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.

NOT: fareler kullanarak deneyler kurumsal hayvan refahı komitesi (IACUC) yönergeleri uygun olarak yapıldı. (Enfeksiyon Üç gün önce) I n Vitro kültürlü Th17 hücreleri ile Fare - / - 1. Rag-1 yeniden kurgulamak <ol><li> Çözünür mevcudiyetinde tek başına U dipli, 96 gözlü levhalar, ya da 3 x 104 CD4 + CD25 + Foxp3 + Tregs ile birlikte ko-kültür bunları Th17 hücreleri, kültür, CD4 + CD44low CD62Lhigh CD25 naiv T hücreleri (x 104 3) oluşturulması için α-CD3 (1 ug / ml), α-CD28 (2 ug / ml), Th17 koşulları altında antikorlar ve antijen sunan hücreler (IL-6 (25 ng / ml), TGF-β kullanılarak polarize (2 ng / ml) , α-IFN-γ (2 ug / ml) ve α -IL-4 (2 ug / ml)) 3-4 gün karıştırılmıştır. Not: Saf hücreleri ve T STüz THP kullanılarak dizildi (FACS) birManyetik hücre izolasyon prosedürleri, d ve kültüre daha önce 19-22 açıklandığı gibi. </li><li> 4. günde, pipet kullanarak Th17 hücrelerin tekrar asılı tutulmasından sonra, kültürler bunları toplamak ve steril koşullar altında santrifüj. <ol><li> Bunların canlılığı ve fenotipleme incelemek için, bu hücrelerin küçük bir kısım al. Propidyum iyodür (200 ng / ml) ekleyin ve akış sitometrisi ile hemen uygulanabilirliğini değerlendirmek, RPMI ortamında 500 ul bunları yeniden süspanse edin. Yeniden uyarıldıktan sonra IL-17A üretiminin değerlendirilmesi flow sitometri ile fenotiplendirilmesini gerçekleştirin (bakınız bölüm 6.3). </li></ol></li><li> Aksi belirtilmediği sürece, santrifüj ve tüm adımlar 4 ° C&#39;de 6 dakika boyunca 480 xg&#39;de steril PBS içerisinde hücrelerin büyük bir kısmını yıkayın. </li><li> 6-8 haftalık CD45.2 Rag1 içine evlatlık transferi için bu hücreleri kullanın - / - immünyetmezligi fareler. <ol><li> Soğuk steril PBS kullanılarak 1 x 10 7 hücre / ml hücre yoğunluğunda tekrar süspansiyon hücreleri. </li><li> Intrap hücrelere 100 ul enjekteeritoneal enjeksiyon, bir fare 1 x 10 6 hücre aldığı şekilde, 1 ml tüberkülin şırınga 25 G iğne kullanarak. Bazı fareler sadece PBS veya Th17 hücreleri alır ve diğer fareler, regülatör T hücreleri ile birlikte kültürlendiğinde, Th17 hücreleri alır. NOT: aseptik tüm adımları uygulayın. </li></ol></li></ol> 2. C Büyüyen enfeksiyon için albicans (bir gün Enfeksiyon önce) <ol><li> Aşılamadan önce, CAF2-1 C&#39;de beş koloni dağıtmak albicans laboratuvar steril PBS süspansiyon 100 ul zorlanma, ve steril suyu için süspansiyon ekleyin. </li><li> C. 5 koloni inoküle maya azot baz, 50 ml albicans (amino asit içermeyen YNB) / Pepton / dekstroz et suyu, orta ve 130 rpm&#39;de 15-18 saat süre ile 30 ° C&#39;de çalkalamalı bir inkübatör içinde inkübe edilir. </li><li> Bu mantar büyümesi için bir gösterge olarak, bulutluluk için suyu izleyin. </li></ol> 3. CandidaHasat ve (Enfeksiyon gününde) Sayım İşlemi <ol><li> 15 ml tüpler Candida suyu 10 ml toplayın. Daha büyük miktarlar için, 50 ml tüpler içinde Candida toplamak. </li><li> Aksi belirtilmediği sürece, tüm adımlarda oda sıcaklığında 5 dakika boyunca 1900 x g&#39;de blastosporlar santrifüjleyin. Üstte kalan sıvı kısmın çıkarılması ve ardından santrifüjle blastosporlar, pelet. </li><li> Birden fazla tüp olması durumunda, topaklar, steril PBS ilave, çok sayıda boruların Candida blastosporlar havuz ve sayım için, 10 ml PBS içinde tekrar süspansiyon haline bunları. </li><li> 1.5 ml mikrosantrifüj tüplerine blastosporlar 20 ul al 2X paraformaldehit 20 ul ve 15-20 dakika boyunca oda sıcaklığında inkübe edilir. Mikroskop altında hemasitometre kullanarak sabit blastosporlar sayın. NOT: Paraformaldehyde kanserojen. Sadece davlumbaz sulandırılmamış çözüm açın. </li><li> Bu arada, t santrifüjle, en az iki kez blastosporlar yıkama tekrarPBS hem, ve blastosporlar peletleme. Enfeksiyonu için hazır ABSL1 başlığı içinde, oda sıcaklığında 15 ml tüpler peletlerin tutun. </li><li> Enfeksiyon için blastosporlar resuspending RT&#39;de bazı steril PBS bırakın. </li><li> Sayımının ardından, maya hücre / ml&#39;lik 2 x10 8 maya blastospor hücre sayısını ayarlamak için blastospor pelet steril PBS ilave edin. Bu fareleri enfekte etmek için kullanılacak blastospor süspansiyonudur. NOT: aseptik tüm adımları uygulayın. </li></ol> 4. Fare Enfeksiyon (Hücre Transferi sonrası 3-5 gün) NOT: Daha önce açıklandığı gibi 19,23-25 ​​temel enfeksiyon prosedürü yapılır. <ol><li> / - - PBS veya üç gün önce hücreleri alınan fareler Rag-1 tartılır. </li><li> Kendi vücut ağırlığını kullanarak anestezik ajanların dozu hesaplayın. Intr göre, 1 mi tüberkülin şırınga 25 G iğne kullanılarak, ketamin / ksilazin karışımı (16.1 mg / ml ve 1.6 mg / ml) uygulanması ile fareler anesteziaperitoneal enjeksiyonu. <ol><li> Vücut ağırlığının her 10 gramı başına 50 ul uygulayın. (Örneğin, ketamin, 0.8 mg ve vücut ağırlığının ksilazin 10 g / 0.08 mg ya da ketamin 80 mg ve vücut ağırlığındaki her bir kg başına ksilazin 8 mg) mevcuttur. Ayak tutam yanıt anestezi sonrası her 15 dakika gözlemleyin. NOT: Bu dozaj enfeksiyon prosedürü için yeterli anestezi 60-90 dakika, neden olacaktır. </li><li> Kurumasını önlemek için kornealar farelerin gözünde oftalmik yağ merhemi sürün. Göz yağlayıcı kurumasına gibi, oftalmik yağlama her 45 dakikada bir tekrarlayın. </li></ol></li><li> Anestezi sırasında farelerin rehidrasyon yardımcı, ön ayakları ile deri altından geri bitişik tuzlu su (% 0.9 NaCl) ile 1 ml enjekte edilir. </li><li> Yeni, temiz bir kafes edinin. Bir kez enfekte yeni kafesin içine fare her yerleştirerek, bir defada enfeksiyon prosedürü bir fare izleyin. İlk PBS / sahte enfeksiyonu gerçekleştirin ve sonra Candida enfeksiyonu Gro devamgüç kaynağı. </li><li> Anestezi fare Pick up ve dil tabanı ortaya çıkarmak için geniş ağzını açın. <ol><li> Dil altından, 90 dakika için ağız boşluğu içinde, PBS veya blastospor süspansiyonu, 50 ul ile doyurulmuş 3 mm çapında pamuk yerleştirin. Pamuk topları hareket etmiyor sağlanması, akciğer tıkanıklığı önlemek için geri farelere her 15 dk ön çevirin ve. </li></ol></li><li> Ağız boşluğu Sham veya Candida aşılamadan 90 dakika sağlamak için her fare için bir zamanlayıcı ayarlayın. </li><li> Isı lambası (4 metre uzaklıkta) altında bir kafes ve ısı jel yastıkları her fare tutun. </li><li> Dil dil laserasyon yaralanmasıdır diş önlemek için, diş içinde ve uzak çekilir emin olun. </li><li> 90 dakika sonunda, fare ağızdan pamuk çıkarın. 75 dakikadan daha kısa sürede anestezi kurtarmak herhangi fare izleyin. Böyle bir durumda, sadece, ketamin (40 mg / kg) ile yeniden anestetize. </li></ol> 5. Mesaj prosedüründee (90 dk Anestezi sırasında) İzleme <ol><li> 90 dakika anestezi sırasında ısı jel pedleri kullanın. <ol><li> Vücut ısısını korumak için ve boğulma önlemek için, fareler normal mısır koçanı fare yatak üzerinde kurtarmak için izin vermez. </li></ol></li><li> Anesteziden çıktıktan sonra, arka iki konumda steril% 0.9 NaCl, deri altından ilave 1 ml yönetmek. </li><li> Beş PBS (plasebo) kafes başına aşılanmış farelerde kadar tutun. Kafes başına Evi sadece 1-2 enfekte fareler. </li><li> Alışkanlıkları ve genel sağlık bakım, vücut ağırlığı değişiklikleri not etmek işlem sonrası her gün baş harfleri ile prosedür kartını doldurun. </li></ol> 6. Enflamasyon değerlendirilmesi 6.1) Kilo kaybı <ol><li> Enfeksiyon prosedür öncesi gün ile başlayarak, enfeksiyon sırasında, her gün fareler tartılır. Not: Genellikle, Th17 hücreleri olmadan ve T gruplar gruplar enjekte bağışıklık yetersizliği olan farelere 20 yenikNedeniyle -30% kilo kaybı mantar yükünü 19 artmış ve inflamasyon şiddetlenir. </li></ol> Dil 6.2) Histoloji puanlama <ol><li> Servikal dislokasyon CO2 asfiksiyasyonu ile fareler kurban. </li><li> Çeneleri açın ve makas ve forseps ile dilini incelemek. Dilini tutmayı künt forseps kullanın ve makas ağız arkasına ulaşmak kullanarak dilin arka uç kesilirken. </li><li> Imüno hematoksilin ve eosin dil dokuların (H &amp; E) boyama için, buz gibi soğuk PBS ile dil dokuları yıkayın. 15 ml&#39;lik tüplerde / N 2-3 dil için% 10 formalin 5 ml ekleyin ve O bunları düzeltmek. <ol><li> Sonraki gün, formalin çıkarın ve hiper tespit önlemek için% 70 etanol içinde 5 ml doku bırakın. Kesit ve parafin kesitlerin boyanması, parafin gömme devam etmek ticari hizmet için bunları gönderin. NOT: Ticari histoloji hizmeti adımları gerçekleştirmek için kullanılır. </li&gt; </ol></li><li> Slaytlar geri ticari histoloji hizmetinden kez, sınıf bir ışık mikroskobu altında doku bölümleri gözlemleyerek iltihabı. <ol><li> 0 hiçbir iltihap olan, ve 5 en şiddetli inflamasyon olmak, 0-5 puan. Tablo 1 kullanılarak iltihabı Puan. </li></ol></li></ol> Th17 hücreleri tarafından 6.3) sitokin üretimi Not: Aşırı TNF-α üretimindeki azalma, aşırı immunopatolojilerinde için okumalar biridir. Farenin adoptif Th17 hücreleri ile transfer edildiğinde, sadece, bunun Th17 hücrelerinde aşırı TNF-α üretimi ile ilişkilidir Immunopathology neden olur. <ol><li> Daha önce açıklanan yöntemleri kullanarak 19.26 dalak, servikal lenf nodları, aksiller lymphnodes ve dil tek bir hücre süspansiyonu, toplayın. </li><li> Brefeldin A (10 ug / mi) ile 4 saat için forbol miristat asetat (PMA) (50 ng / ml) ve iyonomisin (500 ng / ml) ile hücrelerin yeniden uyarmak l eklendiast 2 saat. PBS ile hücreleri yıkayın ve üreticinin talimatlarına göre ticari Tespit / permeabilizasyon kiti kullanılarak onları düzeltmek. </li><li> Daha önce tarif edildiği gibi 19 florokrom konjuge anti-TNF-α, anti-IL-17A ve ROR-γt antikorları kullanılarak hücre içi sitokin boyama yapın. </li><li> / Ml nihai konsantrasyon 3 ug - Kısaca anlatmak gerekirse anti-TNF-α, anti-IL-17A ve ROR-γt antikorlar, 2 de her bir antikorun bir kokteyli ile 1X permeabilizasyon tampon maddesi içinde tekrar süspansiyon hücreleri. </li><li> Oda sıcaklığında 1 saat süre ile inkübe hücreleri. </li><li> İnkübasyondan sonra, 1X permeabilizasyon tampon maddesi ile hücreleri yıkayın. </li><li> Akışkan sitometri analizi için% 0.5 sığır serum albümini ile PBS içinde 500 ul hücre pelletini. </li></ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th17+cells+and+IL-17+receptor+signaling+are+essential+for+mucosal+host+defense+against+oral+candidiasis.">Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis.</a> J Exp Med. 206 (2), 299-311 (2009).</li><li>Kamai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+model+of+oropharyngeal+candidiasis+in+mice.">New model of oropharyngeal candidiasis in mice.</a> Antimicrobial Agents And Chemotherapy. 45 (11), 3195-3197 (2001).</li><li>Pandiyan, P., Bhaskaran, N., Zhang, Y., Weinberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+T+cells+from+mouse+oral+tissues.">Isolation of T cells from mouse oral tissues.</a> Biological Procedures Online. 16 (1), 4 (2014).</li><li>Gladiator, A., Wangler, N., Trautwein-Weidner, K., LeibundGut-Landmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+IL-17-secreting+innate+lymphoid+cells+are+essential+for+host+defense+against+fungal+infection.">Cutting edge: IL-17-secreting innate lymphoid cells are essential for host defense against fungal infection.</a> J Immunol. 190 (2), 521-525 (2013).</li><li>Leppkes, M., Roulis, M., Neurath, M. F., Kollias, G., Becker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+functions+of+TNF-alpha+in+the+regulation+of+the+intestinal+epithelial+response+to+inflammation.">Pleiotropic functions of TNF-alpha in the regulation of the intestinal epithelial response to inflammation.</a> International Immunology. 26 (9), 509-515 (2014).</li><li>Bishu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adaptor+CARD9+is+required+for+adaptive+but+not+innate+immunity+to+oral+mucosal+Candida+albicans+infections.">The adaptor CARD9 is required for adaptive but not innate immunity to oral mucosal Candida albicans infections.</a> Infection and Immunity. 82 (3), 1173-1180 (2014).</li><li>Quintin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+role+of+NK+cells+against+Candida+albicans+infection+in+immunocompetent+or+immunocompromised+mice.">Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice.</a> Eur J Immunol. 44 (8), 2405-2414 (2014).</li><li>Ramirez-Garcia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+and+cancer:+Can+this+yeast+induce+cancer+development+or+progression.">Candida albicans and cancer: Can this yeast induce cancer development or progression.</a> Critical Reviews In Microbiology. , 1-13 (2014).</li></ol>

The authors declare that they have no competing financial interests.

Bu model, oral C uyarıcı dayanmaktadır albicans enfeksiyonu bağımlı Th17 iltihabı. Çünkü T regs yokluğu, Th17 hücre kaynaklı inflamasyon sınırsız ve zayıf çözülmesi İmmünopatolojiye yol açar. In vitro Th17 hücreleri evlatlık transferi için kullanılan olarak polarize naif CD4 hücreleri elde edilen. 40 - kültürlenmiş CD4 + hücrelerinin% 50 3. günde yaklaşık keşfedilebilir IL-17A ifade (Th17 hücreleri) gösterir ve bu nedenle, fareler enjeksiyon iç...

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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		<tr height="25">
			<td height="25" style="height:25px;width:380px;">CAF-2</td>
			<td style="width:411px;">University of Pittsburgh (Sarah Gaffen)</td>
			<td style="width:119px;">-</td>
			<td style="width:380px;">Candida culture</td>
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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 height="20" style="height:20px;">a-CD3&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-0031-85</td>
			<td>Polarization of cells to Th17 conditions</td>
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			<td height="20" style="height:20px;">a-CD28&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-0281-85</td>
			<td>Polarization of cells to Th17 conditions</td>
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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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		<tr height="20">
			<td height="20" style="height:20px;">h-TGF-b&#160;</td>
			<td style="width:411px;">R&#38;D</td>
			<td style="width:119px;">240-B</td>
			<td>Polarization of cells to Th17 conditions</td>
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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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		<tr height="20">
			<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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		<tr height="20">
			<td height="20" style="height:20px;width:380px;">&#945;-TNFa-PE-Cy7</td>
			<td>eBiosciences</td>
			<td>25-7423-41</td>
			<td>Used for cell culture - 1:100</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:380px;">&#945;-RORgt PE&#160;</td>
			<td>eBiosciences</td>
			<td>12-6981-82</td>
			<td>Used for cell culture - 1:50</td>
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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>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">15 ml tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">BT888-SY</td>
			<td>Used for cell culture and candida growth</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">50 ml tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">CT 788-YS</td>
			<td>Used for cell culture and candida growth</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table top Centrifuge</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">82017-654</td>
			<td>For pelleting candida -900 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Allegra Centrifuge</td>
			<td style="width:411px;">Beckman Coulter</td>
			<td style="width:119px;">392302</td>
			<td>Cell culture - 480 g</td>
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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>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Ketamine/xylazine&#160;</td>
			<td style="width:411px;">Case Western Reserve University - Animal Resource Center</td>
			<td style="width:119px;">-</td>
			<td>Obtained from ARC approved protocol for anesthesia</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic lubricant ointment&#160;</td>
			<td style="width:411px;">Allergan</td>
			<td style="width:119px;">-</td>
			<td>Eye ointment for animals to prevent dryness</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Saline (0.9% NaCl)</td>
			<td style="width:411px;">G Biosciences</td>
			<td style="width:119px;">786-561</td>
			<td>Administerd to animals to prevent dehydration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-mm-diameter cotton wool ball&#160;</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">BP7603</td>
			<td>3 mm balls for candida infection</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heat gel pads</td>
			<td style="width:411px;">Case Western Reserve University - Animal Resource Center</td>
			<td style="width:119px;">-</td>
			<td>for maintaining the body temperaturre and fast recovery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin and Eosin staining</td>
			<td style="width:411px;">Histoserv - MD</td>
			<td style="width:119px;">-</td>
			<td>Tissue histology</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% formalin&#160;</td>
			<td style="width:411px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">JC1111/MC</td>
			<td>Tissue histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70% ethanol&#160;</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">97064-490</td>
			<td>Sterilization purpose</td>
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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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ionomycin</td>
			<td style="width:411px;">Life Technologies</td>
			<td style="width:119px;">124222 1mg</td>
			<td>Restimulation of cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fixation permeabilization kit</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">E16913-106</td>
			<td>Fixing cells for Flow cytometry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Tuberculin syringes</td>
			<td>BD Biosciences</td>
			<td align="right">309659</td>
			<td>Mice injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G x 3/8 needles</td>
			<td>BD Biosciences</td>
			<td align="right">309626</td>
			<td>Mice injection</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Rag1 -/- mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 002216</td>
			<td>Recipient mice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD45.1 congenic mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no:002014&#160;</td>
			<td>Donor Th17 cells</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:380px;">CB17-SCID mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 001303</td>
			<td>Recipient mice</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:380px;">Balb/c mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 000651</td>
			<td>Donor Th17 cells</td>
		</tr>
	</tbody>
</table>

th17 inflammation model of oropharyngeal candidiasis in immunodeficient mice

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

Bu modelde, Rag-1 de, Candida ile enfekte olan fareler ve enfekte edilmemiş fareler, her iki - / - bağışıklık yetersizliği olan arka plan adoptif enfeksiyona 3-5 gün önce, Th17 hücreleri ile transfer edilmiştir. 10-12 farelerin toplam 2, her bir Sahte enfekte edilmiş gruptaki farelerin ve Candida enfekte gruplarının her biri 4-5 ile, deneyler için kullanılmıştır. Enjekte Th17 hücreleri, sırasıyla (Şekil 1A) Thy1.1 veya CD45.1 boyaması kullanılarak <e...

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.

Immünyetmezligi Farelerde Orofaringeal kandidiyazis ve Th17 İltihabı Modeli

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

Video: Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

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

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

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

Bağırsak Manipülasyon bir Standart Model Analizleri: Kemirgenler Barsak Motilite ve Gut Duvar İnflamasyon İşlevsel Değerlendirilmesi

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

Tıp

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.

Bağışıklık yetersizliği olan farelere subkutan olarak ve intrahepatik insan hepatoselüler karsinoma ksenograftlarının Üretimi

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.

Kombine<em&gt; In vivo</em&gt; Optik ve uCT Görüntüleme Farelerde bir Ortopedik İmplant Enfeksiyon Enfeksiyon, Enflamasyon ve Kemik Anatomisi Monitör

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 İskemi Reperfüzyon Hasarı: Yaralanma Fare Modeli ve Rejenerasyon

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.

Geri dönüşümsüz ve Tersinir Tek taraflı Üreterik Obstrüksiyon bir fare modeli

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.

Yenidoğan Farelerde Miyokard Enfarktüsü, Kalp Rejenerasyon Bir Model

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.

Farelerde Multipl Skleroz EAE Modeli Optik Nörit ve Beyin Enflamasyon Biyoparlaklık ve Yakın-Kızılötesi Görüntüleme

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.

Kuru Toz ve Eczacılık Nebulize Aerosol Soluma bir Burun sadece Pozlama Sistemi Kullanarak Fare teslim

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.

Fare Oropharyngeal aspirasyon modeli Ventilatör ilişkili ve hastane edinilen bakteriyel pnömoni

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.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

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