[Prognostic model for assessing the human glioma cell malignancy grade based on MDM2, MELK, SOX2, CDK4, DR5 and OCT4 gene expression]. / Prognosticheskaya model' dlya otsenki stepeni zlokachestvennosti kul'tury kletok gliomy cheloveka na osnovanii issledovaniya ekspressii paneli genov MDM2, MELK, SOX2, CDK4, DR5 i OCT4.

Glioma cell cultures are used in basic researches of tumor processes, personalized medicine for selecting treatment regimens depending on individual characteristics of patients and pharmacology for assessing the effectiveness of chemotherapy. Suppression of glioma culture growth without reduction of malignancy grade is common. Drug cancellation may be followed by substitution of precursor cells by more malignant clones. Therefore, analysis of culture cell malignancy grade is important. In the future, intraoperative analysis of glioma cell malignancy grade can be used to select individual therapy. OBJECTIVE: We analyzed the relationship between expression of marker genes TUBB3, CD133, CDK4, CDK6, CIRBP, DR4, DR5, EGFR, FGFR, FSHR, GDNF, GFAP, L1CAM, LEF1, MAP2, MDM2, MELK, NANOG, NOTCH2, OCT4, OLIG2, PDGFRA, PDGFA, PDGFB and SOX2 and glioma cell malignancy grade, as well as created appropriate prognostic model. MATERIAL AND METHODS: We analyzed expression of 25 marker genes in 22 samples of human glioma cultures using quantitative real-time PCR. Statistical analysis was performed using the IBM SPSS Statistics 26.0 software. We used the Kolmogorov-Smirnov and Shapiro-Wilk tests to assess distribution normality. Nonparametric Jonckheere-Terpstra and Spearman tests were applied. RESULTS: We obtained a prognostic model for assessing the grade III and IV glioma cell malignancy based on expression of marker genes MDM2, MELK, SOX2, CDK4, DR5 and OCT4. Predictive accuracy was 83% (Akaike information criterion -55.125).

Neural progenitor and hemopoietic stem cells inhibit the growth of low-differentiated glioma.

The effects of neural progenitor and hemopoietic stem cells on C6 glioma cells were studied in in vivo and in vitro experiments. Considerable inhibition of proliferation during co-culturing of glioma cells with neural progenitor cells was revealed by quantitative MTT test and bromodeoxyuridine incorporation test. Labeled neural progenitor and hemopoietic stem cells implanted into the focus of experimental cerebral glioma C6 survive in the brain of experimental animals for at least 7 days, migrate with glioma cells, and accumulate in the peritumoral space. Under these conditions, neural progenitor cells differentiate with the formation of long processes. Morphometric analysis of glioma cells showed that implantation of neural progenitor and hemopoietic stem cells is accompanied by considerable inhibition of the growth of experimental glioma C6 in comparison with the control. The mechanisms of tumor-suppressive effects of neural and hemopoietic stem cells require further investigation.

Activation of expression of brain-derived neurotrophic factor at the site of implantation of allogenic and xenogenic neural stem (progenitor) cells in rats with ischemic cortical stroke.

Ischemic stroke was modeled in the sensorimotor zone of the brain cortex in adult rats. Rat embryonic nervous tissue, neural stem cells from human olfactory epithelium, and rat fibroblasts (cell control) were implanted into the peri-infarction area of rats of different groups immediately after stroke modeling. Expression of BDNF mRNA was analyzed 7 days after surgery by real-time PCR. BDNF expression in cell preparation before their implantation was minimum. The expression of BDNF mRNA increased by 5-6 times in the areas of implantation of rat fibroblasts and human olfactory epithelium and by 23 times in the area of implantation of rat embryonic nervous tissue compared to periinfarction areas without cell implantation. These findings confirm the possibility of realization of the therapeutic effects of neural stem cells via expression of trophic factors.

Reaction of oligoglia to spinal cord injury in rats and transplantation of human olfactory ensheathing cells.

In experiments on rats with lateral TVIII hemisection of the spinal cord and transplantation of ensheating olfactory cells, we studied structural changes at the lesion site and adjacent rostral and dorsal regions of the spinal cord. The state of oligodendrocytes and myelin fibers and motor function in experimental animal were analyzed. Open field testing (BBB test) showed that motor functions steadily increased (by 13% on average) within the interval from day 21 to day 53 after transplantation. Histological examination showed that groups of transplanted cells carrying human nuclear marker (HNu(+)cells) were still present at the lesion site 30 days after surgery. Some of these cells migrated in the rostral and caudal directions from the injection site to a distance up to 6 mm. At the initial period after hemisection, the number of oligodendrocytes (O4(+)-cells) in the immediate vicinity to the lesion site decreased 2-fold, but no significant changes in the number of neurons were found in the rostral and dorsal fragments of the spinal cord compared to the corresponding parameter in controls. Sixty days after transplantation, the cross-section area in the rostral part of the spinal cord at a distance of 3 mm from damage site increased by 15.3% compared to the control. The number of O4(+)-cells at the lesion site and in adjacent rostral and caudal parts of the spinal cord by 22.8% surpassed that in the control group. The number of remyelinated axons also increased. These findings suggest that the absence of pronounced structural changes in the rostral and caudal parts of the spinal cord compared to lesion site at early stages after damage and cell transplantation. At the same time, pronounced activation of oligodendrocytes in this region suggests their involvement together with Schwann cells into remyelination of regenerating axons, which can serve as a factor of partial restoration of motor functions after spinal cord injury.

Study of the efficiency of transplantation of human neural stem cells to rats with spinal trauma: the use of functional load tests and BBB test.

Human ensheating neural stem cells of the olfactory epithelium were transplanted to adult male rats immediately after contusion trauma of the spinal cord at T9 level rostrally and caudally to the injury. Voluntary movements (by a 21-point BBB scale), rota-rod performance, and walking along a narrowing beam were monitored weekly over 60 days. In rats receiving cell transplantation, the mean BBB score significantly increased by 11% by the end of the experiment. The mean parameters of load tests also regularly surpassed the corresponding parameters in controls. The efficiency of transplantation (percent of animals with motor function recovery parameters surpassing the corresponding mean values in the control groups) was 62% by the state of voluntary motions, 37% by the rota-rod test, and 32% by the narrowing beam test. Morphometry revealed considerable shrinking of the zone of traumatic damage in the spinal cord and activation of posttraumatic remyelination in animals receiving transplantation of human neural stem cells.

[Influence of the neural stem cell transplantation on the restoration of CNS functions in rats with cortical stroke].

Ischemic stroke was modeled in white pedigreeless rats by the superficial blood vessel devascularization in the sensorimotor cortex. The preparations of neural progenitors--rat embryonic neural stem cells (rENSC) and human olfactory epithelium-derived neural stem cells (hOENSC) and differentiated fibroblasts ("cell control") were transplanted at the perimeter of the devascularized region. These cells marked with vital tracer stayed alive in the brain parenchyma for at least 16 days. The monitoring of contralateral forepaw motor deficit during 8 weeks demonstrated that only rats with rENSC transplantation had the stable and significant improvement of performance in cylinder test and swimming test (forepaw inhibition test) in comparison to "cell controls" and rats without cell transplantation. The maximal difference in the relative values (the efficacy) was 25% to the end of the experiment. There was no difference in the indicators of vibrissae-elicited forelimb placing test between experimental groups. The methodological approach used makes it possible to broaden the study of mechanisms of neural stem cells' therapeutic effect in stroke.

[Peculiarities of hemostasis, metabolic activity of platelets, and rate of aspirin resistance in patients with chronic heart failure after aorto-coronary bypass surgery].

Aim of the study was to investigate in dynamics peculiarities of hemostasis including platelet aggregation and activity of NADP-dependent dehydrogenases in platelets, as well as prevalence of resistance to aspirin in patients with functional class II-III chronic heart failure (CHF). We determined parameters of vascular thrombocytic and coagulative hemostasis, the state of intracellular metabolism of platelets as assessed by activity of NADP-dependent dehydrogenases in 46 men (age 45 - 72 years) with NYHA class II (n=16) and III (n=30) CHF before and in 12 - 14 days after coronary artery bypass grafting (CABG). After CABG all patients received aspirin (75 - 150 mg/day). Patients with ischemic CHF had moderate activation of intravascular coagulation, endotheliosis, elevation of fibrinogen and plasminogen levels. These processes were significantly augmented after CABG and were most pronounced in class II CHF. At the background of therapy with aspirin in 36 patients (78.3%) ADP and adrenaline induced platelet aggregation was reduced 2 - 3 times. In 10 patients (21.7%) aggregation remained at initial level or even increased what evidenced for resistance to aspirin. Among patients with functional classes II and III CHF portion of resistant to aspirin was 12.5 and 26.6%, respectively. In these patients most striking changes in intracellular metabolism of platelets were revealed. These changes manifested as derangements of energetic and plastic processes in the cell. Thus aspirin resistant patients with CHF comprise a group with risk of development of atherothrombosis of coronary arteries, arteriovenous grafts and arterial conduits.

Immunoliposomal containers as systems of directed transport of minor interfering RNA into Schwann cells.

Immunoliposomal container system for targeted delivery of minor interfering RNA into Schwann cells was developed. Monoclonal antibodies to myelin basic protein served as the vector. Analysis by sandwich ELISA of myelin basic protein showed specific suppression of the target protein gene expression in Schwann cells after incubation with PEG-conjugated immunoliposomes loaded with minor interfering RNA blocking the synthesis of myelin basic protein. Prospects of effective use of this system for targeting of minor interfering RNA for the treatment of diseases of genetic etiology are discussed.

Spontaneous neural differentiation of stem cells in culture of human olfactory epithelium.

We studied differentiation of stem cells in dissociated cultures of olfactory epithelium. Staining with anti-nestin antibodies revealed stem cells in the primary monolayer culture of the olfactory epithelium from adult human. Proliferation of these cells during culturing in serum-containing medium in the presence of nerve growth factors FGF2 and NGF led to the formation of neurospheres freely floating in the medium or attached to the substrate. Further long-term culturing and cloning of dissociated cells from these neurospheres in media not containing nerve growth factors led to spontaneous neural differentiation of the olfactory epithelium stem cells. The cells with phenotypic signs of differentiated neurons were stained with antibodies against beta-tubulin and neurospecific enolase. Differentiated neurons formed diffuse and spatially organized neuronal networks. We hypothesized that factors triggering neural differentiation of olfactory epithelium stem cells are produced byastrocytes present in these cultures.

Multipotent stem and progenitor cells of the olfactory epithelium.

In recent decades, a wide spectrum of fetal and embryonic stem and progenitor cells were used for cell therapy of diseases of the central nervous system, but the olfactory glial ensheathing cells exhibited certain advantages due to their biological properties and capacity to stimulate regeneratory processes in spinal injury. The therapeutic effect of a heterogeneous complex of olfactory epithelial cells is more pronounced; apart from glial ensheathing cells, this complex includes fibroblasts, Schwann cells, stem and progenitor cells of this structure. The use of minimally invasive methods for isolation of human olfactory epithelial tissue is important for clinical practice, because they provide cells for autologous transplantation and rule out graft rejection immune reaction and the risk of transmission viral infection and transfer of genetic defects, which can be associated with allotransplantation.

Culturing of specialized glial cells (olfactory ensheathing cells) of human olfactory epithelium.

A monolayer of dissociated glial cells of human olfactory epithelium was cultured in Petri dishes and 12-well plates using a polylysine-laminin substrate. Primary cultures were subcultured after 10-15 days. The cell cultures were analyzed by phase contrast microscopy at all stages of culturing. A cytological study involved histological methods (trypan blue staining) and immunocytochemical visualization of GFAP, nestin, and low-affinity nerve growth factor receptors. At the final stage of culturing (5 passages) the monolayer cultures included 2 types of cells: GFAP- and p75-positive glial cells and nestin-positive fibroblasts.

[Criteria of efficiency of transplantation of embryonic nervous tissue preparations in rats with 6-OHDA-impaired dopaminergic nigrostriatal system]. / Kriterii éffektivnosti transplantatsii preparatov émbrional'noi nervnoi tkani u krys s povrezhdeniem dofaminergicheskoi nigrostriarnoi sistemy 6-gidrooksidofaminom (6-OHDA).

Effectiveness of transplantation of cells from embryonal nervous tissue of the ventral mesencephalon (VM ENT) and striatum (STR ENT) by apomorphin-induced motor asymmetry (APO-test), consolidation of the transplant (the degree of glyal reaction and amount of dopaminergic neurons) and blood serum levels of GFAP was studied for 3 months in Wistar rats with 6-OHDA-impaired dopaminergic nigrostriatal system. Marked therapeutic effectiveness was registered in VM ENT transplantation in the denervated striatum and in combined transplantation of VM ENT into the lateral cerebral ventricle simultaneously with STR ENT transplantation in the striatum. Separate transplantation of VM ENT in the lateral ventricle and STR ENT in the striatum had no positive effect on recovery of the dopaminergic nigrostriatal system. A correlation was found between the degree of glial reaction of ENT transplants, severity of rotation asymmetry and serum levels of gliofibrillary protein (GFAP). GFAP in the serum for lifetime assessment of transplant consolidation and prognosis of neurotransplantation efficiency was assayed.

Comparison of the efficacy of cell preparations from embryonic ventral mesencephalon of various prenatal age transplanted intrastriatally to rats with 6-OHDA-induced Parkinsonism.

Cell preparations of ventral mesencephalon obtained from 8-, 14-, and 16-17-day rat embryos were stereotactically transplanted to homologous rats with 6-hydroxydopamine-induced hemiparkinsonism. Automated analysis of apomorphine-induced motor asymmetry for 3 months after neurotransplantation revealed higher efficacy of cell preparations from 8- and lower from 16-17-day-old embryos. These data correlated with histomorphological findings, in particular, with the size of grafts, glial reaction, and the number of dopaminergic neurons in the grafts.

[Possibilities of magnetic resonance tomography in pancreatic cancer diagnosis]. / Vozmozhnosti magnitorezonansnoi tomografii v diagnostike raka podzheludochnoi zhelezy.

There was conducted examination of 27 patients with pancreatic cancer using clinico-laboratory and instrumental methods: ultrasonic investigation, computeric tomography (CT), magnetic-resonance tomography (MRT). MRT was uninformative when pancreatic tumor was up to 3 cm in diameter. In diagnosis of cystadenocarcinoma, affection of common biliary duct and vessels and in small focal metastatic hepatic affection the results of MRT showed enhanced precision in comparison with CT. Informativity of MRT and CT in diagnosis of metastases in lymph nodes was equal.

Immunoenzyme assay of glial fibrillary acidic protein for evaluation of functional activity of cell grafts from embryonic ventral mesencephalon in rats with experimental hemiparkinsonism.

The relationship between the release of glial fibrillary acidic protein (GFAP) into systemic circulation and the efficacy of transplantation of embryonic nervous tissue was studied on rats with 6-OHDA-induced hemiparkinsonism. It was found that intrastriatal transplantation of cell preparations from embryonic ventral mesencephalon significantly attenuated apomorphine-induced rotation, which points to functional recovery of the dopaminergic nigrostriatal system. The degree of this recovery depends on reactive astrogliosis around the graft and survival of dopaminergic neurons. Analysis of GFAP concentration revealed significant elimination of this antigen into the circulation 7 and 14 days after transplantation. In rats with good consolidation of the graft without pronounced reactive gliosis, the concentration of GFAP reached 253.99+/-79.30 ng/ml on week 4 after transplantation and decreased to 8.2+/-3.3 ng/ml 8-12 weeks after transplantation. In rats with poor graft consolidation associated with death of transplanted neurons and gliosis in the graft and surrounding tissue the concentration GFAP increased to 476.4+/-111.0 ng/ml within 4 weeks after transplantation and remained elevated (235.0+/-44.8 ng/ml) for 12 weeks. Thus, monitoring of serum GFAP concentrations allows in vivo evaluation of the functional state of intracerebral graft and the level of reactive gliosis. This test can be used for the prognosis of transplantation efficacy.

Catecholamines and their metabolites in the brain and urine of rats with experimental Parkinson's disease.

The content of catecholamines and their metabolites in the brain and the relationship between cerebral catecholamine levels and their urinary excretion were studied in rats with 6-OHDA-induced hemiparkinsonism. 6-OHDA reduced brain concentrations of dopamine, DOPAC, and homovanilic acid and urinary excretion of dopamine, dioxyphenilalanine, and DOPAC by more than 90%. A positive correlation was found between the concentrations of these metabolites in the urine and striatum. Measurement of urinary catecholamines and their metabolites is a perspective test for evaluating the status of the dopaminergic nigrosostriate system of the brain in experimental parkinsonism.

Complex analysis of efficiency of transplantation of embryonic nerve tissue to rats with hemiparkinsonism.

Effect of transplantation of embryonic ventral mesencephalon preparation containing dopaminergic neurons on repair of the dopaminergic nigrostriatal system was studied in rats with hemiparkinsonism induced by 6-hydroxydopamine. Transplantation of embryonic ventral mesencephalon into denervated striatum led to a more than 50% decrease in apomorphine-induced rotation, recovery of dopamine and DOPAC levels in the brain, and to an increase in DOPAC excretion and the DOPAC-dopamine ratio in daily urine of rats with hemiparkinsonism. Dopaminergic neurons of the transplant survived, forming a network of tyrosine hydroxylase-positive processes growing beyond the transplant and reinnervating the adjacent compartments of the striatum. A positive correlation between urinary excretion of DOPAC and brain concentration of dopamine was revealed in denervated rats after transplantation of ventral mesencephalon. Intrastriatal transplantation of cell preparations of embryonic striatum containing no dopaminergic neurons and isolated local injury to the striatum did not affect regeneration of the denervated nigrostratal system.

[Possibilities of x-ray methods in diagnosis of an acute pancreatitis and its local complications]. / Vozmozhnosti luchevoi diagnostiki lokal'nykh oslozhnenii ostrogo pankreatita.

The ray methods of investigation were applied in 182 patients with an acute pancreatitis (AP). In 28% of patients local complications of AP were revealed: pseudocyst, abscess, pancreatic gland phlegmon, the gastric wall infiltration and ulceration, paracolic infiltrate and colonic obstruction, biliary hypertension, portal hypertension, subcapsular and parenchymatous splenic hematoma.