Bu çalışma, Amerikan Gastroenteroloji Derneği (AGA) Araştırma Alimler Ödülü ve Crohn ve Amerika (CCFA) (ECS) (SZS için) Kariyer Geliştirme Ödülü NIH NIDDK F30 DK089692 Kolit Vakfı ve Gastrointestinal Biyoloji Kuzey Carolina Merkezi&#39;nin Üniversitesi tarafından desteklenen ve Hastalık Hibe P30 DK34987 (Histoloji Çekirdek). UNC Sitometrisi Çekirdek Tesisi UNC Lineberger Kapsamlı Kanser Merkezi&#39;nde bir NCI Merkezi Çekirdek Desteği Grant (P30CA016086) tarafından kısmen desteklenmektedir. Biz histopatolojik analiz ve immunohistokimyasal ile yaptığı yardım için Veteriner North Carolina State University College Luke B. Borst teşekkür ederim.

NOT: Tüm hayvan protokolleri ile ve Laboratuvar Hayvanları Bakım ve Kullanım Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) yönetmelik ve Ulusal Araştırma Konseyi&#39;nin Kılavuz uygun onaylanmış emin olun. Verici fareler erkek veya kadın olabilir, ancak alıcı fareler erkek olmalıdır. Dişi alıcı kullanılacak ise, verici fareler 5 kadın olmalıdır. Böylece, farelerin 5,6 kolit fenotip, bu intestinal mikrobiyota etki edilebilir, düzenli olmayan steril yatak ve asitli-olmayan su kullanılarak koloni bakımı ve. 1. Deneysel hazırlanması <ol><li> Buz soğukluğunda ortamı ve tamponlar kullanın. Deney boyunca buz üzerinde hücreleri tutun. </li><li> Steril tekniği kullanarak steril biyolojik tehlike kaput deney yapın. </li></ol> Dalak T hücrelerinin 2. izolasyonu <ol><li> Donör fare / CO fareler servikal dislocatio 2 odacık ardından euthanizen. % 70 etanol ile karın püskürtün. </li><li> Karın yatay bir kesi yapmak ve periton maruz cilt soymaya. Uzak forseps ile iç organlardan periton tutun ve ortaya çıkarmak ve dalağı tüketim sol karın periton bir kesi yapmak. </li><li> Petri kabındaki tam ortam, 10 ml dalak yerleştirin. Çıkarın ve dalak aşırı doku atın. </li><li> Ezmek ve tek hücre süspansiyonu içine dalak ayrı kızdırmak için 2 steril cam slaytlar kullanın. Filtre hücresi 50 ml konik tüp içine 70 mikron süzgeç vasıtasıyla süspansiyon ve tam Medya 5 ml süzgeç durulayın. Bir 50 ml konik tüp içinde 5 dalak kadar yerleştirin. </li><li> 7 dakika için 450 xg&#39;de Santrifüj hücreleri. Kapalı dökerek ya da atık kabına vakum emme ile süpernatant atın. </li><li> Yavaşça, oda sıcaklığında 10 dakika boyunca, kırmızı kan hücrelerinin lize etmek için Lizis Tamponu dalak başına 5 ml süspansiyon hücrelerin. Tüpe tam ortam içinde eşit hacimde (dalak başına 5 mi) eklenir. </li><li&gt; Santrifüj hücreleri 7 dakika için 450 xg&#39;de. Kapalı dökerek ya da atık kabına vakum emme ile süpernatant atın. </li><li> Yavaşça Etiketleme Tampon, 10 ml hücreler tekrar süspansiyon. </li><li> Tripan mavi dışlama hücreleri saymak. <ol><li> Hücre süspansiyonu 20 ul çıkarın ve bir mikrofüj tüpüne 180 ul, tripan mavisi ekleyin ve iyice karıştırın. 5 dakika sonra, hemositometrede ve mikroskop altında olmayan mavi hücreleri saymak için hücreleri etiketli 10 ul ekleyin. Canlı hücrelerin toplam sayısını belirleyin. Hücre / tripan mavi karışımı atın. </li></ol></li><li> 7 dakika boyunca 450 xg&#39;de Etiketleme Tampon, 10 ml santrifüj hücreleri. Kapalı dökerek ya da atık kabına vakum emme ile süpernatant atın. </li></ol> CD4 + T hücreleri 3. zenginleştirilmesi NOT: Bu bölümde kullanılan belirli ürünler için üreticinin talimatlarına uyun. <ol><li> Yavaşça soğuk etiket 20 x 10 6 hücre / ml &#39;lik bir tek hücre süspansiyonuna hücreleri tekrar süspansiyoning Tampon. </li><li> Biyotinlenmiş CD4 T hücresi zenginleştirme antikor 1 x 10 6 hücre başına 5 ul ekleyin. 15 dakika boyunca buz üzerinde kuluçkaya bırakılır. </li><li> Etiketleme Tampon 10x hacim ekleyin. 7 dakika 450 × g Santrifüj hücreleri. Dikkatle atık kabına vakum emme kullanarak tüm süpernatant aspire. </li><li> İyice streptavidin-konjuge manyetik parçacıklar girdap. 1 x 10 6 hücre başına parçacıkların 5 ul ekle. </li><li> İyice fakat nazikçe karıştırın. 30 dakika boyunca, 6-12 ° C&#39;de bir karışım tutun. </li><li> 20-80 x 10 6 hücre / ml &#39;lik bir konsantrasyona kadar Etiketleme Tamponu ekleyin. 12 x 75 mm yuvarlak alt test tüpüne başına hücreleri etiketli 1.0 ml kadar aktarın (&quot;pozitif kesir tüp&quot; olarak anılacaktır). </li><li> 6-8 dakika mıknatıs her olumlu-fraksiyon tüpü yerleştirin. </li><li> Mıknatıs hala pozitif kesir tüpleri ile dikkatlice (yeni steril 50 ml konik tüp cam Pasteur pipet ile pozitif fraksiyon tüpten referre süpernatant aktarmak&quot;zenginleştirilmiş fraksiyon&quot;) olarak d. Bu zenginleştirilmiş fraksiyon, CD4 + T hücrelerini içerir. Mıknatıs çekti etiketli hücreleri bozmak için değil dikkatli olun. </li><li> Süspanse edin hücreleri aşağı yukarı şiddetle pipetleme ve adım 3.6 gibi Etiketleme Tampon aynı hacimde pozitif kesir tüplerde bıraktı. 6-8 dakika geri mıknatıs üzerinde pozitif kesir tüpü yerleştirin. </li><li> Mıknatıs hala pozitif kesir tüpleri ile dikkatlice mıknatıs bağlı hücrelerin aksatmadan 50 ml Aşama 3,8 konik tüp, steril cam Pasteur pipet ile pozitif fraksiyon tüp süpernatant (zenginleştirilmiş fraksiyonu, CD4 +) aktarın. </li><li> Tekrar Elde edilen CD4 + T hücrelerinin verimini artırmak için 3,9-3,10 adımları tekrarlayın. Zenginleştirilmiş fraksiyon (CD4 + hücreleri) kullanılarak protokolü devam edin. </li></ol> 4. Etiketleme ve sıralama Hücreler 7 <ol><li> Santrifüj 7 dakika için 450 xg&#39;de hücreleri zenginleştirilmiş. Veya vakum emilmesini kapalı dökerek süpernatant atınatık kabına iyon. </li><li> Etiketleme, 1 ml tampon içinde süspanse hücreleri. Saymak ve aşama 2.9 olarak tripan mavisi eksklüzyonu ile hücre canlılığı değerlendirmek için hücreler kısım çıkarın. </li><li> 10 x 10 6 hücre / ml Etiketleme getirme tamponunun bir hacminin ilave et; hücrelerin önceden ise &lt;10 x 10 6, 7 dakika boyunca 450 x g&#39;de santrifüj, atık kabı içine dökülen ya da vakum emiş ile süpernatan atılır ve 10 x 10 6 hücre / ml Etiketleme Tamponu hacmi ekleyin. <ol><li> Mikrofüj tüplerinde izotip lekeli, boyanmadan ve tek boyanmış kontrol hücreleri için yaklaşık 5-10 x 10 5 hücre, her biri ayrı ayrı alikotları ayarlayın. </li></ol></li><li> Hücrelere 5 ug / ml CD4-FITC ve 1 ug / ml CD45RB-PE ekleyin. Mikrofüj tüplerinde alikotları mülk aynı konsantrasyonda izotip kontrol lekeleri ve tek lekeler ekleyin. Iyi bir şekilde fakat nazikçe karıştırın ve 30 dakika için ışıktan korunması buz üzerinde inkübe edilir. </li><li> Hücre ve kontrolü Etiketleme Tampon 10x hacmi ekles. 7 dakika Santrifüj 450 xg. Atık kabına vakum emme ile süpernatant atın. </li><li> Adım 4.5 hacme Etiketleme Tampon süspanse. 7 dakika Santrifüj 450 xg. Atık kabına vakum emme ile süpernatant atın. </li><li> 10 x 10 6 hücre / ml Etiketleme Tamponu içinde yeniden süspanse edin. FACS tüpüne 70 mikron süzgeç vasıtasıyla hücrelere geçerler. FACS için hazır olana kadar ışıktan korunan buz üzerinde tutun. </li><li> Set up ve boyanmamış hücreler ve tek lekeli kontrolleri ile hücre sıralayıcı uygun tazminat belirler. </li><li> Ileriye ve yan dağılım yolluk (Şekil 1A) ile cansız hücreleri hariç. Izotip-lekeli kontrolleri ile CD4 + ve CD45RB + hücreleri için yolluk ayarlayın. CD4 + nüfus Kapı hücreleri. </li><li> Tam ortam, 2 ml tüplere PE-boyanmış hücreler için basit bir histogram kullanarak yüksek CD45RB ve CD45RB alt popülasyon kriteri CD4 + hücreleri. Yüksek CD45RB </sus&gt; nüfus CD4 + CD45RB + hücreleri (yüksek CD45RB) en yüksek% 40 temsil eder ve CD45RB alt popülasyonu, CD4 + CD45RB + hücrelerinin en% 20 (alt CD45RB, Şekil 1B). </li></ol><img alt="Şekil 1," src="/files/ftp_upload/52533/52533fig1.jpg" /> Şekil 1: FACS analizi sırasında, CD4 + CD45RB, T hücre popülasyonlarının sitometrisi lekeleri Örnek akımı (A - C), FITC CD4 ve PE içine FACS ile sınıflandırılmıştır verici, C57BL / 6 farelerinden alınan splenositler CD45RB lekeli CD4 + CD45RB yüksek ve CD4 +. CD45RB alt T hücresi popülasyonu. (A) Doublet olayları ileri dağılım arsa üzerinde dışı bırakıldı. (B) lenfositler ileri ve yan dağılım arsa kapılı edildi. (C), CD4 + </destek&gt; T hücreler geçitlendi ve (D) CD4 + CD45RB + T hücreleri, PE karşı etkinlikler histogram çizilmiştir. CD4 + CD45RB alt hücreler, CD45RB + hücrelerinin en% 20 olarak kabul edildi. CD4 + CD45RB, yüksek hücre CD45RB + hücrelerinin en yüksek% 40 olarak tayin edilmiştir. <ol start="11"><li> Popülasyonlarının saflığı analiz etmek üzere FACS makinede her bir hücre popülasyonunun bir kısım çalıştırın. </li><li> Santrifüj 7 dakika için 450 xg&#39;de hücreleri sıralanır. 1 ml PBS içinde yeniden süspanse. Saymak ve aşama 2.9 olarak tripan mavisi eksklüzyonu ile hücre canlılığı değerlendirmek için hücreler kısım çıkarın. </li></ol> Alıcılar içine Hücreleri 5. Enjeksiyon <ol><li> Süspanse 4 x 10 6 hücre / ml (CD45RB yüksek) veya PBS 2 x 10 6 hücre / ml (düşük CD45RB) hücreleri sıralanır. </li><li> Yeni steril tüpe alıcı başına CD45RB yüksek hücrelerin 100 ul aktarın. PBS Başı 100 ul ekleyinbu tüp alıcısı. Bu nedenle, hayvan başına toplam enjeksiyon hacmi 200 ul ve alıcı için hücrelerin toplam miktarı, 4 x 10 5, CD4 + CD45RB yüksek naiv T hücreleri. </li><li> Regülatör T hücreleri alan deney grubu arzu edildiği takdirde, yeni bir steril tüpe alıcı için CD45RB, yüksek hücre 100 ul aktarın. Aynı tüp alıcı başına CD45RB düşük hücrelerin 100 ul ekleyin. Hayvan başına toplam enjeksiyon hacmi 200 ul; CD45RB yüksek oranı: CD45RB alt hücreler 2: 1 dir. </li><li> Yüksek / düşük CD45RB CD4 + hücreleri intraperitoneal her alıcının karın (200 ul toplam) sağ ve sol tarafına CD45RB yüksek veya CD45RB 100 ul enjekte edilir. </li></ol> Alıcı Hayvanlarda Hastalık ilerleme 6. İzleme <ol><li> Alıcı hayvanların klinik durumunu değerlendirmek aşağıdaki Paramete için toplam klinik skorları atamak içinEnjeksiyon gününde rs 8 haftalık sonra, ve kurban sırasında: <ol><li> Kilo kaybı ölçerek israf belirleyin: 0 - hayır kilo kaybı; 1 - İlk vücut ağırlığının% 0.1-10 kaybı; 2 - Başlangıç ​​vücut ağırlığı (Şekil 2A), ve% 10&#39;dan daha fazla zarar. </li></ol></li></ol><img alt="Şekil 2," src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> Şekil 2: inflamasyonun klinik ve toptan patolojik işaretler Rag1 içine yabani tip CD4 + CD45RB yüksek T hücrelerinin transferinden sonra meydana - / - 11 (A) &#39;NRDKO alıcıları 5 ile başlangıç ​​vücut ağırlıkları 10 ortalama% kayıp NRDKO alıcı fareler. haftalar sonrası transfer, Rag1 ise- / - Alıcıları, şu anda, hastalığın klinik belirtilerini göstermemiştir. Her nokta ± SEM kohort için başlangıç ​​vücut ağırlığının ortalama yüzdesini temsil eder. ** P &lt;0.005. Rektal prolapsus varlığı ile gösterildiği gibi (B) Bazı fareler, şiddetli bağırsak iltihabı geliştirdi. Bu NRDKO alıcı fare rektal prolapsus temsili resim. (C) Büyük oranda, gelen iki nokta üst üste, hem Rag1 - / - ve NRDKO alıcı fareler Rag1 gelen iki nokta üst üste ile karşılaştırıldığında kalınlaşmış ve kısaltılmış - / - ve T hücre evlatlık transferi olmadan NRDKO fareler. NRDKO alıcı farelerin kolonlar şiddetli inflamasyon ve artan kolon ağırlıkları (veriler gösterilmemiştir) gösterir. <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <ol><li><ol start="2"><li> O geçene kadar temiz bir kaba hayvan yerleştirerek dışkı kalitesini belirleyinDışkı: 0 - yok; 1 - yumuşak dışkı; 2 - sulu ve / veya kanlı dışkı. </li><li> Hastalığın belirtileri aşağıdaki varlığı ile hayvanın genel sağlık belirleyin: 0 - hayır kambur duruş, kürk, ya da cilt lezyonları kıllı; 1 - Aşağıdaki mevcut herhangi bir: kambur duruş, kürk, deri lezyonları veya kıllı. </li><li> Rektal prolapsus varlığı belirleyin: 0 - devamsızlık; 1 - Mevcut (Şekil 2B). </li></ol></li><li> Bu, başlangıç ​​vücut ağırlığına göre% 20 kayıp ya da hangisi daha önce arzu edilen bir zaman noktasında, var olduğunda CO, sonra servikal dislokasyon 2 inhalasyon hayvanlar kurban. Klinik hastalık haftada 5 sonrası dolgunluk başlayan tipik açıktır. </li><li> Daha önce açıklandığı gibi 5,7 hastalık şiddeti değerlendirmek. <ol><li> Adım 6.1 8 gibi klinik skorları atayın. </li><li> Kolon uzunluğu ve ağırlığı (Şekil 2C) ölçün. </li><li> Inflamasyon histolojik analiz gerçekleştirin5. </li><li> Bağırsak doku eksplant kültürlerinde 9 spontan sitokin ifadesini belirleyin, mezenterik lenf düğümleri 10, ve / veya serum 11. <ol><li> Kısaca, eksplant kültürler 9, uzunlamasına açık, kurban sonrası iki nokta üst üste kaldırmak ve PBS ile temiz. Oda sıcaklığında 30 dakika boyunca tam ortam içinde 250 rpm&#39;de bir orbital çalkalayıcı üzerinde iki nokta üst üste inkübe edin. </li><li> Küçük parçalar halinde doku doğranır ve 24 saat süre ile tam ortam içinde, 37 ° C&#39;de inkübe edildi. Süpernatan toplamak ve ELISA ile 100 mg doku başına düşen sitokinlerin miktarının belirlenmesi için kullanılır. </li></ol></li><li> T hücre fenotipleri ve / veya fonksiyon 10 ex vivo karakterizasyonu gerçekleştirin. </li></ol></li></ol>

<ol>
	<li>Xavier, R. J., Podolsky, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unravelling+the+pathogenesis+of+inflammatory+bowel+disease.">Unravelling the pathogenesis of inflammatory bowel disease.</a> Nature. 448 (7152), 427-434 (2007).</li><li>Cho, J. H., Brant, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+the+genetics+of+inflammatory+bowel+disease.">Recent insights into the genetics of inflammatory bowel disease.</a> Gastroenterology. 140 (6), 1704-1712 (2011).</li><li>Jostins, L., 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=Host-microbe+interactions+have+shaped+the+genetic+architecture+of+inflammatory+bowel+disease.">Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.</a> Nature. 491 (7422), 119-124 (2012).</li><li>Powrie, F., Leach, M. W., Mauze, S., Caddle, L. B., Coffman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypically+distinct+subsets+of+CD4++T+cells+induce+or+protect+from+chronic+intestinal+inflammation+in+C.+B-17+scid+mice.">Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice.</a> Int Immunol. 5 (11), 1461-1471 (1993).</li><li>Ostanin, D. V., 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=T+cell+transfer+model+of+chronic+colitis:+concepts,+considerations,+and+tricks+of+the+trade.">T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade.</a> Am J Physiol Gastrointest Liver Physiol. 296 (2), 135-146 (2009).</li><li>Ma, B. W., 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=Routine+habitat+change:+a+source+of+unrecognized+transient+alteration+of+intestinal+microbiota+in+laboratory+mice.">Routine habitat change: a source of unrecognized transient alteration of intestinal microbiota in laboratory mice.</a> PLoS One. 7 (10), e47416 (2012).</li><li>Read, S., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+inflammatory+bowel+disease+in+immunodeficient+mice+by+depletion+of+regulatory+T+cells.">Induction of inflammatory bowel disease in immunodeficient mice by depletion of regulatory T cells.</a> Curr Protoc Immunol. Chapter 15 (Unit 15 13), (1999).</li><li>Maillard, M. 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=The+Wiskott-Aldrich+syndrome+protein+is+required+for+the+function+of+CD4(+)CD25(+)Foxp3(+)+regulatory+T+cells.">The Wiskott-Aldrich syndrome protein is required for the function of CD4(+)CD25(+)Foxp3(+) regulatory T cells.</a> J Exp Med. 204 (2), 381-391 (2007).</li><li>Hegazi, R. A., 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=Carbon+monoxide+ameliorates+chronic+murine+colitis+through+a+heme+oxygenase+1-dependent+pathway.">Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway.</a> J Exp Med. 202 (12), 1703-1713 (2005).</li><li>Kole, A., 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=Type+I+IFNs+regulate+effector+and+regulatory+T+cell+accumulation+and+anti-inflammatory+cytokine+production+during+T+cell-mediated+colitis.">Type I IFNs regulate effector and regulatory T cell accumulation and anti-inflammatory cytokine production during T cell-mediated colitis.</a> J Immunol. 191 (5), 2771-2779 (2013).</li><li>Kobayashi, T., 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=NFIL3-deficient+mice+develop+microbiota-dependent,+IL-12/23-driven+spontaneous+colitis.">NFIL3-deficient mice develop microbiota-dependent, IL-12/23-driven spontaneous colitis.</a> J Immunol. 192 (4), 1918-1927 (2014).</li><li>Steinbach, E. C., 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=Innate+PI3K+p110delta+Regulates+Th1/Th17+Development+and+Microbiota-Dependent+Colitis.">Innate PI3K p110delta Regulates Th1/Th17 Development and Microbiota-Dependent Colitis.</a> J Immunol. 192 (8), 3958-3968 (2014).</li><li>Kobayashi, T., 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=NFIL3+is+a+regulator+of+IL-12+p40+in+macrophages+and+mucosal+immunity.">NFIL3 is a regulator of IL-12 p40 in macrophages and mucosal immunity.</a> J Immunol. 186 (8), 4649-4655 (2011).</li><li>Leach, M. W., Bean, A. G., Mauze, S., Coffman, R. L., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease+in+C.B-17+scid+mice+reconstituted+with+the+CD45RBhigh+subset+of+CD4++T+cells.">Inflammatory bowel disease in C.B-17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells.</a> Am J Pathol. 148 (5), 1503-1515 (1996).</li><li>Powrie, F., 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=Inhibition+of+Th1+responses+prevents+inflammatory+bowel+disease+in+scid+mice+reconstituted+with+CD45RBhi+CD4.">Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4.</a> T cells. Immunity. 1 (7), 553-562 (1994).</li><li>Read, S., Malmstrom, V., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+lymphocyte-associated+antigen+4+plays+an+essential+role+in+the+function+of+CD25(+)CD4(+)+regulatory+cells+that+control+intestinal+inflammation.">Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation.</a> J Exp Med. 192 (2), 295-302 (2000).</li><li>Rogers, G. B., 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=Functional+divergence+in+gastrointestinal+microbiota+in+physically-separated+genetically+identical+mice.">Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice.</a> Sci Rep. 4, 5437 (2014).</li><li>Fukata, 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+myeloid+differentiation+factor+88+(MyD88)+is+required+for+CD4++T+cell+effector+function+in+a+murine+model+of+inflammatory+bowel+disease.">The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease.</a> J Immunol. 180 (3), 1886-1894 (2008).</li><li>Kurtz, C. C., 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=Extracellular+adenosine+regulates+colitis+through+effects+on+lymphoid+and+nonlymphoid+cells.">Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells.</a> Am J Physiol Gastrointest Liver Physiol. 307 (3), 338-346 (2014).</li><li>Naganuma, 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=Cutting+edge:+Critical+role+for+A2A+adenosine+receptors+in+the+T+cell-mediated+regulation+of+colitis.">Cutting edge: Critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis.</a> J Immunol. 177 (5), 2765-2769 (2006).</li><li>Ranatunga, D. C., 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=A+protective+role+for+human+IL-10-expressing+CD4++T+cells+in+colitis.">A protective role for human IL-10-expressing CD4+ T cells in colitis.</a> J Immunol. 189 (3), 1243-1252 (2012).</li><li>Srikrishna, 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=Carboxylated+glycans+mediate+colitis+through+activation+of+NF-kappa.">Carboxylated glycans mediate colitis through activation of NF-kappa.</a> B. J Immunol. 175 (8), 5412-5422 (2005).</li><li>Wang, F., 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=IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction.">IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction.</a> Gastroenterology. 131 (4), 1153-1163 (2006).</li></ol>

The authors declare that they have no competing financial interests.

Burada bağışıklık yetersizliği olan farelere, CD4 + CD45RB + T hücrelerinin adoptif nakli ile farelerde kolon inflamasyonu yol açan bir adım adım protokol açıklar. Alıcı fareler başka soybaşları rağmen (örneğin, BALB / c, 129S6 / SvEv, obez olmayan diyabetik (NOD)) immün yetmezlik ve genetik modelleri (örneğin, SCID, Rag2 - / - Bu, C57BL / 6 verici dalak ve genetik olarak özdeş Rag1 kullanılan - / -) da 4,14-16...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

İnsan inflamatuar barsak hastalıklarının (IBD), kendi avantajları ve dezavantajları her patogenezi çalışmak için birçok farklı hayvan modelleri vardır. Burada T ve B hücresi eksik alıcı farelere singeneik dalak CD4 + CD45RB yüksek T hücrelerinin adoptif nakli ile başlatılır deneysel kolit modelini açıklar. Büyük ölçüde naif efektör hücrelerin oluşur CD4 + T hücre CD45RB yüksek popülasyonu yakından insan IBD&#39;nin kilit yönlerini benzeyen, kronik bağırsak inflamasyonu uyarma yeteneğine sahiptir. Bu yöntem, hastalığın başlangıcında ve ilerlemesinde yönlerini incelemek için manipüle edilebilir. Ayrıca bağırsak iltihabı yani doğuştan gelen, adaptif fonksiyonlarını ve düzenleyici immün hücre popülasyonları ve çevresel maruziyet rolü, Mikrobiyota, incelemek için kullanılabilir. Bu yazıda, bir adım-adım protokolü ile Kolit için bir metodoloji göstermektedir. Bu incidarecisi başarıyla araştırma amaçlı deneysel kolit bu fare modeli geliştirilmesi için gerekli anahtar teknik yönlerini bir video gösterisi.

Yaklaşık yetişkin, C57BL / 6 verici fareden 10 dalaklarından 10 x 10 6, CD4 + T hücreleri, CD45RB yüksek güvenilir izole edilir. Bu sayı yaşla ve gerginlik verici fare ve araştırmacı yeterliliğine bağlı olarak değişecektir. Ne zaman 4 x 6 CD4 + T hücreleri CD45RB yüksek C57BL / 6 Rag1 aktarılır / 10 5 C57BL - / - alıcı fareler, hastalığın klinik belirtileri hafta 5 sonrası dolgunluk etrafında ortaya veya far...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Efektör CD4 Evlatlık transferi ile Kemirgen Bağırsak Enflamasyon indüksiyonu<sup&gt; +</sup&gt; CD45RB<sup&gt; Yüksek</sup&gt; Bağışık yetmezliği olan farelere içine T Hücreleri

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

16.2K Görüntüleme.  University of North Carolina at Chapel Hill. Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Video: Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Deneysel metastaz ve CTL Kabul edilmiş Transfer İmmünoterapi Fare Modeli

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

İmmünoloji ve Enfeksiyon

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Bağışıklık Hücreleri murin aorta içinde Sitometrisi Analizi

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Diyabetojenik CD4 Kabul edilmiş Transfer ile Farelerde Hızlandırılmış Tip 1 Diyabet İndüksiyon + T Hücreleri

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Hematopoetik progenitörlerin Evlatlık Transferi kullanarak Peter Parçacıklannda Murin plazmasitoid dendritik hücreleri Gelişimini değerlendirilmesi

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Bakım Glioblastoma Standardı Kapsamında Adoptif Tedavisi için CAR T hücrelerinin üretilmesi

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analizi<em&gt; Yersinia enterocolitica</emAna Hücreleri içine&gt; Efektör Translokasyon Beta-laktamaz Effector Fusions kullanma

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Fare Dalak Değişmeyen Doğal Öldürücü T Hücreleri İzolasyon ve Yaygınlaştırılması için optimize Yöntemi

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Farelerde Deneysel Bakteriyel Pnömoni İndüksiyonunun ve fenotipleme bir Non-invaziv ve Teknik Olmayan yoğun Yöntem

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Felç ve görsel sistem yaralanma tarafından Nöromyelitis Optica Autoantigen Aquaporin-4 T hücreleri belirli farelerde indüksiyon

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Orthotopic insan Glioblastoma Multiforme Xenografts fare modelleri sitotoksik bağışıklık hücrelerinin stereotaksik evlat edinen Transfer

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...