The Effect of Therapy Regimen on Antitumor Efficacy of the Nanosomal Doxorubicin against Rat Glioblastoma 101.8.

One of the key problems of glioblastoma treatment is the low effectiveness of chemotherapeutic drugs. Incorporation of doxorubicin into PLGA nanoparticles allows increasing the antitumor effect of the cytostatics against experimental rat glioblastoma 101.8. Animal survival, tumor volume, and oncogene expression in tumor cells were compared after early (days 2, 5, and 8 after tumor implantation) and late (days 8, 11, and 14) start of the therapy. At late start, a significant increase in the expression of oncogenes Gdnf, Pdgfra, and Melk and genes determining the development of multidrug resistance Abcb1b and Mgmt was revealed. At early start of therapy, only the expression of oncogenes Gdnf, Pdgfra, and Melk was enhanced. Early start of treatment prolonged the survival time and increased tumor growth inhibition by 141.4 and 95.7%, respectively, in comparison with the untreated group; these differences were not observed in the group with late start of therapy. The results indicate that the time of initiation of therapy is a critical parameter affecting the antitumor efficacy of DOX-PLGA.

Comparative Molecular and Biological Characteristic of the Systemic Inflammatory Response in Adult and Old Male Wistar Rats with Different Resistance to Hypoxia.

Morphological, molecular, and biological features of the systemic inflammatory response induced by LPS administration were assessed in adult and old male Wistar rats with high and low resistance to hypoxia. In 6 h after LPS administration, mRNA expression levels of Hif1a, Vegf, Nfkb, and level of IL-1ß protein in old rats were higher than in adult rats regardless of hypoxia tolerance. The morphometric study showed that the number of neutrophils in the interalveolar septa of the lungs was significantly higher in low-resistant adult and old rats 6 h after LPS administration. Thus, in old male Wistar rats, systemic inflammatory response is more pronounced than in adult rats and depends on the initial tolerance to hypoxia, which should be considered when developing new approaches to the therapy of systemic inflammatory response in individuals of different ages.

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.

Role of Neurohumoral Imbalance at Post-Traumatic Stress Disorder in the Antitumor Immune Response. Experimental Study.

The aim of the study is to investigate the growth and development of B16 melanoma in mature male C57Black/6 mice with a post-traumatic stress disorder (PTSD) model. Behavioral, immunohistochemical, morphometric methods, enzyme immunoassay were used. A forced decrease in the level of corticosterone, which is characteristic for PTSD, was established, followed by intensification of the production of increased concentrations of pro-inflammatory interleukins by the cells of the immune system and, at the same time, a decrease in the secretion of anti-inflammatory cytokines. Priority data were obtained: the neurohumoral imbalance that develops in PTSD is a limiting factor to the growth of B16 melanoma, at least at the initial stages of the oncological process.

Morphological and Molecular Biological Features of the Systemic Inflammatory Response in Old Wistar Rats with High and Low Resistance to Hypoxia.

Morphological and molecular biological features of LPS-induced systemic inflammatory response were assessed in old male Wistar rats with high (HR) and low (LR) resistance to hypoxia. The systemic inflammatory response was modeled by intraperitoneal injection of E. coli O26:B6 LPS; the animals were sacrificed after 6 h. In histological sections, the number of neutrophils in the interalveolar septa and the area of necrosis in the liver were determined. The expression levels of Hif1a, Hif2a, Nfkb, Vegf, Il1b, Il6, Il10, and Tgfb mRNA in the liver and serum concentrations of HIF-1α and IL-1ß were determined. In 4-6 h after LPS injection, 3 (43%) of 7 HR rats died, whereas no deaths were observed among LR rats. At 6 h after LPS injection, the number of neutrophils in the interalveolar septa of the lungs in LR rats was significantly higher than in HR rats, while the area of necrosis in the liver did not differ. At the same time, the mRNA expression levels of the proinflammatory cytokine genes Il1b and Il6 increased in the liver of both HR and LR rats, whereas the expression of Il10 increased only in HR rats. The expression of the Hif1a gene in the liver was higher in LR rats, but the content of HIF-1α protein in blood serum was higher in HR animals. These data should be taken into account when developing new approaches to the therapy of systemic inflammatory response in infectious and inflammatory diseases in the elderly.

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.

Morphological and molecular-biological features of glioblastoma progression in tolerant and susceptible to hypoxia Wistar rats.

Hypoxia is a major pathogenetic factor in many cancers. Individual resistance to suboptimal oxygen availability is subject to broad variation and its possible role in tumorigenesis remains underexplored. This study aimed at specific characterization of glioblastoma progression in male tolerant and susceptible to hypoxia Wistar rats. Hypoxia resistance was assessed by gasping time measurement in an 11,500 m altitude-equivalent hypobaric decompression chamber. Based on the outcome, the animals were assigned to three groups termed 'tolerant to hypoxia' (n = 13), 'normal', and 'susceptible to hypoxia' (n = 24). The 'normal' group was excluded from subsequent experiments. One month later, the animals underwent inoculation with rat glioblastoma 101.8 followed by monitoring of survival, body weight dynamics and neurological symptoms. The animals were sacrificed on post-inoculation days 11 (subgroup 1) and 15 (subgroup 2). Relative vessels number, necrosis areas and Ki-67 index were assessed microscopically; tumor volumes were determined by 3D reconstruction from histological images; serum levels of HIF-1α, IL-1ß, and TNFα were determined by ELISA. None of the tolerant to hypoxia animals died of the disease during observation period, cf. 85% survival on day 11 and 55% survival on day 15 in the susceptible group. On day 11, proliferative activity of the tumors in the tolerant animals was higher compared with the susceptible group. On day 15, proliferative activity, necrosis area and volume of the tumors in the tolerant to hypoxia animals were higher compared with the susceptible group. ELISA revealed no dynamics in TNFα levels, elevated levels of IL-1ß in the susceptible animals on day 15 in comparison with day 11 and tolerant ones. Moreover, there were elevated levels of HIF-1α in the tolerant animals on day 15 in comparison with day 11. Thus, the proliferative activity of glioblastoma cells and the content of HIF-1α were higher in tolerant to hypoxia rats, but the mortality associated with the tumor process and IL-1ß level in them were lower than in susceptible animals. Specific features of glioblastoma 101.8 progression in tolerant and susceptible to hypoxia rats, including survival, tumor growth rates and IL-1ß level, can become the basis of new personalized approaches for cancer diseases treatment in accordance to individual hypoxia resistance.

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.

About 4-Day Rhythm of Proliferative Activity of L-929 Cells in Culture Correlates with the Intensity of Secondary Cosmic Radiation Fluctuations.

The dynamics of proliferative activity of the L-929 cell culture of mouse fibroblast-like cells in the phase of logarithmic growth was compared with some heliogeophysical parameters (Ap and ULF indexes of geomagnetic activity, vertical component of the interplanetary magnetic field, and intensity of fluctuations of secondary cosmic radiation estimated by the neutron monitoring near the Earth's surface). Among the considered heliogeophysical parameters, only the magnitude of fluctuations of minute-to-minute changes in the neutron monitor indicator reliably and negatively correlates with the rate of cell culture reproduction. Considering that the amplitude of secondary cosmic fluctuations is about 5%, which is 0.1% of the total ray flux, and proliferative activity varies in the range of 30-50%, the probability of a direct biotrophic effect of this physical factor is extremely low. It seems likely that proliferative activity of L-929 cell culture is directly affected by another environmental factor, the marker of which is the intensity of neutron counting rate fluctuations.

[Nitric oxide donor nitrosorbide potentiates the antitumor effect of doxorubicin against experimental glioblastoma]. / Donor oksida azota izosorbida dinitrat povyshaet protivoopukholevyi effekt doksorubitsina pri khimioterapii eksperimental'noi glioblastomy.

BACKGROUND: Doxorubicin is a well-known antitumor drug that is not employed for chemotherapy of brain tumors. Indeed, doxorubicin does not penetrate across the blood-brain barrier in therapeutic concentrations. OBJECTIVE: To study the antitumor effect of doxorubicin combined with nitrosorbide on intracranial experimental glioblastoma 101/8 in rats. MATERIAL AND METHODS: Male Wistar rats (n=86) with intracranial implanted glioblastoma 101/8 received doxorubicin (i.v. 1.5 mg/kg thrice) alone or in combination with nitrosorbide (i.v or orally, 0.5 mg/kg thrice) in 2, 5 and 8 days after implantation. Efficacy was assessed considering survival and brain tumor volume in 14 days after tumor implantation. RESULTS: Combination of doxorubicin and nitrosorbide significantly increased survival (57% and 155%, respectively) and slowed down tumor growth (16±12 and 8±6 mm3, respectively) compared to doxorubicin alone. Effectiveness of nitrosorbide alone was trivial. CONCLUSION: Nitric oxide donor nitrosorbide considerably potentiated the antitumor effect of doxorubicin against intracranial 101/8 glioblastoma in rats.

Morphological and Biochemical Characteristics of Prostate Hyperplasia during Sulpiride Treatment.

We studied morphological changes in the prostate ventral lobe, proliferative activity of the epithelium in prostate acini, and the levels of prolactin and prostate-specific antigen in the blood serum of Sprague-Dawley rats after repeated injections of sulpiride in a dose of 40 mg/ kg over 30 and 60 days and in 10 and 30 days after withdrawal. Morphological and morphometrical analysis of hyperplastic changes in the prostate ventral lobe was performed. Ki-67+ proliferating epithelial cells in the acini were counted. The dynamics of serum concentrations of prolactin and prostate-specific antigen was evaluated by ELISA. Morphological and morphometrical analysis and evaluation of the content of Ki-67+ cells demonstrated epithelium hyperplasia in the prostate ventral lobe after sulpiride treatment for 30 or 60 days and in 10 days after withdrawal, but serum level of prostate-specific antigen did not differ from the control. After 60-day sulpiride treatment and in 30 days after withdrawal, pronounced hyperplastic changes of prostate and elevated concentrations of prostate-specific antigen (but not prolactin) were observed. Thus, administration of sulpiride (40 mg/kg) to Sprague-Dawley rats for 60 days allows, by morphological criteria and serum level of prostate-specific antigen, to model stable hyperplastic changes in the prostate corresponding to benign prostatic hyperplasia in humans.

Morphological Characteristic of Melanoma B16 Progression in C57BL/6 Mice with High and Low Resistance to Hypoxia.

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.

[Morphology of the thymus and the specific features of its cellular composition in experimental acute and chronic ulcerative colitis]. / Morfologiia timusa i osobennosti ego kletochnogo sostava pri éksperimental'nom ostrom i khronicheskom iazvennom kolite.

OBJECTIVE: To investigate morphological changes in the thymus, the subpopulation composition of lymphocytes and its non-lymphoid cells in dextran-induced experimental acute ulcerative colitis and in different periods of chronic ulcerative colitis. MATERIAL AND METHODS: Acute and chronic ulcerative colitis was simulated in C57BL/6 mice, by replacing drinking water with a 1% aqueous dextran sulfate sodium solution. Thymic changes were morphometrically assessed; the number and absolute area of thymic corpuscles and epithelial cells were calculated; and the subpopulation composition of lymphocytes and thymic stromal cells was determined using flow cytofluorimetry; the Kruskal-Wallis test and the Mann-Whitney test were used to compare the groups. RESULTS: In acute catarrhal and ulcerative colitis, there was acute accidental thymic involution with devastation of the cortical substance and with a decline in its volume fraction, with an increase in the levels of cells dying through the mechanism of apoptosis, and with a decrease in the absolute number of lymphocytes, T-helper cells, cytotoxic T-cells, regulatory T-lymphocytes, B-lymphocytes, and dendritic cells, with a rise in the index of the area of thymic corpuscles and in the content of late-phase corpuscles among them, and with the appearance of thymic corpuscles as cyst-like cavities. In chronic ulcerative colitis, the cortex was expanded and the area of thymic corpuscles and the count of medullary epithelial cells increased. The cyst-like thymic corpuscles formed clusters, the count of dendritic cells increased in early-stage chronic ulcerative colitis, but the levels of macrophages decreased in both periods of its development. CONCLUSION: There is acute accidental involution and thymic hyperplasia with an increase in medullary epithelial cells and thymic corpuscles consisting of cytokeratin 19+ in the epithelial cells in experimental acute and chronic ulcerative colitis, respectively. The more pronounced epithelial cell response found in end-stage experimental chronic ulcerative colitis reflects the enhanced differentiation of regulatory T-lymphocytes and the larger number of which is observed in peripheral blood and in the focus of inflammation in patients with ulcerative colitis, according to the literature.

Histophysiology of the immune system of Wistar rats at delayed terms after ovariectomy.

We studied histophysiological changes in organs of the immune system, liver, and lungs of Wistar rats at delayed terms after ovariectomy. On day 52 after surgical castration leading to a decrease in estradiol, progesterone, and testosterone levels, activation of the immune system was revealed characterized by stage I accidental thymus involution, while pulp depletion in the spleen, and enhanced IL-2 and IFN-γ production. We also observed small-droplet degeneration of hepatocytes and reactive changes in the lungs manifested in increased number of neutrophils in the interalveolar septa.

Histophysiology of immune system and barrier organs in the late period of orchiectomy in Wistar rats.

We studied the histological and physiological changes in the organs of the immune system, liver, and lungs as well as colon microflora in the late period after orchiectomy in Wistar rats. It was shown that on day 52 after surgical castration leading to a sharp decline in androgens, activation of the immune system was detected with enhanced production of IL-12 and IL-6 and accumulation of neutrophils in the interalveolar septa of the lungs. The level of aspartate aminotransferase was within the normal range. Qualitative and quantitative changes of microflora were associated with increased number of opportunistic bacteria.

[Comparative characteristic of the hormonal profile in men with stable obstructive pulmonary disease and smokers].

Comparative analysis of the hormonal profile was performed in 12 and 16 patients with mild and moderately severe chronic obstructive pulmonary disease (COPD) respectively, in 25 patients with chronic bronchitis, 13 smokers without respiratory symptoms and dysfunction of external respiration, and in healthy non-smokers. Patients with moderately severe COPD had lower concentrations of total and free testosterone and DHEA than smokers with chronic bronchitis and mild COPD. Blood androsterone level positively correlated with the forced expiratory volume during the first second and inversely related to the duration of smoking and severity of coughing.

Gender differences in pulmonary and immune response in acute experimental endotoxicosis.

Differences in the immune and inflammatory response were revealed in the lungs of male and female Wistar rats on day 1 after administration of LPS in a dose inducing the development of acute bacterial endotoxemia. Females showed more pronounced morphofunctional signs of immune system activation than males: this was characterized by more severe accidental involution of the thymus, devastation of the splenic white pulp, and enhanced production of IL-4, IL-12, and TNF-α by splenocytes. In males, production of the above cytokines decreased and inflammation in the lungs was more pronounced at these terms.