We thank Dr. Helene Bernstein for providing us with access to her flow cytometer. We also thank CFAR flow cytometry facility for the flow cytometry services. This project was in part supported by CTSC core utilization funding and STERIS corporation/University Hospitals-Division of Infectious diseases grant to PP.

HINWEIS: Die Experimente mit Mäusen wurden in Übereinstimmung mit der institutionellen Tierschutzausschusses (IACUC) Richtlinien durchgeführt. 1. Reconstituting der Rag-1 - / - Mäuse, die mit I n vitro kultiviert Th17-Zellen (drei Tage vor der Infektion) <ol><li> Zum Herstellen Th17 Zellen, der Kultur CD4 + CD25- CD44low CD62Lhigh naiven T-Zellen (3 x 104) in U-Boden-Platten mit 96 Vertiefungen allein oder Cokultur sie zusammen mit 3 x 104 CD4 + CD25 + Foxp3 + Treg in Gegenwart löslicher α-CD3 (1 &amp; mgr; g / ml), α-CD28 (2 ug / ml) Antikörper und Antigen-präsentierenden Zellen unter Bedingungen, Th17 (polarisiert mit IL-6 (25 ng / ml), TGF-β (2 ng / ml) , α-IFN-γ (2 ug / ml) und α-IL-4 (2 &amp; mgr; g / ml)) für drei bis vier Tage. HINWEIS: Naive Zellen und T regs wurden mittels Fluoreszenz-aktivierter Zellsortierung sortiert (FACS) eind magnetische Zellisolierungsverfahren und kultiviert wie zuvor 19 bis 22 beschrieben. </li><li> Am Tag 4 nach Resuspendieren der Th17-Zellen mit der Pipette, sammeln sie aus Kulturen und zentrifugieren unter sterilen Bedingungen. <ol><li> Nehmen Sie eine kleine Teilmenge dieser Zellen, um ihre Lebensfähigkeit und zur Phänotypisierung zu untersuchen. Resuspendieren sie in 500 ul RPMI-Medium, fügen Propidiumiodid (200 ng / ml) und eine Bewertung ihrer Rentabilität sofort mittels Durchflusszytometrie. Führen Phänotypisierung mittels Durchflusszytometrie Beurteilung der IL-17A Produktion nach Restimulation (siehe Abschnitt 6.3). </li></ol></li><li> Zentrifuge und wäscht die Hauptmasse der Zellen in sterilem PBS bei 480 × g für 6 Minuten bei 4 ° C in allen Stufen, wenn nicht anders angegeben. </li><li> Verwenden Sie diese Zellen für die adoptive Transfer in 6-8 Wochen alte CD45.2 Rag1 - / - immundefizienten Mäusen. <ol><li> Resuspendieren der Zellen in 1 x 10 7 Zellen / ml Zelldichte mit kaltem sterilem PBS. </li><li> Injizieren 100 ul der Zellen durch intraperitoneal Injektion, unter Verwendung von 25 G Nadeln auf 1 ml Tuberkulin-Spritze, derart, dass eine Maus erhält 1 x 10 6 Zellen. Einige Mäuse PBS oder Th17 Zellen nur zu empfangen, und andere Mäuse Th17 Zellen, co-kultiviert mit Treg-Zellen wurden erhalten. HINWEIS: Führen Sie alle Schritte unter aseptischen Bedingungen. </li></ol></li></ol> 2. Wachsende C. albicans für eine Infektion (einen Tag vor der Infektion) <ol><li> Vor der Inokulation zerstreuen fünf Kolonien der CAF2-1 C. albicans Laborstamm in 100 ul sterilem PBS-Suspension, und fügen Sie die Suspension auf die sterile Brühe. </li><li> Impfen 5 Kolonien der C albicans in 50 ml des Hefe-Stickstoff-Base (YNB ohne Aminosäuren) / Peptone / Dextrose-Broth-Medium und Inkubation in einem Schüttelinkubator bei 30 ° C für 15-18 h bei 130 Umdrehungen pro Minute. </li><li> Überwachen Sie die Brühe für Bewölkung, denn das ist ein Indiz für das Wachstum des Pilzes. </li></ol> 3. CandidaErnte und Counting-Verfahren (Am Tag der Infektion) <ol><li> Sammeln Sie 10 ml Candida Brühe in 15 ml Tuben. Für größere Volumina, sammeln Candida in 50 ml Tuben. </li><li> Zentrifugieren Blastosporen bei 1900 × g für 5 min bei RT in allen Stufen, wenn nicht anders angegeben. Pellet die Blastosporen durch Zentrifugation, gefolgt von der Entfernung des Überstandes. </li><li> Wenn es mehr als ein Rohr Vereinige die Candida Blastosporen aus mehreren Rohren, Zugabe von sterilem PBS auf die Pellets und Resuspendieren sie in 10 ml PBS für die Zählung. </li><li> Nehmen Sie 20 ul Blastosporen in 1,5 ml Mikrozentrifugenröhrchen, fügen Sie 20 ul der 2X Para und Inkubation bei Raumtemperatur für 15-20 min. Zählen Sie die Fest Blastosporen mit dem Hämozytometer unter dem Mikroskop. HINWEIS: Paraformaldehyd ist krebserregend. Öffnen Sie die unverdünnte Lösung nur im Abzug durchführen. </li><li> In der Zwischenzeit wiederholt wenigstens zweimal Waschen der Blastosporen durch Zentrifugieren them in PBS, und Pelletieren der Blastosporen. Halten Sie die Pellets in 15-ml-Röhrchen bei Raumtemperatur, in der ABSL1 Haube, bereit für die Infektion. </li><li> Lassen Sie etwas sterilem PBS bei RT Resuspendieren der Blastosporen für eine Infektion. </li><li> Nach dem Zählen hinzufügen sterilem PBS in die Blastospore Pellet um die Zellzahlen Hefe Blastospore bis 2 x10 8 von Hefezellen / ml einzustellen. Dies ist der Blastosporen Suspension, verwendet, um die Mäuse zu infizieren wird. HINWEIS: Führen Sie alle Schritte unter aseptischen Bedingungen. </li></ol> 4. Mäuse Infektion (3-5 Tage nach der Zellübertragung) HINWEIS: Die Grund Infektion Verfahren durchgeführt wird, wie oben beschrieben, 19,23-25. <ol><li> Wiegen Sie die Rag-1 - / - Mäuse, die PBS oder die Zellen drei Tage zuvor erhalten. </li><li> Berechnen Sie die Dosis des Narkosemitteln mit ihrem Körpergewicht. Anesthetize der Mäuse durch die Verabreichung von Ketamin / Xylazin-Mischung (16,1 mg / ml und 1,6 mg / ml), unter Verwendung von 25 G Nadeln auf 1 ml Tuberkulin-Spritze durch intraperitoneal Injektion. <ol><li> Gabe von 50 &amp; mgr; l pro 10 g Körpergewicht. (Das heißt, 0,8 mg Ketamin und 0,08 mg Xylazin / 10 g Körpergewicht oder 80 mg Ketamin und 8 mg Xylazin pro kg Körpergewicht beziehungsweise). Beachten Sie für toe Prise Reaktion alle 15 min nach der Narkose. HINWEIS: Diese Dosis wird 60-90 min der Anästhesie, die genug für die Infektion Verfahren einzuleiten. </li><li> Übernehmen Sie die Augen Schmiermittel Salbe in den Augen der Mäuse, die Hornhaut vor dem Austrocknen zu verhindern. Da das Auge Schmiermittel kann austrocknen, wiederholen Augen Schmierung alle 45 min. </li></ol></li><li> Injizieren 1 ml Salzlösung (0,9% NaCl) subkutan am Rücken benachbart dem Vorderbein zu Rehydration der Mäuse während der Anästhesie zu helfen. </li><li> Besorgen Sie sich einen neuen, sauberen Käfig. Folgen Sie der Infektion Verfahren eine Maus zu einem Zeitpunkt, indem jede Maus in den neuen Käfig einmal infiziert. Führen Sie die PBS / Schein-Infektion zuerst, und dann auf die Candida-Infektion gro gehenups. </li><li> Nehmen Sie den narkotisierten Maus und öffnen Sie den Mund weit auf, um die Basis der Zunge zeigen. <ol><li> Legen Sie eine 3 mm Durchmesser Wattebausch mit 50 ul PBS oder Blastospore Aussetzung gesättigten, sublingual in der Mundhöhle für 90 min. Drehen Sie die Mäuse alle 15 min vor und zurück zu Staulunge zu vermeiden, sicherstellen, dass die Watte nicht zu bewegen. </li></ol></li><li> Einrichten eines Zeitgebers für jede Maus 90 Minuten von Sham oder Candida Inokulation in der Mundhöhle zu gewährleisten. </li><li> Halten Sie sie in einem Käfig unter der Wärmelampe (4 Meter), und jede Maus auf Hitze Gel-Pads. </li><li> Stellen Sie sicher, dass die Zunge in und von den Zähnen zurückgezogen, um die Zähne zerreißt die Zunge zu vermeiden. </li><li> Am Ende der 90 Minuten, entfernen Sie den Wattebausch aus dem Mund der Maus. Sehen Sie für jede Maus, die aus der Narkose erholen kann früher als 75 Minuten. In einem solchen Fall zu betäuben sie wieder mit Ketamin (40 mg / kg) allein. </li></ol> 5. Beitrag procedure Monitoring (während der 90 Minuten Anästhesie) <ol><li> Verwenden Sie Hitze Gel-Pads während der 90 Minuten Narkose. <ol><li> Um die Körpertemperatur zu halten und zum Ersticken zu vermeiden, darf nicht zulassen, dass Mäuse, um auf den regulären Maiskolben Maus Betten erholen. </li></ol></li><li> Nach der Erholung von der Anästhesie verabreichen zusätzliche 1 ml steriler 0,9% NaCl subkutan an zwei Stellen auf der Rückseite. </li><li> Bleiben Sie auf dem fünf PBS (Schein) geimpft Mäusen pro Käfig. Haus nur 1-2 infizierten Mäusen pro Käfig. </li><li> Füllen Sie das Verfahren Karte mit Initialen jeden Tag nach dem Eingriff zu notieren, die Veränderungen im Körpergewicht, Körperpflege Gewohnheiten und die allgemeine Gesundheit. </li></ol> 6. Die Beurteilung der Entzündung 6.1) Gewichtsverlust <ol><li> Wiegen Sie die Mäuse jeden Tag während der Infektion, beginnend am Tag vor der Infektion Verfahren. HINWEIS: In der Regel werden die immundefizienten Mäuse mit Th17 Zellen und ohne T regs injiziert erliegen 20-30% Gewichtsverlust aufgrund erhöhter Pilzbelastung 19 und verschlimmert die Entzündung. </li></ol> 6.2) Histologie Scoring der Zunge <ol><li> Opfern die Mäuse durch CO 2 Ersticken durch Genickbruch folgte. </li><li> Die Backen öffnen und sezieren die Zunge mit einer Schere und Pinzette. Verwenden stumpfen Pinzette, um den Mund zu halten und mit der Schere erreichen, um den hinteren Teil des Mundes, um das hintere Ende der Zunge einzuschneiden. </li><li> Für immunzytochemische Hämatoxylin und Eosin (H &amp; E) Färbung der Zunge Gewebe, spülen Sie die Zunge Gewebe mit eiskaltem PBS. 5 ml 10% Formalin für 2-3 Zungen und befestigen Sie O / N in 15 ml Tuben. <ol><li> Am nächsten Tag, entfernen Sie das Formalin und tauchen das Gewebe in 5 ml 70% Ethanol, um hyper-Fixierung zu verhindern. Senden Sie sie aus, um kommerziellen Dienst und Paraffineinbettung weiter, Schneiden und Färben von Paraffinschnitten. HINWEIS: Kommerzielle Histologie Service wird verwendet, um diese Schritte durchführen. </li&gt; </ol></li><li> Sobald die Dias aus dem Handels Histologie Service zurück, Grad der Entzündung durch die Beobachtung der Gewebeschnitte unter einem Lichtmikroskop. <ol><li> Score von 0 bis 5, wobei 0 keine Entzündung und 5 die schwerste Entzündungen. Ergebnis der Entzündung mit Tabelle 1. </li></ol></li></ol> 6.3) Zytokin-Produktion von Th17-Zellen HINWEIS: Übermäßiger TNF-α Produktion ist eine der Ablesungen für eine übermäßige Immunpathologie. Wenn Mäuse adoptiv mit Th17 Zellen nur übertragen, verursacht es Immunpathologie, die mit übermäßiger TNF-α-Produktion in Th17-Zellen assoziiert ist. <ol><li> Sammeln Sie eine Einzelzellsuspension aus Milz, Lymphknoten, axillären Lymphknoten, und der Zunge, mit zuvor beschriebenen Verfahren 19,26. </li><li> Restimulieren der Zellen mit Phorbolmyristatacetat (PMA) (50 ng / ml) und Ionomycin (500 ng / ml) für 4 h mit Brefeldin A (10 ug / ml) in der l hinzugefügtast 2 Std. Waschen Sie die Zellen mit PBS und fixieren Sie diese mit einem handelsüblichen Fixierung / Permeabilisierung Kit gemäß Herstellerangaben. </li><li> Führen intrazelluläre Cytokin Färbung mit dem Fluorochrom konjugierten anti-TNF-α, Anti-IL-17A und ROR &amp; ggr; t-Antikörper wie zuvor beschrieben 19. </li><li> Kurz gesagt, die Zellen werden in dem 1X Permeabilisierung Puffer mit dem Cocktail von Anti-TNF-α, Anti-IL-17A und ROR &amp; ggr; t-Antikörper, jedes Antikörpers mit einem 2 - 3 ug / ml Endkonzentration. </li><li> Inkubieren der Zellen für 1 h bei RT. </li><li> Nach der Inkubation, waschen Sie die Zellen mit 1X Permeabilisierung Puffer. </li><li> Zellpellet in 500 ul PBS mit 0,5% Rinderserumalbumin für die Durchflußzytometrie-Analyse. </li></ol>

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F., Kollias, G., Becker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+functions+of+TNF-alpha+in+the+regulation+of+the+intestinal+epithelial+response+to+inflammation.">Pleiotropic functions of TNF-alpha in the regulation of the intestinal epithelial response to inflammation.</a> International Immunology. 26 (9), 509-515 (2014).</li><li>Bishu, S., 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+adaptor+CARD9+is+required+for+adaptive+but+not+innate+immunity+to+oral+mucosal+Candida+albicans+infections.">The adaptor CARD9 is required for adaptive but not innate immunity to oral mucosal Candida albicans infections.</a> Infection and Immunity. 82 (3), 1173-1180 (2014).</li><li>Quintin, J., 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=Differential+role+of+NK+cells+against+Candida+albicans+infection+in+immunocompetent+or+immunocompromised+mice.">Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice.</a> Eur J Immunol. 44 (8), 2405-2414 (2014).</li><li>Ramirez-Garcia, A., 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=Candida+albicans+and+cancer:+Can+this+yeast+induce+cancer+development+or+progression.">Candida albicans and cancer: Can this yeast induce cancer development or progression.</a> Critical Reviews In Microbiology. , 1-13 (2014).</li></ol>

The authors declare that they have no competing financial interests.

Dieses Modell basiert auf Induktion von oraler C basiert albicans-Infektion abhängig Th17 Entzündung. Wegen der Abwesenheit von regulatorischen T ist die Th17 Zelle induzierte Entzündung ungebremst und führt zu schlecht aufgelöst Immunpathologie. In vitro abgeleitet naiven CD4-Zellen polarisiert Th17 Zellen wurden für die adoptive Übertragung verwendet. 40 bis 50% des gezüchteten CD4 + Zellen zeigen nachweisbare IL-17A-Expression um Tag 3 (Th17-Zellen), und wurden daher für...

Oral infections and inflammation have been related to cancer and cardiovascular diseases, and have dramatic impact on overall human health2,3. Opportunistic infections and inflammation caused by C. albicans are associated with primary immunodeficiencies (PID)4,5, inflammatory disorders such as periodontitis 6,7, Sjogren&#8217;s syndrome, and salivary gland disease8,9, as well as oral squamous cell carcinoma 10-12. C. albicans is a dimorphic commensal fungus that colonizes the mouths of 60% of healthy humans asymptomatically, yet it is the most common fungal pathogen causing infections when the host defense is weakened13-15. It causes recurring and chronic infections and inflammation in patients with AIDS and PID, and also in other immunocompromised individuals. As a commensal, its colonization load is associated with the change in the diversity of the overall oral microbiome16. As a pathogen it causes several forms of oropharyngeal candidiasis such as acute pseudomembranous, acute atrophic, chronic atrophic, chronic hypertrophic/hyperplastic, and angular cheilitis. 
Protection against C. albicans is determined not only by host immune resistance, but also by the ability to appropriately control Candida-induced immunopathology. Although commensals such as C. albicans contribute to modulation and exacerbation of other oral inflammatory conditions, the mechanisms by which dysbiosis occur during opportunistic infections are unclear. Besides the known role of adaptive Th17 cells in memory response to C. albicans17, their role in initiation and perpetuation of inflammation pathology during chronic infections remain unclear. Furthermore, oral inflammatory diseases such as Sjogren&#8217;s syndrome and periodontitis are associated with Th17 mediated pathology. Interestingly, these diseases are also strongly associated with frequent OPC. However, the interactions among Th17 cells, oral immunopathology of OPC and other oral inflammatory diseases are unstudied. 
Although mouse models of primary and secondary infection of oral candidiasis are available, a mouse model to study Candida infection associated Th17 inflammation, especially in the context of immunodeficiency is unavailable. This study presents a method for establishing oral Th17 inflammation associated with oral Candida infection in mice. Candida infection in mice is characterized by fungal lesions, inflammation in the tongue, decreased food intake, weight loss and eventually a moribund state. Oral pathology resembles chronic candidal infection lesions, as well as epithelial dysplasia in mouse oral cancer models12,18.

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			<td height="25" style="height:25px;width:380px;">CAF-2</td>
			<td style="width:411px;">University of Pittsburgh (Sarah Gaffen)</td>
			<td style="width:119px;">-</td>
			<td style="width:380px;">Candida culture</td>
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			<td>Polarization of cells to Th17 conditions</td>
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			<td>Polarization of cells to Th17 conditions</td>
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			<td>Polarization of cells to Th17 conditions</td>
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			<td>Polarization of cells to Th17 conditions</td>
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			<td>Polarization of cells to Th17 conditions</td>
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			<td height="20" style="height:20px;width:380px;">&#945;-RORgt PE&#160;</td>
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			<td height="25" style="height:25px;">YNB w/o amino acids)/Peptone/Dextrose broth medium&#160;</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">S507.SIZE</td>
			<td>Mediium for candida growth</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Shaker incubator&#160;</td>
			<td style="width:411px;">New Brunswick Scientific</td>
			<td style="width:119px;">Innova 4300</td>
			<td>Incubation growth for candida</td>
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			<td height="24" style="height:24px;">15 ml tubes</td>
			<td style="width:411px;">Bio Basic</td>
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			<td height="22" style="height:22px;">50 ml tubes</td>
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			<td>Used for cell culture and candida growth</td>
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			<td height="20" style="height:20px;">Table top Centrifuge</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">82017-654</td>
			<td>For pelleting candida -900 g</td>
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			<td height="20" style="height:20px;">Allegra Centrifuge</td>
			<td style="width:411px;">Beckman Coulter</td>
			<td style="width:119px;">392302</td>
			<td>Cell culture - 480 g</td>
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		<tr height="25">
			<td height="25" style="height:25px;">1.5 ml eppendorf tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">BT620-NS</td>
			<td>Preparing final concentration of candida</td>
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		<tr height="24">
			<td height="24" style="height:24px;">2x paraformaldehyde</td>
			<td style="width:411px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>Fixing candida for count</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">15170-172</td>
			<td>Counting cells</td>
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		<tr height="20">
			<td height="20" style="height:20px;">PBS - Phosphate Buffered Saline</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">PD8117</td>
			<td>Preparation of buffers</td>
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			<td height="23" style="height:23px;">Ketamine/xylazine&#160;</td>
			<td style="width:411px;">Case Western Reserve University - Animal Resource Center</td>
			<td style="width:119px;">-</td>
			<td>Obtained from ARC approved protocol for anesthesia</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic lubricant ointment&#160;</td>
			<td style="width:411px;">Allergan</td>
			<td style="width:119px;">-</td>
			<td>Eye ointment for animals to prevent dryness</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Saline (0.9% NaCl)</td>
			<td style="width:411px;">G Biosciences</td>
			<td style="width:119px;">786-561</td>
			<td>Administerd to animals to prevent dehydration</td>
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		<tr height="20">
			<td height="20" style="height:20px;">3-mm-diameter cotton wool ball&#160;</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">BP7603</td>
			<td>3 mm balls for candida infection</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heat gel pads</td>
			<td style="width:411px;">Case Western Reserve University - Animal Resource Center</td>
			<td style="width:119px;">-</td>
			<td>for maintaining the body temperaturre and fast recovery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin and Eosin staining</td>
			<td style="width:411px;">Histoserv - MD</td>
			<td style="width:119px;">-</td>
			<td>Tissue histology</td>
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		<tr height="24">
			<td height="24" style="height:24px;">10% formalin&#160;</td>
			<td style="width:411px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">JC1111/MC</td>
			<td>Tissue histology</td>
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		<tr height="20">
			<td height="20" style="height:20px;">70% ethanol&#160;</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">97064-490</td>
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		<tr height="20">
			<td height="20" style="height:20px;">PMA - Phorbol 12-myristate 13-acetate</td>
			<td style="width:411px;">Sigma-Aldrich</td>
			<td style="width:119px;">P1585-1MG</td>
			<td>Restimulation of cells</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ionomycin</td>
			<td style="width:411px;">Life Technologies</td>
			<td style="width:119px;">124222 1mg</td>
			<td>Restimulation of cells</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Fixation permeabilization kit</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">E16913-106</td>
			<td>Fixing cells for Flow cytometry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Tuberculin syringes</td>
			<td>BD Biosciences</td>
			<td align="right">309659</td>
			<td>Mice injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G x 3/8 needles</td>
			<td>BD Biosciences</td>
			<td align="right">309626</td>
			<td>Mice injection</td>
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		<tr height="39">
			<td height="39" style="height:39px;">Rag1 -/- mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 002216</td>
			<td>Recipient mice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD45.1 congenic mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no:002014&#160;</td>
			<td>Donor Th17 cells</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:380px;">CB17-SCID mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 001303</td>
			<td>Recipient mice</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:380px;">Balb/c mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 000651</td>
			<td>Donor Th17 cells</td>
		</tr>
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th17 inflammation model of oropharyngeal candidiasis in immunodeficient mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

In diesem Modell werden sowohl die Candida infizierten Mäusen und nicht-infizierte Mäuse in Rag-1 - / - immundefizienten Hintergrund wurden adoptiv mit Th17 Zellen 3-5 Tage vor der Infektion übertragen. Insgesamt 10-12 Mäuse wurden für die Experimente verwendet, mit 2 Mäuse in jeder Sham infizierten Gruppen und 4-5 in jedem der Candida infizierten Gruppen. Naive Zellen wurden von kongenen Thy1.1 oder CD45.1 Mäusen stammten, damit injiziert Th17 Zellen wurden in vivo unter Ver...

Watch this Scientific Journal Video about Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice at JoVE.com

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

Th17 Entzündung Modell Oropharyngeale Candidiasis in immundefizienten Mäusen

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

th17-inflammation-model-oropharyngeal-candidiasis-immunodeficient

Research

JoVE Journal

Medicine

9.9K Aufrufe.  Case Western Reserve University. Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice. 

Serie: Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Functional Assessment of Darmmotilität und Darmwand Entzündung in Nagetiere: Analysen in einem standardisierten Modell der intestinalen Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Forschung

Medizin

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Generierung von subkutanen und Intrahepatische Menschenleberzellkarzinom Xenografts in immundefizienten Mäusen

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Kombiniert<em&gt; In-vivo-</em&gt; Optische und μCT Imaging-Infektion, Entzündung und Knochenanatomie in einem Orthopädie Implantat-Infektion in Mäuse Monitor

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renale Ischämie Reperfusionsschaden: Ein Maus-Modell der Schädigung und Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Ein Mausmodell der irreversiblen und reversiblen Einseitige Ureterobstruktion

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Ein Repetitive erschütternder Head Injury-Modell in Mäusen

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induktion von beschleunigter Atherosklerose bei Mäusen: Das "Wire-Injury"-Modell

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Biolumineszenz und Nah-Infrarot-Imaging von Neuritis und Gehirnentzündung im EAE-Modell der Multiplen Sklerose in Mäusen

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Trockenpulver und Vernebelte Aerosol Inhalation von Arzneimitteln geliefert an Mäusen Mit einem Nose-only Belichtungssystem

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

Murine oropharyngealen Aspiration Modell der Ventilator-assoziierten und nosokomiale Bakterielle Pneumonie

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...