Usage of plasmid vector carrying vegf and fgf2 genes after spinal cord injury in rats

Using rat model of spinal cord contusion injury at TVIII, we compared the effectiveness of immediate single transplantation of human mononuclear umbilical cord blood cells transfected with pBud-VEGF-FGF2 plasmid and immediate direct injection of the same plasmid into the lesion area. The results suggest that the delivery of therapeutic genes vegf and fgf2 in cells is more effective than direct injection of plasmid DNA with the same genes (judging from the number of myelinated fibers). Better tissue preservation and motor function recovery in experiments with direct injection of plasmid pBud-VEGF-FGF2 suggest that direct gene therapy seems to be an effective additional procedure to the method of gene delivery with transfected stem and progenitor cells. © 2013 Springer Science+Business Media New York

Adenoviral vector carrying glial cell-derived neurotrophic factor for direct gene therapy in comparison with human umbilical cord blood cell-mediated therapy of spinal cord injury in rat

© 2016 International Spinal Cord Society.Study design:Experimental study.Objective:To evaluate the treatment of spinal cord injury with glial cell-derived neurotrophic factor (GDNF) delivered using an adenoviral vector (AdV-GDNF group) in comparison with treatment performed using human umbilical cord blood mononuclear cells (UCB-MCs)-transduced with an adenoviral vector carrying the GDNF gene (UCB-MCs+AdV-GDNF group) in rat.Setting:Kazan, Russian Federation.Methods:We examined the efficacy of AdV-GDNF and UCB-MCs+AdV-GDNF therapy by conducting behavioral tests on the animals and morphometric studies on the spinal cord, performing immunofluorescence analyses on glial cells, investigating the survival and migration potential of UCB-MCs, and evaluating the expression of the recombinant GDNF gene.Results:At the 30th postoperative day, equal positive locomotor recovery was observed after both direct and cell-based GDNF therapy. However, after UCB-MCs-mediated GDNF therapy, the area of preserved tissue and the number of spared myelinated fibers were higher than those measured after direct GDNF gene therapy. Moreover, we observed distinct changes in the populations of glial cells; expression patterns of the specific markers for astrocytes (GFAP, S100B and AQP4), oligodendrocytes (PDGFαR and Cx47) and Schwann cells (P0) differed in various areas of the spinal cord of rats treated with AdV-GDNF and UCB-MCs+AdV-GDNF.Conclusion:The differences detected in the AdV-GDNF and UCB-MCs+AdV-GDNF groups could be partially explained by the action of UCB-MCs. We discuss the insufficiency and the advantages of these two methods of GDNF gene delivery into the spinal cord after traumatic injury

Adenoviral vector carrying glial cell-derived neurotrophic factor for direct gene therapy in comparison with human umbilical cord blood cell-mediated therapy of spinal cord injury in rat

© 2015 International Spinal Cord Society Study design:Experimental study.Objective:To evaluate the treatment of spinal cord injury with glial cell-derived neurotrophic factor (GDNF) delivered using an adenoviral vector (AdV-GDNF group) in comparison with treatment performed using human umbilical cord blood mononuclear cells (UCB-MCs)-transduced with an adenoviral vector carrying the GDNF gene (UCB-MCs+AdV-GDNF group) in rat.Setting:Kazan, Russian Federation.Methods:We examined the efficacy of AdV-GDNF and UCB-MCs+AdV-GDNF therapy by conducting behavioral tests on the animals and morphometric studies on the spinal cord, performing immunofluorescence analyses on glial cells, investigating the survival and migration potential of UCB-MCs, and evaluating the expression of the recombinant GDNF gene.Results:At the 30th postoperative day, equal positive locomotor recovery was observed after both direct and cell-based GDNF therapy. However, after UCB-MCs-mediated GDNF therapy, the area of preserved tissue and the number of spared myelinated fibers were higher than those measured after direct GDNF gene therapy. Moreover, we observed distinct changes in the populations of glial cells; expression patterns of the specific markers for astrocytes (GFAP, S100B and AQP4), oligodendrocytes (PDGFαR and Cx47) and Schwann cells (P0) differed in various areas of the spinal cord of rats treated with AdV-GDNF and UCB-MCs+AdV-GDNF.Conclusion:The differences detected in the AdV-GDNF and UCB-MCs+AdV-GDNF groups could be partially explained by the action of UCB-MCs. We discuss the insufficiency and the advantages of these two methods of GDNF gene delivery into the spinal cord after traumatic injury.Spinal Cord advance online publication, 29 September 2015; doi:10.1038/sc.2015.161

Possible specialization of motoneuron axonal compartments in synthesis of particular proteins

Spontaneous quantal neurotransmitter release and its modulation was studied on neuromuscular preparations of rat soleus from intact animals and from animals in which colchicine had been applied to the sciatic nerve to block the axonal transport. After six days of colchicine application, neither the spontaneous quantal secretion nor its reaction to potassium-induced membrane depolarization or to activation of the presynaptic acetylcholine receptors with carbachol were disturbed in any way. Keeping in mind the relatively short half-life of proteins that take part in exocytosis and its regulation, it may be concluded that the functioning of the terminal neurosecretory apparatus does not depend on the state of axonal transport. These data are consistent with the earlier hypothesis that some proteins performing their functions in nerve terminals are synthesized directly at the site of their operation, rather than in the perikaryon as traditionally assumed. © 2010 Pleiades Publishing, Ltd

Extracellular vesicles derived from Acholeplasma laidlawii PG8

Extracellular vesicle production is believed to be a ubiquitous process in bacteria, but the data on such a process in Mollicutes are absent. We report the isolation of ultramicroforms - extracellular vesicles from supernatants of Acholeplasma laidlawii PG8 (ubiquitous mycoplasma; the main contaminant of cell culture). Considering sizes, morphology, and ultrastructural organization, the ultramicroforms of A. laidlawii PG8 are similar to membrane vesicles of Gram-positive and Gram-negative bacteria. We demonstrate that A. laidlawii PG8 vesicles contain genetic material and proteins, and are mutagenic to lymphocytes of human peripheral blood. We show that Mycoplasma gallisepticum S6, the other mycoplasma, also produce similar structures, which suggests that shedding of the vesicles might be the common phenomenon in Mollicutes. We found that the action of stress conditions results in the intensive formation of ultramicroforms in mycoplasmas. The role of vesicular formation in mycoplasmas remains to be studied. ©2011 with author. Published by TheScientificWorld

Survival and differentiation of endogenous Schwann cells migrating into spinal cord under the influence of neurotrophic factors

Schwann cells are a major figure in the process of regeneration in the peripheral nervous system. They migrate into the injury region of spinal cord, which are involved in remyelination and are regarded as the source of numerous molecular signals that could potentially support the growth of axons in the central nervous system. In the present work we describe the behavior of migrating into the injury dosed region spinal cord Schwann cells under the influence of neurotrophic factors - vascular endothelial growth factor (VEGF) and fibroblast growth factor 2(FGF2), delivered by direct introduction of «naked» plasmid DNA and by transplantation of genetically modified human umbilical cord blood mononuclear cells. Using immunohistochemical detection of markers of S100, GFAP, Krox20 and HSP25 identified different phenotypes of migrating into the spinal cord of endogenous Schwann cells. Found that greatest influence on their numbers in the injury region provides local delivery of genes vegf and fgf2 by human umbilical cord blood mononuclear cells. However, the direct introduction of the same plasmid may also be promising in the case of synthetic platforms that enhance its transfection activity

Unadapted and adapted to starvation Acholeplasma laidlawii cells induce different responses of Oryza sativa, as determined by proteome analysis

For the first time, we studied the phytopathogenicity toward Oryza sativa L. of unadapted and adapted to unfavorable environment (starvation) cells of Acholeplasma laidlawii PG8 - ubiquitous mycoplasma found in the soil, waste waters, tissues of the highest eukaryotes and being the basic contaminant of cell cultures and a causative agent of phytomycoplasmoses. The features of morphology, ultrastructural organization and proteomes of unadapted and adapted cells of the mycoplasma and infected plants were presented. Using 2D-DIGE and MS, 43 proteins of O. sativa L. that were differentially expressed in the leaves of plants cultivated in media with A. laidlawii PG8 were identified. The qualitative and quantitative responses of the plant proteome toward adapted and unadapted mycoplasma cells differed. That may be explained by differences in the virulence of the corresponding bacterial cells. Using 2D-DIGE and MS, 82 proteins that were differentially expressed in adapted and unadapted mycoplasma cells were detected. In adapted cells of the mycoplasma, in comparison with unadapted ones, a significant increase in the expression of PNPase - a global regulator of virulence in phytopathogenic bacteria occurred; there was also decreased expression of 40 proteins including 14 involved in bacterial virulence and the expression of 31 proteins including 5 involved in virulence was not detected. We propose that differences in the phytopathogenicity of adapted and unadapted A. laidlawii PG8 cells may be related to features of their proteomes and membrane vesicles. © 2011 Elsevier B.V

Structure-Sensitive Mechanism of Nanographene Failure

The response of a nanographene sheet to external stresses is considered in terms of a mechanochemical reaction. The quantum chemical realization of the approach is based on a coordinate-of-reaction concept for the purpose of introducing a mechanochemical internal coordinate (MIC) that specifies a deformational mode. The related force of response is calculated as the energy gradient along the MIC, while the atomic configuration is optimized over all of the other coordinates under the MIC constant-pitch elongation. The approach is applied to the benzene molecule and (5, 5) nanographene. A drastic anisotropy in the microscopic behavior of both objects under elongation along a MIC has been observed when the MIC is oriented either along or normally to the C-C bonds chain. Both the anisotropy and high stiffness of the nanographene originate at the response of the benzenoid unit to stress.Comment: 19 pages, 7 figures 1 tabl

Characterization of spinal cord glial cells in a model of hindlimb unloading in mice

© 2014 IBRO. Exposure to microgravity has been shown to result in damaging alterations to skeletal muscle, bones, and inner organs. In this study, we investigated the effects of microgravity by using a hindlimb unloading model (HUM) in mice. The characteristics of the lumbar spinal cords of HUM mice 30. days after hindlimb unloading were examined. Morphometric analysis showed reductions of the total area, gray matter, and white matter by 17%, 20%, and 12%, respectively. Myelinated fibers in the white matter showed prominent myelin destruction. Analysis of the number of glial fibrillary acidic protein (GFAP+)/S100 calcium-binding protein B (S100B-), GFAP+/S100B+, and GFAP-/S100B+ astrocytes in the ventral horn (VH), central channel area (CC), dorsal root entry zone (DREZ), main corticospinal tract (CST), and ventral funiculi (VF) showed that the number of GFAP+/S100B- astrocytes was increased in the DREZ and CST of HUM mice. Additionally, GFAP+/S100B+ cell numbers were significantly decreased in the VH and CST but did not differ in the CC or DREZ of HUM mice, as compared with the control. The numbers of GFAP-/S100B+ cells were significantly reduced only in the VH of HUM mice. Moreover, the number of ionized calcium-binding adaptor molecule 1 (Iba1+) microglia cells was significantly increased in the CC and DREZ of HUM mice. In control mice, homeobox protein HoxB8 (HoxB8+) cells were found only in the CC; in contrast, HoxB8+ cells were observed in all studied areas in HUM mice, with the greatest number found in the CC. Genome-wide transcriptome analysis of the lumbar spinal cords of HUM mice showed decreased expression of genes encoding myelin, extracellular matrix, cytoskeleton, and cell adhesion proteins. Real-time polymerase chain reaction (PCR) confirmed reductions in the expression of mpz, pmp2, pmp22, and prx genes, which are involved in myelination, as well as decreases in the levels of genes encoding extracellular matrix molecules, including glycoproteins (matrix gla protein (MGP), osteoglycin (OGN), microfibrillar associated protein 5 (MFAP), and collagen, type IV, alpha 1 (COL4A)), proteoglycans (perlecan (heparan sulfate proteoglycan) (HSPG)), and metalloproteinases (lysyl oxidase (LOX)). Thus, our results showed that hindlimb unloading caused decreases in gray and white matter areas, changes in gene expression, alterations in myelination, and phenotypic modifications in glial cells in the lumbar spinal cords of mice

Evaluation of direct and cell-mediated triple-gene therapy in spinal cord injury in rats

© 2017 Elsevier Inc.Current treatment options for spinal cord injury (SCI) are scarce. One of the most promising innovative approaches include gene-therapy, however no single gene has so far been shown to be of clinical relevance. This study investigates the efficacy of various combinations of vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG) and neuronal cell adhesion molecule (NCAM) in rats. Multiple therapeutic genes were administered intrathecally either via adenoviral vectors or by using genetically modified human umbilical cord blood mononuclear cells (hUCBMCs). Following the induction of SCI, serial assessment of cord regeneration was performed, including morphometric analysis of gray and white matters, electrophysiology and behavioral test. The therapeutic gene combinations VEGF + GDNF + NCAM and VEGF + ANG + NCAM had positive outcomes on spinal cord regeneration, with enhanced recovery seen by the cell-based approach when compared to direct gene therapy. The efficacy of the genes and the delivery methods are discussed in this paper, recommending their potential use in SCI