Este trabalho foi financiado pela Agência de Pesquisa esloveno (projeto J2-9770, IP-0510 centro de infra-estrutura e programa P2-0249). Este vídeo representa material suplementar para o &quot;Eletroporação baseado em Tecnologias e Tratamentos&quot; workshop científico e pós-graduação, organizado pela Faculdade de Engenharia Elétrica da Universidade de Ljubljana, na Eslovénia. Autores agradecem também DUSA Hodzic por ter gentilmente cedido DNA plasmidial.

1. Cultura de células, plasmídeos e preparação de buffer para o experimento <ol><li> Neste experimento células de ovário de hamster chinês (CHO-K1) são utilizados. As células são cultivadas em uma mistura de nutrientes HAM-F12 (PAA), suplementado com 2 mM L-glutamina, 10% de soro fetal bovino, 400 mL / l de gentamicina (todos da Chemie GmbH Sigma-Aldrich, Deisenhofen, Alemanha), e 1 ml / l crystacilin (Pliva, Zagreb, Croácia). As células são mantidas a 37 ° C em 5% CO 2 umidificado atmosfera na incubadora por 24h. </li><li> Amplificar plasmídeo pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, EUA), codificação de uma proteína fluorescente verde (GFP) em tensão DH5α de Escherichia coli e isolá-lo com o Kit Maxi HiSpeed ​​Plasmid (Qiagen, Hilden, Alemanha). Concentração de DNA plasmidial (plasmídeo dissolvido em tampão TE) deve ser determinada espectrofotometricamente a 260 nm e confirmada por eletroforese em gel. </li><li> Prepare tampão fosfato de sódio isoosmolar (10 mM Na 2 HPO 4, 10 mM NaH 2 PO 4, 1 mM MgCl 2, 250 mM de sacarose, pH 7,4). </li><li> No dia do experimento preparar suspensão celular por tripsinização com 0,25% de tripsina / EDTA (Sigma-Aldrich Chemie GmbH, Deisenhofen, Alemanha). Células centrifugar por 5 min a 1000 rpm (180 xg) a 4 ° C (Sigma, Alemanha) e ressuspender pellet celular em tampão fosfato de sódio isoosmolar a uma densidade de células de 5 × 10 6 células / ml. </li></ol> 2. Equipamentos de hardware <ol><li> Células são expostas ao campo elétrico na ponta da pipeta (Figura 1) com eletrodos integrados ligado a um gerador de protótipos de alta tensão. A ponta do eletrodo ea geometria permite a aplicação de relativamente homogênea campo elétrico e do gerador permite a entrega de pulsos elétricos em direções diferentes. A ponta eo gerador foram desenvolvidos no Laboratório de Biocybernetics, Faculdade de Engenharia Elétrica, Universidade de Ljubljana 1. </li></ol><img src="/files/ftp_upload/3309/3309fig1.jpg" alt="Figura 1"/> Figura 1. Vertical e horizontal (a) seção transversal e fotografia (b) da ponta da pipeta com eletrodos integrados. Na cruz de cor cinza seção é usada para a caixa de plástico e preto para os eletrodos. A ponta da pipeta com eletrodos integrados é composta por quatro eletrodos haste cilíndrica. Os eletrodos são feitos de aço inoxidável; seu diâmetro é de 1,4 mm, eletrodos adjacentes são de 1 mm, com eletrodos opostos são 2 mm. Os eletrodos são colados na ponta de plástico em paralelo e seu comprimento aplicável é de 30 mm 2. 3. Gene electrotransfer protocolo <ol><li> Adicionar plasmídeo pEGFP-N1 a uma suspensão de células em concentração de 10 mg / ml. </li><li> Incubar a mistura por 2-3 minutos em temperatura ambiente, antes da aplicação de impulsos elétricos. </li><li> Aspire mL 100 de suspensão de células para a ponta da pipeta com eletrodos integrados. </li><li> Para alcançar eficácia melhor electrotransfer gene e manter a viabilidade celular, os parâmetros ideais de pulsos elétricos devem ser usados. Neste experimento um trem de pulsos retangulares de 8 (cada uma com duração de 1 ms, amplitude 225 V na freqüência de 1 repetição Hz) é aplicado a cada amostra, utilizando alta tensão do gerador protótipo. Dois diferentes protocolos de campo elétrico (Figura 2) são usados: no primeiro protocolo todos os pulsos são entregues na mesma direção, enquanto que no segundo protocolo pulsos são entregues mudando alternadamente a direção do campo elétrico e de orientação. O segundo protocolo só pode ser usado com gerador de pulsos adequado, que permite a aplicação de impulsos elétricos em direções diferentes. </li><li> Imediatamente após a aplicação do pulso de transferência de células de ponta da pipeta em 6 prato bem e adicionar soro fetal bovino (FCS-Sigma, EUA) (25% do volume da amostra). </li><li> Incubar as células por 5 minutos a 37 ° C para permitir a recolagem membrana celular. </li><li> Adicionar 2 ml de HAM-F12 para cada amostra em 6 bem e incubar as células por 24h a 37 ° C em um 5% CO 2 umidificado atmosfera na incubadora. </li></ol><img src="/files/ftp_upload/3309/3309fig2.jpg" alt="Figura 2"/> . Figura 2 protocolos de campo elétrico: (a) todos os pulsos são entregues na mesma direção, (b) pulsos são entregues mudando alternadamente a direção do campo elétrico e de orientação. 4. Aquisição de imagem e determinação do gene eficácia electrotransfer <ol><li> Eficácia do gene electrotransfer é determinada como a porcentagem de células expressando GFP 24h após a aplicação do pulso. </li><li> As células são observados usando um microscópio de fluorescência (no nosso caso Zeiss 200, Axiovert, ZR Alemanha), com luz de excitação a 488 nm gerada com um sistema monocromador (policromos IV, Visitron, Alemanha) e de emissões é detectada em 507 nm. As imagens são gravadas utilizando imagens do sistema (Metamorph imagem do sistema, Visitrna, Alemanha), mas o software de aquisição semelhante também pode ser usado. </li><li> Adquirir pelo menos cinco imagens (contraste de fase e fluorescência verde) com ampliação de 20x objetivo. </li><li> Contagem de células à imagem de contraste de fase e células que estão expressando GFP à imagem de fluorescência verde. Determinar a percentagem de eficácia electrotransfer gene pela divisão do número de células que estão expressando GFP com o número de todas as células em cada imagem correspondente (Figura 3). </li></ol> 5. Resultados representativos: <img src="/files/ftp_upload/3309/3309fig3.jpg" alt="Figura 3"/> Figura 3. A porcentagem de células expressando GFP, quando todos os pulsos são entregues na mesma direção e quando os pulsos são entregues mudando alternadamente a direção do campo elétrico e de orientação é apresentada. As células foram expostas a um trem de oito pulsos com amplitude 225 V, 1 ms de duração e freqüência de repetição de 1 Hz. Resultados foram obtidos por meio de microscopia de fluorescência. Cada valor no gráfico representam a média de três experimentos independentes ± desvio padrão. Mudando a direção do campo elétrico e orientação a porcentagem de células expressando GFP aumenta.

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	<li>Rebersek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporator+with+automatic+change+of+electric+field+direction+improves+gene+electrotransfer+in-vitro.">Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.</a> Biomed Eng Online. 6, 25-25 (2007).</li><li>Trontelj, K., Rebersek, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip+electrode+chamber+for+small+volume+electroporation,+electrofusion,+and+gene+transfection.">Tip electrode chamber for small volume electroporation, electrofusion, and gene transfection.</a> 18, (2006).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-841 (1982).</li><li>Daud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+trial+of+interleukin-12+plasmid+electroporation+in+patients+with+metastatic+melanoma.">Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.</a> J Clin Oncol. 26, 5896-5903 (2008).</li><li>Pavlin, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+conductivity+of+a+suspension+of+permeabilized+cells:+a+theoretical+analysis.">Effective conductivity of a suspension of permeabilized cells: a theoretical analysis.</a> Biophys J. 85, 719-729 (2003).</li><li>Xie, T., Sun, L., Zhao, H., Fuchs, J., Tsong, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+mechanisms+of+electric+field-induced+DNA+transfection.+IV.+Effects+of+DNA+topology+on+cell+uptake+and+transfection+efficiency.">Study of mechanisms of electric field-induced DNA transfection. 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Não há conflitos de interesse declarados.

Gene electrotransfer é uma técnica de biotecnologia versátil que permite a transferência de DNA em células por meio de aplicação de curto, pulsos de alta tensão elétrica 3 e representa uma alternativa mais segura para vetores virais, devido à sua eficácia, segurança e facilidade de aplicação. Embora electrotransfer gene hoje é amplamente utilizado para transfecção todos os tipos de células e primeira fase I de ensaios clínicos utilizando este método tem sido relatada 4, os mecan...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> HAM-F12 </td><td> PAA </td><td> E15-016 </td><td> meio de cultura </td></tr><tr><td> L-glutamina </td><td> Sigma-Aldrich </td><td> G7513 </td><td></td></tr><tr><td> soro fetal bovino </td><td> PAA </td><td> A15-151 </td><td></td></tr><tr><td> gentamicina </td><td> Sigma-Aldrich </td><td> G1397 </td><td> antibiótico </td></tr><tr><td> crystacilin </td><td> Pliva </td><td> 625110 </td><td> antibiótico </td></tr><tr><td> pEGFP-N1 </td><td> Clontech Laboratories </td><td> 6085-1 </td><td> DNA plasmidial </td></tr><tr><td> Kit Maxi HiSpeed ​​Plasmid </td><td> Qiagen </td><td> 12662 </td><td></td></tr><tr><td> Na 2 HPO 4 </td><td> Merck </td><td> F640786 933 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> TKI Hrastnik </td><td> 0795 </td><td></td></tr><tr><td> MgCl 2 </td><td> Sigma-Aldrich </td><td> M-8266 </td><td></td></tr><tr><td> sacarose </td><td> Sigma-Aldrich </td><td> 16104 </td><td></td></tr><tr><td> tripsina / EDTA </td><td> Sigma-Aldrich </td><td> T4174 </td><td></td></tr><tr><td> pipeta ponta </td><td> Feitos </td><td></td><td></td></tr><tr><td> elétrica do gerador de pulsos </td><td> Feitos </td><td></td><td></td></tr><tr><td> 6 Placa bem </td><td> TPP </td><td> 92406 </td><td></td></tr><tr><td> 15 tubo de centrifugação ml </td><td> TPP </td><td> 91015 </td><td></td></tr><tr><td> 75 centímetros frasco de cultura 2 </td><td> TPP </td><td> 90076 </td><td></td></tr></tbody></table>

changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro

Gene electrotransfer é um método físico utilizado para entregar genes dentro das células através da aplicação de curto e intenso pulsos elétricos, que causam a desestabilização da membrana celular, tornando-a permeável a pequenas moléculas e permite a transferência de grandes moléculas como o DNA. Ela representa uma alternativa aos vetores virais, devido à sua eficácia, segurança e facilidade de aplicação. Para o gene electrotransfer diferentes protocolos de pulso elétrico são utilizadas a fim de alcançar transfecção gênica máximo, um deles está mudando a direção do campo elétrico e orientação durante a entrega de pulso. Mudando de direção e orientação do campo elétrico aumentar a área de membrana competentes para a entrada de DNA na célula. Neste vídeo, demonstramos a diferença na eficácia electrotransfer gene, quando todos os pulsos são entregues na mesma direção e quando os pulsos são entregues mudando alternadamente a direção do campo elétrico e de orientação. Para esta dica propósito com eletrodos integrados e de alta tensão do gerador protótipo, que permite a mudança do campo elétrico em diferentes direções durante a aplicação do pulso elétrico, foram usados. Gene electrotransfer eficácia é determinada 24h após a aplicação do pulso como o número de células expressando a proteína fluorescente verde dividido com o número de todas as células. Os resultados mostram que a transfecção de genes é aumentada quando a orientação do campo elétrico durante o parto pulso elétrico é alterado.

Watch this Scientific Journal Video about Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro at JoVE.com

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Transfecção gênica por eletroporação é melhorada aproximadamente duas vezes quando a orientação do campo elétrico é alterado durante a aplicação do pulso, enquanto que a viabilidade das células não é afetado. O aumento da transfecção do gene é causada pelo aumento da área de membrana que é feito competentes para a entrada de DNA na célula.

Mudando a direção e orientação do campo elétrico Durante Elétrica Aplicação Pulsos melhora a transferência de Gene Plasmid In vitro</em

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

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Research

JoVE Journal

Medicine

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

10.4K visualizações.  University of Ljubljana. Transfecção gênica por eletroporação é melhorada aproximadamente duas vezes quando a orientação do campo elétrico é alterado durante a aplicação do pulso, enquanto que a viabilidade das células não é afetado. O aumento da transfecção do gene é causada pelo aumento da área de membrana que é feito competentes para a entrada de DNA na célula.

Vídeo: Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Estabelecimento e propagação de Tumores Humanos em Retinoblastoma Imune camundongos deficientes

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Medicina

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Transferência de genes para insuficiência cardíaca isquêmica em um modelo pré-clínica

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Produção e Detecção de Espécies Reativas de Oxigênio (ROS) em Câncer

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Neo-Islet Formação em fígado de ratos diabéticos por Helper dependente de Transferência de Gene adenoviral Vector Mediada

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Vascular transferência de genes de Metallic Stent superfícies usando Adenoviral Vetores Tethered através hidrolisável reticulantes

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Técnica Gene Transferência e<em&gt; In vitro</em&gt; Ensaios baseados em células para o Estudo da perda óssea em Distúrbios Osteomusculares

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

O aumento de artéria pulmonar fluxo pulsátil melhora Hypoxic Hipertensão Pulmonar em Leitões

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can improve strength in individuals with MS, however; it remains unclear the duration of RST that is needed to make strength gains and how to adapt hip strengthening exercises for individuals of varying strength using only resistance bands. This paper describes the methodology to set up and implement an adapted resistance strength training program, using resistance bands, for individuals with MS. Directions for pre- and post-strength tests to evaluate efficacy of the strength-training program are included. Safety features and detailed instructions outline the weekly program content and progression. Current evidence is presented showing that significant strength gains can be made within 8 weeks of starting a RST program. Evidence is also presented showing that resistance strength training can be successfully adapted for individuals with MS of varying strength with little equipment.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis. Isolated muscle strengthening is a useful method to target specific weaknesses. This protocol describes a progressive resistance-training program using exercise bands to increase hip muscle strength.

Treinamento da resistência adaptada melhora a força em oito semanas em indivíduos com esclerose múltipla

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...