Yazarlar Amanda Tardo, Dr. Mostafa Bouljihad Stephanie Feely Carol Coyne, Dr. Erin Ribka, Dr. Pete Didier ve Dorothy Kuebler, teknik yardım için teşekkür etmek istiyorum. Bu çalışma NIH hibe R01-DK076653 ve KS 3R01 DK076653-02S1 ve TNPRC RR000164 temel işletim hibe tarafından desteklenmiştir.

1. Cilt biyopsisi örnek toplama <ol><li> Önce cilt biyopsisi işlemi, 2,5 mg / tiletamine hidroklorür ve zolazepam hidroklorür telazol karışımı (Fort Dodge Hayvan Sağlığı, Fort Dodge, IA) kg intramusküler hayvanları uyutmak. Uzanma kadar anestezik yönetimi hayvanlar izleyin ve sonra da onların muhafaza kaldırmak. </li><li> Ilgilendiren bir boyutu 40 bıçak (Oster Profesyonel Ürünler, McMinnville, TN) ile bir Oster Altın A5 Tek Hız Veteriner Clipper kullanan deri alanından saç çıkarın ve aseptik betadin fırçalayın ve alkol alternatif hazırlamak. Seçilen biyopsi sitesi üzerinden steril bir Fenestre asmak sabitleyin. </li><li> Alet hafif basınç saat yönünde ve saat yönünün tersine 180 derece dönerken punch biyopsi subkutan ti dermal katmanlardan ile transects kadar steril bir tekniği kullanarak, deri karşı 4,0 mm Miltex Punch Dermal Biyopsi aleti (Miltex York, PA)ssue. Biyopsi örneği çıkarın ve forseps ve deri altı doku ile cilt transeksiyon kısmını kavramak. </li><li> 3-0 naylon sütür ile çapraz desen 3 / 8 daire kesici iğne (Ethilon, Ethicon, Johnson &amp; Johnson Medikal Limited, Berkshire, İngiltere) bağlı cilt defekti kapatın. Bütün hayvanların post operatif analjezi için 0.01 mg / kg intramusküler buprenorfin hidroklorür (Hospira, Lake Forest, IL) verin. </li></ol> 2. Örnek işleme <ol><li> Kronik dermatit ve sağlıklı kontrol deri biyopsi örnekleri ile İş rhesus makaklarda. 02:58 (çapı 4 mm) her hayvan numuneleri biyopsi alın. </li><li> 24 saat boyunca çinko formalin (Z-fix, Anatech Ltd, Battle Creek, MI) Fix ilk örnek, ASP300 Leica doku işlemci (Leica Microsystems Inc, 30 dakika süreyle suda yıkayın% 70 etanol içinde kısa bir süre yıkama yeri ve Buffalo Grove, KS doku,% 70,% 80,% 95 ve% 100 etanol 48 artan notları ile susuz)ksilen (Fisher) tarafından iki değişiklik her (Fisher Scientific, Pittsburgh, PA), dk. </li><li> Oda sıcaklığında uzun vadeli depolama için, parafin medya (Balıkçı) gömün. Kesit için 20 dakika önce-20oC yerleştirin. 6 mikron bölümlerde döner mikrotom (HM325, Microm International, Waldorf, Almanya) kullanarak hazırlayın. Ücret slaytlar (Balıkçı) ve bir gecede 60 ° C kuru hava yerleştirin bölümleri. Leke ile hematoksilen ve eozin (H &amp; E) standart yöntemi (aşağıda açıklanmıştır). </li><li> Fix ikinci örnek oda sıcaklığında 30 dakika süreyle% 2 paraformaldehid (USB Corp, Cleveland, Ohio), fosfat üç defa yıkamak salin (PBS, Gibco-Invitrogen, Carlsbad, CA)% 30 sakaroz (Balıkçı), tamponlu 4 saat ve Ekim dondurma orta (Sakura Finetek, Torrence, CA) gömmek. Kesit için 20 dakika önce-80oC tutun. 15 mikron bölümleri Kriyostat (HM560, Thermo Scientific, Kalamazoo, MI) kullanarak hazırlayın. </li></ol> 3. H &amp; E boyama <ol><li> Embed Deparaffinizeüç değişiklik% 100,% 95 iki değişiklik, bir değişim ve% 80 distile su, 2 dakika her üç xylene (Balıkçı) değişiklikleri ve kademeli ethanols yoluyla rehidrate ile ded kesitler. </li><li> Bir dakika için hematoksilen (Richard Allan Bilimsel, Kalamazoo, MI) Leke ve musluk suyu çalıştırarak kısa bir yıkama tarafından takip. </li><li> Eozin (Sigma) ile 6 dakika Counterstain. </li><li> 2 dakika her iki değişiklikler ve ardından xylene üç değişikliklere açık, her biri 3 dakika,% 95 ve mutlak etanol ile lekeli dokuları dehydrate. </li><li> H &amp; E boyalı doku bölümleri üzerinde Cytoseal Montaj Orta kullanarak Dağı coverglass (Richard Allan Bilimsel). </li></ol> 4. Histopatolojik değerlendirme Bir SPOT Insight dijital fotoğraf makinesi (Dijital Instruments Inc, Michigan, ABD) ile 40x büyütme ve Leica mikroskop - 4 kullanarak ışık mikroskobu altında H &amp; E boyalı biyopsi cilt bölümleri inceleyin. 5. Immunofluorescent staining <ol><li> Immunofluorescent boyama OCT gömülü cilt bölümleri ile çalışın. Boyamadan önce, 20 dakika PBS içinde% 0.1 ile% 0.2 Triton X-100 (Sigma) OCT kaldırmak ve doku permeabilize balık derisi jelatin (FSG, Sigma) içeren bekletin. </li><li> Slaytları yıkadıktan sonra, oda sıcaklığında 1 saat süreyle PBS-FSG seyreltilmiş% 10 normal keçi serumu (NGS, Sigma) ile nemlendirilmiş bir slayt odası, kuluçkaya yatmaktadır. </li><li> IgA ve tTG2 antikorlar birikimi görselleştirmek için, 1 saat süreyle% 10 NGS-PBS-FSG sulandırılmış anti-rhesus IgA ve tTG2 birincil antikorlar, slaytlar kuluçkaya yatmaktadır. </li><li> 10 dakika, 10 dakika süreyle PBS-FSG tarafından takip PBS-FSG-Triton X-100 (Sigma) ile yıkayın. </li><li> 1:1,000 seyreltme Alexa Fluor florokrom 488 (yeşil), 568 (kırmızı) veya 633 (mavi) (Moleküler Probları-Invitrogen) ile etiketli ikincil, izotip-özgül antikorlar ile inkübe edin. ToPro-3 boya, nükleer DNA görselleştirmek için kullanın. Antikor seyreltici olarak% 10 NGS-PBS-FSG kullanın. </li><li> Lekeli doku kesitlerinde monte etmek için anti-su verme solüsyonu (Sigma) kullanın. </li></ol> 6. Konfokal görüntüleme Argon-Kripton lazer 488 nm (yeşil), 568 nm dalga boyunda bir Kripton lazer (üç lazerler (kullanılabilir uyarma altı satırlık) ile donatılmış bir Leica TCS SP2 Gerçek Konfokal Lazer Tarama Mikroskobu (Leica, Wetzlar, Almanya) ile görüntüleme gerçekleştirin kırmızı) ve görünür spektrumun uzak kırmızı tarafına yayılmış 633 nm (mavi), helyum-neon lazer. Etiketli mimari dokusu değerlendirilmesi için Kayıt diferansiyel girişim kontrast (DIC) görüntüleri. 7. Temsilcisi Sonuçlar Şekil tTG2 ve IgA antikorları hem de ETG pozitif tanıma bir örnek gösterilmiştir. 3A. Tutarlı insan hastalarda durum ve rhesus makaklarda de gluten duyarlılığı CD-DH-benzeri dermatit daha çok daha düşük yaygınlık nedeniyle, deri biyopsi örneklerinin en sho bekleniyorw, ancak tTG2 + IgA + çift pozitif hücreler (Şekil 4) varlığı olmadan tTG2 antikorları ile pozitif reaksiyon. Mikrobiyal kökenli dermatit vakalar da dahil olmak üzere bu durumlarda, DH-ilgisiz, epitel altında dağınık bazı tek pozitif IgA + hücreler ortaya ama tTG2 ve IgA etiketli belirteçleri (Şekil 3B ve 4) arasında spektral örtüşme. Bu nedenle, histopatolojik değerlendirme (Şekil 2) ve başka daha yaygın dermatit sebep olan ajanların yalnızca tespit edilebilir, konfokal tarafından klinik muayene (Şekil 1) ile başlayan, önerilen test prosedürleri düzeni sağlamak için önemlidir. tTG2 ve IgA için lekeli cilt biyopsi mikroskop incelemesi garantilidir. Tek başına klinik ve / veya histopatolojik değerlendirme, bu durumun olumlu bir tanı için yeterli değildir. Bu durumlarda çift-pozitif (tTG2 + IgA +), DH-benzeri hücreler dermal papilla alanı çevresinde tespit dermatit tanısı verilebilir. Bu gibi durumlarda diyet gluten, yoksunlukve dermatit semptomları hafifletmek için glutensiz bir diyet uygulaması tavsiye edilir. <img src="/files/ftp_upload/3154/3154fig1.jpg" alt="Şekil 1" /> Şekil 1. önce EM96 makak Kasık bölge (A) ve sonrası (B) 5 hafta boyunca diyet gluten çekilmesi. Diyet gluten yeniden yerleştirilmesi dermatit yeniden ortaya çıkma (C) ile çakıştı. <img src="/files/ftp_upload/3154/3154fig2.jpg" alt="Şekil 2" /> Şekil 2 HE, önce toplanan EM96 makak deri biyopsi boyama (A) ve sonrası (B) 9 hafta boyunca diyet gluten çekilmesi . A: Epitel hiperkeratotik hücreleri kalın bir yüzeysel kabuk yozlaşan nötrofiller ile infiltre (7-15 kat epitel hücrelerinin) kalındır. Nötrofiller epitel içinde de görünür ve lenfositler, plazma hücreleri ve üst dermiste etrafındaki etrafında histiyositler orta numaraları ile karıştırılır. B: Bazı hyperkeratosis hala mevcut, üst dermiste minimal lenfoplazmositik infiltrasyon epitel kalınlığı (4-7 katman) normalize edilir. C: hiçbir lenfoplazmositik infiltrasyon FR56 makak Normal karın cilt, karşılaştırma için yer almaktadır. Büyütme 20x. <img src="/files/ftp_upload/3154/3154fig3.jpg" alt="Şekil 3" /> Şekil 3, önce toplanan EM96 makak deri biyopsi mikroskopisi görüntüleri (A) ve sonrası (B) 9 hafta boyunca diyet gluten çekilmesi . A: IgA boyama (yeşil), tTG2 (kırmızı) ve ToPro-3 nükleer DNA marker (mavi). IgA ve tTG2 Co-lokalizasyon dermal papilae alanı etrafında sarı görünür. B: Cilt örneği 9 hafta sonra alınan bazı subepitelyal IgA, (yeşil) hala tTG2 (kırmızı) ve ToPro-3 nükleer DNA marker (mavi) mevcut ise glutensiz diyet epitel içinde hiçbir IgA gösterir. <img src="/files/ftp_upload/3154/3154fig4.jpg" alt="Şekil 4" /> Şekil 4. </strong&gt; GI24 Rhesus makak cilt biyopsi mikroskopisi görüntüleri toplanan hayvan kronik dermatit (DH-ilgisiz) belirtileri gösteren iken. IgA boyama, kırmızı ve ToPro-3 nükleer DNA marker mavi. Yeşil, tTG2 TTG2 + hücreleri, epitel içinde birkaç IgA + epitel altında hücreleri, kırmızı ve yeşil işaretleri arasında geniş spektral örtüşme görülüyor. <table border="1"><tbody><tr><td> Hayvan Etiketi </td><td> Seks [E / K] </td><td> Yaş [Yıllar] </td><td> Klinik Belirtileri </td><td> Plazma tTG2 antikorlar </td><td> Diyet [1,2] * </td></tr><tr><td> HJ71 </td><td> F </td><td> 1,3 </td><td> Sağlıklı </td><td> - </td><td> 1 </td></tr><tr><td> HD13 </td><td> M </td><td> 1,4 </td><td> Sağlıklı, zaman zaman ishal </td><td> + </td><td> 1 </td></tr><tr><td> FR56 </td><td> M </td><td> 5,4 </td><td> Kronik idiopatik diyare </td><td> - </td><td> 1 </td></tr><tr><td> EM96 </td><td> F </td><td> 5,0 </td><td> Kronik dermatit </td><td> + / - (Sınırda) </td><td> 1 ve 2 </td></tr><tr><td> GI24 </td><td> F </td><td> 4,6 </td><td> Kronik dermatit </td><td> - </td><td> 1 </td></tr></tbody></table> (Tablo 1). TNPRC rhesus deri biyopsi toplama için kullanılan makaklarda * Diyet 1 - Düzenli maymun chow (gluten içeren) Diyet 2 - glutensiz diyet

<ol>
	<li>Diosdado, B., Wijmenga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+the+adaptive,+innate+and+regulatory+immune+responses+in+the+intestinal+mucosa+of+celiac+disease+patients.">Molecular mechanisms of the adaptive, innate and regulatory immune responses in the intestinal mucosa of celiac disease patients.</a> Expert. Rev. Mol. Diagn. 5 (5), 681-700 (2005).</li><li>Schuppan, D., Dennis, M. D., Kelly, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celiac+disease:+epidemiology,+pathogenesis,+diagnosis,+and+nutritional+management.">Celiac disease: epidemiology, pathogenesis, diagnosis, and nutritional management.</a> Nutr. Clin. Care. 8 (2), 54-69 (2005).</li><li>Sardy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+transglutaminase+(TGase+3)+is+the+autoantigen+of+dermatitis+herpetiformis.">Epidermal transglutaminase (TGase 3) is the autoantigen of dermatitis herpetiformis.</a> J. Exp. Med. 195 (6), 747-757 (2002).</li><li>Rose, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+against+epidermal+transglutaminase+are+a+sensitive+diagnostic+marker+in+patients+with+dermatitis+herpetiformis+on+a+normal+or+gluten-free+diet.">Autoantibodies against epidermal transglutaminase are a sensitive diagnostic marker in patients with dermatitis herpetiformis on a normal or gluten-free diet.</a> J. Am. Acad. Dermatol. 61 (1), 39-43 (2009).</li><li>Bethune, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+transport+and+enzymatic+detoxification+of+gluten+in+gluten-sensitive+rhesus+macaques.">Transepithelial transport and enzymatic detoxification of gluten in gluten-sensitive rhesus macaques.</a> PLoS ONE. 3 (3), e1857-e1857 (2008).</li><li>Mazumdar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+transepithelial+passage+of+the+immunogenic+33-residue+peptide+from+&#x03B1;-2+gliadin+in+gluten-sensitive+macaques.">Visualization of transepithelial passage of the immunogenic 33-residue peptide from &#x03B1;-2 gliadin in gluten-sensitive macaques.</a> PLoS ONE. 5 (4), e10228-e10228 (2010).</li><li>Schuppan, D., Junker, Y., Barisani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celiac+disease:+from+pathogenesis+to+novel+therapies.">Celiac disease: from pathogenesis to novel therapies.</a> Gastroenterol. 137 (6), 1912-1933 (2009).</li><li>Hunt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+identified+genetic+risk+variants+for+celiac+disease+related+to+the+immune+response.">Newly identified genetic risk variants for celiac disease related to the immune response.</a> Nature Genet. 40 (4), 395-402 (2008).</li><li>Abadie, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+genetic+and+immunological+insights+into+a+model+of+celiac+disease+pathogenesis.">Integration of genetic and immunological insights into a model of celiac disease pathogenesis.</a> Annu. Rev. Immunol. 29, 493-525 (2011).</li></ol>

Çıkar çatışması ilan etti.

Gluten hassasiyeti yeni kurulan insan dışı primat modeli son zamanlarda diyet gluten 5,6 ile tetiklenen bağırsak geçirgenliği değişiklikleri incelemek için kullanılır. Plazma tTG2 antikorlar yanı sıra bağırsak tTG2 + IgA + mevduat glutene duyarlı bazı makaklarda tespit edildi. Son zamanlarda rhesus koloni hiçbir olağan (metabolik / enfeksiyöz) etiyoloji kurulamadı makaklarda, idiyopatik dermatit nadir olayların tespit yılından bu yana, bu çalışmanın amacı değerlendirmek için is...

<table border="1"><tbody><tr><td> Reaktif veya ekipman Adı </td><td> Şirket </td><td> Katalog Numarası / Ürün Kodu </td><td> Not </td></tr><tr><td> Anti-İnsan IgA-FITC * </td><td> Sigma, St Louis, MO </td><td> F-5259 </td><td> 1:100 çalışma seyreltme </td></tr><tr><td> Anti-İnsan tTG2 </td><td> Thermo Scientific, Kalamazoo, MI </td><td> MS-300-P1ABX </td><td> 1:100 çalışma seyreltme </td></tr><tr><td> Anti-Mouse IgG1 ** </td><td> Invitrogen, Carlsbad, CA </td><td> A-21124 </td><td> 1:1,000 çalışma seyreltme </td></tr><tr><td> ToPro-3 DNA prob *** </td><td> Invitrogen, Carlsbad, CA </td><td> T-3605 </td><td> 1:1,000 çalışma seyreltme </td></tr><tr><td> Formalin Çinko (Z-fix) </td><td> Anatech Ltd Battle Creek, MI </td><td> 171 </td><td></td></tr><tr&gt; <td> Ekim Donma Orta </td><td> Sakura Finetek, Torrence, CA </td><td> 4583 </td><td></td></tr><tr><td> Cytoseal Montaj Orta </td><td> Richard Allan Bilimsel, Kalamazoo, MI </td><td> 8312-4 </td><td></td></tr><tr><td> Anti söndürme Çözüm </td><td> Sigma, St Louis, MO </td><td> P 3130 </td><td></td></tr><tr><td> Ethilon (Naylon Sütür) </td><td> Ethicon, Johnson &amp; Johnson, Berkshire, İngiltere </td><td> 663G </td><td></td></tr><tr><td> Miltex Punch Dermal Biyopsi Enstrüman </td><td> Miltex York, PA </td><td> NA </td><td></td></tr><tr><td> Oster Altın A5 Tek hızlı bir boyut bıçak ile 40 Veteriner Clipper </td><td> Oster Ürünler, McMinnville, TN </td><td> 78005-050 </td><td></td></tr><tr><td> Leica Doku İşlemci </td><td> Leica Microsystems IncBuffalo Grove, KS </td><td> ASP300 </td><td></td></tr><tr><td> Döner Mikrotom </td><td> Microm Uluslararası Waldorf, Almanya </td><td> HM325 </td><td></td></tr><tr><td> Kriyostat </td><td> Thermo Scientific, Kalamazoo, MI </td><td> HM560 </td><td></td></tr><tr><td> Konfokal Mikroskop </td><td> Leica, Wetzlar, Almanya </td><td> TCS SP2 </td><td></td></tr></tbody></table> * Floresein izotiyosiyanat TTG2 primer antikor sonra kullanılan ** İkincil (Alexa Fluor 568-konjuge) antikor ** Boya

recognition of epidermal transglutaminase by iga and tissue transglutaminase 2 antibodies in a rare case of rhesus dermatitis

Doku transglutaminazı 2 (tTG2) zaten kısmen sindirilmiş diyet gluten örneğin gliadin peptitleri deamidates bir bağırsak sindirim enzimi. Genetik yatkınlığı olan bireylerde, tTG2 tTG2 antikorların üretimi ve ince bağırsak duvarının 1,2 içine doğrudan birikimi ile karakterize otoimmün yanıtları tetikler. Bu antikorların varlığı çölyak hastalığı (CD) en önemli işaretlerinden birini oluşturmaktadır. Epidermal transglutaminaz (ETG), CD hastaların küçük bir azınlık bir otoantijen olarak da işlev görebilir transglutaminaz ailenin bir başka üyesi. Bu nispeten nadir durumlarda, ETG klinik olarak herpetiform dermatit (DH) olarak bilinen bir otoimmün reaksiyonu (deri döküntüsü) tetikler. CD ve DH patogenez mekanizması tam olarak bilinmemekle birlikte, bilindiği tTG2 ve CD ve DH hastalarda 3,4 hem de serum antikorları tarafından kabul edilebilir hisse antijenik epitoplar ETG (macaca mulatta) (Tablo 1, Şekil 1 ve 2) deri lezyonları biyopsi örneklerinin konfokal mikroskop incelemesi etkilenen dokuların çalışmak için kullanılmıştır. Bir hayvan (EM96) IgA ve tTG2 antikorlar (Şekil 3) spektral örtüşme olduğu gösterilmiştir. Çift pozitif tTG2 varlığı + IgA + hücreler dermal papilla çevresindeki derin epidermis duruldu. Bu lezyonlar DH hastaların 3 açıklanan tutarlı. EM96 bir glutensiz diyet, dermatit, yanı sıra yerleştirildi tTG2 + IgA + mevduat kayboldu ve artık (Şekil 1-3) saptanabilir edildi. Dermatit EM96 diyet gluten (gösterilmemiştir) yeniden giriş dayalı, ancak ortaya çıkıverdi. Hayvan ile ilgisiz dermatit de dahil olmak üzere diğer makaklarda, tTG2 + IgA + mevduat tespit edilmedi. TTG2, varlığı ile birlikte EM96 dermatit Glutensiz diyet bağımlı remisyon + IgA +, cilt hücreleri öneririz otoimmün bir, DH-gibiBu durumun geliştirilmesi için mekanizma. Bu herhangi bir insan dışı primat DH-benzeri dermatit ilk rapor.

Watch this Scientific Journal Video about Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis at JoVE.com

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) IgA ve epidermal transglutaminaz (ETG) arasında otoimmün bir reaksiyon ile karakterize kronik inflamatuar bir durumdur. DH gluten duyarlı ve / veya çölyak hastaların çok küçük bir kısmında gelişir. Bu çalışmanın sonuçları, DH de Rhesus maymun konak idiyopatik dermatit semptomları ile geliştirebilir göstermektedir.

Nadir bir Case IgA ve Doku Transglutaminaz 2 Antikorlar Epidermal Transglutaminaz tanınması Rhesus Dermatit

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

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JoVE Journal

Immunology and Infection

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

24.4K Görüntüleme.  Tulane National Primate Research Center. Dermatitis herpetiformis (DH) IgA ve epidermal transglutaminaz (ETG) arasında otoimmün bir reaksiyon ile karakterize kronik inflamatuar bir durumdur. DH gluten duyarlı ve / veya çölyak hastaların çok küçük bir kısmında gelişir. Bu çalışmanın sonuçları, DH de Rhesus maymun konak idiyopatik dermatit semptomları ile geliştirebilir göstermektedir.

Video: Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

Rhesus Makak maymunlarının Hücresel Bağışıklık Tepkisi Multicolor Sitometrisi Analizleri

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

İmmünoloji ve Enfeksiyon

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IGY Teknoloji: Yumurta Sarısı Tavuk Antikorlar çıkarımı Polietilen Glikol (PEG) Yağış

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

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 ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNA gen Sıralama ve MALDI-TOF MS tarafından Nadir Bakteriyel patojenlerin tanımlanması

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

Antibody Binding Specificity for Kappa (V&amp;#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Kappa (VK) için Antikor Bağlama Özgünlük Hafif Zincir içeren İnsan (IgM) Antikorlar: Polysialic Asit (PSA) bir Vaka Çalışması olarak NCAM Bağlı

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Hibridoma teknolojisi ile murin monoklonal antikorlar üretilmesi

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Maymun bağışıklık eksikliği virüsüne özgü CD8 analizi<sup&gt; +</sup&gt; Peptid MHC-I tetramer boyama ile Rhesus macaque maymunlarının T-hücrelerinin

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Formaldehit maruz bir fare modeli Atopik dermatit yenidoğan kapsaisin tedavi tarafından indüklenen etkileri

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Grip Hemagglutinin özel antikorlar tarafından indüklenen Fc-aracılı efektör fonksiyonları değerlendirmek için bir yöntem

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...