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

Bu eser Associazione Italiana per la Ricerca sul Yengeç, Üniversite İtalyan Bakanlığı ve Araştırma İlkeleri projeleri ve Progetti di Ricerca di Ateneo, hibe N ° CPDA137871, Fondazione Cariplo tarafından desteklenen N MdB için 2011-0485 ° ve Finanziamento Giovani tarafından hibe Gaia Codolo için Studiosi, Padua Üniversitesi,.

Hayvan taşıma için tüm işlemler İtalyan Sağlık Bakanlığı (DM 204/2011-B) tarafından onaylanmıştır. 1. Hayvanlar <ol><li> Microisolation filtreleri ile ayrı ayrı havalandırmalı kafeslerde yaşı 8 hafta C57BL6 / J dişi fareler büyütün. Not: Onların dış ürogenital aygıtının anatomik konformasyon erkeklerde daha kateterizasyon kolay işler çünkü dişiler tercih edilir. </li></ol> 2. Hücre Kültürü <ol><li> Nemlendirilmiş bir% 5 CO2 içinde 37 ° C&#39;de,% 10 ısı ile inaktive edilmiş fetal büyükbaş hayvan serumu ve 50 ug / ml gentamisin ile takviye edilmiş RPMI 1640 ortamı içinde, fare ürotelyal karsinoma hücre hattı MB49 koruyun. </li><li> % 80 izdiham T-75 şişelerinde MB49 hücreleri büyütün. Trypsinize ve 0.5 × 10 5 de tekrar süspansiyon - farelerde implantasyon öncesi PBS 150 ul başına 0,5 x 10 6 hücre. </li></ol> 3. İntravezikal Tümör İmplant <ol><li> (Sırasıyla, 33 mg / kg ve 20 mg / kg), anestetikler zoletil ve xylor bir karışımını kullanarak fareler anestezi, karın içinden enjekte edilmiştir. </li><li> Bir yatar pozisyonda hayvanlar koyun ve kapaklar ile ped arka ayakları düzeltmek. Pençeler yeterince iyi görülebilir ve kateterin yerleştirilmesi için kolaylıkla erişilebilir üretral meatusun işlemek için ayrılması var. </li><li> Not: kateterizasyon, bir 24 G kateter çocuk 8-10 haftalık fareler için uygundur. Kateterin bir seçim üretral duvar ve kateter arasındaki sıvı sızmasını önlemek için çok önemlidir. Büyük kalibreli kateterler daha büyük veya daha büyük fareler için kullanılmalıdır. </li><li> Küçük bir cımbız kullanarak, üretranın dış kısmının bir kenarı çimdik ve farelerin &quot;baş ucuna&quot; doğru çok hafifçe bükün. Bu eylem üretral meatus ortaya çıkarır. </li><li> 45 ° &#39;lik bir açı ile üretra ikinci kateterin iç iğne çıkarın ve ekleme, hafif yönlendirilmişFarelerin &quot;kuyruk sonunda&quot; doğru. Kateterin giriş zorlamayın; Bu üretranın içine fişleri kadar direnç durumunda, sadece, çok yavaşça kateter bükün. Kateterin yaklaşık 0.5-0.8 cm idrar olduğunda, dikkatli bir şekilde farelerin kuyruk kateter paralel yer tamamen yerleştirin. </li><li> Kateter takılı olması gereken bir iğne olan bir 1 ml şırınga kullanarak, steril PBS 200-300 ul enjekte edilerek kalan idrar mesane yıkama ve mesane tüm sıvı aspire. </li><li> Not: PBS enjekte ederken, mesane dolduracaktır ve bir şişme farelerin arka bacaklarının arasına görünür olacaktır; Bu kateterizasyon doğru olduğunu teyit etmektedir. Çoğu kateterler enjeksiyon hacmi hesaplanmasında dikkate alınması gereken bir ölü hacme sahip. </li><li> 1 ml steril bir şırınga kullanılarak, 0.1 N HCI, 200 ul enjekte edilir. Onun tüm duvar yakmak için mesaneyi doldurun. 10 saniye sonra, her asidi çıkarmak hemen neutralisteril bir şırınga ile 0.1 N NaOH, 200 ul enjekte edilerek ze. En yüksek hücre sayısı (0.5 x 10 6) enjeksiyonu durumunda, asitleme aşaması gerekli değildir. </li><li> Aspire tüm artık sıvı hemen steril PBS içinde 300 ul boşluğu yıkayın. </li><li> Mesane içine - (0.5 x 10 6 aralık 0.5 × 10 5) aspirasyonu ile mesaneyi boşaltın ve MB49 hücreleri içeren PBS 150 ul enjekte edilir. Hücrelerin sızmasını önlemek için kateter monte şırınga bırakın. Bu kapaklar ile pad sabitlemek şırıngayı sabitlemek için iyidir. </li><li> 1 saat sonra, yavaşça üretradan kateter ayıklamak ve onlar uyanık kadar bir akkor lambası altında, kafes içinde fareler yerleştirin: bu genellikle tedavinin bitiminden 1 ila 2 saat oluşur. </li></ol> HP-NAP 4. ntravezikal Teslimat <ol><li> Not: Hücre uygulamasından sonra en az 3 gün, bu başlamak mümkündürTedavi. </li><li> Adımlar 3,1-3,4 açıklandığı gibi kateterize. Bu aşamada, bu HCI ile mesane duvarı yakmak için gerekli değildir. </li><li> 1 ml şırınga kullanarak, steril PBS 200-300 ul enjekte edilerek kalan idrar mesane yıkama ve mesane tüm sıvı aspire. </li><li> Mesane içine 150 ul PBS başına HP NAP 50 ug enjekte edilir. Protein çözeltisi akıntısını önlemek için kateter takılı şırınga bırakın. Kapaklar ile pad sabitlemek şırıngayı sabitleyin. </li><li> Onlar uyanık kadar bir akkor lambası altında, 1 saat sonra, yavaşça üretradan kateter ayıklamak ve kafese fareler yerleştirin. </li></ol> 5. Tümör Eksplantlarındaki ve Histolojik Analiz <ol><li> Tedavi sonunda, hayvanlar kurban yatar pozisyonda yerleştirin ve kapaklar ile ped arka ayakları düzeltmek. </li><li> Cerrahi neşter kullanılarak, cilt ve içinden yaklaşık 1.52 cm uzunluğunda dikey bir kesi yapmakPeriton, sadece fareler &quot;baş ucuna&quot; dış genital üzerinde başlayan. Mesane zarar riskini en aza indirmek için, kesme derinliği özellikle dikkat. </li><li> Mesane kesim merkez sol yerleştirilmiş olacak ve tümör koyu pembe / mor kitle olarak görülebilir. Kesildikten sonra, histolojik kafese Bütün mesane yerleştirin ve parafin içine yerleştirilmeden önce,% 10 tamponlu formalin içinde sabitlenir. </li><li> Histolojik ve immün histokimyasal analizi için, standart bir mikrotom kullanılarak mesane (46 um kalınlığında) seri kesitler hazırlamak. </li><li> Hematoksilin / eozin ile bölümleri Leke ve tümör büyüklüğü (D xd 2) ve bir bilgisayar destekli görüntü analiz cihazı kullanarak enine kesit alanı başına nekroz oranını belirlemek hesaplama ile devam eder. </li><li> Damar sayımı, standart bir immünoperoksidaz tekniği kullanılarak, endotelyal işaretleyici CD31 / PECAM için bölümleri boyama ile devam edin. Bölümleri tara BirEn yüksek vasküler yoğunluğu bölgeyi tanımlamak için düşük güç (4 ×) t. Bu Bölgede, yüksek güç (20 ×) beş ayrı rastgele alanlarda bireysel mikrodamarlar saymak. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarını olduğunu beyan ederim.

kanser tedavisinde ilerlemelere çoğunluğu klinik denemeler başlamadan önce hayvan modellerinde test gerektirir. in vivo tümör biyolojisi okuyan hayvan modellerinde yararlanarak olasılığı, farklı tedavi yaklaşımları değerlendirmesini sağlayan kanser patogenezinde araştıran araştırmacılar için önemli bir araç temsil eder. Ortotopik modeller altın standart 14-15 kalır, hem de çünkü tümör modeli kurmak ve mevcut hücre hatlarının büyük miktarda doğal olarak oluşan t?...

Mesane kanseri ABD&#39;de 1 yaklaşık 75.000 yeni vaka her yıl, ürogenital sistem en yaygın kanserlerden biridir. Nüks oranlarının yüksek mesane kanseri tedavisinde costliest kanserlerin biri yapar ömür boyu takip gerektirir. yüksek dereceli NMIBC için altın standart tedavi BCG intravezikal immünoterapi ardından, trans-üretral rezeksiyon olduğunu. BCG, anti-tümör aktivitesi kesin mekanizması tam olarak aydınlatılamamıştır olmasına rağmen, bu T yardımcı (Th) 1 ve sitotoksik T (Tc), 1 hücreleri, zenginleştirilmiş bir hücre-aracılı bağışıklık karşılığının harekete geçirmek için çok önemli olduğu kabul edilmektedir tedavinin 2. başarısı. BCG NMIBC için seçilecek tedavi olmaya devam olmasına rağmen, tedaviye yanıt vermeyen hastalarda yüksek oranda; Ayrıca, yan etkilerin bir neden olabilir: tedavi edilen tümörlerin yaklaşık% 70 kas-invaziv forma ~% 15 ilerleme bir süre sonra nüksetme vehastalığı. BCG tedavisi ile ilişkili diğer yan etkiler dizüri, sistit ve böbrek enfeksiyonu 3-6 arasındadır. yeni tedavi stratejilerinin geliştirilmesi, ilk in vitro değerlendirmenin ardından, dikkate klinik öncesi modellerinin kullanımını almalıdır; Bu kimin mikroçevresinin ölçüde tedaviye gelişimlerini ve yanıt etkileyebilecek tümörlerde özellikle önemlidir. Son on yılda, biz, HP-NAP, bakteri Helicobacter pylori tarafından üretilen bir proteinin bağışıklık modüle aktivitesinin birden yönlerini ele alınmıştır, başlangıçta polimorfonükleer hücreler (PMN) 7 endotel yapışmasını teşvik yeteneğine olarak belirledi. Yapısal olarak, HP-NAP hasret koşulları (DPS) ailesi 8 altında DNA koruyucu protein aittir ve bir dodecameric kabuk düzenlenmiş 12 özdeş alt birimden oluşmaktadır. Biz, HP-NAP olduğunu göstermiştirT farklılaşmasını tahrik sorumlu güçlü bir immüno-modüle edici aktivitesi olan bir Toll benzeri reseptör (TLR) 2 agonisti, in vitro ve in vivo 9-10 hem de TH1 fenotipine doğru lenfosit. Bu aktivite sayesinde HP, NAP-alerjik astım 11 bir murin modelinde daha faydalı Th1 yanıt olarak Th2 immun cevabı yönlendirmek edebilmektedir. HP-NAP Th1 bağımlı anti-tümör potansiyelini değerlendirmek için, BCG yönetiminin etkisini değerlendirmek için O&#39;Donnel ve arkadaşları 12 tarafından birkaç yıl önce geliştirilen mesane kanseri bir fare modelinde yararlandı. Bu protokol ile, HP-NAP mesane kanserine karşı güçlü bir anti-tümör potansiyele sahip olduğunu ve HP-NAP yönetiminin etkinliği (interferon üreten hem Th1 ve Tc1 lenfositlerin, tümör ve bölgesel lenf düğümleri içinde önemli birikimi ile paralellik göstermektedir IFN) 13 -γ </&gt; sup. HP NAP-ile tedavi edilmiş farelerde izole edilmiş tümörler fazla nekroz ve muamele edilmemiş muadili daha az vaskülarizasyon göstermiştir. Bu rapor, hayvanların hazırlanması, bunların üretral kateter, mesane mukozasının hücrelerin bağlanması için gerekli olan kimyasal yanma ve tümör hücrelerinin enjeksiyonu ayrıntılı bir adımadım protokolü sağlar. Ayrıca mesane kanseri tedavisi için geliştirilmiş herhangi bir terapötik maddelerin bir prototip olarak kabul edilebilir, HP-NAP lokal olarak uygulanmasını tarif eder. Kontrol ve HP-NAP-tedavi edilen hayvanlardan izole edilen tümör karşılaştırarak elde edilen deliller, sadece HP NAP mesane kanseri immünoterapi için iyi bir aday değil, aynı zamanda kurmak deneysel genel etkinliği olabileceği gerçeğini vurgulamaktadır.

<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.

Şekil 1, kateterizasyon tekniği göstermektedir; Fare kateterize ve 0.5 × 10 6 MB49 hücreleri ile damlatılır. HP-NAP ile tedavi mesane duvarına yapışır ve çoğalırlar sağlayacak, 3 gün, tümör hücrelerinin enjeksiyonundan sonra başlatılır. Kontrol grubuna ait bütün hayvanlar tümör geliştirme; bunların bazıları üretra tıkanıklığı 13 günlük sürenin bitiminden önce ölebilir. HP-NAP ile ilk enjeksiyondan sonra, hayvanlar dört enj...

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.

Etkinliğinin Değerlendirilmesi<em&gt; H. pylori</emOrtotopik Fare Modelinde Mesane Kanseri Tedavisinde Terapötik Aracı Olarak&gt; Protein HP-NAP

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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JoVE Journal

Medicine

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 Görüntüleme.  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.

Bir Fare Ortotopik Mesane Tümör Modeli ve Tümör Tespit Sistemi

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 ...

Kanser Araştırmaları

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.

Fare mesane Duvar Enjeksiyon

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

Tıp

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.

Manyetik rezonans görüntüleme değerlendirme kanserojen kaynaklı fare mesane Tümörlerinin

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.

Bir Ortotopik Mesane Tümörü Model ve intravezikal Sarna Tedavisinin Değerlendirilmesi

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.

Gene Teslim Çalışmaları Ortotopik Mesane Kanseri Modeli

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.

Polianhidrit IL-1α Nanopartiküllerinin In vivo Antitümör Aktivitesinin Değerlendirilmesi

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.

Bağışıklık Sağlam İnsan MUC1 Transgenik Farelerde İnvaziv Geçici Hücre Mesane Karsinomu indüksiyon: immünoterapi Kalkınma İçin Bir Model

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.

Tek adımlı olumsuz Kromatografik arıtma<em&gt; Helicobacter pylori</em&gt; Nötrofil-aktive protein aşın<em&gt; Escherichia coli</em&gt; Toplu Modunda

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

İmmünoloji ve Enfeksiyon

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.

Fare modelleri Helicobacter enfeksiyonu ve gastrik patolojiler

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 ...

Etkinliğinin Değerlendirilmesi

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