Este trabalho foi suportado por concessões do NIH: R03 DK089061-01 (VKY); NIH: K08 DK068391 (VKY), o Diabetes e Endocrinologia Centro de Pesquisa (DERC - P30DK079638) no Baylor College of Medicine, uma bolsa de piloto e de viabilidade de do DERC (VKY); Juvenile Diabetes Research Foundation: JDRF Prêmio # 5-2006-134 (VKY).

<ol>
	<li>Parks, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+helper-dependent+adenovirus+vector+system:+removal+of+helper+virus+by+Cre-mediated+excision+of+the+viral+packaging+signal.">A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.</a> Proc. Natl. Acad. Sci. U. S. A. 93, 13565-13570 (1996).</li><li>Kim, I. H., Jozkowicz, A., Piedra, P. A., Oka, K., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+correction+of+genetic+deficiency+in+mice+with+a+single+injection+of+helper-dependent+adenoviral+vector.">Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13282-13287 (2001).</li><li>Belalcazar, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stable+expression+of+human+apolipoprotein+A-I+mediated+by+helper-dependent+adenovirus+gene+transfer+inhibits+atherosclerosis+progression+and+remodels+atherosclerotic+plaques+in+a+mouse+model+of+familial+hypercholesterolemia.">Long-term stable expression of human apolipoprotein A-I mediated by helper-dependent adenovirus gene transfer inhibits atherosclerosis progression and remodels atherosclerotic plaques in a mouse model of familial hypercholesterolemia.</a> Circulation. 107, 2726-2732 (2003).</li><li>Oka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stable+correction+of+low-density+lipoprotein+receptor-deficient+mice+with+a+helper-dependent+adenoviral+vector+expressing+the+very+low-density+lipoprotein+receptor.">Long-term stable correction of low-density lipoprotein receptor-deficient mice with a helper-dependent adenoviral vector expressing the very low-density lipoprotein receptor.</a> Circulation. 103, 1274-1281 (2001).</li><li>Nomura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+receptor+gene+therapy+using+helper-dependent+adenovirus+produces+long-term+protection+against+atherosclerosis+in+a+mouse+model+of+familial+hypercholesterolemia.">Low-density lipoprotein receptor gene therapy using helper-dependent adenovirus produces long-term protection against atherosclerosis in a mouse model of familial hypercholesterolemia.</a> Gene Ther. 11, 1540-1548 (2004).</li><li>Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-efficiency+Cre/loxP-based+system+for+construction+of+adenoviral+vectors.">A high-efficiency Cre/loxP-based system for construction of adenoviral vectors.</a> Hum. Gene Ther. 10, 2667-2672 (1999).</li><li>Palmer, D., Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+system+for+helper-dependent+adenoviral+vector+production.">Improved system for helper-dependent adenoviral vector production.</a> Mol. Ther. 8, 846-852 (2003).</li><li>Oka, K., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper-Dependent+Adenoviral+Vectors.">Helper-Dependent Adenoviral Vectors.</a> Current Protocols in Molecular Biology. , 16.24.1-16.24.23 (2005).</li><li>Yechoor, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin3+is+sufficient+for+transdetermination+of+hepatic+progenitor+cells+into+neo-islets+in+vivo+but+not+transdifferentiation+of+hepatocytes.">Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes.</a> Dev. Cell. 16, 358-373 (2009).</li><li>Yechoor, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+with+Neurogenin+3+and+Betacellulin+Reverses+Major+Metabolic+Problems+in+Insulin-Deficient+Diabetic+Mice.">Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice.</a> Endocrinology. , (2009).</li></ol>

Não há conflitos de interesse declarados.

HDAds têm sido desenvolvidas para ultrapassar a fraqueza de anúncios geração inicial e para aproveitar para aplicação de terapia génica. No entanto, os desafios técnicos permanecem. Por exemplo, requer HDAD HV para embalagens HDAD e amplificação vector não é tão eficiente como os anúncios geração antecipada. HV é um anúncio de primeira geração e de qualquer contaminação de compromisso da eficácia da HV HDAD. Portanto, transfecção altamente eficiente e as condições ideais para cada passagem de série são críticos. Um outro parâmetro crítico para a produção de vector é que a passagem (P1-P4) deve ser utilizada para a passagem subsequente de 5, que é directamente utilizado como inoculo para as células em suspensão. Para a experiência, os melhores resultados são obtidos utilizando a passagem pela qual HDAD proporção vector é dramaticamente aumentada na seguinte passagem (P3 na Figura 2). O rendimento de vectores HDAD depende cassetes transgene. Durante a produção de vector, ambos os transgenes são expressos porque ambos os genes estão sobpromotor onipresente. Ngn3 é um factor de transcrição e BTC é um factor de crescimento, o que sugere que o factor de transcrição HDAD vector expressando o que pode influenciar a linhagem de células inibe a amplificação vector, enquanto que a hormona do crescimento expressando ajuda na replicação do vector e embalagem. Com diabetes assumindo proporções epidêmicas, novas abordagens para restaurar b célula de massa são necessários. Neste relatório, nós descrevemos métodos para aproveitar as vantagens de vetores HDAD para efetuar a transferência de genes de ilhota linhagem definir fator de transcrição, Ngn3 juntamente com o fator de crescimento ilhota, betacelulina para induzir neogênese ilhota em regiões periportal do fígado. Para avaliar a eficácia do presente, é importante escolher os ratos com hiperglicemia estável e assegurar que os controlos apropriados são sempre incluídas. Para estas experiências de transferência de genes, o vector vazio trataram ratos diabéticos deve sempre ser utilizado. Além disso, utilizando-HDAD Ngn3 e HDAD-Btc tratadas individualmente diabética mice serve para testar a contribuição desses dois genes em ilhota neogênese. Como os nossos dados demonstram que Ngn3 por si só é suficiente para induzir a ilhota neogénese, mas a adição do factor de crescimento, BTC, serve para aumentar a resposta levando à indução de ilhota neogénese robusto. Também é importante para testar se o vector de expressão é de facto conseguida no tecido alvo, o fígado e também para demonstrar que a insulina testados no plasma de ratinhos tratados não é proveniente de ilhotas residuais no pâncreas, demonstrando a ausência de pâncreas ilhotas nos ratos diabéticos. Em resumo, a vantagem de o sistema HDAD vector para a transferência de gene é a sua capacidade de clonagem elevada, a transdução eficiente e da expressão do gene de longa duração no fígado com toxicidade crónica mínimo, assim como a sua natureza de não integração do genoma do vector no hospedeiro cromossomo. As limitações principais são os passos complexos envolvidos na sua produção e do seuaplicação in vivo é essencialmente limitada ao fígado, sendo o mais popular Ad serotipo 5. Islet neogénese podem ser induzidas a restaurar completamente a insulina no plasma e de tolerância à glucose em ratos diabéticos através da indução de neogénese ilhota no fígado, por transferência de genes de factor de transcrição ilhota linhagem definidora, Ngn3 juntamente com o factor de crescimento das ilhotas, betacelulina. Neste relatório, nós apresentamos o protocolo ideal para gerar alta qualidade HDAD-Ngn3 e HDAD-BTC, e demonstrar técnicas para induzir e avaliar neogênese ilhota nos fígados dos camundongos diabéticos para reverter a hiperglicemia. Nota de rodapé: Os vectores virais e as linhas de células descritas aqui estão disponíveis a partir do núcleo Vector Laboratory Produção, Diabetes Research Center, Baylor College of Medicine ( <a href="http://www.bcm.edu/mcb/index.cfm?pmid=7731" target="_blank">http://www.bcm.edu/mcb/index.cfm?pmid=7731</a> ). Alguns kits comerciais também estão disponíveis para a geração de vírus (por exemplo, HDAD Microbix biosystems Inc.).

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> ProFectionR kit Transfecção mamíferos </td><td> Promega </td><td> E1200 </td></tr><tr><td> Kit Blood &amp; Tissue Dneasy (50) </td><td> Qiagen </td><td> 69504 </td></tr><tr><td> Perfecta SYBR Green Supermix, ROX </td><td> Quanta Biociências </td><td> 95055-500 </td></tr><tr><td> Deoxicolato de sódio </td><td> Sigma </td><td> D6750-25G </td></tr><tr><td> MEM em pó </td><td> Invitrogen </td><td> 61100087 </td></tr><tr><td> Estreptomicina penicilina </td><td> Sigma </td><td> 15140122 </td></tr><tr><td> FBS </td><td> Atlanta Biologicals </td><td> S11150 </td></tr><tr><td> L-glutamina </td><td> Invitrogen </td><td> 25030-081 </td></tr><tr><td> Higromicina B </td><td> Sigma </td><td> H0654-1G </td></tr><tr&gt; <td> MEM ÁGUIA Joklik </td><td> Sigma </td><td> M0518-10L </td></tr><tr><td> Rnase </td><td> Roche </td><td> 10109169001 </td></tr><tr><td> DNase I, grau II </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> Estreptozocina </td><td> Sigma </td><td> s0130 </td></tr><tr><td> Vidro giratório frascos </td><td> Corning </td><td> 4500-3L </td></tr><tr><td> Vidro giratório frascos </td><td> Corning </td><td> 4500-250 </td></tr><tr><td> Deslize A-lyzer Casset </td><td> PIERCE CH </td><td> PI66380 </td></tr><tr><td> Tubo optiseal poli allomer, 11,2 ml </td><td> Beckman Coulter </td><td> 362181 </td></tr><tr><td> Cloreto de césio 1 kg </td><td> JT4042-2 </td><td> VWR </td></tr><tr><td> Beckman LE-80K </td><td> Beckman Coulter </td><td> Ultracentrífuga LE-80K ideal </td></tr><tr><td> Filtrar </td><td> VWR </td><td> 28143-338 </td></tr><tr><td> centrifugar m tubo de 500l </td><td> Corning </td><td> 431123 </td></tr><tr><td> tailveiner limitador </td><td> Braintree científica, INC </td><td> tv-150 </td></tr><tr><td> A insulina, Rato ELISA </td><td> Mercodia </td><td> 10-1247-01 </td></tr><tr><td> microvette CB300 </td><td> Sarstedt </td><td> 16.443.100 </td></tr><tr><td> D-glucose </td><td> Sigma </td><td> G8270 </td></tr><tr><td> Rato peptídeo C ELISA Kit </td><td> Wako Pure Chemical Industries, Ltd </td><td> # 631-07231 </td></tr><tr><td> cobaia anticorpo anti-insulina </td><td> Abcam </td><td> ab7842 </td></tr><tr><td> anticorpo de cabra anti-Pdx1 </td><td> presente do Dr. Christopher Wright </td><td></td></tr><tr><td> rato anti Ngn3 anticorpo </td><td> Consórcio de Biologia das células beta, Univ. da Pensilvânia </td><td> AB2013 </td></tr><tr><td> rato anti Nkx6.1 anticorpo </td><td> Consórcio de Biologia das células beta, Univ. de Pennsylvania </td><td> F64A6B4 </td></tr><tr><td> anticorpo anti-betacelulina </td><td> Cell Sciences </td><td> PAAQ1 </td></tr><tr><td> ALT (TGP) SET Reagente de Cor. </td><td> Teco Diagnóstico </td><td> A526 - 120 </td></tr><tr><td> AST / (TGO), Cor conjunto de reagentes Endpoint </td><td> Teco Diagnóstico </td><td> A561-120 </td></tr></tbody></table> Tabela 2. Reagentes e equipamentos específicos.

neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer

A diabetes tipo 1 é causada pela destruição mediada por células T auto-imune das células produtoras de insulina no pâncreas. Até agora, a reposição de insulina é ainda a terapêutica maior, porque transplante de ilhotas foi limitada pela disponibilidade de dadores e pela necessidade de imunossupressão a longo prazo. Induzida por ilhota neogénese por transferência de genes de Neuogenin3 (Ngn3), a linhagem de ilhotas de definição factor de transcrição específico e betacelulina (BTC), um factor de crescimento de ilhotas tem o potencial de curar a diabetes do tipo 1. Vectores adenovirais (Ads) são vectores de transferência de genes altamente eficiente, no entanto, os anúncios geração iniciais têm várias desvantagens para a utilização in vivo. Ajudante de dependentes de Anúncios (HDAds) são os anúncios mais avançadas que foram desenvolvidos para melhorar o perfil de segurança da primeira geração de anúncios e para prolongar transgene expressão 1. Eles não têm a toxicidade crônica porque faltam seqüências virais codificação 2-5 e manter somente o anúncio cis elements necessárias para a replicação do vector e embalagem. Isto permite a clonagem de genes de até 36 kb. Neste protocolo, descrevemos o método para gerar HDAD-Ngn3 e HDAD-BTC e entregar esses vetores em ratos STZ-induzidos diabéticos. Os nossos resultados mostram que a co-injecção de HDAD-Ngn3 e &quot;ilhéus&quot; neo-HDAD Btc induz no fígado e inverte a hiperglicemia em ratos diabéticos.

Watch this Scientific Journal Video about Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer at JoVE.com

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

Nós descrevemos a formação de neo-hepática ilhota em STZ (estreptozotocina) induzida ratos diabéticos por transferência de genes de Neurogenin3 (Ngn3) e betacelulina (BTC), utilizando ajudante dependente do vector adenoviral (HDAD) e reversão da hiperglicemia. O nosso método tem vantagens do ajudante dependente de vectores adenovirais com a sua grande eficácia na transdução in vivo e a expressão do gene de longa duração.

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 ...

neo-islet-formation-liver-diabetic-mice-helper-dependent-adenoviral

Research

JoVE Journal

Medicine

11.7K visualizações.  Baylor College of Medicine. Nós descrevemos a formação de neo-hepática ilhota em STZ (estreptozotocina) induzida ratos diabéticos por transferência de genes de Neurogenin3 (Ngn3) e betacelulina (BTC), utilizando ajudante dependente do vector adenoviral (HDAD) e reversão da hiperglicemia. O nosso método tem vantagens do ajudante dependente de vectores adenovirais com a sua grande eficácia na transdução in vivo e a expressão do gene de longa duração.

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

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

A utilização de uma suspensão do sistema de peso para isquemia hepática em ratos

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

Medicina

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 ...

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 ...

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 ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Ligadura do ducto biliar em ratos: Indução de Inflammatory Lesão hepática e fibrose por colestase obstrutiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

Fígado Preservação Human Funcional e cobrança através de Subnormothermic Máquina de Perfusão

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

Na Vivo Transferência de genes para o endotélio da artéria carótida comum de coelho

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Protocolo para criar feridas crônicas em camundongos diabéticos

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Particulas de Lentivirus terapia gênica mediada por vetor de hepatócitos Ex Vivo para transplante autólogo em suínos

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

Indução da Inflamação Intestinal por Transferência Adotiva de Células CD4+ T Transgênicas CBir1 T para camundongos imunodeficientes

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...