[Safe thyroidectomy for thyroid and parathyroid diseases]. / Puti osvoeniya bezopasnoi tekhniki tireoidektomii pri zabolevaniyakh shchitovidnoi i okoloshchitovidnykh zhelez.

OBJECTIVE: To offer the ways for safe thyroidectomy aimed at prevention of damage of recurrent laryngeal nerve in patients with thyroid and parathyroid diseases. MATERIAL AND METHODS: We analyzed postoperative outcomes after thyroidectomy in 342 patients aged 20-80 years. Topography of recurrent laryngeal nerves was studied on 20 laryngeal-tracheal complexes of deceased patients. Technique of visualization of various segments of recurrent laryngeal nerve was worked out. RESULTS AND DISCUSSION: Thyroidectomy was performed in 342 patients with thyroid and parathyroid diseases. Thyroidectomy was performed in accordance with recommendations described by F.W. Lahey, W.B. Hoover (1938) and H. Malcolm, M.D. Wheeler (1998). Location of recurrent laryngeal nerve varied in patients with nodular, retrosternal goiter and parathyroid gland adenoma. Comparison of intraoperative and morphological data on recurrent laryngeal nerve visualization showed possible risks of its damage during manipulations on thyroid gland, esophagus and trachea. Our study confirmed the need for visualization and mobilization of recurrent laryngeal nerve in all procedures on thyroid and parathyroid glands. Introduction of the described technique of thyroidectomy and training sessions for recurrent laryngeal nerve mobilization on laryngeal-tracheal complexes reduced postoperative incidence of phonation disorders from 21.6% to 0.98%. CONCLUSION: Thyroidectomy may be a safe procedure if surgeons are familiar with the details of surgical technique and prevent damage to adjacent structures.

[Features and choice of surgical strategy in patients with gastrointestinal fistulas]. / Osobennosti taktiki i vybora sposoba khirurgicheskogo lecheniya patsientov so svishchami zheludochno-kishechnogo trakta.

OBJECTIVE: To evaluate the features and choice of surgical strategy in patients with gastrointestinal fistula based on classification of their types. MATERIAL AND METHODS: There were 398 patients with gastrointestinal fistula. Fistula type 1 was found in 126 (31.7%) cases, type 2 - 38 (9.6%) cases, type 3 - 73 (18.3%) cases, type 4 - 26 (6.5%) patients, type 5 - 135 (33.9%) cases. One-stage and two-stage treatment was applied in patients with fistula type 1, two-stage treatment only - for fistula type 2. In patients with fistula type 3, treatment strategy depended on timing of fistula formation, its level and amount of intestinal chymus loss. In case of fistula type 4, radical treatment is difficult. However, surgery is safer when adhesions between intestinal loops are not yet dense enough. Indeed, dissection is associated with less risk of their damage. Reconstructive procedures were applied for fistula type 5 depending on its localization. RESULTS: The causes of gastrointestinal fistula were complications after surgery for acute ileus in 73 patients (17 ones died), blunt abdominal trauma in 81 (8), open abdominal trauma with cold weapons in 39 (6) and firearms in 11 cases (2), mesenteric thrombosis in 33 patients (8), pancreatic necrosis in 25 cases (9), abdominal hernia in 15 cases (4), acute appendicitis in 40 patients (3), colonic diverticulosis in 24 patients (1), urological diseases in 5 cases, colon perforation by a foreign body in 3 cases, colonoscopy in 5 patients, Hirschsprung's disease in 2 patients, Crohn's disease in 11 cases (3), colon polyps in 4 patients, intestinal tuberculosis in 1 case (1), small bowel resection for obesity in 1 patient and gynecological diseases in 25 patients (2). Fistulas type 1 and 4 were followed by the highest postoperative mortality since these interventions are associated with the most severe changes in abdominal cavity. Low mortality was observed in patients with fistula type 5, no abdominal inflammation and normalized intestinal passage. The overall mortality in patients with gastrointestinal fistulas was 16.1%. CONCLUSION: Treatment strategy in patients with gastrointestinal fistula primarily depends on the type of fistula that requires emergency, urgent, delayed or reconstructive surgery. Staged approach in patients with gastrointestinal fistulas can improve treatment outcomes.

[Prospects of combined anterior prosthetic hernia repair in treatment of large and giant ventral hernias]. / Peredniaia proteziruiushchaia gernioplastika kombinirovannym sposobom pri bol'shikh i gigantskikh ventral'nykh gryzhakh.

AIM: To evaluate clinical efficacy of combined anterior prosthetic hernia repair in treatment of large and giant ventral hernias. MATERIAL AND METHODS: Patients with large and giant hernias have been analyzed. In the main group (n=675) combined methods of hernia gates repair were applied, in control group (n=257) - stretching repair including prosthesis deployment. Surgeons (n=22) were interviewed for learning curve, safety, limitations and reliability of combined methods. RESULTS: Combined method of hernia repair in patients with large and giant hernias reduces overall postoperative morbidity (p<0.001), wound complications (p<0.05) and incidence of recurrent hernia (p<0.001). Questionnaire data showed the possibility of learning for the method by the most of surgeons to treat these patients. As a result, some practical recommendations are presented for successful procedures and satisfactory results of treatment.

A defensin fragment regulates CHO-K1 cell spreading and induces renewal of fatty acid composition in cell membrane phospholipids.

A tetrapeptide defensin fragment has been shown to stimulate the spreading of CHOK1 cells. The tetrapeptide investigated had virtually no effect on the composition of cell membrane phospholipids but participated in the regulation of the renewal of fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Incubation of cells with the peptide resulted in a change in the composition of the unsaturated fatty acid residues in the phospholipids investigated: specifically, the content of monoenoic and/or dienoic acids increased and that of polyenoic acids decreased. The possible role of the peptide investigated (1) in the regulation of the functional activity of integrin receptors, and (2) in changes in the packing density of the phospholipid acyl chains in cell membrane microdomains, which affects the rates of integrin clustering and adhesion complex formation, is discussed.

[BDNF secretion in human mesenchymal stem cells isolated from bone marrow, endometrium and adipose tissue].

The ability of mesenchymal stem cells (MSCs) to differentiate into neuronal lineage determines the potential of these cells as a substrate for a cell replacement therapy. In this paper we compare the neurogenic potential of MSCs isolated from bone marrow (BMSC), subcutaneous adipose tissue (AD MSC) and menstrual blood (eMSC). It was found that the native eMCSs, BMSCs and AD MSCs express neuronal marker ß-III-tubulin with a frequency of 90, 50 and 14%, respectively. We also showned that eMSCs have a high endogenous level of brain-derived neurotrophic factor (BDNF), whereas the BMSCs and the AD MSCs are characterized by low basal BDNF levels. As induction of neuronal differentiation in the studied MSCs using differentiation medium containing B27 and N2 supplements, 5-azacytidine, retinoic acid, IBMX and dbcAMF caused changes in the cells morphology, the increased expression of ß-III-tubulin, and the appearance of neuronal markers GFAP, NF-H, NeuN and MAP2. BDNF secretion during differentiation was significantly enhanced in the BMSCs and decreased in the eMSCs cultures. However, no correlation between the basal and induced levels of the neuronal markers expression and BDNF secretion in the studied MSCs has been established.

[Mesenchymal stem cells of human endometrium do not undergo spontaneous transformation during long-term cultivation].

Mesenchymal stem cells isolated from human endometrium (eMSC) are perspective source of stem cells for regenerative medicine. Large amount of these cells accumulated by in vitro cultivation is usually required for transplantation into patients. We established several cell eMSC lines and cultivated them during long period of time to examine the possibility of their spontaneous transformation. All cell lines demonstrate limited lifespan, undergo replicative senescence and die. Karyotypic analysis on different passages reveals that most cells display karyotypic stability. Thus, extended in vitro cultivation of eMSCs does not lead to spontaneous transformation that makes therapeutic application of these cells safety for patients. During long-term cultivation eMSCs sustain the expression of surface markers.

[The effect of the collagen tripeptide fragment (GER) on the adhesion and spreading of fibroblasts depends on the properties of adhesive surface].

The effect of collagen tripeptide fragment GER on the adhesion and spreading of mouse embryonic fibroblasts STO to different substrates--polystyrene plastic and immobilized on plastic poly-L-lysine, fibronectin or gelatin was studied. Tripeptide GER has been found to participate in the regulation of fibroblast adhesion and spreading. Therewith, the tripeptide effect value on cell response was dependent both on the mode of tripeptide addition to culture medium and on the type of used substrate. During coincubation of fibroblasts with the tripeptide the stimulation of cell attachment and spreading to untreated plastic and plastic coated with fibronectin or gelatin was observed. At the same time the tripeptide did not change cell adhesion to immobilized poly-L-lysine. Preincubation of cells with the tripeptide resulted in partial inhibition of fibroblast adhesion and spreading on fibronectin- and gelatin-coated substrata. In was shown that the extent of activation and inhibition of adhesive processes on fibronectin was higher than such ones on gelatin after tripeptide treating. The data obtained support the assumption about concerted action of tripeptide GER (activity of which was dependent both on the used concentration of the tripeptide and on the mode of tripeptide addition to culture medium) and chemical characteristics of substrate (polymers of styrene and L-lysine, ECM proteins in native (fibronectin) or partly denatured (gelatin) form) on the cell adhesion and spreading. The main targets on which the GER peptide may affect during the formation of cell-substrate interactions are discussed.

[Examination of cytotoxic effect of anti-cancer drug doxorubicin on human embryonic stem cells].

Cytotoxic effect of anti-cancer drug, doxorubicin (DR), has been examined on human embryonic stem cells (ESC) C910 and fibroblasts spontaneously differentiated from these cells. The fibroblasts retained diploid karyotype. It was found that ESC are more sensitive to DR than fibroblasts: DR dose killing 20% cells was 0.01 and 0.1 microg/ml, respectively. DR induced ESC apoptotic death and reduced both ESC and fibroblast proliferation. Unlike fibroblasts DR reversibly inhibited ESC proliferation. Thus, we have demonstrated that ESC and their differentiated derivates differ their sensitivity and response to the genotoxic agent.

[Generation of dopamine neurons from human embryonic stem cells in vitro].

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.

[Novel human embryonic stem cell lines C612 and C910].

Novel human embryonal stem cell lines C612 and C910 have been established from hatching blastocytes. Cells were cultivated in mTeST medium on mouse fibroblast feeder-layers. They express common pluripotent markers such as alkaline phosphatase, Oct 3/4, SEEA-4, Nanog, Rex1. Immunophenotyping of these cells by flow cytometry revealed expression of CD90 (Thy-1) and CD117 (c-kit) antigens and weak or no expression of CD13, CD34, CD45, CD130, HLA class I and HLA class II antigens. This pattern of surface antigen expression is common for human embryonic stem cells. G-banding assay of C612 and C910 metaphase plates showed that karyotypic structure of these cells was normal both in chromosome number and structure. The cells are pluripotent because of their capability to generate embryoid bodies, undergo spontaneous differentiation and express markers of all germ layers: nestin, keratin, vimentin (ectoderm), alpha-fetoprotein (entoderm), and muscle alpha-actinin (mesoderm). Thus, C612 and C910 cells have all attributes of typical human embryonic stem cells (diploid, capable of self-renewal, express pluripotent markers and differentiate into three germ layers) and may be of potential use for fundamental and regenerative medicine researches.

[The role of the collagen tripeptide fragment GER in the adhesion activation and modification of fatty acid composition in membrane phospholipids of CHO-K1 cells].

It has been found that multiply repeated tripeptide fragment GER (Gly-Glu-Arg) from different collagen types stimulates nonspecific adhesion of CHO-K1 cells. Activation of cell adhesion is accompanied by modifications in fatty acid composition of cell membrane phospholipids. Cell incubation with the synthetic peptide increases the unsaturation indexes of phosphatidylcholin (PC), phosphatidylethanolamine (PEA) and phosphatidylinositol (PI). Arachidonic (C20:4omega6) acid is mainly contributed to the increased unsaturation index of PI. In the case of PC and PEA not only arachidonic acid but also other unsaturated fatty acids: docosatetraenoic (C22:4omega6), docosapentaenoic (C22:5omega3) and docosahexaenoic (C22:6omega3) acids are implicated in the index increasing. Besides, the elevation of relative content of molecules with polyenoic fatty acids in the group of PI molecules is accompanied by decrease in monoenoic fatty acids caused mainly by decrease in the oleic (C18:1) acid level. The role of the investigated peptide: 1) in the activation of cell adhesion as a regulator of active or non active state of integrin receptors: 2) in the alterations of fatty acid composition in main classes of phospholipids as modulator of fluidity level in annular lipid zones around these adhesive molecules is discussed.

[Complex treatment of the patients with postoperative ventral hernia].

Overall 605 patients with postoperative ventral hernia underwent plasty of anterior abdominal wall by combined methods and on-lay or in-lay disposition of synthetic implant. Concomitant diseases were diagnosed 432 (71.4%) patients that required 495 simultaneous operations at 283 (43.8%) patients. Wound complications after surgery were seen at 21 (3.47%) patients. Long-term results were followed-up to 11 years: recurrences of hernia were diagnosed at 12 (1.9%) patients, 3 (0.5%) patients died due to pulmonary embolism. It is concluded that the treatment of patients with postoperative ventral hernia requires complex approach and leads to good short- and long-term results.

[Effects of synthetic fragments of defensins on aggregation and adhesion of epitheliolike cells].

Normal course of processes of regeneration and epitheliazation of damage tissues has been shown to be based on the capability of cells participating in these processes for selective adhesion. In the case of the complete or partial absence of this capability in the cells-participants of the wound healing process, the so-called non-healing wounds appear. In this connection, it remains actual to search for natural agents promoting healing of chronic non-healing wounds. In the present work, we studied effects of synthetic fragments of leukocytic antimicrobial peptides defensines--GER, FGER, and GERA--on aggregation and adhesion of epitheliolike cells of the CHO-K1 line. These peptides have been established to have aggregate-stimulating properties; besides, they enhance adhesion of the cells to the untreated plastic and inhibit fibronectinmediated cell adhesion. Possible pathways of regulation by peptides of processes of intercellular and cell-matrix interaction are discussed as well as ways of release of these compounds in an organism and their functional role in an organism.

Redox environment in stem and differentiated cells: A quantitative approach.

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

[Cytophotometric determination of non-heme iron content in hepatocytes. I. Effect of cell separation techniques on iron content]. / Tsitofotometricheskoe opredelenie soderzhaniia negemovogo zheleza v gepatotsitakh I. Vliianie uslovii izoliatsii kletok na soderzhanie v nikh zheleza.

To study conditions of the preservation of non-heme iron (3+) in hepatocytes, experiments were performed on rats fed with the diet supplemented with 2% carbonyl iron. The cells were isolated by a collagenase method (an enzymatic method) or by a treatment of the tissue with the phosphate buffer (a nonenzymatic method). When using the enzymatic method, the main steps of obtaining preparations-smears were analysed: perfusion of the organ, subsequent washing out of the cells from collagenase, mounting of the smear on the object glass. When using the non-enzymatic method, such steps were an incubation of the tissue pieces in the isolation solution and mounting of the smears. It has been found that the enzymatic isolation method results in practically no losses of iron from the cells if the perfusion lasts for 20 min. A slight loss (10-12%) of the Perls'-stained iron can occur during the initial 30 min of the washing out of the cells from collagenase. This step is not accompanied by any morphological changes of the cells; their viability, according to the trypan test, is 70-87%. The iron release from the cells rises with decrease in the viability of hepatocytes. It has been shown that the greatest losses of iron can occur at the mounting step when the cells are submitted to a substantial mechanical effect. When the nonenzymatic method is applied, the incubation of the cells in the phosphate buffer for up to 2 hr causes no marked morphological changes revealed in the light microscope; however, the cell viability is very low (about 1%). The preservation of iron in the cells is lower when using the nonenzymatic than the enzymatic method. Thus, for cytophotometric determinations of the iron content in hepatocytes, the collagenase method of isolation of cells is recommended.

[The kinetics of rhodamine 123 efflux from cells with multiple drug resistance under the action of energy metabolism inhibitors]. / Kinetika vykhoda rodamina 123 iz kletok s mnozhestvennoi lekarstvennoi ustoichivost'iu pri deistvii ingibitorov énergeticheskogo metabolizma.

A study was made of the effects of inhibitors of ATP synthesis on the process of rhodamine 123 (R-123) release from sensitive sp2/0-Ag14 cells, multidrug-resistant mouse myeloma spEBR-5 cells and hybridoma IF7, derived from spEBR-5 cells. It has been shown that IF7 cells are cross-resistant to ethidium bromide, colchicine, actinomycin D and adriamycin. However, hybridoma IF7 cells, compared to parental spEBR-5 cells, show a lower resistance index. When studying the dependence of the R-123 efflux rate on glycolysis intensity (effect of 2 mM 2-deoxyglucose) and on the level of oxidative phosphorylation activity (effect of 2 mM KCN and 30 microM dinitrophenol), the following distinctive properties of the R-123 transport system of IF7 cells (compared to spEBR-5 cells) were detected: 1) uptake of R-123 into IF7 cells is similar to that observed for the sensitive sp2/0-Ag14 cells; 2) efflux of R-123 from IF7 cells takes place more intensely; 3) R-123 transport is dependent on the rate of glycolysis and may be inhibited by KCN. It is found that 2,4-dinitrophenol inhibits the R-123 efflux from all the cells. Verapamil reverses the multidrug resistance both in spEBR-5 and IF7 cells. The mechanisms of multidrug resistance of cells are discussed.

[The genome structure and phenotypic characteristics of murine hybridoma 1F7 cells selected in the presence of adriamycin and ethidium bromide]. / Issledovanie struktury genoma i fenotipicheskikh osobennostei kletok myshinoi gibridomy 1F7, otselektirovannykh v prisutstvii adriamitsina i bromistogo étidiia.

A study was made of geno- and phenotypic changes, associated with of multidrug resistance development in murine 1F7 hybridoma cells, selected for adriamycin and ethidium bromide resistance (1F7-EBR and 1F7-ADR). In both cell lines overexpression of mdr1 gene was observed, while amplification of mdr1 gene was detected only in 1F7-ADR cells. Karyotypic analysis revealed in resistant cells the presence of a specific marker M45 absent from parental cells, thus suggesting its selective importance. The M45 length instability, as well as the presence of a distal typical homogeneously stained region in it provide an evidence for a link between M45 and mdr1 gene amplification. The frequency of double minute chromosomes in parental lines was the same as in multidrug resistant lines. In 1F7-ADR cells, in contrast with 1F7-EBR cells, the enhancement of immunoglobulin production and the increase in immunoglobulin gamma 2b heavy chain gene expression were observed, which correlated with a decline in DNA-topoisomerase II activity.

[Effect of ectopic superexperssion of the anti-apoptotic gene bcl-2 on the level and character of karyotypic instability in the CHLL V-79 RJK Chinese hamster cell line]. / Vliianie éktopicheskoi sverkhékspressii antiapoptoticheskogo gena bcl-2 na uroven' i kharakter kariotipicheskoi nestabil'nosti v kletkakh kitaiskogo khomiachka linii CHL V-79 RJK

Karyotypic destabilization in cells of Chinese hamster fibroblasts CHL V-79 RJK with ectopically overexpressed antiapoptotical human bcl-2 gene from pSFFV-bcl-2 vector has been analysed. Analysis of G-banded metaphase chromosomes from 4 clones with different levels of bcl-2 expression revealed an increased level of chromosomal instability in bcl-2-transfected cells. Besides, an increased percentage of aneu- and polyploid cells and high level of cells with different chromosomal aberrations was observed. The degree of karyotypic instability positively correlated with the level of bcl-2 expression in bcl-2-transfected cells. Cells of a clone with the highest bcl-2 expression at the 13th passage of cultivation displayed an almost 100% polyploidization and the presence of specific aberrations and a tricentric marker chromosome. Selection of cells with non-random specific chromosome changes was observed in pSFFV-bcl-2-transfected CHL V-79 RJK cells in the process of their long-term cultivation. By contrast, cells of the parental cell line, as well as the control pSFFV-neo transfectants, displayed a stable karyotype throughout the long period of cultivation. It is important that the presence of morphological markers of gene amplifications--DOO, DM, MH--was observed in bcl-2-transfected cells. These findings suggest that the overexpression of antiapoptotic human bcl-2 gene may result in destabilization of the karyotype structure in cells of Chinese hamster fibroblasts CHL V-79 RJK. The character and level of destabilization correlate with the level of ectopic overexpression of this gene.

[The isolation and characteristics of monoclonal antibodies to the HNK-1 antigen of natural killers]. / Poluchenie i kharakteristika monoklonal'nykh antitel k HNK-1-antigenu estestvennykh killerov.

Monoclonal antibodies to the HNK-1 differentiated antigen of natural killer cells have been obtained. A glycoprotein of the white human brain connected with myelin was used as an antigen for immunization of mice. The monoclonal antibodies obtained are shown to reduce the cytotoxic activity of a human blood mononuclear fraction as much as by 65% in relation to the human lymphoblastoma cell culture K-562. Their interaction with the surface antigen of mononuclears was shown by immunofluorescent method. Monoclonal antibodies belong to the class of immunoglobulins M.

Resistance to adriamycin in human chronic promyeloleukemia line K562 correlates with directed genome destabilization--amplification of MDR1 gene and nonrandom changes in karyotype structure. / Ustoichivost' kletok khronicheskogo promieloleikoza cheloveka linii K562 k adriamitsinu korreliruet s napravlennoi destabilizatsiei genoma--amplifikatsiei gena MDR1 i ne sluchainymi izmeneniiami v strukture genotipa.

Three independent subclones (B2, B3 and C9) of human myelogenous leukemia cell line K562 selected with adriamycin (ADM) were analysed. Cross-resistance of these ADM-resistant cells was examined for a resistance to the number of drugs including colchicine, actinomycin D and ethidium bromide. MDR 1 gene amplification in B3 and C9 subclones was detected using Southern-hybridization with specific probe. Additional genetical material was found in genomes of resistant cells by analysis of G-banded metaphase chromosomes. An extraordinarily long marker chromosome was observed in every C9 metaphase plate. The character of this chromosome G-banding suggests that it may be a derivative of chromosome 5 containing a large homogeneously staining region (HSR) in locus 5q15. Both B2 and B3 subclones expressed double minute chromosomes (DMs) in 5% of cells. In the course of a prolonged cultivation (about 2 years) of C9 cells in the presence of ADM a progressive karyotype destabilization was observed: the frequency of new markers formation in C9 cells increased, cells having additional copies of marker chromosome with HSR appeared, the length of HSR varied, coexistence of HSR and DMs being found in several C9 cells. These karyotypical changes may be regarded as patterns of genome destabilization due to the multidrug resistance of K562/ADM cells.