Hyaluronic Acid as a LYVE-1 Receptor Ligand in the Lymphatic System of Healthy Human Skin.

Immunohistochemical detection of the LYVE-1 marker in healthy human full-thickness skin (the epidermis and the dermis) was carried out. LYVE-1 expression was found in the endothelium of lymphatic capillaries located in the papillary dermis, in the endothelium of larger lymphatic vessels of the reticular dermis, and in fibroblasts, which indicates their joint participation in hyaluronan metabolism. LYVE-1+ staining detected for the first time in cells of the stratum basale, the stratum spinosum, and the stratum granulosum of healthy human epidermis indicates their participation in hyaluronan metabolism and allows us to consider the spaces between epidermis cells as prelimphatics.

[Expression of markers of epithelial-mesenchymal transition in breast cancer]. / Ekspressiya markerov epitelial'no-mezenkhimal'nogo perekhoda pri rake molochnoi zhelezy.

OBJECTIVE: To study of the expression of epithelial-mesenchymal transition markers in various molecular subtypes of cancer and in benign breast diseases with different risks of malignant transformation. MATERIAL AND METHODS: An immunohistochemical study of the expression of E-cadherin, collagen II, integrin 1ß, cyclin D1 was carried out in breast tissue samples: 58 with invasive breast carcinoma of no special type and 17 with benign breast diseases with a high and low risk of malignant transformation. RESULTS: Patients with a triple negative molecular subtype are characterized by lower expression of collagen II compared with individuals with luminal A and B+ subtypes. A distinctive feature of patients with the HER2+ subtype was increased expression of cyclin D1 compared with the luminal A subtype, and in patients with the luminal B+ subtype, the expression of integrin 1ß is higher than in the luminal B- and HER2+ subtypes. The expression of cyclin D1 in patients of both groups with benign diseases was lower than in all groups with invasive carcinoma, but at the same time, in patients with a low risk of malignant transformation, the expression of cyclin D1 was higher than in those with an increased risk. CONCLUSION: The study revealed the relationship between the expression of markers and molecular subtypes of breast tumors, in addition, some patients with benign diseases were found who need further more careful monitoring and may be at risk for the development of malignancy.

Effect of Melatonin on the Expression of LYVE-1 Receptor in Liver Sinusoidal Endothelial Cells of Mice with the Light-Induced Functional Pinealectomy Model.

Intragastric administration of melatonin in physiological doses (1 mg/kg body weight) for 14 days to C57BL/6 mice with light-induced functional pinealectomy model (24-h lighting for 14 days) results in an increase in the LYVE-1 expression area by 2.4 times and a significant increase in receptor concentration (1.6% decrease in staining brightness) in liver sinusoidal endothelial cells in comparison with animals kept under continuous lighting and not treated with the hormone, which indicates the formation of stability of the endothelial barrier in the organ. Melatonin treatment also enhanced lymphatic drainage in all it links (including interstitial non-vascular pathways and lymphatic vessels) and improved structural and functional parameters of blood circulation and lymph flow in the organ, which created conditions for reducing metabolic load on structural elements of the liver.

Light-Induced Functional Pinealectomy: Expression of MT2 Receptors in Liver Cells of C57BL/6 Mice after Melatonin Treatment.

We performed an immunohistochemical study of MT2 melatonin receptor expression in the liver of C57BL/6 mice with modeled light-induced functional pinealectomy and after melatonin administration by the indirect avidin-biotin peroxidase ABC method. The animals were kept for 14 days under constant lighting. Intragastric administration of melatonin in physiological doses (1 mg/kg body weight for 14 days) to mice with light-induced functional pinealectomy resulted in a 2-fold increase in the relative expression area of MT2 receptors in liver cells in comparison with that in animals kept under standard lighting conditions, 24-h lighting for 14 days, or 24-h lighting receiving placebo (intragastric administration of 200 ml distilled water). Melatonin treatment had practically no effect on MT2 staining intensity. Our results attest to the important role of MT2 receptors in melatonin synthesis disorders and can serve as the basis for the development of therapeutic strategies aimed at melatonin receptors.

Effect of Melatonin on Expression of Apoptosis Regulator Proteins Bcl-2 and Bad in Ovarian Follicular Apparatus after High Temperature Exposure.

Expression of proapoptotic Bad and antiapoptotic Bcl-2 proteins in ovarian follicular apparatus of Wistar rats was evaluated on days 3, 7, and 14 after single experimental hyperthermia (EH) followed by therapeutic correction with subcutaneous melatonin (0.1 mg) dissolved in 0.2 ml physiological saline (PS) injected daily for 3 days. Duration of EH was less than 17 min; it was terminated when the rectal temperature attained 43.5°C. In acute posthyperthermia period (on experimental day 3), Bcl-2 and Bad expression area in folliculocytes of the experimental (EH+melatonin) and reference rats simultaneously increased in comparison with the corresponding values in control rats. At this, Bcl-2/Bad ratio of expression areas in experimental and reference rats did not differ from the control level. During the recovery period (on posthyperthermia day 7), Bad protein expression area significantly decreased in experimental rats resulting in elevation of Bcl-2/Bad ratio in comparison with control and reference groups on days 3 and 7. On day 14, Bcl-2 and Bad expression areas and Bcl-2/Bad ratio restored in experimental animals to the corresponding values assessed in control and reference groups. Thus, melatonin produced no effect on Bcl-2/Bad protein expression ratio during acute posthyperthermia period, but reduced the expression area of proapoptotic Bad protein and increased Bcl-2/Bad ratio on posthyperthermia day 7. Thus, melatonin can inhibit apoptosis via mitochondrial pathway in ovarian follicles on posthyperthermia day 7.

Personalized Approach to Determination of Histidine-Rich Glycoprotein and E-Cadherin in Supernatants of Immunocompetent Blood Cells and Breast Biopsy Specimens in Breast Malignant and Non-Malignant Disease.

The material of patients with invasive carcinoma of no special type (ICNT) and nonmalignant diseases (ND) of the mammary gland was studied. When comparing the concentrations of histidine-rich glycoprotein (HRG) and E-cadherin (CDH1), statistically significant differences between ICNT and ND by HRG in the supernatant of blood cells and its spontaneous production by biopsies and by CDH1 at its induced production, as well as by influence indices of polyclonal activators on the production of CDH1 were found. When comparing the expression of immunohistochemical markers, no statistically significant differences between ICNT and ND were obtained.

Expression of Apoptosis Regulator Proteins Bcl-2 and Bad in Rat Ovarian Follicular Apparatus during Recovery after Extreme Hypothermia.

The expression of molecular and cellular regulators of apoptosis (proapoptotic protein Bad and antiapoptotic protein Bcl-2) was measured in the follicular apparatus of rat ovaries during the recovery period (days 7 and 14) after hyperthermia (up to rectal temperature 43.5°C). The Bcl-2/Bad index was calculated. The expression of Bcl-2 in the follicular apparatus of rat ovaries increased on day 7 after the exposure. The Bcl-2/Bad index also increased, which suggests that the development of apoptosis by the mitochondrial pathway in follicles was limited at this term after hyperthermia. On day 14 after hyperthermia, the area of immunohistochemical staining for the antiapoptotic protein Bcl-2 significantly decreased in cells of the ovarian follicular epithelium, but the expression of the proapoptotic protein Bad significantly increased; these changes led to a decrease in Bcl-2/Bad index, which attested to weakening of the antiapoptotic defense and activation of oocyte apoptosis by the mitochondrial pathway.

Effect of Linagliptin on the Ratio of Apoptosis Regulators in the Model of Non-Alcoholic Fatty Liver Disease in db/db Mice.

We studied the effects of dipeptidyl peptidase 4 (DPP4) inhibitor linagliptin on the expression of apoptosis regulator proteins Bcl-2 and Bad in the liver of db/db mice with genetically determined obesity and type 2 diabetes mellitus. The mice received daily linagliptin or saline (placebo) by gavage from week 10 to week 18 of life. In the liver of non-treated mice, the area positively stained for Bad was greater than the area of Bcl-2 expression, which created the conditions for apoptosis activation in liver at this age. Administration of linagliptin decreased Bad stained area and increased Bcl-2 stained area in the liver cells. At the same time, Bad stained area remained larger in treated mice than the area of Bcl-2 expression area, which attested to partial normalization of pro- and antiapoptotic protein balance.

Personalized Approach to Assessing mRNA Expression of Histidine-Rich Glycoprotein and Immunohistochemical Markers in Diseases of the Breast.

Biopsy material of patients with malignant and benign breast diseases was examined. HRG mRNA expression was detected in 70% of cases in biopsy material obtained from patients with nonspecific invasive carcinoma and in 66.7% of cases in biopsy material of patients with benign breast diseases. Immunohistochemical analysis revealed expression of collagen II, the beta-1 integrin, and E-cadherin-markers of epithelial-mesenchymal transition. The use of RT-qPCR combined with immunohistochemical study made it possible to identify atypical cells, which can be regarded as precancerous changes, in individual patients.

Analysis of IL-1α, bFGF, TGF-ß1, IFNγ, MMP-1, and CatD Expression in Multinuclea Macrophages In Vitro.

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.

Expression of Bcl-2 Family Proteins in the Ovarian Follicular Apparatus in the Acute Period after Experimental Hyperthermia.

The expression of apoptosis regulators (proapoptotic protein Bad and anti-apoptotic protein Bcl-2) was analyzed and Bcl-2/Bad ratio in the follicular apparatus of the rat ovary was determined on day 3 after hyperthermia (rectal temperature 43.5°C). Hyperthermia in the catabolic phase leads to different degrees of activation of the molecular "switches" of apoptosis in cells of ovarian follicular epithelium. This was seen from increased intensity of immunohistochemical staining for Bad protein against the background of more pronounced expression of Bcl-2 protein. On day 3 after exposure to hyperthermia, Bcl-2/Bad ratio increased, which reflects antiapoptotic protection of cells and conditions for blockade of mitochondrial pathway of apoptosis in the follicular apparatus of the ovaries during the acute period after hyperthermia.

Melatonin-Aluminum Oxide-Polymethylsiloxane Complex on Apoptosis of Liver Cells in a Model of Obesity and Type 2 Diabetes Mellitus.

We studied the effects of a melatonin-aluminum oxide-polymethylsiloxane complex (complex M) on the expression of apoptosis regulators Bcl-2 and Bad in the liver of homozygous db/db BKS.Cg-Dock7m+/+Leprdb/J mice with obesity and type 2 diabetes. Complex M or placebo was administered daily through the gastric tube during weeks 8-16 of life. In mice with type 2 diabetes mellitus receiving placebo, enhanced immunohistochemical reactions for proapoptotic Bad protein and weak response for anti-apoptotic Bcl-2 protein were observed. Administration of complex M shifted the ratio of apoptosis regulators: the area of Bcl-2 expression significantly increased and against the background of reduced Bad expression area. These findings attest to antiapoptotic effect of complex M in the liver on the model of type 2 diabetes mellitus.

In Vitro Study of Cytophysiological Characteristics of Multinuclear Macrophages from Intact and BCG-Infected Mice.

Peritoneal macrophages were isolated from intact and BCG-infected BALB/c mice and explanted in vitro. Multinuclear macrophages formed in these cultures differed by the number of nuclei, expression of apoptosis inductors and regulators (TNF-α, p53 protein, caspase 3, and Bcl-2 protein), and cytophysiological characteristics (phagocytic activity, ROS generation, and antimycobacterial properties). Our results indicate that the formation of multinuclear macrophages is accompanied by induction of apoptosis (p53 signaling pathway) and appearance of multinuclear macrophage-derived cells characterized by high phagocytic and antimycobacterial activity.

Effects of Melatonin, Aluminum Oxide, and Polymethylsiloxane Complex on the Expression of LYVE-1 in the Liver of Mice with Obesity and Type 2 Diabetes Mellitus.

The effects of melatonin, aluminum oxide, and polymethylsiloxane complex on the expression of LYVE-1 (lymphatic vessel endothelial hyaluronan receptor) in the liver were studied in db/db mice with experimental obesity and type 2 diabetes mellitus. The complex or placebo was administered daily by gavage from week 8 to week 16 of life. The animals receiving the complex exhibited enhanced, in comparison with the placebo group, immunohistochemical LYVE-1+ staining of endothelial cells in sinusoids. Enhanced expression of LYVE-1 was associated with less pronounced dilatation of interlobular arteries, veins, and lymphatic vessels. Thee findings suggest a protective effect of the complex towards structural changes in the liver of mice with obesity and type 2 diabetes.

Changes in Peripheral Blood Monocytes and Liver Macrophages in Male Rats after Benzo(a)pyrene Injection.

Intraperitoneal injections of benzo(a)pyrene to male rats in a total dose of 60 mg/kg modified the production of ROS and the phagocytic potential of blood monocytes by modulating their potential bactericidal activity. The lysosomal system (particularly the secondary lysosomes) of liver macrophages was activated, which promoted fusion of the hydrolytic potentials of macrophages and monocytes. These results indicated that the toxin modulated the cellular immune homeostasis and the level of general nonspecific resistance.

Modifying Effects of Nanosized Diamonds on Hydrolytic Potential of Macrophages In Vitro.

The effects of nanosized particles (4-6 nm) of synthetic diamonds on mouse macrophage expression and secretion of lysosomal cathepsins (B and D) and MMP-1 and MMP-9 were studied in vitro. Culturing of peritoneal macrophages in medium with diamond nanoparticles led to an increase in the counts of macrophages expressing the above enzymes and to stimulation of their secretion. However, the manifestations of these effects varied significantly for various enzymes. The data indicate modulation of macrophage functions by nanodiamonds. These results help better understand the possible role of the "corpuscular" xenobiotic factors in the pathogenesis of diseases associated with macrophage capturing of these factors irrespective of their chemical "activity".

In vitro effects of nanosized diamond particles on macrophages.

The effects of synthetic diamond nanoparticles (4-6 nm) on mouse macrophage biotropism and biocompatibility and the modulation of the macrophage functions (expression of IL-1α, TNF-α, GM-CSF, bFGF, and TGF-ß) by nanoparticles in different concentrations were studied in vitro during exposure of different duration. Macrophage endocytosis of nanodiamonds increased with increasing the concentration of nanoparticles in culture and incubation time. Nanodiamonds exhibited high biotropism and biocompatibility towards macrophages; in doses of 10-20 µg/ml, they induced expression of GM-CSF and TGF-ß, inhibited expression of bFGF, and did not stimulate IL-1α and TNF-α. These data indicate that nanodiamond capture by macrophages in the studied experimental model led to modulation of the functional status of macrophages that determine their capacity to stimulate reparative processes without increasing proinflammatory and profibrogenic status.

Effectiveness of composition based on oxidized dextran in the treatment of grade IIIB skin burns.

Grade IIIB skin burns were treated with a composition based on oxidized dextran with a molecular weight of 40 kDa (oxidation of 7% glucose residues). On day 32 after burn infliction and from the start of the treatment, the area of skin defect in rats was 30% less than in the group without treatment and by 2.3 times less than in rats treated with panthenol. In rats treated with dextran-based composition or panthenol, the eschar was absent on day 21 after the start of the treatment; by day 32, we found cells of surface epithelium, hair follicles, and sebaceous glands above the scar tissue that were absent in untreated animals; in rats treated with the composition, their number was higher by 2.5 times than in animals treated with panthenol. Treatment with the composition increased volume density (by 2.5 times) and numerical density (by more than 3 times) of blood vessels in the wound and reduced signs of inflammation and fibroplastic activity of fibroblasts in comparison with the corresponding parameters in untreated animals or animals treated with panthenol.

Hepatocyte apoptosis in rats exposed to benzo(a)pyrene.

We studied the effect of benzo(a)pyrene on activity of nuclear endonucleases and expression of molecular regulator of apoptosis Bcl-2 in liver cells in rats. Intraperitoneal injection of benzo(a)pyrene (in a total dose of 60 mg/kg body weight) reduced activity of nuclear endonucleases in the liver cells, which attests to inhibition of apoptosis by the nuclear pathway. Injection of the toxicant enhanced the expression of intracellular molecular regulator of apoptosis Bcl-2 protein in the liver cells, which attested to triggering of proapoptotic signaling in these cells and organism's attempts to limit the development of apoptosis by the mitochondrial mechanism via activation of Bcl-2-dependent anti-apoptotic defense.

In vitro expression of IL-1α, GM-CSF, and TNF-α by multinucleated macrophages from BCG-infected mice.

Peritoneal cells from intact and BCG-infected mice were explanted in vitro. In these cultures, multinucleated macrophages in different number of nuclei were formed. The intensity of multinucleated cell formation was higher in cultures from BCG-infected mice. Increasing role of amitosis in the formation of multinucleated macrophages with relatively high number of nuclei was noted with presumable domination of cell fusion mechanism. Relatively high level of IL-1α expression was noted only in the population of binucleated macrophages of BCG-infected mice in comparison with mononuclear cells. It was found macrophages from BCG-infected mice demonstrate a kind of "lineage commitment" towards multinucleated cells, which manifested in culture in initially high and increasing (with increasing the number of nuclei in cells) expression of granulocyte-macrophage CSF and TNF-α as well as initially high amitotic activity of macrophages.