Vol 9, No 3 (2014)

The article is dedicated to the outstanding Russian scientist A.J. Friedenstein, a founder of the hematopoietic organs' stroma doctrine, a discoverer of mesenchymal stem cells (multipotent mesenchymal stromal cells, MMSC), an initiator of the tests to determine the «verity» of stem stromal cells. Discoveries of the scientist and scientific school he created are used both in the research and clinical practice all over the world.

Chondroitinsulfate proteoglycans (CSPG) play a crucial role in the inhibition of posttraumatic axonal regeneration in the central nervous system. Understanding of the mechanism of the CSPG action on axonal regeneration is important for treatment of patients after the brain and spinal cord injuries. Here we make a review of recent studies that discovered CSPG receptors acting in the plasma membrane of neurons.

Halloysite clay mineral is found in kaolin deposits and is a multilayered nanotube with external diameter of 50 nm inner diameter 15 nm and length1-2 micron. Halloysite is a rolled kaolin and it has found applications in fine porcelain, polymeric composites, tissue engineering, antimicrobial coatings and in cosmetics. Halloysite is also applied as microvesicles to carry drugs and enzymes. It is used as an additive to improve the mechanical strength of polymers. Halloysite is non-toxic to living organisms and the environmentally safe. This review summarizes recent data on the structure of halloysite and its applications.

The review provides modern data and the results of author's research on physiological and pathological roles of the new gasotransmitter - hydrogen sulfide (H2S) in the central and peripheral nervous system. H2S is synthesized by three enzymes: cystathionine p-syntase, cystathionine y-lyase and 3-mercaptopiruvat sulftransferase/cysteine aminotransferase. In nerve systems the main source of synthesis H2S is cystathionine p-syntase and high level enzyme expression observed in the embryonic and early postnatal period of organism development that is apparently necessary for the growth and maturation of neural networks for the protection of neurons and astrocytes in the conditions of oxidative stress. Cystathionine p-syntase gene mutation in humans leads to an autosomal recessive metabolic diseases, mental dysfunction, vascular lesions and hyperhomocysteinemia. The aim of this review is to present the currents data about the effects of H2S on ion channels, transmitter release, its participation in the pathology of various neurodegenerative diseases, as well as its antioxidative and neuroprotective action in central and peripheral nervous systems.

The transplanted allogeneic multipotent mesenchymal stromal cells (MMSC) were previously thought to be poorly recognized by host immune system; the prolonged survival of these cells in host tissues was explained by their privileged immune status. As long as this concept is currently being revised, the understanding of MMSC routes should be reconsidered given the emerging role of host immune system in their gradual elimination. The study was focused upon elimination of PKH26-labeled MMSC, derived from umbilical cord, analyzed in animal models for two distinct pathologies: subtotal liver resection and critical skeletal muscle ischemia. Specific patterns of PKH26-positive macrophages (defined as CD68+ cells) were described for intact spleen and regenerating liver, and for the ischemic skeletal muscle, respectively. The PKH26-positive cells were observed in spleen of the subtotally hepatectomized model animals at 24 h. after surgery combined with MMSC transplantation; 83,2±4,6% of these were CD68+; the ratio reached 100% 3 days after transplantation. The PKH26-positive cells were also detected in regenerating liver starting from 3 days after transplantation, the great majority of them were CD68+ (96,8±2,2% and 96,3±2,6% for 3 and 10 days after transplantation, respectively). A different sort of host environment was provided by the damaged skeletal muscle model: productive phase of aseptic inflammation triggered by ischemia. The PKH26-positive fraction in the pool of macrophages significantly increased from 48,1 ±3,2% 3 days to 76,2±3,9% 30 days after transplantation. Thus, transplanted allogeneic MMSC are recognized and eliminated by host immune system. The rates of elimination depend on site of injection and time elapsed since the injection; the efficacy may reach 100%. The presence of РКН26 vital label (as well as any other exogenous label) in living cell can by no means solely prove its exogenous origin. The massive elimination of MMSC by host macrophages leads to impregnation of the latter with the dye that is masking the true presence of the former. The study accentuates the need of additional criteria for correct data interpretation.

There were proposed several methods for stimulation of skin wound repair over the last few decades. The most perspective among them are gene and stem cell therapy. In our experiments we combined both approaches by application of human cord blood mononuclear cells (hUCB-MC) transfected with pBud-VEGF165-FGF2 plasmid to enhance healing of full thickness skin wounds in rats. Dual expression cassette plasmid pBud-VEGF165-FGF2 encodes both VEGF and FGF2 therapeutic genes, expressing pro-angiogenic growth factors. Our results showed that in 2 weeks after transplantation some transplanted cells still retain expression of stem cell and hematopoietic markers C-kit and CD34. Other transplanted cells could be found among keratinocytes, hair follicle cells, endothelial cells and in derma. Study of PCNA expression revealed that application of transfected cells terminate proliferative processes in regenerating wound earlier then application of untransfected cells.

This study demonstrates the possibility of using cell-based therapies for the treatment of the longterm intergrows fractures and pseudoarthrosis in dogs. Multipotent mesenchymal stem cells (MMSCs) were isolated from adipose tissue of an adult dog. Multipotent cells was confirmed by the ability to differentiate into 3 main directions (osteogenic, adipogenic and chondrogenic), as well as the expression of the markers CD73, CD105, vimentin. Clinical application received MMSCs in the treatment of pseudoarthrosis of tibia in dogs stimulated bone formation processes. The result of cell therapy was callus formation and recovery of the support function of the injured extremity.

The proliferative process intensification in the disrupted organs is very important in reparative regeneration. But the possibility of pancreas cells proliferation during damage is not been studied. The aim of our work was to examine the proliferation activity of pancreas during the experimental alloxan diabetes. The work was made on 33 rats. Blood glucose levels were measured. Also the expression of nuclear antigen of proliferating cells PCNA was studied in pancreas cells. After the day of the experimental hyperglycemia PCNA expression in islet and acinar cells of pancreas was found, which was persisted at all stages of the experiment. After 2 days of the experiment single PCNA-positive cells with the cytoplasmic staining were found in the interstitium, near vessels and ducts. Proliferating cells, which produce insulin were found in islets during double staining. Thus, there is the proliferation in islet and acinar cells of pancreas during experimental diabetes type I after alloxan damage. Also, the results of our study confirms the ability of p-cells and progenitor pancreas cells proliferation.

The effect of sodium hydrosulfide (NaHS) - donor of hydrogen sulfide (H2S) on the force of contraction of isolated mouse atrium was studied. Cumulative application of NaHS in concentrations 100, 200 and 300 ^M induced dose-dependent decrease of the force of contraction, the maximum velocity of contraction and relaxation of the myocardium. A substrate of H2S synthesis - L-cysteine in concentrations 1, 10, 50 uM also had the negative inotropic action, whereas a blocker of H2S synthesis - p-cyano alanine caused an increase of the force of contraction. Inhibition of K-channels by tetraethylammonium (2 mM) caused the increase of the amplitude of contraction and the reduction of negative inotropic effect of NaHS in all used concentrations. After the inhibition of ATP-dependent K-channels by glibenclamide NaHS action was prevented in concentration 100 uM, significantly decreased in concentration 200 uM and didn't changed in concentration 300 uM. Activation of ATP-dependent K-channels by diazoxide did not affect the negative inotropic effect of NaHS. The obtained data suppose that in the mouse atrium exogenous and endogenous H2S causes a reduction of the force of contraction, which is mediated by the activation of ATP-dependent, calcium-activated or voltage-dependent K-channels.

The aim was to elucidate impact of gene therapy with plasmid encoding vegf165 on the muscle tissue pathohistology of patients with peripheral arterial diseases. Twice repeated intramuscular injections of plasmid («Neovaskulgen», RN LP-000671 from 28.09.2011) were performed to 6 patients (ischemia grade IIb-III by Pokrovsky-Fontaine) according to specification. Standart tredmill test, ankle-brachial index estimation were performed. Histological study of injured muscle biopsies taken before and 3 months after injection was performed. In intact muscles therapy with vegf165 leads to increase of mean cross-sectional muscle fiber area without significant angiogenic effect. In muscles with decreased capillary density this therapy leads to blood supply improvement promoting regeneration of muscles by myosatellites proliferation and increase of mean cross-sectional muscle fiber area and connective tissue degradation. Treadmill test showed painless walking distance increased by 31,74% on average (from 94,96±49,79 m to 139,11±60,78 m, p<0,05) in all patients. There was correlation of pathohistological analysis with clinical data.

Platelet factor 4 (PF4) pertains to a family of CXC chemokines released by activated platelets. PF4 has a broad spectrum of effects on different cell types, including modulation of the immune response. In this study, we explore effects of PF4 on the morphological and biochemical signs of apoptosis in human T-lymphocytes in vitro. T-lymphocytes were isolated from peripheral blood of healthy donors using negative immunomagnetic separation and cultured in the complete RPMI 1640 medium for 24 hours in the absence and presence of PF4 added at a final concentration of 2 /ug/ml or 100 ug/ml. After 2, 4, 6, 12 and 24 hours of incubation the cells were studied with transmission electron microscopy and Western blot analysis with respect to potential apoptotic changes. The electron microscopy of control T-lymphocytes showed that the vast majority of the cells had a morphology characteristic of apoptosis at different stages. Adding PF4 at a concentration of 2 ug/ml reduced the number of cells at the late stages of apoptosis, while maintaining the signs of the early apoptosis in most of the T-lymphocytes. In the presence of 100 ug/ml PF4 nearly all of the cells kept a typical morphology of normal T-cells throughout the time of cultivation. The morphological apoptotic changes correlated well with expression of caspase-3, which was clearly detected in the control cells and cells treated with 2 ug/ ml PF4, but was almost abolished in the cells treated with 100 ug/ml PF4. Our results provide direct evidence for the dose-dependent anti-apoptotic effects of PF4 on T-cells, suggesting that PF4 sustains an immune response by extending T-lymphocyte survival.

Most of the fundamental studies in the field of developing new methods for treating liver diseases in regenerative medicine are performed with haematopoietic and mesenchymal stem cells. At the same time more and more importance is gaining need for finding new approaches enabling stimulation of regional stem cell compartment and using regional stem cells which are possibly represented by hepatic stellate cells. But studies aimed at their transplantation are rare. Previously, we have established the possibility of these cells to differentiate into hepatocytes after transplantation to rats with partial hepatectomy. In our present research we studied liver regeneration after transplantation of hepatic stellate cells on partial hepatectomy with 2-acetylaminofluorene damage model in rats. 2-acetylaminofluorene blocks hepatocyte proliferation. Results of study confirmed that hepatic stellate cells have the ability to differentiate into hepatocyte and stimulate it's regeneration without the threat of fibrosis after transplantation in rats with partial hepatectomy and administration of 2-acetylaminofluorene.

The article analyzes the existing ideas about the specific mechanisms of therapeutic resistance of glial tumors of the brain, systematized the main trends in modern chemotherapy glial tumors, an attempt to justify the new bioengineering approaches to the creation of personalized cell preparations for therapy of glial tumors based on molecular - biological characteristics of tumor stem cells. It is shown that the main tool of the therapeutic effects can be own stem cells of cancer patients and tumor stem cell proteome can be considered as the primary target cell therapy.

Polymeric capsules (containers) are widely used now in researches connected to the design of new pharmaceutical preparations. Here we present data allowing to consider these objects also as essential parts of biocomputers, capable, in particular, to carry out targeted delivery and triggered start of the «main program» (encapsulated specific molecules) to the areas with damaged functioning of the system, resulted from undesirable reactions. We also consider the use of cells as program package, capable to perform parallel functions in biocomputers when delivered in a proper place and started functioning in a proper moment.

This review represents ongoing clinical studies devoted to recovery of muscle tissue using cell technologies, as well as analysis of applied populations of cells.

There are medicines known as adrenoreceptors ligands, which influences a variety of lipid exchange, therefore the study of their mutagenic and antimutagenic potentials, their genotoxicity, is of great interest. The study are fullfiled using C57B4/6 mice (males) of 20 g and 1,5-2 months of age. Mutagenic and antimutagenic effects of the ligands were evaluated using in vivo tests: mouse brain skeleton cells (a number of chromosomal aberrations) and mouse peripheral red blood cells peripheral (a number of cells with micronuclei from 2000 analyzed cells). 6 male mice were used for every experimental and control variant. All medicines studied - epinephrine, phenylephrine, orciprenalin, proroxan, and propranolol (besides epinephrine, 50 mg/kg) - in a majority of doses used don't increase the level of chromosomal aberrations and the number of red blood cells with micronuclei.Epinephrin, phenylephrine, orciprenalin in doses studied demonstrated a protector effect and decreased a level of chromosomal aberrations and a number of red blood cells with micronuclei induced by cyclophosphamide administration. Proroxan and propranolol didn't change a level of aberrations. It was shown that adrenoreceptor ligands decreasing or increasing lipid level did not express mutagenic effect, and, in opposite, expressed antimutageni which testify that the system participating simultaneously in a realization of lipid exchange and mutagenesis/ antimutagenesis exists. An action of the system is realized via cell membrane receptors. According mechanism of action, the system is polyreceptoric (a- and р-adrenoreceptors take a part) and polyvalent, namely, receptor stimulators and blocators take a part in a formation of cell antimutagenesis.

Lipids can be bound to nucleic acids forming coding sequence at genomic DNA, and participating in realization of language of genome. Interaction of DNA oligonucleotide(A)20-(T)20 is studied with by moleculat dynamics method. The complex is prepared as a result of molecular docking, phospholipid molecule being located at DNA minor groove. Binding energy is equal to - 6.3 kcal/mol. Formation of hydrogen binding, interactions leading to those bonds, and some other interactions between DNA and phospholipid. The number of those interactions due to molecular dynamics is varied between 175 and 337. The key role in stabilization of DNA-phospholipid complex play also van-der-vaals and hydrophobic interactions along with hydrogen binding. The role of ethanolamine grouping in complexation between DNA and phospholipid is demonstrated.

Here we discuss the ability of Proteus mirabilis 5127-1 and Morganella morganii ZM to invade human cervical epithelial adenocarcinoma cells (HeLa-M) and to cause cytotoxicity in those cells. We found that ability of bacteria to invade HeLa-M cells depended on the multiplicity of infection for both strains. Cytotoxicity of P. mirabilis on HeLa-M cells was evaluated through quantification of eukaryotic cell detachment from plastic. We hypothesized that cytotoxicity of P.mirabilis could be due its extracellular proteolytic and hemolytic activities. In contrast, no cytotoxicity was detected in HeLa-M cells infected with M.morganii.

To overcome the effects of neurodegeneration and stimulating neuroregeneration in the spinal cord transgenic mice with model of amyotrophic lateral sclerosis were injected intraspinally with umbilical cord blood mononuclear cells (UCBMCs) transduced with adenoviral vectors encoding for vascular endothelial growth factor, glia-derived neurotrophic factor and neural adhesion molecule. Two weeks after transplantation UCBMCs were localized in the site of injection and at a distance from it, confirming the migration capacity of the cells in spinal cord parenchyma. Using triple immunofluorescence staining UCBMCs expressing therapeutic genes were found. The study results suggest the expedience of given gene-cell construct to stimulate the neuroregeneration in central nervous system after neurotrauma, ischemic stroke and in neurodegenerative diseases.

On the model of rat spinal cord dosed contusion at T8 level studied the effect of delivery into the area of damage of the cell-mediated and direct gene delivery GDNF on maintaining a population of glial cells. Delivery into the area of damage adenoviral vector with gene GDNF (AdV-gdnf) using human umbilical cord blood mononuclear cells has a greater influence the amount of Ch47+-cells in the ventral horn (VH). Direct delivery of AdV-gdnf influences the amount of Ch47+-cells in dorsal roots entry zone (DREZ). Cell-mediated gene delivery GDNF causes the most pronounced changes in the expression of marker proteins astrocyte GFAP, S100B, and AQP4 in the ventral funiculus (VF) of white matter. Cell-mediated and direct delivery of gene GDNF support population GFAP+/S100B+-cells. The results indicate that the direct and cell-mediated gene delivery GDNF into spinal cord injury have different effects on the populations of glial cells in specific areas of spinal cord, that is important for the optimal method of delivery of therapeutic genes to stimulate posttraumatic neuroregeneration.

In frog neuromuscular junction, muscarine, exogeneous acetylcholine and acetylcholinesterase inhibitor proserine reduced the intensity of calcium-sensitive dye fluorescence (calcium transient) at low frequency of nerve stimulation, suggesting that calcium ions entry into nerve ending was decreased. M2 muscarinic receptor blocker methoctramine prevented the action of muscarine. The amplitude of endplate currents was reduced in presence of muscarine at low frequency nerve stimulation, and atropine abolished this effect. Amplitudes of endplate currents evoked by high frequency stimulation were enhanced in presence of methoctramine, and synaptic depression was less pronounced, probably due to elevated calcium concentration in nerve ending. Thus, activation of presynaptic muscarinic receptors predominantly of M2 subtype reduces the intensity of quantal acetylcholine release in frog neuromuscular synapses that may be associated with decreased level of calcium ions in the nerve ending to provide the modulation of postsynaptic currents amplitude at high frequency firing.

Alzheimer's disease is progressive incurable neurodegenerative disease, which is manifested mainly by dementia. One of the most promising directions in development of Alzheimer's disease treatment is use of gene-cell technologies. The aim of current study was to perform transplantation of wild-type or EGFP expressing umbilical cord blood mononuclear cells (МКПК) to APP/PS1 transgenic mice with Alzheimer's disease model with further evaluation of transplantation impact with behavioral (T-maze, plus maze, open field) and immunohistochemical methods. It was found that МКПК transplantation significantly ameliorates behavioral performance of APP/PS1 mice: improves spatial memory, decreases anxiety and non-specific excitability, increases the efficacy of exploratory behavior. Grafted cells were found in cortex and hippocampus of mice even 3 months after МКПК transplantation, herewith EGFP expression in grafted cells was found at early stages after transplantation. Thus, use of МКПК-based gene-cell constructs represents a promising direction in development of Alzheimer's disease therapy.

It was investigated the effect of secreted proteinases Bacillus pumilus 7P belonging to different classes of proteolytic proteins on matrix metalloproteinases of mouse fibroblast (3T3BalbSV40), multipotent mesenchymal stromal and cells Hela-M. Matrix metalloproteinases of all cell lines was inactivated by glutamyl-specific endopeptidase. Subtilisin-like proteinase and adams-like metalloproteinase of B. pumilus did not change the activity of matrix metalloproteinases of differentcell lines.

The results of the comparative study of the diagnostic efficacy of the method of isolation of street rabies virus using rat Gasser‘s ganglion neurinoma cell culture (RGGN-1) and biological tests on white mice are presented. It is shown that the method of virus isolation using RGGN-1 cell culture is not inferior in the sensitivity to the classic bioassay on white mice and has obvious advantages, such as reducing the time of diagnosis (up to 3 days using RGGN-1 compared to 30 days using white mice) and its cost-effectiveness. Method of cell line RGGN-1infection is recommended for inclusion in the Standards for the rapid diagnosis of rabies and isolation street rabies virus, which should be considered as a possible replacement of bioassays on white mice. Rapid diagnosis of rabies using RGGN-1 cell culture will provide early diagnosis of rabies in animals to reduce the risk of disease in human and animals.

We studied the lymphocyte apoptosis in 15 infants with late neonatal sepsis, whereas 8 of them (53.3%) were considered as a preterm infants. Etiology of sepsis was identified in 4 cases (26.7%): in 1 case of each, the cause of the disease was Klebsiella pneumoniae, Pseudomonas koreenses, Candida and St. agalactae. Control group consisted of 5 healthy newborns. Apoptosis analysis was conducted by measuring the amount of hypodiploid cells by using a propidium iodide (Sigma Aldrich) DNA staining procedure and flow cytometry (FACsCanto II, Becton Dickinson). We observed an elevated numbers of apoptotic cells in all cases of neonatal sepsis. The most significant difference was observed when the lymphocytes were cultured for 3 and 5 days. For example, on day 3 of culture the numbers of apoptotic cells in patients with neonatal sepsis vs control group were 19.6% and 5.13%, respectively. Absolute lymphopenia was noted in 26.7% of cases with neonatal sepsis. No difference in the lymphocyte apoptosis between term and preterm infants was observed. An increased amount of lymphocyte apoptosis was not associated with C-reactive protein level during the whole time-points of the experiment: at 1, 3 and 5 days. Acute phase of neonatal sepsis is associated with an increased apoptosis of peripheral blood lymphocytes.

Normally, the human body maintains a certain balance of micronutrients. The danger of a constant inflow of toxic elements from the environment caused by the inability to selfdestruction and elimination from the body, so the search for an optimal balance correction of metals is carried out. By AAS definition of the nine most common toxic and essential trace elements in hair of children in all 11 children's clinics service areas was held: Pb, Cu, Zn, Cr, Sr, Mn, Ni, Fe, Cd. Six different types of biopolymer ehnterosorbents: pectin, alginates, chitosan, microcrystalline cellulose and chitin were studied in the proposed model. A method of produce and combination of biopolymer and mineral enterosorbents with qualitatively new properties and lower cost compared to monosorbents was developed. 11 most common toxic and essential trace elements in hair of children were analyzed. The most variable and requiring of geographically differentiated correction elements in areas of research were determined. The most effective biopolymer enterosorbents and their combinations with mineral enterosorbents were discovered. Experimentally a method of producing composite enterosorbents based colloids was created. The highest sorption capacity related to of Zn, Cd, Fe and Cu requiring geographically differentiated correction was noted in combination of octahedral smectite and chitosan tests.

Staphylococcus aureus is widespread bacterium that causes inflectional diseases of humans and animals. Currently, there is growing incidence of methicillin-resistant strains of Staphylococcus aureus. Resistance of Staphylococcus aureus strains to methicillin is detected by the presence in the genome of staphylococcal chromosomal cassette of SCCmec type. In this work 60 clinical strains of MRSA isolated from the skin and mucous of patients with allergic pathology were examined. The analysis of Staphylococcus aureus strains was performed in differential mediums. Additionally, specific identification was carried out by MALDI-TOF. Specific regions of the genes of mec gene complex were identified by PCR. As a result of typing we determined that 47% of tested strains were resistant to oxacillin phenotypically, but did not contain the specific primer sites related to mec- gene complex types I-IV. The remaining 53% of the strains were contained primer-specific amplicons. These strains were also phenotypically resistant to oxacillin. This study showed that MRSA strains as part of its genetic apparatus are several different types of SCCmec cassettes. This property allows them to express multidrug resistance.

In the last several years, there has been an increase in the use of plant materials in clinical development of new drugs and methods in the treatment of neoplastic diseases. In this regard, the current study was aimed to investigate the cytotoxic activity of Nigella sativa (NS), Salvia officinalis (SO) plant oils and peptide metabolites (PM) of Streptomyces mirabilis FK749 on the viability of Hela and MCF-7 cancer cells. To achieve this objective, cells were exposed to 0, 20, 40, 60, 80 and 100 mg/ml of NS, SO plant oils and PM S. mirabilis FK749. After 24 h. incubation the effect of plant oils and PM on the viability and apoptotic activity of the cancer cells were assessed by using trypan blue and TMRE assays. Morphological changes were examined. Whereas, the half-maximal inhibitory concentrations of these oils and PM were assessed after 72 h. incubation. The results of this study showed that treatment Hela and MCF-7 cells with NS, SO and PM at 40 mg/ml and above concentrations up to 100 mg/ml were found to be cytotoxic and leads to a decrease in the cell viability. The morphological changes observed in Hela and MCF-7 cells exposed to different concentrations of NS SO and PM were found to be in a concentration dependent manner. Hela cells were shown to be more resistant against oil components and PM than MCF-7 cells.

This article describes a clinical case of treating the patient with injury of ulnar nerve using a “gold standard” of microsurgery, the autonerve grafting, and transplantation of autologous cells of stromal vascular fraction (SVF) of adipose tissue. Nerve defect in 5 cm was replaced by three autonerve grafts of the sural nerve, and then autologous cells of SVF in the fibrin glue were transplanted. 1 year after operation, the patient has resumed the function of bringing the fingers, there have not been any neurogenic strain, muscle atrophy of the hand, as well as restoration of sensitivity in the autonomous zone of innervation of the ulnar nerve has been observed.