Name: mass isolation and in vitro cultivation of intramolluscan stages of the human blood fluke schistosoma mansoni
Uploaded: 2018-01-14T13:00:00.000+00:00
Duration: 12 min 5 s
Description: Watch this Scientific Journal Video about Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni at JoVE.com

Kısmen NIH grant RO1AI015503 tarafından finanse edilmektedir. Paraziti enfekte fareler, BEI kaynaklar üzerinden dağıtım için NIH NIAID sözleşme HHSN272201000005I NIAID sistozomyas Kaynak Merkezi Biyomedikal Araştırma Enstitüsü (Rockville, MD) tarafından verilmiştir.

Tüm hayvan bakımı ve deneysel prosedürler kurumsal hayvan bakım ve kullanım Komitesi, Wisconsin-Madison Üniversitesi iletişim kuralı altında tarafından onaylanmış yok. V005717. Bu protokol için tasvir paraziti aşamaları hiçbiri insanlar ya da diğer memeliler infektif olmasına rağmen aşağıdaki protokol bir seviye 2 (BSL2) tesis insan patojenler için çalışan içerir. Risk grubu 2 (RG2) insan patojenleri işlemek için kurumsal ilkeleri uygulayın.
Not: Biz böylece daha büyük yumurta yükleri verimli enfeksiyon10, onların daha yüksek duyarlılık nedeniyle kadın Swiss-Webster fareler (6 - eski hafta) kullanın. Tucker ve ark. bu modeli sistem11' kullanılan fare ve salyangoz enfeksiyon iletişim kurallarını açıklar. Malzemeler tablo tüm özel ekipman, malzeme, reaktifler ve hayvansal kaynaklar bu deneysel protokol taşımak için gerekli listeler.
1. enfekte karaciğerleri işlenmesi
<ol><li>CO2 boğulma (dakika başına odası 's hacmi 10-%30 oranı) tarafından fareler, 6-7 hafta sonrası enfeksiyon, ötenazi. Fareler solunum ve kalp atışı kesilmesi için izleyin. Not: Genellikle, belirli bir zamanda 20 fareler kadar işlenmiş.</li>
	<li>Ötenazi fareler bir biyogüvenlik kabini aktarma sonra fare sırtlarını yerleştirin ve onların ventral yüzeyler % 70 etanol ile sprey. 1 dk. için emmek izin.</li>
	<li>Çimdik ve alt karın deri çekin ve boğumlu cilt Bankası arasında steril Cerrahi makas ile karın en yakın kesti. Çekin kesilen deri anteriorly (Yani, kafa doğru) karaciğer ortaya çıkarmak için. Karaciğeri genişlemiş ve bir yumurta kaynaklı Granülomatöz nedeniyle yanıt (şekil 2A) benekli olduğunu unutmayın.</li>
	<li>Karaciğer çıkarın ve 200 mL steril % 1.2 NaCl çözüm içeren kalem/strep içeren bir ölçek yerleştirin.</li>
	<li>1.3 ve 1.4 kalan fareler ile adımları yineleyin. Not: Yirmi fareler genellikle tek bir hasat işlenir.</li>
	<li>Karaciğer içinde steril Petri kabına yerleştirin ve sigara-karaciğer yağ/bağ dokusu ile steril forseps kaldırın.</li>
	<li>Kesilmiş/temizlenmiş karaciğerleri ~ 200 mL steril % 1.2 NaCl çözeltisi ile kalem/strep içeren bir steril 250 mL santrifüj şişe aktarın.</li>
	<li>Şiddetle karaciğerleri içeren şişe sallamak ve steril bir tek kullanımlık pipet kullanarak, köpük/yüzen enkaz fazla yüzey kaldırın. Yavaş yavaş bir konteyner içine kalan tuz solüsyonu dökün veya içeren % 1 çamaşır suyu (dezenfektan) batmak.</li>
	<li>Karaciğer için ~ 200 mL taze tuz çözeltisi ekleyin ve 1.8 üç kez daha tekrarlayın.</li>
</ol>2. karaciğer çıkarma ve miracidial yalıtım
<ol><li>Karaciğerleri küçük steril paslanmaz çelik blender fincan içine yerleştirin ve ~ 2 mL % 1.2 NaCl çözeltisi (şekil 2B) ekleyin.</li>
	<li>Kapak folyo ve sızıntıları önlemek ve 1 dk. için düşük ve yüksek hız (20 s düşük hız/10 s yüksek hızlı; tekrar) alternatif tarafından karışımı bir petri kupa (şekil 2C).</li>
	<li>(4 şişe 20 karaciğerleri kullanmak için) steril 250 mL santrifüj şişe karıştırılan karaciğer süspansiyon dağıtabilmenizi.</li>
	<li>Antibiyotik steril serum fizyolojik ~ 200 mL toplam birim/şişe için ekleyin. Tartmak/şişe Santrifüjü önce denge.</li>
	<li>Santrifüj 290 x g 4 oC. yavaşça, 15 dakika, karışım karaciğerleri supernatants atarak önce çamaşır suyu ile bir kaba dökün.</li>
	<li>2.4-2,5 bir kez yineleyin. Santrifüjü önce karaciğer tortu iyice resuspend emin olun.</li>
	<li>Son serum discarding sonra yıkayın (Adım 2.6), 200 mL steril Göl suyu ile kalem/strep pelleted karaciğer dokusu, tortu resuspend ve tamamen alüminyum ile kapsadığı steril 1-L volumetric flask süspansiyon aktarmak için sallamak için ekleyin folyo, üst boyun (şekil 2B) 3 cm dışında. Not: 1-L şişe iki şişe (10 karaciğerleri =) kullanın. Göl suyu miracidial damızlık yumurtadan uyarmak için gerekli doğal tatlı su ortamına benzeyen.</li>
	<li>Steril Göl suyu kullanarak, yaklaşık 3 cm boyunda kapsayan folyo yukarıda şişeye doldurun. Sonra gecikmeden (miracidia yakında başladığınızda yumurtadan), kalan köpük ve karaciğer dokusu yüzeye hızla ~ 12 mL kadar su birikintisi Su enkaz içeren pipetting tarafından yüzen kaldırmak ve ayrı bir atarak at tüp. Taze steril Göl suyu ile değiştirin.</li>
	<li>Tekrar temizlik adım 2.8, üç kez, miracidia at tüp varlığı için kontrol edin. Tekrarlama miracidia yıkama çözümde görünüyorsa, adım Temizleme keserler.</li>
	<li>Son yıkama sonra ~ 12 mL sıcak steril gölet su (30 oC önceden ısındı) üstüne ılık suyla temiz, steril bir sıcaklık gradyanı oluşturmak için yavaş yavaş ekleyin. Not: Bu en iyi yavaş yavaş karıştırma önlemek için şişe Boyun kenarındaki su boşaltma tarafından gerçekleştirilir.</li>
	<li>Balonun açılış üzerinde bir küçük Petri kabına kapağını yerleştirin ve şişeye su üst yüzeyi aydınlatmak için folyo kapak yukarıda üstünde parlak bir ışık. Not: Genellikle 5-10 dk içinde miracidia, ışığa, çekici yüzme çok sayıda Göl suyu (şekil 3A) içinde ilk 2-3 cm odaklanacaktır.</li>
</ol>3. Miracidial hasat ve işleme
<ol><li>10 dakika sonra yüzeyde miracidia topladığı zaman, steril transfer damlalıklı kullanarak ~ 8 mL Göl suyu ve steril 15 mL santrifüj tüpü transfer kaldırın. Miracidia buz üzerinde içeren tüp yerleştirin.</li>
	<li>Sıcak steril Göl suyu volumetric flask iç boyunca arka 8 mL ekleyin ve başka bir 10 dakika bekleyin.</li>
	<li>Her seferinde yeni borular kullanarak, 3.1 ve 3.2 üç kez daha arasındaki adımları yineleyin. 4th toplandıktan sonra buz miracidia yerleşmek izin vermek 10 dakika için son tüp tutun. Tüpler bu dizi ilk hasat oluşmaktadır.</li>
	<li>Miracidia 290 x g 4 oC önceden soğutulmuş soğutmalı santrifüj, 1 dk. için de içeren santrifüj tüpleri.</li>
	<li>Hemen parazit Pelet (şekil 3B) bozmamaya dikkat edin süpernatant (~ 7 mL) çoğunu kaldırın.</li>
	<li>Tüm miracidia gelen bu ilk hasat içine bir tüp, tüm orijinal tüpleri ~ 1 mL steril su birikintisi su ile durulayın ve "havuz" tüp eklemek Havuzu.</li>
	<li>Parazitler buza ~ 5 min için yerleşmek izin verir ve tekrar 290 x g 4 oC., 1 dk. için parazitlere santrifüj kapasitesi</li>
	<li>Dikkatle süpernatant kaldırın ve 6-8 mL steril Chernin'ın dengeli tuz solüsyonu (CBSS +; bkz: malzemeler tablo) içeren kalem/strep ekleyin.</li>
	<li>Hafifçe miracidia ve onlara bir 24-şey kültür plaka (iyi başına 1 mL), takip tüp 6-8 mL CBSS + durulama ve her şey için durulama 1 mL ekleyerek aliquot askıya alma. 26 oC normoxic koşullar altında parazitlere kuluçkaya. İzole miracidia konsantrasyonları değişir, ancak genellikle ~ 7000/mL ortalamasını alır.</li>
	<li>Parazitler hala ışık kaynağından konsantre oldular adımları 3.1 3.9 için tarama geç miracidia hasat devam etmek için tekrar ancak 15-20 dk koleksiyonları arasındaki zaman aralığını artırın. Not: İkinci hasat tüpler bu yeni kümesini temsil eder. Ancak, miracidial numaraları genellikle düşük olduğundan, larvaları üzerinden tüm tüpler genellikle de 2 mL CBSS + içeren tek bir kültürün içine havuza.</li>
	<li>Yavaşça 24 h hasat sonrası ve yetiştirme, onların Wells sporocysts pipetting tarafından resuspend.</li>
	<li>Parazitler kuyuları altına yerleşmek izin vermek için birkaç dakika bekleyin. Bir kez onlar yerleşmiş, larva dönüştürme sırasında miracidia tarafından döken siliyer epidermal plakaları çoğunu içeren süpernatant (~1.5 mL), dikkatli bir şekilde çıkarın. Bu "dönüşüm" süpernatant ya atılan veya olabilir larva salgı ürünleri takip çalışmaları dahil.</li>
	<li>Taze CBSS + 1,5 mL her şey ve yavaşça pipet sporocysts resuspend ekleyin. Daha fazla yıkama veya farklı bir orta olarak değiştirmek isterseniz 3.12 adımları yineleyin.</li>
</ol>

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Miracidia izole ve burada, açıklandığı gibi manipüle S. mansoni, sadece salyangoz ara ana bilgisayar enfekte olabilir ve bu nedenle insan bir biohazard larva gelişme bu aşamada temsil etmemektedir. Ancak, yanlışlıkla pozlama/in helezon bulaşmasını önlemek için duyarlı Biomphalaria salyangoz türleri mevcut veya tutulan nerede olabilir alanlardan farklı bir konumda miracidial izolasyonların gerçekleştirmek için özen gösterilmelidir. BSL2 alanı kayıtlı ayrı bir oda önerilir. B...

İnsan flukes Schistosoma spp. kan, şistozomiyazis, ihmal edilen tropikal bir hastalık bir tahmini 230 milyon kişi dünya çapında1etkileyen etken ajanlardır. En yaygın tür, Schistosoma mansoni, Güney Amerika, Karayipler adalar, Orta Doğu ve alt-Sahra Afrika2coğrafi olarak dağıtılır. S. mansonive diğer schistosomes yaşam döngüsünün bir memeli kesin ana bilgisayar ve zorunlu ara konukçu olarak hizmet tatlı su Biomphalaria cinsi salyangoz içeren karmaşık bir şey.
S. mansoni erkek ve dişi yetişkin bağırsak mukoza küçük venüller içinde teslim olmak embryonated yumurta (ova) sürümü sonuçlanan yaşayan memeli ev sahibi çoğaltmak, posterior Mezenterik damarlarında çiftleri solucan. Yumurta sonra tatili gemi duvarları üzerinden Mukozal doku ile göç ve sonunda nerede dışkı ile hükümsüz intestinal Lümen girin. Ova tatlı su ve free-swimming larva (miracidia) kapağı girdiğinizde, onlar kısa sürede yaşam döngüsünün devam edebilmek için bulaştırmak için uygun bir Biomphalaria salyangoz bulmalısınız. Bu enfeksiyon işlem salyangoz'ın dış gövdesi yüzey aktif penetrasyon ve larva giriş ana bilgisayar takip miracidial eki içerir. Yakında girmesinden sonra miracidium sonraki larva sahne alanı, birincil ya da anne sporocyst'ın yeni dış yüzey (tegument) olacak bir syncytium formlar onun dış siliyer epidermal plakaları döken başlar. Eşeysiz üreme, her birincil sporocyst üretir ve ikinci nesil sporocysts, ikincil olarak adlandırdığı veya sırayla, oluşturmak ve son intramolluscan aşaması, cercaria3 çok sayıda yayın kızı sporocysts bültenleri . Salyangoz ana bilgisayardan kaçış, free-swimming cercariae için bağlama ve doğrudan bir insan veya diğer uygun memeli ana cildin derinlemesine yeteneğine sahiptirler. Yeni ev sahibi girdiği, üzerine cercariae parazit schistosomula Sahne Alanı'na dönüştürmek ve damar sistemi istila. Onlar, buna karşılık, akciğer ve sonra nerede yetişkin solucanlar için olgun, erkek ve dişi çiftleri oluşturmak ve onların yaşam döngüsünü tamamlamak için Mezenterik damarların seyahat hepatik ven göç.
Başarılı intramolluscan veya larva schistosomes gelişimi "Salyangoz aşama" için insan ana bilgisayar ana bilgisayar veri aktarımı döngüsünün devamlılık esastır. Salyangoz ev sahibi ve birincil sporocyst sahneye erken dönüştürmenin içine free-swimming miracidium dönem aşağıdaki girdiyi kritik önem taşımaktadır. Fizyolojik ve immünolojik arasında uyumluluğu sağlamak bağlı olarak ev sahibi ve parazit, larva gelişme bu aşamada o ilk başarı veya başarısızlık enfeksiyonlarına eğilimli kararlı4,5,6 kurmak için öyle . Birincil sporocysts sonra insan Enfektif cercariae oluşturmak, ikincil sporocysts eşeysiz üretebilen sonraki gelişimi daha fazla gereken tüm asıl olayın asalağın büyüme için sağlar bir keyfi fizyolojik ana ortamı ve üreme ihtiyacı var.
Bugüne kadar küçük temel hakkında fizyolojik, biyokimyasal bilinir ve moleküler süreçler, paraziti-salyangoz etkileşimleri kontrol özellikle molekülleri ve yollar larva gelişme ve üreme düzenleyen veya oransal olarak dahil ana bilgisayar bağışıklık yanıtı. Larva bu aşamaların deneysel düzenleme izni vitro kültür koşullar altında çok sayıda hazır erişim büyük gelişim yolları keşfi ve hücre büyüme, farklılaşma temel mekanizmaları kolaylaştırmak ve üreme. Kritik parazit yolları veya vitro deneyler tespit mekanizmaları, sonra larva büyüme/üreme salyangoz konak bozabilir ya da salyangoz konak immün mekanizmalar tanıma ve ortadan kaldırılması yer belirlemek için kullanılabilir miracidial veya sporocyst aşamaları.
Bu makale ve video sunumu, free-swimming S. mansoni çok sayıda izole bir yöntem ayrıntılı bir açıklama sağlamak için giriş sonra takip için deneysel tabi vitro kültür içine miracidia manipülasyon (iletişim kuralı iş akışı şematik bir bakış için bkz: şekil 1 ). Her ne kadar benzer yordamlarda açıklandığı daha önce7,8,9, biz ayrıntılı bir protokol bu modeli hala cevaplanmamış soruları ile ilgili gidermek için istihdam etmek isteyen araştırmacılar için yararlı olacağını hissettim intramolluscan larva geliştirme, hücresel proliferasyonu ve paraziti-salyangoz bağışıklık etkileşimleri. Buna ek olarak, bu yaklaşım kolayca mesane-konut S. haematobium, karaciğer flukes veya akciğer kuyruk gibi tıbbi açıdan önemli diğer trematodes yumurtadan yalıtım için adapte.

<table><tbody><tr><td>&lt;strong&gt;Chernin&#039;s balanced salt solution (CBSS+)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 1L of solution&lt;/strong&gt;</td></tr><tr><td>2.8 g sodium chloride</td><td>Fisher Scientific</td><td>S271-3</td><td>Dissolve salts, except calcium chloride, and&amp;nbsp;</td></tr><tr><td>0.15 g of potassium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P5405</td><td>sugars in 800 mL ddH2O</td></tr><tr><td>0.07 g sodium phosphate, dibasic anhydrous</td><td>Fisher Scientific</td><td>S374-500</td><td>Dissolve calcium chloride separately in 200&amp;nbsp;</td></tr><tr><td>0.45 g magnesium sulfate heptahydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M1880</td><td>mL ddH2O</td></tr><tr><td>0.53 g calcium chloride dihydrate&amp;nbsp;</td><td>Mallinckrodt</td><td>4160</td><td>Slowly add calcium soln to the salt/sugar soln with&amp;nbsp;</td></tr><tr><td>0.05 g sodium bicarbonate&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific</td><td>S233-3</td><td>with constant mixing</td></tr><tr><td>1 g glucose</td><td>MP Biomedicals</td><td>152527</td><td>Adjust to pH 7.2 and filter sterilize using a&amp;nbsp;</td></tr><tr><td>1 g trehalose</td><td>Sigma-Aldrich</td><td>T0167</td><td>0.22 &amp;micro;m disposable bottle-top filter</td></tr><tr><td>10 mL 100X penicillin/streptomycin</td><td>Hyclone</td><td>SV30010</td><td>Add filtered penicillin and streptomycin soln&amp;nbsp;&lt;br/&gt; prior use</td></tr><tr><td>&lt;strong&gt;Incomplete Bge&amp;nbsp; medium (Ibge)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 900 mL solution&lt;/strong&gt;</td></tr><tr><td>220 mL Schneider&amp;rsquo;s Drosophila medium modified</td><td>Lonza</td><td>04-351Q</td><td>Mix Schneider&#039;s medium with 680 mL ddH2O</td></tr><tr><td>4.5 g lactalbumin enzymatic hydrolysate</td><td>Sigma-Aldrich</td><td>L9010</td><td>Add lactalbumin hydrolysate and galactose</td></tr><tr><td>1.3 g galactose</td><td>Sigma-Aldrich</td><td>G0625</td><td>Adjust to pH 7.2 and filter sterilize using a 0.22 &amp;micro;m&lt;br/&gt; pre-sterilized disposable bottle-top filter</td></tr><tr><td>&lt;strong&gt;Complete Bge medium (cBge)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 100 mL of solution&lt;/strong&gt;</td></tr><tr><td>90 mL Incomplete Bge medium</td><td></td><td></td><td>To heat-inactivate FBS: Incubate thawed FBS in&amp;nbsp;</td></tr><tr><td>9 mL heat-inact. fetal bovine serum (FBS) (Optima)</td><td>Atlanta Biologicals</td><td>S12450</td><td>waterbath at 60&amp;deg;C for 1 hr while gently&amp;nbsp;</td></tr><tr><td>1 mL 100X penicillin/streptomycin</td><td>Hyclone</td><td>SV30010</td><td>swirling the bottle every 10 min&lt;br/&gt; Aliquot heat-inactivated FBS into 15-mL tubes&amp;nbsp;&lt;br/&gt; and store at -20&amp;deg;C.&amp;nbsp; Mix medium + FBS and&lt;br/&gt; filter sterilize using a 0.22 &amp;micro;m pre-sterilized&amp;nbsp;&lt;br/&gt; disposable bottle-top filter&amp;nbsp;&lt;br/&gt; Add penicillin and streptomycin prior to use</td></tr><tr><td>&lt;strong&gt;Pond water (stock solution)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1L of stock solution&lt;/strong&gt;</td></tr><tr><td>12.5 g calcium carbonate</td><td>Fisher Scientific</td><td>C64-500</td><td>Mix all salts in 1L of ddH2O</td></tr><tr><td>1.25 g magnesium carbonate</td><td>Fisher Scientific</td><td>M27-500</td><td>Note that the salts will not have completely&amp;nbsp;</td></tr><tr><td>1.25 g&amp;nbsp; sodium chloride</td><td>Fisher Scientific</td><td>S271-3</td><td>dissolved.&amp;nbsp; Shake vigorously to suspend&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>0.25 g&amp;nbsp; potassium chloride</td><td>Sigma-Aldrich</td><td>P5405</td><td>salts prior to making the working soln&amp;nbsp;&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Pond water (working solution)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1.5L of solution&lt;/strong&gt;</td></tr><tr><td>0.8 mL stock solution pond water (shake prior use) in&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td>Mix stock to ddH2O</td></tr><tr><td>1500 mL of ddH2O</td><td></td><td></td><td>Sterilize pond water by autoclaving (slow&amp;nbsp;cycle)</td></tr><tr><td>1.5 mL of 100X penicillin/streptomycin&amp;nbsp;</td><td>Hyclone</td><td>SV30010</td><td>Add penicillin and streptomycin prior use</td></tr><tr><td>&lt;strong&gt;Saline solution (1.2% NaCl)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1.5L of solution&lt;/strong&gt;</td></tr><tr><td>18 g sodium chloride in 1500 mL of ddH2O</td><td>Fisher Scientific</td><td>S271-3</td><td>Autoclave saline solution to sterilize</td></tr><tr><td>1.5 mL of 100X penicillin/ streptomycin</td><td>Hyclone</td><td>SV30010</td><td>Add penicillin and streptomycin prior use</td></tr><tr><td>&lt;strong&gt;Additional equipment and material:&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>7-L mouse euthanizing chamber</td><td></td><td></td><td>Following approved IACUC protocol no. V001551</td></tr><tr><td>Mice</td><td>Taconic Biosciences</td><td></td><td>Swiss-Webster, female, 6-wk old, murine&amp;nbsp;&amp;nbsp;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; tank and regulator</td><td></td><td></td><td>pathogen-free</td></tr><tr><td>24-well tissue culture plate</td><td>TPP</td><td>92424</td><td></td></tr><tr><td>1-L volumetric flasks</td><td></td><td></td><td></td></tr><tr><td>Light source (150W)</td><td>Chiu Tech Corp</td><td>Model F0-150</td><td></td></tr><tr><td>Centrifuge, refrigerated, swinging bucket</td><td>Eppendorf</td><td>Model 5810R</td><td></td></tr><tr><td>Centrifuge bottles (250 mL)</td><td>Nalgene</td><td></td><td></td></tr><tr><td>15-mL centrifuge tubes, sterile</td><td>Corning</td><td>430053</td><td></td></tr><tr><td>Sterile disposable transfer pipets&amp;nbsp;</td><td>Fisher Scientific</td><td>1371120</td><td></td></tr><tr><td>0.22 &amp;micro;m pre-sterilized disposable bottle-top filter&amp;nbsp;</td><td>EMD Millipore</td><td>SCGPS05RE</td><td></td></tr><tr><td>Stainless steel blender</td><td>Waring Commercial</td><td>Model 51BL31</td><td></td></tr><tr><td>Blender cup, 100 mL capacity</td><td>Waring Commercial</td><td></td><td></td></tr><tr><td>Inverted compound microscope</td><td>Nikon Instruments&amp;nbsp;</td><td>Eclipse TE300</td><td></td></tr></tbody></table>

mass isolation and in vitro cultivation of intramolluscan stages of the human blood fluke schistosoma mansoni

İnsan kan flukes, Schistosoma spp., bir salyangoz ara ve memeli son ev sahibi içinde eşeysiz ve cinsel Gelişim aşamaları sırasıyla içeren karmaşık bir yaşam döngüsü var. İzole etmek ve farklı yaşam döngüsü aşamaları vitro kültür koşullar altında sürdürmek yeteneği büyük ölçüde araştırmalar parazit büyüme, gelişme ve ana bilgisayar düzenleyen hücresel, biyokimyasal ve moleküler mekanizmaları kolaylaştırdı etkileşimler. Sistozomyas iletimini eşeysiz üreme ve gelişme salyangoz ana bilgisayar içinde birden çok larva aşamaları gerektirir; Enfektif miracidium, insanlara infektif son cercarial aşaması için birincil ve ikincil sporocysts aracılığıyla. Bu yazıda biz kitle tarama ve izole Schistosoma mansoni miracidia yumurtadan karaciğerleri virüslü fareler ve onların sonraki giriş vitro kültür içine elde edilmesi için adım adım bir protokolü mevcut. Detaylı iletişim kuralı yeni araştırmacılar meşgul ve bu önemli alan paraziti araştırma genişletmek için teşvik edecek öngörülmektedir.

Miracidia büyük çoğunluğu genellikle ilk iki "Hasat" toplanmış. CBSS + izole miracidia kuluçka (Şekil 4A- C) miracidium sporocyst dönüşüm sürecini tetikler. İlk bir saat içinde CBSS +, yüzme miracidia kes (şekil 4A) çoğunluğu içeren aşağıdaki kültür aktarmak kuyuları. 6 h kültür, miracidia altındadır aktif onların siliyer epidermal plakaları (şekil 4B</stron...

Watch this Scientific Journal Video about Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni at JoVE.com

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Bu makalede büyük ölçekli axenic yalıtım ve insan kan fluke Schistosoma mansoni free-swimming miracidia toplanması için bir protokol ve onların sonraki işlem vitro kültür içine giriş için.

Kitle yalıtım ve Intramolluscan insan kan Fluke Schistosoma Mansoni aşamalarında Vitro ekimi

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

mass-isolation-vitro-cultivation-intramolluscan-stages-human-blood

Research

JoVE Journal

Developmental Biology

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

9.1K Görüntüleme.  University of Wisconsin, School of Veterinary Medicine. Bu makalede büyük ölçekli axenic yalıtım ve insan kan fluke Schistosoma mansoni free-swimming miracidia toplanması için bir protokol ve onların sonraki işlem vitro kültür içine giriş için.

Video: Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Kemirgen Sıtma Parazitler Genetik Haç üretimi için protokol

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

İmmünoloji ve Enfeksiyon

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

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 ...

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 ...

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Echinococcus granulosus'un Strobilated Formlarının Geçici Transdüksiyonu

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

Genetik

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Kuruluş ve Eğitim için bir Yüksek Verimli Kur Optimizasyonu<em&gt; Staphylococcus epidermidis</em&gt; Ve<em&gt; Mycobacterium marinum</em&gt; Enfeksiyon İlaç Keşif için bir model olarak

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Canlı zayıflatılmış intradermal aşılama sonrasında Fare Deri ve akan lenf düğümü gelen Myeloid Hücre İzolasyonu<em&gt; Plasmodium</em&gt; Sporozoitler

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Kullanımı Çevre Örneklerinde Yeni Faustoviruses İzolasyonunda bir Hızlı Stratejisi<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

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 ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Echinococcus granulosus'tan Küçük Hücre Dışı Veziküllerin İzolasyonu ve Dendritik Hücre Alımı

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Kitle yalıtım ve Intramolluscan insan kan Fluke Schistosoma Mansoni aşamalarında Vitro ekimi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Kemirgen Sıtma Parazitler Genetik Haç üretimi için protokol

Cercarial Dönüşüm ve In vitro Yetiştiriciliği Schistosoma mansoni Schistosomules

Gelen Yumurta kullanma

Echinococcus granulosus'un Strobilated Formlarının Geçici Transdüksiyonu

Kuruluş ve Eğitim için bir Yüksek Verimli Kur Optimizasyonu

Canlı zayıflatılmış intradermal aşılama sonrasında Fare Deri ve akan lenf düğümü gelen Myeloid Hücre İzolasyonu

Kullanımı Çevre Örneklerinde Yeni Faustoviruses İzolasyonunda bir Hızlı Stratejisi

Yalıtım ve yetişkin Schistosoma japonicum üzerinden ekstraselüler veziküller karakterizasyonu

Echinococcus granulosus'tan Küçük Hücre Dışı Veziküllerin İzolasyonu ve Dendritik Hücre Alımı

A Whole Dağı