bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of regional state aid in Slovakia: A focus on the least developed districts
Authors: Košdy Martina
Published: June 2023

This paper examines the geographical distribution of regional state aid in Slovakia between 2004 and 2021, while focusing on projects realized in the least developed districts. The purpose is to answer the following research question: how much investment support is provided to areas with high rates of long-term unemployment to promote local economic activity? The investigation was conducted using a spatial distribution analysis and descriptive statistical methods. The findings demonstrate that the level of support in less developed districts is below the level of aid directed into more developed regions not only in terms of the number of supported projects, but also regarding the total amount of aid and the number of created jobs. Out of the 20 least developed districts we monitored, only the results for Košice-okolie significantly outperformed the results of the other districts. This article provides possible explanations for these findings and contributes to the literature by providing insights into the practical application of state aid in Slovakia.
APPLICATION OF FUZZY LOGIC IN THE ROBOT CONTROL FOR MECHANICAL PROCESSING
Authors: Khoi Phan Bui
Published: June 2023

Robot application in mechanical machining is growing day by day because it has many advantages over conventional machines such as high flexibility, large working space, and high repeatability. Many degrees of freedom of motion give robots the ability to perform complex technological operations, but also because of that, methods of controlling robots based on dynamic models have difficulties. Applying fuzzy logic to robot control can partially or completely exclude the calculation of the robot's dynamic model as well as overcome other uncertainties of the whole technological system. The variables and parameters of the fuzzy logic-based controller are modeled in a linguistic form, called linguistic variables, and are defined by the linguistic semantic values. Fuzzy rules are an important basis for the performance of operations defining the control quantities of the controller. Fuzzy rules are constructed by natural human inference and are based on expert intelligence. The main tasks of applying fuzzy logic control include “Fuzzification” to determine fuzzy parameters in the form of fuzzy sets of input-output data; “Fuzzy Rules and Fuzzy Inference Mechanism” to perform fuzzy operations defining control quantities, and finally “Defuzzification” to convert control quantities from linguistic values to physical values for controller operation. There have been many types of research on applying fuzzy logic to control robots in general, but the percentage of fuzzy control research work for mechanical machining robots is still limited.

The article is based on published works on fuzzy control for robots in general and mechanical processing robots to analyze the applicability of fuzzy control for mechanical machining robots. The article provides detailed information on fuzzy controller design, on determining input and output variables, proportional mapping to determine the number of fuzzy sets and the corresponding type of membership function. The construction of fuzzy rule base system and fuzzy inference mechanism is presented, and finally defuzzification. Prospects for the use of methods to perfect and develop fuzzy control systems for mechanical machining robots are also presented. Collectively, the information in this document is intended to guide the implementation of fuzzy logic-based controller designs for application to machining robots, as well as to general robot control.
Between Sunnah Currency and Sufi Doctrine: Mediating and Negotiating the Gold Dinar and Silver Dirham on Digital Platforms by the Indonesian Murabitun
Authors: Ahmadi Rizqa , Hefni Wildani , Nafis Muhammad Muntahibun
Published: June 2023

The Murabitun World Movement promotes the gold dinar and silver dirham as authentic Islamic currency tools. However, their vision of establishing an independent Islamic currency is rejected by the socio-political dynamics of the communities’ home countries. Using Talal Asad’s theory of Islam as a discursive tradition, the study investigates the local practices of the Murabitun among Indonesian Muslims, with particular attention to their cultural identity. This study relies much on online data and cyber resources. The Indonesian Murabitun actively promoted the circulation of the gold dinar and silver dirham as an Islamic currency at the Pasar Muamala (exchange market) in Madiun and Yogyakarta. The Indonesian Murabitun movement was initially established in Depok, West Java by Zaim Saidi with his creation of the Pasar Muamala. After Zaim Saidi was formally charged with violating the currency law, his movement lose much its influence. However, utilizing the dynamics of the socio-political landscape this movement has continued to be actively involved in promoting the gold dinar and silver dirham currency—as an alternative transaction tool alongside the regular currency—on digital media platforms.
Chronobiological approach to study the physiological activity of Candida species
Authors: Nikolenko M. V. , Baryshnikova N. V. , Malishevskaya O. I. , Vaseva E. M.
Published: June 2023

Rythmometric markers can be identified within a day during the study of biological characteristics in order to be used for differential diagnostics of pathogens of different patients’ physical condition. These principles are based on analysis of clinical isolates С. albicans, С. tropicalis and C. krusei allocated from the vaginal microbiota at Candida dysbiosis condition. Control examples were the master samples from the American Type Culture Collection (ATCC). Detailed research was conducted on physiological characteristics through the formation of biofilms by yeast pathogens. Biological activity of Candida sp. biofilming was observed within 2 days with 4 hours interval in winter. Daily cultures were used for the experiment to correspond to their maximum adhesion to the glass surface. It was important to obtain 6 measurements per day with 3-5 times repetition of experiment conditions during the specified timeline. In order to determine the periodicity of the parameters studied, the data was statistically processed by Student’s t-test, using Mann–Whitney criteria and nonparametric method of least square method.

It was found out that biofilming activity during 24 hours (р < 0.05) of fungi exists and that all species have many principles in common. It was attested that the main rhythmometric parameters of diagnostic significance are the rhythm period and amplitude-phase stability. It was found that the daily dynamics of C. albicans 24433 biofilm formation from American Type Culture Collection was characterized by an ultradian (about 12-hours) contribution of the rhythm in the morning – 4 A.M and in the evening – 4 P.M. Significant circadian (approx. daily) rhythms of adhesion glass surface activity were revealed in C. non-albicans from American Type Culture Collection. The dynamic of biofilm formation isolates of yeast from female reproductive organs with Candida pathology was characterized by reliable ultradian (about 12-hour) harmonics which biological significance defines resistance to external impact and the ability to adaptively respond to periodic stimuli.

To sum up, implementation of the chronobiological approach has opened up new prospects for studying the physiology of Сandida sp., as it enables us to predict the dynamics of microbial states and takes into account the specificity of emergency and long-term adaptation to different environmental factors. The detection of the circadian rhythm of biofilm formation activity of different Candida sp. strains provides the possibility to manage the vitality of the Society of Bacteria and Fungi and predicts its resistance to various antibiotics.
Coordination of NK cell markers expression and IgG response in hCMV infection
Authors: Ustiuzhanina M. O. , Vavilova Ju. D. , Alekseeva N. A. , Lutcenko G. V. , Chudakov D. M. , Kovalenko E. I.
Published: June 2023

Human cytomegalovirus (hCMV) is a prevalent virus that affects a large proportion of the population worldwide. Natural Killer (NK) cells are essential immune cells that play a crucial role in controlling hCMV infection. Despite the wide spread of hCMV infection, there is still not enough data related to the association between innate and adaptive immunity. This study investigated the coordination between some of the NK cell markers expression and humoral immune response during hCMV infection. Thirty-three samples obtained from different healthy donors were investigated. The anti-hCMV IgG antibody titer was measured in serum samples, and expression of NKG2C, HLA-DR, CD57, KIR2DL2/DL3, and KIR2DL1 were analyzed in CD56+CD3- cells in PBMC samples by flow cytometry. To evaluate the dependence of proportions of different NK cell subsets on IgG titers, cluster analysis was first performed on all the obtained data, resulting in the identification of four main clusters. The identified clusters demonstrated a dependence on the levels of hCMV antibodies, according to which clusters corresponding to seronegative and low-positive were grouped. The results confirmed that hCMV infection leads to an expansion of NK cell populations expressing the NKG2C marker, which correlates with higher levels of IgG response to hCMV. Besides, we identified increased HLA-DR+ and decreased of KIR2DL1+ NK cells proportions in the middle anti-CMV-IgG level group compared to samples obtained from seronegative and low-positive donors. Moreover, the statistically significant negative correlation was found between KIR2DL1+NK cell percentage and anti-CMV IgG antibody titer, while the positive correlation between HLA-DR+NK cell proportion and the IgG level was noticed only without the cluster corresponded to high level of anti-hCMV IgG. In this cohort, we did not find any association between KIR2DL3 and CD57 expression in NK cells and levels of IgG response to hCMV. This may indicate that different subsets of NK cells may have distinct roles in regulating humoral immunity to hCMV. Overall, the results of the study provide valuable insights into the coordination of NK cell marker expression and IgG response in hCMV infection.
Development of a vaccine adjuvant based on squalene and study of its adjuvant properties
Authors: Volosnikova E. A. , Shcherbakov D. N. , Ermolaev V. V. , Volkova N. V. , Kaplina O. N. , Borgoyakova M. B. , Danilenko E. D.
Published: June 2023

The use of modern subunit vaccines involves adjuvant introduction into their composition. Currently, the search for new and improvement of existing adjuvant systems is actively underway. Squalene- based adjuvants are well-known and approved in a number of countries for clinical use in influenza vaccines. Our study was devoted to the development of an adjuvant composition on the basis of squalene. The resulting adjuvants were composed in a form of oil emulsion containing a hydrophilic and hydrophobic phase. The stability of the emulsion was achieved by treating it with ultrasound at a frequency of 22 kHz. Particle sizes of the obtained emulsions were examined with the use of an electron microscope. The particle size was calculated to be 50-80 nm for the majority of particles (84%). Adjuvant activity was evaluated in 100 male Balb/C mice, weighing 16-18 g. To assess the humoral immune response, immunization was performed twice, with a 14-day interval, by intramuscular injection of 200 mL per animal. The receptor-binding domain (RBD) of the surface S protein of the severe acute respiratory syndrome coronavirus 2 (Delta variant (B.1.617.2)) or ovalbumin (OVA) from chicken eggs were used as antigens. RBD was administered at a dose of 50 mg/animal; OVA was administered at two doses (1 mg or 5 mg/animal). An antigen with aluminum hydroxide was used as a positive control; a saline solution was used as a negative control. The effectiveness of the obtained adjuvants was determined by measuring the titers of specific antibodies in mouse sera in ELISA assays using the recombinant RBD of SARS-CoV-2 S-protein or ovalbumin from chicken eggs. It was shown that the use of squalene-based adjuvants increased the antigens’ immunogenicity. The average titers of specific antibodies against RBD in the experimental group were 4 times higher than in the group immunized with RBD adjuvanted with aluminum hydroxide. An increase in immunogenicity of the antigen adjuvanted with squalene was also observed in the experimental OVA-group. Thus, it was shown that the developed squalene-based adjuvant compositions could be an alternative to the traditional adjuvants based on aluminum salts.
Experimental application results of mesenchymal stem cell microvesicles in the mouse model of acute renal failure
Authors: Ivanova I. P. , Seledtsova G. V. , Seledtsov V. I. , Khabalova T. S. , Dorzhieva A. B.
Published: June 2023

An important role in restoration of damaged organs and tissues is played by mesenchymal stem cells (MSCs) and microvesicular particles (MV) produced by them. They can be a source of cytokines, anti- apoptotic and growth stimulating factors. In addition, MVs carry out transport of mRNA, miRNA, and signal proteins into damaged tissues. This increases the ability of cells to regenerate and to inhibit apoptosis, promote to angiogenesis and stimulate cell proliferation. The aim of our research was to study the immunoregulatory and pro-regenerative properties of mesenchymal stem cell microvesicles (MSC-MV) in a model of glycerol- induced acute renal failure (ARF) in mice. The experiments were carried out on CBA mice aged 3-4 months. AKI was induced by a single intramuscular injection of 50% glycerol. MSCs were obtained from the bone marrow of healthy animals and cultivated under standard conditions. Microvesicles were obtained by centrifugation at 12000g of MSC supernatant after induction of their apoptosis by culturing under oxygen deprivation conditions and in serum-free medium. MSC-MV was injected intravenously into the retroorbital sinus one day after induction of ARF. The MV dose was calculated as equivalent to (derived from) 1 million MSCs, which was 100 mL per mouse. Animals were taken out of the experiment on days 4 and 11 after MSC-MV injection. Blood plasma was taken to determine the level of creatinine, urine – for albumin analysis, kidneys – for histological examination. It has been shown that MVs induced by MSCs dose-dependently stimulated splenocyte proliferation in both spontaneous and Con-A induced tests. The addition of MV caused a decrease in doxorubicin-induced apoptosis of splenic lymphocytes in mice. Probably, in this case, MV produced by MSCs had an immunostimulatory and antiapoptotic effect. Also, MVs had a positive impact on the restoration of structure and function kidneys in a model of ARF in mice. The use of MSC-MV in treatment of acute renal failure induced by a single injection of 50% glycerol contributed to decrease albumin level urine and restoration of creatinine level in blood serum of animals. Morphological studies have shown decrease in the height cell and collecting duct diameter in the medulla and a decrease in the largest transverse diameter of superficial glomeruli in the renal cortex of sick mice. Thus, the obtained results indicate significant therapeutic and pro-regenerative properties of MSC-MV, which require further study.
Effect of Bifidobacterium bifidum supernatant on the morphological and functional characteristics of human fibroblasts in real time during an in vitro experiment
Authors: Markov A. A. , Kostolomova E. G. , Timokhina T. Kh. , Solovyev G. S. , Paromova Ya. I. , Polyanskih E. D. , Voronin K. A.
Published: June 2023

Currently, there is an active search for exogenous stimulators of repair and regeneration processes. In the recent decades, some data on the immunotropic activity of bifidobacteria have been accumulated. The key role in healing of wound defects belongs to fibroblasts due to the secretion of the extracellular matrix components, metabolites, signal factors for the surrounding cells, and tissue metabolism regulation. The paper presents the results of the study of the effect of Bifidobacterium bifidum supernatant (10 ml/mL) on the morphological and functional properties of human fibroblasts in real time during the in vitro experiment. In our work, we used the reference strain B. bifidum 791 (All-Russian Collection of Industrial Microorganisms of the State Research Institute for Genetics and Selection of Industrial Microorganisms “Genetika”, Deposit No. AS-1247) used in the production of the probiotic product “Bifidumbacterin” (ZAO “Ecopolis”, the city of Kovrov), and adult human fibroblasts (cell line LECH-4 (81)) (laboratory of cell cultures ENIIVI, the city of Yekaterinburg). Structural and functional studies were conducted on co-culture days 1, 3, 7, 14, 21, and 28. The products of B. bifidum secondary metabolism have a stressful effect on the morphological and functional state of fibroblasts on the first day. The processes of proliferation are stimulated in the culture in the experiment (2.67±0.24) compared with the control group (0.75±0.15) (p < 0.01) without blocking apoptosis in the cell. This leads to the increase in the production of extracellular matrix proteins, both collagen (pg/mL) (400±19 against 110±25 in the control group), and elastin (ng/mL) 395±30 and 125±29). Co-culture of fibroblasts within 24 hours in the experimental sample leads to a massive “release” of the CD44 receptor (p < 0.05), compared to the control group which is confirmed by phenotypic changes (r = 0.66). The decrease of CD105+, CD44+ receptors (p < 0.05), compared with the control group and the increase of CD29+ expression (p < 0.05) is observed on days 1 and 3. Activated fibroblasts have an altered secretory phenotype that produces cytokines of various types such as TGF-b (r = 0.78), IL-6 (r = 0.57), IL-1b (r = 0.75), IL-8 (r = 0.63). The maximum adaptation of cells in the experimental system is registered on the 7th day, which correlates with morphometric (r = 0.59) and cytometric (r = 0.71) studies. The received data contribute to understanding of the mechanisms of the immunoregulatory influence of normal biota (in the bifidobacteria model) on the repair and regeneration processes.
Effect of endomorphins on humoral immune response, Th1/Th2/Th17 cytokine production and CD4+, CD8+ lymphocyte apoptosis in vivo
Authors: Kadochnikova Ya. A. , Gein S. V.
Published: June 2023

Endogenous opioid peptides are a large group of physiologically active compounds with a pronounced affinity for opioid-type receptors, capable of showing pronounced analgesic activity, as well as having additional effects on the periphery, due to their wide distribution on the cells of many organs and tissues. Little studied representatives of this group are endomorphins, which due to their structure and properties, are capable of producing a strong antinociceptive effect after central administration, which means that, in the future, they can be considered as potential substitutes for low molecular weight opiates. The aim of this study is to evaluate the effect of endomorphins on the humoral immune response, the production of Th1/Th2/Th17 cytokines and apoptosis of CD4+, CD8+ lymphocytes in vivo. The splenocytes of Swiss white mice were used as the object of the study. The number of antibody-forming cells in the spleen was assessed using the method of local hemolysis in agarose gel according to Jerne. Quantitative determination of cytokines was carried out by enzyme-linked immunosorbent assay using kits (R&D, USA) according to the method proposed by the manufacturer. Apoptosis was assessed using Annexin V-FITC/7-AAD kit reagents (Beckman Coulter, USA) according to the manufacturer’s instructions by flow cytometry on a CytoFLEX S flow cytometer (Beckman Coulter, USA). In the course of the study, it was found that endomorphins enhance the antibody genesis of the spleen, and the preliminary blockade of opiate receptors with naloxone led to the cancellation of the stimulating effect of peptides. Endomorphins didn’t affect splenocyte production of IL-2, IL-4, and IFNg, however, the introduction of endomorphin-2 naloxone-independent enhanced the induced production of IL-17. Evaluation of the effect of endomorphins on apoptosis of splenocytes in 24-h cultures showed that endomorphin-2 in unstimulated cultures of naloxone-dependently increased the percentage of late apoptosis of CD8+ lymphocytes, however, in stimulated cultures, both endomorphins increased the apoptotic activity of CD8+ lymphocytes, regardless of the preliminary blockade of opioid receptors. In summary, we can say that in the in vivo system, endomorphins have a wide range of multidirectional immunomodulatory effects, which may be of interest for practical use in the future.
Evaluation of the long-term memory T cell in mice after immunization with a live tularemia vaccine
Authors: Kartseva A. S. , Silkina M. V. , Titareva G. M. , Kombarova T. I. , Mironova R. I. , Firstova V. V.
Published: June 2023

The vaccine strain F. tularensis 15 NIIEG induces long-lived cell-mediated immunity but exhibits a certain reactogenicity and genetic instability. Progress in development of a vaccine against tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. The BALB/c mouse is the most commonly used animal to study tularemia due to its relatively low cost, well-characterized genetics, available immunological tools and mouse infection with virulent F. tularensis recapitulates human disease.

CD4+ and CD8+T cells are known to be critical for the formation of protective immunity but the relative roles of memory T cell subpopulations in long lived protection against virulent strains of F. tularensis are not well established. We hypothesized that this immunity depends on central (TCM) and effector memory (TEM) T cells and their functional activity. In this study we have dissected the T cell immune response in BALB/c mice 30, 60 and 90 days after subcutaneous vaccination with 15 NIIEG.

Multiparametric flow cytometry were used to characterize in vitro recall responses of splenocytes to F. tularensis antigen. TEM cells were identified as CD3+CD4+CD44+CD62L- and CD3+CD8+CD44+CD62L-, TCM cells as CD3+CD4+CD44+CD62L+ and CD3+CD8+CD44+CD62L+, respectively. The functional activity of memory T cells was assessed by the following parameters: the level of expression of the activation marker CD69 and cytokine-producing activity by staining with the intracellular cytokines IFNg and TNFa.

Thus, development of a long-lived vaccine directed against F. tularensis is dependent on identifying not only the correlates of immunity present early after vaccination, but also those that persist in the host after the effector phase has ended. The maintenance of long-term protective immunity initiated by vaccination with F. tularensis strain 15 NIIEG has been shown to require the presence of antigen-specific CD4+ and CD8+ memory T cells producing IFNg and TNFa and expressing the activation marker CD69. A decrease in count and functional activity of CD8+TCM and CD8+TEM was detected in the long term after vaccination. The detected parameters of functional activity of memory T cells can be used as criteria for evaluation of protective immunity against virulent strains of F. tularensis.
Galectin-9 influences the Tim-3 molecule expression in natural killer different subpopulations
Authors: Orlova E. G. , Loginova O. A. , Gorbunova O. L. , Shirshev S. V.
Published: June 2023

Galectin-9 is a b-galactoside binding lectin with expressed immunoregulatory activity. During pregnancy galectin-9 is produced by trophoblast cells and regulates the function of natural killer (NK) cells at the maternal-fetal interface via binding to Tim-3 (T-cell Ig and mucin domain-containing protein 3) molecules. Natural killer (NK) lymphocytes belong to the innate lymphoid cells, which have a cytotoxic effect on target cells and are capable of producing a large number of regulatory factors (cytokines, chemokines). Decidual NK have a tolerant phenotype and play a leading role in the regulation of invasive trophoblast growth and provide peripheral immune tolerance in the area of uteroplacental contact. Peripheral NK cells express Tim-3 molecules. Galectin-9 concentration is increased in peripheral blood during physiologic pregnancy. At pregnancy phenotype and functions of peripheral NK cells are changed to maintain the maternal–fetal immune tolerance. Peripheral NK cells migrate to the maternal-fetal interface and are transformed into a decidual NK-like phenotype cells. Galectin-9 concentration is decreased in women with a  complicated  pregnancy and miscarriage. However the galectin-9 effects on different NK cell subpopulations of peripheral blood are not investigated. Therefore, we studied the galectin-9 influence on phenotype transformation and Tim-3 expression of NK cells isolated from peripheral blood of healthy non-pregnant fertile women. CD56+NK cells were obtained by immunomagnetic separation and cultivated in vitro during 72 hours with cytokines (IL-2 and IL-15). Galectin-9 (5 ng/mL) and anti-Tim-3 (10 mg) antibodies were added to the NK cultures. Galectin-9 concentration is corresponded to its level during first trimester of physiologic pregnancy. The number of regulatory NK (CD16-CD56bright), cytotoxic NK (CD16+CD56dim/-) cells and Tim-3 expression on different NK subpopulations were assessed by flow cytometry. It was found that Tim-3 was expressed on all subpopulations of peripheral blood NK cells (CD16-CD56brightNK, CD16+CD56dimNK, CD16+CD56-NK). Incubation with galectin-9 increased the expression of Tim-3 on regulatory CD16-CD56brightNK cells and did not change on cytotoxic CD16+CD56dim/-NK cells. Galectin-9 reduced the percentage of cytotoxic CD16+CD56dimNK in culture, but did not influence the number of regulatory CD16-CD56bright NK and cytotoxic CD16+CD56-NK cells. Thus, galectin-9 regulates Tim-3 molecule expression and NK cell subpopulation distributions in vitro culture.
HDL apoprotein immunization induces T cell-mediated venulitis and inflammation in aorta
Authors: Sidorov А. Yu. , Fomina K. V. , Beduleva L. V.
Published: June 2023

The hypothesis that atherosclerotic processes are mostly caused by immune (autoimmune) mechanisms has recently been gaining attraction. At the same time, the autoimmune hypothesis of atherogenesis has not become generally accepted and requires additional evidence. Previously, we were able to induce changes in the aortic wall similar to those observed in the early stages of human atherosclerosis, and also to produce visceral obesity in normocholesterolaemic Wistar rats by a single immunization with human native high- or low-density lipoproteins. We also found that the immune response to native human HDL causes atherosclerosis-like lesions in the rabbit aorta, such as adipocyte and chondrocyte metaplasia, proteoglycan deposits, and leukocyte infiltration. Atherosclerosis-like lesions developed in the aorta of hnHDL-immunized rabbits against a background of normal blood LDL-cholesterol level. Thus, an immune response against HDL or LDL may be an independent cause of atherogenesis. The aim of this study was to test whether immunization with human HDL apoproteins (apoA1 and apoE proteins) would induce atherosclerosis-like lesions in the aorta of normocholesterolemic Wistar rats. HDL apoproteins were isolated from human or rat plasma. Wistar rats (n = 5) aged 2 months were used for immunization with human HDL apoproteins. HDL apoproteins were administered as a single intradermal injection of 100 mg per rat in incomplete Freund’s adjuvant. Control rats were injected subcutaneously with incomplete Freund’s adjuvant (n = 5). Rats were dissected 25 weeks after immunization. Rat aorta sections were stained with hematoxylin and eosin for light microscopy. T lymphocytes infiltration was determined by immunohistochemical staining with FITC-labeled antibodies specific to rat CD3. CD3+T lymphocytes were detected using an Olympus BX53 fluorescent microscope. The level of antibodies to human and rat HDL apoproteins was determined by indirect enzyme-linked immunosorbent assay. Immunization with HDL apoproteins induced a T cell mediated immune response without production of autoantibodies to HDL apoproteins. The aortic intima and adventitia were infiltrated with T lymphocytes in rats immunized with HDL apoproteins. Pronounced T lymphocytic infiltration was found in all layers of the vein wall in rats immunized with human HDL apoproteins. Thus, immunization with HDL apoproteins causes T cell mediated inflammation of the aorta and venulitis.
Immunological aspects of the use of melatonin in experimental thermal trauma
Authors: Osikov M. V. , Ageeva A. A. , Boyko M. S. , Ageev Yu. I.
Published: June 2023

The prevalence of thermal trauma, the high risk of infectious and non-infectious short- and long- term complications, and the limited effectiveness of the therapeutic approaches used are prerequisites for the search and pathogenetic justification of new therapies, among which the endogenous homeostasis regulator with pleiotropic properties melatonin attracts attention.

The aim of the work is to investigate the immunological aspects of intraperitoneal use of melatonin (MT) in experimental thermal trauma (TT).

The work was performed on 158 rats of the Wistar line, grade III TT and a relative area of 3.5% were simulated by skin immersion in water at 98-99 °C for 12 s. MT was administered intraperitoneally daily at a dose of 10 mg/kg for 5 days. The quantitative composition of blood cells was evaluated on a hematological analyzer. Plasma concentrations of IL-4, TNFa, IFNg, and CRP were determined on an automatic enzyme immunoassay using rat-specific test systems, and MT by capillary electrophoresis.

With experimental TT, against the background of a progressive increase in the number of leukocytes in the blood from 5 to 20 days due to neutrophils, monocytes, basophils, the number of lymphocytes decreases. With TT, the concentration of CRP increases in serum on days 5 and 10. The content of TNFa, IL-4 increases on days 5, 10 and 20 in the absence of significant changes in the concentration of IFNg. The concentration of serum MT does not change significantly. Intraperitoneal use of MT in TT leads to a partial restoration of the number of lymphocytes in the blood on day 5. Evaluation of the cytokine profile in serum revealed a decrease in the concentration of TNFa on days 10 and 20, no significant changes in the concentration of IL-4 and IFNg were recorded, the concentration of CRP decreased on day 5. The concentration of serum MT increases by 5 days.

With TT on the 5th, 10th, 20th day of the experiment, the number of neutrophils, monocytes, basophils in the blood increases, decreases – lymphocytes, the serum content of CRP, TNFa, IL-4 increases, the content of IFNg and melatonin does not change. Intraperitoneal use of MT in TT partially restores the number of lymphocytes in the blood, the concentration of CRP, TNFa. A decrease in serum concentrations of TNFa and CRP in TT under the conditions of MT use suggests a limitation of the acute phase response as a consequence of the antioxidant, anti-inflammatory effect of MT, which can accelerate healing and reduce the area of the lesion of TT.
Investigation of local expression of NLRP3 inflammasome complex genes in modeling retinal degeneration in vivo
Authors: Neroeva N. V. , Svitich O. A. , Neroev V. V. , Kinkulkina A. R. , Balatskaya N. V. , Sorozhkina E. S.
Published: June 2023

Neurodegenerative ophthalmopathology is one of the main causes of irreversible blindness and disability in the world. In the pathogenesis of diseases of this group, more and more attention has recently been paid to the role of local inflammation caused by the activation of innate immunity and the mechanisms of its genetic regulation. In recent years, works have appeared in the field of experimental ophthalmology that have demonstrated the possibility of NLRP1, NLRP3 inflammasome complexes assembling when exposed to hyperglycemia, oxygen deprivation of retinal cells, as well as modeling compressive stress similar to that in glaucoma [15]. However, the mechanism of inflammasome involvement in the development of neurodegenerative eye diseases remains unclear. The aim of the study was to investigate the local expression of genes encoding proteins of the NLRP3 inflammasome complex (NLRP3, CASP-1) in an experimental model of retinal degeneration in rabbits. The studies were performed on samples of tissue complex (TC) of the retina/retinal pigment epithelium (RPE) (retina/RPE TC), isolated from the eyes of 14 New Zealand albino rabbits, in which degenerative retinal lesion was modeled by a single subretinal injection of 0.01 mL of 0.9% sodium chloride solution, and 7 healthy rabbits without eye damage. The formation of retinal degeneration was judged on the basis of changes in morphofunctional parameters obtained during specialized ophthalmological research methods (optical coherence tomography, fundus autofluorescence, electroretinography) at follow-up periods of 1, 3 and 6 months. The level of expression of NLRP3 and CASP-1 genes in the retina/RPE TC was evaluated by reverse transcription polymerase chain reaction (RT-PCR). According to the results of the study, a statistically significant increase in NLRP3 gene expression (p < 0.001) was noted in the retina/RPE TC of experimental animals, which may indicate the involvement of NLRP-3 inflammasome components in the development of neurodegenerative retinal lesions. At the same time, the expression of the gene encoding CASP-1 was detected only in the retina/RPE TC of experimental eyes and is probably due to local inflammatory mechanisms in the retinal tissue.

The high level of NLRP3, CASP-1 mRNA, detected in all retina/RPE TC samples of experimental eyes at late stages of the experiment (3 and 6 months), allows us to assume the formation of mechanisms (for example, activated glial phenotype) that support inflammation in retinal tissue. This should be taken into account in actively developing transplantation methods for the treatment of retinal degeneration.
Influence of microbial polyamines on the IL-10 production by peripheral blood leukocytes of healthy donors
Authors: Godovalov А. Р. , Morozov I. А.
Published: June 2023

As is known, bacterial polyamines, which include cadaverine and putrescine, are capable of influencing the activity of immunocompetent cells in many ways. In particular, this situation is observed in long-term inflammatory diseases, especially with intensive reproduction of microorganisms capable of producing polyamines. It is of interest to study the production of one of the main anti-inflammatory cytokines, IL-10, under the influence of bacterial polyamines. For research, a population of mononuclear leukocytes was isolated from the peripheral blood of healthy donors by gradient centrifugation. The cell suspension was placed in a round-bottomed plates with preliminarily added polyamines at concentrations of 5, 25, 50, 75, and 100 mmol/L. Wells not containing polyamines were used as a control. After incubation for 72 h at 37 °C and 5% CO2, the supernatants were harvested and used to determine the concentration of IL-10. We used a kit for determining the concentration of IL-10 using the enzyme immunoassay method (Russia). Statistical analysis was performed using the Statistica 6.0 software package. In the case of a distribution close to normal, Student’s t-test was used; in the rest, the Mann–Whitney test was used to assess the significance of differences. Studies have shown that leukocytes in the presence of concanavalin A produce IL-10 at a concentration of 17.13±6.08 pg/mL. It has been established that under the influence of polyamines of bacterial origin, the production of IL-10 is enhanced only if putrescine and cadaverine are at concentrations of 50 mmol/L and higher. At low concentrations of polyamines, no significant increase in IL-10 production was detected. Since IL-10 is an anti- inflammatory cytokine, for which the analgesic effect is also known, it should be expected that with an increase in its concentration in the focus of invasion of opportunistic bacteria, the inflammatory process will develop latently, when the symptoms are mild. In general, it can be expected that polyamine-producing bacteria will contribute to the maintenance of few symptomatic inflammation.
NK cells expansion in vitro is followed by loss of inhibitory KIR expression
Authors: Alekseeva N. A. , Ustyuzhanina M. O. , Streltsova M. A. , Grechikhina M. V. , Lutsenko G. V. , Kovalenko E. I.
Published: June 2023

NK cells are innate lymphocytes that are able to eliminate altered cells, which makes them promising for the immunotherapy of viral diseases and tumors. The NK cell population is characterized by high phenotypic and functional diversity. In particular, in the pool of highly differentiated NK cells in the presence of cytomegalovirus (HCMV), a population of adaptive cells can be formed, characterized by a high lifespan and high cytotoxicity. However, in order to carry out a cytotoxic reaction, a NK cell must undergo a licensing process, during which it acquires the expression of NKG2A and KIRs. Currently, there are many effective methods of NK cell accumulation for subsequent use in therapy, one of them is the stimulation with IL-2 and K562-mbIL21 feeder cells. Highly differentiated adaptive-like NK cells are able to expand in respond to such stimulation. However, the phenotype of actively expanding NK cells dynamically changes. Loss of inhibitory KIR expression during intense proliferation of NK cells may adversely affect their cytotoxic potential. This work shows that highly differentiated CD56dimNKG2C+ NK cells from HCMV-seropositive individuals have a high proportion of KIR2DL2/3+ cells. This may indicate a high stability of KIR receptor expression in this population. We have shown that CD56dimNKG2C+ clonal cultures obtained by stimulation with IL-2 and K562- mbIL21 are characterized by high stability of KIR2DL2/3 expression compared to NKG2C-negative and less differentiated CD56brightNKG2C+. Also, in heterogeneous cultures of adaptive NK cells precursors CD57- CD56dimNKG2C+, a higher expression level of KIR2DL2/3 was observed in comparison with NKG2C-negative cultures of CD57-CD56dimNKG2C-. Thus, the accumulation of NK cells upon stimulation with IL-2 and K562- mbIL2 feeder cells can lead to loss of expression of KIR receptors and a decrease in their functional activity. However, cultures of highly differentiated NK cells of HCMV-seropositive individuals CD56dimNKG2C+, as well as cultures of precursors of adaptive NK cells CD57-CD56dimNKG2C+, are characterized by a greater stability of KIR2DL2/3 expression. As a result, stimulation with IL-2 and K562-mbIL21 feeder cells can be used to accumulate adaptive-like cells and their progenitors with stable inhibitory KIR expression and high cytotoxic potential.
Phagocytic activity of peripheral blood monocytes under in vivo and in vitro hypoxia conditions in tolerant and susceptible to oxygen deficiency rats
Authors: Dzhalilova D. Sh. , Kosyreva A. M. , Tsvetkov I. S. , Makarova O. V.
Published: June 2023

It is known that there are individual differences in resistance to hypoxia, which can determine the predisposition to the development and severity of various diseases, including infectious, inflammatory and tumor. There are no standardized methods for assessing resistance to hypoxia in experimental animals and humans without hypoxic exposure. The search for molecular-biological markers, identifying people with different resistance to oxygen deficiency under normoxic conditions or under moderate hypoxic exposure is undoubtedly efficient. It is possible that the assessment of the basic resistance to hypoxia can help to predict the development and severity of the course of diseases, the mechanisms of which are associated with oxygen deficiency. One of the methods to assess organism resistance to hypoxia without exposure in a decompression chamber or in highland conditions can be modeling hypoxia in vitro. The aim of the study was to characterize the phagocytic activity of peripheral blood monocytes in tolerant and susceptible to hypoxia Wistar rats under normoxic conditions, as well as after hypoxic exposure in vitro and in vivo. The resistance of rats to hypoxia was determined by the gasping time at an altitude of 11.500 m in a decompression chamber. A month after determining the resistance to hypoxia, one group of rats was placed in a decompression chamber at an altitude of 5,000 m for 1 hour to simulate the hypoxic state in vivo. Blood from the tail vein of the other group of rats was placed in 1% oxygen for 1 hour to simulate the hypoxic state in vitro. The phagocytic activity of peripheral blood monocytes was assessed by flow cytometry. It was demonstrated that phagocytic activity of monocytes did not differ in tolerant and susceptible to hypoxia rats under normoxic conditions. The phagocytic activity of monocytes after in vitro and in vivo hypoxic exposure was higher in tolerant to hypoxia animals in comparison to susceptible ones. An increase in the phagocytic activity of monocytes compared to normoxia conditions was observed only in tolerant rats under in vitro conditions of hypoxic exposure. The obtained results indicate that tolerant and susceptible to hypoxia organisms differ in the phagocytic activity of monocytes under conditions of oxygen deficiency, which can determine the course of inflammatory and tumor diseases. The data obtained will be the basis for further experimental investigations organism hypoxia resistance markers.
Spatial characteristics of neutrophils and eosinophils in conducting airway mucosa of mice with induced allergic airway inflammation
Authors: Shevchenko M. A. , Murova D. E. , Servuli E. A.
Published: June 2023

Daily inhaled antigens induce cellular immune response in the airways. In case of allergens, allergic airway inflammation is usually represented by eosinophils, however, neutrophil infiltration is also observed during severe asthma. Animal models contribute to investigation of the mechanisms that involve the switching to eosinophil- or neutrophil-mediated inflammation. Data about the spatial location of eosinophils and neutrophils in the airways are necessary for both the understanding of allergic airway inflammation mechanisms and the drag potential estimation, however, not completely investigated. In the present study, we characterized the model of Aspergillus fumigatus extract-induced allergic airway inflammation that allowed investigating the early stage of inflammation development. The model adequacy was confirmed according to the blood and bronchoalveolar lavage eosinophilia. Using immunohistochemical staining of conducting airway as a whole-mount and confocal laser scanning microscopy, we estimated neutrophil and eosinophil spatial location: in the luminal side of the epithelium, in the airway wall or in the submucosal compartment close to the smooth muscle layer. An allergic airway response activation was detected upon significant elevation of blood eosinophil percentage compared to intact mice. Simultaneously, the number of eosinophils in the bronchoalveolar lavage was also significantly increased compared to the intact mice. At this time point, eosinophils predominated both in bronchoalveolar lavages and in conducting airway mucosa compared to neutrophils. Spatial location of conducting airway mucosal cell analysis demonstrated that eosinophils mostly located in the submucosal compartment, in a lesser extent in the airway wall, and a few eosinophils were detected in the luminal side of the epithelium. Neutrophils mainly infiltrated the luminal side of the epithelium, and a few neutrophils were detected in the submucosal compartment, while no neutrophils were detected in the airway wall. The data suggests that in response to the further allergen challenge, evidently eosinophils but not neutrophils will migrate through the airway wall to the airway lumen. Thus, eosinophils can be expected to damage airway epithelium in allergic airway inflammation development. Simultaneously, neutrophils located in close proximity to the smooth muscle layer together with eosinophils can contribute to bronchoconstriction induction.
Study of phenotypic and cytotoxic properties of erythroid cells of the spleen under hematopoiesis-stimulating effects
Authors: Shevchenko Yu. A. , Nazarov K. V. , Sennikov S. V.
Published: June 2023

In recent years, research has revealed a wide variety of erythroid cell functions, including modulation of innate and adaptive immune responses. Anemic or hypoxic stress stimulates a physiological response in the form of stress erythropoiesis, aimed at increasing oxygen delivery to tissues. Stress erythropoiesis activates progenitor cells and uses mechanisms that differ from stationary bone marrow erythropoiesis. To consider the role of erythroid cells in the regulation of hematopoiesis, hematopoiesis-activating states were modeled: chemically induced hemolytic anemia, acute blood loss, hypoxia. A series of experiments was carried out on first-generation hybrid mice CBA C57Bl6. Isolation of erythroid cells was performed using magnetic separation for the CD71 marker. The stages of differentiation of erythroid cells were determined by the combination of expression of TER-119 and CD71 markers and direct light scattering parameters in the population of both CD45-positive and CD45-negative spleen cells. To study the immunoregulatory activity of erythroid cells, we investigated the mediated cytotoxicity of splenocytes against tumor cells of the mouse melanoma B78 line after cultivation with conditioned spleen media after various hematopoiesis-stimulating effects. With various hemopoiesis-stimulating effects, the quantitative and qualitative composition of the spleen cells is reorganized depending on the compensatory mechanism for restoring homeostasis. An analysis of the cellular composition of the spleen showed that under hematopoiesis-stimulating effects, a redistribution of populations with the CD45 marker occurs: during hypoxia, the number of CD45-negative cells sharply decreases and the number of CD45-positive cells increases. The population of basophilic erythroblasts is the least susceptible to quantitative changes under all hematopoiesis-stimulating effects. During hypoxia, the most noticeable change in the cellular composition of the spleen is observed due to the increased accumulation of CD45-positive erythroid cells in the spleen. Mediators of erythroid cells of the spleen of mice after hypoxia do not lead to an increase in the cytotoxic proapoptotic effect of splenocytes on tumor cells, in contrast to the erythroid cells of the normal spleen, spleen with anemia and blood loss. Thus, it is tissue hypoxia that is the process that not only stimulates erythropoiesis, but also leads to the maximum change in the suppressive properties of surrounding cells. We assume that the implementation of compensatory mechanisms under the studied hematopoiesis-stimulating effects is aimed at activating the mechanisms of innate immunity and local immunosuppression to prevent local inflammation, accumulate nutrients, and attract cellular elements to the focus of hematopoiesis to restore homeostatic functions.