Gli autori riconoscono il supporto da finanziamenti della Deutsche Forschungsgemeinschaft SI-1281/4-1 e BIOS della Facoltà di Medicina dell&#39;Università di Friburgo (a CS).

1. Preparazione di anticorpi patogeni Nota: Purificazione e concentrazione degli anticorpi deve essere eseguito lo stesso giorno, come anticorpi non devono essere immagazzinati in 0,1 M glicina pH 2,5-3 (tampone di eluizione) per tutta la notte (ON). Purificazione di affinità di IgG: utilizzare 25 ml di siero di coniglio immune: <ol><li> Scongelare il siero di coniglio ON a 4 ° C, mescolare 1:1 con 1xPBS (tampone di corsa) e centrifugare a 1260 xg per 10 min a 20 ° C. Facoltativamente, una fase di filtrazione con filtro di carta può essere incluso dopo centrifugazione nel caso il siero è grasso. </li><li> Nel frattempo, lavare la proteina G matrice colonna riempita con 10 volumi del letto (volume del letto = volume matrice) di 1XPBS (tampone di corsa). </li><li> Applicare il siero diluito e filtrato alla colonna e incubare per 1 ora sulla piattaforma oscillante a temperatura ambiente (RT). </li><li> Raccogliere il flow-through (FT) in un tubo da 50 ml Flacon. Non gettarlo fino alla concentrazionee titolo del IgG purificata è noto. </li><li> Lavare la matrice con 5 volumi di letto 1XPBS e nel frattempo preparare 50 ml provette Falcon per la raccolta della frazione eluita IgG. Pipettare 1 ml di tampone di neutralizzazione pH: 1 M TRIS pH 10 in ciascun tubo (altrimenti la proporzione di 1:20 tampone TRIS rispetto alla quantità di eluato da raccogliere). </li><li> Applicare 100-150 ml tampone di eluizione: 0,1 M glicina pH 2,5-3 alla colonna e raccogliere 50 ml frazione (s). Capovolgere il tubo 2-3x, misurare il pH e aggiungere tampone neutralizzante fino a quando hai raggiunto il pH 7,2-7,4. </li><li> Raccogliere gocce di eluato IgG e con il tampone di eluizione come etalon misurare la densità ottica a 280 nm. Se le OD è &lt;0,1 raccolta stop. </li><li> Rigenerare la matrice proteina G e conservarlo secondo le istruzioni del produttore. </li></ol> Concentrazione mediante ultrafiltrazione Amicon con tubi: <ol><li> Lavare il Amicon Ultra (15 ml / 30kDa) tubi di ultrafiltrazione con 1XPBS mediante centrifugazione 15-20 mina 3220 xg a 4 ° C. Eliminare il FT dal Amicon. </li><li> Riempire i Amicons con 15 ml di eluito frazione IgG e centrifugare per 25-30 min a 3220 xg e 4 ° C, il tempo di centrifugazione dipende sempre dal contenuto proteico dell&#39;eluato. </li><li> Eliminare FT e ripetere fino a ultrafiltrazione non avete frazione eluita a sinistra. </li><li> Lavare il concentrato IgG ampiamente, per due volte, con 1XPBS 25-30 minuti per centrifugazione. </li><li> Raccogliere il &quot;appiccicoso&quot;, soluzione giallastra anticorpo dal filtro Amicon in un volume finale di 1,500-2,000 pl di 1XPBS. </li><li> Misura la concentrazione di preparazione IgG utilizzando uno spettrofotometro o NanoDrop: Conc.. (Mg / ml) = A (280 nm) x fattore di diluizione / 1,4 </li><li> Lavare e conservare i Amicons seguendo le istruzioni del produttore. </li></ol> 2. L&#39;iniezione di collagene VII-IgG specifiche in topi Prima di iniziare la procedura attuale sperimentale, assicurarsi che il pro sperimentaleprotocollo è scritto e tutti i materiali sono preparati per l&#39;esperimento. <ol><li> Preparare i fogli di dati per il punteggio malattia, ELISA, immunofluorescenza (IF), l&#39;analisi e la mieloperossidasi (MPO) test 4. </li><li> Etichettare le provette di sangue e di organi, cryomolds nonché istologia di trasformazione e Cassette per inclusione. </li><li> Le soluzioni di anticorpo deve essere preparato fresco o scongelato dalle scorte e caratterizzato per quanto riguarda la loro reattività (titolo da microscopia IF e / o ELISA). La soluzione filtrata sterile anticorpo deve essere diluito in PBS in modo che la dose richiesta per l&#39;iniezione varia tra 250-1000 microlitri. Assicurarsi di avere abbastanza IgG per eseguire l&#39;intero esperimento. </li><li> Preparare fresco anticoagulante: eparina 20 U / ml e 8,7 mg / ml di ketamina / 1,3 mg / ml miscela xilazina per anestesia. Quando sacrificare i topi hanno etichettato i tubi con il 3,7% di formaldeide preparato. </li><li> Sterilizzare gli strumenti chirurgici (bisturi, forbici, punto fine e Curvforcipes DE) e preparare siringhe (siringhe da insulina, 1 e 2 siringhe ml), aghi, fotocamera digitale e scheda di memoria aggiuntiva. </li></ol> Nota: Eseguire tutte le procedure sui topi narcotizzata ketamina / xilazina o isoflurano isoflurano è l&#39;opzione preferita per l&#39;anestesia dei controlli della pelle ogni giorno, come topi recuperare in fretta.. Tuttavia, quando si scattano fotografie, 87 mg / kg di ketamina e 13 mg / kg di xilazina di solito è iniettato per via sottocutanea. Per l&#39;eutanasia ketamina / xilazina dosaggio viene aumentato a 130 mg / kg e 20 mg di ketamina / xilazina kg. <ol><li> Controllare gli animali già segnalati per la loro condizione di salute generale, la pelle e l&#39;aspetto di pelliccia, li pesare e misurare il loro spessore orecchio (sempre rispettare lo stesso orecchio). </li><li> Registrati valori osservati e dei cambiamenti nella scheda di valutazione clinica. </li><li> Raccogliere 20-30 microlitri (2-3 gocce) sangue dalla vena della coda in una siringa contenente lo stesso volume di anticoagulante. </li><li> Disinfettare la pelliccia epelle utilizzando 80% di etanolo. </li><li> Iniettare la soluzione di anticorpi per via sottocutanea nella schiena utilizzando una siringa da insulina. (Per alcuni siti (ad esempio, le orecchie) solo piccoli volumi (10-50 microlitri) sono realizzabili da iniettare.) </li></ol> Le iniezioni devono essere ripetute ogni due giorni, 4 volte. Balb / c e C57BL6 topi di peso corporeo di circa 20 g iniziare a sviluppare la malattia 3-4 giorni dopo la prima somministrazione di 400-500 fino a 750 mcg / g di peso corporeo / iniezione autoanticorpi. Quando si finisce l&#39;esperimento: <ol><li> Mettere i topi sotto narcosi profonda, iniettando 130 mg / kg e 20 mg / kg di ketamina / xilazina per via sottocutanea, li exsanguinate dalla vena cava posteriore e tagliare il cuore. </li><li> Raccogliere campioni di tessuti / organi come la pelle: coda, l&#39;orecchio, il sangue e l&#39;esofago e metterli in tubi precedentemente etichettati e PBS o 3,7% di formaldeide riempito. </li></ol> Quando si ritorna allaboratorio: <ol><li> Centrifugare il sangue a 1.200 xg, 10 min a RT per separare il plasma, trasferire il plasma per tubi etichettati e conservare di conseguenza. </li><li> Incorporare il biopsia cutanea in ambiente ottimale Temperatura Tagliare con cryomolds correttamente, etichette, e conservarli a -80 ° C. </li><li> Posizionare i campioni di tessuto destinati istologia nelle formelle etichettati e tenerli nel 3,7% di formaldeide fino inclusione in paraffina. </li></ol> 3. La valutazione clinica della gravità della malattia I topi devono essere esaminati tutti i giorni per le lesioni della cute e delle mucose ed i risultati devono essere registrati utilizzando i moduli appropriati. I criteri per eutanasia precoce è lesioni cutanee colpiscono oltre il 20% della superficie del corpo e una perdita di peso del 5-10% del peso corporeo totale in tre giorni consecutivi, contando per un punteggio malattia di 5 (Tabella 1). <ol><li> Posizionare il mouse sul ventre. Inizia con la zona della testa, vale a dire con la destraorecchio e verificare la presenza di bolle, erosioni, crosta su quello interno, nonché sul lato esterno. Proseguire allo stesso modo con l&#39;orecchio sinistro. </li><li> Gli occhi destro e sinistro sono controllati per i segni di eritema, alopecia, erosioni e / o in crosta. </li><li> Fronte e la zona muso sono spazzolati fino al successivo, continuando con la parte ventrale del muso, labbra e la mucosa della bocca. </li><li> Esaminare la gamba anteriore destra a partire dalla zampa, in movimento verso l&#39;alto, passare alla zampa anteriore sinistra. Stessa procedura con le gambe posteriori destro e sinistro. </li><li> Spennellare con il pelo sul collo e sulla schiena zona con una pinza curva punta fine. </li><li> Accendere il mouse sul dorso e controllare la parte ventrale e ai lati ventrali degli arti allo stesso modo come in precedenza. </li><li> Controllare la coda. </li><li> Scattare foto di parti del corpo in questione, qualora i topi sviluppano la malattia. È necessario prestare attenzione alla qualità e quantità delle immagini. </li><li> Calcolare i punteggi clinici di malattia. </li></ol> 4. Analisi di campioni di pelle e Plasma <ol><li> Preparare, archiviare e / o elaborare tutti i tessuti raccolti secondo il piano. </li><li> Tagliare sezioni di tessuto congelato per IF rilevamento di anticorpi di coniglio e mouse componenti del sistema del complemento. </li><li> Analizzare il diverso contenuto di proteine ​​e di enzimi (dosaggio MPO) di tessuti congelati e / o estratti di organi. </li><li> Sezione la paraffina incorporato tessuto e macchia con H &amp; E di visualizzare l&#39;entità del danno tissutale. </li><li> Analizzare il plasma mediante ELISA, immunoblot. </li></ol>

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Il trasferimento passivo di anticorpi in animali sperimentali è un approccio importante per dimostrare la patogenicità 7, -12. Modelli animali ottenuti mediante questo metodo, oltre ad essere la prova indiretta per autoimmunità 13, permettono l&#39;indagine della fase efferente del meccanismo patogenetico. Il modello di trasferimento passivo di anticorpi indotta granulociti-dipendente pelle vescicole di epidermolisi bollosa acquisita (EBA) è stato utilizzato per analizzare i meccanismi di danno...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> antigene ricombinante </td><td></td><td></td><td> La codifica plasmidi per le forme ricombinanti di murino collagene VII sono disponibili presso l&#39;autore corrispondente. </td></tr><tr><td> siero di coniglio immune </td><td> <a href="http://www.eurogentec.com/" target="_blank">www.eurogentec.com</a> </td><td></td><td> Avevamo conigli bianchi New Zealand immunizzati con 200 mg di antigene, 3 volte ad intervalli di 2 settimane. A questo scopo abbiamo utilizzato i servizi di Eurogentec SA, Belgio. </td></tr><tr><td> Proteine ​​G agarosio </td><td> Roche Applied Science </td><td> 11243233001 </td><td></td></tr><tr><td> Topi BALB / c </td><td> Charles River Laboratories </td><td></td><td></td&gt; </tr><tr><td> Ottobre composto, Tissue-Tek </td><td> Sakura Finetek </td><td> 4583 </td><td> Ottobre, temperatura di taglio ottimale </td></tr><tr><td> Cryomold di serie </td><td> Sakura Finetek </td><td> 4557 </td><td> 25 millimetri × 20 mm × 5 mm </td></tr><tr><td> Cryomold intermedio </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> Istologia trasformazione e Cassette per inclusione </td></tr><tr><td> Spezial-Tatowierfarbe Schwarz </td><td> H. Hauptnerund &amp; Richard Herberholz GmbH &amp; Co. KG </td><td> 71492000 </td><td> tatuaggio pasta </td></tr><tr><td> Tatowierzange TZ1 </td><td> EBECO E. Becker GmbH &amp; Co </td><td></td><td> tatuaggio dispositivo </td></tr><tr><td> Eparina </td><td> Carl Roth GmbH &amp; Co. </td><td> 7.692,1 </td><td></td></tr><tr><td> Formaldeide 37% </td><td> Carl Roth GmbH &amp; Co. </td><td> 7386 </td><td></td></tr><tr><td> Cloridrato di ketamina </td><td> Sigma-Aldrich Chemie GmbH </td><td> K2753-1G </td><td></td></tr><tr><td> Xylazina cloridrato </td><td> Sigma-Aldrich Chemie GmbH </td><td> X1251-1G </td><td></td></tr><tr><td> PBS sterile </td><td> Biochrom </td><td> L182-50 </td><td></td></tr><tr><td> macchina fotografica digitale </td><td> Nikon </td><td> Coolpix 5400 </td><td></td></tr><tr><td> Siringa guidato unità filtro da 0,45 micron </td><td> Millipore </td><td></td><td></td></tr><tr><td> Pinza </td><td> Mitutoyo 7309 </td><td> 1667338 </td><td> Farnell (distributore) </td></tr><tr><td> malattia punteggio foglio </td><td></td><td></td><td> esempio custodie insed </td></tr></tbody></table>

granulocyte-dependent autoantibody-induced skin blistering

Fenomeni autoimmuni si verificano in individui sani, ma quando auto-tolleranza non riesce, la risposta autoimmune può portare a patologia specifica. Secondo Witebsky postulati, uno dei criteri di diagnosi di una malattia autoimmune, come è la riproduzione della malattia in animali da esperimento dal trasferimento passivo di autoanticorpi. Per epidermolisi bollosa acquisita (EBA), un prototipo di organo-specifica malattia autoimmune della pelle e le mucose, diversi modelli sperimentali sono stati di recente istituzione. Nel modello animale descritto nel nostro lavoro attuale, purificati anticorpi IgG contro un tratto di 200 aminoacidi (aa 757-967) del collagene VII vengono iniettate più volte in topi che riproducono il fenotipo vesciche così come le caratteristiche del isto-e immunopatologiche caratteristiche per la salute umana EBA 1. Conclamata malattia diffusa è visto solitamente 5-6 giorni dopo la prima iniezione e l&#39;estensione della malattia correla con la dose somministrata di collagen VII-IgG specifiche. Il danno tissutale (formazione di bolle) nel sperimentale EBA dipende il reclutamento e l&#39;attivazione di granulociti da parte del tessuto-bound autoanticorpi 2, -4. Pertanto, questo modello consente la dissezione del granulociti-dipendente percorso infiammatorie coinvolte nel danno tissutale autoimmune, come il modello riproduce soltanto la cellula T-indipendente fase della risposta autoimmune efferente. Inoltre, il suo valore è sottolineata da una serie di studi che dimostrano il blister che inducono potenziale di autoanticorpi in vivo e indagare il meccanismo di formazione in blister EBA 1,3, -6. Infine, questo modello agevolerà notevolmente lo sviluppo di nuove terapie anti-infiammatorie in autoanticorpi malattie dovute al. Nel complesso, il passivo modello animale trasferimento di EBA è un modello di malattia accessibile e istruttivo e consentirà ai ricercatori di analizzare non solo patogenesi EBA, ma di rispondere a fondamentali biologicamente e clinicamenteautoimmunità domande essenziali.

Il trasferimento passivo di anticorpi antigene specifici risultati in una malattia in piena regola nei topi, che assomigliano a livelli clinici, istologici e immunopatologiche l&#39;EBA umano. Blister, croste, erosioni e alopecia sviluppano sulle orecchie, muso, zampe, gambe, schiena e intorno agli occhi dei topi. I primi segni clinici della malattia probabilmente apparirà sulle orecchie e / o della zona testa. Deposizione di specifiche IgG di coniglio, e il mouse del complemento C3 viene rilevato da diretta IF all&#39...

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Granulocyte-dependent Autoantibody-induced Skin Blistering

Nel modello animale descritto nel nostro lavoro attuale, purificati anticorpi IgG contro un tratto di 200 aminoacidi (aa 757-967) del collagene VII vengono iniettate più volte in topi che riproducono il fenotipo vesciche così come le caratteristiche del isto-e immunopatologiche caratteristiche per la salute umana epidermolisi bollosa acquisita (EBA)<sup&gt; 1</sup&gt;.

Granulociti-dipendente indotta da autoanticorpi pelle vescicole

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 Views.  University of Freiburg. Nel modello animale descritto nel nostro lavoro attuale, purificati anticorpi IgG contro un tratto di 200 aminoacidi (aa 757-967) del collagene VII vengono iniettate più volte in topi che riproducono il fenotipo vesciche così come le caratteristiche del isto-e immunopatologiche caratteristiche per la salute umana epidermolisi bollosa acquisita (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.

Leishmaniosi cutanea nella pelle dorsale di criceti: ​​un modello utile per lo screening delle droghe Antileishmanial

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

Ricerca

Immunologia e infezioni

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.

Imaging di HIV-1 Envelope indotta Synapse virologica e segnalazione sui doppi strati lipidici sintetici

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.

Il trapianto di coda della pelle per studiare allogenico di cellule T CD4 risposte in topi

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.

Linfociti Isolamento da Pelle umana per l&#39;analisi fenotipica e<em&gt; Ex Vivo</em&gt; 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 ...

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.

Preparazione di cella singola sospensioni per analisi citofluorimetrica provenienti da diverse regioni della pelle del mouse

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.

Isolamento di leucociti infiltranti da mouse pelle utilizzando enzimatica Digest e Separazione Gradient

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.

Mieloide isolamento delle cellule da topo pelle e drenante linfonodi seguito intradermica immunizzazione con vivo attenuato<em&gt; Plasmodium</em&gt; sporozoiti

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.

Vasodilatazione dei vasi isolati e l&#39;isolamento della matrice extracellulare di topi Tight-pelle

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.

Murine tutto spessore trapianto della pelle

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.

Sviluppo di un sistema di consegna economico DNA da &quot;Acufection&quot; e la sua applicazione alla ricerca della pelle

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