[Therapeutic Effects of Multipotent Mesenchymal Stromal Cells after Irradiation].

Multipotent mesenchymal stromal cells (MSC) are now considered to be a perspective multifunctional treatment option for radiation side effects. At present.a great number of sufficient evidence has been collected in favor of therapeutic effects of MSCs in acute radiation reactions. It has been shown that MSC-based products injected locally or systemically have therapeutic effects on irradiated organs and tissues. This review presents summarized experimental and clinical data about protective and regenerative effects of MSCs on different radiation-injured organs and tissues; the main probable therapeutic mechanisms of their action are also discussed.

[Structural changes of the adrenal cortex in experimental genital herpes virus infection].

The reactive changes in the adrenal gland cortex were studied in mature female guinea pigs (n=5) in an experimental model of acute genital herpes virus infection. The methods of light and transmission electron microscopy were used. To confirm the presence of viral antigen in the corticosterocytes (CSC), the methods of immunfluorescence and electron microscopic immunocytochemistry were used. It was shown that at day 7 of an acute process, focal CSC reactive changes appeared in the glomerular zone - at the light microscopic level, CSC had intact nuclei and optically empty cytoplasm, while at the electron microscopic level, these CSC demonstrated the damaged membranous organelles, and various membranous structures which were not found in the normal cells. The aggregates of hypertrophied CSC were found in the fasciculate zone. The changes described were reversible, as they practically disappeared by the onset of spontaneous recovery (day 21 after inoculation). The regeneration of CSC of glomerular and fasciculate zones of the adrenal cortex involves both intracellular and cellular mechanisms.

Comparative characteristics of platelet lysates from different donors.

We studied the effect of platelet lysates from different donors on fibroblast growth in culture. In most samples (40 of 50), the growth-stimulating characteristics were greater than in 10% FCS, but every ninth sample exhibited low mitogenic activity. A weak dependence between platelet concentration and total protein content was noted, but no correlation was found between these parameters and fibroblast growth in culture.

[Allocation and identification of fibrocytes from human peripheral blood].

Fibrocytes are the cells circulating in peripheral blood that synthesize a big number of various factors and take part in the start of reclaiming processes. The wound healing is a result of activity of fibrocytes. It is known that they participate in formation of hypertrophic and kelloid scars. The purpose of the present work was to research specific properties of fibrocytes in vitro. The data obtained testify that these cells really have hematopoietic origin and are undifferentiated. In this connection, while cultivating fibrocytes it is necessary to keep to some specific conditions: the use of the medium specific for stem cells and very high density of cultivation. In 10 days of culturing, the fibrocytes differentiate into fibroblasts. From the general pool of peripheral blood mononuclear cells, only fibrocytes are capable of DNA synthesis but in spite of it proliferative potential of these cells is very low.

[Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins].

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.

[Changes in human burn fluid biological activity during normal burn healing]. / Izmenenie biologicheskoî aktivnosti ozhogovogo ékssudata cheloveka v khode zazhivleniia ozhoga.

The main goal of this work was monitoring the changes occurring in human burn fluid biological activity during normal burn healing. The fluid available in the burn until healing makes a good material for controlling biochemical microenvironment of burn cells. This environment involves factors, such as extracellular matrix proteins and matrix metalloproteinases. In this work our previous studies of the influence of wound and burn fluids on the functional activity of cells were extended to include the effect of burn fluid on fibroblasts and keratinocytes, i. e. human skin cells present in the wound and involved in wound healing. It was shown that human burn fluid biological activity depends on the time that passed after burning, and on the correctness of healing. Migration of human fibroblasts becomes more intensive under the influence of such a fluid independently on the time of fluid sampling. Unlike, keratinocyte migration was inhibited by burn fluid sampled 1-3 days after burning but was enhanced by fluids sampled 6 days following burning. The obtained data are to be necessarily taken into consideration at burn treatment and also at transplantation of cells for healing of wounds of different nature.

[Enhancement of fibroblast growth promoting activity of human blood after its irradiation in vivo (transcutaneously) and in vitro with visible and infrared polarized light]. / Povyshenie rostostimuliruiushchei aktivnosti krovi cheloveka dlia fibroblastov posle ee oblucheniia in vivo (transkutanno) i in vitro vidimym i infrakrasnym poliarizovannym svetom.

Visible and infrared (IR) irradiation of laser and non-laser sources has a pronounced wound-healing effect promoting tissue repair without hyperproduction of connective tissue elements. This effect develops as a consequence of local and systemic light effects, but many aspects of their mechanism have been yet unclear. In the present work, we have shown that in 0.5 h after irradiation of a small area of the volunteers' body surface with polychromatic visible + IR light (400-3400 nm, 95% polarization, 12 J/cm2) the amounts of PDGF and TGF-beta 1 in the blood serum increase, on average, by 20 and 43%, respectively. This effect is preserved for at least 24 h to be recorded only in volunteers with the initially normal and decreased levels of the growth factors; the initially elevated content of PDGF-AB decreases. Addition of such a plasma (2.5%) to the nutrient medium of primary cultures of human embryonal fibroblasts stimulates cell proliferation, on average, by 10 and 17%, but only in the case if the initial growth-promoting (GP) blood activity was low. Similar changes occur in parallel experiments following irradiation of blood samples of the same volunteers in vitro, as well as at mixing irradiated and non-irradiated autologous blood at the ratio 1:10 (v/v), i.e. at modeling a situation in the vascular bed, when the transcutaneously photomodified blood contacts with the rest of its volume. Similar changes in the blood GP activity under conditions in vitro were recorded as well after 4-9 daily phototherapy sessions. This allows us to suggest that changes in GP activity of circulating blood of the irradiated volunteers may be, to a large extent, the consequence of effect exerted on the blood by small amounts of transcutaneously photomodified blood. The obtained results are discussed in terms of light effect on wound healing and scar tissue formation, with regard to the authors' previous data on much higher GP of the irradiated blood in respect to keratinocytes, the fast decrease in proinflammatory cytokine levels, and the increase in IFN-gamma content.

[Beta-1 and beta-4 integrins, and laminin 67 kDa receptors in interaction between A431 cells and laminin isoforms ]. / Vklad integrinovykh retseptorov s beta-1 i beta-4 subedinitsami i retseptora laminina s molekuliarnoi massoi 67 kDa vo vzaimodeistvie kletok linii A431 s izoformami laminina.

Laminins, as basal membrane glycoproteins, are able to stimulate cell adhesion and migration, and to influence gene expression. The laminin molecule has a set of bioactive sites that interact with different integrin and nonintegrin receptors, and, as a result, the reaction of the same cell type to different laminin isoforms may be different. The aim of this study was to determine the contributions of both integrins with beta 1 and beta 4 chains and 67 kDa laminin receptor in the interaction of A431 cells with two laminin isoforms: laminin-1 and laminin-2/4. The obtained data show that integrin alpha 6 beta 4 is more specific for interaction with laminin-2/4 than with laminin-1 and takes part in the stage of attachment of A431 cells to laminin. 67 kDa receptor promotes cell spreading on laminin-2/4 and inhibits cell spreading on laminin-1. An assumption was made about the complex action of receptors for interaction of A431 cells with laminins ("integrin alpha 6 beta 4--67 kDa receptors" complex).

[Enhancement of growth promoting activity of human blood on keratinocytes after its irradiation in vivo (transcutaneously) and in vitro with visible and infrared polarized light]. / Povyshenie rostostimuliruiushchei aktivnosti krovi cheloveka dlia keratinotsitov posle ee oblucheniia in vivo (transkutanno) i in vitro vidimym i infrakrasnym poliarizovannym svetom.

To stimulate wound healing, current medicine uses various methods of phototherapy. The induced activation of proliferative processes in the wound occurs due to development of not only local, but also systemic processes, whose nature remains largely uninvestigated. The present work provides evidences that as early as 30 min after irradiation of a small area of the volunteer's body surface with polychromatic visible light + infrared polarized light (400-3400 nm, 95% of polarization) at a therapeutic dose (12 J/cm2), soluble factors appear in the circulating blood, which are able to stimulate proliferation of human keratinocytes in primary culture. A similar effect was also revealed after a direct blood irradiation. A proof is provided in favor of a hypothesis that a rapid rise of growth promoting activity of the entire circulating blood may be a consequence of transcutaneous photomodification of the small amount of light-modified blood in superficial skin vessels, and of the effect of such blood on its entire circulating volume. A possibility of a release into plasma of growth factors from blood cells and complexes with alpha 2-macroglobulin is discussed.

Effect of melanins from black yeast fungi on proliferation and differentiation of cultivated human keratinocytes and fibroblasts.

The effects of melanin preparations from black yeast fungi (BYF) on the proliferation and differentiation of normal cultivated human skin keratinocytes and embryonic pulmonary fibroblasts have been investigated. Melanin preparations in the range of 5-0.1 microg/ml were optimally active, with a more pronounced effect on keratinocyte than on fibroblast proliferation. Of 17 dihydroxynaphthalene (DHN) natural melanin preparations and two commercial dihydroxyphenylalanine (DOPA) melanin preparations, only one preparation--DOPA melanin (of animal origin) significantly stimulated proliferation of keratinocytes at 5 microg/ml; four preparations (DHN melanin from BYF) significantly inhibited proliferation of these cells at 5 or 1 microg/ml. The remaining preparations had no significant effect. Similarly, of the 17 preparations of DHN melanin from BYF, one preparation significantly stimulated fibroblast proliferation, and four significantly inhibited proliferation at 5 microg/ml, one at all the concentrations, and three from 1 down to 0.1 microg/ml. These melanin preparations were also shown to affect the in vitro differentiation of keratinocytes.

[Differences in the character of interaction of normal and transformed human keratinocytes with laminin isoforms]. / Razlichiia v kharaktere vzaimodeistviia normal'nykh i transformirovannykh keratinotsitov cheloveka s izoformami laminina.

Laminins constitute a family of heterotrimeric glycoproteins of basement membranes. Laminins promote cell adhesion, migration, growth, and differentiation. So far, at least 12 different isoforms of laminin have been known. However, no sufficient knowledge is available on the nature of cell response on different laminins. The study was aimed to compare adhesive properties of two laminin isoforms, laminin-1 and laminin-2/4, with respect to normal (freshly isolated keratinocytes) and transformed (A-431) human skin cells. We have used the following assays: cell adhesion to the substrate covered with laminin isoformes, interaction of latex beads (D = 1 micron) coated with the same proteins with cells in suspension, and a comparative study of the cytoskeleton structure of cells spread on the immobilized laminins. It was demonstrated that laminin-2/4 is a more effective potent promotor of adhesion for both normal keratinocytes and transformed A-431 cells, compared with laminin-1. A comparison of many attached protein-covered beads allowed to estimate a relative quantity of cell surface receptors to laminin isoforms in different cell types. The relative number of receptors to laminin-2/4 on the keratinocyte surface is 7 times higher than that to laminin-1 after a 30 min incubation with cells, and is 6 times higher after 1 hour. As for A-431 cells, their attachment to laminin-2/4 beads is 5 times higher than that to laminin-1-beads after a 1 min incubation, but as early as after 5 min this distinction disappeared, owing to bead internalization. The presence of a specific receptor to laminin-2/4 but not to laminin-1 on the keratinocyte surface has been suggested. Keratin differences in cytoskeleton organization in normal and transformed skin cells spread on the substrates covered with laminin-1 and laminin-2/4 were demonstrated.

[Cell interaction with extracellular proteins during two-phase polymer system formation]. / Vzaimodeistvie kletok s belkami vnekletochnogo matriksa pri formirovanii dvukhfaznoi polimernoi sistemy.

A modified method of investigation of surface properties of cells and proteins with the help of a two-phase polymer system dextran-500/polyethylenglycol-6000 was used. This method is based on changing the kinetics of two-phase system partitioning into phase on adding cells or macromolecules. These changes were registered by measuring the top phase optical density during the system partitioning at 500 nm. Cell lines L (NCTC clone 929), LS and A431, human keratinocytes and platelets, collagen I, laminin-1, laminin-2/4, and fibronectin were studied. The interaction between collagen and all cell types with the formation of complexes takes place during co-partitioning of cells and proteins in the two-phase system. Laminins differ in surface properties and in interaction with cells. Laminin-1 makes preferable complexes with cells of monolayer subline L (NCTC clone 929), but not with cells of suspension subline LS. No interaction of laminin-2/4 with L cells was detected, but, in contrast to laminin-1, this protein has the affinity to A431 cells. No interaction of L cells with fibronectin were detected.

[Effect of melanins from black yeast fungi on cultured human cells. I. Proliferation of keratinocytes and fibroblasts]. / Deistvie melaninov iz chernykh drozhzhevykh gribov na kul'tiviruemye kletki cheloveka. I. Proliferatsiia keratinotsitov i fibroblastov.

Results of screening of the influence exerted by yeast black melanin on the proliferation of human skin keratinocytes and embryonic fibroblasts are presented. The optimal concentration of the investigated melanins was found to be within 0.005 and 0.0001 mg/ml. 17 samples of DHN-melanin from black yeast and 2 commercial samples of [symbol: see text]OPA-melanin (natural and synthetic) were investigated. It was established that keratinocyte proliferation was inhibited by 3 black yeast melanin samples; the influence of other 14 samples was the same as in the control. Keratinocyte proliferation was stimulated only by a commercial sample of natural [symbol: see text]OPA-melanin at concentration 0.005 mg/ml. The synthetic melanin at concentrations 0.005 and 0.001 mg/ml inhibited keratinocyte proliferation. Of the 17 investigated black yeast melanin samples, only one sample stimulated fibroblast proliferation at concentration 0.005 mg/ml. Three other samples inhibited the proliferation; of these one sample did it at all used concentrations, and two samples at concentration 0.0001 mg/ml. The rest 13 samples of black yeast DHN-melanins and the synthetic [symbol: see text]OPA-melanin did not differ in either action from the control.

[Effect of melanins from black yeast fungi on cultured human cells. II. Differentiation of keratinocytes in vitro]. / Deistvie melaninov iz chernykh drozhzhevykh gribov na kul'tiviruemye kletki cheloveka. II. Differentsirovka keratinotsitov in vitro.

Data on the influence of the black yeast melanin (3 samples) on the in vitro differentiation of human keratinocytes are presented. The effect of melanins was estimated by the morphological state of keratinocytes using electron microscopy. The obtained differences in the state of the formed multilayer keratinocyte sheets depended on the melanin sample.

[Various effects of laminin-1 and laminin-2/4 on adhesion and migration of cultured human keratinocytes]. / Razlichnoe vliianie laminina-1 i laminina-2/4 na adgeziiu i migratsiiu kul'tiviruemykh keratinotsitov cheloveka.

The cell-matrix interaction is one of the factors defining the cell behavior in normal and wounded tissues. To determine the function of laminin-2/4, one of components of the skin basement membrane in the process of reepithelization, we studied its interaction with human keratinocytes. The adhesive properties of laminin-2/4 and its effect on keratinocytes migration in vitro were analysed. For comparison with our present investigation, we used the earlier studied laminin-1 from EHS mouse sarcoma. Laminin-2/4 appeared to be a good substrate for human keratinocytes, and this correlates with a greater number of cell surface receptors compared with laminin-1. Laminin-2/4 alone does not stimulate keratinocyte migration, but, in contrast to laminin-1, supports EGF-mediated migration. The obtained results give an insight into the function of laminin-2/4 in normal skin and during wound healing.

Cell type-dependent collagen-type recognition by cell receptors.

Affinity chromatography of a number of cell types on collagens I and III reveals three proteins with M(R)of 250, 170 and 140 kDa. These proteins are able to discriminate between types I and III, but not types III and IV. Collagen-type recognition is therefore characteristic for cells of connective tissue origin. Polyclonal antibodies (Ab) raised against 170 and 140 kDa polypeptides and used in immunofluorescence show membrane localisation for both, with their distribution being similar to each other and to the distribution of the integrin beta1 chain. Ab p140 and commercial monoclonal antibodies against alpha(2)chain stain a band of the same molecular mass as from purified collagen binding proteins from liver cells, indicating that the 140 kDa protein is probably the alpha(2)integrin chain. The alpha(2)chain containing integrins are therefore able to discriminate collagen types I and III and collagen type recognition by this receptor is cell-type dependent.

[Morphofunctional characteristics of fibroblasts in basal cell nevus and Cockayne syndrome]. / Morfofunktsional'nye kharakteristiki fibroblastov pri bazal;nokletochnom nevuse i sindrome Kokeina.

Some morphofunctional characters of fibroblasts in two genetic disorders--Cockayne syndrome (CS) and Basal cell naevus syndrome (BCNS) have been examined. The size of nucleus in BCN1SP line has been shown to be about 1.5 times less as well as the total size of nucleoli per nucleus, while the number of nucleoli was 2 times more compared with other cell lines investigated. Using the method of silver staining numerous nucleoli were shown to contain active loci of the nucleolus organizer regions. With the help of hybridization in situ the number of transcripts of 18S RNA molecules was shown to be 5 times more in BCN1SP cell line, and about 2.8 times more than in the other cell lines tested. The data obtained may be interpreted as a suggestion in favour of a greater activity of the nucleolus organizer regions in BCN1SP cell line followed by the disturbance in protein homeostasis of the cells.