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.

NOTA: Os experimentos com ratos foram realizados de acordo com o animal institucional comitê de bem-estar (IACUC) orientações. 1. Reconstituição da Rag-1 - / - Os ratos com células I n Vitro Cultivadas Th17 (três dias antes da infecção) <ol><li> Para o estabelecimento de células Th17, cultura de células CD4 + CD44low CD62Lhigh CD25- células T ingénuas (3 x 104) de fundo em U em placas de 96 poços, sozinho ou co-cultura eles, juntamente com 3 x 104 células T CD4 + CD25 + Foxp3 + Tregs na presença de solúvel α-CD3 (1 ug / ml), α-CD28 (2 ng / ml) de anticorpos e células apresentadoras de antigénio, sob condições Th17 (polarizadas usando IL-6 (25 ng / mL), TGF-β (2 ng / ml) , α-IFN-γ (2 ug / ml) e α -IL-4 (2 ng / ml)) durante três a quatro dias. NOTA: células ingênuo e regs T foram classificados usando fluorescência de células activadas (FACS) umad procedimentos de isolamento de células magnético e cultivadas como descrito anteriormente 19-22. </li><li> No dia 4, depois de re-suspender as células Th17, utilizando a pipeta, recolher-los a partir de culturas, e centrifugar-las em condições estéreis. <ol><li> Tomar uma pequena alíquota destas células para examinar a sua viabilidade e para fenotipagem. Ressuspender-los em 500 ul de meio RPMI, adicionar iodeto de propídio (200 ng / ml) e avaliar a sua viabilidade imediatamente por citometria de fluxo. Execute fenotipagem por citometria de fluxo avaliação da produção de IL-17A após restimulação (ver secção 6.3). </li></ol></li><li> Centrifugar e lavar o grosso das células em PBS estéril a 480 xg durante 6 minutos a 4 ° C em todas as etapas, a menos que especificado de outra forma. </li><li> Use essas células para transferência adotiva em 6-8 semanas de idade CD45.2 RAG1 - / - ratos imunodeficientes. <ol><li> Ressuspender as células a 1 x 10 7 células / ml de densidade celular utilizando PBS estéril frio. </li><li> Injectar 100 ul de células por intraperitoneal injecção, utilizando agulhas 25 G em seringas de 1 ml de tuberculina, de tal modo que um ratinho receber 1 x 10 6 células. Alguns ratos receberão células Th17 PBS ou somente, e outros ratos receberão células Th17 que foram co-cultivados com células Treg. Observação: executar todos os passos de forma asséptica. </li></ol></li></ol> 2. Crescer C. albicans para a infecção (um dia antes da infecção) <ol><li> Antes da inoculação, dispersar cinco colônias do CAF2-1 C. albicans estirpe laboratorial em 100 ul de suspensão de PBS estéril e adicionar a suspensão ao caldo estéril. </li><li> Inocular 5 colônias de C. albicans em 50 ml de base de azoto de levedura (YNB sem aminoácidos) / peptona / meio de caldo de dextrose e incubar numa incubadora com agitação a 30 ° C durante 15-18 horas a 130 rpm. </li><li> Monitorar o caldo para a opacidade, uma vez que é uma indicação para o crescimento do fungo. </li></ol> 3. CandidaColheita e Counting Procedimento (No dia da infecção) <ol><li> Recolha de 10 ml de caldo de Candida em tubos de 15 ml. Para volumes maiores, coletar Candida em tubos de 50 ml. </li><li> Centrifugar os blastosporos a 1900 xg durante 5 min à temperatura ambiente em todas as etapas, a menos que especificado de outra forma. Agregar as blastosporos por centrifugação, seguida pela remoção do sobrenadante. </li><li> Se houver mais do que um tubo, reunir os blastosporos Candida a partir de múltiplos tubos, adicionando PBS estéril para os peletes e ressuspender-los em 10 ml de PBS para contagem. </li><li> Tomar 20 ul de blastosporos em tubos de microcentrífuga de 1,5 ml, adicionar 20 uL de paraformaldeído a 2X e incubar à temperatura ambiente durante 15-20 min. Contagem blastosporos fixos usando o hemocitômetro sob o microscópio. NOTA: O paraformaldeído é cancerígeno. Abrir a solução não diluída apenas na hotte. </li><li> No entretanto, repetir a lavagem dos blastosporos pelo menos duas vezes, por centrifugação them em PBS, e peletização blastosporos. Manter os peletes em tubos de 15 ml à temperatura ambiente, na capa ABSL1, prontos para a infecção. </li><li> Deixar alguns PBS estéril à temperatura ambiente durante ressuspensão dos blastosporos para a infecção. </li><li> Após a contagem, adicionar PBS estéril para o sedimento blastosporo para ajustar o número de células de levedura blastosporo a 2 x10 8 de levedura células / ml. Esta é a suspensão blastosporo que será utilizado para infectar os ratinhos. Observação: executar todos os passos de forma asséptica. </li></ol> 4. Ratos Infection (3-5 dias após a transferência celular) NOTA: O procedimento básico infecção é realizada como descrito anteriormente 19,23-25. <ol><li> Pesar o Rag-1 - / - ratos que receberam PBS ou as células três dias antes. </li><li> Calcula-se a dose de agentes anestésicos, utilizando o seu peso corporal. Anestesiar os ratos por administração de cetamina / xilazina mistura (16,1 mg / ml e 1,6 mg / ml), utilizando agulhas 25 G em seringas de 1 ml de tuberculina, por intrinjeção aperitoneal. <ol><li> Administrar 50 ul por 10 gramas de peso corporal. (Ou seja, 0,8 mg de cetamina e 0,08 mg de xilazina / 10 g de peso corporal ou 80 mg de cetamina e de xilazina 8 mg por kg de peso corporal, respectivamente). Observar o desenvolvimento de resposta toe pitada a cada 15 minutos após a anestesia. NOTA: Esta dosagem irá induzir 60-90 min da anestesia, o que é suficiente para o procedimento de infecção. </li><li> Aplicar a pomada lubrificante oftálmico aos olhos dos ratinhos para evitar que as córneas de secar. Como o lubrificante ocular pode secar, repita lubrificação oftálmica a cada 45 min. </li></ol></li><li> Injectar 1 mL de solução salina (NaCl a 0,9%) por via subcutânea no lado de trás do membro anterior, para ajudar a re-hidratação dos ratos durante a anestesia. </li><li> Obter uma nova gaiola, limpo. Siga o procedimento de uma infecção do mouse em um momento, colocando cada rato na gaiola nova, uma vez infectado. Realize a infecção PBS / sham em primeiro lugar, e depois prosseguir para as gro infecção por Candidaups. </li><li> Pegue o rato anestesiado e abra a boca para revelar a base da língua. <ol><li> Coloque uma bola de algodão 3 mm de diâmetro saturado com 50 ul de PBS ou blastosporo suspensão, sublingualmente na cavidade oral durante 90 min. Virar os ratos a cada 15 minutos frente e de trás para evitar congestão pulmonar, garantindo que as bolas de algodão não se movem. </li></ol></li><li> Configurar um temporizador para cada rato para garantir 90 min de Sham ou Candida inoculação na cavidade oral. </li><li> Mantê-los em uma gaiola sob a lâmpada de calor (4 metros de distância), e cada mouse sobre almofadas de gel de calor. </li><li> Certifique-se de que a língua é retirado dentro e longe dos dentes, para evitar dentes dilacerante da língua. </li><li> No final de 90 minutos, retire a bola de algodão da boca do mouse. Preste atenção para qualquer rato que pode recuperar da anestesia mais cedo do que 75 minutos. Em tal caso, anestesiar-los novamente com cetamina (40 mg / kg) apenas. </li></ol> 5. Publicar Procedure Monitoramento (durante a anestesia 90 min) <ol><li> Use almofadas de gel de calor durante a anestesia 90 min. <ol><li> Para manter a temperatura do corpo e para evitar sufocamento, não permitem que os ratos se recuperar no mouse cama sabugo de milho regular. </li></ol></li><li> Após a recuperação da anestesia, administrar um adicional de 1 ml de NaCl a 0,9% esterilizado subcutaneamente em dois locais no dorso. </li><li> Mantenha-se cinco PBS (sham) camundongos inoculados por gaiola. Casa apenas 1-2 camundongos infectados por gaiola. </li><li> Preencha o cartão de procedimento com as iniciais todos os dias após o procedimento para anotar as mudanças no peso corporal, higiene hábitos e saúde em geral. </li></ol> 6. Avaliação da Inflamação 6.1) A perda de peso <ol><li> Pesar os ratinhos todos os dias durante a infecção, começando no dia anterior ao processo de infecção. NOTA: Normalmente, os ratos imunodeficientes injetados com células Th17 e sem regs T sucumbir a 20-30% De perda de peso devido ao aumento da carga fúngica 19 e exacerbado inflamação. </li></ol> 6.2) pontuação Histologia da língua <ol><li> Sacrifício dos ratinhos por asfixia com CO 2, seguida por deslocação cervical. </li><li> Abra as mandíbulas e dissecar a língua com tesouras e pinças. Use uma pinça rombo para manter a língua, e usando a tesoura chegar à parte de trás da boca para entalhar a extremidade traseira da língua. </li><li> Para hematoxilina immunocytochemical e eosina (H &amp; E) a coloração dos tecidos da língua, lavar os tecidos língua com PBS gelado. Adicionar 5 ml de formol a 10% por 2-3 línguas e corrigi-los O / N em tubos de 15 ml. <ol><li> No dia seguinte, remover a formalina e mergulhar os tecidos em 5 ml de etanol a 70% para evitar a hiper-fixação. Enviá-los para o serviço comercial para continuar com parafina, corte e coloração de cortes de parafina. NOTA: serviço histologia comercial é usado para executar estes passos. </li&gt; </ol></li><li> Uma vez que as lâminas estão de volta a partir do serviço histologia comercial, grau de inflamação, observando as secções de tecido sob um microscópio de luz. <ol><li> Pontuação de 0 a 5, sendo 0 sem inflamação, e 5 sendo o mais grave inflamação. Pontuação da inflamação usando Tabela 1. </li></ol></li></ol> 6.3) A produção de citocinas pelas células Th17 NOTA: A produção excessiva de TNF-α é uma das leituras de imunopatologia excessiva. Quando os ratos são transferidos adoptivamente com células Th17 só, faz com que a imunopatologia que está associado com a produção excessiva de TNF-α em células Th17. <ol><li> Coletar uma suspensão de células individuais do baço, linfonodos cervicais, linfonodos axilares, e língua, utilizando métodos previamente descritos 19,26. </li><li> Reestimular as células com miristato acetato de forbol (PMA) (50 ng / ml) e ionomicina (500 ng / ml) durante 4 h com Brefeldina A (10 ug / ml) adicionada no last 2 h. Lavam-se as células com PBS e corrigi-los utilizando um kit de fixação / permeabilização comercial de acordo com as instruções do fabricante. </li><li> Realização da coloração de citocinas celular intra utilizando o anti-TNF-α conjugado com fluorocromo, anti-IL-17A e anticorpos ROR-γt como anteriormente descrito 19. </li><li> Resumidamente, ressuspender as células em tampão de permeabilização 1X com o cocktail de anticorpos anti-TNF-α, anti-IL-17A e anticorpos ROR-γt, cada anticorpo a 2-3 ug / ml de concentração final. </li><li> Incubam-se as células durante 1 h à TA. </li><li> Após a incubação, lavar as células com tampão de permeabilização 1X. </li><li> Ressuspender o sedimento celular em 500 ul de PBS com 0,5% de albumina de soro bovino para análise de citometria de fluxo. </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.

Este modelo é baseado na indução C. bucal infecção albicans dependente inflamação Th17. Devido à ausência de regs T, a inflamação induzida por células Th17 desprendido e leva a imunopatologia mal resolvidas. In vitro derivados de células T CD4 + naive polarizadas como células Th17 foram utilizados para a transferência adoptiva. 40 - 50% das células CD4 + cultivadas células mostram detectável expressão de IL-17A por volta do dia 3 (células Th17), e por isso foram ...

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>Polarization of cells to Th17 conditions</td>
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			<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>Polarization of cells to Th17 conditions</td>
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			<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>Used for cell culture - 1:50</td>
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			<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>
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			<td>12-6981-82</td>
			<td>Used for cell culture - 1:50</td>
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			<td height="25" style="height:25px;">YNB w/o amino acids)/Peptone/Dextrose broth medium&#160;</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">S507.SIZE</td>
			<td>Mediium for candida growth</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Shaker incubator&#160;</td>
			<td style="width:411px;">New Brunswick Scientific</td>
			<td style="width:119px;">Innova 4300</td>
			<td>Incubation growth for candida</td>
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		<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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			<td height="20" style="height:20px;">Allegra Centrifuge</td>
			<td style="width:411px;">Beckman Coulter</td>
			<td style="width:119px;">392302</td>
			<td>Cell culture - 480 g</td>
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		<tr height="25">
			<td height="25" style="height:25px;">1.5 ml eppendorf tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">BT620-NS</td>
			<td>Preparing final concentration of candida</td>
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		<tr height="24">
			<td height="24" style="height:24px;">2x paraformaldehyde</td>
			<td style="width:411px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>Fixing candida for count</td>
		</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>
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		<tr height="20">
			<td height="20" style="height:20px;">PBS - Phosphate Buffered Saline</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">PD8117</td>
			<td>Preparation of buffers</td>
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		<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic lubricant ointment&#160;</td>
			<td style="width:411px;">Allergan</td>
			<td style="width:119px;">-</td>
			<td>Eye ointment for animals to prevent dryness</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Saline (0.9% NaCl)</td>
			<td style="width:411px;">G Biosciences</td>
			<td style="width:119px;">786-561</td>
			<td>Administerd to animals to prevent dehydration</td>
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		<tr height="20">
			<td height="20" style="height:20px;">3-mm-diameter cotton wool ball&#160;</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">BP7603</td>
			<td>3 mm balls for candida infection</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heat gel pads</td>
			<td style="width:411px;">Case Western Reserve University - Animal Resource Center</td>
			<td style="width:119px;">-</td>
			<td>for maintaining the body temperaturre and fast recovery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin and Eosin staining</td>
			<td style="width:411px;">Histoserv - MD</td>
			<td style="width:119px;">-</td>
			<td>Tissue histology</td>
		</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.

Neste modelo, tanto os camundongos infectados por Candida e camundongos não infectados em Rag-1 - / - fundo imunodeficientes foram adotivamente transferidos com células Th17 3-5 dias antes da infecção. Um total de 10-12 ratinhos foram utilizadas para as experiências, com dois ratos em cada Sham grupos infectados e 5/4 em cada um dos grupos infectados Candida. Células naive foram derivadas de Thy1.1 ou CD45.1 ratinhos congénicos, de modo que as células Th17 injectados foram rastreados...

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 Inflamação Modelo de orofaringe Candidíase em imunodeficientes 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 ...

th17-inflammation-model-oropharyngeal-candidiasis-immunodeficient

Research

JoVE Journal

Medicine

9.9K visualizações.  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. 

Vídeo: 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.

Avaliação Funcional da motilidade intestinal e inflamação da parede do intestino em Roedores: Análises em um modelo padronizado de Manipulação Intestinal

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

Medicina

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.

Geração de subcutânea e intra-hepática Humanos carcinoma hepatocelular Xenoenxertos em Immunodeficient Ratos

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.

Combinado<em&gt; In vivo</em&gt; Optical e μCT imagem para monitorar infecções, inflamações, and Bone Anatomy em uma infecção Orthopaedic Implant em Ratos

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.

Renal Isquemia Reperfusão: um modelo do rato de lesão e regeneração

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.

Um modelo murino de irreversível e reversível obstrução ureteral unilateral

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.

Infarto do miocárdio em ratos neonatais, um modelo de regeneração cardíaca

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.

Bioluminescência e Near-infrared imagem de neurite óptica e Cérebro Inflamação no modelo de EAE de esclerose múltipla em ratinhos

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.

Pó seco e nebulizada de inalação de aerossóis de produtos farmacêuticos entregue no Ratos Usando um nariz somente Sistema de Exposição

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.

Modelo murino aspiração orofaríngea da Pneumonia bacteriana associada e hospitalares

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.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

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