EB, KMB, e HX contribuiscono allo stesso modo. Questo studio è stato sostenuto dal SPORE NCI in tumori gastrointestinali P50 CA062924, CA148964 NIH K23, Fondazione Lustgarten, Viragh Foundation, Investigator Award ASCO giovane, NIH CA0090701 5T32-28 Training Grant, Sol Centro pancreatico Goldman Cancer, e della Fondazione Nazionale Pancreas.

B7-H1/PD-L1, un membro della famiglia B7-immune di regolamentazione proteine ​​di superficie cellulare, svolge un ruolo importante nella regolazione negativa di risposte immunitarie mediate da cellule attraverso la sua interazione con il suo recettore, morte programmata-1 (PD -1) 1,2. Sovraespressione di B7-H1 da cellule tumorali è stato osservato in un certo numero di tumori umani, compreso il melanoma, glioblastoma, e carcinomi del polmone, seno, colon, ovaio, e cellule renali, e ha dimostrato di alterare anti-tumorali T- cella immunità 3-8. Recentemente, B7-H1 espressione tessuti adenocarcinoma pancreatico è stato identificato come un potenziale marcatore prognostico 9,10. Inoltre, il blocco di B7-H1 in un modello murino di cancro pancreatico ha dimostrato di produrre una risposta antitumorale 11. Questi dati suggeriscono l&#39;importanza di B7-H1 come un potenziale bersaglio terapeutico. Anti-B7-H1 anticorpi bloccanti sono quindi in fase di sperimentazione in studi clinici per l&#39;uomo piùtumori solidi tra cui il melanoma e tumori del polmone, del colon, rene, stomaco e pancreas 12. Per essere successivamente in grado di identificare i pazienti che beneficeranno B7-H1 di targeting terapie, è fondamentale per indagare la correlazione tra espressione e la localizzazione delle B7-H1 e la risposta del paziente al trattamento con anticorpi B7-H1 blocco. Esaminando l&#39;espressione di B7-H1 in tessuti umani adenocarcinoma pancreatico attraverso immunoistochimica darà una migliore comprensione di come questa co-inibitrice molecola di segnalazione contribuisce alla soppressione dell&#39;immunità antitumorale in microambiente del tumore. L&#39;anticorpo anti-B7-H1 monoclonale (clone 5H1) sviluppato da Chen e colleghi ha dimostrato di produrre risultati affidabili in colorazione criosezioni di più tipi di tessuti neoplastici umani 4,8, ma macchie nei paraffinati diapositive era stata una sfida fino recentemente 13-18. Abbiamo sviluppato la B7-H1 stainiprotocollo ng per le diapositive incluse in paraffina di tessuti adenocarcinoma pancreatico. La B7-H1 protocollo di colorazione qui descritto produce coerente colorazione membranosa e citoplasmatica di B7-H1 con po &#39;di storia. 1. De-parafinization <ol><li> Bake scorre a 55 ° C per 20 min. </li><li> In una cappa chimica, immergere i vetrini in xilolo per 10 min. </li><li> Immergere i vetrini in xilene fresco per 10 minuti. </li><li> Immergere i vetrini in etanolo al 100% per 5 min. </li><li> Immergere i vetrini in etanolo al fresco 100% per 5 min. </li><li> Immergere i vetrini in etanolo al 95% per 5 min. </li><li> Immergere i vetrini in etanolo al 80% per 5 min. </li><li> Immergere i vetrini in acqua deionizzata per 5 min. </li></ol> 2. Antigen Retrieval <ol><li> Immergere i vetrini in tampone Tris-EDTA (pH 9,0) e riscaldare in una pentola a pressione a 125 ° C per 30 sec, quindi 90 ° C per 10 sec. </li><li> Lasciare raffreddare i vetrini per 60 min. </li><li> Immergere i vetrini in TBST per 5 min; rEPEal doppio. </li></ol> 3. Colorazione <ol><li> Pulire soluzioni in tutto il tessuto nelle diapositive. Segnare un cerchio intorno al tessuto con una penna idrofobo. </li><li> Porre i vetrini in una camera umida ed evitare l&#39;essiccamento del tessuto sul vetrino durante tutto il processo di colorazione intero. Porre una goccia (circa 200 pl o più per coprire la zona di tessuto completamente) del blocco perossidasi all&#39;interno del cerchio idrofobico sul vetrino ed incubare i vetrini in blocco perossidasi per 5 min. </li><li> Sciacquare i vetrini con TBST, poi mettere in TBST per 5 min. </li><li> Porre una goccia di BLOCK ACE tampone di bloccaggio su ogni diapositiva e incubare per 15 minuti. </li><li> Sciacquare i vetrini con TBST, poi mettere in TBST per 5 min. </li><li> Mettere una goccia di soluzione di avidina su ogni diapositiva e incubare per 15 minuti. </li><li> Sciacquare i vetrini con TBST, poi mettere in TBST per 5 min. </li><li> Mettere una goccia di soluzione di biotina su ogni diapositiva e incubare per 15 minuti. </li><li> Risciacquare slides con TBST, poi mettere in TBST per 5 min. </li><li> Incubare vetrini con diluizione di 1:1000 topo anti-B7-H1 anticorpo monoclonale (clone 5H1) o un mouse IgG1 κ controllo isotipico in 200 microlitri TBST a 4 ° C per una notte per 22 ore + / - 1 h. La concentrazione finale di anticorpo in TBST è 2,57 mg / ml. </li><li> Sciacquare i vetrini con TBST, poi mettere in TBST per 5 min. Ripetere due volte. </li><li> Incubare vetrini con biotina coniugata ratto anti-topo IgG1 anticorpo secondario ad una concentrazione 1:500 (in 200 ul di soluzione al 2% di siero di capra) per 30 min. </li><li> Sciacquare i vetrini con TBST, poi mettere in TBST per 5 min. Ripetere due volte. </li><li> Una goccia di streptavidina-biotina Complex (deve essere preparata almeno 30 minuti prima dell&#39;uso) su ciascun vetrino e incubare per 15 min. </li><li> Sciacquare i vetrini con TBST, poi mettere in TBST per 5 min. Ripetere due volte. </li><li> Porre una goccia di reagente di amplificazione (diluito 2:1 con TBST) su ogni diapositiva e incubare per 4 min. </li><li> Sciacquare i vetrini con TBST, quindi posto in TBST per 5 min. Ripetere due volte. </li><li> Porre una goccia di streptavidina-HRP in ogni diapositiva e incubare per 15 minuti. </li><li> Sciacquare i vetrini con TBST, poi mettere in TBST per 5 min. Ripetere due volte. </li><li> Una goccia di 3,3-diaminobenzidina (DAB) e substrato cromogeno su ciascun vetrino e sviluppare il colore per 2 min. </li><li> Subito dopo lo sviluppo del colore, lavare i vetrini con DDH 2 O per 1-2 min. </li><li> Incubare vetrini con una goccia di ematossilina per 90 sec. </li><li> Lavare con DDH 2 O per 1-2 min. </li><li> Lavare con acqua corrente e sapone per 1-2 min. </li><li> Lavare con DDH 2 O per 1-2 min. </li></ol> 4. Disidratazione e montaggio <ol><li> Immergere i vetrini in etanolo al 80% per 3 min. </li><li> Immergere i vetrini in etanolo al 95% per 3 min. </li><li> Immergere i vetrini in etanolo al 100% per 5 min. Ripeti. </li><li> Immergere i vetrini in xilene per 5 min. Ripeti. </li><li> Aggiungere una goccia diCytoseal 60 a diapositiva e il luogo del coprioggetto. </li></ol> La Tabella 1 elenca i reagenti utilizzati nelle procedure sopra descritte.

<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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Nessun conflitto di interessi dichiarati.

Immunoistochimica del B7-H1 sulla criosezioni è stato riportato in studi precedenti per una varietà di tumori 3,4. Tuttavia, clinici campioni tumorali di solito sono disponibili solo in forma di paraffina-incastonati blocchi. L&#39;anticorpo monoclonale 5H1 è recentemente stato applicato con successo per la colorazione di tessuti inclusi in paraffina tumorali compresi carcinoma renale, carcinoma della cervice, carcinoma della vescica e melanoma 13-18. B7-H1 colorazione su inclusi in paraffina <s...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> CSA Sistema </td><td> Dako </td><td> K1500 </td><td> Comprende i seguenti reagenti di cui al protocollo: blocco perossidasi, streptavidina-biotina complessi, reagenti di amplificazione, streptavidina-HRP, ed il substrato cromogeno DAB e </td></tr><tr><td> Avidina / biotina Blocco Kit </td><td> Vettore </td><td> SP-2001 </td><td></td></tr><tr><td> Blocco ACE </td><td> Serotec </td><td> BUF029 </td><td></td></tr><tr><td> Biotina anti-topo IgG1 </td><td> BD </td><td> 553441 </td><td></td></tr><tr><td> IgG1 di topo K Isotype controllo </td><td> eBioscience </td><td> 14-4714-85 </td><td></td></tr><tr><td> TBS (Tris-salina tamponata), 10X </td><td> Cellgro </td><td> 46-012-CM </td><td> 10xTBS contiene 80 g / L di NaCl e 24,2 g / L Tirs; Diluito a 1X con ddH2O per lavaggi; </td></tr><tr><td> Tween 20 </td><td> Sigma-Aldrich </td><td> P1379 </td><td> 1 ml aggiunto a 1 L di 1X TBS per fare TBST </td></tr><tr><td> Target Retrieval, 20x </td><td> Celerus Onda </td><td> 014-3000-000 </td><td> Diluito a 1x con DDH 2 O </td></tr><tr><td> Alcool etilico 190 Proof </td><td> Pharmco-Aaper </td><td> 111000190 </td><td></td></tr><tr><td> Alcool etilico 200 Proof </td><td> PHARMO-Aaper </td><td> 111000200 </td><td></td></tr><tr><td> Xilene </td><td> VWR </td><td> 95057-827 </td><td></td></tr><tr><td> Ematossilina </td><td> Richard-Allan scientifico </td><td> 72804 </td><td></td></tr><tr><td> Cytoseal 60 </td><td> Richard-Allan scientifico </td><td> 8310-4 </td><td></td></tr><tr><td> Coprioggetto </td><td> Corning </td><td> 2940-245 </td><td></td></tr><tr><td> Umidità camera </td><td> Sigma </td><td> H6644 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella 1. Reagenti e apparecchiature. </td></tr></tbody></table>

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

B7-H1/PD-L1, un membro della famiglia B7-immune di regolamentazione proteine ​​di superficie cellulare, svolge un ruolo importante nella regolazione negativa di risposte immunitarie mediate da cellule attraverso la sua interazione con il suo recettore, morte programmata-1 (PD -1) 1,2. Sovraespressione di B7-H1 da cellule tumorali è stato osservato in un certo numero di tumori umani, compreso il melanoma, glioblastoma, e carcinomi del polmone, seno, colon, ovaio, e cellule renali, e ha dimostrato di alterare anti-tumorali T- cella immunità 3-8. Recentemente, B7-H1 espressione tessuti adenocarcinoma pancreatico è stato identificato come un potenziale marcatore prognostico 9,10. Inoltre, il blocco di B7-H1 in un modello murino di cancro pancreatico ha dimostrato di produrre una risposta antitumorale 11. Questi dati suggeriscono l&#39;importanza di B7-H1 come un potenziale bersaglio terapeutico. Anti-B7-H1 anticorpi bloccanti sono quindi in fase di sperimentazione in studi clinici per il mulgestire più tumori umani solidi tra cui il melanoma e tumori del polmone, del colon, rene, stomaco e pancreas 12. Per essere successivamente in grado di identificare i pazienti che beneficeranno B7-H1 di targeting terapie, è fondamentale per indagare la correlazione tra espressione e la localizzazione delle B7-H1 e la risposta del paziente al trattamento con anticorpi B7-H1 blocco. Esaminando l&#39;espressione di B7-H1 in tessuti umani adenocarcinoma pancreatico attraverso immunoistochimica darà una migliore comprensione di come questa co-inibitrice molecola di segnalazione contribuisce alla soppressione dell&#39;immunità antitumorale in microambiente del tumore. L&#39;anticorpo anti-B7-H1 monoclonale (clone 5H1) sviluppato da Chen e colleghi ha dimostrato di produrre risultati affidabili in colorazione criosezioni di più tipi di tessuti neoplastici umani 4,8, ma macchie nei paraffinati diapositive era stata una sfida fino recentemente 13-18. Abbiamo devfuggita la B7-H1 protocollo di colorazione per le diapositive incluse in paraffina di tessuti adenocarcinoma pancreatico. La B7-H1 protocollo di colorazione qui descritto produce coerente colorazione membranosa e citoplasmatica di B7-H1 con po &#39;di storia.

Un successo colorazione immunoistochimica di B7-H1 dimostrerà l&#39;espressione eterogenea di B7-H1 (marrone segnali di sviluppo di colore DAB) in adenocarcinoma pancreatico. Alcune cellule pancreatiche adenocarcinoma hanno espressione prevalentemente membranosa B7-H1 (Figura 1A), mentre altri hanno espressione sia prevalentemente citoplasmatica di B7-H1 o espressione poco B7-H1 (figure 2 e 3). Epitelio dotto pancreatico normale esprime poco B7-H1 (Figura 4). Colorazio...

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) e il suo legame al PD-1 forniscono una maggiore tumore segnale indotto immunosoppressiva nel microambiente del tumore. Una tecnica di colorazione immunoistochimica per caratterizzare l&#39;espressione e la localizzazione di B7-H1 in adenocarcinoma pancreatico è descritto qui.

Colorazione immunoistochimica di B7-H1 (PD-L1) in paraffina incorporate diapositive di tessuto pancreatico Adenocarcinoma

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 Views.  The Johns Hopkins University School of Medicine. B7-H1 (PD-L1) e il suo legame al PD-1 forniscono una maggiore tumore segnale indotto immunosoppressiva nel microambiente del tumore. Una tecnica di colorazione immunoistochimica per caratterizzare l'espressione e la localizzazione di B7-H1 in adenocarcinoma pancreatico è descritto qui.

Video: 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.

Protocolli di colorazione di isole pancreatiche umane

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

Ricerca

Medicina

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.

Formazione di epitelio prostatico umano utilizzando tessuti ricombinazione dei roditori urogenitale Mesenchima Sinus e cellule staminali umane

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.

Bioluminescente modello ortotopico di cancro al pancreas Progressione

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.

Preparazione e respirometrici valutazione dei mitocondri isolati dal tessuto muscolare scheletrico Ottenuto dalla biopsia percutanea

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.

Applicazione dell'anastomosi to-end in robotica Pancreatectomy 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 ...

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 trapianto di isole pancreatiche nel modello del topo

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.

Immunofluorescenza Etichettatura delle trappole extracellulari di neutrofilo umano e murino nel tessuto incorporato di paraffina

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.

Enucleazione robotica di un Insulinoma intra-pancreatico nella testa pancreatica

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.

Misurazione del contenuto di ferro non eme tissutale utilizzando un saggio colorimetrico basato su bathofenantrolina

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.

Predizione farmacologica di rete e validazione sperimentale del meccanismo d'azione di Trichosanthes-Fritillaria thunbergii contro l'adenocarcinoma polmonare