EB, KMB und HX tragen gleichermaßen. Diese Studie wurde von der NCI SPORE in Gastrointestinal Cancers P50 CA062924, NIH K23 CA148964, Lustgarten Foundation, Viragh Foundation, ASCO Young Investigator Award, NIH 5T32 CA0090701-28 Training Grant, Sol Goldman Pancreatic Cancer Center und National Pancreas Foundation unterstützt.

B7-H1/PD-L1, ein Mitglied der B7-Familie von immunregulatorische Zelloberflächenproteinen, spielt eine wichtige Rolle bei der negativen Regulation von zellvermittelten Immunantworten durch seine Wechselwirkung mit seinem Rezeptor, programmierten Zelltod-1 (PD -1) 1,2. Überexpression von B7-H1 durch Tumorzellen wurde in einer Reihe von menschlichen Krebsarten, einschließlich Melanom, Glioblastom und Karzinomen der Lunge, Brust, Dickdarm, Eierstock und Nierenzellen bemerkt worden, und wurde gezeigt, dass anti-Tumor-T beeinträchtigen Zell-Immunität 3-8. Vor kurzem hat B7-H1 Expression von Pankreaskarzinom Geweben als potentieller prognostischer Marker 9,10 identifiziert worden. Zusätzlich hat Blockade von B7-H1 in einem Mausmodell von Bauchspeicheldrüsenkrebs gezeigt worden, um eine Anti-Tumor-Antwort 11 zu erzeugen. Diese Daten legen nahe, wie wichtig B7-H1 als ein potenzielles therapeutisches Ziel. Anti-B7-H1-Blockade Antikörper sind daher in klinischen Studien für mehrere Menschen getestetsolider Tumoren wie Melanomen und Karzinomen der Lunge, Dickdarm, Niere, Magen und Bauchspeicheldrüse 12. Um schließlich in der Lage sein, um die Patienten, die von B7-H1 Targeting-Therapien profitieren zu identifizieren, ist es wichtig, den Zusammenhang zwischen Expression und Lokalisation von B7-H1 und Ansprechen des Patienten auf die Behandlung mit B7-H1-Blockade Antikörper zu untersuchen. Untersuchen der Expression von B7-H1 in menschlichen Pankreaskarzinom Gewebe durch Immunhistochemie wird ein besseres Verständnis, wie diese mit-inhibitorische Signalmolekül trägt zur Unterdrückung der Antitumor-Immunität in Mikroumgebung des Tumors. Die Anti-B7-H1-Antikörper (Klon 5H1) von Chen und Mitarbeitern entwickelt wurde gezeigt, zuverlässig Färbeergebnisse in Kryoschnitten mehrerer Arten von menschlichen neoplastischen Geweben 4,8 zu produzieren, aber auf Anfärben Paraffin eingebetteten Folien war eine Herausforderung, bis kürzlich 13-18. Wir haben die B7-H1 staini entwickeltng Protokoll für Paraffin eingebettete Folien Pankreaskarzinom Gewebe. Die B7-H1 Färbeprotokoll hier beschriebenen konsistente membranöse und zytoplasmatische Färbung von B7-H1 mit wenig Hintergrund. Ein. De-parafinization <ol><li> Bake gleitet bei 55 ° C für 20 min. </li><li> In einer chemischen Haube, Folien tauchen in Xylol für 10 min. </li><li> Objektträger in frischem Xylol für 10 min. </li><li> Objektträger in 100% Ethanol für 5 min. </li><li> Objektträger in frisch 100% Ethanol für 5 min. </li><li> Objektträger in 95% Ethanol für 5 min. </li><li> Objektträger in 80% Ethanol für 5 min. </li><li> Objektträger in entionisiertem Wasser für 5 min. </li></ol> 2. Antigen Retrieval <ol><li> Objektträger in Tris-EDTA-Puffer (pH 9,0) und Wärme in einem Dampfkochtopf bis 125 ° C für 30 sec, dann 90 ° C für 10 sek. </li><li> Lassen Dias cool für 60 min. </li><li> Objektträger in TBST für 5 min; repebei zweimal. </li></ol> 3. Färbung <ol><li> Wischen Sie Lösungen rund um das Gewebe auf den Folien. Markieren Sie einen Kreis um das Gewebe mit einer hydrophoben Stift. </li><li> Objektträger in einer feuchten Kammer und vermeiden das Trocknen des Gewebes auf der Folie über den gesamten Färbeprozesses. Einen Tropfen (etwa 200 ul oder mehr, um das Gewebe vollständig zu bedecken Bereich) von Peroxidase Block innerhalb des hydrophoben Kreis auf der Rutsche und inkubieren in Peroxidase Block für 5 min. </li><li> Die Objektträger mit TBST, dann in TBST für 5 min. </li><li> Geben Sie einen Tropfen BLOCK ACE Blocking-Puffer auf jeder Folie und Inkubation für 15 min. </li><li> Die Objektträger mit TBST, dann in TBST für 5 min. </li><li> Geben Sie einen Tropfen Avidin-Lösung auf jeder Folie und Inkubation für 15 min. </li><li> Die Objektträger mit TBST, dann in TBST für 5 min. </li><li> Geben Sie einen Tropfen Biotin-Lösung auf jeder Folie und Inkubation für 15 min. </li><li> Spülen slides mit TBST, dann in TBST für 5 min. </li><li> Die Objektträger mit 1:1000 Verdünnung von Maus-Anti-B7-H1-Antikörper (Klon 5H1) oder einer Maus IgG1 κ Isotypenkontrolle in 200 ul TBST bei 4 ° C über Nacht für 22 h + / - 1 h. Die endgültige Antikörperkonzentration in TBST ist 2,57 ug / ml. </li><li> Die Objektträger mit TBST, dann in TBST für 5 min. Zweimal wiederholen. </li><li> Die Objektträger mit einem Biotin konjugiertes Ratte-anti-Maus-IgG1-Antikörper in einer sekundären 1:500 Konzentration (in 200 ul 2% Ziegenserum-Lösung) für 30 min. </li><li> Die Objektträger mit TBST, dann in TBST für 5 min. Zweimal wiederholen. </li><li> Geben Sie einen Tropfen Streptavidin-Biotin-Komplex auf jeder Folie und Inkubation für 15 min (muss mindestens 30 min vor Gebrauch vorbereitet werden). </li><li> Die Objektträger mit TBST, dann in TBST für 5 min. Zweimal wiederholen. </li><li> Geben Sie einen Tropfen Amplifikationsreagenz (verdünnt 2:1 mit TBST) auf jeder Folie und Inkubation für 4 min. </li><li> Die Objektträger mit TBST, dann in TBST für 5 min. Zweimal wiederholen. </li><li> Geben Sie einen Tropfen Streptavidin-HRP auf jeder Folie und Inkubation für 15 min. </li><li> Die Objektträger mit TBST, dann in TBST für 5 min. Zweimal wiederholen. </li><li> Setzen Sie einen Tropfen 3,3-Diaminobenzidin (DAB)-Substrat und Chromogen auf jeder Folie entwickeln und die Farbe für 2 min. </li><li> Unmittelbar nach der Farbentwicklung, waschen Folien mit ddH 2 O für 1-2 min. </li><li> Die Objektträger mit einem Tropfen Hämatoxylin für 90 sec. </li><li> Waschen mit ddH 2 O für 1-2 min. </li><li> Waschen mit Seifenwasser Leitungswasser für 1-2 min. </li><li> Waschen mit ddH 2 O für 1-2 min. </li></ol> 4. Dehydration und Montage <ol><li> Eintauchen der Objektträger in 80% Ethanol für 3 min. </li><li> Tauchen Sie die Folien in 95% Ethanol für 3 min. </li><li> Eintauchen der Objektträger in 100% Ethanol für 5 min. Wiederholen. </li><li> Eintauchen der Objektträger in Xylol für 5 min. Wiederholen. </li><li> Einen TropfenCytoseal 60 auf den Schlitten und Ort das Deckglas. </li></ol> Tabelle 1 listet die Reagenzien in den oben beschriebenen Verfahren verwendet.

<ol>
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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. 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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. 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Immunhistochemische Färbung von B7-H1 auf Kryoschnitten In früheren Studien wurde für eine Vielzahl von Krebsarten 3,4 gemeldet. Jedoch klinischen Tumorproben üblicherweise nur in Form von in Paraffin eingebetteten Blöcke verfügbar. Die monoklonalen Antikörper 5H1 hat neuerdings erfolgreich an die Färbung von Paraffin eingebettet Tumorgeweben einschließlich Nierenzellkarzinom, Gebärmutterhalskrebs, Blasenkrebs und Melanom 13-18 angewandt. B7-H1 Färbung auf Paraffin eingebettete, 9,1...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> CSA-System </td><td> Dako </td><td> K1500 </td><td> Enthält die folgenden Reagenzien in dem Protokoll genannt: Peroxidase Block, Streptavidin-Biotin-Komplex, Amplifikationsreagens, Streptavidin-HRP und DAB Substrat und Chromogen </td></tr><tr><td> Avidin / Biotin Blocking Kit </td><td> Vektor </td><td> SP-2001 </td><td></td></tr><tr><td> Block ACE </td><td> Serotec </td><td> BUF029 </td><td></td></tr><tr><td> Biotin Anti-Maus-IgG1 </td><td> BD </td><td> 553441 </td><td></td></tr><tr><td> Mouse IgG1 K Isotypkontrolle </td><td> eBioscience </td><td> 14-4714-85 </td><td></td></tr><tr><td> TBS (Tris-gepufferte Salzlösung), 10X </td><td> Cellgro </td><td> 46-012-CM </td><td> 10xTBS enthält 80 g / l NaCl und 24,2 g / L Tirs; Verdünnt 1X mit ddH2O für Waschmittel; </td></tr><tr><td> Tween 20 </td><td> Sigma-Aldrich </td><td> P1379 </td><td> 1 ml zugesetzt, um 1 l 1X TBS zu machen TBST </td></tr><tr><td> Target Retrieval, 20x </td><td> Celerus Welle </td><td> 014-3000-000 </td><td> Verdünnt auf 1x mit ddH 2 O </td></tr><tr><td> 190 Proof Ethylalkohol </td><td> Pharmco-Aaper </td><td> 111000190 </td><td></td></tr><tr><td> 200 Proof Ethylalkohol </td><td> Pharmo-Aaper </td><td> 111000200 </td><td></td></tr><tr><td> Xylol </td><td> VWR </td><td> 95057-827 </td><td></td></tr><tr><td> Hematoxylin </td><td> Richard-Allan Scientific </td><td> 72804 </td><td></td></tr><tr><td> Cytoseal 60 </td><td> Richard-Allan Scientific </td><td> 8310-4 </td><td></td></tr><tr><td> Deckgläser </td><td> Corning </td><td> 2940-245 </td><td></td></tr><tr><td> Feuchte Kammer </td><td> Sigma </td><td> H6644 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabelle 1. Reagenzien und Ausrüstung. </td></tr></tbody></table>

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

B7-H1/PD-L1, ein Mitglied der B7-Familie von immunregulatorische Zelloberflächenproteinen, spielt eine wichtige Rolle bei der negativen Regulation von zellvermittelten Immunantworten durch seine Wechselwirkung mit seinem Rezeptor, programmierten Zelltod-1 (PD -1) 1,2. Überexpression von B7-H1 durch Tumorzellen wurde in einer Reihe von menschlichen Krebsarten, einschließlich Melanom, Glioblastom und Karzinomen der Lunge, Brust, Dickdarm, Eierstock und Nierenzellen bemerkt worden, und wurde gezeigt, dass anti-Tumor-T beeinträchtigen Zell-Immunität 3-8. Vor kurzem hat B7-H1 Expression von Pankreaskarzinom Geweben als potentieller prognostischer Marker 9,10 identifiziert worden. Zusätzlich hat Blockade von B7-H1 in einem Mausmodell von Bauchspeicheldrüsenkrebs gezeigt worden, um eine Anti-Tumor-Antwort 11 zu erzeugen. Diese Daten legen nahe, wie wichtig B7-H1 als ein potenzielles therapeutisches Ziel. Anti-B7-H1-Blockade Antikörper sind daher in klinischen Studien für die mul geprüfttiple humanen soliden Tumoren wie Melanomen und Karzinomen der Lunge, Dickdarm, Niere, Magen und Bauchspeicheldrüse 12. Um schließlich in der Lage sein, um die Patienten, die von B7-H1 Targeting-Therapien profitieren zu identifizieren, ist es wichtig, den Zusammenhang zwischen Expression und Lokalisation von B7-H1 und Ansprechen des Patienten auf die Behandlung mit B7-H1-Blockade Antikörper zu untersuchen. Untersuchen der Expression von B7-H1 in menschlichen Pankreaskarzinom Gewebe durch Immunhistochemie wird ein besseres Verständnis, wie diese mit-inhibitorische Signalmolekül trägt zur Unterdrückung der Antitumor-Immunität in Mikroumgebung des Tumors. Die Anti-B7-H1-Antikörper (Klon 5H1) von Chen und Mitarbeitern entwickelt wurde gezeigt, zuverlässig Färbeergebnisse in Kryoschnitten mehrerer Arten von menschlichen neoplastischen Geweben 4,8 zu produzieren, aber auf Anfärben Paraffin eingebetteten Folien war eine Herausforderung, bis kürzlich 13-18. Wir haben developed die B7-H1 Färbeprotokoll für Paraffin eingebettete Folien Pankreaskarzinom Gewebe. Die B7-H1 Färbeprotokoll hier beschriebenen konsistente membranöse und zytoplasmatische Färbung von B7-H1 mit wenig Hintergrund.

Eine erfolgreiche immunhistochemische Färbung von B7-H1 demonstrieren die heterogene Expression von B7-H1 (braun Signale DAB Farbentwicklung) in Pankreaskarzinom. Einige Pankreaskarzinom-Zellen haben überwiegend membranöse Expression von B7-H1 (Abbildung 1A), während andere entweder vorwiegend zytoplasmatische Expression von B7-H1 oder wenig Expression von B7-H1 (Abbildungen 2 und 3). Normale Pankreasgangepithel drückt etwas B7-H1 (Abbildung 4). Anfärbung mit eine...

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) und seine Bindung an PD-1 einen großen tumorinduzierte immunsuppressiven Signal in Mikroumgebung des Tumors. Eine immunhistochemische Färbetechnik, die Expression und Lokalisation von B7-H1 in Pankreaskarzinom zu charakterisieren ist hier beschrieben.

Immunhistochemische Färbung von B7-H1 (PD-L1) auf Paraffin-embedded Slides der Pankreaskarzinom Tissue

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 Aufrufe.  The Johns Hopkins University School of Medicine. B7-H1 (PD-L1) und seine Bindung an PD-1 einen großen tumorinduzierte immunsuppressiven Signal in Mikroumgebung des Tumors. Eine immunhistochemische Färbetechnik, die Expression und Lokalisation von B7-H1 in Pankreaskarzinom zu charakterisieren ist hier beschrieben.

Serie: 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.

Färbeprotokollen for Human Pankreasinseln

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

Forschung

Medizin

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 of Human Prostate Epithel Mit Tissue Rekombination von Nagetier Urogenitalsinus Mesenchym und menschlichen Stammzellen

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

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Bioluminescent orthotopen Modell von Bauchspeicheldrüsenkrebs Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

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.

Vorbereitung und Respirometrische Bewertung der Mitochondrien isoliert aus dem Skelettmuskelgewebe durch perkutane Nadelbiopsie Erhalten

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.

Anwendung von End-to-End-Anastomose in Roboter-zentrale Pankreatektomie

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

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal Transplantation der Bauchspeicheldrüse Inselchen im Mausmodell

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

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.

Immunfluoreszenz-Kennzeichnung von humanen und murinen neutrophilen extrazellulären Fallen in Paraffin-Embedded Tissue

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.

Robotische Enukleation eines intrapankreatischen Insulinoms im Bauchspeicheldrüsenkopf

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.

Messung des Gewebe-Nicht-Häm-Eisengehalts mit einem Bathophenanthrolin-basierten kolorimetrischen Assay

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

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and experimental verification. The effective components and potential targets of Trichosanthis and Fritillaria thunbergii were collected by high-throughput experiment and reference-guided (HERB) database of traditional Chinese medicine and a similarity ensemble approach (SEA) database, and the LUAD-related targets were queried by the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. A drug-component-disease-target network was constructed by Cytoscape software. Protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted to obtain core targets and key pathways. An aqueous extract of Trichosanthes-Fritillaria thunbergii and A549 cells were used for the subsequent experimental validation. Through the HERB database and literature search, 31 effective compounds and 157 potential target genes of Trichosanthes-Fritillaria thunbergii were screened, of which 144 were regulatory targets of Trichosanthes-Fritillaria thunbergii in the treatment of lung adenocarcinoma. The GO functional enrichment analysis showed that the mechanism of action of Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma is mainly protein phosphorylation. The KEGG pathway enrichment analysis suggested that the treatment of lung adenocarcinoma by Trichosanthes-Fritillaria thunbergii mainly involves the PI3K/AKT signaling pathway. The experimental validation showed that an aqueous extract of Trichosanthes-Fritillaria thunbergii could inhibit the proliferation of A549 cells and the phosphorylation of AKT. Through network pharmacology and experimental validation, it was verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

This study reveals the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma based on network pharmacology and experimental verification. The study also demonstrates that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

Netzwerkpharmakologische Vorhersage und experimentelle Validierung des Wirkungsmechanismus von Trichosanthes-Fritillaria thunbergii gegen Lungenadenokarzinome