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.

הערה: הניסויים באמצעות עכברים בוצעו בהתאם לועדת רווחת בעלי החיים המוסדיים (IACUC) הנחיות. 1. לשחזר את סמרטוט-1 - / - העכברים עם תאים אני n חוץ הגופייה בתרבית TH17 (שלושה ימים לפני זיהום) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להקמת תאי TH17, תרבות תאי T מסוג CD4 + הנאיבי CD44low CD62Lhigh CD25- (3 x 104) ב 96 צלחות U-תחתונה גם לבד, או שיתוף תרבותם יחד עם 3 x 104 CD4 + CD25 + Foxp3 + Tregs בנוכחות מסיסה α-CD3, α-CD28 (2 מיקרוגרם / מיליליטר) נוגדנים ותאי הצגת אנטיגן, בתנאי TH17 (מקוטב באמצעות IL-6 (25 ng / ml), β TGF- (2 ng / ml) (1 מיקרוגרם / מיליליטר) , Α-IFN-γ (2 מיקרוגרם / מיליליטר) ו-IL-4 α (2 מיקרוגרם / מיליליטר)) לשלוש עד ארבעה ימים. הערה: תאים נאיביים ובתקנות T מוינו באמצעות תא קרינה המופעל מיון (FACS)ד נהלי בידוד תא מגנטי ותרבותי כפי שתואר לעיל 19-22. </li><li style=";text-align:right;direction:rtl"> ביום 4, לאחר resuspending תאי TH17 באמצעות פיפטה, לאסוף אותם מתרבויות, וצנטריפוגות בתנאים סטריליים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קח aliquot קטן של תאים אלה לבחינת הכדאיות שלהם ועבור phenotyping. Resuspend אותם 500 μl של מדיום RPMI, להוסיף יודיד propidium (200 ng / ml) ולהעריך את הכדאיות שלהם באופן מיידי על ידי cytometry זרימה. בצע phenotyping ידי cytometry זרימת ההערכה של ייצור IL-17 א לאחר restimulation (ראה סעיף 6.3). </li></ol></li><li style=";text-align:right;direction:rtl"> צנטריפוגה ולשטוף את הנפח העיקרי של התאים בPBS סטרילי ב 480 XG במשך 6 דקות ב 4 ° C בכל השלבים, אלא אם כן צוין אחרת. </li><li style=";text-align:right;direction:rtl"> להשתמש בתאים אלה להעברת מאמצת לCD45.2 Rag1 6-8 שבוע ישן - / - עכברי immunodeficient. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Resuspend התאים בצפיפות תאי 1 x 10 7 תאים / מיליליטר באמצעות PBS סטרילי הקר. </li><li style=";text-align:right;direction:rtl"> הזרק של התאים 100 μl ידי intrapהזרקת eritoneal, באמצעות 25 מחטים G על 1 מיליליטר מזרקים טוברקולין, כך שעכבר אחד מקבל 1 x 10 6 תאים. עכברים מסוימים יקבלו תאי PBS או TH17 בלבד, ועכברים אחרים יקבלו תאי TH17 שהיו שיתוף תרבותי עם תאי Treg. הערה: בצע את כל שלבי סביבה נקיה מחיידקים. </li></ol></li></ol> 2. גידול C. אלביקנס לזיהום (יום אחד לפני זיהום) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני החיסון, לפזר חמש מושבות של ג CAF2-1 זן אלביקנס מעבדה בהשעית PBS סטרילי 100 μl, ומוסיף את ההשעיה למרק סטרילי. </li><li style=";text-align:right;direction:rtl"> לחסן 5 מושבות של ג אלביקנס ב 50 מיליליטר של בסיס חנקן שמרים (YNB ללא חומצות אמינו) / Peptone / בינוני דקסטרוז מרק ודגירה בחממה שייקר ב 30 מעלות צלזיוס למשך 15-18 שעות ב 130 סל&quot;ד. </li><li style=";text-align:right;direction:rtl"> לפקח על המרק לעננות, כפי שהיא אינדיקציה לצמיחה של הפטרייה. </li></ol> 3. קנדידהקציר וסדר ספירה (ביום של זיהום) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף 10 מיליליטר של מרק קנדידה ב 15 מיליליטר צינורות. לנפחים גדולים יותר, לאסוף קנדידה בצינורות 50 מיליליטר. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה blastospores ב1,900 XG במשך 5 דקות בRT בכל השלבים, אלא אם כן צוין אחרת. גלולה blastospores ידי צנטריפוגה, ואחריו הסרת supernatant. </li><li style=";text-align:right;direction:rtl"> אם יש צינור אחד או יותר, בריכת blastospores קנדידה מצינורות מרובים, הוספת PBS סטרילי לכדורים וresuspend אותם 10 מיליליטר של PBS לספירה. </li><li style=";text-align:right;direction:rtl"> קח 20 μl של blastospores בצינורות 1.5 מיליליטר microcentrifuge, להוסיף 20 μl של paraformaldehyde 2X ולדגור על RT במשך 15-20 דקות. רוזן blastospores הקבוע באמצעות hemocytometer מתחת למיקרוסקופ. הערה: Paraformaldehyde הוא מסרטנת. פתח את הפתרון חי רק במנדף. </li><li style=";text-align:right;direction:rtl"> בינתיים, לחזור על שטיפת blastospores לפחות פעמיים, על ידי צנטריפוגה tמכפלת בPBS, וpelleting blastospores. שמור את כדורים ב 15 מיליליטר צינורות ב RT, בשכונה ABSL1, מוכן לזיהום. </li><li style=";text-align:right;direction:rtl"> להשאיר כמה PBS סטרילי בRT לresuspending blastospores לזיהום. </li><li style=";text-align:right;direction:rtl"> לאחר ספירה, להוסיף PBS סטרילי לגלולת blastospore להתאים את מספרי תא השמרים blastospore עד 2 x10 8 תאים / מיליליטר שמרים. זה ההשעיה blastospore שישמש להדביק עכברים. הערה: בצע את כל שלבי סביבה נקיה מחיידקים. </li></ol> 4. זיהום עכברים (3-5 ימים לאחר העברת Cell) הערה: הליך הזיהום הבסיסי מבוצע כפי שתואר לעיל 19,23-25. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשקול את סמרטוט-1 - / - העכברים שקיבלו PBS או תאי שלושה ימים קודם לכן. </li><li style=";text-align:right;direction:rtl"> לחשב את המינון של חומרי הרדמה באמצעות משקל גופם. להרדים את העכברים על ידי מתן קטמין / תערובת xylazine (16.1 מ&quot;ג / מיליליטר ו -1.6 מ&quot;ג / מיליליטר), באמצעות 25 מחטים G על 1 מיליליטר מזרקים טוברקולין, על ידי מילת קריאההזרקת aperitoneal. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנהל 50 μl לכל 10 גרם של משקל גוף. (כלומר, 0.8 מ&quot;ג של קטמין ו0.08 מ&quot;ג של xylazine / 10 גרם של משקל גוף או 80 מ&quot;ג של קטמין ו -8 מ&quot;ג xylazine לכל קילוגרם של משקל גוף בהתאמה). שים לב לתגובת קמצוץ הבוהן כל 15 דקות לאחר הרדמה. הערה: מינון זה יגרום 60-90 דקות של הרדמה, וזה מספיק להליך הזיהום. </li><li style=";text-align:right;direction:rtl"> החל המשחה הסיכה עיניים בעיניים של העכברים כדי למנוע את הקרניות מהתייבשות. כחומר הסיכה העין עלולה להתייבש, לחזור על שימון עיניים כל 45 דקות. </li></ol></li><li style=";text-align:right;direction:rtl"> הזרק 1 מיליליטר של תמיסת מלח (0.9% NaCl) תת עורי בגב הצמוד לforelimb, כדי לסייע להחזרת נוזלים של העכברים במהלך הרדמה. </li><li style=";text-align:right;direction:rtl"> להשיג כלוב חדש, נקי. בצע את עכבר הליך אחד זיהום בזמן, הצבת כל עכבר לכלוב החדש הנגוע פעם. בצע את PBS / זיהום הדמה ראשון, ולאחר מכן להמשיך לGro זיהום קנדידהעליות. </li><li style=";text-align:right;direction:rtl"> להרים את העכבר הרדים ולפתוח את הפה הרחב כדי לחשוף את בסיס הלשון. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח כותנה קוטר כדור 3 מ&quot;מ רווי עם 50 μl של PBS או blastospore השעיה, sublingually בחלל הפה למשך 90 דקות. הפוך את העכברים כל חזית 15 דקות וחזרתי למניעת גודש ריאה, על מנת להבטיח שכדורי צמר גפן לא זזו. </li></ol></li><li style=";text-align:right;direction:rtl"> הגדרת טיימר לכל עכבר כדי להבטיח 90 דקות של חיסון שאם או קנדידה בחלל הפה. </li><li style=";text-align:right;direction:rtl"> שמור אותם בכלוב תחת מנורת החום (4 מטרים), וכל עכבר על כריות ג&#39;ל חום. </li><li style=";text-align:right;direction:rtl"> ודא כי הלשון תבוטל בתוך ומחוץ לשיניים, כדי למנוע את השיניים ופצעו את הלשון. </li><li style=";text-align:right;direction:rtl"> בתום 90 דקות, להסיר את כדור צמר גפן מפיו של העכבר. Watch עבור כל עכבר שעשוי להתאושש מהרדמה מוקדם מ -75 דקות. במקרה כזה, להרדים אותם שוב עם קטמין (40 מ&quot;ג / קילוגרם) בלבד. </li></ol> 5. ההודעה Procedurדואר ניטור (בהרדמת 90 דקות) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש ברפידות ג&#39;ל החום במהלך ההרדמה 90 דקות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לשמור על טמפרטורת הגוף ולמנוע חנק, אל תאפשר עכברים להתאושש על מצעי עכבר התירס של הרגילים. </li></ol></li><li style=";text-align:right;direction:rtl"> לאחר ההתאוששות מהרדמה, לנהל 1 מיליליטר נוסף של 0.9% מתחת לעור NaCl סטרילי בשני מקומות על הגב. </li><li style=";text-align:right;direction:rtl"> שמור עד חמש PBS (דמה) עכברים מחוסן בכל כלוב. בית עכברים נגועים רק 1-2 בכל כלוב. </li><li style=";text-align:right;direction:rtl"> מלא את כרטיס ההליך בראשי תיבות בכל יום לאחר ההליך כדי לרשום את השינויים במשקל הגוף, טיפוח הרגלים ועל בריאות כללית. </li></ol> 6. הערכת הדלקת משקל 6.1) הפסד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשקול את העכברים בכל יום במהלך הזיהום, החל מהיום לפני הליך זיהום. הערה: בדרך כלל, עכברי immunodeficient הוזרקו תאי TH17 וללא את תקנות T להיכנע עד 2030% ירידה במשקל עקב העלייה פטרייתי נטל 19 והחריפה את דלקת. </li></ol> 6.2) ניקוד היסטולוגיה של הלשון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להקריב את העכברים על ידי CO 2 מחנק ואחריו נקע בצוואר הרחם. </li><li style=";text-align:right;direction:rtl"> פתח את הלסתות ולנתח את הלשון עם מספריים ומלקחיים. שימוש במלקחיים בוטים להחזיק את הלשון, ובאמצעות המספריים להגיע לחלק האחורי של הפה כדי לחתוך את הקצה האחורי של הלשון. </li><li style=";text-align:right;direction:rtl"> לhematoxylin immunocytochemical וeosin מכתים (H &amp; E) של רקמות הלשון, לשטוף את רקמות לשון עם קרח הקר PBS. הוסף 5 מיליליטר של פורמלין 10% במשך 2-3 לשונות ולתקן אותם O / N ב 15 מיליליטר צינורות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> למחרת, להסיר את פורמלין ולטבול את הרקמות ב 5 מיליליטר של אתנול 70% כדי למנוע היפר-קיבעון. שלח אותם לשירות מסחרי להמשיך עם הטבעת פרפין, חתך וצביעת חלקי פרפין. הערה: שירות היסטולוגיה מסחרי משמשת כדי לבצע שלבים אלה. </li&gt; </ol></li><li style=";text-align:right;direction:rtl"> ברגע השקופיות חזרו משירות היסטולוגיה המסחרי, הכיתה הדלקת על ידי התבוננות בסעיפי הרקמות תחת מיקרוסקופ אור. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ציון 0-5, עם 0 להיות לא דלקת, ו -5 להיות הדלקת החמורה ביותר. ציון הדלקת באמצעות טבלה 1. </li></ol></li></ol> 6.3) ייצור ציטוקינים על ידי תאי TH17 הערה: ייצור TNF-α מופרז היא אחד מהקריאות לimmunopathology מוגזם. כאשר עכברים מועברים adoptively עם תאי TH17 רק, זה גורם immunopathology המשויך לייצור TNF-α מוגזם בתאי TH17. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף השעיה תא בודדת מטחול, בלוטות לימפה בצוואר הרחם, lymphnodes בבית השחי, ולשון, תוך שימוש בשיטות שתוארו קודם לכן 19.26. </li><li style=";text-align:right;direction:rtl"> Restimulate התאים עם אצטט myristate phorbol (PMA) (50 ng / ml) וionomycin (500 ng / ml) במשך שעה 4 עם Brefeldin (10 מיקרוגרם / מיליליטר) הוסיף בlAST שעה 2. לשטוף את התאים עם PBS ולתקן אותם באמצעות ערכת הקיבוע / permeabilization מסחרית בהתאם להוראות יצרן. </li><li style=";text-align:right;direction:rtl"> לבצע מכתים ציטוקינים סלולריים תוך שימוש באנטי TNF-α fluorochrome מצומדות, אנטי-IL-17 א ונוגדני ROR-γt כפי שתואר לעיל 19. </li><li style=";text-align:right;direction:rtl"> בקצרה, resuspend תאי חיץ permeabilization 1X עם הקוקטייל של אנטי TNF-α, אנטי-IL-17 א ונוגדני ROR-γt, כל נוגדן ל 2 - 3 מיקרוגרם / מיליליטר ריכוז סופי. </li><li style=";text-align:right;direction:rtl"> דגירה התאים עבור שעה 1 ב RT. </li><li style=";text-align:right;direction:rtl"> לאחר הדגירה, לשטוף את התאים עם חיץ permeabilization 1X. </li><li style=";text-align:right;direction:rtl"> Resuspend התא גלולה ב 500 μl של PBS עם 0.5% אלבומין בסרום שור לניתוח cytometry זרימה. </li></ol>

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L., Daniels, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+candidiasis+in+Sjogren's+syndrome:+prevalence,+clinical+correlations,+and+treatment.">Oral candidiasis in Sjogren's syndrome: prevalence, clinical correlations, and treatment.</a> Oral Surgery, Oral Medicine, And Oral Pathology. 68 (3), 324-329 (1989).</li><li>Gall, F., 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+spp.+in+oral+cancer+and+oral+precancerous+lesions.">Candida spp. in oral cancer and oral precancerous lesions.</a> The New Microbiologica. 36 (3), 283-288 (2013).</li><li>Grady, J. F., Reade, 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=Candida+albicans+as+a+promoter+of+oral+mucosal+neoplasia.">Candida albicans as a promoter of oral mucosal neoplasia.</a> Carcinogenesis. 13 (5), 783-786 (1992).</li><li>Dwivedi, P. P., Mallya, S., Dongari-Bagtzoglou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+immunocompetent+murine+model+for+Candida+albicans-promoted+oral+epithelial+dysplasia.">A novel immunocompetent murine model for Candida albicans-promoted oral epithelial dysplasia.</a> Medical Mycology. 47 (2), 157-167 (2009).</li><li>Cheng, S. C., Joosten, L. A., Kullberg, B. J., Netea, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Candida+albicans+and+the+mammalian+innate+host+defense.">Interplay between Candida albicans and the mammalian innate host defense.</a> Infection and Immunity. 80 (4), 1304-1313 (2012).</li><li>Marodi, L., Johnston, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+Candida+species+disease+in+infants+and+children:+occurrence,+risk+factors,+management,+and+innate+host+defense+mechanisms.">Invasive Candida species disease in infants and children: occurrence, risk factors, management, and innate host defense mechanisms.</a> Current Opinion In Pediatrics. 19 (6), 693-697 (2007).</li><li>Kumamoto, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+gastrointestinal+Candida+colonization.+Current+Opinion+In.">Inflammation and gastrointestinal Candida colonization. Current Opinion In.</a> Microbiology. 14 (4), 386-391 (2011).</li><li>Kraneveld, E. 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=The+relation+between+oral+Candida+load+and+bacterial+microbiome+profiles+in+Dutch+older+adults.">The relation between oral Candida load and bacterial microbiome profiles in Dutch older adults.</a> PloS One. 7 (8), e42770 (2012).</li><li>Hernandez-Santos, N., 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=Th17+cells+confer+long-term+adaptive+immunity+to+oral+mucosal+Candida+albicans+infections.">Th17 cells confer long-term adaptive immunity to oral mucosal Candida albicans infections.</a> Mucosal Immunology. 6 (5), 900-910 (2013).</li><li>Hasina, 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=ABT-510+is+an+effective+chemopreventive+agent+in+the+mouse+4-nitroquinoline+1-oxide+model+of+oral+carcinogenesis.">ABT-510 is an effective chemopreventive agent in the mouse 4-nitroquinoline 1-oxide model of oral carcinogenesis.</a> Cancer Prevention Research. 2 (4), 385-393 (2009).</li><li>Pandiyan, P., 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=CD4(+)CD25(+)Foxp3(+)+regulatory+T+cells+promote+Th17+cells+in+vitro+and+enhance+host+resistance+in+mouse+Candida+albicans+Th17+cell+infection+model.">CD4(+)CD25(+)Foxp3(+) regulatory T cells promote Th17 cells in vitro and enhance host resistance in mouse Candida albicans Th17 cell infection model.</a> Immunity. 34 (3), 422-434 (2011).</li><li>Pandiyan, P., 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+role+of+IL-15+in+activating+STAT5+and+fine-tuning+IL-17A+production+in+CD4+T+lymphocytes.">The role of IL-15 in activating STAT5 and fine-tuning IL-17A production in CD4 T lymphocytes.</a> J Immunol. 189 (9), 4237-4246 (2012).</li><li>Pandiyan, P., Lenardo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+CD4+CD25+Foxp3++regulatory+T+cell+survival.">The control of CD4+CD25+Foxp3+ regulatory T cell survival.</a> Biology Direct. 3 (6), (2008).</li><li>Pandiyan, P., Zheng, L., Ishihara, S., Reed, J., Lenardo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)CD25(+)Foxp3(+)+regulatory+T+cells+induce+cytokine+deprivation-mediated+apoptosis+of+effector+CD4(+)+T+cells.">CD4(+)CD25(+)Foxp3(+) regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4(+) T cells.</a> Nat Immunol. 8 (12), 1353-1362 (2007).</li><li>Conti, H. R., Huppler, A. R., Whibley, N., Gaffen, S. L., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+candidiasis.">Animal models for candidiasis.</a> Current Protocols In Immunology. 105, 11-19 (2014).</li><li>Conti, H. 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=Th17+cells+and+IL-17+receptor+signaling+are+essential+for+mucosal+host+defense+against+oral+candidiasis.">Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis.</a> J Exp Med. 206 (2), 299-311 (2009).</li><li>Kamai, Y., 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=New+model+of+oropharyngeal+candidiasis+in+mice.">New model of oropharyngeal candidiasis in mice.</a> Antimicrobial Agents And Chemotherapy. 45 (11), 3195-3197 (2001).</li><li>Pandiyan, P., Bhaskaran, N., Zhang, Y., Weinberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+T+cells+from+mouse+oral+tissues.">Isolation of T cells from mouse oral tissues.</a> Biological Procedures Online. 16 (1), 4 (2014).</li><li>Gladiator, A., Wangler, N., Trautwein-Weidner, K., LeibundGut-Landmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+IL-17-secreting+innate+lymphoid+cells+are+essential+for+host+defense+against+fungal+infection.">Cutting edge: IL-17-secreting innate lymphoid cells are essential for host defense against fungal infection.</a> J Immunol. 190 (2), 521-525 (2013).</li><li>Leppkes, M., Roulis, M., Neurath, M. 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.

מודל זה מבוסס על גרימת ג האוראלי זיהום אלביקנס דלקת TH17 תלויה. בגלל העדר את תקנות T, דלקת תא TH17 המושרית היא חסרת מעצורים ומובילה לimmunopathology גרוע נפתר. במבחנה נגזרת תאי CD4 נאיביים מקוטבים כמו תאי TH17 שמשו להעברת המאמצת. 40 - 50% מCD4 + תאים בתרבית להראות ביטוי...

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.

<table border="0" cellpadding="0" cellspacing="0" width="1289">
	<tbody>
		<tr height="25">
			<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>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">U-bottom 96 well plates&#160;</td>
			<td style="width:411px;">Fisher</td>
			<td style="width:119px;">055588</td>
			<td>Used for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-CD3&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-0031-85</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-CD28&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-0281-85</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">m-IL-6</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">RC232</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">h-TGF-b&#160;</td>
			<td style="width:411px;">R&#38;D</td>
			<td style="width:119px;">240-B</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-IFN-g&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-7311-85</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-IL-4&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-7041-85</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">&#945;-IL17A ef660</td>
			<td style="width:411px;">eBiosciences</td>
			<td>50-7177-82</td>
			<td>Used for cell culture - 1:50</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">&#945;-TNFa-PE-Cy7</td>
			<td>eBiosciences</td>
			<td>25-7423-41</td>
			<td>Used for cell culture - 1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">&#945;-RORgt PE&#160;</td>
			<td>eBiosciences</td>
			<td>12-6981-82</td>
			<td>Used for cell culture - 1:50</td>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">15 ml tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">BT888-SY</td>
			<td>Used for cell culture and candida growth</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">50 ml tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">CT 788-YS</td>
			<td>Used for cell culture and candida growth</td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="23">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td>Sterilization purpose</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
	</tbody>
</table>

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.

במודל זה, שני עכברי קנדידה -infected ועכברים נגוע בסמרטוט-1 - / - רקע immunodeficient הועבר adoptively עם תאי TH17 3-5 ימים לפני הזיהום. בסך הכל 10-12 עכברים שימש לניסויים, עם 2 עכברים בכל קבוצות שאם נגוע, ו4-5 בכל אחת מהקבוצות נגועות קנדידה. תאים נאיביים נגזרו מעכברי Thy1.1 או CD45.1 conge...

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 דגם דלקת של לוע תחתון פטרת בעכברים immunodeficient

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

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Research

JoVE Journal

Medicine

9.9K Views.  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. 

Video: 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.

הערכה תפקודית של תנועתיות מעיים ודלקת דופן מעי במכרסמים: ניתוח בדגם סטנדרטי של מניפולצית מעיים

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

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.

דור של תת עורי וintrahepatic אדם xenografts קרצינומה hepatocellular בעכברים עם מערכת חיסון חלשים

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  &amp;#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.

בשילוב<em&gt; בvivo</em&gt; אופטי וμCT הדמיה למעקב אחר זיהום, דלקת, ועצם אנטומיה בזיהום אורטופדיים שתל בעכברים

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.

כליות איסכמיה reperfusion פגיעה: במודל עכבר של הפגיעה והתחדשות

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.

דגם Murine של חסימת Ureteric חד-צדדית בלתי הפיכה והפיכה

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

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

אוטם שריר לב עכברים ילודים, מודל התחדשות לב

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

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.

הדמית פליטת אור קרוב אינפרא אדום של אופטיק בעצב המוח דלקת במודל EAE של טרשת נפוצה בעכברים

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.

אבקת ניקוי ו nebulized תרסיס שאיפת תרופות ביצוע עכברים באמצעות מערכת חשיפת אף בלבד

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.

השאיפה הלוע התחתון מאתר מודל של הנשמה-הקשורים ו רכשה החולים בדלקת ריאות חיידקית

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Contractility איזומטרי מדידה של עורק מצע המעי העליון העכבר באמצעות חוט Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...