Neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands.

Stem/progenitor cells are thought to have the potential in the treatment of severe neurodegenerative diseases. Recently, sympathoadrenal progenitors expressing specific markers of neural crest derivatives and capable to differentiate into neurons were discovered in adult bovine and human adrenal glands, but there was no reported data on cryopreservation of sympathoadrenal progenitors. The aim of the present study was to examine the neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands. Considering impact of various initial state of frozen biomaterial on cell recovery, we carried out a comparative estimation of cryopreservation outcome both for adrenal tissue fragments and isolated primary cells. The estimation consisted of determining cell yield, viability, ability to adhere, proliferate and differentiate in vitro. Cells isolated from the fresh adrenal glands were cultured until confluence. A formation of sympathoadrenal progenitors-embedded spherical cell colonies, whose cells are differentiated then into ßIII-tubulin-positive cells with neuron-like morphology, was observed on the monolayer. The colonies were well preserved after cryopreservation of cell culture with a cooling rate of 1 °C/min in the cryoprotectant media containing 5-15% of dimethylsulfoxide. Adrenal tissue fragments were cryopreserved in the presence of 10% dimethylsulfoxide at the cooling rates of 0.3; 1: 5; 40 and > 100 °C/min. Sympathoadrenal progenitors were recovered after cryopreservation with 0.3 °C/min cooling rate but not higher.

Adhesion, growth, and proliferation of endothelial cells on biopolymer extracellular film matrices.

We analyzed the influence of initial properties of biopolymer fi lm extracellular matrices made of chitosan on adhesion of swine embryonic kidney epithelium-like cells, their morphology, growth, and proliferation. It was found that adhesion index of cells on the studied matrices was similar, but cell morphology had significant qualitative differences. Round non-flattened cells were described in endothelial cell cultures on chitosan-based matrices. Local variations in charge density on matrices induced by the presence of chitin nanofibers led to the appearance of flattened spindle-shaped and non-flattened sphericalal cells. Long-term culturing on biopolymer extracellular fi lm matrices was associated by the formation of spatial conglomerates of sphericalal cells.

[Proteasomes on thyroid tissue allotransplantation under induction of donor specific tolerance in rats].

The proteasomes in the liver of August rats (RT1C) were investigated 30 days after the allotransplantation of Wistar rat (RT1u) thyroid tissue under renal capsule with/without induction of donor specific tolerance by donor splenocyte intraportal administration. The level of the total proteasome pool, immune proteasomes containing the LMP2 and/or LMP7 subunits, proteasome 19S- and 11S-regulators was defined. The intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups that indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of non-tolerated rats compared to tolerated animals. In the liver of tolerated rats with survived transplants, the quantity of mononuclear cells, expressing the immune subunit LMP2, greatly increased in comparison with control and non-tolerated animals. Study of the survived transplants showed the increase of the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them compared to the tissue replacing the rejected transplants. In the control intact thyroid tissue, the immune proteasomes were almost not revealed, while 19S/11S ratio was maximal. Thus, the development of the immune reaction or its suppression is accompanied by change of the balance between different proteasome forms. The immune subunit LMP7 and 11S regulator are connected with the response against donor tissue. On the contrary, the immune subunit LMP2 and 19S regulator are likely to be important for the immune tolerance development and survived tissue functioning. The low content of the immune proteasomes in the follicle cells was found by immunofluorescence assay. The formation of antigens for major histocompatibility complex class I molecules was impaired by low immune proteasome content that led to immunological tolerance to hormone-producing follicle cells.

[Characteristics of primary cell culture from neonatal thyroid grand of pigs: folliculogenesis, hormone and growth].

In the study, comprehensive assessment of proliferative and hormonal activity of primary cell cultures derived from neonatal pig thyroid has been carried out for the first time. We have evaluated the basal and TSH-stimulated secretion of thyroxine and morphological features of culture, depending on the initial state of the material placed in culture: in the form of single cells or follicular conglomerates. Folliculogenesis and formation of dome structures were observed in culture spontaneous and under chronic TSH stimulation. The ability of the cells to expression of ß-III-tubulin during prolonged cultivation in the presence of NGF has been demonstrated in the study.

[Changes in the proteasome function after induction of donor-specific tolerance in rats with ovarian allograft].

Induction of donor-specific tolerance in a recipient is one of the methods for enhancing acceptance of the grafts of endocrine glands in the absence of immunodepressants, which interfere with hormone production. This paper describes changes in the proteasome pool in the rat liver, spleen, and graft during the development of donor-specific tolerance after intraportally infusing the recipient with donor splenocytes with subsequent allografting of ovarian tissue into the renal capsule. It has been demonstrated that the shift in the balance in the liver and graft proteasome pools towards the variants with the LMP2 subunit determines the development of immunological tolerance and graft retention. On the contrary, an increase in the forms with the LMP7 subunit induces the immune response and graft rejection.

Outcome of adrenal tissue fragments allotransplantation: the impact of cryopreservation.

Cryopreservation is thought to have the potential to preserve tissue for transplantation. In addition, it can also be used for decreasing tissue immunogenicity, which might be important for prolonging allograft survival. In the present study we examined the impact of cryopreservation at various cooling rates on the outcome of allotransplantation of murine adrenal tissue fragments (ATFr). ATFr were cryopreserved with a cooling rate at 1; 10; 40 and more than 100 °C/min. After thawing it was found that the number of the cells expressing markers of dendritic cells (CD11c) and macrophages (CD11b) in the suspension obtained from ATFr decreased with increasing cooling rate. After allotransplantation the survival rates of adrenalectomized mice and the blood serum levels of corticosterone were higher in recipients of cryopreserved ATFr. By immunohistochemistry, cryopreserved allografts displayed a decreased infiltration by CD4+ and CD8+ T-lymphocytes as compared to fresh grafts. These findings suggest that cryopreserved allografts cause a less severe rejection by decreasing graft immunogenicity.

[Effect of some drugs on the intensity of lipid peroxidation in surrounding tissues in the local treatment of suppuration]. / Vliianie nekotorykh preparatov na intensivnost perekisnogo okisleniia lipidov v perifokalnykh tkaniakh pri mestnom lechenii gnoinogo ochaga.

The effect of some drugs in their local use for treatment of purulent wounds on the intensity of peroxide oxidation of lipids in the perifocal tissues of an experimental suppurative focus was studied. The increased antioxidant and antiinflammatory effect in use of lyposomal form of gentamycin with the ectericide-modified membranes versus ectericide solution as well as more pronounced antioxidant and antiinflammatory effect of ferecterisole vs. cimesole was revealed. The results obtained indicate that the use of ferecterisole and lyposomal forms of some antibiotics for local treatment of purulent wounds has good prospects.

[Permeability of liposomes with different cholesterol content as affected by low temperature]. / Pronitsaemost' liposom s razlichnym soderzhaniem kholesterina pod vliianiem nizkikh temperatur.

Membrane permeability is found to decrease in the range of 0+ -25 degrees C with an increase of the cholesterol content in liposomes from 0 to 50%. A higher stability of liposomes at low temperatures is observed under hypertonic conditions as compared with hypotonic and isotonic conditions, the cholesterol content being 20-50%. The obtained results demonstrate the cholesterol effect on structural changes in the liposome membrane under supercooling and important role of cholesterol in the liposome stability to environmental changes.

[Effect of excess cooling on rat liver mitochondria: release of K+ and malate dehydrogenase at lipid phase transition temperatures]. / Vliianie pereokhlazhdeniia na mitokhondrii pecheni krysy: osvobozhdenie kaliia i malatdegidrogenazy pri temperaturakh fazovykh perekhodov lipidov.

The effects of cooling within the temperature range of 0-25 degrees on the state of mitochondrial membranes was studied. The increase in membrane permeability for K+ and malate dehydrogenase which is localized in mitochondrial matrix, served as an index of membrane structural damage within the given temperature interval. In order to prevent crystallization of the aqueous phase upon cooling ethylene glycol was added to the medium to reach a final concentration of 40%. The decrease of temperature resulted both in the release of K+ and in the increase of the activity of free malate dehydrogenase. A drastic increase in the mitochondrial membrane permeability to K+ and malate dehydrogenase was observed within the temperature range of -6 -- -14 degrees, showing a maximum at -10 degrees. At -14 degrees the rate of K+ and malate dehydrogenase release was lower; however, further cooling (down to -20 degrees) induced another increase of permeability. The disruption of mitochondrial at negative temperatures after addition of 1.25 M NaCl to the incubation mixture caused a temperature-dependent release of the proteins into the supernatant. After ultracentrifugation of the membrane fragments at 140 000 g for 60 min the soluble proteins were detected in the samples coled down to -10 degrees. It was concluded that the changes in the state of mitochondrial membranes are due to the phase transitions of the inner membrane lipids.

[Effect of low temperatures on the phospholipase A2 activity of rat liver mitochondria]. / Bliianie nizkikh temperatur na aktivnost' fosfolipazy A2 mitokhondrii pecheni krys.

Phospholipase A2 is shown to be activated by freezing-thawing, possibly due to changes in the state of lipid bilayer under the effect of both the temperatures themselves and physicochemical factors formed in the low-temperature range.

[Effect of freezing-thawing on the lipid composition of mitochondria]. / Vliianie zamorazhivaniia-ottaivaniia na lipidnyi sostav mitokhondrii.

The effect of near- and subzero temperatures on alterations in the lipid composition of albino rat liver mitochondrial membranes has been studied. Low temperature effect has been shown to intensify peroxide processes and to activate A2 phospholipase in mitochondrial membranes. This is followed by the decrease in lecithin, ethanolaminophosphatide, cardiolipin and cholesterol contents as well as by the accumulation of free fatty acids in the different temperature regions. The dependence of lipid mitochondrial membrane modification intensity on freezing and rewarming rates has been determined.

[Changes in phospholipid composition of mitochondria after freezing-warming in the presence of Na2S204]. / Izmeneniia fosfolipidnogo sostava mitokhondrii posle zamorazhivaniia--otogrevaniia v prisutsvii ditionita.

The phospholipid composition of mitochondria membranes subjected to rapid freezing (300-400 degrees C per 1 min) to -196 degrees C and subsequent slow warning (at 20 degrees C) was determined by the method of chromatography in the thin layer of silicagel. Under such conditions of freezing and warming a significant decrease in lecithin and ethanolamine phosphatide content is observed in the mitochondria membranes. When freezing the suspension of mitochondria in the medium containing Na2S2O4 in a concentration of 0.05 M only the lipid component changes slightly.

[Changes in the lipid composition of mitochondrial membranes following exposure to low temperatures]. / Zminy lipidnoho skladu mambran mitokhondrii pislia diï nyz'koï tmeperatury

The phospholipid composition of the membranes of the albino rat liver mitochondria was studied by the method of thin-layer chromatography before and after rapid freezing down to --196 degree C and rapid (at 37 degree C) and slow (at room temperature) thawing. Significant reduction of lecithine and ethanolamine phosphatide composition is observed in the mitochondria exposed to rapid freezing and slow thawing. A simultaneous accumulation of diglycerides is found in the membranes. No reliable changes in phospholipid composition of the mitochondria membranes are observed after exposure to rapid freezing and rapid thawing.