Vol 164, No 4 (2018)

Pumping function of the heart ventricles under conditions of electrical stimulation was examined in adult dogs and rabbits. Pacing induced different changes in intracardiac hemodynamics manifested in impairment of pumping function of the right ventricle, which is largely determined by the functional state of the left ventricle. Initially high HR in rabbits more deeply limited functional reserve of the myocardium in response to electrical stimulus and was accompanied by more pronounced disturbances of pumping function of both ventricles.

A quantitative study of lymphoid cells in the B- and T-cell-dependent areas of intestinal lymphoid nodules and mesenteric lymph nodes in behaviorally passive and active rats was performed at various periods after acute stress on the model of 1-h immobilization with simultaneous electrocutaneous stimulation. Stress exposure is accompanied by a decrease in the number of lymphoid cells in immunogenic structures of the gastrointestinal tract. Post-stress changes in the cytoarchitectonics of B- and T-cell-dependent areas in mesenteric lymph nodes of animals are less pronounced than in lymphoid nodules. Quantitative changes in lymphoid cells of B-cell-dependent areas in the small intestine of rats are greater than in T-cell-dependent areas. Changes in the cellular composition of immunogenic structures in the digestive system are most significant at the early stages of the post-stress period (1st week). Passive rats are characterized by significant changes in the cytoarchitectonics of B- and Tcell-dependent areas in the small intestine after extreme exposure, which illustrates functional exhaustion of the lymphoid tissue in stress-predisposed specimens.

Echocardiographic parameters were assessed in patients with non-ST segment elevation acute coronary syndrome, who underwent emergency percutaneous coronary intervention followed by various outpatient physical cardiac rehabilitation programs. The patients underwent physical rehabilitation for 3 months under conditions of diagnostic centre in the rehabilitation unit according to the standard program including in treadmill or bicycle exercise in the exercise therapy room or with Nordic walking in the main training block. After rehabilitation course, the left ventricular mass index significantly decreased and systolic volume and left ventricular ejection fraction significantly increased in both groups. Nordic walking training for 3 months non-ST segment elevation acute coronary syndrome induced similar positive shifts in the parameters of intracardiac hemodynamics, as standard treadmill or bicycle training program, which allows considering it as an alternative cardiac rehabilitation method.

Sex differences in the expression of iodide transporter SLC5A5 and thyroid peroxidase in thyroid follicular epithelium and thyroid serum profile were assessed in pubertal rats exposed to endocrine disruptor DDT starting from the first postnatal day. It was found that exposure to DDT reduced expression of SLC5A5 in peripheral regions of thyroid lobes in males and in central regions in females. The most pronounced sex differences were observed in thyroid peroxidase expression that remained sensitive to thyroid stimulating hormone regulation in males and lost sensitivity to pituitary stimulation in females after exposure to disruptor, which determines more pronounced hypothyroidism in females.

We studied the effect of α-tocopherol on gentamicin-induced morphological and functional changes in the kidneys of Wistar rats. Special attention was paid to the ability of α-tocopherol administered in combination with gentamicin to correct ultrastructural changes in the glomerular basal membrane and tubules. Combined treatment with α-tocopherol (100 mg/kg) and gentamicin (100 mg/kg) led to correction of histopathological and biochemical changes and oxidative injury to the kidneys induced by this antibiotic.

The incidence of mono- and multinuclear cells and their expression of pro- and antifibrotic factors were studied in cultured peritoneal macrophages from intact and BCG-infected mice. Generally, the expression of factors increased with an increase in the number of nuclei per cell. However, the expression was higher in macrophages from BCG infected mice, except the cells with 3 and more nuclei, extremely rarely expressing IL-1α in cultures from intact and BCG-infected animals. The number of macrophages with 3 and more nuclei, expressing CatD, was comparable with the number of mono- and binuclear macrophages. Presumably, this was determined by various mechanisms of formation of multinuclear (3-5 and more nuclei) macrophages, for example, by amitosis.

We studied the role of endogenous melatonin in the development and functioning of T cells that produce IL-17 (Th17) and regulatory T cells (Treg) during pregnancy. The study was performed ex vivo and in vitro with auto-serum as the source of endogenous melatonin under conditions of blockade of melatonin-dependent signaling. Participation of the hormone in the regulation of differentiation of both CD4⁺RORγt⁺ and CD4⁺FoxP3⁺T cells and their key products IL-17A and TGF-β was demonstrated. It is known that the normal gestational process is accompanied by a decrease in Th17/Treg ratio due to hormonal changes. The sensitivity of the studied subpopulations to melatonin during pregnancy can affect its outcome.

Posttraumatic stress disorders were induced in Wistar rats by modeling predatory stress. Animals tested in elevated plus-maze demonstrated signs of high anxiety and difficulties in space orientation. Behavioral disorders were paralleled by degenerative changes in hepatocytes. A course of low-dose high-molecular heparin after stress exposure promoted effective adaptation and normalization of behavioral and morphofunctional disorders. Analysis of the results indicated good prospects of heparin as an effective drug under conditions of exposure to extreme factors of psychotraumatic type.

We studied peculiarities of the structure of human entorhinal cortex at weeks 20-26 of gestation (10 hemispheres). The samples were Nissl-stained and immunohistochemically treated with antibodies to parvalbumin, calretinin, calbindin, and cytoskeleton proteins (MAP2 and N200). 3D-reconstruction of the entorhinal cortex from serial sections was performed, caudomedial and rostrolateral areas were isolated. Parvalbumin⁺ cells in layer I, discrete distribution of layer II cells with colocalization of MAP2 and calretinin at the border with layer I, and two sublayers Va and Vb with MAP2⁺ neurons were typical for the caudomedial area. Rostrolateral area was characterized by the homogenous layer II with big amount of cells, high density of MAP2⁺ neurons only in layer III, and the unique layer V. Reelin⁺ Cajal—Retzius cells and N200⁺ fiber plexus in layer I were observed in the caudomedial and rostrolateral areas of the entorhial cortex. Layer IV was represented by a cell-free desiccant.

The study examined the myocardial ultrastructural alterations in rats maintained on various atherogenic diets. It revealed the complex ultrastructural alterations of cardiomyocytes and endotheliocytes (including the lytic and destructive changes of the intracellular organelles, upregulation of the autophagocytosis in the cardiomyocytes, and necrobiosis with apoptosis of endotheliocytes) reflecting the cytopathic features of circulating cholesterol and lipoproteins, whose elevation determined the intensity of destructive processes. The revealed peculiarities in the changes of lipid inclusions (their osmiophilic transformation) in cardiomyocytes can be provoked by entry of cholesterol into the cells and its further metabolic modifications. During moderate dyslipidemia, the cardiomyocytes demonstrated the ultrastructural signs of induction of intracellular regeneration (marked with the clusters of polysomes in the intermyofibrillar and subsarcolemmal spaces with appearance of neogenic myofilaments) and upregulated pinocytotic activity. In all cases, up-regulated autophagocytosis in cardiomyocytes was accompanied by accumulation of myelin- and vacuole-like structures in the intercellular spaces and capillary lumens paralleled with appearance of activated forms of macrophages and fibroblasts in the myocardium.

We performed screening of nootropic properties of 10 new derivatives of quinolizidine alkaloid (-)-cytisine. Compounds with β-endo stereochemistry were more active than α-endo-isomers. Under stress conditions (3aR,4S,8S,12R,12aS,12bR)-10-methyl-2-phenyloctahydro-1H-4,12a-etheno-8,12-methanopyrrolo[3’,4’:3,4]pyrido[1,2-a] [1,5]diazocine-1,3,5(4H)-trione enhanced memory and had a positive effect on cognitive functions of rats. According to molecular docking data, the nootropic activity of the compound can be associated with its affinity for the glutamate-binding subunits GluK1 and GluR2 of the kainate and AMPA receptor, respectively.

We studied the effect of autologous and allogeneic lymphocytes on multipotent mesenchymal stromal cells in co-culture. It is shown that changes in multipotent mesenchymal stromal cells and in lymphocytes did not depend on the source of lymphocytes. Contact with lymphocytes triggers expression of HLA-DR molecules on multipotent mesenchymal stromal cells and these cells lose their immune privilege. In multipotent mesenchymal stromal cells, the relative level of expression of factors involved in immunomodulation (IDO1, PTGES, and IL-6) and expression of adhesion molecule ICAM1 increased, while expression of genes involved in the differentiation of multipotent mesenchymal stromal cells remained unchanged. Priming of multipotent mesenchymal stromal cells with IFN did not affect these changes. In turn, lymphocytes underwent activation, expression of HLA-DR increased, subpopulation composition of lymphocytes changed towards the increase in the content of naïve T cells. These findings are important for cell therapy.

We studied the influence of the type and structure of polyethyleneimine on bioavailability and expression of plasmid DNA carrying IGF-1 gene. Polymers with different molecular weights (2.5, 10, 25, and 60 kDa) of linear and branching structure were studied. It was found that the time of polyplex circulation in the blood did not exceed 24 h and the maximum concentration of plasmid DNA was attained with complexes with a molecular weight of 60 kDa. Analysis of liver samples showed that administration of 60-kDa branched polyethyleneimine complex provides DNA protection from degradation for 4 h; in 24 h from the start of the experiment, its concentration was significantly higher than the concentration of other studied polyethyleneimines. The expression of plasmid IGF-1 DNA for this complex attained maximum in 4 h and was equal to 15.50 (7.98; 21.98) arb. units/ml. These results allow us to recommend using polyethyleneimines with branched structure and a molecular weight of 60 kDa for improving plasmid DNA protection and bioavailability.

The effect of apoptotic endonuclease EndoG on alternative splicing of mRNA of human telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) and telomerase activity in normal human lymphocytes were studied. Human CD4⁺, CD8⁺, B, and NK cells were transfected with a plasmid pEndoG-GFP containing EndoG gene or control plasmid pGFP. The levels of mRNA of EndoG or hTERT splicing variants were analyzed by real-time PCR. Protein content was assessed by Western blotting. Telomerase activity was measured by the telomere repeats amplification protocol. EndoG overexpression reduced the expression of active full-length hTERT and increased the expression of inactive splice variant. Shifted balance of hTERT splice variants in cells led to a significant decrease in telomerase activity within 72 h after transfection.

Immunohistochemical assay was employed to determine localization of MMP-2 in cardiomyocytes of WAG rats and changes in MMP-2 expression during modeled cardiomyopathy induced by single intraperitoneal injection of cyclophosphamide (125 mg/kg) alone or in combination with preventive intraperitoneal administration of an equal dose of asparcam-L (potassium-magnesium asparaginate) 30 min prior to the cytostatic. In the myocardium of control and experimental rats, MMP-2 was mostly located in cardiomyocyte nuclei. During the development of cyclophosphamide-induced cardiomyopathy (in 3 days after injection), the index of MMP-2-positive cardiomyocyte nuclei increased by 76%. In contrast to control hearts, MMP-2 was also expressed in the cardiomyocyte sarcoplasm. Preventive injection of asparcam-L moderated the cardiotoxic effect of cyclophosphamide, which manifested in less pronounced increase in the volume density of cardiomyocytes with lytic changes (by 42%) and index of MMP-2⁺ cardiomyocyte nuclei (by 23%) in comparison with the rats exposed to cyclophosphamide alone.

Indocyanine green fluorescence was used for evaluation of the degree of trachea revascularization after its autotransplantation in rabbits (transplantation of 1- and 2-cm segment of the trachea). Intravenous administration of indocyanine green was followed by a significant fluorescence of the substance in microvessels of the trachea over 30-40 sec. Immediately after surgery, fluorescence in the implanted segment was absent, but within 7 days it was completely restored in rabbits receiving transplantation of a 1-cm segment. After transplantation of 2-cm segment of the trachea, fluorescence did not recover, which corresponded to the clinical picture and autopsy results. Thus, fluorescent angiography is an informative method for evaluation of trachea revascularization.

We developed an optimal protocol for preparing and culturing of olfactory ensheathing cells from human olfactory mucosa. Using this protocol, we obtained a culture enriched with human olfactory ensheathing cells. Immunofluorescence analysis by simultaneous expression of GFAP and p75NTR markers showed that the content of ensheathing cells was maximum in passage 3 and 4 cultures (94 and 89.5%, respectively). The developed protocol can be recommended for obtaining autologous preparations of human ensheathing cells for cell therapy of spinal cord injuries.

We developed and tested a method for fabrication of a biosensor chip based on native human whole blood lymphocyte and a titanium tipless cantilever. The biosensor can be used for measuring intermolecular adhesion forces in the cell—cell system by using atomic force spectroscopy. The developed biosensor chip was applied for measuring adhesion force between lymphocyte and granulocyte and between lymphocyte and erythrocyte in healthy individuals and in patients with acute lymphoblastic leukemia (before treatment, during standard treatment, and during relapse). It was found that adhesion force between lymphocyte and granulocyte and between lymphocyte and erythrocyte increased almost twice during relapse, which is an important diagnostic marker of early cytological abnormalities indicating progression of the disease.

We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures at early passages exceeds 90%. Typical morphofunctional signs of myogenic differentiation disturbances were identified and their dynamics was studied. Signs of alternative adipogenic and chondrogenic differentiation of cells were revealed. Based on these data, limitations for the use of myoblast cultures of certain passages for biomedical research and cell therapy were evaluated.

We studied the expression of MMP-2, MMP-9, and inhibitor TIMP-1 in myocardial autopsy samples from subjects of different age and in cardiomyocyte cultures in the norm and in dilated cardiomyopathy. In autopsy samples of normal myocardium and in cardiomyocyte cultures, expression of molecules involved in extracellular matrix remodeling did not change during aging. In dilation cardiomyopathy, expression of MMP-2, MMP-9, and TIMP-1 and their ratios in autopsy material and in cultures was elevated by 1.5-9 times. Remodeling of extracellular matrix plays an important role in the pathogenesis of dilated cardiomyopathy. MMP-2, MMP-9, and their inhibitor TIMP-1 and the MMP/TIMP ratios can be regarded as promising predictors of dilated cardiomyopathy and used for evaluation of the effectiveness of treatment of this conditions in patients of different ages.

We isolated and characterized cultures of bone and cartilage tissue cells of laboratory minipigs. The size and morphological features of adherent osteogenic and chondrogenic cells were specified. During long-term culturing under standard conditions, the studied cultures expressed specific markers that were detected by immunohistochemical staining: alkaline phosphatase and calcium deposits in osteoblasts and type II collagen and cartilage extracellular matrix in chondrogenic cells. Proliferative potential (mitotic index) of both cell types was 4.64% of the total cell number. Cell motility, i.e. the mean velocity of cell motion was 49 pixels/h for osteoblasts and 47 pixels/h for chondroblasts; the mean migration distance was 2045 and 2118 pixels for chondroblasts and osteoblasts, respectively. The obtained cell lines are now used as the control for evaluation of optimal biocompatibility of scaffold materials in various models. Characteristics of the motility of the bone and cartilage tissue cells can be used for modeling and estimation of the rate of cells population of 3D scaffolds made of synthetic and biological polymers with different internal structure and physicochemical properties during designing in vitro tissue implants.

We compared survival of bone marrow mesenchymal stem cells after compressor, ultrasound, and mesh nebulization of the cell suspension over 10 min. Viability of stromal cells was best preserved after compressor nebulization (72%). Cell survival after ultrasonic nebulization was significantly lower (20%). After mesh nebulization, no live cells were found. Thus, compressor nebulization is the most preferable method of the production of cell aerosol for their delivery to the lower respiratory tract.