Bu çalışma Sağlık (NIH) AI091759 ve Crohn ve Amerika&#39;nın William ve Shelby Modell Aile Vakfı Araştırma Ödülü (MN) arasında Kolit Vakfı Ulusal Sağlık Enstitüleri tarafından desteklenmiştir. Şiştozom-enfekte farelerde NIAID şiştozom Kaynak Merkezi (NIAID Sözleşme N01 A130026) tarafından sağlandı.

1. Schistosoma mansoni Yumurta Arındırıcı <ol><li> Schistosoma yaşam döngüsü 8 bulaşıcı aşamasıdır şiştozom cercariae ile İsviçre-Webster fareler bulaştırmak. Alternatif olarak, NIAID Schistosomiasis Kaynak Merkezi (gelen şiştozom bulaşmış farelerde elde <a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/~~V</a> ). Olduğu zaman noktası tarafından, yumurta karaciğer mevcut olan ve aşağıda açıklandığı gibi (bakınız Şek. 1) elde edilebilir, enfeksiyon sonrası 6-7 hafta boyunca fareler bırakın. </li><li> CO 2 fareler Euthanize, ve% 70 etanol ile ıslatın. Arka fare yerleştirin. Deri ve altındaki periton kesip künt forseps ve makas kullanın. Göğüs kafesi ve diyafram altında yer alan Özel Tüketim karaciğer,. </li><li> Ince karaciğerler kıyma ve kolajenaz / dispase (0.5 mg / mL), penisilin (100 U / ml) ve PBS içinde streptomisin (100 ug / ml) oluşan sindirim karışımının karaciğer başına 20 mL, ekleyin. Mikst koyun50 ml Falcon tüp ure ve 37 ° C shaker&#39;da gece inkübe edin. </li><li> 400 xg / 3 dakika / 20 ° C&#39;de karışım santrifüjleyin Nazikçe aşırı sıvı kapalı dökün ve PBS ile tüp doldurun. Tekrar santrifüj ve PBS yıkama tekrarlayın. Pastör pipetiyle pelet 25 ml PBS içinde, son santrifüj ardından. </li><li> Ince bir elekten (ince elekten karışımı itmek için 10 ml şırınga kullanmak yoluyla karışımı geçirerek takip standart bir mutfak metalik süzgeç (bir cam behere aracılığıyla lapa karaciğer parçaları için 50 ml şırınga pistonu kullanın) aracılığıyla yeniden süspanse pelet Gerinim İkinci bir cam behere). </li><li> 50 ml Falcon tüp içinde gergin karışımı koyun. 400 xg / 5 at Santrifüj min / 20 ° C Pelet kaybetmeden süpernatant dikkatlice dökmek. 3 ml PBS içinde Pastör pipetiyle pelet. </li><li> Bindirme% 20 Percoll üzerine nazikçe karıştırarak, 8 mL Percoll ve 32 mL 0.25M sukroz (FW = 342,3 böylece 8,56 g sucrose/100 mL H 2 0), 40 mL gradyanı. </li><li> 10 dakika / 800 xg / 20 ° C Santrifüj Bir off Pipetid karaciğer hücrelerinin jelatinimsi üst kat atın. Bir transfer pipetle altındaki yumurta pelet çıkarın ve 15 ml Falcon tüp içine yerleştirilmiş. </li><li> 10 ml PBS / 1 mM EDTA / 1 mM EGTA kullanılarak santrifüj ayarlarda yıkama pelet 3X: 3 dakika / xg 30/20 ° C. 500 uL PBS içinde süspanse edin. </li><li> 10 dakika / 800 xg / 20 ° C: yeni bir% 25 Percoll, santrifüj ayarları kullanarak 10 ml degrade (2.5 mL Percoll ve 7.5 mL 0.25M sakkaroz) üzerinde Yerleşimi mix </li><li> Santrifüj ayarları kullanarak 10 ml PBS içinde 3X yıkayın: 3 dak / 30 xg / 20 ° C </li><li> Son 10 ml yıkama olarak, yumurta saymak için 100 uL kaldırın. Yumurta hızlı bir şekilde çözmek, Falcon tüp örnek çıkarmadan önce iyice karıştırılmalıdır yüzden. 900 uL PBS ile 100 uL örnek sulandırınız. Karışımı 100 uL bir diseksiyon mikroskobu (1:10 seyreltme) kullanarak bir mikroskop lamı üzerine damlacıklar olarak sayılmalıdır. Geniş çaplı iğne ucu kullanılmalıdır. </li><li> 50.000 yumurta / mL PBS içinde yumurta süspanse edin. Yumurta -80 ° C&#39;de birkaç ay depolanır ve bir veya TWIC çözülmüş olabilirkullanımı için e. Kullanmadan önce, yumurta (bkz. Şekil 1). Hala sağlam olduğundan emin olmak için mikroskop altında incelemek. </li></ol> 2. S. ile intraperitoneal Duyarlılık mansoni Yumurta: Günlük 0 <ol><li> 5 ml snapcap tüp içinde 5,000 eggs/100 uL PBS yumurta hazırlayın. Enjeksiyon için gerekli% 50 daha fazla yumurta tahmin ediyoruz. </li><li> Bir 23 gauge 3/4inch iğne ve fare başına yumurta süspansiyon 100 mcL ile 1 ml şırınga yükleyin. Yumurta kullanımdan hemen önce yumurta ile, bu nedenle yük iğne yerleşirler. </li><li> Önce her enjeksiyon için yumurtayı karıştırın ve fare başına süspansiyon intraperitoneal 100 uL enjekte etmek için ileri geri Kaya şırınga. </li></ol> 3. S. ile retroorbital İntravenöz Mücadelesi mansoni Yumurta: 14. Gün <ol><li> 5000 eggs/100 uL itibariyle yukarıda yumurta hazırlayın. </li><li> Ksilazin (10 mg / kg) ve ketamin (120 mg / kg) ile (% 4) izofluran veya intraperitonal olarak uzman odacık içinde farelerin uyuşturmak. Anest Derinliğihesia ayak yastığı çimdirilmesine ve herhangi bir fiziksel tepki sağlanması ile belirlenir. </li><li> Yumurta ile 1 ml şırınga ve 23 gauge 3/4inch iğne yüklemesini takiben, açı içine aşağı bakacak şekilde konik ile 90 ° açı ile iğne bükmek için forseps kullanabilir. Hiçbir kabarcıkları olduğundan emin olun. </li><li> Bir tarafta fare yerleştirin. Göz çıkıntı ve burun 45 ° açıyla gözün arkasındaki damarların içine 100 uL enjekte böylece cildin geri çekin. </li><li> Iğneyi çıkartınız ve kanama kontrolü için hafif basınç uygulayın. </li></ol> Tüm bu prosedürlerde dikkatli yapılmalıdır ve bunlar anestezi kurtarmak kadar farelerin izlenmesi gerekir. Bu protokoller Pennsylvania Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) Üniversitesi tarafından onaylanmıştır. 4. Deneysel Hasat: 22 Gün <ol><li> CO 2 ile fareler Euthanize. </li><li> Arka% 70 etanol ve yerine ıslak fare. </li><li> Künt Forc kullanmaeps, fare karın kavramak ve deride küçük bir kesi yapmak. Dikkatle makasla cilt kaldırmak ve göğüs kafesi ve karın zarı ortaya. </li><li> Periton açın kesin. Bir Pasteur pipeti ile kan kurtarmak için abdominal aort kesin. Buz üzerinde saklayın. </li><li> Diyafram maruz karaciğer aşağı hareket ettirin. Diyafram kesmek için ince makas kullanın. Dikkatle akciğer dokusunun açığa göğüs kafesinin açık kesti. </li><li> Ince forseps ile akciğer drenaj parathymic lenf düğümleri kurtarın. Bunlar timus 9 her iki tarafında, göğüs kafesinin altında sıkışmış vardır. 1mL steril ortam içinde buz üzerinde deposu (DMEM Invitrogen tüm% 10 ısı-inaktive edilmiş fetal buzağı serumu, 100 U / ml penisilin, 100 ug / ml streptomisin, 2 mM L-glutamin, 50 uM 2-merkaptoetanol, takviye). </li><li> BAL kurtarmak ve histolojik analiz için akciğerlerini şişirmek için, zor entübasyon yapılmalıdır. Tarafına tükürük bezlerinin hareket ettirerek trakea Açığa ve placitrakea altında ng ince forseps. Ince makas kullanarak, trakeanın ortasında bir delik ve ciğerlerine tüp yerleştirin. Cerrahi iplik ile bağlayarak boru sabitleyin. </li><li> Boru için PBS dolu 1 ml şırınga takın ve 1 mL PBS ile akciğerlerini şişirmek. Dikkatle şırınga ile BAL yıkama almak. Buz üzerinde saklayın. </li><li> Boru için PBS dolu 1 ml şırınga içinde% 4 paraformaldehit (PFA) takın ve akciğerleri şişirmek. </li><li> Tüp çıkarın ve PFA sızmasını önlemek için cerrahi iplik sıkın. </li><li> Dikkatle 5 ml% 4 PFA ile 50 ml Falcon tüp akciğer dokusu ve yerde dışarı incelemek. </li></ol> 5.. Kurtarılan Dokular hazırlanması <ol><li> Serum: Kan pıhtılaşma için 30 dakika için 2 saat süreyle buz üzerinde aşağıdaki depolama, serumu (13000 x g / dak 10/4 ° C) santrifüjle kandan geri kazanılır. Serum -20 ° C&#39;de muhafaza edilir ve sitokin ELISA&#39;lar ya S. için kullanılabilir mansoni yumurta antijen spesifik IgG izotip ELISA&#39;larla <sup&gt; 6, 10. </li><li> Lenf düğümleri: Tek hücre süspansiyonları hazırlandı ve hücre sayımı, bağışıklık cevabı büyüklüğü değerlendirmek için kullanılmıştır. Th2&#39;nin kutuplaşma incelemek için, hücrelerin S. ile tekrar uyarılır 72 saat mansoni yumurta antijen. Intracellar sitokin boyama akış sitometresi ile incelendi edilebilir, ve süpernatanlar Th2 sitokinlerin ELISA (IL-4, IL-5, IL-13) 6 kurtarılmış ve -20 ° C&#39;de saklanabilir. </li><li> BAL: Hücreler (400 xg / 5 dak / 4 ° C) pelet vardır. BAL sıvısı ELISA ile Th2 sitokin analizi için iyileşti ve -20 ° C&#39;de saklanır. BAL hücre sayımı hava yolu iltihabı için bir gösterge olarak kullanılır. 10,000-100,000 hücreleri (500 devir / dk 5/4 ° C) arasında Cytocentrifuge preparatlar, Diffquik boyama takiben inflamatuar sayımı 4, 6 infiltrasyonu izin verir. Alternatif olarak, BAL hücreler flow sitometri 6 tarafından muayene edilebilir. </li><li> Akciğer dokusunun: Akciğer dokusunun stor olduğunu4 &#39;de bir gece ed ° C. Sabit doku parafine gömülü, kesitli ve histolojik ve immünfloresan analiz için kızaklar üzerinde monte edilebilir. </li></ol> 6. Temsilcisi Sonuçlar Bu protokol detayları (i) in vivo kullanımı için Schistosoma mansoni yumurta (Şekil 1), (ii) duyarlı ve bu yumurta ile farelerin meydan saflaştırılmış ve (iii) akciğer inflamatuar yanıtın incelenmesi için organlarını kurtarmak hazırlamak için gerekli tüm adımları. Bu in vivo modelde tekrarlanabilir güçlü bir Th2 akciğer enflamatuvar yanıt yönlendirir. Bu gibi BAL hücre sayımı ve cytocentrifuge preparatları (Şekil 2) görselleştirilmiş hava yolu inflamasyonu ve eozinofili ile gösterilir. H &amp; E boyalı akciğer bölümleri yumurta bağlı granülomlar (Şekil 3a) için muayene ve akciğer bölümlerinin immünofloresan boyama alternatif aktive makrofajlar ve bu tür dirençler gibi Th2 sitokin kaynaklı genlerin görselleştirme sağlar edilebilirkalay gibi molekülü (AHLAK) α (Şekil 3b). Antijene spesifik CD4 Th2 sitokin yanıtı, IL-5 ELISA&#39;lar tarafından incelenmiştir, IL-4 ve antijen-uyarılmış parathymic lenf düğümü hücreleri IL-13 (Şekil 4). Edilir <img alt="Şekil 1" src="/files/ftp_upload/3905/3905fig1.jpg" /> Şekil 1 Schistosoma mansoni yumurta enjeksiyon modeli Temsilcisi yüksek S.mansoni-yumurta yüklerin karakteristik granülomatöz karaciğerinin resim, ve S.mansoni yumurta gösterilir -.. Ortalama büyüklüğü (L) (W) 50 mikron ile 120 mikron. <img alt="Şekil 2" src="/files/ftp_upload/3905/3905fig2.jpg" /> Şekil 2. S. mansoni yumurta enjeksiyon hava yolu inflamasyonu sürücüler. A. Naif (N) veya S.mansoni (Sm) yumurta enjekte edilen farelerin BAL hücre sayıları (x10 5). * P &lt;0.05. BAL hücrelerinin B. Cytocentrifuge hazırlanması. Mac, MAKROFAJ; Eos, eozinofil; LENF, lenfosit. Bar, 10 mikron. <img alt="Şekil 3" src="/files/ftp_upload/3905/3905fig3.jpg" /> Şekil 3. Pulmoner granülomlar ve S.mansoni yumurta çevresindeki alternatif aktive makrofajların görselleştirilmesi. A. Sm yumurta (siyah ok) çevreleyen Pulmoner granülom H &amp; E boyalı akciğer bölümlerde görüntülendi. RELMα B. immünofloresan boyaması (yeşil), mannoz reseptörü (kırmızı) ve DAPI (mavi) autofluorescent Sm yumurta (siyah ok) çevreleyen granüloma alternatif aktive makrofajlar (beyaz ok) açığa çıkarır. Bar, 50 mikron. <img alt="Şekil 4" src="/files/ftp_upload/3905/3905fig4.jpg" /> Şekil 4. S.mansoni yumurta antijen spesifik Th2 sitokin yanıtı. Yumurta enjekte naif (N) veya Sm farelerden parathymic lenf hücrelerinin boşaltılması Sm ile uyarıldı &lt;/ Em IL-4, IL-5, IL-13 için süpernatan ELISA ardından 72 saat boyunca&gt; yumurta antijeni,. Ölçek, ng / ml. * P &lt;0.05.

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Çıkar çatışması ilan etti.

Burada, tip 2 akciğer iltihabı teşvik için Schistosoma mansoni yumurta kullanan bir model açıklanmıştır. Bu modelin karakteristik özellikleri güçlü Th2 immün yanıt, hava yolu inflamasyonu ve pulmoner granülom oluşumu vardır. Bu parametreler CD4 Th2 hücre yanıtlarının 11 bağımlı olmaları nedeniyle, bu Th2 hücre yanıtları protein özgü veya soy-spesifik delesyonlar etkilerini araştırmak için yararlı bir modeldir. Burada okuma antijen spesifik Th2 sitokinlerin ölçüm?...

<table border="1"><tbody><tr><td> Reaktif </td><td> Boyutu / Açıklama </td><td> Şirket </td><td> # Kataloğa </td></tr><tr><td> Kollajenaz / Dispase </td><td> 500 mg </td><td> Roche Applied Science </td><td> 11 097 113 001 </td></tr><tr><td> Diff Quik Hema 3 Leke </td><td> Paketlemek </td><td> Fisher Scientific Co LLC </td><td> 122-911 </td></tr><tr><td> DMEM </td><td> Sıvı </td><td> Gibco </td><td> 11965 </td></tr><tr><td> Falcon 50mL </td><td> Paketlemek </td><td> Şahin </td><td> 352070 </td></tr><tr><td> Intramedic PE Boru </td><td> 0.58 mm </td><td> Becton Dickinson and Co </td><td> 427410 </td></tr><tr><td> Izofluran </td><td> 250 mL </td><td> Abbot Hayvan Sağlığı </td><td> 52600405 </td></tr><tr><td> Ketamin </td><td> 100 mg / mL </td><td> Fort Dodge, Hayvan Sağlığı </td&gt; <td> N / A </td></tr><tr><td> Manoz alıcı antikorunu </td><td> Biotin </td><td> AbD Serotec </td><td> MCA2235B </td></tr><tr><td> Metalik Süzgeç </td><td> Standart mutfak süzgeci </td><td> Hedef </td><td> Laurentiis </td></tr><tr><td> Paraformaldehyde </td><td> % 16 </td><td> Elektron Microsc. Bilimler </td><td> 15710 </td></tr><tr><td> Penisilin / streptomisin </td><td> 100x </td><td> Gibco </td><td> 15070 </td></tr><tr><td> Percoll </td><td> 1 Litre </td><td> GE Healthcare Biosciences </td><td> 17 01 0891 </td></tr><tr><td> RELMα antikoru </td><td> 50 mcg </td><td> Peprotech </td><td> 500-P214 </td></tr><tr><td> Cerrahi Konu </td><td> 2.0 USP 100 yds </td><td> Cerrahi Uzmanlıkları Corp </td><td> SP118 </td></tr><tr><td> Şırınga İğnesi </td><td> 23g, 3/4 inç </td><td> BD Vacutainer Lab. Med. </td><td>305143 </td></tr><tr><td> ABD Standart Test Elek </td><td> 150 um, 0,0059 &quot; </td><td> WS Tyler, Inc </td><td> ASTME-11, Spec. 100 sayılı </td></tr><tr><td> Ksilazin </td><td> 20 mg / ml </td><td> Baş uşak </td><td> N / A </td></tr></tbody></table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

Schistosoma parazitleri dünyada tahminen 200 milyon kişi 1 enfekte kan kurtları vardır. Schistosoma, karaciğer fibroz ve splenomegali de dahil olmak üzere ciddi patoloji, kronik enfeksiyon oldukça parazitin kendisini 2 den parazit yumurtaları için bağışıklık yanıtı kaynaklanır. Parazit yumurtaları, IL-4 üretimini, IL-5, IL-13, makrofaj alternatif aktivasyonu ve eozinofillerin alımı ile karakterize edilen, Th2 tepkisinin uyarılmasından. Burada, parazit özgü akciğer Th2 sitokin yanıtları ve lenf düğümleri, yumurta çevreleyen pulmoner granuloma oluşumu ve hava yolu inflamasyonu incelemek için bir model olarak Schistosoma mansoni yumurta enjeksiyonu açıklar. Intraperitoneal hassasiyete ve intravenöz meydan, S. ardından mansoni yumurta onlar akciğer parankimi içinde sıkışıp kalırlar pulmoner arterler yoluyla akciğere taşınırlenfositler, eozinofiller ve alternatif aktive makrofajlar 3-6 oluşan granülomlarlakarakterize. Bronko-alveolar alanlarda granülom oluşumu, inflamasyon ile ilişkili, lenf düğümleri ve CD4 genişleme T hücre aktivasyonu görülebilir. Burada ayrıntı, enfekte ciğeri (7 değiştirilerek) den Schistosoma mansoni yumurta tecrit fareler hassasiyet ve zorlu ve analizi için organların (bronko-alveolar lavaj (BAL), akciğer ve lenf düğümleri) kurtarmak için protokol. Biz de temsilcisi histolojik ve ek immünolojik analizi için immünolojik veriler ve öneriler içerir. Genel olarak, bu yöntem, in vivo modelde, bir parazit enfeksiyonu, fibrotik hastalıkları, alerjik enflamasyon ve astım da dahil olmak üzere Th2 enflamatuar hastalıkların çalışması için genel olarak uygulanabilir olan, akciğer parazit kaynaklı immünolojik yanıtları araştırmak için sağlamaktadır. Tipik bir çalışma için, bu modelde avantajlarıAkciğer e 2 iltihabı akciğer güçlü bir Th2 inflamatuvar yanıt tekrarlanabilirlik ve lenf düğümleri, yumurta çevreleyen granülomların histolojik inceleme ile inflamasyonun değerlendirilmesi kolaylığı ve parazit uzun süreli depolama için potansiyel bulunmaktadır yumurta.

Watch this Scientific Journal Video about Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation at JoVE.com

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni yumurta T hücre tip 2 (Th2) immün yanıt, parazit enfeksiyonu karakteristik, astım ve allerjik inflamasyonun güçlü uyarıcılar vardır. Bu protokol S. kullanır CD4 Th2 sitokin bağlı inflamatuar yumurtanın etrafında akciğer granülom oluşumu ile karakterize, akciğerin yanıt, eozinofili ve makrofaj aktivasyonu alternatif oluşturmak için mansoni yumurta enjeksiyon.

Gelen Yumurta kullanma<em&gt; Schistosoma mansoni</em&gt; Bir şekilde<em&gt; In vivo</emHelmint bağlı Akciğer iltihabı ve&gt; Model

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

using-eggs-from-schistosoma-mansoni-as-an-vivo-model-helminth-induced

Research

JoVE Journal

Immunology and Infection

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

13.7K Görüntüleme.  University of Pennsylvania. Schistosoma mansoni yumurta T hücre tip 2 (Th2) immün yanıt, parazit enfeksiyonu karakteristik, astım ve allerjik inflamasyonun güçlü uyarıcılar vardır. Bu protokol S. kullanır CD4 Th2 sitokin bağlı inflamatuar yumurtanın etrafında akciğer granülom oluşumu ile karakterize, akciğerin yanıt, eozinofili ve makrofaj aktivasyonu alternatif oluşturmak için mansoni yumurta enjeksiyon.

Video: Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Cercarial Dönüşüm ve In vitro Yetiştiriciliği Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

İmmünoloji ve Enfeksiyon

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host hastalığı indüksiyonu ve<em&gt; In Vivo</emBir MHC-eşlemeli murin Model Kullanma&gt; T Hücre İzleme

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo Görüntüleme Yöntemi Akut ve Kronik İltihap ayırt

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Bir<em&gt; Ex vivo</em&gt; Akut Beyin Dilimleri bir Oligodendrosit yönelik T-Hücre Saldırı Modeli

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Embriyone Tavuk Yumurta Grip Virüsü Yayılım

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

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 ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Roman bakteri özel optik tarama Ex Vivo insan Akciğer doku Confocal lazer Endomicroscopy tarafından probları

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Sitotoksik değerlendirilmesi ve insan hassas kesim akciğer dilimleri maddelerin etkileri Immunomodulatory

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Yalıtım ve yetişkin Schistosoma japonicum üzerinden ekstraselüler veziküller karakterizasyonu

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...