Chromatographic purification of plasmid DNA for clinical applications (gene therapy)

For the successful application of plasmid vectors in gene therapy protocols it is necessary to develop methods for purification of highly homogeneous preparations of recombinant DNA that do not contain contaminants, primarily chromosomal DNA, bacterial proteins, RNA and endotoxins. In the course of our study we performed optimization of the purification of plasmid supercoiled DNA by three chromatographic steps from an alkaline lysate of bacterial strain of E. coli. We determined an optimal conditions for alkaline lysis step in order to increase the yield and minimize the duration of the plasmid purification by gel filtration

The Temperature-Dependent Selectivity of Potential Interaction Partners for the Small Heat Shock Protein IbpA from Acholeplasma laidlawii

© 2016, Springer Science+Business Media New York.Small heat shock proteins (sHsps) of α-crystalline type play a key role in the cell survival under stress conditions by preventing irreversible denaturation and aggregation of proteins. In contrast to most Mollicutes (mycoplasmas) where no sHsps were found, recently, some sHsp homologs were identified in the Acholeplasmataceae family, including Acholeplasma laidlawii, the only representative of Mollicutes that is known to survive in a host-free environment. Using pull-down followed by LC-MS, we identified the potential target proteins co-eluting with IbpA from the A. laidlawii cell extracts after exposition to low- and high temperatures. 308 and 464 proteins were co-eluted with IbpA from the cold- and heat-treated extracts, respectively, while only 240 of them were co-eluted with sHsp independently of the temperature. Most of potential IbpA targets were identified as enzymes involved in biosynthetic cycles and energy metabolism. We show IbpA specificity for target proteins on the incubation temperature. Significant differences between protein pools co-eluting specifically with IbpA at either 4 or 46 °C could be observed in terms of their aliphatic index, charge, molecular weight, and isoelectric point. Interestingly, only the isoelectric point distribution significantly differed between the protein pool co-eluting with IbpA under cooling (4 °C) and the entire proteome. In contrast, significant discrepancies in the distributions of aliphatic index, charge, hydropathy, molecular weight, and isoelectric point could be observed between the pool of proteins co-eluting with IbpA under heating (46 °C) and the entire proteome, indicating that there is likely a complex selective mechanism for proteins interaction with IbpA under heat shock conditions

Combined treatment of throphic ulcer of the heel using vacuum therapy with direct gene therapy: Case report

In this article we describe a clinical case of successful treatment of neurotrophic ulcers of heel region using classical surgical techniques such as debridement and autodermoplastics combined with vacuum therapy and injection of double cassette expression plasmid encoding cDNAs of vascular endothelial growth factor and fibroblast growth factor 2. The outcome of therapy opens new horizons in the treatment of such diseases in short period of time. © Human stem cells institute, 2013

Human adipose derived stem cells do not alter cytokine secretion in response to the genetic modification with pEGFP-N2 plasmid DNA

Adipose tissue contain progenitor cells with regenerative (angiogenic, neuroprotection, trophic etc.) potential and treatment based on adipose tissue-derived stem cells (ADSCs) transplantation may constitute a promising therapy. Genetic modification of stem cells with genes encoding growth factors and other biologically active molecules might further increase therapeutic efficiency. However, there is a question about the advantages and disadvantages of such gene/stem cell therapy. For a better understanding of the mechanisms occurring in organism after cell transplantation we should first study cell behavior in vitro. Here we report results of ADSCs transfection with plasmid DNA pEGFP-N2 and subsequent study of cytokines and chemokines secretion using Luminex technology. We demonstrated that genetic modification of ADSCs with commonly used control plasmid vector, encoding enhanced green fluorescent protein (EGFP), did not affect secretion of cytokines/chemokines IFN-{filled circle}, IL-1{filled circle}, IL-2, IL-8, IL-10, IL-12, MCP-1. Thus, genetic modification procedure on its own might have little effect on stem cell properties, making it useful for gene-stem cell therapy applications. © IDOSI Publications, 2013

Effects of transplantation of human cord blood mononuclear cells expressing the recombinant VEGF and FGF2 genes into spinal cord traumatic injury sites in rats

A model of dosed TVIII spinal cord contusion trauma in rats was used to study the effects of immediate single-dose transplantation of human cord blood mononuclear cells transformed with the recombinant genes for neurotrophic factors - vascular endothelia growth factor (VEGF) and fibroblast growth factor 2 (FGF2) - into the injury zone. A further group of animals, in the same conditions, received the same cells transfected with plasmid pEGFP-N2. EGFP-labeled cells were detected in the white matter for 21 days after transplantation at distances of at least 10 mm in the rostral and caudal directions from the administration point. By 30 days after transplantation with cells transfected with plasmid pBud-VEGF-FGF2, the area of intact gray matter 3 mm from the trauma epicenter increased by more than 60%. By this time, the outer areas of the white matter in animals of this group, 1.5 cm from the trauma epicenter, showed an average 30% increase in the number of perivascular cells expressing platelet-derived growth factor β receptors (PDGFβR). Addition of therapeutic genes VEGF an FGF2 to the trauma injury zone and their expression in carrier cells stimulated vascularization and post-traumatic regeneration of the spinal cord. © 2013 Springer Science+Business Media New York

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

Resistência à seca em seringueira. II. Crescimento e partição de assimilados em clones submetidos a déficit hídrico

Plants from IAN 717, IAN 873, IAN 2903, IAN 3087, IAN 6323 and Fx 3899 rubber tree (Hevea spp.) clones grown in a greenhouse were subjected to cycles of water deficit with the objective of evaluating the effects on growth and assimilation partitioning. After 185 days and five cycles of stress, a reduction in the number of leaves, in the number of flushes, and in the length and diameter of the shoots occurred in the majority of the clones. The IAN 2903 progeny showed an increase in the number of leaves (4.7%) and in the number of flushes (10%). Shoots of the IAN 717 and IAN 3087 progenies were the least affected in their growth. The assimilation partitioning showed a modification in relation to the sinks occurring with the deficit; the prefered sinks were the root in the Fx 3899 and 873 progenies the stem in the IAN 3087 and IAN 6323 progenies and the leaf in the IAN 717 and IAN 2903 progenies. The ratio of the aerial part to the root system was greater in the IAN 2903 progeny and smaller in the Fx 3899 progeny. Under water stress no significant changes occurred in the specific leaf area production values for leaf area and in the leaf area ratio, but the net assimilation value and the accumulation and production of dry matter decreased differentially when submitted to water deficit. The clone least affected by the stress was IAN 3087. The greatest reductions on studied parameters occurred in Fx 3899. The results observed in each clone were discussed in relation to each parameter studied.Plantas de seringueira (Hevea spp) oriundas dos clones IAN 717, IAN 873, IAN 2903, IAN 3087. IAN 6323 e Fx 3899 foram cultivadas em casa-de-vegetação e submetidas a ciclos de estresse hídrico, com objetivo de avaliar comparativamente os efeitos sobre o crescimento e a partição de assimilados. Após 185 dias e cinco ciclos de estresse, o número de folhas, número de lançamentos, comprimento e diâmetro das brotações na maioria dos clones diminuíram. O IAN 2903 aumentou o número de folhas (4,7%) e o de lançamento (10%). As brotações do IAN 717 e IAN 3087 foram as menos afetadas no seu crescimento. A partição de assimilados mostrou uma modificação na preferência dos drenos com o déficit, sendo que alocação preferencial para a raiz foi do Fx 3899 e IAN 873, para o caule do IAN 3087 e IAN 6323 e para a folha do IAN 717 e IAN 2903. A relação parte aérea/sistema radicular foi maior no IAN 2903 e menor no Fx 3899. O déficit não provocou mudanças significativas na área foliar específica e na razão de peso foliar. Entretanto, a área foliar total, taxa de produção de área foliar, razão de área foliar, taxa assimilatória líquida, acúmulo e produção de matéria seca decresceram diferencialmente quando submetidos à seca. O clone menos afetado pelo estresse foi o IAN 3087. As maiores reduções nos parâmetros estudados ocorreram no Fx 3899. São discutidos os resultados de cada clone com relação a cada parâmetro estudado

Post-Traumatic Changes in the Spinal Cord in Rats after Transplantation of Mononuclear Cells from Human Umbilical Blood Modified with the vegf and fgf2 Genes

Experiments were carried out using 25 white laboratory rats. A model of dosed contusional trauma to the spinal cord at the T VIII level in rats was used to measure the areas of pathological cavities and the numbers of myelinated fibers in the outer zones of the white matter were counted after single rapid doses of human umbilical blood mononuclear cells transfected with a plasmid carrying the vegf and fgf2 genes into the injured area. Animals of the control group received the same cells in analogous conditions, but transfected with plasmid pEGFP-N2, carrying the gene for enhanced green fluorescent protein (egfp). By postadministration day 30, the total area of pathological cavities in the outer zones of the white matter on transverse sections of the spinal cord 3 mm from the trauma epicenter in the caudal direction was more than two times smaller in animals of the experimental group than in controls. The numbers of myelinated fibers in the same white matter zones at the same distance from the trauma epicenter in the caudal and rostral directions were an average of 20% greater than in controls, while at a distance of 5 mm in the rostral direction the number was 40-70% greater. Administration of the therapeutic genes vegf and fgf2 into the injured area decreased cavitation, restricted the processes of secondary degeneration and maintained the number of myelinated fibers in the damaged spinal cord. © 2012 Springer Science+Business Media New York

Correction of the face soft tissue defect using autologous fat tissue enriched by cells of stromal-vascular fraction

The article presents a clinical case of successful surgical treatment of a patient with idiopathic progressive hemifacial atrophy. We have used lipofilling with autologous fat tissue enriched by stromal-vascular fraction cells. Observation of the patient within two years suggests that this method of treatment is safe and highly effective method of face soft tissue defect substitution of such pathology

Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

© 2014 Elsevier Inc. All rights reserved. Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis