reactive oxygen species (ROS) by leukocytes can cause damage to intrinsic tissues. The pathogenesis of sepsis is based on an excessive inflammatory response of the body. Several studies have reported the inhibitory effect of lidocaine on neutrophilic granulocytes. AIM: This study aimed to analyze the effect of lidocaine on the oxidative activity of phagocytes. MATERIALS AND METHODS: Blood from 16 healthy donors was used in this study. Leukocyte mass was extracted using spontaneous sedimentation. Half of the leukocyte samples were incubated in buffered physiological saline with lidocaine. The other half of the leukocyte samples were incubated in physiological saline without lidocaine. The generation of ROS was studied using two methods. Method 1 included a nitro blue tetrazolium (NBT) test), which is based on the ability of ROS to reduce NBT to insoluble diformazan. Method 2 was based on the chemiluminescence reaction. A culture of S. Aureus was used to induce the production of ROS. RESULTS: NBT test revealed a decrease in the oxidative activity of leukocytes in the presence of lidocaine by 18% (p 0.05). The study of luminol-dependent chemiluminescence of leukocyte suspension in the presence of lidocaine revealed a significant 2-fold decrease in both spontaneous and stimulated respiratory activity of cells. CONCLUSIONS: After incubation with lidocaine, phagocytes generated ROS to a significantly lower extent. However, their complete blockade was not recorded. This property of lidocaine may be used in clinical practice to treat an excessive inflammatory response in sepsis.

The nature of metabolic and functional state of the innate antimicrobial protection factors in persons with rhinosinusitis

The purpose is to study the features of metabolic and functional activity of cellular factors of innate immunity in nasal secretion in persons with rhinosinusitis. Material and methods. We studied the qualitative and quantitative composition of leukocytes of the nasal secretion of patients with rhinosinusitis, their viability, phagocytic and lysosomal activity, oxygen-dependent metabolism of neutrophilic granulocytes in the NBT test. Results. It was demonstrated that in patients with rhinosinusitis, an increase in the absolute and relative number of viable neutrophilic granulocytes, an increase in their lysosomal activity, a decrease in the activity and intensity of phagocytosis, and inhibition of biocidal properties according to the spontaneous NBT test are recorded. Conclusion. The revealed increase in the number of viable neutrophilic granulocytes, an increase in their lysosomal activity, a traced decrease in the activity and intensity of phagocytosis with inhibition of biocidal properties confirm the need to search for additional methods of rhinosinusitis therapy.

Clinical observation of Сhronic granulomatous disease in a 6-year-old child

Chronic granulomatous disease (CGD) is a hereditary disease caused by a genetic defect of violations of oxygen — dependent mechanisms of phagocytosis. Clinical manifestations of the disease are recurrent bacterial or fungal infections of the skin, hepatic abscesses, pneumonia, osteomyelitis, sepsis, meningitis et al. Most available laboratory method for the diagnosis of CGD is the test of histochemical nitro blue tetrazolium recovery (NBT-test). Allogeneic hematopoietic stem cell transplantation is considered a radical treatment for chronic granulomatous disease. The article presents a clinical observation of the manifestation of chronic granulomatous disease with an unfavorable outcome in a child aged 6 years.

PROOXIDANT AND ANTIOXIDANT BLOOD SYSTEMS AND CYTOKINE PROFILE IN PATIENTS WITH ABDOMINAL SEPSIS

Aim. to study the dynamics of clinical and laboratory parameters of endogenous intoxication and prooxidant and antioxidant blood systems in patients with abdominal sepsis. Material and methods. 57 patients with abdominal sepsis were examined. The inclusion criteria for the study were the presence of a clinic of abdominal sepsis and changes in the parameters of the prooxidant and antioxidant blood systems and blood cytokines. Results. In patients with abdominal sepsis, signs of oxidative damage to lipids and proteins were found, as evidenced by a significant increase in the MDA level up to 5.8 μmol / L, catalase content in erythrocytes 94 mmol / min*gHb, spontaneous NBT test activity up to 95% and a decrease in ceruloplasmin in the blood up to 194 mg / l. With abdominal sepsis, a significant increase in the level of TNF-82.7 pkg/ml, IL-10 to 167.6 pkg/ml, IL-6 to the maximum values in this study up to 286.4 pg/ml and IL to 34 pkg was /ml observed. Conclusion. It was found that in patients with abdominal sepsis there is an increase in lipid peroxidation indicators - malondialdehyde in the blood plasma, the activity of the spontaneous NBT test, depending on the phase of the disease and the presence of complicated forms. Changes in the antioxidant defense of the body in abdominal sepsis are manifested by inhibition of the plasma antioxidant ceruloplasmin with a simultaneous increase in catalase in erythrocytes, depending on the phase of the disease and also on the presence of complicated forms of the disease. As a result of this study, increased production of a complex of both pro-inflammatory and anti-inflammatory cytokines with their rapid and excessive entry into the peripheral blood was revealed.

COMPARISON OF THREE DIFFERENT TECHNIQUES FOR ISOLATION OF NEUTROPHILS FROM BLOOD AND THEIR UTILITY IN PERFORMING NITROBLUE TETRAZOLIUM TEST

Background & Objectives: Assays for neutrophils constitute an important component of screening tests in clinical immunology. There are no standard protocols for performing many of these tests and procedures vary from one laboratory to another. In addition, normal ranges for these assays in healthy Indian population have not been defined. Hence, an attempt is made to evaluate and present a simple technique for WBC isolation and NBT test. Methods: The study involved participation of 30 healthy adult volunteers. Ten of blood sample collected from each subject was subjected to three different procedures for isolation of WBCs - Ficoll-Hypaque gradient, dextran sedimentation and gelatin sedimentation methods. Cells isolated from these procedures were then used to perform NBT test. Smears were prepared, stained with Giemsa and results were expressed as % of stimulated and unstimulated cells. Results: The mean cell yield from both dextran and gelatin methods was comparable (2921.67cells vs 2806.67cells/cu mm). The cell yield from Ficoll-Hypaque method was much lower (1408.33 cells/cu mm). In NBT test, the mean readings of stimulated (61%) and unstimulated cells (18%) were almost similar in all three procedures of cell isolation. Conclusions: Comparison of procedures show that gelatin and dextran sedimentation methods yield high amount of relatively purified WBCs. The efficacy of cells isolated from all three procedures in NBT test was almost similar. The range of stimulated and unstimualted cells in the subjects were within expected levels. Gelatin sedimentation is economical, easy to perform and can be adopted to any clinical laboratory for WBC isolation.

Effect of new bedding material on morpho-biochemical and immunological factors of gosling blood

The purpose of research and production experiment in the conditions of Akhmetov farm located in the Vysokogorsky district of the Republic of Tatarstan was to study the harmlessness of new sanitary bedding material and the effect on gosling broilers. The experiment evidenced that a new sanitary bedding material made of woodworking waste, thermochemically and biotechnologically treated, does not have an irritating effect on the skin and mucous membranes of the eye, nor does it provoke allergic reactions in rabbits, but, on the contrary, improves certain morphobiochemical and immunological factors of gosling blood. Ultimately, this resulted in accelerated red blood cells by 6.94 %, hemoglobin – 5.20 %, total protein – 6.30 %, γ-globulin – 9.38 %, total calcium – 6.14 %, inorganic phosphorus – 5.31 %, glucose – 5.38 %, increased phagocytic activity by 2.24 %, phagocytic number – 4.37 %, and increased number of S. Marcescens-stimulated cells through the NBT test by 5.17 % compared with the control, respectively. The data obtained are consistent with the advanced livestock viability in the experiment by 1.8 %, as compared with the control.

FUNCTIONAL PROPERTIES OF BLOOD NEUTROPHILS AND PERITONEAL EXUDATE IN PATIENTS WITH PERITONITIS

The aim of the study is to investigate the functional properties of neutrophils in the blood and peritoneal exudate in patients with peritonitis. Materials and methods. The metabolic activity of neutrophils determined in patients with peritonitis and in patients of the control groups in the blood, peritoneal exudate and effusion of the abdominal wall on the 1–2 day of the postoperative period in the Sp and St test with nitro blue tetrazolium (NBT), as well as reserve indicator (RI) of metabolic activity. We studied the percentage of active neutrophils in the phagocytosis –PhI ( index) and the number of absorbed Candida albicans particles on average by one neutrophil –PhN(number). Results and their discussion.The peculiarities of the cellular factors of local protection decreased metabolic activity of neutrophils in the abdominal cavity in the Sp NBT test compared to blood neutrophils and an increased reserve during stimulation. For the comparisons which included the activity of neutrophils abdominal contents in patients with peritonitis versus in control established that neutrophils in the abdominal cavity with peritonitis have a reduced intensity of phagocytosis. Patients with pancreatic peritonitis have inverse correlations between the amount of NBT-positive blood NG in the SP NBT test and IR of blood NG and between the amount of NBT-positive blood NG in the Sp NST test and PhI of blood NG, while patients with abdominal peritonitis do not have correlations between indicators of innate defense of the body. Conclusions. Peritonitis was associated with functional disorders of NG. The leading areas of disorders are significant activation of metabolic processes of NG and inhibition of the phagocytic function of NG blood and peritoneal exudate. Patients with pancreatic peritonitis had inverse correlations between the indicators of the innate defense of the body, while patients with abdominal peritonitis did not have correlations.

Adenosine-related mechanisms in the pathogenesis of chronic obstructive pulmonary disease in patients with pulmonary tuberculosis

Актуальность. Учитывая важность пуриновой регуляции воспалительного процесса, раскрытие её участия в патогенезе хронической обструктивной болезни лёгких (ХОБЛ) может дать дополнительную информацию о патофизиологических механизмах воспаления и компенсации, приводящих к развитию ХОБЛ на фоне хронического воспаления, поддерживаемого туберкулёзной инфекцией. Цель настоящего исследования - выявить связь параметров аденозинового метаболизма с характеристиками окислительного взрыва, генерацией оксида азота и функциональными показателями внешнего дыхания у больных туберкулёзом лёгких в сочетании с ХОБЛ. Материалы и методы. В исследование включены мужчины активные курильщики с верифицированным диагнозом туберкулёз лёгких (ТЛ) и ТЛ в сочетании с ХОБЛ (ТЛ+ХОБЛ). Пуриновый метаболизм оценивали по активности аденозиндезаминазы (АДА-1 и АДА-2) в сыворотке крови (экто-АДА), мононуклеарах (мн) и нейтрофилах (нф), уровню экто-5’-нуклеотидазы (экто-5’-НК) в сыворотке крови, CD26 (дипептидилпептидазы-4, ДПП-4) в сыворотке и мононуклеарах. Окислительный взрыв фагоцитов оценивали по тесту восстановления нитросинего тетразолия (НСТ-тесту). Генерацию оксида азота - по концентрации метаболитов NO в сыворотке крови, мононуклеарах и нейтрофилах. Результаты. У больных ТЛ и ТЛ+ХОБЛ получены разнонаправленные изменения концентрации внеклеточного аденозина (рост активности экто-АДА-2, уровня экто-5’-НТ, снижение активности экто-АДА-1), в то время как внутриклеточные концентрации этого медиатора могут быть повышены (снижение активности АДА-1 и CD26 (ДПП-4) мононуклеаров). У больных ТЛ+ХОБЛ выявлено усиление респираторного взрыва мононуклеаров и нейтрофилов. У больных ТЛ регистрировали усиление продукции реактивных радикалов кислорода только в индуцированном НСТ-тесте нейтрофилов. В обеих группах отмечено значимое снижение продукции нитритов и нитратов как в моноцитах, так и нейтрофилах. У больных ТЛ параметры функции внешнего дыхания (ФВД) были связаны с активностью экто-5’-НТ и неспецифической пептидазы CD26 (ДПП-4), с продукцией нитритов нейтрофилами и моноцитами. В то время как у больных ТЛ+ХОБЛ показатели ФВД были ассоциированы с активностью экто-АДА-1 и АДА-1 в моноцитах, с сывороточной продукцией нитратов и продукцией нитритов нейтрофилами. Выводы. У больных ТЛ нарушение ФВД связано с избыточным образованием аденозина при чрезмерной активации формирующих его ферментов, а также с продукцией нитритов нейтрофилами и моноцитами, активных участников бактерицидных реакций, направленных против микобактерии туберкулеза (МБТ). Тогда как при ТЛ+ХОБЛ ведущим является нарушение деградации аденозина при снижении активности АДА-1, развитие эндотелиальной дисфункции и изменение функционального состояния нейтрофилов. Таким образом, разработка целенаправленных воздействий, приводящие к нормализации пуринергического обмена у больных ТЛ, в частности, к увеличению активности изоформы АДА-1, сможет обеспечить либо превенцию с ХОБЛ, либо, у лиц с уже развывшейся патологией, привести к стабилизации процесса. Background: Purine regulation plays an important role in inflammation. Therefore, disclosing the role of purine regulation in the pathogenesis of chronic obstructive pulmonary disease (COPD) may provide additional information about inflammation pathophysiology and compensation, which lead to COPD in chronic inflammation supported by tuberculosis infection. The aim of this study was to identify the relationship of adenosine metabolic indexes with characteristics of an oxidative burst, nitric oxide generation and functional parameters of external respiration (ER) in patients with pulmonary tuberculosis in combination with COPD. Materials and methods. The study included male active smokers with a verified diagnosis of pulmonary tuberculosis (PT) and PT in combination with COPD (PT+COPD). Purine metabolism was evaluated by adenosine deaminase (ADA-1 and ADA-2) activity in serum (ecto-ADA), mononuclear cells (mnc), and neutrophils (nph); serum concentration of ecto-5’-nucleotidase (ecto-5’-NT); and serum and mnc concentrations of CD26 (dipeptidyl peptidase-4, DPP-4). Oxidative burst in phagocytes was evaluated by the nitroblue tetrazolium conversion test (NBT-test). Nitric oxide generation - by concentrations of NO metabolites in blood serum, mnc, and nph. Results. Patients with PT and PT+COPD had multidirectional changes in extracellular adenosine concentration (increased activity of ecto-ADA-2, level of ecto-5’-NT, decreased activity of ecto-ADA-1). At the same time, intracellular adenosine concentrations could be increased (decreased mnc activities of ADA-1 and CD26 (DPP-4)). In patients with PT+COPD, the respiratory burst was observed only in mnc and nph. In patients with PT, increased production of reactive oxygen species was observed only in nph in the inductive NBT-test. In both groups, the nitrite and nitrate production significantly decreased both in monocytes and nph. In patients with PT, parameters of external respiration (ER) were linked to ecto-5’-NT and nonspecific peptidase CD26 (DPP-4) activities and to nitrite production by nph and monocytes. At the same time, in patients with PT+COPD, ER indexes were linked to monocyte activities of ecto-ADA-1 and ADA-1 and to serum nitrate and nph nitrite. Conclusion. In PT patients, impaired ER was associated with excessive formation of adenosine and excessive activation of adenosine-forming enzymes, as well as with nitrite production by nph and monocytes, which are active participants in bactericidal reactions directed against mycobacterium tuberculosis (MBT). However, in PT+COPD, the leading factor is impaired adenosine degradation with decreased ADA-1 activity, development of endothelial dysfunction, and changed nph functionality. Therefore, development of targeted means for normalizing the purinergic metabolism in patients with PT, specifically, by increasing the ADA-1 isoform activity, may provide either prevention in COPD or stabilization of the process in patients with already developed pathology.

OXYGEN-DEPENDENT MECHANISMS AND OXIDATIVE STRESS IN CHILDREN FOR FEMORAL HEAD OSTEOCHONDROPATHY

Degenerative-dystrophic processes in the articular tissues are accompanied by the changes in the values of congenital immunity, being a result of immune responses to aseptic inflammation. The purpose of this study - to investigate the oxygen-dependent mechanisms of neutrophil phagocytic activity (NBT-test, myeloperoxidase activity), the biochemical values of lipid peroxidation, the oxidative modification of proteins and the activity of enzyme antioxidant protection in children (at the age of 12-16 years) with osteochondropathy of the femoral head.The increase in spontaneous NBT-test and myeloperoxidase content in neutrophils, the decrease in NBT stimulation index, as well as the statistically significant increase in the activity of superoxide dismutase, the main producer of hydroperoxide, was revealed in the group examined. At the same time, no increase in catalase activity and in the content of lipoperoxidation products was observed, however, redistribution of the products of oxidative modification of proteins with ketone predominance was found. In case of osteochondropathy development in children the activation of the oxygen-dependent mechanisms of neutrophil phagocytic activity is observed, as well as the increase in the number of the secondary products of protein peroxidation - ketones. An increase in SOD activity, with a simultaneous and significant decrease in catalase activity evidence of accumulation of hydroperoxide causing neutrophil activation and chemotaxis. The study data can be used for predicting osteonecrosis development as additional criteria when decision-making of the advisability of performing reconstructive surgeries of the joint.

Study of innate factors in the local immune defense of the genital organs of dogs and cats

Immune system of reproductive organs of animals is represented by a number of cellular and humoral protection factors. Recently, the attention of scientists was attracted by the role of immunocompetent cells, which integrally provide immune homeostasis at all stages of the reproductive function. The purpose of the work was to study the cellular factors of immune defense of mucous membranes of genital organs of dogs and cats. The cytological composition and determination of immunological parameters of vaginal microscope slide were determined by patented methods. Local immunity was determined by the parameters of extracellular protection mechanisms, which consisted in the study of the Oxygen-dependent potential of neutrophil granulocytes (NBT-test) and their ability to form extracellular traps (NETs). Our studies have found that in different stages of the sexual cycle in beach and cats, along with cytological changes in epithelial cells, changes occur in phagocytic protection. In particular, in the post-infusion period there was an increase in the number of intermediate and basal epithelial cells on the surface of which were adhered microorganisms. Along with epithelial cells, activated phagocytes were grouped. Neutrophils showed cytochemical reactivity in the NBT-test, and also formed NETs. At the same time, activated phagocytes absorbed microorganisms, and some epithelial cells formed specific cytoplasmic inclusions, which obviously also has an important role in the antimicrobial immunity of the mucosa. Some neutrophilic granulocytes were in a state of apoptosis. During the sexual cycle in the organism of dogs and cats there are morphology and functional changes in the genital organs, which was accompanied by a change in the cytological picture of vaginal microscope slides. It was established that the antimicrobial protection system plays an important role in the neutrophilic granulocytes, the functional activity of which also has a change. The main mechanisms of extracellular antimicrobial protection of microphages are realized with the involvement of Oxygen species in the excretion system of active forms and the formation of neutrophilic extracellular traps. Epithelial cells are also involved in the formation of immune homeostasis of genital mucous membranes.