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

Effects of ionizing radiation on proteins in lyophilized or frozen demineralized human bone

OBJECTIVE: The aim was to study the effects of application of ionizing radiation (gamma and electrons) as sterilizing agents at doses of 15 kGy, 25 kGy and 50 kGy, on lyophilized or frozen demineralized bone tissue for use in transplants. METHODS: Five human femoral diaphyses from different donors of musculoskeletal tissue were demineralized and preserved as lyophilized or frozen at -80 °C. The samples were divided into two groups: non-irradiated (control) and irradiated by means of gamma rays or an electron beam. The bone proteins were extracted and used to determine the concentrations of total protein and BMP 2 and 7. RESULTS: Decreases in total protein and BMP 2 and 7 concentrations were observed. The decreases in total protein concentrations, in comparison with the respective control groups, were significant in the lyophilized and frozen samples that were irradiated at a dose of 50 kGy of gamma radiation and electron beam, with reductions of more than 30%. Significant decreases in the levels of BMP 2 and 7 were also observed at higher doses and especially through use of the electron beam. CONCLUSION: The reductions in the concentrations of total proteins and osteoinductive proteins (BMP 2 and 7) were related to the radiation dose, i.e. they increased with higher doses of ionizing radiation type and the type of bone preservation. The largest reductions in concentrations were observed in the bones irradiated by means of an electron beam and at a dose of 50 kGy. However, this type of radiation and this high dose are not usual practices for sterilization of bone tissue

Radio-synthesized protein-based nanoparticles for biomedical purposes

Protein-crosslinking whether done by enzymatic or chemically induced pathways increases the overall stability of proteins. In the continuous search for alternative routes for protein stabilization we report a novel technique - radio-induced synthesis of protein nanoparticles - to achieve size controlled particles with preserved bioactivity. Papain was used as model enzyme and the samples were irradiated at 10. kGy in a gammacell irradiator in phosphate buffer (pH=7.0) and additives such as ethanol (0-40%) and sodium chloride (0-25%). The structural rearrangement caused by irradiation under defined conditions led to an increase in papain particle size as a function of the additive and its concentration. These changes occur due to intermolecular bindings, of covalent nature, possibly involving the aromatic amino acids. Ethanol held major effects over papain particle size and particle size distribution if compared to sodium chloride. The particles presented relative retained bioactivity and the physic-chemical characterization revealed similar fluorescence spectra indicating preserved conformation. Differences in fluorescence units were observed according to the additive and its concentration, as a result of protein content changes. Therefore, under optimized conditions, the developed technique may be applied for enzyme nanoparticles formation of controllable size and preserved bioactivity.Fil: Varca, Gustavo H. C.. Comissao Nacional de Energia Nuclear. Centro de Lasers e Aplicacoes. Instituto de Pesquisas Energéticas e Nucleares; BrasilFil: Ferraz, Caroline C.. Comissao Nacional de Energia Nuclear. Centro de Lasers e Aplicacoes. Instituto de Pesquisas Energéticas e Nucleares; BrasilFil: Lopes, Patricia S.. Universidade de Sao Paulo; BrasilFil: Mathor, Monica Beatriz. Comissao Nacional de Energia Nuclear. Centro de Lasers e Aplicacoes. Instituto de Pesquisas Energéticas e Nucleares; BrasilFil: Grasselli, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Lugão, Ademar B.. Comissao Nacional de Energia Nuclear. Centro de Lasers e Aplicacoes. Instituto de Pesquisas Energéticas e Nucleares; Brasi

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.CNPq (National Counsel of Technological and Scientific Development)National Counsel of Technological and Scientific Development (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior

<b>Evaluation of <i>in vitro</i> biological properties of <i>Senna occidentalis</i> (L.) Link

Senna species have been widely used by American, African and Indian ethic groups mainly in the treatment of feebleness, constipation, liver disorders and skin infections. Senna occidentalis (L.) Link is a perennial shrub native to South America and indigenous to tropical regions throughout the world. Current study evaluated the antimicrobial activity of aqueous and hydroalcoholic extracts from S. occidentalis prepared from different parts of the plant. Antimicrobial activity was assessed against standard pharmaceutical microorganisms by spectrophotometry and microdilution technique. Escherichia coli was sensitive only to compounds extracted from seeds which may be proteinaceous. A broader antimicrobial spectrum was demonstrated by the hydroalcoholic extract of seeds, mostly against Pseudomonas aeruginosa. The in vitro toxicity using mouse fibroblasts indicated that the extract might be a biocompatible ingredient for topical formulations, while the hydroalcoholic extract of aerial parts demonstrated to be potentially cytotoxic

Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes

The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia

Ultrastructural evaluation of human keratinocyte growth and differentiation on a fibrin substrate Avaliação ultraestrutural do crescimento e da diferenciação de queratinócitos sobre um substrato de fibrina

PURPOSE: In order to circumvent several difficulties that have been met in the routine use of the in vitro keratinocyte cultures using the standard procedure described by Rheinwald and Green, and obtain a more resilient and the least possible immunogeneic skin substitute for a future clinical application, this work studied a new keratinocyte culture system, which envisages the utilization of a fibrin substrate in association with high densities of human keratinocytes. METHODS: Through light and transmission electron microscopy and immunohistochemical assays, long-term proliferative and differentiative characteristics of keratinocytes cultured onto a fibrin gel under immerse and air-liquid interface culture conditions were evaluated. RESULTS: Despite the absence of a dermal substitute, the results demonstrated that the proposed composite was constituted of a transparent and elastic fibrin film covered by a well-attached, multistratified epithelium with morphological characteristics that resemble human epidermis, including the neoformation, albeit incomplete, of the basement membrane. CONCLUSIONS: Increased mechanical resistance due to the presence of an easy handling substrate, the delivery of nonclonfluent keratinocytes as well as the removal of animal-derived cells from the culture system suggest its potential use for future transplantation purposes.<br>OBJETIVO: Com o intuito de contornar diversas dificuldades encontradas no uso rotineiro de queratinócitos cultivados in vitro pela técnica descrita por Rheinwald e Green, e obter um substituto cutâneo mais resistente e o menos imunogênico possível para futuras aplicações clínicas, este trabalho avaliou um novo sistema de cultura de queratinócitos que prevê a utilização de um substrato de fibrina em associação com queratinócitos humanos em alta densidade. MÉTODOS: Através de microscopia óptica e eletrônica e análise imunohistoquímica, foram avaliadas as características proliferativas e de diferenciação em longo prazo de queratinócitos cultivados em condição imersa e na interface ar-líquido. RESULTADOS: Apesar da ausência de um substituto dérmico, foi demonstrado que o composto proposto constituiu-se de um substrato de fibrina transparente e elástico coberto por epitélio multi-estratificado, bem aderido, com características morfológicas semelhantes à epiderme humana, incluindo a neo-formação, embora incompleta, da membrana basal. CONCLUSÕES: A maior resistência mecânica com a presença de um substrato de fácil manuseio, a possível liberação de queratinócitos não-confluentes, e a remoção de células com origem animal dos sistemas de cultura sugerem que o composto proposto neste estudo apresenta grande potencial para uso clínico futuro

Influence of gamma radiation onto polymeric matrix with papain

Papain is a proteolytic enzyme that has been widely used as debridement agent for scars and wound healing treatment. However, papain presents low stability, which limits its use to extemporaneous or short shelf-life formulations. The purpose of this study was to entrap papain into a polymeric matrix in order to obtain a drug delivery system that could be used as medical device. Since these systems must be sterile, gamma radiation is an interesting option and presents advantages in relation to conventional agents: no radioactive residues are formed: the product can be sterilized inside the final packaging and has an excellent reliability. The normative reference for the establishment of the sterilizing dose determines 25 kGy as the inactivation dose for viable microorganisms. A silicone dispersion was selected to prepare membranes containing 2% (w/w) papain. Irradiated and non-irradiated membranes were simultaneously assessed in order to verify whether gamma radiation interferes with the drug-releasing profile. Results showed that irradiation does not affect significantly papain release and its activity. Therefore papain shows radioresistance in the irradiation conditions applied. In conclusion, gamma radiation can be easily used as sterilizing agent without affecting the papain release profile and its activity onto the biocompatible device is studied. (C) 2009 Elsevier Ltd. All rights reserved