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.

REMARQUE: Les expériences utilisant des souris ont été effectués conformément au comité de protection des animaux institutionnelle (IACUC) des lignes directrices. 1. La reconstitution de l&#39;Rag-1 - / - souris avec des cellules I n vitro de culture Th17 (Trois jours avant l&#39;infection) <ol><li> Pour l&#39;établissement des cellules Th17, culture CD4 + CD44low CD62Lhigh CD25 des lymphocytes T naïfs (3 x 104) en U à fond plaques à 96 puits seuls ou co-culture eux avec 3 x 104 cellules CD4 + CD25 + Foxp3 + Treg en présence d&#39;soluble α-CD3 (1 pg / ml), α-CD28 (2 pg / ml) d&#39;anticorps et de cellules présentatrices d&#39;antigène, dans des conditions Th17 (polarisées à l&#39;aide d&#39;IL-6 (25 ng / ml), TGF β (2 ng / ml) , α-IFN-γ (2 ug / ml) et α -IL-4 (2 ng / ml)) pendant trois à quatre jours. REMARQUE: les cellules T naïves et regs ont été triés à l&#39;aide cellulaire activé par fluorescence (FACS) und procédures d&#39;isolement cellulaire magnétique et cultivées comme décrit précédemment 19-22. </li><li> Le jour 4, après remise en suspension des cellules Th17 utilisant la pipette, les recueillir à partir de cultures, et les centrifuger dans des conditions stériles. <ol><li> Prenez une petite aliquote de ces cellules à examiner leur viabilité et pour le phénotypage. Remettre en suspension dans les 500 pi de milieu RPMI, ajouter l&#39;iodure de propidium (200 ng / ml) et d&#39;évaluer leur viabilité immédiatement par cytométrie en flux. Effectuer phénotypage en cytométrie de flux évaluation de la production d&#39;IL-17A après restimulation (voir rubrique 6.3). </li></ol></li><li> Centrifugeuse de lavage et l&#39;essentiel des cellules dans du PBS stérile à 480 g pendant 6 min à 4 ° C dans toutes les étapes sauf indication contraire. </li><li> Utilisez ces cellules pour le transfert adoptif dans la vieille 6-8 semaines CD45.2 Rag1 - / - souris immunodéficientes. <ol><li> Remettre en suspension les cellules à une densité cellulaire 1 x 10 7 cellules / ml en utilisant froide PBS stérile. </li><li> Injecter 100 ul de cellules par intraperitoneal injection, en utilisant 25 g d&#39;aiguilles de seringues de 1 ml de tuberculine, de telle sorte que une souris reçoit 1 x 10 6 cellules. Certaines souris recevront PBS, les cellules Th17 ou uniquement, et d&#39;autres souris recevront Th17 cellules qui étaient co-cultivées avec des cellules Treg. REMARQUE: Effectuez toutes les étapes de manière aseptique. </li></ol></li></ol> 2. Croissance C. albicans l&#39;infection (un jour avant l&#39;infection) <ol><li> Avant l&#39;inoculation, disperser cinq colonies de la CAF2-1 C. albicans souche de laboratoire dans 100 pi de suspension PBS stérile, et ajouter la suspension au bouillon stérile. </li><li> Inoculer cinq colonies de la C. albicans dans 50 ml de la base azotée de levure (YNB sans acides aminés) / peptone / bouillon dextrose et incuber dans un incubateur agitateur à 30 ° C pendant 15 à 18 heures à 130 rpm. </li><li> Surveiller le bouillon de trouble, car ce est une indication de la croissance du champignon. </li></ol> 3. CandidaRécolte et de la procédure de comptage (Le Jour des maladies infectieuses) <ol><li> Recueillir 10 ml de bouillon Candida dans 15 ml tubes. Pour des volumes plus importants, de recueillir Candida dans 50 ml tubes. </li><li> Centrifuger les blastospores à 1900 xg pendant 5 min à température ambiante dans toutes les étapes sauf indication contraire. Pellet les blastospores par centrifugation, suivie par l&#39;élimination du surnageant. </li><li> Se il ya plus d&#39;un tube, mutualiser les blastospores de Candida à partir de tubes multiples, ajoutant PBS stérile pour les boulettes et les remettre en suspension dans 10 ml de PBS pour le comptage. </li><li> Prenez 20 pi de blastospores dans 1,5 ml microtubes, ajouter 20 pi de la paraformaldéhyde 2X et incuber à température ambiante pendant 15 à 20 min. Comptez les blastospores fixes utilisant l&#39;hémocytomètre sous le microscope. REMARQUE: Paraformaldéhyde est cancérogène. Ouvrez la solution non diluée seulement dans la hotte. </li><li> En attendant, répéter le lavage des blastospores au moins deux fois par centrifugation tourlet dans du PBS, et la granulation blastospores. Gardez les pastilles dans 15 ml tubes à température ambiante, dans la hotte ABSL1, prêt pour l&#39;infection. </li><li> Laissez un peu de PBS stérile à température ambiante pendant remise en suspension des blastospores de l&#39;infection. </li><li> Après comptage, ajouter du PBS stérile à la pastille de blastospores à ajuster les nombres de cellules de levure à deux blastospores de levure x10 8 cellules / ml. Ce est la suspension de blastospores qui sera utilisé pour infecter les souris. REMARQUE: Effectuez toutes les étapes de manière aseptique. </li></ol> 4. Souris Infection (3-5 jours après transfert de cellules) NOTE: La procédure de l&#39;infection de base est effectuée comme décrit précédemment 19,23-25. <ol><li> Peser le Rag-1 - / - souris qui ont reçu PBS ou les cellules trois jours plus tôt. </li><li> Calculer la dose à l&#39;aide d&#39;agents anesthésiques leur poids corporel. Anesthésier les souris par administration de kétamine / xylazine mélange (16,1 mg / ml et 1,6 mg / ml), en utilisant 25 g d&#39;aiguilles sur les seringues de tuberculine de 1 ml, par intraperitoneal injection. <ol><li> Administrer 50 pl par 10 g de poids corporel. (Ce est-0,8 mg de kétamine et 0,08 mg de xylazine / 10 g de poids corporel ou 80 mg de kétamine et de xylazine 8 mg par kg de poids corporel respectivement). Observer la réponse orteil pincée toutes les 15 min après l&#39;anesthésie. NOTE: Ce dosage va induire 60-90 min de l&#39;anesthésie, ce qui est suffisant pour la procédure d&#39;infection. </li><li> Appliquer le lubrifiant pommade ophtalmique dans les yeux des souris pour éviter les cornées de se dessécher. Comme le lubrifiant oculaire peut se dessécher, répétez la lubrification ophtalmique toutes les 45 min. </li></ol></li><li> Injecter 1 ml de solution saline (NaCl 0,9%) sous-cutanée sur le dos à côté de la patte avant, pour aider à la réhydratation de la souris pendant l&#39;anesthésie. </li><li> Obtenir un nouveau, cage propre. Suivre la procédure infection une souris à la fois, en plaçant chaque souris dans la nouvelle cage une fois infecté. Effectuez la / infection simulacre PBS abord, et ensuite procéder aux gro d&#39;infection à Candidaups. </li><li> Ramassez la souris anesthésiée et ouvrez la bouche pour révéler la base de la langue. <ol><li> Placer une balle de coton diamètre de 3 mm saturé avec 50 pl de PBS ou blastospore suspension, sublinguale dans la cavité buccale pendant 90 min. Retournez la souris tous les fronts 15 min et à l&#39;arrière pour éviter une congestion pulmonaire, veiller à ce que les boules de coton ne se déplacent pas. </li></ol></li><li> Mettre en place une minuterie pour chaque souris 90 min pour se assurer de Sham ou Candida inoculation dans la cavité buccale. </li><li> Gardez-les dans une cage sous la lampe de chaleur (4 mètres), et chaque souris sur coussinets de gel de chaleur. </li><li> Assurez-vous que la langue est retirée à l&#39;intérieur et à l&#39;écart des dents, pour éviter dents lacérants la langue. </li><li> A la fin de 90 min, retirer la boule de coton à partir de l&#39;embouchure de la souris. Surveillez toute souris qui peut remettre de l&#39;anesthésie plus tôt que 75 minutes. Dans un tel cas, anesthésier à nouveau avec de la kétamine (40 mg / kg) seul. </li></ol> 5. Poster PROCEDURe surveillance (pendant les 90 min anesthésie) <ol><li> Utilisez coussinets de gel de chaleur au cours de la 90 min anesthésie. <ol><li> Pour maintenir la température du corps et pour prévenir la suffocation, ne permettent pas de récupérer souris sur la literie de la souris épi de maïs régulier. </li></ol></li><li> Après la récupération de l&#39;anesthésie, administrer un 1 ml supplémentaires de NaCl 0,9% stérile voie sous-cutanée à deux endroits sur le dos. </li><li> Gardez jusqu&#39;à cinq PBS (sham) de souris inoculées par cage. Maison seulement 1-2 souris infectées par cage. </li><li> Remplissez la carte de procédure avec les initiales tous les jours après la procédure de noter les changements dans le poids du corps, les habitudes et la santé globale de toilettage. </li></ol> 6. Évaluation de l&#39;inflammation 6.1) La perte de poids <ol><li> Peser la souris tous les jours pendant l&#39;infection, à partir du jour avant l&#39;intervention de l&#39;infection. NOTE: En règle générale, les souris immunodéficientes injectées avec des cellules Th17 et sans regs T succombent à 20-30% De perte de poids due à l&#39;accroissement de la charge fongique 19 et exacerbé l&#39;inflammation. </li></ol> 6.2) histologie notation de la langue <ol><li> Sacrifier les souris par asphyxie au CO2, suivie par dislocation cervicale. </li><li> Ouvrir les mâchoires et disséquer la langue avec des ciseaux et des pinces. Utilisez le forceps émoussé pour tenir la langue, et l&#39;aide des ciseaux tendre la main à l&#39;arrière de la bouche à inciser la fin arrière de la langue. </li><li> Pour hématoxyline immunocytochimique et de l&#39;éosine (H &amp; E) coloration des tissus de la langue, rincer les tissus de la langue avec PBS glacé. Ajouter 5 ml de formol à 10% pendant 2-3 langues et les fixer O / N en tubes de 15 ml. <ol><li> Le lendemain, éliminer le formol et plongez les tissus dans 5 ml d&#39;éthanol à 70% pour éviter l&#39;hyper-fixation. Envoyez-les en service commercial de poursuivre inclusion dans la paraffine, la coupe et la coloration des coupes en paraffine. NOTE: le service d&#39;histologie commerciale est utilisée pour effectuer ces étapes. </li&gt; </ol></li><li> Une fois les diapositives sont de retour à partir du service d&#39;histologie commerciale, la qualité de l&#39;inflammation en observant les sections de tissu sous un microscope optique. <ol><li> Score de 0 à 5, 0 étant pas d&#39;inflammation, inflammation et 5 étant le plus sévère. Note l&#39;inflammation en utilisant le tableau 1. </li></ol></li></ol> 6.3) La production de cytokine par les cellules Th17 REMARQUE: Une production excessive de TNF-α est l&#39;une des lectures pour immunopathologie excessive. Lorsque les souris sont transférées de manière adoptive avec des cellules Th17 seulement, il provoque l&#39;immunopathologie qui est associée à une production excessive de TNF-α dans les cellules Th17. <ol><li> Recueillir une suspension cellulaire unique à partir de la rate, les ganglions cervicaux, axillaires ganglions lymphatiques, et de la langue, en utilisant des méthodes décrites précédemment 19,26. </li><li> Restimuler les cellules avec du phorbol myristate acétate (PMA) (50 ng / ml) et de l&#39;ionomycine (500 ng / ml) pendant 4 heures avec Brefeldine A (10 ug / ml) a été ajouté dans la last 2 h. Laver les cellules avec du PBS et fixer à l&#39;aide d&#39;un kit de fixation / de perméabilisation commerciale selon les instructions du fabricant. </li><li> Effectuer intra coloration de cytokine cellulaire en utilisant l&#39;anticorps anti-TNF-α conjugués à un fluorochrome, anti-IL-17A et d&#39;anticorps ROR-yt 19 comme décrit précédemment. </li><li> En bref, remettre en suspension les cellules dans le tampon de perméabilisation 1X avec le cocktail d&#39;anticorps anti-TNF-α, l&#39;anti-IL-17A et ROR-yt anticorps, chaque anticorps à 2-3 ug / ml de concentration finale. </li><li> Incuber les cellules pendant 1 heure à température ambiante. </li><li> Après l&#39;incubation, laver les cellules avec le tampon de perméabilisation 1X. </li><li> Remettre en suspension le culot cellulaire dans 500 pi de PBS avec 0,5% de sérum albumine bovine pour l&#39;analyse de cytométrie en flux. </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.

Ce modèle est basé sur l&#39;induction par voie orale C. albicans infection inflammation Th17 dépendante. En raison de l&#39;absence de T regs, l&#39;inflammation cellulaire Th17 induite est effrénée et conduit à l&#39;immunopathologie mal résolu. In vitro dérivés des cellules CD4 naïfs polarisés que les cellules Th17 ont été utilisés pour le transfert adoptif. 40-50% de la culture CD4 + cellules montrent détectable expression de l&#39;IL-17A autour de 3 jours (cellules Th17),...

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 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>
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			<td>Polarization of cells to Th17 conditions</td>
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			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-0281-85</td>
			<td>Polarization of cells to Th17 conditions</td>
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		<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>
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			<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>
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			<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>
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			<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>
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			<td height="20" style="height:20px;width:380px;">&#945;-IL17A ef660</td>
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			<td>50-7177-82</td>
			<td>Used for cell culture - 1:50</td>
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			<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>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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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>
		</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>
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		<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>
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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>
		</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>
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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>
			<td>Sterilization purpose</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>
		</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.

Dans ce modèle, les deux souris infectées par Candida et les souris non infectées dans Rag-1 - / - fond immunodéficientes ont été adoptive transféré avec des cellules Th17 3-5 jours avant l&#39;infection. Un total de 10 à 12 souris ont été utilisés pour les expériences, avec deux souris dans chaque groupe Sham infecté et 4-5 dans chacun des groupes infectés Candida. Cellules naïves ont été obtenues à partir de souris congéniques CD45.1 ou Thy1.1, de sorte que les cellules...

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 Inflammation modèle de la candidose oropharyngée chez des souris immunodéficientes

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 vues.  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. 

Vidéo: 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.

D&#39;évaluation fonctionnelle de la motilité intestinale et inflammation de la paroi intestinale chez les rongeurs: analyses dans un modèle standardisé de la manipulation intestinale

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

Recherche

Médecine

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.

Génération de sous-cutanée et intra-hépatique droits de carcinome hépatocellulaire xénogreffes chez la souris immunodéficientes

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.

Combiné<em&gt; In vivo</em&gt; Optique et μCT imagerie pour surveiller l&#39;infection, l&#39;inflammation, et Bone Anatomy dans une infection orthopédique Implant souris

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.

Rénale ischémie reperfusion: un modèle de souris de la blessure et de la régénération

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.

Un modèle murin d&#39;irréversible et réversible unilatérale obstruction urétérale

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.

Infarctus du myocarde chez les souris néonatales, Un modèle de régénération cardiaque

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.

Bioluminescence et proche infrarouge Imaging de névrite optique et inflammation du cerveau dans le modèle EAE de la sclérose en plaques chez des souris

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.

Poudre sèche et aérosol nébulisée L&#39;inhalation de produits pharmaceutiques à des souris Livrés L&#39;utilisation d&#39;un nez seul système d&#39;exposition

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.

Modèle murin Aspiration oropharyngée de pneumonie bactérienne associée à et nosocomiales

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.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

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