Bu çalışma, R01 AI32556 (NIAID, Ulusal Sağlık Enstitüsü) tarafından desteklenmiştir. Bu çalışma ayrıca, Louisiana Aşı Merkezi ve Güney Louisiana Louisiana Regents Kurulu tarafından desteklenen Enfeksiyon Hastalıkları Araştırma Enstitüsü tarafından desteklenmiştir.

1. Candida albicans ile Vajinal aşılama <ol><li> Aşılama Üç gün önce, karın ortaya çıkarmak için hayvan frenleyici, alt karın bölgesinde subkütan 0.1-0.5 mg β-östradiol içeren susam yağı enjekte 100 ul. Enjeksiyon yerinden sızıntı en aza indirmek için cilde yan 5 ila 10 mm iğne ilerletin. Alt karın östrojen subkutan uygulanması, genital sistem yakınlığı nedeniyle bu model en uygunudur. Etkili dozlarda fare suşlar, yaşları ya da östrojen türevleri göre değişebilir. CBA-J (H-2 κ), C3H/HeN (H-2 κ), C57BL / 6 (H-2 b), Balb / c (H-2 d) Daha önceki çalışmalarda, DBA / 2 (H- 2 d), yaş 6-8 hafta SJL (H-2 s) farelerde, 0.1 mg/100 ul etkili bulunmuştur vajinal duvar kalınlaşması ile kanıtlanır, vajinal mukus ve azaltılmışepitel hücre dökülmesi arttı. Yukarıdaki bu konsantrasyonda östrojen ile tedavi edilen farelerde, Candida ile tutarlı vajinal kolonizasyon sergilerler . Diğer suşları ve yaşları farelerde aşılama için bir pilot çalışma değiştirilmiş koşullar altında östrojen etkinliğini sağlamak ve gerekirse östrojen dozu artırmak için önerilir. Östrojen çözüm enjeksiyon gününde her zaman taze olarak hazırlanması gerekir. Susam yağı östrojen tam çözünürlük sağlamak için, iyice bir vorteks mikser ve ısı 37 ° aralıklı olarak kullanarak çözüm karıştırmak ° C Haftalık çalışma süresi boyunca enjeksiyon işlemi tekrarlayın. </li><li> Inokulum hazırlamak için, C. loopful ekleyin 10 ml% 0.1 glukoz ile desteklenen Phytone-pepton suyu içine Sabouraud dekstroz agar (SDA) bir son altkültürü hazırlık albicans blastoconidia. 25 ° C sallayarak bir su banyosunda 18 saat süreyle sabit faz suyu kültür inkübe edin. </li><li> Inkübasyon sonrasında, inci toplamake 5 dakika boyunca 800 xg&#39;de 15 ml konik boru ve santrifüj içine suyu kültürü. Pelet steril PBS ile iki kez yıkayın. </li><li> Tripan mavi boya dışlama hemasitometre canlı blastoconidia numaralandırır. Hücre konsantrasyonu 2.5 x 10 6 / ml steril PBS (ya da istediğiniz bir inokulum konsantrasyonu) ayarlayın. </li><li> Fare stabilize etmek için, iki parmak ile kuyruk tabanı tutun ve vajinal açıklık (Şekil 1A) dönüktür kalça yukarı kadar kaldırın. Fare, fare kuyruğu kısıtlama karşı direnç sağlar, böylece, düz bir rendelenmiş yüzey (örneğin kafesinin üst) yerleştirilir ise idealdir. </li><li> Pipet 20 ul vajinal lümen (Şekil 1B) derinliklerine yaklaşık 5 mm pipet ucu takarak inokulum süspansiyon (veya istenen hacmi 20 ul geçmemek üzere). Fare sıkıntı olduğunca çabuk ve nazik bir şekilde mümkün olan en aza indirmek için bu adımı tamamlayın. </li></ol> 2. Vajinal lavaj <ol><li> Aşağıdaki ötenazi (ya da genel anestezi), hvajinal açılış maruz olur böylece iki parmak ile kuyruk tabanı tarafından aşağı eski fare. </li><li> Bir pipet ucu ile tekrar aspirasyon ve ajitasyon ile steril PBS 100 ul getirerek vajinal lümen Lavaj. Pipet ucu hücreleri ile tıkanmış olabilir. Bu durum ortaya çıkarsa, engelleyen hücreleri dağıtmak ve lavaging vajina içinde kalan PBS ile devam edin. Bir mikrosantrifüj tüp içine lavaj sıvısı toplayın. </li><li> Alternatif olarak, vajinal lavaj izofluran uçucu anestezi ile anestezi fareler üzerinde yapılabilir. Bunun için, tam olarak (~ 30 sn.) Sedasyon kadar izofluran buharlaşmış farelerin maruz kalmaktadır. Farelerin kuyruk tabanı tarafından aşağı doğru tutun ve yavaşça 50 ul steril PBS kullanarak vajinal lümen lavajı. Bu işlem sırasında vajina travmayı en aza indirmek için bir pipet ucu ile sert bir ajitasyon önlemek için emin olun. Sedatize fareler ortam havasına maruz kalma 30 saniye içinde anestezi kurtarmak gerekir. İsofluranınizofluran bir buharlaştırıcı ve O 2 (tercih edilen) veya buharlaştırıcı sistemi (solunum sıkıntısı önlemek için sedasyon ise hayvanın yakın takip gerektirir) olmadan kapalı anestezi odasına standart bir damla sistemi kullanarak buharlaşmış olabilir. Anestezi fareler üzerinde Vajinal lavaj aynı fareler üzerinde uzunlamasına lavaj örnekleri için tercih edilen yöntem olmalıdır. Ardıl lavaging zaman 41 üzerinde mantar yükünün değerlendirilmesi etkilemez. </li><li> Lavaj sıvısı, cam bir slayt üzerine monte ıslak hazırlıkları için 10 ul transferi ve ışık mikroskobu ile 400-1000x büyütme az gözlemlemek. Ayrıca, lavaj sıvısının hücresel kesirler hücre ve nükleer morfolojileri incelemek için lekeli olabilir. Smear hazırlıkları için lavaj sıvısı, cam bir slayt üzerine 10 ul transferi ve dış duvar bir pipet kullanarak hafifçe yayılır. CytoPrep fiksatif ile smear örnekleri korumak ve standart Papanicolaou tekniği ile leke (Pap smear.) 400 × büyütmede ışık mikroskobu ile gözlemleyin. </li></ol> 3. Vajinal mantar yükünün Kantitasyonu <ol><li> 96 yuvarlak alt plaka, lavaj sıvısı, bu sütunun aşağıdaki 5 kuyu (tabak aşağı) içine üst satır ve 180 ul steril PBS iyi birine aktarın. </li><li> Sütun bir sonraki kuyuya 20 ul sıvı aktararak vajinal lavaj sıvısının 01:10 seri dilüsyonları olun. Her aktarmadan önce tekrarlanan aspirasyon ile iyice karıştırın. 12 lavaj örnekleri (tam bir yatay satır) kadar seri dilüsyonları 12-kanallı pipet kullanarak aynı anda yapılabilir. </li><li> Sabouraud dekstroz agar (SDA), en düşük sulandırma ile başlayarak, 10 ul üzerine örnek aktarmak. 36 örnekleri kadar Kaplama, 1 tabak kullanarak kare ızgara ve ayarlanabilir bir boşluk çok kanallı pipet petri kaplarına hazırlanan SDA yapılabilir. </li><li> 34 az inkübasyondan sonra koloni oluşturan birim (CFU) numaralandırır ° C fveya 48 saat </li></ol> 4. Vajinal doku ekstraksiyonu <ol><li> Vajinal lavaj prosedürü izleyerek, sırtında ötenazi fare yattı ve% 70 etanol ile kasık bölgesinde doyurabilecek. Bir çift forseps kullanarak, yukarı vajinal açılış maruz haline gelmesi idrar orifis kaldırın. </li><li> Kavisli bir forseps bir çift vajinal lümen içine yerleştirin ve serviks bulun. Forseps ile sağlam bir tutuş korurken, vajinal boşluğu (Şekil 2A-C) ile serviks ayıklayın. </li><li> Vajina Vergi üssünde vajinal açılış ve ardından cerrahi makas ile vajinadan rahim ağzı çıkarın. Vajinal dokuların (epitel tarafı vajina iç dışa açık) involuted olduğunu aklınızda bulundurun. Doku ya yanal vajinanın başlangıç ​​yönlendirmesinden korumak için ters veya lateral kesi yaparak bir sayfa açılıyordu olabilir. Eksize vajinal dokuların) 1 kullanılabilir lenfosit çekimini takiben kolajhistolojik analizler için 25 Nase sindirim (~ 1 × 10 4 / fare) 40, 2) epitel hücre izolasyonu aşağıdaki dispase sindirim (~ 5 × 10 4 / fare) 28, 3) dondurulmuş veya parafine gömülü preparatlar. </li></ol> 5. Bel lenf nodu eksizyonu <ol><li> Vajinal lavaj prosedürü izleyerek, sırtında ötenazi fare yattı ve% 70 etanol ile karın doyurabilecek. Lateral kesi alt karın göğüs başlangıç ​​yapın ve iç organların maruz. Iki eliyle bir çift forseps kullanarak, merkezi kan damarları görünür hale gelir bağırsak yukarı kadar hareket ettirin. </li><li> Inferior vena kava ve abdominal aorta bulun. Normalde, bir çift lomber lenf nodu, abdominal aort bitişik belirlenen yaklaşık yarım böbrek ve ortak iliak arterlerden 39 kökeni arasında yer olabilir. Bu lenf düğümleri görsel olarak yağ dokusunun elastik doku tarafından ayırt edilebilirve yağ dokusu (Şekil 3) ile karşılaştırıldığında daha açık renkli ve daha opak. Bu lenf düğümleri uninoculated hayvanlara göre, enfekte hayvanların belirgin şekilde daha büyük. </li><li> Vergi sonra lenf düğümü altında microforceps koyarak düğümleri ve doku çevreleyen ayrı hafifçe yukarı çekin. </li></ol> 6. Tek hücre süspansiyonları lenfoid hücrelerin izolasyonu <ol><li> ~ 10 ml Hanks dengeli tuz solüsyonu (HBSS) (Şekil 4) içeren bir steril cam petri içine yerleştirilen steril bir tel örgü ekran (boyut olarak yaklaşık 3 x 3 cm 2) üzerine lenf düğümleri aktarın. </li><li> Hafif eğimli petri ile karşı lenf düğümleri bir şırınga piston kafası ile ekran tuşuna basın. Düğümleri hücresel olmayan bileşenleri (yani, membranlar, stroma, yağ) ekranda kalırken düğümleri hücresel içeriği ekrana geçmesi, böylece tüm lenf düğümleri kırmak için emin olun. </li><li> Aynı piston ve şırınga, aspirat tiçeren hücrelerin HBSS. ~ 5 ml HBSS ile ekran yıkayın ve 15 ml konik bir tüp içine kalan sıvı toplamak. </li><li> 10 dakika için 800 xg&#39;de santrifüjleyin. Sıvı atarak önce bir pipet ile sıvının üst kısmında herhangi bir yağ mevduat aspire. HBSS hücre pelletini üç kez yıkayın. Tripan mavi boya dışlanma ve numaralandırmak canlı hücreler tarafından HBSS 1 ml pelletini tekrar. </li></ol> 7. Temsilcisi Sonuçlar: &gt; 4-günlük inoküle fareler vajinal lavaj sıvısının hücresel kesirler genellikle Candida, epitel hücreleri ve hücresel infiltratlar (Şekil 5) oluşur. Islak-montaj mikroskopi, Candida görsel hifler varlığı yanı sıra maya (Şekil 5A) tarafından tespit edilebilir. Ana hücreleri üç nucle tarafından belirlenen nötrofil olan epitel hücreleri ve infiltre lökositler, vajinal lavaj sıvısının Smear hazırlıkları incelemek için Papanicolaou tekniği ile boyananar loblar (Şekil 5B). Çok az sayıda nötrofil, eğer varsa, 41 uninoculated fareler (Şekil 5C) tespit edilir. Vajinal mantar yükünün bir örnek Şekil 6&#39;da gösterilmiştir. CFU sayımı (Şekil 6A) için belirli bir zaman noktalarında toplanan Vajinal lavaj sıvısı kültürü. Candida vajinal kolonizasyonu olmayan östrojen ile tedavi edilen farelerde (Şekil 6C) inoküle kurmak için başarısız olur, Candida vajinal kolonizasyon / enfeksiyon, östrojen tedavisi inoküle fareler (Şekil 6B) hafta boyunca devam etmektedir . Uninoculated östrojen ile tedavi edilen farelerde süresi (veriler gösterilmemiştir) boyunca Candida negatif kalır. Buna ek olarak, her zaman noktasında veya boyuna aynı farelerde anestezi altında vajinal lavaj ayrı fareler üzerinde ya bir kez gerçekleştirilir olabilir. Lomber lenf düğümleri, genital sistem ve vajinal sorun sistemik bağışıklık yanıtlarını değerlendirmek için en uygun site temel drenaj lenf düğümleri. Note uninoculated fareler normalde oldukça küçük görünen bu lenf düğümleri inoküle farelerde genişlemiş olabilir. Lökosit hücresel kurtarma genellikle 8 aralık × 10 5 / uninoculated fare 5 × 10 6 / inoküle fare. Lomber lenf bezlerine ek olarak, kasık, popliteal ve mezenterik lenf düğümleri de kullanılabilir. <img alt="Şekil 1" src="/files/ftp_upload/3382/3382fig1.jpg" /> Şekil 1. Candida ile Vajinal aşılama. A) fare inokülasyon için ölçülü. Fare, bir tel kafes eklemek yerleştirilir ve bacaklar hafifçe yukarı doğru kaldırın ve vajinal açılma ortaya çıkarmak için, kuyruk tabanı tarafından düzenlenen. Kuyruk kısıtlama direnmeye çalışır fare kalça aynı eli ile stabilize olabilir. B) inokulum vajinal lümen içine giriş. Bir pipet yavaşça vajinal lümen içine derin yaklaşık 5 mm eklenir. Süspansiyon inokulum sonra yatırılır. Şekil 2: Vajinal doku ekstraksiyonu . AB serviks) Ekstraksiyon. Serviks kavisli bir forseps ile yer ve vajinal boşluğu yoluyla dışa maruz kalmaktadır. Bir kez vajinal kavite, serviks, vajina tamamen açığa çıkarmak için daha fazla dışa çekilir. C) vajina Ekstraksiyon. Vajina, vulva makas ile eksize edilir. Müstakil sonra, vajinadan rahim ağzı çıkarın. <img alt="Şekil 3" src="/files/ftp_upload/3382/3382fig3.jpg" /> Şekil 3. lomber lenf nodlarının belirlenmesi. Pelvis çevresinde, çevre organlara / kan damarları arasında lomber lenf nodu yeri gösterilir. A, abdominal aorta. B, mesane. C, iliyak arter. Ben, bağırsak. L, karaciğer. R, rektum. S, dalak. U, Uteri. <img alt="Şekil 4" src="/files/ftp_upload/3382/3382fig4.jpg" /> Şekil 4, bir tel örgü ekran üzerine yerleştirilen lomber lenf düğümleri . Lenf düğümleri, HBSS ile bir petri kabına yerleştirilir ekrana toplanmış. Lenf düğümleri tek hücre süspansiyonları lenfoid hücreleri elde etmek için bir şırınga pistonu ile ekrana bastırılır. <img alt="Şekil 5" src="/files/ftp_upload/3382/3382fig5.jpg" /> Şekil 5 inoküle farelerden alınan vajinal lavaj sıvısı Hücresel kesirler. Vajinal lavaj örneklerinin A) Islak mount ve B) Pap smear preparatları uninoculated farelerden alınan) sonrası aşılama ve C 4 gün toplanan. Görüntüler 1000 × (A) veya 400 × (B, C) büyütme. B eklemek 1000 × vajinal nötrofil nükleer morfoloji göstermektedir. Candida maya (Y) ve hif (H), epitel hücreleri (EC) ve neutrohils (N) gösterilir. <img alt="Şekil 6" src="/files/ftp_upload/3382/3382fig6.jpg" /> Şekil 6: Algılamavajinal mantar yükünün iyon. A) Temsilcisi C. albicans kolonileri SDA plaka üzerinde büyüdü. Neat (N) altı (üst satırda) farklı inoküle fareler lavaj örnekleri seri olarak seyreltilmiş ve CFU sayımı için kültüre edildi. B) vajinal mantar yükünün Niceleme östrojen ile tedavi edilen ve C) non-östrojen ile tedavi edilen farelerde. Inoküle farelerden alınan lavaj sıvısı CFU/100 ul belirtilen zaman noktalarında değerlendirildi.

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Çıkar çatışması ilan etti.

Candida vajinit, deneysel bir fare model kurulmuş ve tarihsel olarak son birkaç yıldır kullanılan 3,4,11,13,16,17,19,21,24 antifungal tedaviler test etmek için Candida mukozal konak yanıtının yanı sıra çalışma olmuştur 25,37. Protokoller, burada verimli ve daha az emek-yoğun yöntemleri dahil, ve Candida vajinit en optimize edilmiş bir model sistemleri bugüne kadar açıklanan gibi görünen sundu. Bu teknikler hızlı kantifikasyon vajinal ö...

<table border="1"><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Kadın CBA / J fareler </td><td> Charles River Laboratuvarları </td><td> 01C38 </td><td> 5-6 hafta </td></tr><tr><td> Candida albicans (3153A) </td><td> Patojenik Mantarlar, Birleşik Krallık Ulusal Koleksiyonu </td><td> NCPF3153 </td><td></td></tr><tr><td> Susam yağı </td><td> Sigma-Aldrich </td><td> S3547 </td><td> Kullanmadan önce ön sterilize edilmek üzere ihtiyaç duymaz </td></tr><tr><td> Β-östradiol 17-valerat </td><td> Sigma-Aldrich </td><td> E1631 </td><td> Susam yağı 0.1-0.5mg </td></tr><tr><td> Phytone pepton </td><td> Becton Dickinson </td><td> 211906 </td><td> % 0.1 glukoz ile Ek </td></tr><tr><td> Tripan mavi çözümü </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Sabouraud dekstroz agar </td><td> Becton Dickinson </td><td> 211584 </td><td></td></tr><tr><td> Kollajenaz tip IV </td><td> Sigma-Aldrich </td><td> C5138 </td><td> % 0.25 </td></tr><tr><td> Dispase </td><td> Invitrogen </td><td> 17105-041 </td><td> 1.7 U / ml </td></tr><tr><td> Tel örgü ekranlar </td><td> TWP </td><td> 060X060S0065W36T </td><td> No 60 mesh, paslanmaz </td></tr><tr><td> Hanks dengeli tuz çözeltisi </td><td> Invitrogen </td><td> 24020-117 </td><td></td></tr><tr><td> CytoPrep fiksatif </td><td> Fisher Scientific </td><td> 12-570-10 </td><td> Smear slaytlar korur </td></tr><tr><td> Leke Papanicolaou EA-65 </td><td> Merck Kimyasallar </td><td> 7054X-85 </td><td></td></tr><tr><td> Leke Papanicolaou RG-6 </td><td> Merck Kimyasallar </td><td> 7052X-85 </td><td></td></tr><tr><td> Harris &#39;Şap hematoksilen </td><td> Merck Kimyasallar </td><td> 638A-85 </td><td></td></tr><tr><td> İsofluranın </td><td> Baxter Healthcare </td><td> NDC 10019-773-60 </td><td> Izofluran buharlaştırıcı ya da bir damla sistemi el anestezi kamara kapalı </td></tr></table>

protocols for vaginal inoculation and sample collection in the experimental mouse model of candida vaginitis

Vulvovajinal kandidiazis (VVC), Candida türlerinin neden olduğu, aksi takdirde sağlıklı kadınların yaklaşık% 75&#39;i üreme yıl boyunca 18,32-34 etkileyen alt dişi genital sistemin bir mantar enfeksiyonu. Predispozan faktörler, antibiyotik kullanımı, kontrolsüz diyabet ve rahatsızlık üreme hormon düzeyleri gebeliğe bağlı, oral kontraseptif veya hormon replasman tedavileri 33,34 içerir . Tekrarlayan VVC (RVVC) yılda üç veya daha fazla bölüm olarak tanımlanan, hiçbir predispozan faktör 33 ile% 8 kadın için ayrı bir 5 etkiler. VVC deneysel bir fare modeli kurulmuş ve patogenezi ve Candida 3,4,11,16,17,19,21,25,37 mukozal konak yanıtı çalışma için kullanılan olmuştur. Bu model aynı zamanda, in vivo 13,24 potansiyel antifungal tedaviler test etmek için işe alındı ​​. Modeli hayvanlar opt pseudoestrus bir devlet muhafaza gerektirirolmuş bölgeler Candida kolonizasyonu / infeksiyonu 6,14,23. Bu koşullar altında, haftalar veya aylar için inoküle hayvanlar vajinal mantar yükünün saptanabilir olacaktır. Geçmişte yapılan çalışmalar, immünolojik ve fizyolojik özellikleri 3,16,21 göre hayvan modeli ve insan enfeksiyonu arasındaki son derece yüksek bir paralellik göstermektedir. Farklılıklar Ancak, normal vajinal flora olarak Candida eksikliği ve farelerde nötr bir vajinal pH içerir. Burada, bir dizi vajinal aşılama, vajinal örneklerin hızlı toplanması, vajinal mantar yükünün değerlendirilmesi ve hücresel çıkarma / izolasyonu için doku hazırlıkları fare vajinit modeli temel yöntemler göstermektedir. Bu vajinal lavaj sıvısı, mantar yükünü, ve drenaj lenf nodu lökosit verim bileşenlerinin temsilcisi sonuçları takip edilmektedir. Anesteziklerin kullanımı ile lavaj örnekleri boyuna değerlendirilmesi için aynı fareler üzerinde birden fazla zaman noktalarında toplananenfeksiyon / kolonizasyon. Ayrıca, tanımlanmış ana koşullar altında immünolojik çalışmalar sağlayan bu model, enfeksiyon başlatmak için herhangi bir immünosupresif ajanlar gerektirir. Son olarak, model ve her iki tekniğin burada potansiyel düşük kadın genital sistem (viral, parazitik, bakteriyel) ve ilgili lokal veya sistemik konak savunmasını diğer bulaşıcı hastalıkları incelemek için metodolojilerin kullanımı doğurabilecek tanıttı.

Watch this Scientific Journal Video about Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis at JoVE.com

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Değerlendirilmesinde kullanılacak kilit teknikleri<em&gt; Candida</em&gt; Vajinit deneysel bir hayvan modeli anlatılmaktadır. Yöntemleri, lomber lenf düğümleri drenaj, vajinal örneklerin ve lenfositler hızlı toplama sağlayacaktır. Bu teknikler, kadın alt genital sistem diğer hastalıkların fare modelleri yol açabilir.

Deneysel Fare Modeli Vajinal Aşılama ve Örnek Toplama Protokolleri Candida Vajinit

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

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Immunology and Infection

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

32.7K Görüntüleme.  Louisiana State University Health Sciences Center. Değerlendirilmesinde kullanılacak kilit teknikleri Candida Vajinit deneysel bir hayvan modeli anlatılmaktadır. Yöntemleri, lomber lenf düğümleri drenaj, vajinal örneklerin ve lenfositler hızlı toplama sağlayacaktır. Bu teknikler, kadın alt genital sistem diğer hastalıkların fare modelleri yol açabilir.

Video: Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

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

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Deneysel Otoimmün hipofizitis İndüksiyon Fare Hipofiz İmmunogen hazırlanması

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Bir odacıklı coverglass Modeli Kullanılarak Biyofilm Kalkınma Sputum Etkilerinin görselleştirme

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

HIV ile ilişkili Karaciğer Patogenezi için Çift İnsanize TK-NOG Fare Modelinin Oluşturulması

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Hemolymph toplama ve Metarhizyum blastosporların omurgasız patoloji çalışmalarına doğru Rhipicephalus Microplus ticks içine inoculation

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

Viral Kaynaklı Nöroinflamatuar Yanıtları İncelemek Için Fare Ayak Takımı Aşılama Modeli

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Deneysel Otoimmün Üveit: Göz İçi İnflamatuar Bir Fare Modeli

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Biliyer atrezi çalışması için kronik karaciğer fibrozisinin bir fare modeli

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Procedure for Swine Burn Wounding, Burn Wound Imaging, Wound Inoculation, and Biopsy Collection

To begin the procedure inside the properly arranged surgery room, place the anesthetized pig in the sternal recumbency position, and ensure all the connected lines and tubes are secured. Remove any residual hairs from the shaved area designated for wounding using hair removal cream. After sterilizing the wound sites, mark the area using a sterile burn template and a surgical skin marker.Set the burner by adjusting the set point on the control unit to 150 degrees celsius. The low set point to 145 degrees Celsius and the high set point to 155 degrees Celsius. Using a heated stainless steel blocks connected to the burn device, create a full thickness burn wound by placing the blocks on the skin for 60 seconds.Take a digital photo of the whole back of the pig, including a placard with the pig's identification time point and date. Then separately take images for each wound along with a ruler and a placard displaying the pig ID, wound ID and time point. To assess the wound microvascular perfusion in real time using laser speckle imaging or LSI, image all the wounds in a single recording using a LASCA blood perfusion imager.To measure trans epidermal water loss or TEWL, place a clean probe cover over the TEWL probe tip, which will be in contact with the wound tissue. Measure each wound five times, first in the center, followed by each corner. Then export all the readings to a spreadsheet.Perform a harmonic ultrasound or HUSD mapping by scanning the wound with an ultrasound probe from the midline or vertebral column, starting from normal skin and moving towards the lateral side of the pig over the wounds until the normal skin is again reached. Follow the scanning pattern for each wound in both B mode and tissue elastography mode. Change the annotation after each wound is imaged in both B mode and elasto mode, which is two recordings per wound.Once all the imaging is complete, cover the burn wounds individually with transparent film dressings or the test dressing. Then place a larger transparent film dressing over the entire wound area. Apply a second layer of roll gauze loosely around the entire trunk of the pig to absorb any fluid exuding from the wounds.Cover the gauze loosely with a layer of flexible elastic bandage. Cover the elastic bandage with a final layer of four inch elastic tape. After discontinuing the anesthetic gas upon completion of the bandaging, allow the pig to remain on oxygen for at least five minutes.To inoculate the burn wounds on day three post burn, disperse the inoculum across the surface of the exposed wounds using a pipette, and spread it evenly using a disposable spreader. To collect the tissue biopsies for analysis following imaging, infiltrate the area around the wound with 0.5%bupivacaine. Using a disposable scalpel with a size 10 blade, cut a 3-4 millimeter wide strip from one edge of the wound to the other, keeping small margins of normal skin on both sides.Cut a 6 millimeter punch biopsy from the wound either from the wound bed or wound edge. Achieve hemostasis after the samples are collected by gently pressing the wound with a sterile gauze. Cover the wound with a non-adherent dressing and bandage it as demonstrated previously.Analysis of the biopsy sample by hematoxylin and Eosin staining showed distortion and necrosis of all the histological skin layers. The established biofilm was further validated at day seven post inoculation by colony forming unit. Scan the electron microscopy imaging, and immunofluorescence staining.The burn wounds showed a thick biogenic membrane on the wound surface by day seven post inoculation, confirming the infection and establishment of biofilm. The wound area started to shrink by day 14, but incomplete healing to about 25%of the original wound size was observed by day 56 indicating wound chronicity. Wound chronicity and impaired wound healing were further confirmed by the TEWL showing high trans epidermal water loss at all measured time points.This was also confirmed by the suppression of the tight junctional proteins, ZO-1 and 2, and the impaired restoration of skin barrier function.