Yazarlar Freiburg Üniversitesi (CS) Tıp Fakültesi Deutsche Forschungsgemeinschaft SI-1281/4-1 ve BIOS&#39;lar hibeleri ile destek için minnettarım.

1. Patojenik antikorun hazırlanması Not: antikorlar gece boyunca 0.1 M glisin pH 2.5-3 (elüsyon tampon) (ON) saklanan olmamak gibi Saflaştırılması ve antikor konsantrasyonu, aynı gün yapılmalıdır. IgG Affinity saflaştırılması: Bağışıklık tavşan serumu 25 ml kullanın: <ol><li> 4. ON tavşan serumu çözülme ° C, 20 ° C de 1xPBS (tampon çalıştıran) ile 1:1 Karışım, 10 dakika boyunca 1260 x g de santrifüjlenir İsteğe bağlı olarak, filtre kağıdı ile filtre adım serum yağ olması durumunda santrifüj sonrası dahil olabilir. </li><li> Bu arada, G matrisi 1XPBS (tampon) çalışan 10 yatak hacmi (yatak hacmi = matriks hacmi) ile sütun doldurulur proteini yıkayın. </li><li> Seyreltildi ve sütuna uygulanır serum filtre edilmiş ve oda sıcaklığında (RT) de sallanan platform üzerinde 1 saat inkübe edilir. </li><li> Collect akan (FT) 50 ml Flakon tüp. Bunu atmayın konsantrasyona kadarve saflaştırılmış IgG titreleri bilinmektedir. </li><li> 1XPBS 5 yatak hacmi ile matris yıkayın ve arada Elüsyonlanan IgG fraksiyonu toplamak için 50 ml Falcon tüpleri hazırlayın. Pipet 1 ml pH nötralize edici tamponu: her bir tüp içinde 1 M Tris pH 10 (aksi takdirde elde edilecek arındırıldı ve miktarına bağlı olarak 1:20 TRIS tampon oranı). </li><li> Sütun 0.1 M glisin pH 2.5-3 ve 50 ml fraksiyon (ler) toplamak: 100-150 ml elüsyon tamponu uygulanır. , Tüp 2-3x çevirin pH ölçümü ve pH 7.2-7.4 ulaşana kadar nötralize tampon eklemek. </li><li> IgG ile arındırıldı ve birkaç damla toplayın ve etalon 280 nm&#39;de OD ölçmek olarak elüsyon tamponu kullanılarak. OD &lt;0.1 durağı koleksiyonculuğu ise. </li><li> Protein G matrisi Regenerate ve üreticinin yönergelerini izleyerek saklayın. </li></ol> Amicon tüpleri ile ultrafiltrasyon ile Konsantrasyon: <ol><li> 15-20 dakika santrifüj edilerek 1XPBS ile Amicon Ultra (15 ml / 30KDa) ultrafiltrasyon tüpler Yıkama4 ° C&#39;de 3.220 x g&#39;da Amicon gelen FT atın. </li><li> 25-30 3.220 xg&#39;de dk ve 4 için yıkandı, IgG fraksiyonu ve santrifüj 15 ml Amicons doldurun ° C; santrifüjleme zamanı hep eluat protein içeriğine bağlıdır. </li><li> FT atın ve sol hiçbir Elüsyonlanan Kesir kadar ultrafiltrasyon tekrarlayın. </li><li> Santrifüjle 1XPBS 25-30 dk, yoğun, iki kez konsantre IgG yıkayın. </li><li> 1XPBS 1,500-2,000 arasında ul nihai hacim içinde Amicon filtreden &quot;yapışkan&quot;, sarımsı bir çözelti antikor toplayın. </li><li> Bir spektrofotometre Nanodrop kullanılarak IgG hazırlık Tedbir konsantrasyonu: Kons. (Mg / ml) = A (280 nm) x seyreltme faktörü / 1,4 </li><li> Üreticinin yönergelerini takip Amicons yıkayın ve saklayın. </li></ol> 2. Fareler içine Kollajen VII-spesifik IgG Enjeksiyon Gerçek deneysel prosedür başlamadan önce, emin olun deneysel proprotokolünü yazılır ve tüm malzemeler deney için hazır edilir. <ol><li> Hastalığın puanlama, ELISA&#39;lar, immünofloresan (IF) analizi ve miyeloperoksidaz (MPO) testi 4 veri sayfalarına hazırlayın. </li><li> Kan ve organlar, cryomolds yanı sıra histolojik işleme ve kasetler gömmek için tüpler etiketleyin. </li><li> Antikor çözeltileri taze hazırlanmış ya da stoklarından çözülmüş ve (IF mikroskopi ve / veya ELISA ile titre) kendi reaktivite bakımından karakterize edilmelidir. Steril filtre edilmiş antikor solüsyon enjeksiyon için gerekli doz ul 250-1000 arasında değişen bir şekilde PBS ile seyreltilmelidir. Eğer tüm deneme gerçekleştirmek için yeterli IgG sahip olduğunuzdan emin olun. </li><li> Hazırlayın taze antikoagülan: Heparin 20 U / ml ve 8.7 mg / ml ketamin / anestezi için 1.3 mg / ml xylazine karışımı. Farelerde hazırlanan% 3.7 formaldehit ile tüpler etiketli ödün zaman. </li><li> (Neşter, makas, ince noktası ve curv cerrahi aletler sterilizeed forcipes) ve şırıngalar (insülin şırıngaları, 1 ve 2 ml şırınga), iğneler, dijital kamera ve ekstra hafıza kartı hazırlayın. </li></ol> Not: ketamin / xylazine veya izofluran uyuşturulur fareler üzerindeki tüm prosedürleri gerçekleştirin fareler hızlı bir şekilde kurtarmak gibi İzofluran, günlük cilt kontrolleri için tercih edilen anestezi seçenektir.. Ancak, resim, 87 mg / kg ketamin ve 13 mg / kg xylazine çekerken genellikle cilt altına enjekte edilir. Ötenazi için ketamin / xylazine doz 130 mg / kg ketamin ve 20 mg / kg xylazine yükselmiştir. <ol><li> Genel sağlık durumu, deri ve kürk görünümü için zaten işaretlenmiş hayvanlar kontrol, onları tartmak ve (her zaman aynı kulak sopa) onların kulak kalınlığını ölçmek. </li><li> Gözlenen değerler ve klinik değerlendirme levha değişiklikleri kaydedin. </li><li> Antikoagülan aynı hacimde içeren bir şırınga içinde, kuyruk veni 20-30 ul (2-3 damla), kan toplamak. </li><li> Kürk Dezenfekte ve% 80 etanol kullanılarak cilt. </li><li> . Subkutan insülin şırınga kullanarak arkasına antikor çözüm enjekte (bazı sitelerde (örneğin, kulaklar) için sadece küçük miktarlarda (10-50 ul) enjekte edilmesi mümkün bulunmaktadır.) </li></ol> Enjeksiyonlar, ikinci günde 4 kez tekrar edilmelidir. Balb / c ve yaklaşık olarak 20 g vücut ağırlığı C57BL6 fareler 3-4 günde 750 mcg / g vücut ağırlığı / enjeksiyon otoantikor için 400-500 ilk uygulamadan sonra hastalık geliştirme başlar. Deney çalışması bittikten sonra: <ol><li> 130 mg / kg ve ketamin / xylazine subkutan 20 mg / kg enjekte ederek derin narkoz altında fareler koyun, posterior vena kava onları exsanguinate ve kalbini sökecek. </li><li> Deri gibi organ / doku örnekleri toplayın: kuyruk, kulak, kan ve özofagus ve önceden etiketlenmiş ve PBS veya% 3,7 formaldehit dolu tüplerin koydum. </li></ol> Dönen zamanlaboratuvar: <ol><li> 1.200 xg, ayrı plazma RT 10 dak kan santrifüj, düzgün etiketli tüplere plazma aktarmak ve onları uygun şekilde saklayın. </li><li> Cilde düzgün cryomolds, etiket kullanarak Optimal Kesme Sıcaklık orta biyopsisi ve -80 saklayın Embed ° C. </li><li> Etiketli gömme kalıplara histoloji içindir doku örnekleri yerleştirin ve parafine gömme kadar% 3,7 formaldehit içinde saklayın. </li></ol> 3. Hastalık Şiddeti Klinik Değerlendirme Fare deri ve mukoza lezyonları için günlük muayene edilmeli ve bulguların uygun formları kullanarak kayıt edilmelidir. Erken ötanazi için kriterler 5 bir hastalık skoru (Tablo 1) sayma, daha o 20 vücut yüzeyinin% ve üç gün üst üste toplam vücut ağırlığının% 5-10 kilo kaybı etkileyen deri lezyonları olduğunu. <ol><li> Karnı üzerinde fare yerleştirin. Sağ, yani baş bölgesi ile başlayınkulak ve kabarcıklar, erozyonlar, iç kabuk üzerinde hem de dış tarafında kontrol edin. Sol kulak ile aynı şekilde devam edin. </li><li> Sağ ve sol gözler alopesi, eritem belirtileri için kontrol, erozyonlar ve / veya kabuk vardır. </li><li> Alın ve burun alan burun, dudak ve ağız mukoza ventral kısmı ile devam eden, önümüzdeki aracılığıyla fırçalanır. </li><li> Yukarı hareketli, pençe itibaren sağ ön bacak inceleyin; sol ön bacağa geçin. Sağ ve sol arka ayakları ile aynı prosedür. </li><li> Kavisli bir ince nokta forseps ile boyun ve sırt alanda kürk ile fırçalayın. </li><li> Sırt üstü çevirin ve fare karın bölümü ile birlikte uzuvların ventral tarafı önceden olduğu gibi aynı şekilde kontrol edin. </li><li> Kuyruk kontrol edin. </li><li> Farelerde hastalığı geliştirmek vücudun ilgili fotoğraflarını çekin. Dikkat resim kalitesi ve miktarına dikkat edilmelidir. </li><li> Klinik hastalık puanları hesaplayın. </li></ol> 4. Cilt ve Plazma Örneklerinin Analizi <ol><li> , Mağaza ve / hazırlayın veya planına göre toplanan tüm dokuları işlemek. </li><li> Dondurulmuş doku kesitlerinde Cut IF tavşan antikorları ve fare kompleman sistem bileşenlerinin tespiti. </li><li> Dondurulmuş doku ve / veya organ ekstre farklı enzim ve protein içeriği (MPO tahlil) analiz edin. </li><li> Bölüm parafine gömülü dokuda ve doku hasarı ölçüde görselleştirmek için H &amp; E ile leke. </li><li> ELISA, immunoblotting tarafından plazma analiz edin. </li></ol>

<ol>
	<li>Sitaru, C., Mihai, S., Otto, C., Chiriac, M. T., Hausser, I., Dotterweich, B. <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+dermal-epidermal+separation+in+mice+by+passive+transfer+of+antibodies+specific+to+type+VII+collagen.">Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen.</a> J. Clin. Invest. 115, 870-878 (2005).</li><li>Kopecki, Z., Arkell, R. M., Strudwick, X. L., Hirose, M., Ludwig, R. J., Kern, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+Flii+gene+increases+dermal-epidermal+blistering+in+an+autoimmune+ColVII+mouse+model+of+epidermolysis+bullosa+acquisita.">Overexpression of the Flii gene increases dermal-epidermal blistering in an autoimmune ColVII mouse model of epidermolysis bullosa acquisita.</a> J. Pathol. 225, 401-413 (2011).</li><li>Kulkarni, S., Sitaru, C., Jakus, Z., Anderson, K. E., Damoulakis, G., Davidson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K&#946;+plays+a+critical+role+in+neutrophil+activation+by+immune+complexes.">PI3K&#946; plays a critical role in neutrophil activation by immune complexes.</a> Sci. Signal. 4, 23-23 (2011).</li><li>Chiriac, M. T., Roesler, J., Sindrilaru, A., Scharffetter-Kochanek, K., Zillikens, D., Sitaru, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+is+required+for+neutrophil-dependent+autoantibody-induced+tissue+damage.">NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.</a> J. Pathol. 212, 56-65 (2007).</li><li>Mihai, S., Chiriac, M. T., Takahashi, K., Thurman, J. M., Holers, V. M., Zillikens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alternative+pathway+of+complement+activation+is+critical+for+blister+induction+in+experimental+epidermolysis+bullosa+acquisita.">The alternative pathway of complement activation is critical for blister induction in experimental epidermolysis bullosa acquisita.</a> J. Immunol. 178, 6514-6521 (2007).</li><li>Sesarman, A., Sitaru, A. G., Olaru, F., Zillikens, D., Sitaru, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Fc+receptor+deficiency+protects+from+tissue+injury+in+experimental+epidermolysis+bullosa+acquisita.">Neonatal Fc receptor deficiency protects from tissue injury in experimental epidermolysis bullosa acquisita.</a> J. Mol. Med. 86, 951-959 (2008).</li><li>Huang, X. R., Holdsworth, S. R., Tipping, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2+responses+induce+humorally+mediated+injury+in+experimental+anti-glomerular+basement+membrane+glomerulonephritis.">Th2 responses induce humorally mediated injury in experimental anti-glomerular basement membrane glomerulonephritis.</a> J. Am. Soc. Nephrol. 8, 1101-1108 (1997).</li><li>Matsumoto, I., Staub, A., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthritis+provoked+by+linked+T+and+B+cell+recognition+of+a+glycolytic+enzyme.">Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme.</a> Science. 286, 1732-1735 (1999).</li><li>Oswald, E., Sesarman, A., Franzke, C., W&#246;lfle, U., Bruckner-Tuderman, L., Jakob, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flavonoid+luteolin+inhibits+Fc&#947;-dependent+respiratory+burst+in+granulocytes,+but+not+skin+blistering+in+a+new+model+of+pemphigoid+in+adult+mice.">The flavonoid luteolin inhibits Fc&#947;-dependent respiratory burst in granulocytes, but not skin blistering in a new model of pemphigoid in adult mice.</a> PLoS One. 7, e31066 (2012).</li><li>Liu, Z., Diaz, L. A., Troy, J. L., Taylor, A. F., Emery, D. J., Fairley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+passive+transfer+model+of+the+organ-specific+autoimmune+disease,+bullous+pemphigoid,+using+antibodies+generated+against+the+hemidesmosomal+antigen,+BP180.">A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.</a> J. Clin. Invest. 92, 2480-2488 (1993).</li><li>Anhalt, G. J., Labib, R. S., Voorhees, J. J., Beals, T. F., Diaz, L. A. <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+pemphigus+in+neonatal+mice+by+passive+transfer+of+IgG+from+patients+with+the+disease.">Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.</a> N. Engl. J. Med. 306, 1189-1196 (1982).</li><li>Toyka, K. V., Brachman, D. B., Pestronk, A., Kao, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myasthenia+gravis:+passive+transfer+from+man+to+mouse.">Myasthenia gravis: passive transfer from man to mouse.</a> Science. 190, 397-399 (1975).</li><li>Rose, N. R., Bona, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+criteria+for+autoimmune+diseases+(Witebsky's+postulates+revisited.">Defining criteria for autoimmune diseases (Witebsky's postulates revisited.</a> Immunol. Today. 14, 426-430 (1993).</li><li>Sitaru, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+epidermolysis+bullosa+acquisita.">Experimental models of epidermolysis bullosa acquisita.</a> Exp. Dermatol. 16, 520-531 (2007).</li><li>Sitaru, C., Kromminga, A., Hashimoto, T., Br&#246;cker, E. B., Zillikens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+to+type+VII+collagen+mediate+Fcgamma-dependent+neutrophil+activation+and+induce+dermal-epidermal+separation+in+cryosections+of+human+skin.">Autoantibodies to type VII collagen mediate Fcgamma-dependent neutrophil activation and induce dermal-epidermal separation in cryosections of human skin.</a> Am. J. Pathol. 161, 301-311 (2002).</li><li>Sesarman, A., Oswald, E., Chiriac, M. T., Csorba, K., Vuta, V., Feldrihan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+human+pemphigoid+autoantibodies+do+not+trigger+disease+by+the+passive+transfer+into+mice.">Why human pemphigoid autoantibodies do not trigger disease by the passive transfer into mice.</a> Immunol. Lett. , (2012).</li><li>Ishii, N., Recke, A., Mihai, S., Hirose, M., Hashimoto, T., Zillikens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibody-induced+intestinal+inflammation+and+weight+loss+in+experimental+epidermolysis+bullosa+acquisita.">Autoantibody-induced intestinal inflammation and weight loss in experimental epidermolysis bullosa acquisita.</a> J. Pathol. 224, 234-244 (2011).</li></ol>

Çıkar çatışması ilan etti.

Deney hayvanlarının içine otoantikorların pasif transfer onların patojenite 7, -12 göstermek için önemli bir yaklaşımdır. Bu yöntemle elde edilen hayvan modelleri üzerinde, otoimmünite 13 için dolaylı kanıt olmanın yanı sıra, patojenik mekanizmasının efferent faz inceleme sağlar. Epidermolizis bülloza (EBA) ve kabarma antikorun neden granülosit-bağımlı cilt pasif transfer modeli otoimmün dermatozlarda doku hasarının mekanizmaları incelemek için kullanılmıştır. B...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> rekombinant antijen </td><td></td><td></td><td> Mürin kolajen VII rekombinant biçimleri için kodlayan plasmidin karşılık gelen yazar temin edilebilir. </td></tr><tr><td> immun tavşan serumu </td><td> <a href="http://www.eurogentec.com/" target="_blank">www.eurogentec.com</a> </td><td></td><td> Biz antijen 200 mikrogram, 2 haftalık aralıklarla 3 kez ile immünize Beyaz Yeni Zelanda tavşan vardı. Bu amaçla Eurogentec SA, Belçika hizmetleri kullandık. </td></tr><tr><td> Protein A agaroz </td><td> Roche Uygulamalı Bilimler </td><td> 11243233001 </td><td></td></tr><tr><td> Balb / c fareleri </td><td> Charles River Laboratories </td><td></td><td></td&gt; </tr><tr><td> Ekim bileşik, Tissue-Tek </td><td> Sakura Finetek </td><td> 4583 </td><td> OCT, optimum kesim sıcaklık </td></tr><tr><td> Cryomold standart </td><td> Sakura Finetek </td><td> 4557 </td><td> 25 mm × 20 mm × 5 mm </td></tr><tr><td> Cryomold ara </td><td> Sakura Finetek </td><td> 4566 </td><td> 15 mm × 15 mm × 5 mm </td></tr><tr><td> Uni-Link-Einbettkassette </td><td> R. Langenbrinck </td><td> 09-0503 </td><td> Histoloji işleme ve gömme kasetleri </td></tr><tr><td> Spezial-Tatowierfarbe Schwarz </td><td> H. Hauptnerund &amp; Richard Herberholz GmbH &amp; Co KG </td><td> 71492000 </td><td> yapıştırma dövme </td></tr><tr><td> Tatowierzange TZ1 </td><td> EBECO E. Becker &amp; Co GmbH </td><td></td><td> Cihaz dövme </td></tr><tr><td> Heparin </td><td> Carl Roth GmbH &amp; Co </td><td> 7692.1 </td><td></td></tr><tr><td> Formaldehit% 37 </td><td> Carl Roth GmbH &amp; Co </td><td> 7386 </td><td></td></tr><tr><td> Ketamin hidroklorür </td><td> Sigma-Aldrich Chemie GmbH </td><td> K2753-1G </td><td></td></tr><tr><td> Ksilazin hidroklorür </td><td> Sigma-Aldrich Chemie GmbH </td><td> X1251-1G </td><td></td></tr><tr><td> steril PBS </td><td> Biochrom </td><td> L182-50 </td><td></td></tr><tr><td> dijital fotoğraf makinesi </td><td> Nikon </td><td> Coolpix 5400 </td><td></td></tr><tr><td> Şırınga filtre ünitesi 0.45 mikron tahrik </td><td> Millipore </td><td></td><td></td></tr><tr><td> Kaliper </td><td> Mitutoyo 7309 </td><td> 1667338 </td><td> Farnell (distribütör) </td></tr><tr><td> hastalığı puanlama sac </td><td></td><td></td><td> Örneğin enclosed </td></tr></tbody></table>

granulocyte-dependent autoantibody-induced skin blistering

Otoimmün fenomenler sağlıklı bireylerde ortaya, ama öz-hoşgörü başarısız olduğunda, otoimmün yanıtı spesifik bir patoloji neden olabilir. Göre Witebsky sitesi, otoimmün bir hastalık tanısında kullanılan kriterlerden biri önermeleri otoantikorların pasif transferi ile deney hayvanlarında hastalık üreme olduğunu. Epidermolizis için bülloza (EBA), deri ve mukoza bir prototipi organ-spesifik otoimmün hastalık, çeşitli deneysel modeller yakın zamanda kuruldu. Kolajen VII 200 amino asit (aa 757-967) bir streç karşı IgG antikorları saflaştırılmış, bugünkü iş tarif hayvan modelinde kabarma fenotipi yanı sıra insan için karakteristik histolojik ve immunopatolojik özellikleri üreten farelere sürekli enjekte edilir İTÇ 1. Tam teşekküllü bir yaygın hastalık genellikle ilk enjeksiyondan sonra 5-6 gün görülen ve hastalığın yaygınlığını yönetilen c dozu ile ilişkilidir edilirollagen VII-spesifik IgG. Deneysel EBA doku hasarı (kabarcık oluşumu) -4 doku bağlı otoantikorlar 2 ile granülosit alımı ve aktivasyon bağlıdır. Örnek efferent otoimmün tepki sadece T hücre-bağımsız bir faz üretir Bu nedenle, bu model, otoimmün doku hasarına neden granülosit-bağımlı enflamatuvar yolunun diseksiyon sağlar. Ayrıca, değeri in vivo otoantikorların blister uyaran potansiyelini gösteren ve İTÇ 1,3, -6 yılında blister oluşumu mekanizması araştıran çok sayıda çalışma tarafından belirlenmektedir. Son olarak, bu model oldukça otoantikor kaynaklı hastalıklarda yeni bir anti-inflamatuar tedavilerin geliştirilmesini kolaylaştıracaktır. Genel olarak, İTÇ pasif transfer hayvan modeli, erişilebilir ve öğretici hastalık modeli ve araştırmacılar İTÇ patogenezi sadece analiz etmek değil, temel biyolojik ve klinik cevap yardımcı olacakesansiyel otoimmünite sorular.

Klinik, histolojik ve immunopatolojik düzeyde insan İTÇ benzeyen farelerde tam şişmiş bir hastalık spesifik antijen antikor sonuçları, pasif transfer. Kabarcıklar, kabuklar, erozyonlar ve alopesi kulakları, burunları, pençeleri, bacaklar, sırt ve farelerin göz etrafında gelişir. Hastalığın ilk klinik bulguların muhtemelen kulak ve / veya kafa alanı görünecektir. Belirli tavşan IgG ve fare kompleman C3 çökelmesi IF perilezyonel deri cryosections içinde dermal epidermal bileşkede doğrudan tar...

Watch this Scientific Journal Video about Granulocyte-dependent Autoantibody-induced Skin Blistering at JoVE.com

Granulocyte-dependent Autoantibody-induced Skin Blistering

Kolajen VII 200 amino asit (aa 757-967) bir streç karşı IgG antikorları saflaştırılmış, bugünkü iş tarif hayvan modelinde kabarma fenotipi yanı sıra insan için karakteristik histolojik ve immunopatolojik özellikleri üreten farelere sürekli enjekte edilir epidermolizis bülloza (EBA)<sup&gt; 1</sup&gt;.

Granülosit-bağımlı otoantikor indüklenen Cilt Kabarcıklanma

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

granulocyte-dependent-autoantibody-induced-skin-blistering

Research

JoVE Journal

Immunology and Infection

10.4K Görüntüleme.  University of Freiburg. Kolajen VII 200 amino asit (aa 757-967) bir streç karşı IgG antikorları saflaştırılmış, bugünkü iş tarif hayvan modelinde kabarma fenotipi yanı sıra insan için karakteristik histolojik ve immunopatolojik özellikleri üreten farelere sürekli enjekte edilir epidermolizis bülloza (EBA) 1.

Video: Granulocyte-dependent Autoantibody-induced Skin Blistering

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Hamsterlar ve Dorsal Skin Kutanöz Leishmaniasis: Antileishmanial Uyuşturucu Tarama için Faydalı Model

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

İmmünoloji ve Enfeksiyon

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Görüntülemede HIV-1 Zarf bağlı Virolojik Synapse ve Sentetik Lipid Bilayers üzerine Sinyal

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Farelerde allojenik CD4 + T hücre Cevapları Eğitim için Kuyruk Cilt Nakli

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Fenotipik Analizi ve İnsan Cilt lenfosit izolasyonu<em&gt; Ex Vivo</em&gt; Hücre Kültürü

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

Farklı Fare Cilt Bölgelerinden sitoflüorimetrik analize için tek hücre süspansiyonları hazırlanması

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Enzimatik Digest ve Gradient Ayırma kullanma Fare Skin gelen sızan lökositlerin İzolasyonu

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Canlı zayıflatılmış intradermal aşılama sonrasında Fare Deri ve akan lenf düğümü gelen Myeloid Hücre İzolasyonu<em&gt; Plasmodium</em&gt; Sporozoitler

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

İzole Kapların vazodilatasyon ve Sıkı cilt Fare Ekstraselüler Matrix İzolasyonu

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Mürin Tam kat Cilt Nakli

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

&quot;Acufection&quot; ve Cilt Araştırmaları onun Uygulaması tarafından bir Ekonomik DNA Yerleştirme Sisteminin Geliştirilmesi

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...