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>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+modulating+activity+of+the+Helicobacter+pylori+HP-NAP:+Friend+or+foe.">The immune modulating activity of the Helicobacter pylori HP-NAP: Friend or foe.</a> Toxicon. 56, 1186-1192 (2010).</li><li>Codolo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neutrophil-activating+protein+of+Helicobacter+pylori+down-modulates+Th2+inflammation+in+ovalbumin-induced+allergic+asthma.">The neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in ovalbumin-induced allergic asthma.</a> Cell Microbiol. 10, 2355-2363 (2008).</li><li>Gunther, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+syngeneic+orthotopic+murine+bladder+cancer+(MB49).">Optimizing syngeneic orthotopic murine bladder cancer (MB49).</a> Cancer Res. 59, 2834-2837 (1999).</li><li>Codolo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HP-NAP+inhibits+the+growth+of+bladder+cancer+in+mice+by+activating+a+cytotoxic+Th1+response.">HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1 response.</a> Cancer Immunol Immunother. 61, 31-40 (2012).</li><li>Chan, E., Patel, A., Heston, W., Larchian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+orthotopic+models+for+bladder+cancer+research.">Mouse orthotopic models for bladder cancer research.</a> BJU Int. 104, 1286-1291 (2009).</li><li>Kubota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+models+of+human+cancer+xenografted+in+the+nude+mouse:+the+importance+of+orthotopic+transplantation.">Metastatic models of human cancer xenografted in the nude mouse: the importance of orthotopic transplantation.</a> J Cell Biochem. 56, 4-8 (1994).</li><li>Loi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+orthotopic+model+to+validate+antivascular+therapies+for+cancer.">The use of the orthotopic model to validate antivascular therapies for cancer.</a> Int J Dev Biol. 55, 547-555 (2011).</li><li>Miyazaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconditioning+methods+influence+tumor+property+in+an+orthotopic+bladder+urothelial+carcinoma+rat+model.">Preconditioning methods influence tumor property in an orthotopic bladder urothelial carcinoma rat model.</a> Mol Clin Oncol. 2, 65-70 (2014).</li><li>Horiguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+orthotopic+mouse+superficial+bladder+tumor+model+for+studies+on+intravesical+treatments.">Establishment of orthotopic mouse superficial bladder tumor model for studies on intravesical treatments.</a> Hum Cell. 21, 57-63 (2008).</li></ol>

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 tümör 16 ortamı taklit çünkü. Burada açıklandığı gibi ikinci xenoimplants tabi bağışıklık eksikliği olan farelerde mümkün değildir çünkü Dahası, sinjenik farelerde kanser ortotopik modellerini kurma, immünoterapi dayalı yeni tedavi stratejilerinin araştırılması amacıyla çalışmaların durumunda özellikle önemlidir. Bu protokolde gösterilen metodoloji t açıklartümör hücrelerinin aşılanmasını ve mesane boşluğuna terapötik maddelerin uygulanmasını hem izin veren bir dişi fare üretranın O kateterizasyon. (yerine örneğin tripsin ya da poli-lisin kullanımı gibi diğer yaklaşımlar, ve, yukarıda tarif edilenler gibi) bir asit yanığı ile mesane duvarı ön muameleden geçirilmesi avantajı, büyük ve oldukça invaziv tümörlerin gelişimini teşvik olmasıdır. Muhtemelen, tümör hücrelerinin 17 canlılığı üzerindeki bu maddelerin kalıntılarının bir zararlı etki için tripsin ve poli-lizin, daha küçük ve daha az invaziv tümörlerde sonuçları ile ön koşullandırma tersine,. İkinci mesane duvarının delinmesi ve periton 18 içine tümörün yayılmasını neden olabilir, çünkü asit yanığı da elektrik koterizasyon dayalı başka önkoşullanma sistemine tercih edilir. protokol kısa süreli çalışmalarda (~ 13 gün) için optimize edilmiştir. Pr implantedaha yüksek bir hücre sayısı nedeniyle geniş bir tümör daha hızlı tümör büyümesi ve muhtemelen hayvanların kaybına neden olur çünkü hücrelerin oper sayısı kritik önem taşır. Kullanıcı tekniği ile emin olana kadar Dahası, böylece hayvanların ölümüne neden üretra veya mesane zarar riskin belli bir derecesi vardır. Hayvanlar yeterli (işlem sırasında ölme ve böylece) işletilen eğer bizim deneyim, ancak onlar deneylerde (13 gün) süresince acı olmadan yaşamak. Ayrıca, enjekte edilen hücrelerin sayısını azaltarak, bu deney süresini uzatmak mümkündür. Biz, daha yüksek bir fayda / yan etkilere sahiptir yenisi ile BCG göre mevcut tedaviyi geliştirmek ve en sonunda yerine amacıyla, Helicobacter pylori tarafından üretilen protein, HP-NAP anti-tümör etkisinin değerlendirilmesi için, bu model kullanılan oranı. Daha genel olarak, bu deneysel yaklaşım tüm ön Clini kabul edilebilirMesane boşluğuna terapötik ajanların teslimini gerektiren cal değerlendirmeler.

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>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&#39;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></td>
			<td></td>
			<td></td>
			<td>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>
Materials C57BL/6J female mice Harlan Italy (Udine, Italy) MB49 Cells Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA RPMI Sigma-Aldrich (St. Louis, Missouri, USA) R8758 FBS Sigma-Aldrich F7524 PBS Sigma-Aldrich D1408 10X, to be diluted in apyrogenic water Flask Becton Dickinson (Franklin Lakes, New Jersey, USA) 353135 Syringe 1ml Becton Dickinson 301358 Trypsin Life Technologies (Waltham, Massachusetts, USA) Gentamycin Life Technologies 15710-049 Xilor Bio 98 s.r.l. (Milano, Italy) 2% Xylazin Zoletil Virbac (Carros, France) 359713301992 5% Zolazepam + 5% Tiletamine 24G Catheter Terumo (Rome, Italy) SR+DM2419PX HCl Carlo Erba Reagents (Milano, Italy) 403871 Liquid NaOH JT Baker (Center Valley, Pennsylvania, USA) 10095011 Powder Equipment Surgical Scalpel Albion Surgical Limited (Sheffield, England) Microtome Leica Microsystem ( Wetzlar, Germany) RM2235 Microscope Slides VWR International (Radnor, Pennsylvania, USA) 631-0108 Image Analyzer Zeiss (Jena, Germany) Cyres System

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 enjeksiyon toplam, her üç günde bir tedavi edilir. Deneyin sonunda, günde on üç, hayvanlar kurban edilmiş ve histolojik mesaneler ve bağışıklık histokimyasal analiz için toplanmıştır. Şekil 2A&#39;da gösterildiği gibi, HP-NAP-muamele edilmiş farelerden alınan mesane içinde tümör hacmi 830 ± 1 v (220 ± 62 mm3, araç ile tedavi edilmiş farelerin tümör önemli ölçüde daha küçüktür80 mm3). İlginç şekilde, araçla tedavi edilen hayvanlarda tümör tümörlü mesane duvarı (Şekil 2B) içinde sınırlı 8&#39;in HP NAP-ile tedavi edilen hayvanların 7&#39;sinde, Altındaki yağ dokusu da yayılmıştır. Son olarak, vaskülarite azalır ve nekroz ölçüde hayvanlar (Şekil 2B ve C) kontrole göre HP NAP-ile tedavi edilmiş farelerde artar. <img alt="Şekil 1" src="/files/ftp_upload/52743/52743fig1.jpg" /> Şekil 1. Fare kateterizasyon. Dişi fareler anestezi ve 24 G steril kanül kullanılarak kateterize. Sağ panelde siyah kutu büyütme olduğunu. <a href="https://www.jove.com/files/ftp_upload/52743/52743fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src = &quot;/ files / ftp_upload / 52743 / 52743fig2.jpg&quot; /&gt; HP UEP Şekil 2. Anti-tümör aktivitesi. Fareler, MB49 hücreleri (0.5 x 10 6) ile 0 implante, 3, 6, 9 ve 50, HP-NAP veya PBS (araç) ug ile muamele edilir 12. Tüm hayvanlar 13. günde (A) kontrol farelerinde ve HP-NAP ile tedavi edilen hayvanlar, ve tümör hacmi ölçümü izole mesanelerde Örnek histolojik analiz ile öldürüldü. Iki grup hayvandan panel A ve nekroz yüzdesi bölümlerinin (B) Büyütme. (C) CD31 / PECAM için lekeli Çıkarılan mesane; Kontrol ve HP-NAP-muamele edilmiş hayvanlardan elde edilen temsili bölümleri gösterilmektedir. Mikrodamar yoğunluğu (C sağ panel) Kantitasyonu; HPF, highpower alanlar. Veriler ortalama ± SD temsil eder; Grup başına n = 8 fare. Istatistiksel farkları, Öğrenci t-testi (** p &lt;0.01) ile tespit edilmiştir. Codolo et değiştirilmiş Şekilark. 13. <a href="https://www.jove.com/files/ftp_upload/52743/52743fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

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 ...

evaluation-efficacy-h-pylori-protein-hp-nap-as-therapeutic-tool-for

Nanyang Technological University

Research

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

An Orthotopic Model of Murine Bladder Cancer

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

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

Murin Mesane Kanseri bir Ortotopik Model

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

Tıp

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

TSSB, Nöral Hematolojik ve Davranış Bağlantılı Revealing için Hayvan Modelinde Biyobelirteçler

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

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 ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

İnsan Prostat Kanser metastaz Ortotopik fare modeli

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

Tedaviye dirençli Majör Depresif Bozukluk için bir tedavi olarak dmPFC-shamı MRI kılavuzluğunda

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

İnsan Prostat Kanseri klinik öncesi Ortotopik murin modeli

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Tavşan sklera bir araç olarak ikinci harmonik üretimi sinyalleri tedavi doku (TXL) için miyopi Cross-linking değerlendirilmesi için

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...