Yazarlar bize modeli öğretmek yardımcı olmak için Dr Jay Pepose teşekkür etmek istiyorum. Bu çalışma Sağlık Hibeler EY11885 (PMS), EY21247 (PMS) ve Göz Hastalıkları Anabilim Körlük Engellenir Araştırma sınırsız ödenekle Ulusal Sağlık Enstitüleri tarafından desteklenmiştir.

Hayvan Bakımı Not: anti-enflamatuar ve böylece örnek kılacak şekilde Analjezikler Bu modelde kullanılan değildir. 1. Virüs hazırlanması <ol><li> 3-4 gün için enfeksiyon, 0.01 çokluğu bir başlangıç ​​dilüsyonu (T-150 şişesi içinde, yaklaşık 5 x 10 5 plak oluşturan birim) kullanılarak Vero hücreleri (% 80 confluent) üzerinde, HSV-1 büyümek bütün hücre kadar yuvarlanır-up ve kolay bir şişeye isabet dışarı çıkarılması edilir. </li><li> 4 steril santrifüj tüplerine ve Sorvall Legend RT santrifüj ortamı çıkarın ° C ve 15 dakika için 3.500 rpm. Ortam toplam 5 ml her biri 50 ml tüp supernatant (1.3) tekrar süspansiyon büyük pelet çıkarıldıktan sonra. İki 30 sn patlamaları 1 dakika sonikasyon. </li><li> 4, bir Beckman ° C J2-21 santrifüjü, 1 saat boyunca, süpernatan ve 9000 rpm&#39;de 50 ml steril Oak Ridge, tüpler ve santrifüj içine koymak çıkarın. Bu küçük bir viral pelet üretmektir. Süpernatantı atın. </li><li> 1.2 zekâ gelen sonikasyon birleştirinh 1.3 pelet ve 45 saniye için tekrar sonikasyon. 4, 5 dakika için 1500 rpm&#39;de Sorvall RT Legend, Santrifüj ° C. Süpernatant kaydedin. Bu, -70 bölünüp depolanan virüs stoku ° C. teşkil </li><li> Vero hücreleri üzerinde titresi virüs. Üzerinden 10 -8 seyreltme (her 10 kat seyreltme olmak üzere) 2 ayrı seyreltme kümelerinden 2x Ben genelde plakalı 10 0. Virüs titrasyonu, 12-kuyu steril plakalar üzerinde gerçekleştirilir. </li></ol> 2. Fare Enfeksiyon <ol><li> Anestetik cocktail/20 g fare vücut ağırlığı için 0.1 ml ile her bir fare enjekte edilir (Ketamin, 60 mg / kg + Xylazine 5 mg / HBSS içinde seyreltilmiş kg). Bunu uygun dozlarda kullanılan ve bu ilacın etkileri çok kısa ömürlü vardır anestezinin en güvenli araçlardan biridir çünkü bu kokteyli kullanılır. </li><li> Fareler anestezi olduğunda kornea delikli olmadığından emin olmak için bir diseksiyon mikroskobu kullanarak bir 30 gauge iğne ile sağ gözün kornea çizmeyin. </li><li> Her fare receBirincil enfeksiyonu esnasında hasara karşı korneanın korunması için, toplanmış insan serumu (1:800 arasında en az% 50 viral nötralizasyon için etkili bir doz ile anti-HSV tepkime Sigma, Temecula, California) 1 ml arasında bir intraperitoneal IVED. </li><li> Kulak klipleri ile veya kulak yumruk sayısı fareler. </li><li> Tüm fareler sayılı edildikten sonra, bir 20 ul pipet kullanılarak, 10 6 PFU (5 ul) ile her bir göz enfekte. Biz sadece bir göz ve genellikle sağ göz bulaştırmak. Biz de onlar anestezi iken nemli tutmak için diğer gözlerine HBSS 5 ul yerleştirin. 5 ul prosedürler az 10 dakika içinde gerçekleştirilir ve hayvanlara göre anestetik ile dozlanan çünkü gözlerin hidrasyon korumak için yeterlidir. Enfeksiyon Vero medya 1 ml sulu bir steril pamuk aplikatör ile farelerde 3 gün sonrası enfeksiyon sürüntü ile doğrulanır. Sonra, 100 ul 48 oyuklu plakalar içinde yetiştirilen Vero hücrelerine ilave edilir. Bu plakalar sitopatik etkilerini günlük izliyor<html> (CPE). </li></ol> 3. Gecikme gelen Mice reaktivasyonu <ol><li> Fare primer enfeksiyonu takiben en az 5 hafta anestezi uygulanmıştır. Biz bir yıl aşağıdaki birincil enfeksiyona fareler kadar yeniden ve yanıt olarak anlamlı farklılıklar gözlenmiştir bulunmadığına dikkat edilmelidir. </li><li> Bir kez anestezi sadece tek bir göz UV-B ışık maruz kaldığı bu gibi 302 nm bir dalga boyunda bir tepe UV-B yayar TM20 Chromato-Vu transilluminator, yerleştirilir. Her bir fare 250 UV-B ışık mJ cm 2 maruz kalmaktadır. </li><li> Aktifleştirdiğinizde ilk 1-7 sonrası UV-B ışınlaması gün sonra 0. günde Vero medya 1 ml yerleştirilir (kendiliğinden shedders kontrol etmek için UV-B maruz kalmadan önce) ve bir steril pamuklu aplikatör ile gözler swabbing ile tespit edilir. 2.5 de tarif edildiği gibi bez malzeme) değerlendirilir. Bulaşıcı virüsün sahip olan çubuklar için, virüs miktarı 12 oyuklu plakalar kullanılarak, standart bir plak deneyi ile tittered edilir. </li></ol>ove_title &quot;&gt; 4. Klinik Değerlendirme <ol><li> Fare bir binoküler mikroskop kullanarak maskeli bir gözlemci tarafından klinik göz hastalığı için değerlendirilir. </li><li> Korneal matlık 0 net stromada belirten 0 4 bir ölçekte, puan, 1 hafif stromal opasite gösteren, 2 ayırt iris özellikleri ile orta opaklık gösterir, 3 gösteren öğrenci marjları ve 4 hariç tanımlanan iris ayrıntı kaybı ile yoğun opaklık gösterir posterior manzaralı toplam opaklık. </li><li> Neovaskülarizasyonunda 4 eşit parçaya bölünür kornea bölünmesi ve bu kadranın her birinde vaskülarizasyonu ölçüde belirleyerek 1 ila 8 arasında bir ölçekte derecelendirilmiştir. </li><li> Blefarit şöyle ölçülür: 0, hiçbir lezyonlar; 1, minimal kapak şişme; 2, orta derecede şişkinlik ve huysuz çapaklanma, 3, şiddetli şişlik, orta perioküler saç dökülmesi ve cilt lezyonları ve 4, gözler kabuklu kapalı şiddetli şişlik, Şiddetli perioküler saç dökülmesi ve cilt lezyonları. </li><li> Ensefalit inclu Kanıtded, geri karıştırdı saç ve belirgin nörolojik anormallikler hunched. Onlar ensefalit önemli belirtileri görüntülediğinizde Fare ötenazi vardır. </li></ol> 5. Temsilcisi Sonuçlar Yukarıda tarif edilen tekrarlayan model birinci Shimeld, et al tarafından yayınlanmıştır. 1-3. Onlar test ve laboratuvar yıl 4-15 boyunca birçok el yazmaları yayımlamak için bu modelin değiştirilmiş bir sürümü kullanıyor. Modeli rolü tanımlamak için kullanılmış olduğunu yineleyen HSV-1 hastalığı 10 sitokin interferon-γ çalış. Şekil 3&#39;te gösterildiği gibi, daha IFNγ kötü gösterilir kornea hastalığı eksik fareler vahşi-tip fareler 14 mi. Aynı zamanda birçok farklı aşı 6,10,11 oluşturur ve tedavi değeri tanımlamak için başarıyla kullanılmıştır. Bir olguda test aşı profilaktik iyi çalıştı ama 6 terapötik açıdan etkili olduğu ispat etmedi unutmayın. Bununla birlikte,Bir çoğaltma-beceriksiz viral aşı test edildiğinde, bu profilaktik olarak sadece etkili değil, ama terapötik zamanda 10,11 çok etkili oldu, referans 10 Şekil 5&#39;e bakınız. Daha yakın bir zamanda cinsiyet farklılıkları tekrarlayan HSK geçiren farelerde tespit edilebilmesine edildi olmadığını belirlemek için bu modeli kullanmışlardır. Şekil 1 görüldüğü gibi, erkek C57BL / 6 fareler dişi C57BL / 6 fareler mutlaka önemli ölçüde daha az korneal opasite gösterilecek. Aynı şekilde, korneal neovaskülarizasyon değerlendirildiğinde, erkek farelerin dişi farelerde (Şekil 2) yaptım daha korneada daha az yeni damar büyüme göstermiştir. Şu anda bu fenotipin farelerin diğer suşları benzer analizler yaparak belirli gerginlik olup olmadığı test edilir. Ayrıca, fark testosteron eksikliği ya da östrojen varlığı yüzünden olup olmadığını belirler. <img alt="Şekil 1" src="/files/ftp_upload/4276/4276fig1.jpg" /> Şekil 1. UV-B ile indüklenen reaktivasyon aşağıdaki erkek ve dişi C57BL / 6 fare suşu içinde kornea hastalığı. Latent enfekte farelerde UV-B radyasyon ve fareler 35 gün boyunca korneal opasite için izlenmiştir ile etkinleştirmek için oluşturuldu. Aşağıdaki gibi bu çalışmalar için kullanılan fareler numaraları vardı: erkek, n = 15; kadın, n = 15. Sonuçlar ortalama ± SEM göstermektedir. * Anlamlı olarak daha fazla virüs kaynaklı hastalık 14-35 gün (P = 0,001 ila 0,01) için dişi C57BL / 6 fareleri içinde yapıldı. <img alt="Şekil 2," src="/files/ftp_upload/4276/4276fig2.jpg" /> Şekil 2. UV-B ile indüklenen reaktivasyon aşağıdaki erkek ve dişi C57BL / 6 fare suşu içinde kornea hastalığı. Latent enfekte farelerde UV-B radyasyon ve fareler 35 gün boyunca korneal neovaskülarizasyon için izlenmiştir ile etkinleştirmek için oluşturuldu. Erkeklerde, n: bu çalışmalar için kullanılan farelerin sayısı aşağıdaki gibidir= 15; kadın, n = 15. Sonuçlar ortalama ± SEM göstermektedir. * Anlamlı olarak daha fazla virüs kaynaklı hastalık 14-28 gün (P = 0,001 ila 0,01) için dişi C57BL / 6 fareleri içinde yapıldı.

<ol>
	<li>Shimeld, C., Hill, T. J., Blyth, B., Easty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+model+of+recurrent+herpetic+eye+disease+in+mice.">An improved model of recurrent herpetic eye disease in mice.</a> Curr. Eye Res. 8, 1193-1205 (1989).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+latent+infection+and+induction+of+recurrent+herpetic+eye+disease+in+mice.">Reactivation of latent infection and induction of recurrent herpetic eye disease in mice.</a> J. Gen. Virol. 71, 397-404 (1990).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+immunization+protects+the+mouse+eye+from+damage+after+herpes+simplex+virus+infection+by+limiting+spread+of+virus+in+the+nervous+system.">Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system.</a> J. Gen. Virol. 71, 681-687 (1990).</li><li>Laycock, K. A., Lee, S. F., Brady, R. H., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+murine+model+of+recurrent+herpes+simplex+viral+keratitis+induced+by+ultraviolet+B+radiation.">Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation.</a> Invest. Ophthalmol. Vis. Sci. 32, 2741-2746 (1991).</li><li>Keadle, T. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+ameliorates+corneal+disease+in+a+mouse+model+of+recurrent+herpetic+keratitis.">IL-10 ameliorates corneal disease in a mouse model of recurrent herpetic keratitis.</a> Microbial Path. 38, 13-21 (2005).</li><li>Keadle, T. L., Laycock, K. A., Miller, J. K., Hook, K. K., Fenoglio, E. D., Francotte, M., Slaoui, M., Stuart, P. M., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+recombinant+glycoprotein+D+subunit+vaccine+on+the+development+of+primary+and+recurrent+ocular+infection+with+herpes+simplex+virus+type+1+in+mice.">Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice.</a> J. Inf. Dis. 176, 331-338 (1997).</li><li>Keadle, T. L., Laycock, K. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinflammatory+cytokines+IL-1+and+TNF-a+are+required+for+recurrent+herpetic+keratitis+in+NIH+mice.">Proinflammatory cytokines IL-1 and TNF-a are required for recurrent herpetic keratitis in NIH mice.</a> Invest. Ophth. Vis. Sci. 41, 96-102 (2000).</li><li>Keadle, T. L., Usui, N., Laycock, K. A., Kumano, Y., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+cytokine+expression+in+a+murine+model+recurrent+herpetic+stromal+keratitis.">Corneal cytokine expression in a murine model recurrent herpetic stromal keratitis.</a> Ocular Immunol. Inflam. 9, 193-205 (2001).</li><li>Keadle, T. L., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++and+CD8++cells+are+key+participants+in+the+development+of+recurrent+herpetic+stromal+keratitis+in+mice.">CD4+ and CD8+ cells are key participants in the development of recurrent herpetic stromal keratitis in mice.</a> Microbial Path. 32, 255-262 (2002).</li><li>Keadle, T. L., Morrison, L. A., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+immunization+with+a+virion+host+shutoff-defective,+replication-incompetent+herpes+simplex+virus+type+1+strain+limits+recurrent+herpetic+ocular+infection.">Therapeutic immunization with a virion host shutoff-defective, replication-incompetent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection.</a> J. Virol. 76, 3615-3625 (2002).</li><li>Keadle, T. L., Laycock, K. A., Morris, J. L., Leib, D. A., Morrison, L. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+vaccination+with+vhs(-)+herpes+simplex+virus+reduces+the+severity+of+recurrent+herpetic+stromal+keratitis+in+mice.">Therapeutic vaccination with vhs(-) herpes simplex virus reduces the severity of recurrent herpetic stromal keratitis in mice.</a> J. Gen. Virol. 83, 2361-2365 (2002).</li><li>Keadle, T. L., Morris, J. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+aminoguanidine+on+primary+and+recurrent+ocular+herpes+simplex+virus+infection.">The effects of aminoguanidine on primary and recurrent ocular herpes simplex virus infection.</a> Nitric Oxide. 13, 247-253 (2005).</li><li>Stuart, P. M., Morris, J. E., Sidhu, M., Keadle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL3+protects+mice+from+corneal+pathology+during+recurrent+HSV-1+infection.">CCL3 protects mice from corneal pathology during recurrent HSV-1 infection.</a> Front. Biosci. 13, 4407-4415 (2008).</li><li>Keadle, T. L., Alexander, D. E., Leib, D. A., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+gamma+is+not+required+for+recurrent+herpetic+stromal+keratitis.">Interferon gamma is not required for recurrent herpetic stromal keratitis.</a> Virology. 380, 46-51 (2008).</li><li>Carr, D. J., Austin, B. A., Halford, W. P., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Interferon-gamma+by+an+adenovirus+vector+blocks+herpes+simplex+virus+Type+1+reactivation+in+vitro+and+in+vivo+independent+of+RNase+L+and+double-stranded+RNA-dependent+protein+kinase+pathways.">Delivery of Interferon-gamma by an adenovirus vector blocks herpes simplex virus Type 1 reactivation in vitro and in vivo independent of RNase L and double-stranded RNA-dependent protein kinase pathways.</a> J. Neuroimmunol. 206, 39-43 (2009).</li></ol>

Çıkar çatışması ilan etti.

Herpetik stromal keratit tekrarlayan modeli (HSK) burada açıklanan araştırmacı insan hastalık sırasında gözlenen edildiği şey ile daha tutarlı bir model insan HSK çalışma sağlar. Böylece, model güçlü hastalığı primer hastalık sırasında uyarılan tarafından zaten bir bağışıklık sistemi bağlamında oluşur vardır. Bu fareler zaten HSV-1 için bir immün yanıt olduğu için, bu bağışıklık yanıtını yeniden faktörler başlangıçta oluşturulan bu olanlardan büyük olasılıkla fa...

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Sigma</td>
			<td>S7023</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-C1</td>
		</tr>
		<tr>
			<td>Pen/ Strep</td>
			<td>Sigma</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290018</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td>Legend RT</td>
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		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>52-21</td>
		</tr>
		<tr>
			<td>Transilluminatior</td>
			<td>UVP</td>
			<td>95-0452-02</td>
		</tr>
		<tr>
			<td>Sonicatior</td>
			<td>Branson</td>
			<td>Sonifier 450</td>
		</tr>
		<tr>
			<td>Oak Ridge Tubes</td>
			<td>Nalgene</td>
			<td>Z 720410</td>
		</tr>
		<tr>
			<td>Flasks- 7150</td>
			<td>Corning</td>
			<td>430823</td>
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		<tr>
			<td>Plates- 12 well</td>
			<td>Corning</td>
			<td>CLS 3513</td>
		</tr>
		<tr>
			<td>Plates- 48 well</td>
			<td>Corning</td>
			<td>CLS 3548</td>
		</tr>
		<tr>
			<td>Sterile Conical Tubes- 30 ml</td>
			<td>Corning</td>
			<td>CLS 430828</td>
		</tr>
		<tr>
			<td>Sterile Cotton-tipped Applicators</td>
			<td>Fisher</td>
			<td>23-400-125</td>
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		<tr>
			<td>30G Needles</td>
			<td>Becton-Dickinson</td>
			<td>305106</td>
		</tr>
		<tr>
			<td>25G Needles</td>
			<td>BD</td>
			<td>305122</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309604</td>
		</tr>
	</tbody>
</table>

recurrent herpetic stromal keratitis in mice, a model for studying human hsk

Herpetik göz hastalığı, adlandırdığı herpetik stromal keratit (HSK), korneanın bir potansiyel olarak kör edici enfeksiyonu olduğunu tedavisi için 300.000 &#39;den fazla klinik ziyaretleri her yıl sonuçları. Klinik hastalığı olanlarda 1 ila 2 oranında enfekte kornea görme kaybı yaşayacaksınız. Bu olguların büyük çoğunluğu herpes latent enfeksiyonun reaktivasyonu sonucu akut hastalık nedeniyle değil ben virüsü yazın ve simpleks. İlginç bir şekilde, akut enfeksiyon, genellikle bu hastalık incelemek için kullanılan bir modeldir. Ancak, HSK tekrarlayan modeli klinik hastalık sırasında ortaya ne daha yansıtıcı olacağını hissettim. HSK için tekrarlayan hayvan modellerinde tavşan ve fareler hem istihdam var. Tavşanların avantajı, ikinci gecikme reaktivasyon, bilinen herhangi bir uyarıcı yokluğunun ortaya çıkmasıdır. O tavşan modelinde immün yok çünkü birçok immünolojik faktörler tekrarlayan HSK oynadığı rol keşfetmek için zor olduğunu söylediFarenin sahip olduğu fizyolojik ve genetik kaynaklar. Reaktivasyonu UV-B ışığa maruz kalma ile tetiklenen çünkü biz orada pek çok kaynak mevcuttur olmanın avantajına sahiptir ve aynı zamanda reaktivasyonu ne zaman ortaya çıkacağını biliyoruz çünkü tekrarlayan HSK için fare modeli kullanmayı seçti. Bugüne kadar, bu model bu hastalık için önemli olan çeşitli immünolojik faktörlerin belirlenmesi amacıyla kullananlar laboratuarlar sağladı. Bu da bize tedavi ve aşı etkinliği hem sınamak için izin verdi.

Watch this Scientific Journal Video about Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK at JoVE.com

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetik kornea hastalığı çalışmaların çoğu birincil enfeksiyon modeli kullanın. Ancak, HSV-1 ile birincil infeksiyon genellikle insan hastalığına yol açmaz. Burada daha yakından insan hastalığı taklit herpetik kornea hastalığı, tekrarlayan modeli açıklamak.

Farelerde Tekrarlayan Herpetik Stromal Keratit, İnsan HSK Eğitim İçin Bir Model

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

recurrent-herpetic-stromal-keratitis-mice-model-for-studying-human

Research

JoVE Journal

Immunology and Infection

11.8K Görüntüleme.  Saint Louis University. Herpetik kornea hastalığı çalışmaların çoğu birincil enfeksiyon modeli kullanın. Ancak, HSV-1 ile birincil infeksiyon genellikle insan hastalığına yol açmaz. Burada daha yakından insan hastalığı taklit herpetik kornea hastalığı, tekrarlayan modeli açıklamak.

Video: Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

<em&gt; Citrobacter rodentium</em&gt; Mouse Model: Bulaşıcı Kolit için çalışılması Patojen ve Host Katılımlar

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

İmmünoloji ve Enfeksiyon

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Bir<em&gt; In vitro</emİnsan Makrofaj Farklılaşma ve Polarizasyon heterojenliği Eğitim için&gt; Model

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D modelli Görüntüleme<em&gt; Citrobacter rodentium</emFarelerde&gt; Enfeksiyonlar

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Kemirgen Lenf Düğümü Stromal Hücrelerinin İzolasyonu

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; İnsan Tüm Kan ve Endotelyumunun Arasındaki Etkileşimler incelenmesi için Model

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Deneysel Enfeksiyon<em&gt; Listeria monocytogenes</em&gt; Ana İnterferon-γ Yanıtları incelenmesi için Bir Model Olarak

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Araştırma yöntemleri<em&gt; B. subtilis</emKüçük Molekül Biyofilm İnhibitörleri Karakterizasyonu için Bir Model Olarak&gt; Biyofilmler

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

İnsan Tüm Kan Natural Killer Hücre Parçalayıcı Faaliyet Akışı Sitometrik Analizi

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analiz 18FDG PET/BT uzmanı Mycobacterium tüberküloz enfeksiyon ve insan olmayan primatlarda tedavisi için bir araç olarak görüntüleme

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Athymic Farelerde İnsan Plevral Mezotelyoma'nın Ortotopik Modelinin PET/BT ile İmplantasyonu ve İzlenmesi

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...