Gli autori desiderano ringraziare Amanda Tardo, il Dr. Mostafa Bouljihad, Stephanie Feely, Carol Coyne, il Dr. Erin Ribka, il Dr. Pete Didier e Dorothy Kuebler per la loro assistenza tecnica. Questo lavoro è stato supportato da sovvenzioni NIH R01-DK076653 e 3R01-DK076653-02S1 a KS e la sovvenzione di funzionamento base del TNPRC RR000164.

1. Biopsia cutanea raccolta dei campioni <ol><li> Prima della procedura di biopsia della pelle, anestetizzare gli animali per via intramuscolare con 2,5 mg / kg di cloridrato tiletamina e cloridrato miscela zolazepam telazol (Fort Dodge Animal Health, Fort Dodge, IA). Monitorare gli animali dalla somministrazione di anestetici fino decubito e poi rimuovere dal loro recinto. </li><li> Rimuovere i capelli dalla zona cutanea di interesse utilizzando un Oster d&#39;Oro Clipper A5 Single Speed ​​veterinaria con una lama taglia 40 (Oster Professional Products, McMinnville, TN) e asetticamente preparare con alternanza di macchia Betadine e alcol. Fissare un telino sterile fenestrato il sito selezionato biopsia. </li><li> Usando una tecnica sterile, posizionare un 4,0 millimetri Miltex strumento Punch biopsia cutanea (Miltex, York, PA) contro la pelle mentre si ruota lo strumento di 180 gradi in senso orario e antiorario con una leggera pressione fino a quando la biopsia transetti attraverso gli strati dermici nella ti sottocutaneossue. Rimuovere il campione bioptico e afferrare la porzione di pelle sezionato con una pinza e libero dai tessuti sottocutanei. </li><li> Chiudere il difetto pelle con 3-0 sutura in nylon collegato a un ago 3 / 8 cerchio di taglio (Ethilon, Ethicon, la Johnson &amp; Johnson Medical Limited, Berkshire, Regno Unito) in uno schema crociato. Dare tutti gli animali 0,01 mg / kg buprenorfina cloridrato (Hospira, Lake Forest, IL) per via intramuscolare per la post operatorio analgesia. </li></ol> 2. Esempio di lavorazione <ol><li> Lavora con biopsia campioni di pelle da dermatiti croniche e sani di controllo macachi rhesus. Ottenere 2-3 (4 mm di diametro) da campioni di biopsie ogni animale. </li><li> Fissare primo campione in formalina di zinco (Z-fix, Anatech Ltd., Battle Creek, MI) per 24 ore, lavare in acqua per 30 minuti, lavare brevemente in etanolo al 70%, e il luogo in ASP300 processore tessuto Leica (Leica Microsystems Inc. , Buffalo Grove, KS), dove il tessuto è disidratato con gradi ascendenti del 70%, 80%, 95% e il 100% di etanolo 48min ciascuno (Fisher Scientific, Pittsburgh, PA), seguita da due cambi di xilene (Fisher). </li><li> Incorporare nei media paraffina (Fisher) a lungo termine conservazione a temperatura ambiente. Posto a -20 ° C per 20 minuti prima di sezionamento. Preparare 6 sezioni micron utilizzando un microtomo rotativo (HM325, Microm internazionale, Waldorf, Germania). Luogo sezioni su vetrini carica (Fisher) e aria secca a 60 ° C durante la notte. Colorare con ematossilina e eosina (H &amp; E) metodo standard (descritta sotto). </li><li> Fix secondo campione nel 2% paraformaldeide (USB Corp, Cleveland, OH) per 30 minuti a temperatura ambiente, lavare tre volte in tampone fosfato salino (PBS, Gibco-Invitrogen, Carlsbad, CA), posto nel 30% di saccarosio (Fisher) per 4 ore, e incorporare nel medio congelamento ottobre (Sakura Finetek, Torrence, California). Mantenere a-80 ° C per 20 minuti prima di sezionamento. Preparare 15 sezioni micron utilizzando il criostato (HM560, Thermo Scientific, Kalamazoo, MI). </li></ol> 3. H &amp; E colorazione <ol><li> Sparaffinare incorporaresezioni ded attraverso tre cambi di xilene (Fisher) e reidratare attraverso etanoli classificato: tre cambi del 100%, due cambi del 95%, una variazione del 80% e acqua distillata, 2 min ciascuna. </li><li> Colorare con ematossilina (Richard Allan-scientifico, Kalamazoo, MI) per un minuti e seguite da un lavaggio breve in acqua corrente. </li><li> Contrasto con eosina (Sigma) per 6 min. </li><li> Disidratare i tessuti colorati con il 95% ed etanolo assoluto, due cambi di 2 minuti ciascuna e quindi in chiaro tre cambi di xilene, 3 minuti ciascuno. </li><li> Monte coprioggetti usando Cytoseal Media di montaggio (Richard Allan-scientifico), il H &amp; E-sezioni di tessuto colorato. </li></ol> 4. La valutazione istopatologica Ispezionare H &amp; E-macchiato sezioni biopsia cutanea sotto un microscopio ottico con il 4 - 40x di ingrandimento e microscopio Leica con una SPOT Insight fotocamera digitale (Digital Instruments Inc., Michigan, USA). 5. Stai immunofluorescenzaNing <ol><li> Lavora con ottobre-embedded sezioni pelle per la colorazione di immunofluorescenza. Prima della colorazione, ammollo per 20 minuti in PBS contenente 0,2% di pesci pelle gelatina (FSG, Sigma) con il 0,1% Triton X-100 (Sigma) per rimuovere il PTOM e permeabilize il tessuto. </li><li> Dopo aver lavato le diapositive, incubare con siero di capra 10% normale (NGS, Sigma), diluito in PBS-FSG per 1 ora a temperatura ambiente, in una camera umidificata scivolo. </li><li> Per visualizzare la deposizione di IgA e gli anticorpi tTG2, incubare diapositive con anti-Rhesus tTG2 IgA e gli anticorpi primari, diluiti in 10% NGS-PBS-FSG per 1 ora. </li><li> Lavare con PBS-FSG-Triton X-100 (Sigma) per 10 minuti, seguita da PBS-FSG per 10 min. </li><li> Incubare con secondaria, isotipo-specifici anticorpi taggati con fluorocromo Alexa Fluor 488 (verde), 568 (rosso) o 633 (blu) (Molecular Probes-Invitrogen) alla diluizione 1:1000. Utilizzare il ToPro-3 colorante per visualizzare il DNA nucleare. Usare il 10% di NGS-PBS-FSG come diluente dell&#39;anticorpo. </li><li> Utilizzare la soluzione anti-spegnimento (Sigma) per montare le sezioni di tessuto colorato. </li></ol> 6. Confocale di imaging Eseguire l&#39;imaging con un microscopio Leica TCS SP2 vera confocale a scansione laser (Leica, Wetzlar, Germania) dotato di tre laser (sei linee di eccitazione disponibile): un argon-krypton laser a 488 nm (verde), un laser kripton a 568 nm ( rosso) e di elio-neon laser a 633 nm (blu), che spaziano dal visibile al far-rosso lato dello spettro. Record contrasto differenziale di interferenza (DIC) immagini per la valutazione della non etichettati architettura dei tessuti. 7. Rappresentante Risultati Un esempio di riconoscimento positivo di ETG sia tTG2 e gli anticorpi IgA è mostrato in fig. 3A. Coerenti con la situazione nei pazienti umani e per la prevalenza molto più bassa di DH-come dermatite di tipo CD sensibilità al glutine anche nei macachi rhesus, la maggior parte dei campioni di biopsia cutanea si prevede che show la reazione positiva con tTG2 anticorpi, ma senza la presenza di tTG2 + IgA + cellule doppio positive (Fig. 4). Questi DH-correlate casi, inclusi i casi la dermatite di origine microbica, rivelerà alcuni single-cellule positive + IgA dispersi sotto l&#39;epitelio ma non sovrapposizione spettrale tra le tTG2 e IgA marcata con marcatori (Fig. 3B e 4). Pertanto, è importante mantenere l&#39;ordine delle procedure di test suggerito, iniziando con l&#39;esame clinico (Fig. 1), seguita dalla valutazione istopatologica (Fig. 2) e solo se nessun altro, che causano dermatiti più comuni agenti possono essere identificati, confocale esame microscopico di biopsie cutanee colorate per tTG2 e IgA è giustificata. Valutazioni cliniche e / o istopatologiche da soli non sono sufficienti per una diagnosi positiva di questa condizione. Nei casi in cui doppio-positivi (tTG2 + IgA +) le cellule sono identificate intorno alla zona della papilla dermica, DH-come la diagnosi di dermatite può essere rilasciato. In questi casi, il ritiro del glutine nella dietae la somministrazione di dieta priva di glutine è consigliata al fine di alleviare i sintomi di dermatite. <img src="/files/ftp_upload/3154/3154fig1.jpg" alt="Figura 1" /> Figura 1. Regione inguinale di EM96 macaco prima (A) e dopo (B) ritiro del glutine nella dieta per 5 settimane. Reintroduzione del glutine nella dieta ha coinciso con la ricomparsa della dermatite (C). <img src="/files/ftp_upload/3154/3154fig2.jpg" alt="Figura 2" /> Figura 2. HE colorazione di EM96 biopsie cutanee macaco raccolti prima (A) e dopo (B) ritiro del glutine nella dieta per 9 settimane. R: Epitelio è addensato (7-15 strati di cellule epiteliali) con una spessa crosta superficiale di cellule infiltrate ipercheratosiche con neutrofili degenerando. I neutrofili appaiono anche dentro l&#39;epitelio e si mescolano con i numeri moderata dei linfociti, plasmacellule, istiociti e in tutto il annessi nel derma superiore. B: Alcuni hyperkeratOsis è ancora presente, lo spessore epiteliale è normalizzato (4-7 strati) con il minimo di infiltrazione linfoplasmacellulare nel derma superiore. C: Normale cute addominale da FR56 macaco, senza alcuna infiltrazione linfoplasmacellulare, si effettua il confronto. Ingrandimento 20x. <img src="/files/ftp_upload/3154/3154fig3.jpg" alt="Figura 3" /> Figura 3. Immagini di microscopia confocale di EM96 biopsie cutanee macaco raccolti prima (A) e dopo (B) ritiro del glutine nella dieta per 9 settimane. R: IgA colorazione (verde), tTG2 (rosso) e ToPro-3 marcatore del DNA nucleare (blu). Co-localizzazione di IgA e tTG2 appare in giallo intorno alla zona di papilae cutanea. B: campione di pelle presa dopo 9 settimane di dieta priva di glutine non mostra IgA all&#39;interno dell&#39;epitelio mentre alcuni sottoepiteliale IgA è ancora presente (verde), tTG2 (rosso) e ToPro-3 marcatore del DNA nucleare (blu). <img src="/files/ftp_upload/3154/3154fig4.jpg" alt="Figura 4" /> Figura 4. </strong&gt; immagini di microscopia confocale di GI24 biopsie cutanee macaco rhesus raccolti durante animale stava esibendo i sintomi della broncopneumopatia cronica (DH-correlati) dermatite. La colorazione IgA è in verde, tTG2 in rosso e ToPro-3 marcatore del DNA nucleare è in blu. Le cellule + tTG2 sono presenti all&#39;interno del epitelio, pochi IgA + cellule sotto l&#39;epitelio, mentre non sovrapposizione ampio spettro tra i marcatori rosso e il verde si vede. <table border="1"><tbody><tr><td> Animal Tag </td><td> Sesso [M / F] </td><td> Età [Anni] </td><td> I sintomi clinici </td><td> Plasma tTG2 anticorpi </td><td> Dieta [1,2] * </td></tr><tr><td> HJ71 </td><td> F </td><td> 1,3 </td><td> Sano </td><td> - </td><td> 1 </td></tr><tr><td> HD13 </td><td> M </td><td> 1,4 </td><td> Sano, diarrea occasionale </td><td> + </td><td> 1 </td></tr><tr><td> FR56 </td><td> M </td><td> 5,4 </td><td> Diarrea cronica idiopatica </td><td> - </td><td> 1 </td></tr><tr><td> EM96 </td><td> F </td><td> 5.0 </td><td> Dermatite cronica </td><td> + / - (Borderline) </td><td> 1 e 2 </td></tr><tr><td> GI24 </td><td> F </td><td> 4,6 </td><td> Dermatite cronica </td><td> - </td><td> 1 </td></tr></tbody></table> Tabella 1. TNPRC macachi rhesus utilizzati per la raccolta di biopsie cutanee * Dieta 1 - regolare scimmia chow (contenenti glutine); dieta 2 - dieta senza glutine

<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>

Nessun conflitto di interessi dichiarati.

La nostra nuova costituzione non umani modello primate della sensibilità al glutine è stato recentemente utilizzato per studiare i cambiamenti della permeabilità intestinale innescato da glutine 5,6. Plasma tTG2 anticorpi così come intestinali tTG2 depositi IgA + + sono stati rilevati in alcuni glutine-sensibile macachi. Da quando abbiamo recentemente identificato episodi rari di dermatiti idiopatiche nella colonia di macachi rhesus, dove non sempre (infettivo / metabolica) eziologia è stato possibile st...

<table border="1"><tbody><tr><td> Nome del reagente o attrezzature </td><td> Azienda </td><td> Numero di catalogo / Codice Prodotto </td><td> Nota </td></tr><tr><td> Anti-Human IgA FITC * </td><td> Sigma, St. Louis, MO </td><td> F-5259 </td><td> 1:100 diluizione di lavoro </td></tr><tr><td> Anti-Human tTG2 </td><td> Thermo Scientific, Kalamazoo, MI </td><td> MS-300-P1ABX </td><td> 1:100 diluizione di lavoro </td></tr><tr><td> Anti-Mouse IgG1 ** </td><td> Invitrogen, Carlsbad, CA </td><td> A-21124 </td><td> 1:1000 diluizione di lavoro </td></tr><tr><td> ToPro-3 *** sonda DNA </td><td> Invitrogen, Carlsbad, CA </td><td> T-3605 </td><td> 1:1000 diluizione di lavoro </td></tr><tr><td> Zinco formalina (Z-fix) </td><td> Anatech Ltd., Battle Creek, MI </td><td> 171 </td><td></td></tr><tr&gt; <td> Ottobre di congelamento Medio </td><td> Sakura Finetek, Torrence, California </td><td> 4583 </td><td></td></tr><tr><td> Cytoseal di montaggio Medio </td><td> Richard Allan-scientifico, Kalamazoo, MI </td><td> 8312-4 </td><td></td></tr><tr><td> Anti-Tempra Soluzione </td><td> Sigma, St. Louis, MO </td><td> P 3130 </td><td></td></tr><tr><td> Ethilon (Sutura in nylon) </td><td> Ethicon, la Johnson &amp; Johnson, Berkshire, Regno Unito </td><td> 663G </td><td></td></tr><tr><td> Miltex Punch Strumento biopsia cutanea </td><td> Miltex, York, PA </td><td> NA </td><td></td></tr><tr><td> Oster Golden A5 singola Vet tagliatore di velocità con una lama taglia 40 </td><td> Prodotti Oster, McMinnville, TN </td><td> 78005-050 </td><td></td></tr><tr><td> Leica tessuto del processore </td><td> Leica Microsystems Inc., Buffalo Grove, KS </td><td> ASP300 </td><td></td></tr><tr><td> Microtomo rotativo </td><td> Microm internazionale, Waldorf, Germania </td><td> HM325 </td><td></td></tr><tr><td> Criostato </td><td> Thermo Scientific, Kalamazoo, MI </td><td> HM560 </td><td></td></tr><tr><td> Microscopio confocale </td><td> Leica, Wetzlar, Germania </td><td> TCS SP2 </td><td></td></tr></tbody></table> * Isotiocianato di fluorescina ** Secondaria (Alexa Fluor 568-coniugato) anticorpo utilizzato dopo tTG2 anticorpo primario ** Dye

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

Transglutaminasi tissutale 2 (tTG2) è un enzima digestivo intestinale che deamidates già parzialmente digerito dietetici senza glutine peptidi della gliadina ad es. Nei soggetti geneticamente predisposti, tTG2 innesca reazioni autoimmuni che si caratterizzano per la produzione di anticorpi tTG2 e la loro deposizione diretta in piccoli 1,2 parete intestinale. La presenza di questi anticorpi costituisce una delle caratteristiche principali della malattia celiaca (MC). Transglutaminasi epidermica (ETG) è un altro membro della famiglia transglutaminasi che può anche funzionare come un autoantigene in una piccola minoranza di pazienti con CD. In questi casi relativamente rari, ETG innesca una reazione autoimmune (rash cutaneo), clinicamente conosciuta come la dermatite erpetiforme (DH). Sebbene l&#39;esatto meccanismo della patogenesi CD e DH non è ben compreso, è noto che tTG2 e ETG epitopi antigenici parti che possono essere riconosciuti dagli anticorpi nel siero dei pazienti sia da CD e DH 3,4. (Macaca mulatta) con dermatite (Tabella 1, fig. 1 e 2) è stato utilizzato per studiare i tessuti colpiti. In un animale (EM96) una sovrapposizione spettrale di IgA e gli anticorpi tTG2 (Fig. 3) è stata dimostrata. La presenza del doppio positivo tTG2 + IgA + cellule è stata focalizzata nell&#39;epidermide profonda, intorno alla papilla dermica. Ciò è coerente con le lesioni descritte in pazienti con DH 3. Quando EM96 è stato posto su una dieta priva di glutine, la dermatite, così come tTG2 + IgA + depositi scomparsi e non erano più rilevabili (fig. 1-3). Dermatite riapparve tuttavia, sulla base di re-introduzione del glutine nella dieta in EM96 (non mostrato). Nei macachi altri tra cui animali con dermatite non correlate, il + tTG2 depositi IgA + non sono stati rilevati. Dieta priva di glutine-dipendente remissione della dermatite in EM96 insieme con presenza di tTG2 + IgA + cellule sua pelle suggeriscono una autoimmune, DH-likemeccanismo per lo sviluppo di questa condizione. Questo è il primo rapporto del DH-come dermatite in qualsiasi primate non umano.

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

Dermatite erpetiforme (DH) è una condizione infiammatoria cronica caratterizzata da una reazione autoimmune tra IgA e transglutaminasi epidermica (ETG). DH si sviluppa in una porzione molto piccola di pazienti glutine-sensibile e / o celiaci. I risultati di questo studio indicano che DH può anche svilupparsi in un ospite scimmia rhesus con i sintomi della dermatite idiopatica.

Riconoscimento della transglutaminasi epidermica da IgA e 2 anticorpi transglutaminasi tissutale in un raro caso di Rhesus Dermatite

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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Research

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 Views.  Tulane National Primate Research Center. Dermatite erpetiforme (DH) è una condizione infiammatoria cronica caratterizzata da una reazione autoimmune tra IgA e transglutaminasi epidermica (ETG). DH si sviluppa in una porzione molto piccola di pazienti glutine-sensibile e / o celiaci. I risultati di questo studio indicano che DH può anche svilupparsi in un ospite scimmia rhesus con i sintomi della dermatite idiopatica.

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.

Multicolor Citometria a Flusso di analisi Cellular risposta immunitaria in macachi Rhesus

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 ...

Ricerca

Immunologia e infezioni

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.

Tecnologia IgY: Estrazione di anticorpi pollo rosso d&#39;uovo con polietilenglicole (PEG) Precipitazioni

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.

Trasformazione e cercarie In vitro La coltivazione di 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.

Identificazione di batteri patogeni rari da 16S rRNA sequenziamento del gene e MALDI-TOF MS

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.

Legame dell&#39;anticorpo Specificità per Kappa (Vκ) umani (IgM) Anticorpi catena contenenti chiari: Polysialic Acid (PSA) Allegato alla NCAM come caso di studio

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.

Generazione di Murine anticorpi monoclonali da ibridomi Tecnologia

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.

L&#39;analisi dei Simian Immunodeficiency Virus specifici CD8<sup&gt; +</sup&gt; T-cellule in Rhesus macachi di peptide-MHC-I Tetramero colorazione

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.

Rilevamento e l&#39;arricchimento di Rare antigene-specifiche cellule B per l&#39;analisi del fenotipo e funzione

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.

Effetti dell'esposizione di formaldeide per un modello del ratto della dermatite atopica indotta dal trattamento neonatale capsaicina

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.

Un metodo per valutare le funzioni effettrici Fc-mediata indotte dagli anticorpi specifici di Hemagglutinin di riossidazione

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