Questo lavoro è stato finanziato dalla sovvenzione NIH P01 AI 068730 (SNC, NS), il NIH concedere CA016520 / TAPITMAT (NS), il finanziamento privato da parte della Fondazione Claneil (NS), e il ovarico SPORE concedere FCCC e la University of Pennsylvania ( P50 CA83638) e il Fox Chase Cancer Center core Grant (P30 CA06927) (DCC). Gli autori ringraziano l&#39;ottima assistenza tecnica dello strumento ottico Core / bioluminescenza diretto dal Dr. EJ Delikatny presso la University of Pennsylvania, Anthony Secreto dalle cellule staminali e Core dello xenotrapianto diretto dal Dott. G. Danet-Desnoyers presso la University of Pennsylvania Cancer Centro per la SNC formazione tecnica di iniezione ortotopica e Denada Dangaj presso l&#39;Università della Pennsylvania / OCRC per assistere sulla chirurgia.

1. Colture Cellulari <ol><li> Prima dell&#39;iniezione ortotopico, cultura MOV1 cellule Kat, derivate da tumori DR26, in un pallone T175 fino a quando non sono al 90% confluenti. Prevede di utilizzare 1-5.000.000 cellule per iniezione, che richiede 1 o 2 T175 fiaschi. </li><li> Il giorno della iniezione, raccogliere le cellule e determinare il numero delle cellule utilizzando un emocitometro. </li><li> Una volta che la concentrazione cellulare è stata determinata, agglomerare le cellule per centrifugazione per 5 minuti a 300 g X a temperatura ambiente. </li><li> Dopo la rotazione, risospendere le cellule di avere 1 milione in 10 microlitri di PBS sterile con EDTA 0,002 M </li></ol> 2. Pre-Surgery <ol><li> Prima dell&#39;intervento, riempire una siringa da insulina 3/10cc con 1 milione di MOV1-Kat cellule in 10 microlitri di PBS EDTA. </li><li> Trasferimento di un mouse isofluorane anestetizzati per una piastra elettrica. Aggiungi unguento oculare per prevenire la disidratazione dell&#39;occhio. Poi, subito inserire la testa dell&#39;animale in un sistema di cono collegato ad un vaporizzatore isoflurano per fornire anestesia durante l&#39;intervento chirurgico. </li><li> Dopo la disinfezione del sito di iniezione con tamponi imbevuti di alcool, iniettare per via sottocutanea 5 mg / kg di ketoprofene, un pre-chirurgica analgesico, con una siringa da insulina 3/10cc. </li><li> Utilizzando Clippers, radersi la porzione caudale sinistra del dorso dalla giunzione toraco-lombare alla base della coda dell&#39;animale. Applicare la crema di rimozione dei capelli per rimuovere completamente i capelli. Quindi, rimuovere l&#39;eccesso con un tovagliolo di carta bagnato. </li><li> Una volta che il pelo è stato rimosso, sterilizzare l&#39;area rasata con povidone-iodio e tamponi imbevuti di alcool. Poi, posto un drappo chirurgico intorno alla zona di incisione. </li></ol> 3. Chirurgia <ol><li> Poco prima dell&#39;intervento, regolare i livelli vaporizzatore isoflurano al 1,5%. </li><li> Verificare che l&#39;animale è completamente anestetizzato pizzicando la zampe. </li><li> Quindi, individuare la milza sotto la pelle. Quindi, utilizzando forbici chirurgiche, fare una incisione dorsolaterale 1-2 cm di lunghezza in alto a destra della milza. </li><li> Sezionare il retroperitoneo. Il cuscinetto che circonda l&#39;ovaio del mouse verrà osservato. Utilizzare pinze curve di cogliere e di esporre il cuscinetto adiposo che circonda l&#39;ovaio mouse. </li><li> Idratare l&#39;organo con qualche goccia di PBS sterile. </li><li> Utilizzare pinze curve di cogliere, ritrattare ... posizione ... e poi sicuro l&#39;ovaio per l&#39;iniezione </li><li> Ferma cogliere il ovario con le pinze, iniettare 10 microlitri di MOV1 cellule tumorali Kat in dell&#39;ovaio. Una solida conoscenza impedisce il rigurgito dei fluidi o perdite. </li><li> Subito dopo l&#39;iniezione, allentare la tensione esercitata dal forcipe. La perforazione nell&#39;ovaio dovrebbe spontaneamente ritrattare e chiudere. </li><li> Utilizzando una sutura assorbibile acido poliglicolico attaccato ad un ago, chiudere il retro-peritoneo ferita. </li><li> Rilasciare l&#39;animale dal cono naso. </li><li> Tendere la pelle e sigillare i lembi della ferita dorsolaterale con qualche goccia di adesivo tissutale. </li><li> Infine, somministrare per via orale 100 microlitri di antibiotici per l&#39;animale. Poi metterlo di nuovo nella sua gabbia e monitor per il recupero. Tenere l&#39;animale sotto antibiotici nell&#39;acqua da bere per una settimana. </li></ol> 4. Imaging in vivo <ol><li> Una settimana dopo l&#39;iniezione ortotopico di MOV1 cellule tumorali Kat, eseguire imaging in vivo. Iniziare con il trasferimento di un mouse isofluorane anestetizzato alla camera di imaging. </li><li> Girare il livello isoflurano vaporizzatore fino al 2%. </li><li> Eseguire imaging in vivo secondo le istruzioni del produttore del sistema di imaging è. In questo video, il sistema Lumina verrà utilizzato. </li><li> Per l&#39;immagine, clicca l&#39;immagine vivente icona del software desktop. Poi, sul pannello di controllo acquisizione IVIS, selezionare &quot;Initialize&quot;. Le impostazioni dello strumento sono analoghi ad una le impostazioni della fotocamera </li><li> Sul pannello di controllo acquisizione impostare i parametri di acquisizione dello strumento. Per fluorescenza, selezionare &quot;fluorescente&quot;. Fare clic sulla casella Fotografia di acquisire una fotografia con ogni immagine. </li><li> Successivamente, impostare il tempo di esposizione come Auto. In &quot;pixel binning o CCD risoluzione&quot;, selezionare &quot;Medio&quot;. Poi sotto F / stop o diaframma, controllare il valore di 2. Avanti, selezionare il filtro di eccitazione 535 e il filtro per le emissioni DsRed. </li><li> In campo di vista, fare clic su View B per l&#39;immagine di un mouse. </li><li> Quindi, fare clic su acquisire per iniziare l&#39;acquisizione di immagini. </li><li> Una volta che l&#39;acquisizione delle immagini è completa, utilizzare la regione di interesse, o ROI, strumento per la misura del segnale. Fare clic sull&#39;icona di misura per liberare i valori del segnale e la zona. </li><li> Infine, fare clic su &quot;salva&quot; per salvare l&#39;immagine nella cartella utente </li><li> Dopo le immagini sono state salvate, interrompere la somministrazione di isoflurano e restituire il mouse per la sua gabbia. Il mouse deve svegliarsi immediatamente. </li></ol> 5. Rappresentante Risultati -Usando questo protocollo, La crescita in vivo di un cancro ovarico ortotopico possono essere monitorati per almeno 3 settimane su una procedura non invasiva. <img src="/files/ftp_upload/2146/2146fig1.jpg" alt="Figura 1" /> Figura 1. MOV1 cellule Kat, o PBS come controllo negativo, sono stati iniettati nel ortotopicamente ovaie di topi soggetti non tumorali (animali e animali a destra e sinistra, rispettivamente). Imaging in vivo è stato effettuato 2 settimane più tardi. L&#39;emissione di fluorescenza generata da MOV1 Kat cellule trapiantate nelle ovaie è stata misurata e confrontata con quella di topo di controllo negativo. <img src="/files/ftp_upload/2146/2146fig2.jpg" alt="Figura 2" /> Figura 2. Sezioni congelate di MOV1 tumori ovarici Kat sono state colorate con biotinilato anti-CD4 mAb seguito da substrato DAB (marrone scuro) per rilevare tumore dei linfociti infiltranti (freccia nera). Le cellule sono state di contrasto con metil-verde per visualizzare nuclei cellulari (blu). Cellule tumorali (frecce rosse) sono stati morfologicamente distinte dalle cellule T. La diapositiva appare ingrandita 40x.

<ol>
	<li>Etzioni, R., 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=The+case+for+early+detection.">The case for early detection.</a> Nat Rev Cancer. 3, 243-252 (2003).</li><li>Sasaroli, D., Coukos, G., Scholler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+CA125:+the+coming+of+age+of+ovarian+cancer+biomarkers.+Are+we+there+yet.">Beyond CA125: the coming of age of ovarian cancer biomarkers. Are we there yet.</a> Future Medicine. 3, 275-288 (2009).</li><li>Anderson, G. 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=Assessing+Lead+Time+of+Selected+Ovarian+Cancer+Biomarkers:+A+Nested+Case+Control+Study.">Assessing Lead Time of Selected Ovarian Cancer Biomarkers: A Nested Case Control Study.</a> J Natl Cancer Inst. 102, 26-38 (2010).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocr Rev 22. , 255-288 (2001).</li><li>Dubeau, 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+cell+of+origin+of+ovarian+epithelial+tumours.">The cell of origin of ovarian epithelial tumours.</a> Lancet Oncol. 9, 1191-1197 (2008).</li><li>Connolly, D. C., 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=Female+mice+chimeric+for+expression+of+the+simian+virus+40+TAg+under+control+of+the+MISIIR+promoter+develop+epithelial+ovarian+cancer.">Female mice chimeric for expression of the simian virus 40 TAg under control of the MISIIR promoter develop epithelial ovarian cancer.</a> Cancer Res. 63, 1389-1397 (2003).</li><li>Hensley, H., 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=Magnetic+resonance+imaging+for+detection+and+determination+of+tumor+volume+in+a+genetically+engineered+mouse+model+of+ovarian+cancer.">Magnetic resonance imaging for detection and determination of tumor volume in a genetically engineered mouse model of ovarian cancer.</a> Cancer Biol Ther. 6, (2007).</li><li>Deliolanis, N. C., 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=Performance+of+the+red-shifted+fluorescent+proteins+in+deep-tissue+molecular+imaging+applications.">Performance of the red-shifted fluorescent proteins in deep-tissue molecular imaging applications.</a> J Biomed Opt. 13, (2008).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+better+fluorescent+protein+for+whole-body+imaging.">A better fluorescent protein for whole-body imaging.</a> Trends Biotechnol. 26, (2008).</li><li>Shcherbo, D., 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=far-red+fluorescent+protein+for+whole-body+imaging.">far-red fluorescent protein for whole-body imaging.</a> Nat Methods. 4, 741-746 (2007).</li><li>Fu, X., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ovarian+carcinoma+metastatic+models+constructed+in+nude+mice+by+orthotopic+transplantation+of+histologically-intact+patient+specimens.">Human ovarian carcinoma metastatic models constructed in nude mice by orthotopic transplantation of histologically-intact patient specimens.</a> Anticancer Res. 13, 283-286 (1993).</li><li>Kiguchi, K., 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=A+patient-like+orthotopic+implantation+nude+mouse+model+of+highly+metastatic+human+ovarian+cancer.">A patient-like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer.</a> Clinical &amp; experimental metastasis 16. , 751-756 (1998).</li><li>Bao, R., 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=Activation+of+cancer-specific+gene+expression+by+the+survivin+promoter.">Activation of cancer-specific gene expression by the survivin promoter.</a> J Natl Cancer Inst. 94, 522-528 (2002).</li><li>Connolly, D. C., Hensley, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+and+Transgenic+Mouse+Models+of+Epithelial+Ovarian+Cancer+and+Non+Invasive+Imaging+Modalities+to+Monitor+Ovarian+Tumor+Growth+In+situ+-Applications+in+Evaluating+Novel+Therapeutic+Agents.+Current+Protocols+in+Pharmacology+45.">Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In situ -Applications in Evaluating Novel Therapeutic Agents. Current Protocols in Pharmacology 45.</a> , 1-36 .</li><li>Milne, K., 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=Systematic+analysis+of+immune+infiltrates+in+high-grade+serous+ovarian+cancer+reveals+CD20,+FoxP3+and+TIA-1+as+positive+prognostic+factors.">Systematic analysis of immune infiltrates in high-grade serous ovarian cancer reveals CD20, FoxP3 and TIA-1 as positive prognostic factors.</a> , (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Chirurgia e iniezioni ortotopico Iniezione ortotopico in borsa ovarica esigenze di formazione e di precisione. Così <ol><li> In caso di scarsa esperienza chirurgica, la pratica di cadaveri per primo. </li><li> Usare preferibilmente donne multipare (uno o due cucciolate), come si sviluppano più grandi ovaie nel tempo che facilita l&#39;iniezione e la sopravvivenza aumentano confrontare con le femmine nullipare. </li><li> A causa delle piccole dimensioni della borsa ovarica del mouse, l&#3...

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an orthotopic model of serous ovarian cancer in immunocompetent mice for in vivo tumor imaging and monitoring of tumor immune responses

Background: Il cancro ovarico è generalmente diagnosticato in fase avanzata in cui il caso / rapporto di mortalità è alta e rimane quindi il più letale di tutte le neoplasie ginecologiche tra Stati Uniti le donne 1,2,3. Tumori sierosi sono le forme più diffuse di cancro ovarico e 4,5 il Tg-MISIIR-Tag transgenici rappresenta l&#39;unico modello del mouse che si sviluppa spontaneamente questo tipo di tumori. Tg-MISIIR-Tag topi esprimere SV40 regione trasformando sotto il controllo del Mulleriani inibitori della sostanza di tipo II Receptor (MISIIR) del gene promotore 6. Ulteriori linee transgeniche sono state identificate che esprimono il transgene TAG SV40, ma non sviluppano tumori ovarici. Topi inclini non tumorali mostrano tipica vita per C57BL / 6 topi e sono fertili. Questi topi possono essere utilizzate come riceventi trapianto allogenico di singenici per le cellule tumorali isolate dai Tg-MISIIR-Tag-DR26 topi. Obiettivo: Sebbene l&#39;imaging del tumore è possibile 7, la diagnosi precoce dei tumori in profondità è una sfida in animali che vivono di piccole dimensioni. Per abilitare studi preclinici in un modello animale immunologicamente intatta per il cancro ovarico sieroso, si descrive un modello di topo singenici per questo tipo di tumore ovarico che permette di imaging in vivo, gli studi del microambiente tumorale e tumore risposte immunitarie. Metodi: Per prima cosa deriva a + TAG cancro linea di topi cellule (MOV1) da un tumore ovarico spontanee raccolte in un 26 settimane di età DR26-Tg MISIIR-Tag femminile. Poi, abbiamo MOV1 cellule trasdotte stabilmente con TurboFP635 vettore Lentivirus mammiferi che codifica Katushka, un far-rosso mutante della proteina fluorescente rossa dal mare anemone Entacmaea quadricolor con eccitazione / emissione di massimi a 588/635 nm 8,9,10. Noi ortotopicamente impiantato MOV1 Kat nell&#39;ovaio 11,12,13,14 della non-tumorale inclini Tg-MISIIR-Tag topi femmina. Progressione del tumore è stata seguita da in vivo imaging ottico e microambiente tumorale è stata analizzata mediante immunoistochimica. Risultati: ortotopicamente MOV1 impiantato cellule Kat sviluppato tumori ovarici sierosi. MOV1 Kat tumori possono essere visualizzate da imaging in vivo fino a tre settimane dopo l&#39;impianto (fig. 1) e sono stati infiltrati con leucociti, come osservato nei tumori ovarici umani 15 (fig. 2). Conclusioni: descrivere un modello ortotopico di carcinoma ovarico adatto per imaging in vivo di tumori presto a causa della elevata pH stabilità e fotostabilità di Katushka nei tessuti profondi. Proponiamo l&#39;utilizzo di questo modello romanzo singenici del carcinoma ovarico sieroso per gli studi di imaging in vivo e il monitoraggio dei tumori risposte immunitarie e le immunoterapie.

Watch this Scientific Journal Video about An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses at JoVE.com

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Per studiare<em&gt; In vivo</em&gt; La crescita tumorale e microambiente tumorale, abbiamo utilizzato un modello murino singenici e ortotopico di carcinoma ovarico negli animali immunocompetenti. Noi trasdotte un tumore linea di topi cellule (MOV1) con una proteina fluorescente Katushka (MOV1<sup&gt; KAT</sup&gt;) E qui vi mostriamo il suo impianto ortotopico in ovaio e<em&gt; In vivo</em&gt; Immagini.

Un modello ortotopico di carcinoma ovarico sieroso in topi immunocompetenti per In vivo Tumore Imaging e monitoraggio del tumore Risposte immunitarie

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

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JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

18.1K Views.  University of Pennsylvania-School of Medicine. Per studiare In vivo La crescita tumorale e microambiente tumorale, abbiamo utilizzato un modello murino singenici e ortotopico di carcinoma ovarico negli animali immunocompetenti. Noi trasdotte un tumore linea di topi cellule (MOV1) con una proteina fluorescente Katushka (MOV1 KAT) E qui vi mostriamo il suo impianto ortotopico in ovaio e In vivo Immagini.

Video: An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modello per la misurazione Risposte immunitarie Malaria nel contesto della coinfezione da HIV

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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Immunologia e infezioni

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Un modello di topo transgenizzato IL-8 per il<em&gt; In Vivo</em&gt; Monitoraggio a lungo termine delle risposte infiammatorie

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Sartoria in vivo saggi di citotossicità per studiare l'Immunodominanza nelle risposte delle cellule CD8+ T specifiche del tumore

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Impianto e monitoraggio da PET/CT di un modello ortotopico di mesotelioma pliologico umano in topi atimici

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

Visualizzazione, quantificazione e mappatura delle popolazioni di cellule immunitarie nel microambiente tumorale

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

Mappatura precisa del cervello per eseguire immagini ripetitive in vivo della dinamica neuroimmune nei topi

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Modelli di co-coltura microfluidica per la dissezione della risposta immunitaria in microambienti tumorali in vitro

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infezione ricorrente del tratto urinario da Escherichia coli innescata dall'esposizione alla vescica di Gardnerella vaginalis nei topi

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.