EB, KMB, et HX contribuent à parts égales. Cette étude a été financée par le NCI SPORE de cancers gastro-intestinaux P50 CA062924, CA148964 NIH K23, Lustgarten Foundation, Viragh Fondation, le Prix du jeune chercheur ASCO, NIH 5T32 CA0090701-28 Subvention de formation, Sol Goldman Centre cancer du pancréas, de la Fondation nationale du pancréas.

B7-H1/PD-L1, un membre de la famille B7 du système immunitaire réglementaires protéines de surface cellulaire, joue un rôle important dans la régulation négative des réponses à médiation cellulaire immunitaire grâce à son interaction avec son récepteur, la mort programmée-1 (PD -1) 1,2. La surexpression de B7-H1 par les cellules tumorales a été observée dans un certain nombre de cancers humains, y compris le mélanome, le glioblastome et les carcinomes du poumon, du sein, du côlon, de l&#39;ovaire, et des cellules rénales, et a été montré pour affecter anti-tumorales T- 3-8 immunité cellulaire. Récemment, B7-H1 expression par les tissus adénocarcinome pancréatique a été identifié comme un marqueur pronostique potentiel de 9,10. De plus, le blocage de B7-H1 dans un modèle murin de cancer du pancréas a été montré pour produire une réponse anti-tumorale 11. Ces données suggèrent l&#39;importance de la B7-H1 comme une cible thérapeutique potentielle. Anticorps blocus anti-B7-H1 sont donc l&#39;objet d&#39;essais cliniques pour l&#39;homme multipledes tumeurs solides, y compris le mélanome et le cancer du poumon, du côlon, du rein, de l&#39;estomac et du pancréas 12. Pour finalement être en mesure d&#39;identifier les patients qui bénéficieront de B7-H1 ciblage des thérapies, il est essentiel d&#39;étudier la corrélation entre l&#39;expression et la localisation de B7-H1 et la réponse du patient au traitement avec des anticorps blocus B7-H1. Examiner l&#39;expression de B7-H1 dans les tissus humains adénocarcinome pancréatique par immunohistochimie vous donnera une meilleure compréhension de la façon dont cette molécule de signalisation co-inhibiteur contribue à la suppression de l&#39;immunité anti-tumorale dans le micro de la tumeur. L&#39;anticorps anti-B7-H1 monoclonal (clone 5H1) développé par Chen et ses collègues a été montré pour produire des résultats fiables dans cryosections coloration de plusieurs types de tissus humains néoplasiques 4,8, mais des taches sur paraffine diapositives a été un défi que récemment 13-18. Nous avons développé le staini B7-H1protocole ng pour les diapositives inclus en paraffine des tissus adénocarcinome pancréatique. Le protocole de coloration B7-H1 décrit ici produit conforme coloration membranaire et cytoplasmique de B7-H1 avec petit retour en arrière. 1. De-parafinization <ol><li> Cuire glisse à 55 ° C pendant 20 min. </li><li> Dans une hotte chimique, immerger les lames dans du xylène pendant 10 min. </li><li> Immerger les lames dans du xylène frais pendant 10 min. </li><li> Plonger les lames dans de l&#39;éthanol à 100% pendant 5 min. </li><li> Plonger les lames dans de l&#39;éthanol 100% frais pendant 5 min. </li><li> Plonger les lames dans de l&#39;éthanol à 95% pendant 5 min. </li><li> Plonger les lames dans l&#39;éthanol à 80% pendant 5 min. </li><li> Plonger les lames dans de l&#39;eau désionisée pendant 5 min. </li></ol> 2. Récupération antigène <ol><li> Immerger les lames dans du Tris-EDTA (pH 9,0) et de la chaleur dans une cocotte-minute à 125 ° C pendant 30 secondes, puis 90 ° C pendant 10 sec. </li><li> Laissez refroidir diapositives pendant 60 min. </li><li> Immerger les lames dans du TBST pendant 5 min; Repemoins deux fois. </li></ol> 3. Tachant <ol><li> Essuyez solutions autour du tissu sur les diapositives. Marquer un cercle autour du tissu avec un stylo hydrophobe. </li><li> Placer les lames dans une chambre humide et éviter le séchage du tissu sur la lame pendant le processus de coloration entier. Déposer une goutte (environ 200 pi ou plus pour couvrir complètement la zone de tissu) du bloc de peroxydase dans le cercle hydrophobe sur la diapositive et diapositives incuber dans le bloc peroxydase pendant 5 min. </li><li> Rincer les lames avec du TBST, puis mettre dans TBST pendant 5 min. </li><li> Déposer une goutte de BLOCK ACE tampon de blocage sur chaque lame et incuber pendant 15 min. </li><li> Rincer les lames avec du TBST, puis mettre dans TBST pendant 5 min. </li><li> Déposer une goutte de solution d&#39;avidine sur chaque lame et incuber pendant 15 min. </li><li> Rincer les lames avec du TBST, puis mettre dans TBST pendant 5 min. </li><li> Déposer une goutte de solution de biotine sur chaque lame et incuber pendant 15 min. </li><li> Rincer slides avec TBST, puis mettre dans TBST pendant 5 min. </li><li> Incuber les lames avec 1:1000 dilution des anticorps de souris anti-B7-H1 anticorps monoclonal (clone 5H1) ou une souris IgG1 κ isotype contrôle dans 200 ul TBST à 4 ° C pendant la nuit pendant 22 heures + / - 1 heure. La concentration finale de l&#39;anticorps dans du TBST vaut 2.57 pg / ml. </li><li> Rincer les lames avec du TBST, puis mettre dans TBST pendant 5 min. Répéter deux fois. </li><li> Incuber les lames avec un rat biotine anti-souris conjugué anticorps IgG1 secondaire à une concentration 1:500 (dans 200 ul solution de chèvre 2% de sérum) pendant 30 min. </li><li> Rincer les lames avec du TBST, puis mettre dans TBST pendant 5 min. Répéter deux fois. </li><li> Déposer une goutte de streptavidine-biotine (doit être préparé au moins 30 minutes avant de l&#39;utiliser) sur chaque lame et incuber pendant 15 min. </li><li> Rincer les lames avec du TBST, puis mettre dans TBST pendant 5 min. Répéter deux fois. </li><li> Déposer une goutte de réactif d&#39;amplification (dilué 2:1 avec du TBST) sur chaque lame et incuber pendant 4 min. </li><li> Rincer les lames avec TBST, puis mettre dans TBST pendant 5 min. Répéter deux fois. </li><li> Déposer une goutte de streptavidine-HRP sur chaque lame et incuber pendant 15 min. </li><li> Rincer les lames avec du TBST, puis mettre dans TBST pendant 5 min. Répéter deux fois. </li><li> Déposer une goutte de 3,3-diaminobenzidine (DAB) et substrat chromogène sur chaque diapositive et développer la couleur pendant 2 min. </li><li> Immédiatement après le développement de la couleur, laver les lames avec ddH 2 O pendant 1-2 min. </li><li> Incuber les lames avec une goutte d&#39;hématoxyline pendant 90 sec. </li><li> Laver avec ddH 2 O pendant 1-2 min. </li><li> Laver à l&#39;eau savonneuse robinet pendant 1-2 min. </li><li> Laver avec ddH 2 O pendant 1-2 min. </li></ol> 4. La déshydratation et de montage <ol><li> Immerger les lames dans l&#39;éthanol à 80% pendant 3 min. </li><li> Immerger les lames dans l&#39;éthanol à 95% pendant 3 min. </li><li> Immerger les lames dans l&#39;éthanol à 100% pendant 5 min. Répéter. </li><li> Immerger les lames dans du xylène pendant 5 min. Répéter. </li><li> Ajouter une goutte deCytoseal 60 à la lame et le lieu de la lamelle. </li></ol> Le tableau 1 dresse la liste des réactifs utilisés dans les procédures décrites ci-dessus.

<ol>
	<li>Flies, D. B., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+B7s:+playing+a+pivotal+role+in+tumor+immunity.">The new B7s: playing a pivotal role in tumor immunity.</a> J. Immunother. 30, 251-260 (2007).</li><li>Dong, H., Zhu, G., Tamada, K., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1,+a+third+member+of+the+B7+family,+co-stimulates+T-cell+proliferation+and+interleukin-10+secretion.">B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion.</a> Nat. Med. 5, 1365-1369 (1999).</li><li>Curiel, T. J., Wei, S., Dong, H., Alvarez, X., Cheng, P., Mottram, P., Krzysiek, R., Knutson, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+B7-H1+improves+myeloid+dendritic+cell-mediated+antitumor+immunity.">Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity.</a> Nat. Med. 9, 562-567 (2003).</li><li>Dong, H., Strome, S. E., Salomao, D. R., Tamura, H., Hirano, F., Flies, D. B., Roche, P. C., Lu, J., Zhu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+B7-H1+promotes+T-cell+apoptosis:+a+potential+mechanism+of+immune+evasion.">Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.</a> Nat. Med. 8, 793-800 (2002).</li><li>Dong, H., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+pathway+and+its+role+in+the+evasion+of+tumor+immunity.">B7-H1 pathway and its role in the evasion of tumor immunity.</a> J. Mol. Med. (Berl). 81, 281-287 (2003).</li><li>Strome, S. E., Dong, H., Tamura, H., Voss, S. G., Flies, D. B., Tamada, K., Salomao, D., Cheville, J., Hirano, F., Lin, W., Kasperbauer, J. L., Ballman, K. V., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+blockade+augments+adoptive+T-cell+immunotherapy+for+squamous+cell+carcinoma.">B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma.</a> Cancer Res. 63, 6501-6505 (2003).</li><li>Hirano, F., Kaneko, K., Tamura, H., Dong, H., Wang, S., Ichikawa, M., Rietz, C., Flies, D. B., Lau, J. S., Zhu, G., Tamada, K., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+B7-H1+and+PD-1+by+monoclonal+antibodies+potentiates+cancer+therapeutic+immunity.">Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity.</a> Cancer Res. 65, 1089-1096 (2005).</li><li>Thompson, R. H., Gillett, M. D., Cheville, J. C., Lohse, C. M., Dong, H., Webster, W. S., Krejci, K. G., Lobo, J. R., Sengupta, S., Chen, L., Zincke, H., Blute, M. L., Strome, S. E., Leibovich, B. C., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Costimulatory+B7-H1+in+renal+cell+carcinoma+patients:+Indicator+of+tumor+aggressiveness+and+potential+therapeutic+target.">Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 17174-17179 (2004).</li><li>Geng, L., Huang, D., Liu, J., Qian, Y., Deng, J., Li, D., Hu, Z., Zhang, J., Jiang, G., Zheng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+up-regulated+expression+in+human+pancreatic+carcinoma+tissue+associates+with+tumor+progression.">B7-H1 up-regulated expression in human pancreatic carcinoma tissue associates with tumor progression.</a> J. Cancer Res. Clin. Oncol. 134, 1021-1027 (2008).</li><li>Loos, M., Giese, N. A., Kleeff, J., Giese, T., Gaida, M. M., Bergmann, F., Laschinger, M., WB, M., Friess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+and+regulation+of+the+costimulatory+molecule+B7-H1+in+pancreatic+cancer.">Clinical significance and regulation of the costimulatory molecule B7-H1 in pancreatic cancer.</a> Cancer Lett. 268, 98-109 (2008).</li><li>Okudaira, K., Hokari, R., Tsuzuki, Y., Okada, Y., Komoto, S., Watanabe, C., Kurihara, C., Kawaguchi, A., Nagao, S., Azuma, M., Yagita, H., Miura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+B7-H1+or+B7-DC+induces+an+anti-tumor+effect+in+a+mouse+pancreatic+cancer+model.">Blockade of B7-H1 or B7-DC induces an anti-tumor effect in a mouse pancreatic cancer model.</a> Int. J. Oncol. 35, 741-749 (2009).</li><li>Wang, S., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+cancer+therapies+targeting+CD137+and+B7-H1/PD-1+cosignal+pathways.">Immunobiology of cancer therapies targeting CD137 and B7-H1/PD-1 cosignal pathways.</a> Curr. Top Microbiol. Immunol. 344, 245-267 (2011).</li><li>Thompson, R. H., Webster, W. S., Cheville, J. C., Lohse, C. M., Dong, H., Leibovich, B. C., Kuntz, S. M., Sengupta, S., Kwon, E. D., Blute, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+glycoprotein+blockade:+a+novel+strategy+to+enhance+immunotherapy+in+patients+with+renal+cell+carcinoma.">B7-H1 glycoprotein blockade: a novel strategy to enhance immunotherapy in patients with renal cell carcinoma.</a> Urology. 66, 10-14 (2005).</li><li>Thompson, R. H., Kuntz, S. M., Leibovich, B. C., Dong, H., Lohse, C. M., Webster, W. S., Sengupta, S., Frank, I., Parker, A. S., Zincke, H., Blute, M. L., Sebo, T. J., Cheville, J. C., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+B7-H1+is+associated+with+poor+prognosis+in+renal+cell+carcinoma+patients+with+long-term+follow-up.">Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up.</a> Cancer Res. 66, 3381-3385 (2006).</li><li>Thompson, R. H., Dong, H., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+B7-H1+expression+in+clear+cell+carcinoma+of+the+kidney+for+prognostication+and+therapy.">Implications of B7-H1 expression in clear cell carcinoma of the kidney for prognostication and therapy.</a> Clin. Cancer Res. 13, 709s-715s (2007).</li><li>Karim, R., Jordanova, E. S., Piersma, S. J., Kenter, G. G., Chen, L., Boer, J. M., Melief, C. J., vander Burg, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-expressed+B7-H1+and+B7-DC+in+relation+to+PD-1++T-cell+infiltration+and+survival+of+patients+with+cervical+carcinoma.">Tumor-expressed B7-H1 and B7-DC in relation to PD-1+ T-cell infiltration and survival of patients with cervical carcinoma.</a> Clin. Cancer Res. 15, 6341-6347 (2009).</li><li>Inman, B. A., Sebo, T. J., Frigola, X., Dong, H., Bergstralh, E. J., Frank, I., Fradet, Y., Lacombe, L., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-L1+(B7-H1)+expression+by+urothelial+carcinoma+of+the+bladder+and+BCG-induced+granulomata:+associations+with+localized+stage+progression.">PD-L1 (B7-H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression.</a> Cancer. 109, 1499-1505 (2007).</li><li>Brahmer, J. R., Drake, C. G., Wollner, I., Powderly, J. D., Picus, J., Sharfman, W. H., Stankevich, E., Pons, A., Salay, T. M., McMiller, T. L., Gilson, M. M., Wang, C., Selby, M., Taube, J. M., Anders, R., Chen, L., Korman, A. J., Pardoll, D. M., Lowy, I., Topalian, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+study+of+single-agent+anti-programmed+death-1+(MDX-1106)+in+refractory+solid+tumors:+safety,+clinical+activity,+pharmacodynamics,+and+immunologic+correlates.">Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.</a> J. Clin. Oncol. 28, 3167-3175 (2010).</li><li>Wang, L., Ma, Q., Chen, X., Guo, K., Li, J., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+of+B7-H1+and+B7-1+expressions+in+pancreatic+carcinoma.">Clinical significance of B7-H1 and B7-1 expressions in pancreatic carcinoma.</a> World J. Surg. 34, 1059-1065 (2010).</li><li>Nomi, T., Sho, M., Akahori, T., Hamada, K., Kubo, A., Kanehiro, H., Nakamura, S., Enomoto, K., Yagita, H., Azuma, M., Nakajima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+and+therapeutic+potential+of+the+programmed+death-1+ligand/programmed+death-1+pathway+in+human+pancreatic+cancer.">Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer.</a> Clin. Cancer Res. 13, 2151-2157 (2007).</li><li>Taube, J. M., Anders, R. A., Young, G. D., Xu, H., Sharma, R., McMiller, T. L., Chen, S., Klein, A. P., Pardoll, D. M., Topalian, S. L., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+inflammatory+response+with+B7-h1+expression+in+human+melanocytic+lesions+supports+an+adaptive+resistance+mechanism+of+immune+escape.">Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape.</a> Sci. Transl. Med. 4, 127ra37 (2012).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

La coloration immunohistochimique de B7-H1 sur cryocoupes a été rapporté dans les études antérieures pour une variété de cancers 3,4. Cependant, cliniques échantillons tumoraux sont généralement disponibles que sous la forme de blocs de paraffine. L&#39;anticorps monoclonal 5H1 a plus récemment été appliquée avec succès à la coloration des tissus tumoraux inclus en paraffine y compris le carcinome à cellules rénales, cancer du col, cancer de la vessie et le mélanome 13-18. B7-H1 ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> CSA Système </td><td> Dako </td><td> K1500 </td><td> Comprend les réactifs suivants mentionnés dans le protocole: bloc peroxydase streptavidine-biotine, complexe, de réactifs d&#39;amplification, la streptavidine-HRP et le substrat chromogène DAB et </td></tr><tr><td> Avidine / biotine Blocage Kit </td><td> Vecteur </td><td> SP-2001 </td><td></td></tr><tr><td> Bloquer ACE </td><td> Serotec </td><td> BUF029 </td><td></td></tr><tr><td> La biotine anti-IgG1 de souris </td><td> BD </td><td> 553441 </td><td></td></tr><tr><td> Mouse IgG1 Isotype contrôle K </td><td> eBioscience </td><td> 14-4714-85 </td><td></td></tr><tr><td> TBS (Tris-buffered saline), 10X </td><td> Cellgro </td><td> 46-012 CM- </td><td> 10xTBS contient 80 g / L de NaCl et 24,2 g / L Tirs; Diluée à 1X avec ddH2O pour les lavages; </td></tr><tr><td> Tween 20 </td><td> Sigma-Aldrich </td><td> P1379 </td><td> Ajouté 1 ml à 1 L d&#39;1X TBS pour faire TBST </td></tr><tr><td> Target Retrieval, 20x </td><td> Celerus Vague </td><td> 014-3000-000 </td><td> Dilué à 1 avec ddH 2 O </td></tr><tr><td> 190 preuve Alcool éthylique </td><td> Pharmco-AAPER </td><td> 111000190 </td><td></td></tr><tr><td> 200 Proof Alcool éthylique </td><td> PHARMO-AAPER </td><td> 111000200 </td><td></td></tr><tr><td> Xylène </td><td> VWR </td><td> 95057-827 </td><td></td></tr><tr><td> Hématoxyline </td><td> Richard-Allan scientifique </td><td> 72804 </td><td></td></tr><tr><td> Cytoseal 60 </td><td> Richard-Allan scientifique </td><td> 8310-4 </td><td></td></tr><tr><td> Lamelles de </td><td> Corning </td><td> 2940-245 </td><td></td></tr><tr><td> Chambre d&#39;humidité </td><td> Sigma </td><td> H6644 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tableau 1. Réactifs et matériel. </td></tr></tbody></table>

immunohistochemical staining of b7-h1 (pd-l1) on paraffin-embedded slides of pancreatic adenocarcinoma tissue

B7-H1/PD-L1, un membre de la famille B7 du système immunitaire réglementaires protéines de surface cellulaire, joue un rôle important dans la régulation négative des réponses à médiation cellulaire immunitaire grâce à son interaction avec son récepteur, la mort programmée-1 (PD -1) 1,2. La surexpression de B7-H1 par les cellules tumorales a été observée dans un certain nombre de cancers humains, y compris le mélanome, le glioblastome et les carcinomes du poumon, du sein, du côlon, de l&#39;ovaire, et des cellules rénales, et a été montré pour affecter anti-tumorales T- 3-8 immunité cellulaire. Récemment, B7-H1 expression par les tissus adénocarcinome pancréatique a été identifié comme un marqueur pronostique potentiel de 9,10. De plus, le blocage de B7-H1 dans un modèle murin de cancer du pancréas a été montré pour produire une réponse anti-tumorale 11. Ces données suggèrent l&#39;importance de la B7-H1 comme une cible thérapeutique potentielle. Anticorps blocus anti-B7-H1 sont donc l&#39;objet d&#39;essais cliniques pour le mulsieurs tumeurs solides humaines, y compris le mélanome et le cancer du poumon, du côlon, du rein, de l&#39;estomac et du pancréas 12. Pour finalement être en mesure d&#39;identifier les patients qui bénéficieront de B7-H1 ciblage des thérapies, il est essentiel d&#39;étudier la corrélation entre l&#39;expression et la localisation de B7-H1 et la réponse du patient au traitement avec des anticorps blocus B7-H1. Examiner l&#39;expression de B7-H1 dans les tissus humains adénocarcinome pancréatique par immunohistochimie vous donnera une meilleure compréhension de la façon dont cette molécule de signalisation co-inhibiteur contribue à la suppression de l&#39;immunité anti-tumorale dans le micro de la tumeur. L&#39;anticorps anti-B7-H1 monoclonal (clone 5H1) développé par Chen et ses collègues a été montré pour produire des résultats fiables dans cryosections coloration de plusieurs types de tissus humains néoplasiques 4,8, mais des taches sur paraffine diapositives a été un défi que récemment 13-18. Nous avons devenfuie le protocole de coloration B7-H1 pour les diapositives inclus en paraffine des tissus adénocarcinome pancréatique. Le protocole de coloration B7-H1 décrit ici produit conforme coloration membranaire et cytoplasmique de B7-H1 avec petit retour en arrière.

Une coloration immunohistochimique succès de B7-H1 fera la démonstration de l&#39;expression hétérogène de B7-H1 (brun signaux développement de la couleur DAB) dans l&#39;adénocarcinome du pancréas. Certaines cellules d&#39;adénocarcinome du pancréas ont une expression essentiellement membranaire de B7-H1 (figure 1A) tandis que d&#39;autres ont une expression soit essentiellement cytoplasmique de B7-H1 ou peu d&#39;expression de B7-H1 (figures 2 et 3). Épithélium pancréatiq...

Watch this Scientific Journal Video about Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue at JoVE.com

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) et sa liaison au PD-1 fournissent une importante induite par la tumeur de signal immunosuppresseur dans le micro de la tumeur. Une technique de coloration immunohistochimique pour caractériser l&#39;expression et la localisation de B7-H1 dans l&#39;adénocarcinome pancréatique est décrite ici.

Coloration immunohistochimique de B7-H1 (PD-L1) sur la paraffine Diapositives de tissus adénocarcinome pancréatique

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

immunohistochemical-staining-b7-h1-pd-l1-on-paraffin-embedded-slides

Research

JoVE Journal

Medicine

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

23.0K vues.  The Johns Hopkins University School of Medicine. B7-H1 (PD-L1) et sa liaison au PD-1 fournissent une importante induite par la tumeur de signal immunosuppresseur dans le micro de la tumeur. Une technique de coloration immunohistochimique pour caractériser l'expression et la localisation de B7-H1 dans l'adénocarcinome pancréatique est décrite ici.

Vidéo: Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocoles de coloration pour des îlots pancréatiques humains

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Recherche

Médecine

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Formation de l&#39;épithélium de la prostate humaine utilisant la recombinaison des tissus de rongeurs urogénital Mesenchyme des sinus et des cellules souches humaines

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Préparation et respirométrique évaluation des mitochondries isolées de tissu musculaire squelettique Obtenu par biopsie percutanée aiguille

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Demande d’une anastomose bout à bout en robotique pancréatectomie centrale

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Immunofluorescence Étiquetage des pièges extracellulaires de l'homme et de la murine Neutrophil dans les tissus paraffins

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Robotic Enucleation of an Intra-Pancreatic Insulinoma in the Pancreatic Head

Pancreatic parenchyma sparing surgery for insulinomas avoids the risk of endocrine and exocrine insufficiency, and potential high-risk anastomoses associated with pancreatic resection. Robotic surgery may be used as an alternative for open pancreatic enucleation without compromising dexterity and 3D-vision.
We present the case of a 42-year old woman who presented with sweating, tremor and episodes of hypoglycemia. A fasting test confirmed endogenic insulin overproduction. After inconclusive CT- and MRI imaging, endoscopic ultrasonography showed a hypoechoic lesion, which was fully within the pancreatic head. Although consent was obtained for pancreatoduodenectomy, robotic enucleation seemed feasible. After mobilization, intraoperative ultrasonography was used to identify the lesion and its relation with the pancreatic duct. Dissection was performed using a traction suture, hot shears and bipolar diathermia. A sealant patch was applied for hemostasis and a drain placed. The patient developed a grade B pancreatic fistula for which endoscopic sphincterotomy was performed; the surgical drain could be removed in the outpatient clinic after 20 days. Prospective studies should confirm the short- and long-term benefits of robotic enucleation of insulinomas.

Here, we present a robotic approach to enucleate an insulinoma in the pancreatic head.

Enucleation robotique d'un insulinome intra-pancréatique dans la tête pancréatique

Pancreatic parenchyma sparing surgery for insulinomas avoids the risk of endocrine and exocrine insufficiency, and potential high-risk anastomoses ...

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Mesure de la teneur en fer non hémique des tissus à l’aide d’un test colorimétrique à base de bathophénanthroline

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

Tissue Embedding and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Intégration tissulaire et coloration par immunofluorescence des manchons myocardiques dans les veines pulmonaires murines

<meta charset="utf8"></head><body>Évaluation de l’épaisseur de la rétine et du nombre de couches cellulaires ganglionnaires dans les globes oculaires des rongeurs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

<meta charset="utf8"></head><body>Préparation et analyse de lames histologiques de globes oculaires de rat et de souris pour évaluer la rétine

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

Préparation et analyse de lames histologiques de globes oculaires de rat et de souris pour évaluer la rétine

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...