Introduction. Sepsis is multiple organ dysfunction in response to the generalization of an infectious process. The likelihood of developing sepsis correlates with the value of biochemical parameters, which can serve as prognostic markers. The endothelium, as the structural basis of the circulatory system, is primarily exposed to pathogenetic factors, performing a protective function. The purpose of this review was to summarize the results of assessing the diagnostic and prognostic effectiveness of molecular markers of endothelial damage in the development of inflammation, sepsis and septic shock. Material and methods. The literature search was conducted using the databases PubMed/Medline, Elsevier.com., ResearchGate, RSCI/elibrary, over the past ten years. Results. Using keywords, 215 articles were found and analyzed, from which 70 of the most significant and reliable sources were selected. It was concluded that it is necessary to develop a panel of markers of endothelial damage, which together will provide high reliability and specificity for assessing the risk of developing sepsis and septic shock.

Поиск специфических прогностических и диагностических маркеров – актуальная задача современной педиатрии. Эндотелий – активная система, которая функционирует как рецепторно-эффекторный орган, реагирующий на экзогенный или эндогенный стимул с выделением разнообразных молекул, призванных восстановить сосудисто-тканевый гомеостаз. Эндотелий, с одной стороны, участвует практически во всех процессах, определяемых как гомеостаз, гемостаз и воспаление, с другой – это первое звено патогенеза различных заболеваний как у взрослых, так и у детей и подростков. К настоящему времени сформировалось представление об эндотелиальной дисфункции как о состоянии, характеризующемся дисбалансом медиаторов, обеспечивающих в норме оптимальное течение эндотелий-зависимых процессов. В связи с доказанной ролью развития эндотелиальной дисфункции в патогенезе заболеваний сердечно-сосудистой, дыхательной систем, при метаболических нарушениях и т.д. в качестве такого прогностического и диагностического маркера эндотелиальной дисфункции может быть использован эндотелин-1 (EТ-1). Цель исследования: анализ и обобщение данных литературы по базам данных WoS, Scopus, РИНЦ. На основе клинических и экспериментальных данных можно заключить, что применение маркеров эндотелиальной дисфункции, включая уровень эндотелина-1 в сыворотке крови, может использоваться для оценки тяжести социально-значимых заболеваний при их манифестации в детском и подростковом возрасте. The search for specific prognostic and diagnostic markers is an urgent task in modern pediatrics. The endothelium is an active system that functions as a receptor-effectors organ that responds to an exogenous or endogenous stimulus with the release of various molecules designed to restore vascular-tissue homeostasis. The endothelium, on the one hand, is involved in almost all processes defined as homeostasis, homeostasis and inflammation, on the other hand, it is the first link in the pathogenesis of various diseases in both adults, children and adolescents. To date, the idea of endothelial dysfunction as a condition characterized by an imbalance of mediators that normally ensure the optimal course of endothelium-dependent processes has been formed. Due to the proven role of the development of endothelial dysfunction in the pathogenesis of diseases of the cardiovascular, respiratory systems, metabolic disorders, etc. Endothelin-1 (ET-1) can be used as such a prognostic and diagnostic marker of endothelial dysfunction. Purpose of the study: analysis and synthesis of literature data using WoS, Scopus, RSCI databases. Based on clinical and experimental data, it can be concluded that the use of markers of endothelial dysfunction, including the level of endothelin-1 in blood serum, can be used to assess the severity of socially significant diseases when they manifest in childhood and adolescence.

Introduction. Spontaneous abortion and preeclampsia (PE) are the most common pregnancy abnormalities in humans. The renin-angiotensin-aldosterone system (RAAS) and the immune system are among the first to recognize pregnancy and play key roles in its maintenance. RAAS dysfunction is one of the causes of insufficient trophoblast invasion and defect in spiral artery remodeling, which both cause PE. Apart from circulating RAAS components, there are local ones, including uteroplacental ones. The study aimed to analyze the renin and angiotensin-converting enzyme 2 (ACE2) expression in the placenta and uterus in PE-associated spontaneous and immune-dependent abortions in mice. Materials and methods. We reached the physiological pregnancy model by ♀CBA × ♂Balb/c mating (control group); spontaneous PE-associated abortions were ♀CBA × ♂DBA/2. Induced abortions were caused by intraperitoneal β-heptylglycoside muramyldipeptide administration to CBA females fertilized by Balb/c males on gestation days (GDs) 5 and 7. Mice were removed from the experiment on GD 14 after the placenta has completely formed. We performed immunohistochemistry (IHC) of the uterus and placenta using rabbit monoclonal antibodies to renin and ACE2 and evaluated the distribution of the renin- and ACE2-positive cells and the intensity of IHC reaction. Results. Compared to the physiological pregnancy model and immune abortion groups, the spontaneous abortion/PE group was characterized by reduced renin expression in trophoblast giant cells (TGC) and its absence in placental vascular endothelium. We noted increased renin expression in the myometrium and its vascular endothelium in the spontaneous abortion/PE and immune-dependent abortion groups. ACE2 expression in TGC and glycogen cells and placental vascular endothelium was lower in spontaneous/PE and immune-dependent abortions than in the physiological pregnancy group. In contrast, higher ACE2 expression was observed in the myometrium and its vascular endothelium in the abortion groups compared to the controls. Conclusion. Reduced renin expression in TGC and its absence in the placental vascular endothelium of spontaneous abortions/PE group reflects trophoblast invasion disorder characteristic of PE. The detected changes in ACE2 expression in the myometrium in pregnancy failure and PE may represent a systemic compensatory mechanism, which does not exclude the negative effects of renin. Keywords: preeclampsia, pregnancy failure, uterus, placenta, renin-angiotensin-aldosterone system

Individual resistance to hypoxia largely determines the severity of inflammatory processes and, probably, can affect the regeneration of the skin wound.The aim was to study reparative and inflammatory changes in the wound in Wistar rats with different resistance to hypoxia.Material and methods. The study was performed on mature male Wistar rats weighing 300–350 g. Before the experiment, the resistance of animals to hypoxia was determined. Rats were classified as highly resistant to hypoxia (HR), in which the time to take a lateral position in the pressure chamber “at a height” of 11,500 m was more than 360 s (n=5), to low-resistant (LR) – less than 40 s (n=5). A full-thickness wound with a diameter of 16 mm was applied to the animals in the interscapular zone of the back. On the 7th day after the infliction of the wound, a histological and morphometric study was performed. The proliferative activity of cells in the granulation tissue of the wound was determined immunohistochemically using the Ki-67 marker. Real-time PCR was used to evaluate the level of expression of the Hif-1α, Nf-κB, and Vegf genes.Results. It was shown that in rats with high resistance to hypoxia, the total number of cells and, among them, the number of inflammatory cells was higher than in rats with low resistance to hypoxia. However, the percentage of inflammatory cells in the compared groups did not differ significantly. Cell proliferative activity, as measured by the number of Ki-67 positive cells, was higher in rats with high resistance to hypoxia. In the granulation tissue of highly resistant to hypoxia rats, the expression of the Vegf gene was statistically significantly higher than in low-resistant animals, while there were no statistically significant differences in the level of expression of the Hif-1α, Nf-κB genes.Conclusion. In rats with high resistance to hypoxia, on the 7th day of healing of a full-thickness skin wound, reparative processes in it are more pronounced. In the horizontal layer of fibroblasts, there are more cells, among them inflammatory ones - histiocytes and macrophages, which is combined with a higher number of proliferating Ki-67 positive cells and the expression level of vascular endothelial growth factor (Vegf).

This study was motivated by the pressing need to obtain monoclonal antibodies (mAb) for research and medical purposes. Screening for mAb-producing hybridomas and evaluating various cell cultures requires a technique that combines the advantages of immunohistochemistry (IHC), heterogeneous enzyme immunoassay (ELISA), and Western blotting. Cellular ELISA (In-Cell ELISA, ICE) is valid for in situ measurement of target analytes in both attached and suspended cells with minimal time, specificity, and reproducibility of ELISA. This method can be successfully used to detect surface and intracellular antigens, as well as to assess phosphorylation, methylation, and acetylation. ICE is a simple, fast, inexpensive, and highly sensitive alternative to various common cyto- and histochemical methods. Here, we explored the potential of ICE for screening and selecting the most promising hybridomas and mAbs for IHC staining. The experiments were carried out with the help of the cultural method, the method of heterogeneous and cellular ELISA, and IHC staining. Comparison of ICE and IHC showed a significant agreement in the intensity and localization of staining with the studied mAbs. The most suitable antibodies for subsequent IHC were selected, saving us much time of selection and the number of expensive mAbs. In heterogeneous ELISA, a negative reaction was noted in some cases. In ICE of whole cells, a pronounced response was detected microscopically. With ICE, it is possible to characterize cell cultures by markers that distinguish one cell line from another, show the stages of cell differentiation, and visualize their structures at different stages of cultivation. In some cases, ICE may be more informative than ELISA for selecting monoclones that produce specific mAbs. A preliminary ICE run can be used to determine the optimal concentration of working dilutions of primary antibodies in a subsequent IHC run.

We studied the expression of pluripotency factor Oct-4 and the intensity of apoptosis in the uterus during spontaneous and immune abortions in mice. Increased expression of factor Bax and reduced protein Bcl-2 synthesis in cells of the decidual membrane and decreased Oct-4 expression in the myometrium and perimetrium were detected. Thus, both spontaneous and immune-dependent abortions impair the apoptosis processes in the decidua and the formation of a pool of Oct-4+ cells in the uterus. In immune-dependent abortions, the intensity of apoptosis of decidual cells was lower than in spontaneous abortion. Low expression of the transcription factor Oct-4 in the myometrium and perimetrium characterizes pregnancy failure irrespective of its mechanisms.

This study was motivated by the pressing need to obtain monoclonal antibodies (mAb) for research and medical purposes. Screening for mAb-producing hybridomas and evaluating various cell cultures requires a technique that combines the advantages of immunohistochemistry (IHC), heterogeneous enzyme immunoassay (ELISA), and Western blotting. Cellular ELISA (In-Cell ELISA, ICE) is valid for in situ measurement of target analytes in both attached and suspended cells with minimal time, specificity, and reproducibility of ELISA. This method can be successfully used to detect surface and intracellular antigens, as well as to assess phosphorylation, methylation, and acetylation. ICE is a simple, fast, inexpensive, and highly sensitive alternative to various common cyto-and histochemical methods. Here, we explored the potential of ICE for screening and selecting the most promising hybridomas and mAbs for IHC staining. The experiments were carried out with the help of the cultural method, the method of hetero-geneous and cellular ELISA, and IHC staining. Comparison of ICE and IHC showed a significant agreement in the intensity and localization of staining with the studied mAbs. The most suitable antibodies for subsequent IHC were selected, saving us much time of selection and the number of expensive mAbs. In heterogeneous ELISA, a negative reaction was noted in some cases. In ICE of whole cells, a pronounced response was detected microscopically. With ICE, it is possible to characterize cell cultures by markers that distinguish one cell line from another, show the stages of cell differentiation, and visualize their structures at different stages of cultivation. In some cases, ICE may be more informative than ELISA for selecting monoclones that produce specific mAbs. A preliminary ICE run can be used to determine the optimal concentration of working dilutions of primary antibodies in a subsequent IHC run.

Introduction. Prostate cancer (PC) leads the world structure of male cancer mortality. Understanding the prognosis for PC patients is vitally important and requires diagnostic enhancements such as developing biomarker panels. The studyaimed to estimate sensitivity, specificity, and prognostic significance of the level of immunoglobulin G (IgG) proteolysis products in the blood serum depending on PC Gleason score and detect how the levels of IgG proteolytic fragments were associated with the diagnostic marker expression. Materials and methods. The study included 90 PC patients. We used the Gleason score to evaluate tumor grade. The level of the IgG proteolysis products and total prostate-specific antigen (PSA) in the serum was detected with ELISA. We performed an immunohistochemical assay with Ki-67, plasminogen-binding protein enolase-1 (ENO-1), and prostate-specific membrane antigen (PSMA). Results. ROC analysis demonstrated 100% specificity of the IgG proteolysis products test regardless of Gleason score, while the sensitivity reached 72.2%, 57.5%, 52.6%, and 46.2% for ISUP stages 1, 2, 3, and 4, respectively. An intergroup comparison showed significant differences between patient groups according to Gleason score, preoperative PSA and IgG proteolysis product serum levels and Ki-67 index. We detected a positive correlation between the level of IgG proteolysis products in the PC patients’ serum and ENO-1 expression and a negative correlation with the Gleason score and PSMA expression. Conclusion. Preoperative detection of high levels of proteolytic IgG fragments in the serum of PC patients even with low Gleason score and PSMA expression may help predict a more aggressive disease course and choose the management strategy. Keywords: prostate cancer, plasminogen activation system, proteolytic fragments of immunoglobulin G, oncomarkers

There are individual differences in the tolerance to hypoxia and stress. Stress can contribute to the development of various diseases, including inflammatory bowel disease. It was found that inflammatory bowel diseases in animals susceptible to hypoxia runs more severe course than in tolerant animals. We studied morphofunctional changes in the colon under conditions of modeled cold stress in male C57BL/6 mice susceptible and tolerant to hypoxia. The animals were daily subjected to cold stress (20 min at -20°C) for 2 weeks. Cold stress was followed by an increase in the volume fraction of goblet cells in the colon and production of mucins by these cells in mice tolerant to hypoxia and an increase in cell content in the lamina propria of the colon mucous membrane in animals susceptible to hypoxia. The number of serotoninproducing endocrine cells increased in both groups, but these changes were more pronounced in mice susceptible to hypoxia.

Introduction. Obtaining primary cultures of fibroblasts is necessary for conducting experimental studies to investigate the basic cell response mechanisms to various stimuli. Despite the widespread use of fibroblast cultures, methods for obtaining them from the skin are not standardized. The aim of the studywas to find the optimal conditions to obtain a fibroblast culture from rat skin using enzymatic tissue disaggregation. Materials and methods. The fibroblast culture was obtained from the dermis of 18 male Wistar rats (N=12, 5–6-week-old, and 60–65 g body weight; N=6, 8–10-week-old rats, and 160–180 g body weight). A series of experiments was carried out to obtain a fibroblast culture with selecting the collagenase type and defining its concentration and exposure time. Results.An optimized protocol to obtain rat dermis fibroblasts is presented. Moreover, the problems of obtaining a culture and practical aspects of its use are discussed. Conclusion. When obtaining a primary culture of rat skin fibroblasts we should consider the following fac-tors: the type of the enzyme, its concentration, and exposure time; the age of the animals; the area of skin graft collection. In young rats, the optimal result was achieved when the cells were isolated from the axillary zone using collagenase type II at a 1 mg/ml concentration during a 90-minute exposure. In adult animals, the enzymatic effect of collagenase type II on the skin graft obtained from the back was optimal at a 5 mg/ml concentration during a 120-minute exposure. Keywords: fibroblast culture, dermis, preparation protocols, rats

We studied daily dynamics of proliferative activity of embryonic fibroblast-like cells in culture during the logarithmic growth phase. Daily increase in cell count in the culture showed a 4-day rhythm that persisted over 3 weeks of culturing. In cultures from different animals, the phase of this rhythm was synchronous and did not depend on the moment of cell isolation. It can be hypothesized that the 4-day biorhythm of proliferative activity of embryonic fibroblast-like cells in culture is determined by external environmental factors, probably of electromagnetic nature.

Introduction. The majority of cases of habitual miscarriage are closely related to impaired maternal immune tolerance. Experimental mice models of abortion, where the critical time points are established to monitor pregnancy progression, are widely used to study the pathogenesis of early reproductive loss. However, the relationship between the embryonic development and extraembryonic structures with the immune microen-vironment is not well understood. The aim of the study is to compare the morphofunctional characteristics of the uteroplacental unit and embryo in early pregnancy in mice with experimental spontaneous, induced, and potentiated abortions. Materials and methods. Allogeneic physiological pregnancy was modeled using ♀CBA × ♂BALB/c mice mating. Spontaneous abortions were reproduced by mating ♀CBA × ♂DBA/2 mice. Induced abortions and potentiated abortions were triggered by intraperitoneal administration of the immunomodulator ß-heptylglycoside muramyl dipeptide (MDP) to pregnant females on gestation day (gd) 5 and gd 7 mated in following combinations ♀CBA × ♂BALB/c and ♀CBA × ♂DBA/2, respectively. Mice were withdrawn from the experiment on gd 8, then uterine horns and uteroplacental units were isolated. The material was fixed in 10% neutral formalin, and standard processing was performed for histological examination. In equatorial sections, the cross-sectional area of uteroplacental unit was measured, and maternal, embryonic, as well as extraembryonic structures were analyzed. An immunohistochemical assay with rabbit polyclonal anti-CD68 antibodies was conducted to detect macrophages, then the density of CD68+ cells per 1 mm2 of the decidual area was determined. Results. On day 8 of pregnancy, in the groups of induced and potentiated abortions cross-sectional area of the uteroplacental units was decreased compared with the groups of physiological pregnancy and sponta-neous abortions. Induced and potentiated abortions demonstrated vasodilation in the mesometric decidua zone and stagnation of blood in the blood vessels, compaction of trophoblast, and hemorrhage in decidua basalis whether spontaneous abortions showed occluded maternal vessels surrounded by edematous and vacuolated trophoblast cells.At the early stages of modeling of spontaneous and immuno-dependent abortions, disturbances in the devel-opment of the embryo and extraembryonic structures were revealed, ranging from initial signs of resorption to the destruction of the embryo and membranes.In viable embryos, density indices of CD68+ macrophages in the decidual membrane of the uteroplacental units did not differ significantly in all miscarriage cases, CD68+ cells were not detected in cases of embryo-nal resorption. Conclusion. The MDP treatment in a period after completion of implantation and before forming a mature placenta (gd 5–7) results in the inducing or the potentiating abortions in CBA females mated with by Balb/c or DBA/2 males, respectively. In both cases, we revealed disturbed chorion morphogenesis and embryonic development to the extent of its complete resorption and the absence of CD68+ macrophages in resorbed embryos’ microenvironment on day 8 of pregnancy. Keywords: miscarriage, models, muramyl dipeptide β-heptylglycoside, uteroplacental unit, macrophages, immunotolerance

The features of B16 melanoma progression in male C57BL/6 mice with initially high and low resistance to hypoxia were studied. To assess the resistance to hypoxia, the mice were placed in a low-pressure chamber at a simulated altitude of 10,000 m. One month after testing, B16 melanoma was inoculated to high- and low-resistant animals. In 19 days after melanoma transplantation, the severity of melanoma progression was assessed by morphological and immunofluorescent methods. The expression of vegf-a and hif-1a in the liver of melanomabearing and control mice was evaluated by real-time PCR. Tumor growth progression was more pronounced in low-resistant mice, which was seen from high weight of the primary tumor node, relative necrosis area, proliferation rates (mitotic index and number of Ki-67+ cells), and expression of vegf-a gene in the liver. In high-resistant to hypoxia animals, the number of caspase-3+ cells dying by apoptosis was higher. The data on more rapid melanoma progression in mice with low resistance to hypoxia should be considered during the search of new prognostic markers and methods for therapy of malignant neoplasms.

The rhythms of short-term arousal episodes, associated with normalization of low body temperature, were studied in hibernating Erinaceus roumanicus. The episodes of body temperature recovery during hibernation were 1.7 times more incident during the acrophase of 4.058-day rhythm of glucocorticoid hormones, detected previously, than during the batiphase of this rhythm. Ultradian rhythm of arousal episodes conformed to a 4-h biorhythm: the maximum number of body temperature resumption episodes was recorded at 00.00-01.00, 04.00-05.00, 08.00-09.00, 12.00-13.00, 16.00-17.00, and 20.00-21.00. These data indicated that in mammals the mechanisms of infradian and ultradian rhythm maintenance were stable and did not depend on body temperature or were determined by external factors with periods of 4.058 days and 4 h.

ФГБНУ «Научно-исследовательский институт морфологии человека», Москва, Россия Введение.Для изучения биохимических и молекулярно-биологических механизмов процессов сперматогенеза в норме и при его нарушениях необходимо получить изолированные клетки сперматогенного эпителия разных стадий дифференцировки.Клетки сперматогенного эпителия соединены между собой плотными контактами и цитоплазматическими мостиками, разрушение которых позволяет получить суспензию клеток.Для разделения клеток используют ряд методов, включая механический и ферментативные.Состав, концентрация ферментов и время воздействия, применяющиеся разными авторами, широко варьируют.Тем не менее работы по сравнительной оценке эффективности методов выделения клеток сперматогенного эпителия в литературе отсутствуют

Post-traumatic stress disorder (PTSD) was modeled by keeping mature male Wistar rats in conditions of unavoidable exposure to the odor of a predator (cat urine) for 10-min periods for 10 days followed by a twoweek period of being kept in standard conditions. Anxiety was assessed by testing in an elevated plus maze and in a novel model allowing the animal to demonstrate voluntary locomotor activity in a social context. The consequences of stress in the short- and long-term periods on behavior, the repertoire of psychoemotional expressions, and the serum corticosterone level were studied. The behavioral and psychoemotional signs of the development of PTSD were accompanied by decreases in corticosterone levels. Important data were obtained on retention of infradian rhythms of locomotor activity during and after exposure to stress.

Background: Estrogen receptors beta (ERβ) are highly expressed in different normal and neoplastic tissues that, until recently, has been considered to be ER-negative based on ERα expression evaluation. Among the genes, regulated via estrogen signaling there is one, coding microtubule protein beta-III tubulin (TUBB3). TUBB3 expression is found in many solid tumors and is linked to poor prognosis and resistance to taxanes. Since it is little known about mechanisms behind TUBB3 expression in non-small cell lung cancer (NSCLC), we decided to find out if there is a correlation between ER and TUBB3 expression in this type of cancer. Methods: 104 surgical samples of NSCLC were converted to single-cell suspension, stained with primary anti-ERα (abSP-1), anti-ERβ (ab14C8), anti-TUBB3 (ab7751) antibodies and secondary fluorescent antibodies. Immunofluorescent estimation was performed using flow cytometry. Expression level was determined as the ratio (%) of specifically fluorescent cells to the number of cells stained with secondary antibodies. Spearman rank correlation was used to test the association between variables. Results: Both ER were revealed in all NSCLC specimens. Mean expression level of ERβ was significantly higher compared with ERα (46,6 ± 17,0% vs 23,2 ± 14,2%, respectively). Mean TUBB3 expression level was 43,1±15,7%. In all the tumors investigated only weak correlation observed between and ER status and TUBB3 expression level (rs = 0,3 and rs = 0,4 for ERα and ERβ, respectively). In the group of squamous cell cancer specimens (n = 68) the association was strong (rs = 0,5 and rs = 0,5 for ERα and ERβ, respectively). In the group of adenocarcinoma specimens (n = 36) the correlation between ERα and TUBB3 was very weak (rs = 0,3) and there was no correlation between ERβ and TUBB3. Conclusions: 1. Strong correlation between TUBB3 and ER expression was found only in squamous cell cancer tissue. 2. The dominant type of estrogen receptors is ERβ. 3. In clinical terms high ERβ expression means that in case of resistance to standart platinum/taxane duplets, patients with high tumor ERβ expression may benefit from antiestrogen therapy. Supported by RFBR grants (№№15-04-06991-а, 16-34-01049-mol-a) and grant of the President of RF МК-7709.2016.7. Legal entity responsible for the study: N.N. Blokhin Russian Cancer Research Center. Funding: Russian Foundation for Basic Research Grants, grant of the President of Russian Federation. Disclosure: All authors have declared no conflicts of interest.