Over expression of the selectable marker blasticidin S deaminase gene is toxic to human keratinocytes and murine BALB/MK cells

Background: the blasticidin S resistance gene (bsr) is a selectable marker used for gene transfer experiments. the bsr gene encodes for blasticidin S (BS) deaminase, which has a specific activity upon BS. Therefore, its expression is supposed to be harmless in cells. the work reported on herein consisted of experiments to verify a possible toxicity of bsr on mammalian cells, which include several cell lines and primary cultures.Results: Murine keratinocyte BALB/MK and human primary keratinocyte cells transduced with the retroviral vector LBmSN, which has an improved expression system of bsr, namely bsrm, died in five days after the transduction. Meanwhile the control vector LBSN, which expresses bsr, did not provoke cell death. the lethal activity of bsrm was observed only in human keratinocytes and BALB/MK cells among the cell types tested here. Death appears to be mediated by a factor, which is secreted by the BALB/MK transduced cells.Conclusion: By our study we demonstrated that the expression of bsrm gene is toxic to human keratinocytes and BALB/MK cells. It is likely over expression of BS deaminase gene is responsible for the death.Universidade Federal de São Paulo, EPM, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, EPM, Ctr Gene Therapy, São Paulo, BrazilIPEN CNEN SP, São Paulo, BrazilUniversidade Federal de São Paulo, EPM, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, EPM, Ctr Gene Therapy, São Paulo, BrazilWeb of Scienc

Autonomous growth of BALB/MK keratinocytes transfected with a retroviral vector carrying the human epidermal growth factor gene

Epidermal growth factor (EGF), which promotes epidermal regeneration and wound closure, is important for the proliferation and differentiation of epidermal and epithelial tissues in animals. Exogenous EGF is a promising therapeutic agent for wound healing, but its general use is restricted by the limited availability of this protein. in this work, we show that the transfection of mouse BALB/MK keratinocytes, which are totally dependent on EGF for growth and migration, with mature cDNA for human EGF via a retroviral vector abolished the cells requirement for exogenous EGF. the transformed cells had normal morphology and a growth rate that varied according to the source of the retroviral vector used. Keratinocyte transfection with EGF cDNA provides a time- and cost-efficient means of culturing keratinocytes and yields cells that may be useful for skin grafting

Evaluation of KIT and PDGFRA mutations in Gastrointestinal Stromal Tumors (GIST)

UNIFESP, Dept Pathol, Guarulhos, BrazilUNIFESP, Dept Pathol, Guarulhos, BrazilWeb of Scienc

Amyloid-like self-assembly of a hydrophobic cell-penetrating peptide and its use as a carrier for nucleic acids

Cell-penetrating peptides (CPPs) are a topic subject potentially exploitable for creating new nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P: proline, F: phenylalanine, V: valine, Y: tyrosine, L: leucine and I: isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity, capacity of nuclear localization, and its small size readily hints suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form non-covalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rod-like crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms non-covalent complexes with nucleic acids that retain -sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-

Mesenchymal Stem Cells Do Not Prevent Antibody Responses against Human alpha-L-Iduronidase when Used to Treat Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease that leads to systemic lysosomal storage, which is caused by the absence of alpha-L-iduronidase (IDUA). Enzyme replacement therapy is recognized as the best therapeutic option for MPSI; however, high titers of anti-IDUA antibody have frequently been observed. Due to the immunosuppressant properties of MSC, we hypothesized that MSC modified with the IDUA gene would be able to produce IDUA for a long period of time. Sleeping Beauty transposon vectors were used to modify MSC because these are basically less-immunogenic plasmids. for cell transplantation, 4x10(6) MSC-KO-IDUA cells (MSC from KO mice modified with IDUA) were injected into the peritoneum of KO-mice three times over intervals of more than one month. the total IDUA activities from MSC-KO-IDUA before cell transplantation were 9.6, 120 and 179 U for the first, second and third injections, respectively. Only after the second cell transplantation, more than one unit of IDUA activity was detected in the blood of 3 mice for 2 days. After the third cell transplantation, a high titer of anti-IDUA antibody was detected in all of the treated mice. Anti-IDUA antibody response was also detected in C57Bl/6 mice treated with MSC-WT-IDUA. the antibody titers were high and comparable to mice that were immunized by electroporation. MSC-transplanted mice had high levels of TNF-alpha and infiltrates in the renal glomeruli. the spreading of the transplanted MSC into the peritoneum of other organs was confirmed after injection of In-111-labeled MSC. in conclusion, the antibody response against IDUA could not be avoided by MSC. On the contrary, these cells worked as an adjuvant that favored IDUA immunization. Therefore, the humoral immunosuppressant property of MSC is questionable and indicates the danger of using MSC as a source for the production of exogenous proteins to treat monogenic diseases