Changes in Oncogene Expression in Experimental Glioblastoma 101.8 Rats during Therapy with PLGA Nanoparticles Loaded with Doxorubicin.

We compared the expression of the main glioblastoma oncogenes during therapy with doxorubicin (Dox) and Dox in nanoparticles based on a copolymer of lactic and glycolic acids (Dox-PLGA) at a delayed start of treatment. Late initiation of Dox-PLGA therapy of glioblastoma showed an increase in the expression of multiple drug resistance genes, such as Abcb1b and Mgmt, and a decrease in Sox2 expression. Increased expression of other oncogenes (Melk, Wnt3, Gdnf, and Pdgfra) were observed during both Dox and Dox-PLGA therapy. These changes indicate increased tumor aggressiveness and its resistance to cytostatics at the late start of therapy.

Morphofunctional Characteristics of Lung Macrophages in Rats with Acute Respiratory Distress Syndrome.

A comprehensive morphofunctional study of the lungs and alveolar macrophages was carried out in Sprague-Dawley rats with acute respiratory distress syndrome (n=10) induced by intratracheal administration of E. coli LPS 0111:B4 in a dose of 15 mg/kg. On the first day after LPS administration, bronchopneumonia was observed in the lungs, the number of macrophages of the bone marrow origin and the number of M1 macrophages with the proinflammatory phenotype in the bronchoalveolar lavage increased, the expression of proinflammatory cytokines increased and the expression of anti-inflammatory cytokines decreased, which was accompanied by an increase in LPS and C-reactive protein in the blood serum. The revealed changes correspond to the development of acute respiratory distress syndrome in humans, and the decrease in the number of macrophages in the lungs and their predominant polarization to the M1-proinflammatory phenotype substantiate the use of cell therapy with reprogrammed M2 macrophages.

Gene Expression Profile of 3D Spheroids in Comparison with 2D Cell Cultures and Tissue Strains of Diffuse High-Grade Gliomas.

The use of relevant, accessible, and easily reproducible preclinical models of diffuse gliomas is a prerequisite for the development of successful therapeutic approaches to their treatment. Here we studied the gene expression profile of 3D spheroids in a comparison with 2D cell cultures and tissue strains of diffuse high-grade gliomas. Using real time PCR, we evaluated the expression of Gfap, Cd44, Pten, S100b, Vegfa, Hif1a, Sox2, Melk, Gdnf, and Mgmt genes playing an important role in the progression of gliomas and regulating tumor cell proliferation, adhesion, invasion, plasticity, apoptosis, DNA repair, and recruitment of tumor-associated cells. Gene expression analysis showed that 3D spheroids are more similar to tumor tissue strains by the expression levels of Gfap, Cd44, and Pten, while the expression levels of Hif1a and Sox2 in 3D spheroids did not differ from those of 2D cell cultures, the expression levels S100b and Vegfa in 3D spheroids was higher than in other models, and the expression levels of Melk, Gdnf, and Mgmt genes changed diversely. Thus, 3D spheroid model more closely mimics the tumor tissue than 2D cell culture, but still is not the most relevant, probably due to too small size of spheroids, which does not allow reproducing hypoxia and apoptotic and necrotic processes in the tumor tissue.

Calculation of the Biological Efficiency of the Proton Component from 14.8 MeV Neutron Irradiation in Computational Biology with Help of Video Cards.

Fast neutron therapy, which previously has demonstrated effective results, but along with a large number of complications, can again be considered a promising treatment method in the treatment of cancer. One of the ways of analyzing the relative biological efficiency and accurate biological dose of fast neutrons in body tissues is to improve the algorithms of computational biology and mathematical modeling. A high-performance computing code was written which allows to estimate in real-time mode the biological dose of the proton component from the action of neutron radiation with an energy of 14.8 MeV. A comparative analysis of the computing performance on various video cards was also performed.

Differential Markers of Subpopulations of Epithelial Cells of the Larynx in Squamous Cell Carcinoma.

In squamous cell carcinoma of the larynx, the population of epithelial cells in the tumor tissue is initially heterogeneous and, in addition to tumor cells invading the organ mucosa, includes normal epithelial cells of protein-mucous glands and cells of the stratified epithelium covering the mucous membrane. A search for differential markers to separate these subpopulations was carried out. The surface marker CD44 and cytokeratins 5 and 17 that are often used to verify carcinoma cells, are common markers for all epithelial cells of the larynx. In highly differentiated carcinoma, subpopulations of normal and tumor epithelial cells can be separated by the level of expression of cytokeratins 10 and 18 and nuclear markers Ki-67 and p63. However, in moderately differentiated carcinoma, tumor cells and normal cells of the basal layer of the stratified epithelium covering the mucous membrane of the larynx have similar phenotypes, which should be taken into account when conducting experimental studies.

Influence of Sucrose on the Efficiency of Cryopreservation of Human Umbilical Cord-Derived Multipotent Stromal Cells with the Use of Various Penetrating Cryoprotectants.

We studied the influence of sucrose applied in combination with different concentrations of penetrating cryoprotectants (DMSO, ethylene glycol, and glycerol) on the efficiency of cryopreservation of umbilical cord-derived multipotent stromal cells (MSC). The results indicate that these cells can be cryopreserved with the use of 5-10% DMSO or ethylene glycol with equal efficiency; addition of 0.2 M sucrose does not affect cell survival after thawing. The efficiency of glycerol as a cryoprotectant increases with increasing its concentration from 5 to 10%, but remains significantly lower than the efficiency of DMSO or ethylene glycol. Addition of sucrose to a final concentration of 0.2 M increases the efficiency of glycerol. The efficiency of combination of 10% glycerol and sucrose was comparable with that of combinations of DMSO and ethylene glycol with sucrose. The mechanism of the observed enhancement is apparently related to the influence of sucrose on the dynamic properties of the lipid membranes and facilitation of glycerol diffusion into the cells.

Expression of Metalloproteinases and Type I and III Collagens during Healing of Excisional Skin Wound on the Abdomen and Back in Rats.

The study was carried out using a novel rat model developed in our laboratory, namely16 mm diameter circular excisional wounds were generated on the abdomen which resulted in minimal scarring. Restoration of the skin integrity was completed by day 60 after the wounding surgery. By this time, regenerates on the abdomen were stronger than on the back (at, respectively, 58 and 17.4 % of the tensile strength of the intact skin at corresponding location) and the ratio of type I and type III collagens in regenerates on the abdomen reached the level of intact skin at the same location. On days 3 to 14, the ratio of Mmp9/Timp1 expression levels on the abdomen was higher than on the back. On days 20 and 30, the Mmp9/Timp1 ratio on the abdomen was identical to the level of intact skin, whereas the increased MMPs expression levels on the back were maintained until day 30. It has been shown for the first time that according to functional and molecular characteristics, wound healing on the abdomen of an adult rat is more similar to complete regeneration than scarring repair of the back skin.

Analysis of the Expression of Regulator Genes in Kupffer Cells and Monocytes.

Differences in the gene expression profiles in resident macrophages (in particular, Kupffer cells) and monocytes were revealed. However, these differences in gene expression profiles do not allow considering resident liver macrophages as purely M2 macrophages and monocytes as purely M1 macrophages. At the same time, a significant number of the genes upregulated in Kupffer cells are associated with normal regulation of liver functions (Arg 1, Flt, iNOs, and Kng). In monocytes, the expression of genes Alox15, Alox12, Tlr2, Tlr4, Tlr7, and Tlr8 (typical functional genes of macrophages) was also upregulated in comparison with Kupffer cells.

Quantitative and Qualitative Characterization of Phagocytic Activity of Macrophages of Bone Marrow and Fetal Origin.

We compared phagocytic activity of macrophages of monocyte origin and Kupffer cells under the influence of M1 and M2 inducers and without activation. Cultures of monocyte-derived macrophages and Kupffer cells were characterized by intensive expression of CD68 that was not affected by activation factors. At the same time, these cultures demonstrated different dynamics of phagocytic activity. Monocyte-derived macrophages initially had more pronounced absorption capacity that gradually increased during the experiment. Kupffer cells were characterized by abrupt fluctuations of phagocytic activity: sharp growth and rapid saturation. Despite these differences, the endosomes produced by monocyte-derived macrophages and Kupffer cells had similar degrees of maturity.

Dynamics of Expression of Cytokine Genes and Macrophage Content in the Lungs and Kidneys after Subtotal Hepatectomy in Rats.

The role of the lungs and kidneys in liver regeneration after subtotal hepatectomy was studied on a rat model. It was found that production of hepatocyte growth factor (HGF) in the lungs and kidneys and expression of cytokine genes Il1b, Il6, Il10, and tnfa significantly increased. Analysis of the dynamics of lung macrophage population showed that accumulation of HGF and the increase in the expression of cytokine genes in the lungs were accompanied by simultaneous increase in the number of CD68+ cells, which attested to the leading role of macrophages in activation of HGF synthesis in the lungs. Macrophage content in the kidneys after subtotal hepatectomy did not increase.

Angiogenic Potential of Multipotent Stromal Cells from the Umbilical Cord: an In Vitro Study.

The mechanisms of proangiogenic activity of multipotent stromal cells from human umbilical cord were analyzed in vitro. The absence of secreted forms of proangiogenic growth factor VEGF-A in the culture medium conditioned by umbilical cord-derived multipotent stromal cells was shown by ELISA. However, the possibility of paracrine stimulation of cell proliferation, mobility, and directed migration of endothelial EA.hy926 cells was demonstrated by using MTT test, Transwell system, and monolayer wound modeling. The capacity of multipotent stromal cells to acquire the phenotype of endothelium-like cells was analyzed using differentiation media of three types. It was found that VEGF-A is an essential but not sufficient inductor of differentiation of umbilical cord-derived multipotent stromal cells into CD31+ cells.

Expression of Cytokine Genes and Growth Factors in Rat Lungs and Kidneys after Subtotal Hepatectomy.

Expression of il1b, il6, il10, tnfa, hgf, tgfb, vegf, and fgf2 genes in the lungs and kidneys was examined on rat model of liver regeneration after subtotal hepatectomy. Enhanced expression of il6, il10, tnfa, hgf, and fgf2 genes was detected at the early terms after 80% liver resection.

Role of Progenitor Cells in Liver Regeneration after Subtotal Resection.

In the liver of rats subjected to subtotal liver resection (80% organ weight), the expression of sox9 gene and SOX9 protein content increased and cells with hepatocyte morphology expressing SOX9 appeared; the proportion of cells expressing cytokeratin-19 also increased. Based on these data, we cannot completely exclude the involvement of resident progenitor cells and hepatocyte reprogramming in liver regeneration after subtotal resection, however, the contribution of these processes seems to be insignificant. The leading mechanism of liver mass recovery after subtotal resection is proliferation of hepatocytes.

Mechanisms of therapeutic activity of multipotent cells in heart diseases.

Analysis of our findings and published reports on possible mechanisms of therapeutic activity of stem/progenitor cell transplantation in cardiovascular pathologies is presented.

Hemopoietic cell proliferation and death in the regenerating fetal rat liver.

The proliferation and death of hemopoietic cells in the regenerating liver of 17-day outbred albino rat fetuses were studied during 2 days after resection of 20% of the organ. The mitotic index of hemopoietic cells in the resected liver increased significantly 24 h after the operation in comparison with the control fetuses. No increase in the counts of apoptotic hemopoietic cells was detected in the regenerating liver. Hence, resection of 20% of the liver in rat fetuses stimulated the proliferation of hemopoietic cells and did not stimulate their apoptosis.

Improvement of cardiac contractile function in rats with postinfarction cardiosclerosis after transplantation of mononuclear and multipotent stroma bone marrow cells.

We compared the efficiency of autologous mononuclear cells and multipotent stromal cells of the bone marrow after their non-selective intracoronary transplantation on day 30 after acute coronary infarction in rats. Improvement of hemodynamic parameters of myocardial contractility (rates of left ventricular pressure rise and drop) in comparison with the initial values and deceleration of postinfarction prolongation of QRS and QT intervals were observed in rats of the experimental group in contrast to controls in 4 weeks after transplantation. These functional changes were more intensive after transplantation of multipotent stromal cells and were accompanied by more pronounced morphological signs of reverse myocardial remodeling: thickening of the scarred left ventricular wall, shrinkage of the scar, and decrease in left ventricular dilatation index.

[Actual problems of cell therapy for cardiac diseases].

This article reviews the literatury and own dates of cell therapy for cardiac diseases. The principal unresolved issues were formulated.

Effect of transplantation of bone marrow monomuclears on angiogenesis in rats.

We studied the effect of non-selective intracoronary transplantation of bone marrow mononuclears on day 30 after acute coronary infarction on angiogenesis in rats. On days 14 and 30 after transplantation of mononuclear cells, stable formation of new vessels was observed. The number of venules considerably increased after transplantation of mononuclear cells, which was seen from increased volume density of blood vessels and their caliber. Stable vascularization after transplantation of mononuclear cells improves blood supply, which is essential for reparation of the myocardium.

Pathways of bone marrow mononuclear cell differentiation after transplantation into postinfarction heart.

We studied homing and differentiation fate of transplanted bone marrow mononuclears after non-selective intracoronary injection on day 30 after acute myocardial infarction in rats. Mononuclear cells migrated to the cicatrix zone where they differentiated into fibroblasts and myofibroblasts. Mononuclear cells did not differentiate into cardiomyocytes, endothelial cells, or smooth muscle cells of vascular media. Stimulation of angiogenesis and reparation of the myocardium was observed under these conditions.

Reparation of the myocardium after transplantation of mononuclear bone marrow cells.

Safety and efficiency of intracoronary transventricular transplantation of autologous mononuclear bone marrow cells in rats with postinfarction cardiosclerosis were studied. The cells migrated to the damaged area and were detected only in the cicatricial tissue; they have fibroblast-like phenotype and some of them were stained with Fapα (marker of reactive fibroblasts). More active proliferation of non-muscular cells and formation and maturation of collagen fibers in the cicatricial tissue were observed after transplantation of mononuclear cells. This led to thickening of the cicatricial wall, but the size of the scar and index of dilatation of the left ventricle remained unchanged. The number and volume density of newly formed blood vessels in the damaged area increased after transplantation, but no labeled cells were seen in the vascular walls. It can be hypothesized that stimulation of neoangiogenesis is mediated by paracrine mechanisms, which also explains improvement of global contractility of the left ventricle (increased contractility index in functional tests). Thus, transplantation of mononuclear bone marrow cells leads to thickening and strengthening of the cicatricial wall, stimulates angiogenesis, and improves global myocardial contractility. However, no morphological signs of reverse remodeling of the left-ventricular myocardium were revealed.