Name: evaluation of the efficacy of the h. pylori protein hp-nap as a therapeutic tool for treatment of bladder cancer in an orthotopic murine model
Uploaded: 2015-05-29T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model at JoVE.com

Este trabalho foi apoiado pela Associazione Italiana per la Ricerca sul Cancro, italiano Ministério da Universidade e da Pesquisa, projetos Prin e Progetti di Ricerca di Ateneo, conceder N ° CPDA137871, Fondazione Cariplo, conceder N ° 2011-0485 para MDB, e por Finanziamento Giovani studiosi, da Universidade de Pádua, para Gaia Codolo.

Todos os procedimentos para lidar com os animais foram aprovados pelo Ministério italiano da Saúde (204/2011-B DM). 1. Os animais <ol><li> Crescer camundongos fêmeas C57BL6 / J 8 semanas de idade em gaiolas ventiladas individualmente com filtros microisolation. Nota: As fêmeas são preferidos devido à conformação anatómica do seu aparelho de uro-genital externa torna o cateterismo mais fácil do que nos machos. </li></ol> 2. Cultura celular <ol><li> Manter a linha celular de carcinoma urotelial rato MB49 em RPMI 1640 meio suplementado com 10% de soro bovino fetal inactivado pelo calor e 50 ng / ml de gentamicina, a 37 ° C em 5% humidificada de CO 2. </li><li> Cultivar células MB49 em frascos T-75 a 80% de confluência. Tripsinizar e ressuspender em 0,5 × 10 5-0,5 x 10 6 células por 150 ul de PBS antes da implantação em ratos. </li></ol> 3. Intravesical tumor Implant <ol><li> Anestesiar ratos, utilizando uma mistura de anestésicos e o Zoletil xylor (33 mg / kg e 20 mg / kg, respectivamente), injectada intraperitonealmente. </li><li> Coloque os animais em decúbito dorsal e corrigir as patas traseiras para a almofada com fita adesiva. Patas têm de ser suficientemente separadas para processar o meato uretral bem visíveis e facilmente acessível para a inserção do cateter. </li><li> Nota: Para a cateterismo, um cateter venoso pediátrica 24 G é adequado para ratinhos 8 a 10 semanas de idade. A escolha do cateter é muito importante a fim de evitar a fuga de líquido entre a parede da uretra e do cateter. Cateteres furo maior deve ser utilizado para ratinhos maiores ou mais velhos. </li><li> Usando uma pequena pinça, beliscar uma borda da parte externa da uretra e torcê-lo muito suavemente em direção ao &quot;fim cabeça&quot; dos ratinhos. Esta ação expõe o meato uretral. </li><li> Retirar a agulha do cateter interno e inserir este último na uretra com um ângulo de 45 °, orientada ligeiramenteno sentido da &quot;extremidade posterior&quot; dos ratinhos. Não force a entrada do cateter; em caso de resistência, basta girar o cateter muito suavemente, até que desliza para dentro da uretra. Quando cerca de 0,5-0,8 cm do cateter na uretra é, colocar cuidadosamente o cateter paralelo para a cauda dos ratos e inseri-lo completamente. </li><li> Usando uma seringa de 1 ml, a agulha de que tem de ser inserido no cateter, lava-se a partir de urina residual da bexiga através da injecção de 200-300 ul de PBS estéril e aspirar o líquido a partir da bexiga. </li><li> Nota: Ao injetar PBS, a bexiga vai encher-se e um inchaço será visível entre as patas traseiras dos ratos; isto confirma que o cateterismo é correto. A maioria dos cateteres têm um volume morto que tem de ser considerada quando se calcula o volume de injecção. </li><li> Usando uma seringa esterilizada de 1 ml, injectar 200 ul de 0,1 N de HCl. Preencha a bexiga a fim de queimar toda a sua parede. Após 10 segundos, retire todo o ácido e imediatamente neutralize por injecção de 200 uL de NaOH 0,1 N, com uma seringa estéril. No caso de injecção de maior número de células (0,5 x 10 6), o passo de acidificação não é necessária. </li><li> Aspire todo o líquido residual e imediatamente lavar a cavidade com 300 ï¿½ de PBS estéril. </li><li> Esvaziar a bexiga por aspiração, e injectar 150 ul de PBS contendo células MB49 (intervalo de 0,5 x 10 5-0,5 x 10 6) no interior da bexiga. Deixar a seringa montada no cateter a fim de evitar a fuga de células. É melhor para garantir a seringa, fixando-lo à almofada com fita adesiva. </li><li> Após 1 hora, extrair suavemente o cateter da uretra, e colocar os murganhos na gaiola, sob uma lâmpada incandescente até despertaram: este ocorre geralmente entre 1 e 2 horas após o fim do tratamento. </li></ol> 4. Entrega de Intravesical HP-NAP <ol><li> Nota: pelo menos 3 dias após a instilação de células, é possível começar atratamento. </li><li> Cateterize como descrito nas etapas 3.1 a 3.4. Nesta fase, não é necessário para queimar a parede da bexiga com HCl. </li><li> Usando uma seringa de 1 ml, lava-se a partir de urina residual da bexiga através da injecção de 200-300 ul de PBS estéril e aspirar o líquido a partir da bexiga. </li><li> Injectar 50 ug de HP-NAP por 150 ul de PBS para dentro da bexiga. Deixar a seringa ligado ao cateter para evitar fugas da solução de proteína. Fixe a seringa, fixando-lo à almofada com fita adesiva. </li><li> Após 1 hora, extrair suavemente o cateter da uretra e colocar os murganhos na gaiola, sob uma lâmpada incandescente, até que acordado. </li></ol> 5. tumor explantes e da análise histológica <ol><li> No final do tratamento, sacrificar os animais, colocá-los em posições supina e corrigir as patas traseiras para a almofada com fita adesiva. </li><li> Usando um bisturi cirúrgico, fazer uma incisão vertical sobre a 1,52 centímetros de comprimento através da pele e doperitônio, começando logo acima genitália externa para o &quot;fim cabeça&quot; dos ratinhos. Para minimizar o risco de danificar a bexiga, preste especial atenção para a profundidade do corte. </li><li> A bexiga será posicionado no centro-esquerda do corte e do tumor será visível como uma massa rosa / violeta escuro. Após a sua excisão, colocar toda a bexiga numa gaiola histológica e fixar em 10% de formalina tamponada antes de ser embutido em parafina. </li><li> Para análise histológica e histoquimica imune, preparar secções em série da bexiga (46 uM de espessura) usando um micrótomo padrão. </li><li> Corar as secções com hematoxilina / eosina e prosseguir com o cálculo do tamanho do tumor (D xd 2) e determinar a porcentagem de necrose por área transversal usando um computador assistida analisador de imagem. </li><li> Para a contagem navio, proceder por coloração das secções para o marcador endotelial CD31 / PECAM, usando uma técnica de imunoperoxidase padrão. Digitalizar as seções umt baixa potência (4 x) para identificar a área de maior densidade vascular. Dentro desta região, contar microvasos individuais em cinco campos aleatórios separados em alta potência (20 ×). </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

A maioria dos avanços na terapia do cancro requerem testes em modelos animais antes de começar testes clínicos. A possibilidade de estudar a biologia do tumor in vivo, aproveitando-se de modelos animais, representa uma ferramenta crucial para os pesquisadores que investigam patogênese do câncer, permitindo a avaliação de diferentes abordagens terapêuticas. Modelos ortotópicos sendo o padrão-ouro 14-15, tanto por causa da enorme quantidade de linhas celulares disponíveis para configurar um ...

O câncer de bexiga é um dos cânceres mais comuns do trato urogenital, com cerca de 75.000 novos casos por ano nos EUA 1. As altas taxas de recorrência ao longo da vida exigem acompanhamento, o que torna o câncer de bexiga um dos cancros mais caras para tratar. O tratamento padrão ouro para o alto grau NMIBC é a ressecção trans-uretral, seguida de imunoterapia intravesical com BCG. Embora o mecanismo exacto da actividade anti-tumoral de BCG continua a ser totalmente elucidado, admite-se que a activação de uma resposta imune mediada por células enriquecida em células T auxiliares (Th) e um T citotóxicas (Tc) um células é crucial para o sucesso da terapia de dois. Apesar do fato que a BCG continua a ser o tratamento de escolha para NMIBC, uma elevada proporção de pacientes não responde à terapia; além disso, pode provocar um número de efeitos secundários: cerca de 70% dos tumores tratados recorrer após algum tempo e ~ 15% para a forma de progresso músculo-invasiva dadoença. Outros efeitos secundários associados com o tratamento com BCG incluem disúria, cistite e infecção renal 3-6. O desenvolvimento de novas estratégias terapêuticas deve levar em consideração a utilização de modelos pré-clínicos, seguindo in vitro de avaliação inicial; isto é particularmente relevante em tumores cuja microambiente pode influenciar significativamente o seu desenvolvimento e capacidade de resposta ao tratamento. Ao longo da última década, têm abordado vários aspectos da actividade de modulação imunitária de HP-PNA, uma proteína produzida pela bactéria Helicobacter pylori, inicialmente identificados como capazes de promover a adesão endotelial de células polimorfonucleares (PMN) 7. Estruturalmente, HP-NAP pertence à proteína-protegendo o DNA sob condições de privação (DPS) da família 8 e é composto por 12 subunidades idênticas dispostas em um shell dodecameric. Nós demonstramos que a HP-NAP éum receptor de tipo Toll (TLR) 2 agonista, com uma forte actividade de imuno-regulação responsáveis ​​por dirigir a diferenciação de linfócitos T no sentido do fenótipo Th1, tanto in vitro como in vivo 9-10. Em virtude desta actividade, HP-NAP é capaz de redirecionar a resposta imune Th2 na resposta mais benéfico Th1 em um modelo murino de asma alérgica 11. Para avaliar o potencial anti-tumoral Th1-dependente de HP-NAP, aproveitamos um mouse modelo de cancro da bexiga desenvolvido há vários anos por O&#39;Donnel e colegas 12 para avaliar o impacto da vacina BCG. Com este protocolo, demonstramos que a HP-NAP tem um potencial anti-tumoral forte contra o cancro da bexiga e que a eficácia da administração de HP-NAP é paralela à acumulação significativa, dentro de nós tumorais e linfonodos regionais, de ambos os linfócitos Th1 e Tc1 produtoras de Interferon ( IFN) -γ 13 </sup&gt;. Os tumores isolados de camundongos tratados com HP-NAP mostrou mais de necrose e menor vascularização do que a contrapartida não tratada. O presente relatório proporciona um protocolo stepbystep, detalhando a preparação dos animais e dos respectivos cateterismo uretral, a queima química necessária para a fixação de células à mucosa da bexiga, e a injecção das células tumorais. Nós também descrevem a administração tópica de HP-PNA que pode ser considerada como um protótipo de quaisquer agentes terapêuticos desenvolvidos para o tratamento de cancro da bexiga. A prova obtida através da comparação de tumores isolados de controle e-tratados com NAP HP animais enfatiza não só o fato de que a HP-NAP pode ser um bom candidato para imunoterapia do cancro de bexiga, mas também a eficácia geral da montagem experimental.

<table><tbody><tr><td colspan="4">Materials</td></tr><tr><td>C57BL/6J female mice</td><td>Harlan Italy (Udine, Italy)</td><td></td><td></td></tr><tr><td>MB49 Cells</td><td></td><td></td><td>Obtained from Prof. O&#039;Donnel, University of Iowa Carver College of Medicine, Iowa, USA</td></tr><tr><td>RPMI</td><td>Sigma-Aldrich (St. Louis, Missouri, USA)</td><td>R8758</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D1408</td><td>10X, to be diluted in apyrogenic water</td></tr><tr><td>Flask</td><td>Becton Dickinson (Franklin Lakes, New Jersey, USA)</td><td>353135</td><td></td></tr><tr><td>Syringe 1ml</td><td>Becton Dickinson</td><td>301358</td><td></td></tr><tr><td>Trypsin</td><td>Life Technologies (Waltham, Massachusetts, USA)</td><td></td><td></td></tr><tr><td>Gentamycin</td><td>Life Technologies</td><td>15710-049</td><td></td></tr><tr><td>Xilor</td><td>Bio 98 s.r.l. (Milano, Italy)</td><td></td><td>2% Xylazin</td></tr><tr><td>Zoletil</td><td>Virbac (Carros, France)</td><td>359713301992</td><td>5% Zolazepam + 5% Tiletamine</td></tr><tr><td>24G Catheter</td><td>Terumo (Rome, Italy)</td><td>SR+DM2419PX</td><td></td></tr><tr><td>HCl</td><td>Carlo Erba Reagents (Milano, Italy)</td><td>403871</td><td>Liquid</td></tr><tr><td>NaOH</td><td>JT Baker (Center Valley, Pennsylvania, USA)</td><td>10095011</td><td>Powder</td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>Surgical Scalpel</td><td>Albion Surgical Limited (Sheffield, England)</td><td></td><td></td></tr><tr><td>Microtome</td><td>Leica Microsystem ( Wetzlar, Germany)</td><td>RM2235</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR International (Radnor, Pennsylvania, USA)</td><td>631-0108</td><td></td></tr><tr><td>Image Analyzer</td><td>Zeiss (Jena, Germany)</td><td>Cyres System</td><td></td></tr></tbody></table>

evaluation of the efficacy of the h. pylori protein hp-nap as a therapeutic tool for treatment of bladder cancer in an orthotopic murine model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

A Figura 1 mostra a técnica de cateterização; o mouse é cateterizada e instilada com 0,5 × 10 6 células MB49. O tratamento com HP-NAP foi iniciado 3 dias após a injecção de células tumorais, para lhes permitir fixar à parede da bexiga e proliferar. Todos os animais que pertencem ao grupo de controlo desenvolver o tumor; alguns deles podem morrer, antes do final do período de 13 dias, devido à oclusão da uretra. Após a primeira injecção com HP-PNA,...

Watch this Scientific Journal Video about Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model at JoVE.com

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Avaliação da Eficácia da<em&gt; H. pylori</em&gt; Protein HP-NAP como uma ferramenta terapêutica para o tratamento de cancro da bexiga em um Modelo murino Orthotopic

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

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Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

7.2K visualizações.  University of Padua. Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Vídeo: Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

Um modelo de tumor de murino de bexiga ortotópico e Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Pesquisa do câncer

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Injeção do mouse parede da bexiga

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Medicina

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Um modelo ortotópico de câncer de bexiga murino

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Um modelo de tumor de bexiga ortotópico e Avaliação de Tratamento de sarna intravesical

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Um modelo de cancro da bexiga para Ortotópico Gene Estudos de entrega

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Indução de carcinoma da bexiga invasivo de células transicionais em imunológico intacto Humanos MUC1 Ratos transgênicos: um modelo de desenvolvimento Imunoterapia

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Minimamente Invasiva Estabelecimento de murino ortotópico bexiga Xenoenxertos

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Um passo de purificação cromatográfica de Negativo<em&gt; Helicobacter pylori</em&gt; Neutrófilos-proteína de activação sobre-expresso em<em&gt; Escherichia coli</em&gt; Em Modo Batch

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Imunologia e Infecção

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

Estabelecendo um modelo de câncer de bexiga superficial murino por meio de uma técnica de administração de células intravesical

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Evaluating the Anti-Tumor Efficacy of a Bacterial Immunomodulatory Protein in a Murine Model

Source: Codolo, G. et al. <a href="https://app.jove.com/v/52743">Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates a technique to assess the anti-tumor activity of Helicobacter pylori neutrophil-activating protein, or HP-NAP, in a mouse bladder cancer model. HP-NAP interacts with tumor-cell neutrophils to produce pro-inflammatory cytokines, inducing the differentiation of na&#239;ve T cells into type 1 T helper (Th1) and type 1 cytotoxic (Tc1) T cell phenotypes. The Th1 and Tc1 cells secrete interferon-gamma, inhibiting tumor angiogenesis and promoting cytotoxic T cell-mediated tumor destruction, reducing tumor volume.

Avaliação da eficácia antitumoral de uma proteína imunomoduladora bacteriana em um modelo murino

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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