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
Duration: 6 min 12 s
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 trabajo fue apoyado por Associazione Italiana per la Ricerca sul Cancro, el Ministerio italiano de la Universidad e Investigación, proyectos Prin y Progetti di Ricerca di Ateneo, conceder N ° CPDA137871, Fondazione Cariplo, concede N ° 2011 hasta 0485 a los BMD, y por Finanziamento Giovani studiosi, Universidad de Padua, a Gaia Codolo.

Todos los procedimientos de manipulación de los animales han sido aprobados por el Ministerio italiano de Salud (204/2011-B DM). 1. Los animales <ol><li> Crecer ratones hembras C57BL6 / J a 8 semanas de edad en jaulas ventiladas individualmente con filtros microaislamiento. Se prefieren las hembras porque la conformación anatómica de su aparato uro-genital externa hace que el cateterismo más fácil que en los hombres: Nota. </li></ol> 2. Cultivo Celular <ol><li> Mantener la urotelial ratón línea celular de carcinoma MB49 en medio RPMI 1640 suplementado con 10% de suero bovino fetal inactivado por calor y 50 mg / ml de gentamicina a 37 ° C en humidificado 5% CO 2. </li><li> Crecer células MB49 en matraces T-75 a 80% de confluencia. Trypsinize y resuspender en el 0,5 × 10 5 a 0,5 × 10 6 células por 150 l de PBS antes de la implantación en ratones. </li></ol> 3. intravesical tumor Implante <ol><li> Anestesiar ratones utilizando una mezcla de la Zoletil anestésicos y xylor (33 mg / kg y 20 mg / kg, respectivamente), inyectado por vía intraperitoneal. </li><li> Coloque los animales en posición supina y fijar las patas traseras a la plataforma con cinta scotch. Patas tienen que ser separados suficientemente para hacer que el meato uretral bien visible y fácilmente accesible para la inserción del catéter. </li><li> Nota: Para el cateterismo, un catéter venoso pediátrico 24 G es adecuado para ratones 8 a 10 semanas de edad. La elección del catéter es muy importante con el fin de evitar fugas de líquido entre la pared uretral y el catéter. Catéteres de calibre más grandes deben ser utilizados para los ratones más grandes o más. </li><li> Utilizando una pequeña pinza, apriete uno de los bordes de la parte externa de la uretra y girar suavemente hacia la &quot;cabecera&quot; de los ratones. Esta acción expone el meato uretral. </li><li> Retire la aguja interna del catéter e insertar este último en la uretra en un ángulo de 45 °, orientado ligeramentehacia la &quot;cola&quot; de los ratones. No fuerce la entrada del catéter; en caso de resistencia, simplemente gire el catéter con mucha suavidad, hasta que se desliza dentro de la uretra. Cuando aproximadamente 0,5-0,8 cm del catéter es en la uretra, colocar cuidadosamente el catéter paralelo a la cola de los ratones y se inserte completamente. </li><li> Usando una jeringa de 1 ml, la aguja de los cuales tiene que ser insertado en el catéter, lavar la vejiga de la orina residual mediante la inyección de 200-300 l de PBS estéril y aspirar todo el líquido de la vejiga. </li><li> Nota: Cuando la inyección de PBS, la vejiga se llenará y una hinchazón será visible entre las patas traseras de los ratones; esto confirma que el cateterismo es la correcta. La mayoría de los catéteres tienen un volumen muerto que debe ser considerado en el cálculo del volumen de inyección. </li><li> Usando una jeringa de 1 ml estéril, inyectar 200 l de HCl 0,1 N. Llene la vejiga para quemar toda su pared. Después de 10 segundos, retire todo el ácido y neutrali inmediatamenteze mediante la inyección de 200 l de NaOH 0,1 N con una jeringa estéril. En el caso de la inyección del mayor número de células (0,5 × 10 6), no se requiere la etapa de acidificación. </li><li> Aspire todo el líquido residual e inmediatamente echar la cavidad con 300 l de PBS estéril. </li><li> Vaciar la vejiga por aspiración, e inyectar 150 l de PBS que contienen células MB49 (rango de 0,5 × 10 5 a 0,5 × 10 6) en la vejiga. Deje la jeringa montada en el catéter para evitar la fuga de células. Es mejor para asegurar la jeringa fijándolo a la plataforma con cinta scotch. </li><li> Después de 1 hora, extraer suavemente el catéter de la uretra, y colocar los ratones en la jaula, bajo una lámpara incandescente hasta que despiertan: esto ocurre generalmente entre 1 y 2 horas después del final del tratamiento. </li></ol> 4. Entrega intravesical de HP-PAN <ol><li> Nota: Por lo menos 3 días después de la instilación de células, es posible comenzar latratamiento. </li><li> Cateterizarse como se describe en los pasos 3.1 a 3.4. En esta etapa, no es necesario para quemar la pared de la vejiga con HCl. </li><li> Usando una jeringa de 1 ml, lavar la vejiga de la orina residual mediante la inyección de 200-300 l de PBS estéril y aspirar todo el líquido de la vejiga. </li><li> Inyectar 50 g de HP-PAN por 150 l de PBS en la vejiga. Deje la jeringa conectada al catéter para evitar la fuga de la solución de proteína. Asegure la jeringa fijándolo a la plataforma con cinta scotch. </li><li> Después de 1 hora, extraer suavemente el catéter de la uretra y colocar los ratones en la jaula, bajo una lámpara incandescente, hasta que se despiertan. </li></ol> 5. tumor explantes y el análisis histológico <ol><li> Al final del tratamiento, sacrificar a los animales, colocarlos en posición supina y fijar las patas traseras a la plataforma con cinta scotch. </li><li> El uso de un bisturí quirúrgico, hacer una incisión vertical sobre 1,52 cm de largo a través de la piel y elperitoneo, empezando justo por encima de los genitales externos de la &quot;cabecera&quot; de los ratones. Para minimizar el riesgo de dañar la vejiga, prestar especial atención a la profundidad del corte. </li><li> La vejiga se colocará en el centro-izquierda de la corte y el tumor será visible como una masa de color rosa / violeta oscuro. Tras su escisión, coloque toda la vejiga en una jaula histológico y fijar en el 10% formalina tamponada antes de la incrustación en parafina. </li><li> Para análisis histológico e histoquímico inmune, preparar secciones en serie de la vejiga (46 micras de espesor) usando un microtomo estándar. </li><li> Tinción de las secciones con hematoxilina / eosina y proceder con el cálculo del tamaño del tumor (D xd 2) y determinar el porcentaje de necrosis por área de sección transversal usando un analizador de imagen asistida por ordenador. </li><li> Para el conteo buque, proceder por la tinción de las secciones para el marcador endotelial CD31 / PECAM, utilizando una técnica de inmunoperoxidasa estándar. Analiza las secciones de unt baja potencia (4 ×) para identificar el área de mayor densidad vascular. Dentro de esta región, cuente microvasos individuales en cinco campos aleatorios independientes a alta potencia (20 ×). </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

La mayoría de los avances en el tratamiento del cáncer requiere pruebas en modelos animales antes de comenzar los ensayos clínicos. La posibilidad de estudiar la biología del tumor in vivo, aprovechando modelos animales, representa una herramienta fundamental para los investigadores que investigan la patogénesis del cáncer, lo que permite la evaluación de los diferentes enfoques terapéuticos. Modelos ortotópico siguen siendo el estándar de oro 14-15, tanto debido a la enorme cantidad de lí...

El cáncer de vejiga es uno de los cánceres más comunes del tracto urogenital, con cerca de 75.000 nuevos casos cada año en los EE.UU. 1. Las altas tasas de recurrencia requieren seguimiento de por vida, lo que hace que el cáncer de vejiga uno de los cánceres más costosas de tratar. El tratamiento estándar de oro para el alto grado CVNMI es la resección transuretral, seguida de inmunoterapia intravesical con BCG. Aunque el mecanismo exacto de la actividad anti-tumor de BCG aún no se ha aclarado completamente, se acepta que la activación de una respuesta inmune mediada por células enriquecido en T helper (Th) 1 y T citotóxicas (Tc) de las células 1 es crucial para el éxito de la terapia 2. A pesar de que la BCG sigue siendo el tratamiento de elección para CVNMI, una alta proporción de los pacientes no responden a la terapia; por otra parte, puede causar una serie de efectos secundarios: alrededor del 70% de los tumores tratados se repita después de algún tiempo y ~ 15% de avance a la forma del músculo invasivo de laenfermedad. Otros efectos secundarios asociados con el tratamiento con BCG incluyen disuria, cistitis y la infección renal 3-6. El desarrollo de nuevas estrategias terapéuticas debe tomar en consideración el uso de modelos preclínicos, tras la evaluación inicial in vitro; esto es particularmente relevante en tumores cuya microambiente puede influir significativamente en su desarrollo y la capacidad de respuesta al tratamiento. Durante la última década, hemos abordado varios aspectos de la actividad inmunomoduladora de HP-PAN, una proteína producida por la bacteria Helicobacter pylori, identificado inicialmente como capaz de promover la adhesión endotelial de las células polimorfonucleares (PMN) 7. Estructuralmente, HP-PAN pertenece a la proteína DNA-protector en condiciones de hambre (AD) de la familia 8 y consta de 12 subunidades idénticas dispuestas en una cáscara dodecameric. Hemos demostrado que HP-NAP esun receptor Toll-like (TLR) 2 agonista, con una fuerte actividad inmuno-modulación responsable de impulsar la diferenciación de los linfocitos T hacia el fenotipo Th1, tanto in vitro como in vivo 9-10. En virtud de esta actividad, HP-PAN es capaz de redirigir la respuesta inmune Th2 en la respuesta más beneficiosa Th1 en un modelo murino de asma alérgica 11. Para evaluar el potencial antitumoral Th1 dependiente de HP-PAN, nos aprovechamos de un modelo de ratón de cáncer de vejiga desarrollado hace varios años por O&#39;Donnel y sus colegas 12 para evaluar el impacto de la administración de BCG. Con este protocolo, nos demuestran que HP-PAN tiene un fuerte potencial antitumoral contra el cáncer de vejiga y que la eficacia de la administración de HP-PAN es paralela a la acumulación significativa, dentro del tumor y los ganglios linfáticos regionales, tanto de Th1 y Tc1 linfocitos que producen interferón ( IFN) -γ 13 </sup&gt;. Los tumores aislados a partir de ratones tratados con HP-NAP mostraron necrosis más y menos vascularización que la contraparte no tratada. El presente informe proporciona un protocolo stepbystep, que detalla la preparación de los animales, su cateterismo uretral, la quema químico requerido para la unión de las células a la mucosa de la vejiga, y la inyección de las células tumorales. También describimos la administración tópica de HP-NAP que se puede considerar como un prototipo de cualesquiera agentes terapéuticos desarrollados para el tratamiento de cáncer de vejiga. La evidencia obtenida mediante la comparación de los tumores aislados de control y HP-PAN-animales tratados enfatiza no sólo el hecho de que HP-PAN podría ser un buen candidato para la inmunoterapia del cáncer de vejiga, sino también la eficacia general del montaje 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.

La Figura 1 muestra la técnica de cateterismo; el ratón se cateterizó y inculca con 0,5 × 10 6 células MB49. El tratamiento con HP-NAP se inicia 3 días después de la inyección de las células tumorales, para que puedan adherirse a la pared de la vejiga y proliferar. Todos los animales pertenecientes al grupo de control se desarrollan el tumor; algunos de ellos pueden morir antes del final del período de 13 días debido a la oclusión de la uretra. Despué...

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.

Evaluación de la eficacia de la<em&gt; H. pylori</em&gt; Proteína HP-PAN como herramienta terapéutica para el tratamiento de cáncer de vejiga en un modelo murino ortotópico

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 vistas.  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.

Video: 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.

Un Modelo de tumor y el tumor murino ortotópico de vejiga Sistema de Detección

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 ...

Investigación

Investigación sobre el cáncer

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.

Vejiga ratón pared de inyección

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

Medicina

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

Evaluación de la proyección de imagen de resonancia magnética de los tumores vesicales murina inducida por carcinógenos

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

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.

Un modelo de tumor vesical ortotópica y la Evaluación de tratamiento intravesical SARNA

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.

Un modelo ortotópico de cáncer de vejiga de Estudios de liberación de genes

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

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Evaluación de la actividad antitumoral in vivo de nanopartículas de polianhídrido IL-1α

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

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.

La inducción de la transición de vejiga invasivo Carcinoma en inmune intacto humanos MUC1 Ratones Transgénicos: Un Modelo para el Desarrollo Inmunoterapia

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

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.

De un solo paso negativo Purificación cromatográfica de<em&gt; Helicobacter pylori</em&gt; La proteína activadora de neutrófilos sobreexpresa en<em&gt; Escherichia coli</em&gt; En 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. ...

Inmunología e Infección

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.

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

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Modelos de ratón de la infección por Helicobacter y patologías gástricas

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Evaluación de la eficacia de la

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