This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

Tüm hayvan deneyleri Texas Tıp Şubesi Üniversitesi Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. Enfekte-Sigara ve BNV ile enfekte fareler ile DH&#39;lere 1. izolasyonu <ol><li> Yaş ve cinsiyet maç 6-10 haftalık, vahşi tip C57BL / 6 (B6) ve MyD88 - / - fareler. Periton boşluğu içine (ip) ve Batı Nil Virüsü NS4B- P38G mutantının 500 plak oluşturan birim (pfu) ile inoküle. / - - CO 2 ile fareler gün 3 sonrası enfeksiyon, B6 ve MyD88 euthanize. Kontroller olarak her iki grupta da enfekte olmayan fareler kullanın. Her gruptan üçer fare kullanın. </li><li> Islak% 70 etanol kullanılarak kurban farenin sol tarafındaki kürk ve ön ve arka bacaklarının arasına yarım hakkında, farenin sol tarafındaki kürk kesip. Vücut boşluğu açmak kesin. Forseps kullanarak dalak çıkarın. RPMI ile Petri kabındaki dalak yerleştirin. </li><li> Göre anti-CD11c manyetik boncuk kullanılarak dalak DC&#39;leri izole edinüreticinin talimatları. </li></ol> 2. Arıtma ve OTII Transgenik Farelerde T hücrelerinin Etiketleme <ol><li> Bir naif OTII fare Hasat bir dalak Yukarıda adım 1.2 tarif ve iki slaytlar buzlu uçları arasında dalak homojenize olarak. 14 ml RPMI orta kadar ekleyin, bir 15 ml konik tüp hücre süspansiyonu aktarın. Süspansiyon 5 dakika oturup yeni bir 15 ml konik tüp bunun 13 ml aktarmak edelim. </li><li> Üreticinin talimatlarına uygun olarak bir CD4 + T hücresi izolasyon kiti kullanılarak dalak CD4 + T hücreleri izole edin. </li><li> 50 ml&#39;lik bir konik tüp içine Aktarım hücreleri, PBS ile iki kez yıkanır. Süspanse ml başına 1 x 10 6 hücre,% 0.5 büyükbaş hayvan serum albümini (BSA) içeren PBS içinde hücreleri ve CFSE 0.5 umol / ml ekleyin. Işıksız bir ortamda 10 dakika boyunca 37 ° C&#39;de inkübe edilir. </li><li> % 10 ısı ile etkisiz hale getirilen fötal sığır serumu, 5 ml soğuk tam bir ortam (RPMI-1640 ekleyin, 1/100 hacimantibiyotikler / antimikotikler, 1/100 hacim L-glutamin ve 1000 x 2-merkaptoetanol, 1 / 1,000 hacim) konuldu. </li><li> 5 dakika boyunca buz üzerinde inkübe edilir. Yıkama Hücreler bir kez PBS ile,% 2 PFA 0.2 x 10 6 etiketli CD4 + T hücreleri saptamak ve bir akış sitometresi üzerinde kazanır. KAKE bazal düzeyini belirlemek için bu örnek kullanın. Tam ortamda etiketli hücrelerin geri kalanını yeniden askıya. </li></ol> Batı Nil Virüsü ile enfekte Fareler DCler ile 3. Ortak kültür OTII T hücreleri <ol><li> Kültür 2 x 10 OTII farelerin 5 saflaştırılmış CD4 + T hücreleri, tek başına ya da vahşi tip veya MyD88 saflaştırılmış DCler ile - / - OVA tortu 323-339, bir 24 oyuklu bir plaka içerisinde fareleri (2 x 10 4) (1 g oyuk başına 1 mi tam bir ortamda / ml). </li><li> 5 gün boyunca 37 ° C&#39;de inkübe hücreleri. Hücreleri, (% 1 FBS, PBS) FACS tamponunda iki sefer yıkama ve 0.25 mi% 2 PFA sabitleyin. Vortex hemen. </li></ol> In Vitro 4. Akım Sitometri Analizi &lt;/ Em&gt; T Hücre Astar <ol><li> Edinimi için, çift yazılım simgesi flow sitometri tıklayın. SSC-A 200.000 FSC-A 200.000&#39;den ve 0 lineer FSC-A vs doğrusal SSC-A yoğunluk arsa, kanalları seçmek için 0. (Şekil 1A bakınız P2) Ardından, canlı hücreleri üzerinde bir kapı oluşturun. </li><li> KAKE floresan yoğunluğunu analiz etmek, FL1 kanalı için histogramı açmak ve kapı nüfus 20.000 olayları kazanır. Tek başına kültürlenen (Şekil 1A) OTII hücreleri için örnekler üzerinde bir işaret ayarlayın. </li><li> Vahşi tip DCler (Şekil 1B) veya MyD88 birlikte kültürlenmiş OTII hücreleri için numuneler elde edin - / - DCler (Şekil 1C) benzer bir şekilde. </li></ol> 5. izolasyonu ve sitokin tahlilleri için splenositler uyarılması <ol><li> Ste de anlatılan ile aynı prosedür kullanılarak Batı Nil Virüsü NS4B-P38G mutant farelere - / - B6 ve MyD88 Infects 1.1. Günde 30 sonrası enfeksiyon anda, yeniden meydan günlerinde 8 ve ikincil enfeksiyonu takiben 21 ilköğretim sonrası Batı Nil Virüsü enfeksiyonu veya 4. günde de yabani tip BNV suşu ip 2000 PFU&#39;nun ile fareler hayatta, dalak toplanması için CO 2 ile fareler euthanize . NOT: Her bir zaman noktası için, grup başına 2-3 fare kullanıldı. </li><li> Aşama 1.2 ve adım 2.2 tarif edildiği gibi dalak tek bir hücre süspansiyonu yapmak. Hemasitometre kullanarak hücrelerin sayısı 10 ml tam orta bunları yeniden askıya. </li><li> 2.5 x 10 6 hücre / ml komple ortam ile hücrelerin seyreltin. 1.5 ml mikro santrifüj tüpü içinde splenositlerin 1 ml ilave edilir. </li><li> DMSO içinde 1 mg / ml Batı Nil Virüsü özgü NS4B ve E peptidler (SSVWNATTA ve IALTFLAV), CD8 + T hücreleri simüle sulandırmak. CD4 + T hücrelerinin uyarılması için, DMSO içinde 1 mg / ml Batı Nil Virüsü özgü NS3 ve E peptidler (RRWCFDGPRTNTILE ve PVGRLVTVNPFVSVA) seyreltin. Followe hücrelere peptidlerin 10 ul ekleBrefeldin bir solüsyonun 1 ul d. Kontroller olarak peptidler, tedavi olmaksızın hücreleri kullanır. Hücreleri karıştırın. </li><li> Tüpün kapağının bir 18 G iğne ile iki delik Punch. 5 saat boyunca 37 ° C&#39;de inkübe hücreleri. </li></ol> 6. Hücre içi Sitokin Boyama <ol><li> Yeni bir mikro-santrifüj tüp hücreleri aktarın. 300-400 xg&#39;de 5 dakika boyunca hücreleri Spin ve süpernatant kapalı dökün. 300-400 xg&#39;de 1 ml FACS tampon, spin 5 dk ekleyin ve 120 ul FACS tamponu ile pipetleme hücreleri askıya yeniden. </li><li> 2 ul Fc engelleyici ilave edin ve 10 dakika boyunca oda sıcaklığında kuluçkalayın. Her bir tüpe 1 mL FACS tampon ekleyin. Spin 300 5 dk - 400 x g. </li><li> 300 ul FACS hücreleri askıya Yeniden tampon ve üç tüpler (tüp başına yaklaşık 0.8 x 10 6 hücre) bölebilirsiniz. Tablo 1 &#39;de gösterildiği gibi, fare IgG-PE boyaması için her bir kültür durumda ilk tüp tutar. İkinci boru için, CD4, anti-CD4 APC 3 ul peptit ile muamele edilmiş hücreler, 3 μCD8 peptidler ile muamele edilmiş hücreler ya da peptidler, tedavi olmaksızın hücrelere iki eden antikorlara karşı anti-CD8 FITC l. Tazminat boyama üçüncü tüp kullanın. Her kültür koşulu için, APC-konjuge CD4, FITC konjuge CD8 veya FL1, FL2 ve FL4 kanalları için tazminat kontrolleri gibi yalnız PE etiketli CD3 antikorları (tüp başına 3 ul) ile boyanmış hücrelerin üç tüpleri kullanın. </li><li> Kısaca girdap hücreleri. 20 dakika boyunca buz üzerinde boş ve ışıktan korur. 1.4 mi FACS tampon ekleyin. 300-400 xg&#39;de 5 dakika Spin ve süpernatant kapalı dökün. </li><li> Hücre pelet (veya kısaca girdap) bozmaya çalkalayın. 250 ul sabitleme / permeabilizasyon solutionby pipetleme hücre pelet yeniden askıya. 20 dakika boyunca oda sıcaklığında inkübe edilir ve ışıktan korur. </li><li> 1.2 mi FACS tampon ekleyin. 300 ul FACS tampon 300-400 xg ve tekrar süspansiyon hücreleri de Spin 5 dakika. NOT: Bu noktada, hücreler u ışıktan daha fazla işlenmek üzere bir BL2 laboratuara aktarılır ve saklanan bir buzdolabı ve korunabilirÜç gün p. </li><li> 500 ul FACS tampon ekleyin. Sıkma 1x permeabilizasyon / yıkama tamponu içinde 500 ul 300-400 xg ve tekrar süspansiyon hücreleri de 5 dak. Sıkma 300-400 x g&#39;de 5 dak. </li><li> Daha önce ayrılmış bir tüp içinde aşama 6.3 ve 3.5 ul fare IgG-PE CD4 antikor ve / veya CD8 T hücresi belirleyicileri ilave boruya 3.5 ul anti-IFNy-PE eklemek, 100 ul 1x Perm / Wash hücrelerin yeniden askıya ve buz üzerinde 25 dakika süre ile, IgG kontrolü için ışıktan koruyunuz. 1x permeabilizasyon / yıkama tamponu 1.4 ml ekleyerek hücreleri yıkayın. Sıkma 300-400 x g&#39;de 5 dak. 1.4 ml 1x permeabilizasyon / yıkama tamponu ekleyerek yıkama adımı tekrarlayın. </li><li> 300-400 xg&#39;de 5 dakika Spin ve süpernatant süzün. FACS tampon 1.4 ml ilave edilmesi ve nihai edinimi için FACS tampon 400 ul yeniden askıya hücreleri yıkayın. </li></ol> Hücre içi Sitokin Boyama 7. Akım Sitometri Analizi <ol><li> Adım 4.1 aynı satın alma ayarları kullanın. Create canlı hücreler üzerinde bir kapı (P2, Şekil 2A &#39;ya bakınız). Her grup için tazminat tüpler kullanılarak gerilimleri ayarlayın. </li><li> Açık iki yeni nokta araziler, bir vs FL1 için toplanan verileri görüntülemek için. FL2 (CD8 vs. IFN-γ), diğeri vs FL4 için toplanan verileri görüntülemek için. FL2 (CD4 vs. IFN-γ). Her numunenin kapılı nüfus için 50.000 olayları Edinme. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

BNV bir BL3 patojendir. Nedeniyle güvenlik yönetmeliklerine, WNV-enfekte örnekleri ile immünolojik testler genellikle BL3 tesisleri veya gerçekleştirmek için daha uzun ve sıkıcı bir ekipman durumu ile sınırlıdır. Yeni bir çalışmada, biz Batı Nil Virüsü enfeksiyonu 5. sırasında hücre aracılı bağışıklık yanıtlarını incelemek için iki akış sitometri tabanlı yöntemler kullanılır. Analizlerde, Batı Nil Virüsü ile enfekte edilmiş hücreler,% 1-2 PFA doğrudan ya da% 4 PFA...

Batı Nil virüsü (BNV), bir nörotrop, artı-hissedilen flavivirüs, gelişmekte olan bir halk sağlığı tehdidi olduğunu. Şu anda, hiçbir aşı beşeri 1 için onaylanmıştır. Yapısal olmayan (NS) 4B protein bir P38G yer değişimine bir zayıflatılmış Batı Nil Virüsü soyu, vahşi tip Batı Nil Virüsü NY99 suşu 2&#39;den farelerde hiçbir farelerde ölümcül bulunmuştur ancak daha yüksek doğal sitokin ve T hücresi tepkilerini teşvik ettiği bilinmektedir. NS4B-P38G mutant ile bağışıklık kazandırılmış fareler öldürücü doğal tipte Virüsü ile ikinci bir sorun korundu. Bu NS4B-P38G mutant ideal aşı adayı için uygun özelliklere sahip olduğunu göstermektedir. NS4B-P38G mutant yüksek koruyucu adaptif bağışıklığı hangi mekanizmalar henüz net olarak anlaşılamamıştır. Patojen ilişkili moleküler desenleri tanımaya Toll benzeri reseptörler (TLR), viral enfeksiyon karşı doğal bağışıklığın başlamasında önemli bir rol oynamaktadır. Çekirdek TLR sinyal yolu miyeloid farklılaşma birincil yanıt ge kullanırBirincil adaptörü 3,4 olarak ne 88 (MyD88). Yeni bir çalışmada, MyD88 sinyal farelerde 5 Batı Nil Virüsü NS4B- P38G mutant enfeksiyonu esnasında hücre aracılı bağışıklığın gelişiminde önemli bir rol oynadığı gösterilmiştir. Dentritik hücreler (DC&#39;ler), viral enfeksiyonun 6,7 sırasında birincil T hücresi tepkilerini başlatmak için benzersiz kapasitesini gösteren en önemli antijen sunan hücreler bulunmaktadır. CD4 + ve CD8 + T hücreleri, uzun süreli koruyucu bağışıklık katkı ve vahşi tip Batı Nil Virüsü enfeksiyonu 8,9 Aşağıdaki ana yaşaması için önemliyse her ikisi. İki immünolojik tahliller NS4B-P38G mutant amacıyla enfeksiyon bulaştırılan farelerde bu hücrelerin fonksiyonlarını belirlemek için bu çalışmada kullanılmıştır. - / - Farelerde İlk olarak, in vitro T hücre prime deney Batı Nil Virüsü ile enfekte edilmiş yabani tip ve MyD88 arasında DC&#39;lerin antijen sunan yeteneği karşılaştırmak için kullanılmıştır. Tahlil, naif CD4 &lt;duyarlılığını artırmak içinCFSE (WNV enfekte edilmiş farelerin 339 DCler saflaştırıldı ve karboksiflüoresein süksinimidil paskalya birlikte kültürlenmiş - destek&gt; + T hücreleri, 323 (OVA) peptidi tavuk ovalbümin için Vα2 / Vβ5 TCR ifade OTII transgenik farelerden izole edilmiştir OVA peptidi varlığında) -etiketli CD4 + T hücreleri. Ko-kültür içinde 5 gün sonra, hücreler hasat edilmiş ve paraformaldehit (PFA) ile tespit edilmiş ve akış sitometrisi ile analiz edilmiştir. Çoğalma tahlilleri geleneksel bir 5-bromo-2&#39;-deoksiüridin (BrdU) veya toz haline getirilmiş timin deoxyriboside (3 HTdr) 10 arasında dahil edilmesi yoluyla gerçekleştirilmiştir. Bununla birlikte, bu deneyler radyoaktif ve / veya BNV çalışmalarının yapıldığı biyogüvenlik düzeyi (BL) 3 tesislerde, özel ekipman ihtiyacı ya vardır. CFSE, daha yaygın hale imünolojik deneylerde kullandı vital boyanın flüoresan yoğunluğu seri yarılanmasına lenfosit çoğalmasının akış sitometrik analizi, boya daha stabil olduğu gibive eşit, hücrelere dahil flow sitometri ile kolayca tespit ve 11 radyoaktif olmayan bir. Tahlil, aynı zamanda, hücre bölünmesini belirlemek için yeteneği vardır. Batı Nil Virüsü çalışmalarda, bu tahlili kullanarak bir büyük avantajı,% 1-2 PFA ile enfekte olmuş hücrelerde tespiti, bir BL2 laboratuarda bir akış sitometresinde numune toplama sağlayacak WNV 12, etkisiz olmasıdır. Daha sonra, bir tadil edilmiş hücre içi sitokin boyama (ICS) prosedürü MyD88 NS4B-P38G mutantı ile enfekte olmuş farelerde Batı Nil Virüsü spesifik T hücre karşılıklarının düzenlenmesinde sinyalleme rolünü incelemek için kullanılmıştır. Bu tahlilde, enfekte farelerinden yalıtılmış splenositlerin Batı Nil Virüsü spesifik peptidler ile in vitro muamele edildi. Brefeldin bir hücre içinde sitokin tutmak için ilave edildi. 5 saat inkübasyondan sonra, hücreler toplandı, yıkandı ve T hücre alt-gruplar için boyandı. Hücreler daha sonra, PFA sabit geçirgenleştirildi ve interferon (IFN) -γ boyanmış ve akış sitometrisi ile analiz edilmiştir.Hücreler tespit ve PFA içeren permeabilizasyon tampon maddesi ile muamele edilmiş bir kez başka akış sitometrisi bazlı bir deneyde olduğu gibi, enfekte örnekleri daha fazla işleme ve analizi için bir BL2 laboratuara aktarılır. Birkaç yayınlanan çalışmalarda, biz WNV-enfekte farelerde 13,14 T hücresi efektör fonksiyonları ölçmek için ICS kullandık. Iyi kurulmuş oluyor rağmen, bu testin bir büyük dezavantajı prosedürü çok uzun ve BL3 tesisleri içinde yapıldığında alıcı daha fazla zaman olabilir olduğunu. Burada, mikro-santrifüj tüpü tabanlı ICS yöntem daha uygun, daha kolay devam etmek ve daha az zaman BL3 laboratuar içinde gerçekleştirilen zaman alıcı olduğu gösterilmiştir.

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			<td height="20" style="height:20px;width:216px;">RPMI 1640</td>
			<td style="width:153px;">Invitrogen</td>
			<td align="right" style="width:119px;">11875</td>
			<td>warm up at 37C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD11c magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-001</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD4 magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-248</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFSE</td>
			<td style="width:153px;">Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OVA residue 323-339</td>
			<td>Genscript Corporation</td>
			<td>RP10610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Peptides</td>
			<td style="width:153px;">Proimmune</td>
			<td>PC0AD-D</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Brefeldin A solution</td>
			<td style="width:153px;">BD Bioscience</td>
			<td align="right">555029</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Mouse Fc Blocker</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>14-0161-85</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">APC-conjugated CD4&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>17-0041-81</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">FITC-conjugated CD8&#160;</td>
			<td style="width:153px;">e- bioscience</td>
			<td>11-0081-82</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Fixation/Permeabilization Solution</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right" style="width:119px;">554722</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Permeabilization/wash buffer</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right">554723</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">anti-IFNg-PE&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>12-7311-82</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Accuri flow cytometer</td>
			<td style="width:153px;">BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

Farelerde, vahşi tip Batı Nil Virüsü daha yüksek doğal sitokin ve T hücresi tepkilerini bağlı bir zayıflatılmış Batı Nil virüsü (WNV), bir yapısal olmayan (NS) 4B-P38G mutantı. Son zamanlarda, miyeloid farklılaşma faktörü 88 (MyD88) sinyal Batı Nil Virüsü NS4B-P38G mutant enfeksiyon sırasında, ilk T hücresi uyarılması ve bellek T hücresi gelişimi için önemli olduğu gösterilmiştir. Bu çalışmada iki sitometri tabanlı yöntemler akış - Bir in vitro T hücre prime deneyi ve bir hücre içi sitokin boyama (ICS), - dendritik hücreler (DC) ve T hücre fonksiyonlarını değerlendirmek için kullanılmıştır. OTII transgenik farelerin, CD4 + T hücreleri etiketli - deneyi emişli T hücresi, hücre çoğalması karboksiflüoresein süksinimidil ester (CFSE) ile farelerin her iki grup DC&#39;lerin ko-kültür, aşağıdaki akış sitometrisi ile analiz edilmiştir. Önemli ölçüde gelenek daha genel duyarlılık geliştirilmiş olan bu yaklaşım, CD4 + T hücreleri çoğalan yüzdesi doğru belirlenmesi sağlanırark radyoaktif reaktifler ile deneyleri. Bir mikrosantrifüj tüpü sistemi ICS protokolünün iki hücre kültürü ve sitokin boyama prosedürleri kullanılmıştır. Geleneksel doku kültürü plaka tabanlı sisteme kıyasla, bu değiştirilmiş prosedür biyogüvenlik düzeyi (BL) 3 tesislerinde gerçekleştirmek için daha kolay oldu. Ayrıca, WNV- enfekte olmuş hücreler BL3 dışındaki daha sonraki analiz etkin analizlerde içinde paraformaldehit ile işleme tabi tutuldu. Genel olarak, bu in vitro bağışıklık tahlilleri, etkili bir Batı Nil Virüsü enfeksiyonu esnasında hücre-aracılı bağışıklık karşılıklarının değerlendirmek için de kullanılabilir.

- / - OVA peptidlerinin varlığında veya yokluğunda, farelerde T hücresi priming tahlilde, CFSE etiketli, CD4 + T hücreleri, NS4B-P38G mutantı ile enfekte edilmiş yabani tip ve MyD88 arıtılmış DCler ile kültürlenmiştir. 5 gün negatif kontroller olarak kullanılmıştır için etiketli T hücreleri ya da OVA olmaksızın tek başına kültürlenmiştir. Şekil 1A&#39;da gösterildiği gibi, toplam T hücre FL1 kanal flüoresans yoğunluğu analizi için kapı edildi. ...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Batı Nil Virüsü Mutant Soy Enfeksiyon MyD88-aracılı Hücresel Bağışıklık Tepki Vitro Analizi

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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Research

JoVE Journal

Immunology and Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

8.1K Görüntüleme.  The University of Texas Medical Branch. Two flow cytometry-based methods – an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Video: In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

Rhesus Makak maymunlarının Hücresel Bağışıklık Tepkisi Multicolor Sitometrisi Analizleri

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

İmmünoloji ve Enfeksiyon

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hücreler stellat Yedi Adımda

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Paraziter Eşek Arıları Bir Giriş<em&gt; Drosophila</em&gt; Ve antiparaziter İmmün Yanıt

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Hazırlama ve: Enfeksiyon Önleme Yeni Bir Yaklaşım olarak PRP<em&gt; In vitro</emPRP&gt; Antimikrobiyal Özellikleri

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Fare Makrofagların yılında Doğuştan Bağışıklık Tepkisi Soruşturma RNA girişimi kullanma

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Bir<em&gt; İn Vitro</em&gt; HIV Co-enfeksiyon Bağlamında Sıtma için Bağışıklık Yanıtları Ölçüm Modeli

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Basit ve Etkin Yaklaşım Mutant Vaccinia Virüs Vektörler Construct

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Bir fare modeli için değerlendirmek doğuştan gelen bağışıklık yanıtı Staphylococcus aureus enfeksiyon

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...