Les auteurs tiennent à remercier le soutien par des subventions du SI-1281/4-1 Deutsche Forschungsgemeinschaft et ESBIO de la Faculté de médecine de l&#39;Université de Fribourg (pour CS).

1. Préparation de l&#39;anticorps pathogène Remarque: La purification et la concentration des anticorps doit être effectuée le même jour, que les anticorps ne doivent pas être stockés dans la glycine 0,1 M pH 2,5 à 3 (tampon d&#39;élution) pendant la nuit (ON). La purification par affinité des IgG: utiliser 25 ml de sérum de lapin immunisé: <ol><li> Décongeler le sérum de lapin ON à 4 ° C, le mélanger 1:1 avec 1xPBS (tampon de migration) et centrifuger à 1260 xg pendant 10 min à 20 ° C. Éventuellement, une étape de filtrage avec du papier filtre pourraient être inclus après centrifugation dans le cas où le sérum est gras. </li><li> Pendant ce temps, lavez la protéine G matrice remplie la colonne avec 10 volumes de lit (volume de lit = volume de la matrice) de 1XPBS (tampon de migration). </li><li> Appliquez le sérum dilué et filtré à la colonne et incuber pendant 1 h sur un agitateur oscillant à la température ambiante (RT). </li><li> Recueillir le flux continu (FT) dans un tube de 50 ml Flacon. Ne pas le jeter jusqu&#39;à ce que la concentrationet le titre des IgG purifiée est connue. </li><li> Laver la matrice avec 5 volumes de lit de 1XPBS et en même temps préparer 50 ml tubes Falcon de collecte de la fraction éluée des IgG. Pipette 1 ml de tampon de neutralisation du pH: Tris 1 M pH 10 dans chaque tube (autrement la proportion de 1:20 tampon TRIS par rapport à la quantité d&#39;éluat à collecter). </li><li> Appliquer 100-150 ml de tampon d&#39;élution: 0,1 M de glycine à pH 2,5-3 et reçois la colonne 50 ml fraction (s). Retournez le tube de 2-3x, mesurer le pH et ajouter tampon de neutralisation jusqu&#39;à ce que vous avez atteint le pH 7,2-7,4. </li><li> Recueillir quelques gouttes d&#39;éluat IgG et en utilisant le tampon d&#39;élution comme étalon de mesurer la DO à 280 nm. Si la DO est la collecte &lt;arrêt 0,1. </li><li> Régénérer la matrice protéine G et le stocker en suivant les instructions du fabricant. </li></ol> Concentration par ultrafiltration Amicon avec des tubes: <ol><li> Laver l&#39;Ultra Amicon (15 ml / 30 kDa) des tubes d&#39;ultrafiltration par centrifugation avec 1XPBS 15-20 minà 3220 xg à 4 ° C. Jeter le FT de la Amicon. </li><li> Remplissez les Amicons avec 15 ml d&#39;IgG fraction éluée et centrifuger pendant 25-30 min à 3220 xg et à 4 ° C, le temps de centrifugation dépend toujours de la teneur en protéines de l&#39;éluat. </li><li> Jeter FT et répétez jusqu&#39;à ce ultrafiltration vous n&#39;avez pas de fraction éluée à gauche. </li><li> Laver le concentré d&#39;IgG largement, à deux reprises, avec 1XPBS 25-30 min par centrifugation. </li><li> Recueillir le &quot;collant&quot;, solution d&#39;anticorps jaunâtre du filtre Amicon dans un volume final de 1500-2000 ul de 1XPBS. </li><li> Mesure de la concentration préparation d&#39;IgG aide d&#39;un spectrophotomètre ou NanoDrop: Conc. (Mg / ml) = A (280 nm) x facteur de dilution / 1,4 </li><li> Nettoyez et rangez les Amicons en suivant les instructions du fabricant. </li></ol> 2. L&#39;injection de collagène VII IgG spécifiques dans Souris Avant de commencer la procédure expérimentale réelle, assurez-vous que le pro expérimentaleprotocole est rédigé et tous les matériaux sont préparés pour l&#39;expérience. <ol><li> Préparer les feuilles de données pour la notation des maladies, ELISA, immunofluorescence (IF) l&#39;analyse et la myéloperoxydase (MPO) test 4. </li><li> Etiqueter les tubes pour le sang et les organes, cryomolds ainsi que l&#39;histologie traitement et l&#39;incorporation de cassettes. </li><li> Les solutions d&#39;anticorps doivent être préparés frais ou décongelé des stocks et caractérisé à l&#39;égard de leur réactivité (titre par microscopie IF et / ou ELISA). La solution filtrée stérile d&#39;anticorps doit être dilué dans du PBS de façon que la dose requise pour l&#39;injection varie entre 250-1000 pl. Assurez-vous que vous avez assez d&#39;IgG d&#39;effectuer toute l&#39;expérience. </li><li> Préparer une nouvelle anticoagulant: héparine de 20 U / ml et 8,7 mg / ml de kétamine / xylazine 1,3 mélange mg / ml pour l&#39;anesthésie. Lorsque le sacrifice des souris ont marqué les tubes avec du formaldéhyde 3,7% préparée. </li><li> Stériliser les instruments chirurgicaux (scalpel, ciseaux, pointe fine et Curvforcipes ndlr) et de préparer les seringues (seringues à insuline, 1 et 2 seringues ml), aiguilles, appareil photo numérique et une carte mémoire supplémentaire. </li></ol> Remarque: Effectuez toutes les procédures de kétamine / xylazine ou à l&#39;isoflurane souris narcotized L&#39;isoflurane est l&#39;option préférée anesthésie pour les contrôles quotidiens de la peau, comme des souris de récupérer rapidement.. Toutefois, lorsque vous prenez des photos, 87 mg / kg de kétamine et 13 mg / kg de xylazine est habituellement injecté par voie sous cutanée. Pour l&#39;euthanasie de la dose de kétamine / xylazine est augmentée à 130 mg / kg de kétamine et 20 mg / kg de xylazine. <ol><li> Vérifiez les animaux déjà marqués pour leur état de santé général, la peau et l&#39;apparence de la fourrure, les peser et mesurer leur épaisseur de l&#39;oreille (toujours s&#39;en tenir à la même oreille). </li><li> S&#39;inscrire valeurs observées et des changements dans la fiche d&#39;évaluation clinique. </li><li> Recueillir 20-30 ul (2-3 gouttes) le sang de la veine de la queue dans une seringue contenant le même volume d&#39;anticoagulant. </li><li> Désinfecter la fourrure etpeau à l&#39;aide d&#39;éthanol à 80%. </li><li> Injecter la solution d&#39;anticorps voie sous-cutanée dans le dos à l&#39;aide d&#39;une seringue à insuline. (Pour certains sites (par exemple, les oreilles) que de faibles volumes (10-50 pi) sont réalisables à injecter.) </li></ol> Les injections doivent être répétées tous les deux jours, 4 fois. Balb / c et souris C57BL6 d&#39;un poids d&#39;environ 20 g de commencer à développer la maladie 3-4 jours après la première administration de 400-500 jusqu&#39;à 750 pg / g de poids corporel / injection auto-anticorps. Lors de la finition de l&#39;expérience: <ol><li> Mettez la souris sous narcose profonde en injectant 130 mg / kg et 20 mg / kg de kétamine / xylazine voie sous-cutanée, les être exsangue de la veine cave postérieure et arracher le coeur. </li><li> Recueillir des échantillons de tissus / organes tels que la peau: queue, les oreilles, le sang et l&#39;œsophage et les mettre dans des tubes préalablement marquées et le formaldéhyde, ou 3,7% de PBS remplie. </li></ol> En revenant à l&#39;laboratoire: <ol><li> Centrifuger le sang à 1200 xg, 10 min à température ambiante pour séparer le plasma, transférer le plasma dans des tubes correctement étiquetés et les stocker en conséquence. </li><li> Intégrer la biopsie cutanée en milieu Cut température optimale en utilisant cryomolds, étiqueter correctement, et de les stocker à -80 ° C. </li><li> Placer les échantillons de tissus destinés à l&#39;histologie dans les moules d&#39;enrobage étiquetés et les conserver dans du formol à 3,7% jusqu&#39;à inclusion en paraffine. </li></ol> 3. Évaluation clinique de la gravité de la maladie Les souris doivent être examinées quotidiennement pour les lésions de la peau et des muqueuses, et les résultats doivent être enregistrés en utilisant les formulaires appropriés. Les critères d&#39;euthanasie précoce les lésions cutanées qui affectent plus de 20% de la surface du corps et une perte de poids de 5-10% du poids total du corps en trois jours consécutifs, comptant pour un score de maladie 5 (Tableau 1). <ol><li> Placez la souris sur le ventre. Commencez avec la région de la tête, à savoir le droitoreille et vérifier les ampoules, les érosions, la croûte sur la face interne ainsi que sur le côté extérieur. Continuer de la même façon avec l&#39;oreille gauche. </li><li> Yeux droit et gauche sont contrôlés pour les signes d&#39;érythème, alopécie, des érosions et / ou croûte. </li><li> Le front et la zone de museau sont brossés prochaine, pour continuer avec la partie ventrale du museau, les lèvres et la muqueuse de la bouche. </li><li> Examiner la patte avant droite à partir de la patte, se déplaçant vers le haut; passer à la patte avant gauche. Même procédure avec les jambes droite et gauche de derrière. </li><li> Brosse à travers la fourrure sur le cou et le dos domaine avec une pince courbe à pointe fine. </li><li> Retournez la souris sur le dos et vérifier la partie ventrale ainsi que les côtés ventrales des membres de la même manière que précédemment. </li><li> Vérifiez la queue. </li><li> Prenez des photos de parties du corps sur lesquelles les souris développent une maladie. Une attention particulière doit être accordée à la qualité et la quantité des images. </li><li> Calculer les scores cliniques de la maladie. </li></ol> 4. Analyse des échantillons de peau et Plasma <ol><li> Préparer, stocker et / ou de traiter tous les tissus prélevés selon le plan. </li><li> Couper les coupes de tissus congelés pour IF détection des anticorps de lapin et de souris composants du système du complément. </li><li> Analyser le contenu en protéines et enzymes différentes (test FTU) de gelée de tissus et / ou des extraits d&#39;organes. </li><li> Section du tissu inclus en paraffine et colorer avec H &amp; E pour visualiser l&#39;étendue des lésions tissulaires. </li><li> Analyser le plasma par ELISA, immuno-empreinte. </li></ol>

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A., Troy, J. L., Taylor, A. F., Emery, D. J., Fairley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+passive+transfer+model+of+the+organ-specific+autoimmune+disease,+bullous+pemphigoid,+using+antibodies+generated+against+the+hemidesmosomal+antigen,+BP180.">A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.</a> J. Clin. Invest. 92, 2480-2488 (1993).</li><li>Anhalt, G. J., Labib, R. S., Voorhees, J. J., Beals, T. F., Diaz, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pemphigus+in+neonatal+mice+by+passive+transfer+of+IgG+from+patients+with+the+disease.">Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.</a> N. Engl. J. 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Today. 14, 426-430 (1993).</li><li>Sitaru, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+epidermolysis+bullosa+acquisita.">Experimental models of epidermolysis bullosa acquisita.</a> Exp. Dermatol. 16, 520-531 (2007).</li><li>Sitaru, C., Kromminga, A., Hashimoto, T., Br&#246;cker, E. B., Zillikens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+to+type+VII+collagen+mediate+Fcgamma-dependent+neutrophil+activation+and+induce+dermal-epidermal+separation+in+cryosections+of+human+skin.">Autoantibodies to type VII collagen mediate Fcgamma-dependent neutrophil activation and induce dermal-epidermal separation in cryosections of human skin.</a> Am. J. Pathol. 161, 301-311 (2002).</li><li>Sesarman, A., Oswald, E., Chiriac, M. 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Aucun conflit d&#39;intérêt déclaré.

Le transfert passif d&#39;anticorps chez des animaux de laboratoire est une approche importante pour démontrer leur pathogénicité 7, -12. Les modèles animaux obtenus grâce à cette méthode, en plus d&#39;être l&#39;une preuve indirecte de l&#39;auto-immunité 13, permettre à l&#39;enquête de la phase efférente du mécanisme pathogène. Le modèle de transfert passif d&#39;anticorps induite par des granulocytes-dépendante la peau des cloques sur l&#39;épidermolyse bulleuse acquise (EBA) ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> antigène recombinant </td><td></td><td></td><td> L&#39;encodage des plasmides pour les formes recombinantes de murin du collagène VII sont disponibles auprès de l&#39;auteur correspondant. </td></tr><tr><td> sérum de lapin immunisé </td><td> <a href="http://www.eurogentec.com/" target="_blank">www.eurogentec.com</a> </td><td></td><td> Nous avons eu lapins New Zealand White immunisées avec 200 pg d&#39;antigène, 3 fois à 2 semaines d&#39;intervalle. À cette fin, nous avons utilisé les services d&#39;Eurogentec SA, Belgique. </td></tr><tr><td> Protéine G agarose </td><td> Roche Applied Science </td><td> 11243233001 </td><td></td></tr><tr><td> Souris Balb / c </td><td> Charles River Laboratories </td><td></td><td></td&gt; </tr><tr><td> Composé OCT, Tissue-Tek </td><td> Sakura Finetek </td><td> 4583 </td><td> PTOM, la température de coupe optimale </td></tr><tr><td> Cryomold norme </td><td> Sakura Finetek </td><td> 4557 </td><td> 25 mm x 20 mm x 5 mm </td></tr><tr><td> Cryomold intermédiaire </td><td> Sakura Finetek </td><td> 4566 </td><td> 15 mm x 15 mm x 5 mm </td></tr><tr><td> Uni-Link-Einbettkassette </td><td> R. Langenbrinck </td><td> 09-0503 </td><td> Traitement de l&#39;histologie et de cassettes d&#39;encastrement </td></tr><tr><td> Spezial-Tatowierfarbe Schwarz </td><td> H. Hauptnerund &amp; Richard Herberholz GmbH &amp; Co. KG </td><td> 71492000 </td><td> tatouage pâte </td></tr><tr><td> Tatowierzange TZ1 </td><td> Ebeco E. Becker &amp; Co GmbH </td><td></td><td> tatouage dispositif </td></tr><tr><td> L&#39;héparine </td><td> Carl Roth GmbH &amp; Co. </td><td> 7692.1 </td><td></td></tr><tr><td> Formaldéhyde 37% </td><td> Carl Roth GmbH &amp; Co. </td><td> 7386 </td><td></td></tr><tr><td> Le chlorhydrate de kétamine </td><td> Sigma-Aldrich Chemie GmbH </td><td> K2753-1G </td><td></td></tr><tr><td> Chlorhydrate de xylazine </td><td> Sigma-Aldrich Chemie GmbH </td><td> X1251-1G </td><td></td></tr><tr><td> PBS stérile </td><td> Biochrom </td><td> L182-50 </td><td></td></tr><tr><td> appareil photo numérique </td><td> Nikon </td><td> Coolpix 5400 </td><td></td></tr><tr><td> Seringue entraînée unité de filtre de 0,45 um </td><td> Millipore </td><td></td><td></td></tr><tr><td> Pied à coulisse </td><td> Mitutoyo 7309 </td><td> 1667338 </td><td> Farnell (distributeur) </td></tr><tr><td> feuille de pointage maladie </td><td></td><td></td><td> Par exemple enclosed </td></tr></tbody></table>

granulocyte-dependent autoantibody-induced skin blistering

Phénomènes auto-immunes surviennent chez des personnes en bonne santé, mais quand la tolérance au soi échoue, la réponse auto-immune peut entraîner une pathologie spécifique. Selon Witebsky postulats, l&#39;un des critères de diagnostic d&#39;une maladie auto-immune est que la reproduction de la maladie chez les animaux de laboratoire par le transfert passif d&#39;autoanticorps. Pour l&#39;épidermolyse bulleuse acquise (EBA), un prototype spécifique d&#39;organe maladie auto-immune de la peau et des muqueuses, plusieurs modèles expérimentaux ont été récemment mis en place. Dans le modèle animal décrit dans notre travail actuel, purifié des anticorps IgG contre un tronçon de 200 acides aminés (aa 757-967) de collagène VII sont injectés à plusieurs reprises dans des souris reproduisant le phénotype des cloques ainsi que les caractéristiques de l&#39;histo-et immunopathologiques caractéristiques pour la santé humaine EBA 1. Véritable maladie répandue est généralement considérée 5-6 jours après la première injection et l&#39;étendue de la maladie est en corrélation avec la dose administrée de la collagen VII IgG spécifiques. Les lésions tissulaires (formation de bulles) dans le groupe expérimental EBA est en fonction sur le recrutement et l&#39;activation des granulocytes par des tissus assortis autoanticorps 2, -4. Par conséquent, ce modèle permet la dissection de la voie de granulocytes-dépendante inflammatoire impliquée dans les lésions tissulaires auto-immunes, comme le modèle ne reproduit que la cellule T-indépendante phase de la réponse auto-immune efférente. De plus, sa valeur est soulignée par un certain nombre d&#39;études démontrant le potentiel blister induisant des anticorps in vivo et étudier le mécanisme de la formation de bulles dans l&#39;EBA 1,3, -6. Enfin, ce modèle va grandement faciliter le développement de nouvelles thérapies anti-inflammatoires dans les maladies provoquées par des auto-anticorps. Dans l&#39;ensemble, le modèle de transfert passif des animaux de l&#39;EBA est un modèle de la maladie accessible et instructif et aidera les chercheurs à analyser non seulement la pathogenèse EBA mais pour répondre fondamentale biologiquement et cliniquementdes questions d&#39;auto-immunité essentielles.

Le transfert passif d&#39;anticorps spécifiques d&#39;antigènes résultats dans une maladie épanouie chez la souris, ressemblant à des niveaux cliniques, histologiques et immunopathologiques l&#39;ABE humaine. Cloques, de croûtes, des érosions et alopécie se développer sur les oreilles, le museau, les pattes, les jambes, le dos et autour des yeux des souris. Les premiers signes cliniques de la maladie sera très probablement apparaître dans les oreilles et / ou de la zone tête. Le dépôt de spécifique IgG de...

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Granulocyte-dependent Autoantibody-induced Skin Blistering

Dans le modèle animal décrit dans notre travail actuel, purifié des anticorps IgG contre un tronçon de 200 acides aminés (aa 757-967) de collagène VII sont injectés à plusieurs reprises dans des souris reproduisant le phénotype des cloques ainsi que les caractéristiques de l&#39;histo-et immunopathologiques caractéristiques pour la santé humaine l&#39;épidermolyse bulleuse acquise (EBA)<sup&gt; 1</sup&gt;.

Granulocytes-dépendante boursouflures la peau induite par autoanticorps

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 vues.  University of Freiburg. Dans le modèle animal décrit dans notre travail actuel, purifié des anticorps IgG contre un tronçon de 200 acides aminés (aa 757-967) de collagène VII sont injectés à plusieurs reprises dans des souris reproduisant le phénotype des cloques ainsi que les caractéristiques de l'histo-et immunopathologiques caractéristiques pour la santé humaine l'épidermolyse bulleuse acquise (EBA) 1.

Vidéo: 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.

La leishmaniose cutanée dans la peau dorsale de Hamsters: un modèle utile pour le dépistage des drogues antileishmanienne

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

Recherche

Immunologie et infection

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.

Imagerie de VIH-1 Enveloppe induite Synapse virologique et de signalisation sur bicouches lipidiques synthétiques

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.

Transplantation de queue peau d&#39;étudier allogéniques CD4 réponses des cellules T chez la souris

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.

Lymphocytes Isolation de la peau humaine pour Analyse phénotypique et<em&gt; Ex Vivo</em&gt; Culture cellulaire

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.

Préparation de Suspensions de cellules uniques pour l&#39;analyse cytofluorimétrie de différentes régions de la peau de souris

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.

Isolement de leucocytes infiltrant de la peau de souris Utilisation de digestion enzymatique et séparation par 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.

Myéloïde cellule d&#39;isolement de la peau de souris et Vidange ganglion Après intradermique immunisation avec Live Atténuée<em&gt; Plasmodium</em&gt; sporozoïtes

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.

Vasodilatation des vaisseaux isolés et l&#39;isolement de la matrice extracellulaire de souris Tight-peau

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 Transplantation de peau pleine épaisseur

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.

Développement d&#39;un ADN Système économique Livraison par « Acufection » et son application à la recherche de la peau

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