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
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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

Ce travail a été soutenu par l&#39;Associazione Italiana per la Ricerca sul Cancro, Ministère italien de l&#39;université et de la recherche, les projets et Prin Progetti di Ricerca di Ateneo, accorder N ° CPDA137871, Fondazione Cariplo, accorder N ° 2011-0485 à MdB, et par Finanziamento Giovani Studiosi, Université de Padoue, à Gaia Codolo.

Toutes les procédures de manipulation des animaux ont été approuvés par le ministère italien de la Santé (DM 204/2011-B). 1. Les animaux <ol><li> Cultivez souris femelles C57BL6 / J 8 semaines d&#39;âge dans les cages individuellement ventilées avec des filtres de microisolation. Note: Les femelles sont préférés car la conformation anatomique de leur appareil uro-génital externe rend le cathétérisme plus facile que chez les hommes. </li></ol> 2. Culture cellulaire <ol><li> Maintenir la lignée cellulaire de carcinome urothélial de la souris MB49 en milieu RPMI 1640 supplémenté avec 10% de sérum bovin fœtal inactivé à la chaleur et 50 ug / ml de gentamicine à 37 ° C humidifié à 5% de CO 2. </li><li> Cultiver les cellules MB49 dans des flacons T-75 à 80% de confluence. Trypsiniser et remettre en suspension à 0,5 x 10 5 à 0,5 x 10 6 cellules par 150 pi de PBS avant l&#39;implantation chez la souris. </li></ol> 3. intravésicale tumeur Implant <ol><li> Anesthésier les souris en utilisant un mélange des anesthésiques et Zoletil Xylor (33 mg / kg et 20 mg / kg, respectivement), injecté par voie intraperitoneale. </li><li> Placez les animaux dans une position couchée et fixer les pattes de derrière pour le tampon avec du scotch. Pattes doivent être suffisamment séparés pour rendre le méat urétral bien visible et facilement accessible pour l&#39;insertion du cathéter. </li><li> Remarque: Pour le cathétérisme, un cathéter veineux pédiatrique 24 G est adapté pour les souris 8 à 10 semaines-vieux. Le choix du cathéter est très important afin d&#39;éviter les fuites de liquide entre la paroi de l&#39;urètre et du cathéter. Cathéters plus larges de forage doivent être utilisés pour les souris plus grands ou plus. </li><li> En utilisant une petite pince, pincez un bord de la partie externe de l&#39;urètre et de le tordre très doucement vers la &quot;tête&quot; de la souris. Cette action expose le méat urétral. </li><li> Retirer l&#39;aiguille du cathéter interne et insérer celle-ci dans l&#39;urètre d&#39;un angle de 45 °, légèrement orientéevers la &quot;queue&quot; de la souris. Ne pas forcer l&#39;entrée du cathéter; en cas de résistance, il suffit de tourner le cathéter très doucement, jusqu&#39;à ce qu&#39;il glisse à l&#39;intérieur de l&#39;urètre. Quand environ 0,5-0,8 cm du cathéter est dans l&#39;urètre, placer soigneusement le cathéter parallèle à la queue des souris et insérez-le complètement. </li><li> En utilisant une seringue de 1 ml, l&#39;aiguille de laquelle doit être insérée dans le cathéter, laver la vessie de l&#39;urine résiduelle en injectant 200 à 300 ul de PBS stérile et aspirer tout le liquide de la vessie. </li><li> Remarque: Lors de l&#39;injection du PBS, la vessie se remplira et un gonflement sera visible entre les pattes arrières des souris; ceci confirme que le cathétérisme est correcte. La plupart des cathéters ont un volume mort qui doit être considéré dans le calcul du volume d&#39;injection. </li><li> En utilisant une seringue de 1 ml stérile, injecter 200 pi de HCl 0,1. Remplissez la vessie afin de brûler toute sa paroi. Après 10 sec, retirer tout l&#39;acide et neutrali immédiatementze par injection de 200 ul de NaOH 0,1 N avec une seringue stérile. En cas d&#39;injection du plus grand nombre de cellules (0,5 x 10 6), l&#39;étape d&#39;acidification est pas nécessaire. </li><li> Aspirer tout le liquide résiduel et rincer immédiatement la cavité avec 300 pi de PBS stérile. </li><li> Vider la vessie par aspiration, et injecter 150 pi de PBS contenant des cellules MB49 (gamme 0,5 x 10 5 à 0,5 × 10 6) dans la vessie. Laisser la seringue monté sur le cathéter afin d&#39;éviter la fuite des cellules. Il est préférable de fixer la seringue en le fixant sur le tampon avec du scotch. </li><li> Après 1 heure, extrait doucement le cathéter à partir de l&#39;urètre, et de placer les souris dans la cage, sous une lampe à incandescence jusqu&#39;à leur réveil: cela se produit habituellement entre une et deux heures après la fin du traitement. </li></ol> 4. Livraison intravésicale de HP-NAP <ol><li> Remarque: Au moins 3 jours après l&#39;instillation de cellules, il est possible de commencer latraitement. </li><li> Cathétériser la manière décrite dans les étapes 3.1 à 3.4. A ce stade, il ne faut pas brûler la paroi de la vessie avec HCl. </li><li> En utilisant une seringue de 1 ml, laver la vessie de l&#39;urine résiduelle en injectant 200 à 300 ul de PBS stérile et aspirer tout le liquide de la vessie. </li><li> Injecter 50 pg de HP-NAP par 150 ul de PBS dans la vessie. Laisser la seringue fixée au cathéter pour éviter une fuite de la solution de protéine. Fixer la seringue en la fixant à la pastille avec du scotch. </li><li> Après 1 heure, extrait doucement le cathéter de l&#39;urètre et de placer les souris dans la cage, sous une lampe à incandescence, jusqu&#39;à ce qu&#39;ils se réveillent. </li></ol> 5. Tumeur explants et de l&#39;analyse histologique <ol><li> A la fin du traitement, sacrifier les animaux, les placer dans les positions couchée et fixer les pattes de derrière pour le tampon avec du scotch. </li><li> Avec un scalpel chirurgical, faire une incision verticale d&#39;environ 1,52 cm de long à travers la peau et de lapéritoine, en commençant juste au-dessus des organes génitaux externes de la &quot;tête&quot; de la souris. Pour minimiser le risque d&#39;endommager la vessie, accorder une attention particulière à la profondeur de la coupe. </li><li> La vessie sera placé dans le centre-gauche de la coupe et la tumeur sera visible comme une masse rose / violet foncé. Lors de son excision, placer toute la vessie dans une cage histologique et fixer 10% de formol tamponné avant de l&#39;intégrer dans de la paraffine. </li><li> Pour l&#39;analyse histologique et histochimique immunitaire, préparer des coupes en série de la vessie (46 um d&#39;épaisseur de) en utilisant un microtome standard. </li><li> Colorer les sections avec hématoxyline / éosine et procéder au calcul de la taille de la tumeur (D xd 2) et déterminer le pourcentage de la nécrose par zone transversale en utilisant un analyseur d&#39;image assistée par ordinateur. </li><li> Pour récipient comptage, procéder par coloration des sections pour le marqueur endothelial CD31 / PECAM, en utilisant une technique d&#39;immunoperoxydase standard. Scannez les sections d&#39;unt faible puissance (4 ×) pour identifier la zone de la densité vasculaire plus élevé. Dans cette région, compter microvaisseaux individuels dans cinq champs aléatoires distincts à haute puissance (20 ×). </li></ol>

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Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrentes.

La majorité des avancées dans le traitement du cancer nécessitent des essais sur des modèles animaux avant de commencer les essais cliniques. La possibilité d&#39;étudier la biologie des tumeurs in vivo, profitant de modèles animaux, représente un outil essentiel pour les chercheurs de l&#39;enquête pathogenèse du cancer, permettant l&#39;évaluation des différentes approches thérapeutiques. Orthotopiques modèles restent l&#39;étalon-or 14-15, à la fois en raison de l&#39;énorme quant...

Cancer de la vessie est l&#39;un des cancers les plus fréquents de l&#39;appareil génito-urinaire, avec près de 75 000 nouveaux cas chaque année aux Etats-Unis 1. Les taux élevés de récidive nécessitent tout au long du suivi, ce qui rend le cancer de la vessie l&#39;un des cancers les plus coûteuses à traiter. Le traitement de référence pour la haute qualité CVSEM est la résection trans-urétrale, suivie par immunothérapie intravésicale avec le BCG. Bien que le mécanisme précis de l&#39;activité anti-tumorale de BCG doit encore être élucidé, il est admis que l&#39;activation d&#39;une réponse immunitaire à médiation cellulaire enrichie en lymphocytes T auxiliaires (Th) et une T cytotoxiques (Tc) une des cellules est essentiel pour la réussite de la thérapie 2. Malgré le fait que le BCG reste le traitement de choix pour CVSEM, une forte proportion de patients ne répondent pas à la thérapie; En outre, il peut causer un certain nombre d&#39;effets secondaires: environ 70% des tumeurs traitées réapparaissent après un certain temps et ~ 15% progrès à la forme du muscle-invasivemaladie. D&#39;autres effets secondaires associés au traitement par le BCG comprennent dysurie, cystite et une infection des reins 3-6. Le développement de nouvelles stratégies thérapeutiques doit prendre en considération l&#39;utilisation de modèles précliniques, après évaluation initiale in vitro; ceci est particulièrement pertinent dans les tumeurs dont microenvironnement peut influencer de manière significative leur développement et leur réponse au traitement. Au cours de la dernière décennie, nous avons abordé plusieurs aspects de l&#39;activité de modulation immunitaire de HP-NAP, une protéine produite par la bactérie Helicobacter pylori, initialement identifiée comme capable de favoriser l&#39;adhésion endothéliale de polynucléaires (PMN) 7. Structurellement, HP-NAP appartient à la protéine d&#39;ADN-protecteur dans des conditions affamés (DPS) de la famille 8 et se compose de 12 sous-unités identiques disposées dans une enveloppe dodecameric. Nous avons démontré que HP-NAP estun récepteur de type Toll (TLR) 2 agoniste, avec une activité immuno-modulation fort responsable de la conduite de la différenciation des lymphocytes T vers le phénotype Th1, à la fois in vitro et in vivo 10/09. En vertu de cette activité, HP-NAP est capable de rediriger la réponse immunitaire Th2 en réponse Th1 la plus bénéfique dans un modèle murin d&#39;asthme allergique 11. Pour évaluer le potentiel anti-tumoral Th1 dépendante de HP-NAP, nous avons profité d&#39;un modèle murin de cancer de la vessie développé il ya plusieurs années par O&#39;Donnel et ses collègues 12 pour évaluer l&#39;impact de l&#39;administration du BCG. Avec ce protocole, nous démontrons que HP-NAP a un potentiel anti-tumoral puissant contre le cancer de la vessie et que l&#39;efficacité de l&#39;administration HP-NAP est parallèle à l&#39;accumulation importante, dans les noeuds de la tumeur et les ganglions lymphatiques régionaux, à la fois des lymphocytes de type Th1 et Tc1 produire de l&#39;interféron ( IFN) -γ 13 </sup&gt;. Les tumeurs isolées de souris HP-NAP-traités ont montré une nécrose plus et moins vascularisation de la contrepartie non traitée. Le présent rapport fournit un protocole de stepbystep, détaillant la préparation des animaux, leur cathétérisme urétral, la combustion chimique nécessaire pour la fixation des cellules de la muqueuse de la vessie, et l&#39;injection de cellules tumorales. Nous décrivons également l&#39;administration topique de HP-NAP qui peut être considéré comme un prototype de tous les agents thérapeutiques développées pour le traitement du cancer de la vessie. Les preuves obtenues en comparant les tumeurs isolées de contrôle et HP-NAP-animaux traités souligne non seulement le fait que HP-NAP pourrait être un bon candidat pour l&#39;immunothérapie du cancer de la vessie, mais aussi l&#39;efficacité générale de l&#39;installation expérimentale.

<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 figure 1 illustre la technique de cathétérisation; la souris est sondé et inculqué avec 0,5 × 10 6 cellules MB49. Le traitement avec HP-NAP est démarré 3 jours après l&#39;injection des cellules tumorales, pour leur permettre de fixer sur la paroi de la vessie et proliférer. Tous les animaux appartenant au groupe de contrôle développer la tumeur; certains d&#39;entre eux peuvent mourir avant la fin de la période de 13 jours en raison de l&#39;occlusion de l&#39;urètre. <p ...

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.

Évaluation de l&#39;efficacité de la<em&gt; H. pylori</em&gt; Protéines HP-NAP comme outil thérapeutique pour le traitement du cancer de la vessie dans un modèle murin orthotopique

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 vues.  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.

Vidéo: 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 système de détection de tumeur modèle et tumeur murine Orthotopique vessie

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 ...

Recherche

Recherche en cancérologie

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.

Injection mur souris vessie

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

Médecine

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.

Évaluation de l’imagerie par résonance magnétique des tumeurs de vessie Murine induite par la substance cancérigène

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 modèle de la vessie orthotopique tumorale et l&#39;évaluation de Sarna traitement intravésicale

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 modèle de cancer de la vessie orthotopique d&#39;études livraison Gene

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.

Evaluation de l’activité antitumorale in vivo des nanoparticules de polyanhydride 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.

Induction du envahissantes Transitional cancer de la vessie cellulaire dans immunitaire humain MUC1 souris transgéniques intactes: un modèle pour le développement d&#39;immunothérapie

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.

Une étape négative de purification chromatographique<em&gt; Helicobacter pylori</em&gt; Activation des neutrophiles protéine surexprimée dans<em&gt; Escherichia coli</em&gt; En mode 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. ...

Immunologie et infection

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.

Modèles de souris de l’Infection à Helicobacter et Pathologies gastriques

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 ...

Évaluation de l'efficacité de la

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Chapitres dans cette vidéo

Un système de détection de tumeur modèle et tumeur murine Orthotopique vessie

Injection mur souris vessie

Évaluation de l’imagerie par résonance magnétique des tumeurs de vessie Murine induite par la substance cancérigène

Un modèle de la vessie orthotopique tumorale et l'évaluation de Sarna traitement intravésicale

Un modèle de cancer de la vessie orthotopique d'études livraison Gene

Evaluation de l’activité antitumorale in vivo des nanoparticules de polyanhydride IL-1α

Induction du envahissantes Transitional cancer de la vessie cellulaire dans immunitaire humain MUC1 souris transgéniques intactes: un modèle pour le développement d'immunothérapie

Une étape négative de purification chromatographique

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

Modèles de souris de l’Infection à Helicobacter et Pathologies gastriques