Bu proje, E. Jolly sağlanan Case Western Reserve Üniversitesi&#39;nde başlangıç ​​fonları tarafından finanse edildi. Enfekte salyangoz şiştozom Kaynak Merkezi, NIH-NIAID (NIAID Sözleşme No HHSN272201000009I) tarafından verilmiştir. Ayrıca Chris King ve Ronald Blanton enfekte şiştozom salyangoz güvence destek ve yararlı tartışmalar için teşekkür ederiz.

Güvenlik önlemleri: Şiştozom cercariae bir kesik veya gözeneklerin ihtiyaçları olmadan doğrudan cilde nüfuz eder ve kronik hastalık schistosomiasis neden olabilir. S. mansoni bir biyogüvenlik düzeyi 2 organizma, bu nedenle uygun kişisel koruyucu ekipman ve enfektif cercaria ile çalışırken uygun güvenlik önlemleri takip edilmelidir. Schistosomes büyümeye dönüştürmek için gerekli ekipmanların çoğu protokol sonunda tabloda belirtildiği gibi birkaç önemli istisnalar dışında, standart. Tüm cercarial çalışma, izole bir odada yapılmalıdır. Schistosomules steril, onaylı biyogüvenlik kaput kültüre olmalıdır. İki kat nitril ya da lateks eldiven giyilmelidir bir laboratuvar önlüğü, su geçirmez bir çift ayakkabı, bilek ve silah için herhangi bir delik veya yırtık ve su geçirmez kol koruyucuları pantolon gerekir. Kazara dökülme durumunda, derhal şiddetle kişi hakkında ise sürtünme,% 70 etanol veya% 10 çamaşır suyu ile kirlenmiş alanda sprey. Bu parazit devre dışı bırakmak için yeterli olacaktır, ancak tüm olası enfeksiyonlara ve ciddiye alınmalıdır. Schistosomules doğuştan enfektif olmasa da, kurumsal kurallar değişir ve BSL2 önlemler noninfeksiyöz yaşam aşamalarında gerekli olup olmadığını belirlemek için danışılmalıdır. Biohazard onaylı bir kapta yapılır ve deney atık atmak sonra herhangi bir potansiyel bulaşıcı madde ile temas eden tüm yüzeyler dezenfekte edin. NOT: Kapak salyangoz ana bidonları, 1 ila 2 gün önce dökülme ve ışıktan korumak için 1. Salyangoz konak cercariae Hasat <ol><li> Yarım distile su ile oda sıcaklığına gelmesi dolu deterjan, 500ml temiz bir behere doldurun. Farklı tarihlerde miracidia ile enfekte konak salyangoz ile çalışıyorsanız, her enfeksiyon tarih için ayrı bir beher kullanmak (salyangoz gelecek deneyler için tekrar olacak, bu özellikle önemlidir). Beher boyutu da kullanıcı deneyimine bağlı olarak daha küçük olabilir. </li><li> Gerektiği gibi net bir standart akvaryum balığı ve genel amaçlı forseps kullanarak beher döken ve dikkatli bir şekilde yatırmak isteyen enfekte salyangoz ev sahipliği toplayın. </li><li> Acil bir dökülme Yeri beher akkor bir ışık kaynağı altında yaklaşık 8 ila 12 inç tepsisi. Işığı altında 1-2 saat bekletin. Parlak ışığa maruz kaldığında Cercariae salyangoz konak çıkın. </li><li> Cercariae karışımı dikkatlice salyangoz dışarı filtre. Karışımının son derece bulaşıcı olduğundan, ekstra güvenlik önlemleri alın. Unutmayın, cercariae doğrudan insan derisi nüfuz edebilir! </li><li> Herhangi bir partikül atık madde yerleşmelerine izin verme, yaklaşık 15 dakika ışık kaynağı altında karışımı değiştirin. </li><li> Cercariae beher altındaki enkaz kaçınarak, bir pipetlemeyin kullanarak temiz bir Erlenmeyer şişesinin içine aktarın. Şişesi 60 dakika, cercariae bir köşeye yerleşmek için izin - 45 için karanlık buz üzerinde yaklaşık 45 derecelik bir açıyla yerleştirin. </li><li> Süpernatantı 75 -% 90% 10 çamaşır suyu ya da% 70 etanol içeren beher kaldırmak ve mevduat, su damlaları pipetlemeyin cercariae damla içeren girmemesine dikkat çekici bir 50 ml pipetlemeyin kullanın. </li><li> Swirl Erlenmeyer şişesinde çözüm cercariae tekrar süspansiyon yavaşça cercariae içeren. Steril 50 ml konik tüpler için örnek aktarmak için bir pipetlemeyin kullanın. Karanlıkta 20 dakika boyunca buz üzerinde tekrar tüpleri yerleştirin. Cercariae karanlıkta tüp alt kısmına doğru geçer. </li></ol> 2. Schistosomules için cercariae Manuel dönüşüm Başlamadan önce: RPMI 1640 (RPMI,% 5 fetal sığır serumu, 1X Pen / Strep) tamamlamak ve 37 ° C&#39;ye kadar sıcak olun. (Kullanılan fetal sığır serumu inkübasyon inaktive edildiğini çok önemlidir 56 ° C 1 saat schistosomes tamamlamak için duyarlı olmak gibi). <ol><li> Biohazard kaput, her biri 50 ml konik bir pipet veya vakum cihazı kullanılarak çözüm 5 ila 10 ml kadar süpernatant çıkarın. </li><li> Döner lok uygun boyutta bir şırınga iğnesi bulamaç, çift uçlu, 22 gauge takın. </li><li> Yavaş yavaş şırınga içine karışımı çizin. Bir şırınga içinde 50 ml tüpler cercariae toplayın. Damlayan dikkatli olun. </li><li> Unattached iğne ucu kadar olduğu gibi üzerinde dikkatle şırınga açın. Bulamaç iğne bu tarafında başka bir şırınga takın. </li><li> Cercariae dönüştürmek için iğne ile toplam 40 kez (20 kez ileri ve geri) Pass karışımı. </li><li> Karışım, alt şırınga bulunan ve üst şırınga boş olduğunu dikey gibi şırınga yerleştirin. Üst şırınga çıkarın ve% 70 Etanol dezenfekte. </li><li> Mevduat karışımı temiz bir yüksek duvarlı 100mm x 50mm kabına ya da kristalize çanak içine damla damla. % 70 Etanol iğne ve şırınga dezenfekte edin. </li><li> 37 ° C RPMI 1640 eksiksiz bir medya gibi ekleyinPetri kabı alt Luria suyu plakası dökme gibi tamamen kaplıdır. </li><li> Petri kabı merkezinde schistosomules toplamak için hafifçe Swirl. Sıçramasını önlemek. Not: Bu adım için daha yeterli merkezinde schistosomules görselleştirmek için kaput ışığı kapatmak için yararlı olabilir. </li><li> Hemen steril bir damlalık kullanarak yeni bir 12-kuyu hücre kültür plakasına konsantre schistosomules yatırın. </li><li> 2.9 ve 2.10 schistosomules artık çanak (yaklaşık 10 kat) merkezi bugünkü kadar tekrarlayın. Sonraki her taze bir kuyu koleksiyonu Depozito. </li><li> Her iyi yarısına kadar (~ 1.5 mL) prewarmed RPMI tam medya ile doldurun. </li><li> Schistosomules varlığı ve cercariae yokluğu doğrulamak için bir inverted mikroskop gözünüzde canlandırın. </li><li> Büyümeye CO 2 inkübatör schistosomules - 37 ° C ve% 5 CO 2. </li></ol> 3. Temsilcisi Sonuçlar: Kültür ortamı haline dönüştürülmüş schistosomules Bekleyen transferi, ters bir parlak alan mikroskobu altında (örneğin ekipman tabloya bakınız) görselleştirme Şekil 1, sadece dönüştürülmüş schistosomules iyi olduğu için benzer bir görüntü elde edilmelidir. Potansiyel kirleticiler, sağlam cercariae, kopmuş cercarial kuyrukları, veya diğer çöküntüleri içerebilir. Hiçbir inverted mikroskop varsa, standart bir mikroskop ya da hatta bir diseksiyon kapsamı schistosomules görselleştirmek için yeterli olacaktır. Bu işlem her zaman% 100 cercariae dönüştürmek unutmayın. <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="Şekil 1"/> Şekil 1: Dönüştürülmüş Schistosomulum <img src="/files/ftp_upload/3191/3191fig2.jpg" alt="Şekil 2"/> Şekil 2: Cercariae ve Severed Tails

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> N Engl J Med. 346, 1212-1220 (2002).</li><li>Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis+and+water+resources+development:+systematic+review,+meta-analysis,+and+estimates+of+people+at+risk.">Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk.</a> Lancet Infect Dis. 6, 411-425 (2006).</li><li>Savioli, L., Albonico, M., Engels, D., Montresor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis.">Progress in the prevention and control of schistosomiasis and soil-transmitted helminthiasis.</a> Parasitol Int. 53, 103-113 (2004).</li><li>King, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+cercarial+responses+to+irradiance+changes.">Schistosoma mansoni: cercarial responses to irradiance changes.</a> J Parasitol. 68, 120-124 (1982).</li><li>Chai, M., McManus, D. P., McInnes, R., Moertel, L., Tran, M., Loukas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+lung+schistosomula,+in+vitro+cultured+schistosomula+and+adult+Schistosoma+japonicum.">Transcriptome profiling of lung schistosomula, in vitro cultured schistosomula and adult Schistosoma japonicum.</a> Cell Mol Life Sci. 63, 919-929 (2006).</li><li>Gobert, G. N., Tran, M. H., Moertel, L., Mulvenna, J., Jones, M. K., McManus, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+changes+in+Schistosoma+mansoni+during+early+schistosomula+development+and+in+the+presence+of+erythrocytes.">Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes.</a> PLoS Negl Trop Dis. 4, (2011).</li><li>Basch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+Schistosoma+mansoni+in+vitro.+I.+Establishment+of+cultures+from+cercariae+and+development+until+pairing.">Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing.</a> J Parasitol. 67, 179-185 (1981).</li><li>Yoshino, T. P. &. a. m. p. ;. a. m. p., Laursen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Schistosoma+mansoni+daughter+sporocysts+from+mother+sporocysts+maintained+in+synxenic+culture+with+Biomphalaria+glabrata+embryonic+(Bge)+cells.">Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells.</a> J Parasitol. 81, 714-722 (1995).</li><li>Colley, D. G., Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+simplified+method+for+the+production+of+schistosomules.">Schistosoma mansoni: simplified method for the production of schistosomules.</a> Exp Parasitol. 35, 44-51 (1974).</li><li>Lazdins, J. K., Stein, M. J., David, J. R., Sher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+rapid+isolation+and+purification+of+schistosomula+of+different+developmental+stages+by+centrifugation+on+discontinuous+density+gradients+of+Percoll.">Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll.</a> Exp Parasitol. 53, 39-44 (1982).</li><li>Cousin, C. E., Stirewalt, M. A., Dorsey, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+ultrastructure+of+early+transformation+of+skin-+and+shear-pressure-derived+schistosomules.">Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules.</a> Exp Parasitol. 51, 341-365 (1981).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. Chapter 19, 1-1 (2001).</li></ol>

Çıkar çatışması ilan etti.

Bazı potansiyel sorunları dönüşüm işlemi sırasında ortaya çıkabilecek; Ancak, bu protokol, bu sorunların en önlemek için tasarlanmıştır. Potansiyel bir tehlike genellikle adım 1.3 &#39;de hasat arta kalan enkaz nedeniyle ortaya çıkar bulamaç iğne, tıkanma. Bu sorunu önlemek için tüm partikül madde, 1.5 adım yerleşmek ve adım 1.6 cercariae dikkatli bir şekilde aktarmak için izin emin olun. Ayrıca, biyolojik güvenlik kabini içinde çalışmak, daha önce açıklandığı gibi, kişisel koruyucu ekipman kullanımı (hatta yüz kalkanlar) ve şırınga aşırı basınç kaçınma tavsiye edilir. Müstakil cercarial kuyrukları 2.9 adım sırasında kötü ayrılması göstergesidir medya, görüntülenebilir. Kuyrukları spazm gibi hareket sergiler ve farklı bir şekil var (bkz. Şekil 2) ve thusly schistosomules kolayca ayırt edilebilir. Schistosomules yerleşmelerine izin tamamlamak RPMI aşan schistosomules yıkama kuyrukları tarafından kaldırılabilir. Kuyrukları kayan kalacak ve kapalı vakumlu olabilir. Sağlam cercariae medya varsa, adım 2.5 dönüşüm düzgün idam değildi. Şekil 3b başvuru için sağlam bir cercaria bir görüntü tasvir edilmiştir. Bu sorun oluşursa, kullanılan bulamaç iğne uygun büyüklükte olduğunu sigorta. Ayrıca, cercarial karışımı iğne geçirilir kez, 40 kez genellikle cercarial kuyrukları% 100 kesme yaklaşım çok daha fazla olmasına rağmen, artmış olabilir. Schistosomules içine cercaria Dönüşümü ilk Colley ve Wikel (13) tarafından 1974 yılında tarif edilmiştir. O zamandan beri, schistosomules hazırlanması, bir iğne ve şırınga ile ya kesme strateji kullanarak Bu çalışmada gösterdiği gibi, bu protokolde tanımlanan veya vorteks bir yaklaşım kullanarak ve Percoll, gradyanlar ile ayrılmış (14, 15) ya da dönen olarak yapılmıştır. Schistosomules hazırlanması için bu iki teknik güzel Fred Lewis (16) ve uzun vadeli büyüme için daha fazla schistosomules kültür için daha fazla bilgi NIAID şiştozom Kaynak Merkezi (bulunabilir tarafından <a href="http://www.schisto-resource.org/">açıklanan www.schisto-</a> resource.org) .

<table border="1"><tbody><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> 22g x 2-7/8 &quot;Bulamaç iğne </td><td> Fisher Scientific </td><td> 14-825-17G </td></tr><tr><td> RPMI 1640 Orta 1X (500ml) </td><td> Invitrogen </td><td> 11835030 </td></tr><tr><td> Fetal sığır serumu (FBS) ısı inaktive </td><td> Invitrogen </td><td> 10082139 </td></tr><tr><td> Penisilin / Streptomisin (100X) </td><td> Invitrogen </td><td> 15070063 </td></tr><tr><td> Kristalize Bulaşık (100mm) </td><td> Fisher Scientific </td><td> 08-741D </td></tr><tr><td> Genel Amaçlı Cımbızlar </td><td> Fisher Scientific </td><td> 10-316C </td></tr><tr><td> Salamura karides için Petco Akvaryum net </td><td> Petco </td><td> SKU: 1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

Şiştozom parazitler etken schistosomiasis kronik zayıflatıcı hastalığı, dünyada 200 milyon insanı etkileyen ve halk sağlığı ve sosyo-ekonomik etkileri (1-4) açısından paraziter hastalıklar arasında sıtma ile ikinci sırada yer alıyor. Şiştozom parazitler karmaşık bir yaşam döngüsü serbest yüzen aşamalarında müdahale molluscan ve memeli ev sahipliği yapan asalak bir yaşam arasındaki ¤ lantılarını trematod solucanlar. Kısaca, serbest yüzen cercariae schistosomules dönüşürken, cercariae kuyrukları kaybetmek ve bu süre boyunca salgılanan proteazlar yardımı ile cilde nüfuz konak memeli bozar. Schistosomules şimdi, konak bağışıklık sistemini kaçınmak, kırmızı kan hücrelerinin bir bağırsak sindirim için geliştirmek ve göç gerekir rağmen akciğer ve karaciğer portal sisteminin nihai hedefe giderken portal dolaşıma ve sonunda mezenterik damarlar (S. mansoni ) erkek ve dişi solucanlar çifti ve arkadaşı, her gün yüzlerce yumurta üretmek. Bazı yumurta, taze su, serbest yüzen miracidia (5-10) içine yumurtadan çıkar yumurta içine vücuttan atılır. Miracidia özel salyangoz türleri enfekte ve anne ve kızı da, yaşam döngüsünü tamamlayarak, enfektif cercariae üretmek sporocysts dönüşmek. Ne yazık ki, tüm şiştozom yaşam döngüsü in vitro kültüre, ancak infektif cercariae schistosomules dönüşebilir ve schistosomules vitro veya mikroarray analizi şiştozom gelişme analizi için hafta boyunca kültüre olabilir. Bu protokol, biz cercarial dönüşüm ve schistosomules in vitro kültür görsel bir açıklama sağlar. Biz salyangoz ana Biomphalaria glabrata bulaşıcı cercariae döken ve elle, bir bulamaç çift uçlu bir iğne kullanarak kendi kuyrukları ayırarak schistosomules haline dönüştürmek. , In vitro cercarial dönüşümü ve schistosomules kültür teknikleri, schistosomes, görselleştirme kolaylaştırır ve deneysel analizi için parazit toplanmasını kolaylaştırır. In vitro dönüşüm ve kültür teknikleri schistosomes yıl için yapılmış bir konak memeli, (11 kullanımını önlemek açıklanan 12), ancak hiçbir görsel protokoller tüm toplum için kullanılabilir olduğunu geliştirilmiştir var.

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Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Biz bir tarif<em&gt; In vitro</em&gt; Flatworm parazit kültür schistosomula yöntemi<em&gt; Schistosoma mansoni</em&gt; Tatlı su salyangozu ara konak Biomphalaria glabrata enfektif cercariae hasat ve dönüşüm yoluyla.

Cercarial Dönüşüm ve In vitro Yetiştiriciliği Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

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Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

18.0K Görüntüleme.  Case Western Reserve University. Biz bir tarif In vitro Flatworm parazit kültür schistosomula yöntemi Schistosoma mansoni Tatlı su salyangozu ara konak Biomphalaria glabrata enfektif cercariae hasat ve dönüşüm yoluyla.

Video: Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

Bakteriyel Yapışma Memeli Epitel Hücreleri üzerine In Vitro Testi

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

İmmünoloji ve Enfeksiyon

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

HIV-1 Çekirdek in vitro Uncoating

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Gelen Yumurta kullanma<em&gt; Schistosoma mansoni</em&gt; Bir şekilde<em&gt; In vivo</emHelmint bağlı Akciğer iltihabı ve&gt; Model

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Nicel<em&gt; In vitro</emStatik Koşullarında Account İlköğretim İnsan mikrovasküler endotel hücreleri için Nötrofil yapışma Tedbir&gt; Testi

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

Yetiştirme<em&gt; Heligmosomoides Polygyrus:</em&gt; Bir immunsupresif Nematodu Parazit ve Salgılanmış Ürünleri

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Fırsatçı mantar patojenlere bir Floresan etiketli geninin biyolistik transformasyonu<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Farelerde oral sonda ardından Probiyotik Maya Dönüşümü ve Gastrointestinal Bağışıklık Dokular Onların Kurtarma

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

Ölçümü<em&gt; İn Vitro</em&gt; HIV-1 Preintegration Kompleksleri Entegrasyon Etkinliği

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Terapötik Antikorlar Arasındaki Hedef Bağlanma ve CDC İndüksiyonunu Karşılaştırmak İçin İn Vitro Yöntemler: Biyoeşdeğerlik Analizindeki Uygulamalar

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Yalıtım ve yetişkin Schistosoma japonicum üzerinden ekstraselüler veziküller karakterizasyonu

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...