Francisco Candal, CDC Teknoloji Transfer Ofisi, Atlanta Georgia HBEC-5i hücreler için teşekkür ederiz. Bu çalışma, Wellcome Trust (AC ve JAR Temel Biyomedikal Bilim Kıdemli Bursu 4 yıl kişilik Doktora öğrenciliğine, hibe sayısı 084.226) tarafından finanse edildi.

Genel öneriler İnsan beyni mikrovasküler endotel hücre (HBEC-5i) kültür daha önce 8, 6 tarif edilmiştir. P. falciparum parazitler 9 kültüre edildi. HBEC-5i ve P. falciparum parazit kültürleri, her zaman steril koşullar tutulmalıdır . Bütün çözeltiler 37 ° C&#39;de önceden ısıtılmış olmalıdır Biz mycoplasma kontaminasyon 10 PCR (Minevera Biolabs, aşağıdaki üreticinin talimatlarına) tarafından düzenli olarak kontrol etmenizi öneririz . Protokolü Şekil 1&#39;de özetlenmiştir. 1. Endotel hücre rutin kültür <ol><li> Gerekli reaktifler hazırlayın. </li></ol><table border="1"><tbody><tr><td> Hazırlamak için Orta </td><td> Reaktifler </td><td> Miktar </td></tr><tr><td> &quot;Eksik DMEM&quot; </td><td> DMEM-F12 Ham </td><td> 500ml </td></tr><tr><td> &amp;nbsp; </td><td> L-glutamin 200mm </td><td> 5ml </td></tr><tr><td></td><td> 100X Penisilin / streptomisin </td><td> 5 ml </td></tr><tr><td></td><td> NaOH 1M </td><td> 1.3 ml (pH 7.4 &#39;e ayarlamak) </td></tr><tr><td> &quot;Tam DMEM&quot; </td><td> &quot;Eksik DMEM&quot; </td><td> 450ml </td></tr><tr><td></td><td> Fetal Bovine Serum ısı ile inaktive </td><td> 50ml </td></tr><tr><td></td><td> Endotelyal hücre büyüme ek </td><td> 5 ml </td></tr></tbody></table> <ol start="2"><li> Kültür HBEC% 5 CO 2 ile 37 ° C inkübatör 10ml DMEM tam orta Bacalı balonuna 25cm 2-5i. </li><li> Passage hücreleri konfluent haline. Emme eski orta çıkarın ve DMEM eksik orta veya doku kültürü sınıf PBS (Ca 2 + ve Mg 2 + free) 37 önceden ısıtılmış ° C iki kez kullanarak yıkayın </li><li> Önceden ısıtılmış deneyin 1ml eklebalonun tamamını kapsayacak ve 37 ~ 2 dakika boyunca inkübe psin-EDTA (% 0.025 tripsin, 0.5mm EDTA), girdap ° C </li><li> Inverted mikroskop altında hücrelerin en az% 90 müstakil olduğunu kontrol edin. Gerekirse, yapışık hücreleri çıkarmak için hafifçe balonun alt çalıyorum. 10ml 15ml konik bir tüp içine tripsin ve transfer hücreleri engellemek için DMEM tam orta ekleyin. 300 gr oda sıcaklığında (RT) 4 dakika santrifüjleyin. </li><li> Süpernatantı atın ve pelet DMEM tam orta 10ml ile tekrar süspansiyon haline getirin. Iyice hücreleri tekrar süspansiyon çözüm yukarı ve aşağı Pipet. </li><li> 1 veya 2 ml hücre süspansiyonu, yeni bir kültür balona ekleyin ve kültürünü korumak için taze orta 8 ml ekleyin. Ters bir mikroskop altında her geçen gün kültür gelişimi değerlendirmek ve sarıya döner her 2 ya da 3 gün önce ortamı değiştirmek. </li></ol> 2. Bir seçim için endotel hücreleri hazırlanması <ol><li> Selectio gün iki gün önce<html> n, bir (veya daha fazla) 60 mm Petri kabı steril PBS (2 mg / cm 2) seyreltilmiş fibronektin ekleyin. 37 5 - 20 dakika için çanak inkübe ° C, daha sonra bir ay boyunca 4 ° C&#39;de saklanır ve bir kez yeniden kullanılmış olabilir fibronektin çözüm kaldırmak. </li><li> Passage hücreleri gibi bölümleri 1.3 nitelendirdi. 1.5. Varsayarsak,% 100 konfluent hücreleri müstakil ve 10ml DMEM tam orta (bölüm 1.6. Confluency düşükse orta eşdeğer bir hacmi ile tekrar süspansiyon confluency% 80 ise, örneğin 8 ml ile tekrar süspansiyon) yeniden süspanse edildi, askıya 1.5ml eklemek fibronektin kaplı Petri kabı ve DMEM tam orta bir başka 1.5ml hücreleri. </li><li> Inkübatör numaralı seribaşı Petri kabı yerleştirin. Not: İdeal olarak confluency, iki gün sonra seçim zamanında yaklaşık% 90 olacaktır. </li><li> İsteğe bağlı. HBEC-5i etkinleştirmek için, seçime 50μg/ml 24 saat önce son bir konsantrasyonda TNF (Tümör Nekroz Faktörü) sitokin ekleyin. </li></ol>E_TITLE &quot;&gt; 3 Plasmodium falciparum rutin kültür <ol><li> Gerekli reaktifler (Aşağıdaki tabloya bakınız) hazırlayın. İnsan kırmızı kan hücreleri (RBC), lökosit tükenmesi filtresi (genel sıtma parazitleri kültür için yayın 11 &quot;Sıtma Araştırma Yöntemleri&quot; bölümüne bakın) geçişi tam kan (grup O +) ayırarak hazırlayın . 400 g de 5 dakika süreyle santrifüj ve 10 ml RPMI eksik resuspending RBC iki kez yıkayın. 4 yıkanmış eritrosit tutun ° C,% 50 hematokrit eksik orta. </li></ol><table border="1"><tbody><tr><td> Hazırlamak için Orta </td><td> Reaktifler </td><td> Miktar </td></tr><tr><td> &quot;Eksik RPMI&quot; </td><td> RPMI 1640 (bikarbonat ile) </td><td> 500ml </td></tr><tr><td></td><td> HEPES 1M </td><td> 12.5ml </td></tr><tr><td></td><td> Glikoz 20% </td><td> 5ml </td></tr><tr><td></td&gt; <td> L-glutamin 200mm </td><td> 5ml </td></tr><tr><td></td><td> Gentamisin 50mg/ml </td><td> 250μl </td></tr><tr><td></td><td> NaOH 1M </td><td> 0.7 ml (7.2 pH ayarı) </td></tr><tr><td> &quot;RPMI tamamlamak&quot; </td><td> &quot;Eksik RPMI&quot; </td><td> 450ml </td></tr><tr><td></td><td> Insan (non-immün) serum Pooled </td><td> 50ml </td></tr></tbody></table> <ol start="2"><li> Kültür P. falciparum 37% 2 hematokrit ve inkübe RPMI tam orta ° C,% 3,% 1 O 2, CO 2 ve% 96 N 2 ile enfekte RBC Parazitler (Şekil 2) geliştirme aşamasında değerlendirmek için Giemsa smear 11 günlük olun. </li><li> Düzenli olarak 12 sorbitol tedavi kültür senkronize (yaklaşık haftada bir kez). Gün önce seçim testi% 5 parasitaemia veya daha fazla aşamalı bir halka kültür amacı (1, en azından ideal% 0). </li></ol> 4. P. Seçimi endotel hücreleri cytoadhesion için falciparum <ol><li> HBEC-5i kültür (ideal% 90)% 50 ila% 100 confluency olmalıdır testinin gününde, parazit kültür pigmentli trophozoite aşamada (Şekil 2) (ideal% 10 parasitaemia) olmalıdır . Parazit kültür 30μl hematokrit HBEC-5i Petri kabı başına ihtiyaç vardır. </li><li> Santrifüj (5 dakika için 500 gr.) 1.5ml parazit kültürü, parazitler tarafından iki kez yıkayın. Süpernatant ve taze yapılmış, ısıtılmış DMEM eksik orta 10ml ile tekrar süspansiyon atın. Yıkama, ikinci kez tekrarlayın. % 1 BSA ile eksik DMEM 1.5ml ile 30μl hematokrit tekrar </li><li> Iki kez aspire orta ve eksik DMEM 3ml ekleyerek HBEC-5i kaplamalı Petri kabı yıkayın. </li><li> 37 HBEC-5i çanağı ve inkübe parazitlerin çözüm ° C 75min için ekleyin. İnkübasyon sırasında (30 ve 60 dk sonra) iki kez parazitlerin yeniden süspansedört yönde, saat yönünde ve saat yönünün yanı sıra çanak hafifçe sallayarak. </li><li> Inkübasyondan sonra, 3 ml sıcak DMEM eksik orta ve yumuşak sallanan eklemek için bir plastik Pasteur pipeti kullanarak, orta aspire çanak 5 kez yıkayın. Birçok yemeğin kullanılıyorsa, bu, 37, su ile dolu büyük bir şişesi ° C, sıcak bir yüzey üzerinde tutmak </li><li> Çanak ters bir mikroskop altında kontrol edin. Birçok bulaşmamış RBC hala görünmüyorsa, yukarıda açıklandığı gibi daha yıkar. Herhangi bir bulaşma riskini önlemek için çok dikkatli bir şekilde Petri kabında tutun. </li><li> Yemeğin orta çıkarın ve taze uRBC 40μl hematokrit ile sıcak RPMI tam orta 3ml eklemek. </li><li> Hava geçirmeyen bir kuluçka odasında çanak, 3 dakika süreyle gaz yerleştirin ve 37 ° C gecede bir kuluçka odasının yerleştirin. </li><li> Gün sonra, benzer bir şekilde, bölüm 4.4 &#39;de açıklandığı gibi RPMI eksik orta yıkama parazitlerin hasat. ama daha güçlü. Keep tüm orta konik tüp 15ml (yeniden süspanse RBC içeren). Inverted mikroskop altında tüm RBC çanak kaldırıldı olduğunu kontrol edin. </li><li> Pelet parazitler, 5ml RPMI tam orta yeri ve normal kültür için bir şişe karışımı tekrar süspansiyon süpernatant, atın. </li></ol> 5. Temsilcisi Sonuçlar Seçilmemiş parazitler HBEC-5i (Şekil 3A) bağlanarak düşük bir seviyede göstermektedir. Böylece, seçim ilk turda sonra, çok az sayıda parazitler hasat olacak ve ikinci turda seçim için yeterli bir parasitaemia ulaşmak için kültür bir ay kadar sürebilir. Her seçim turundan sonra, daha fazla parazitler endotel hücrelerine bağlanır ve seçimleri daha kısa zaman aralıklarında tekrar edilebilir. 4-5 tur seçim ardından, yüksek bağlayıcı parazit popülasyonları (Şekil 3) elde edilir. Bizim ellerde, HB3-HBEC HBEC-5i veya TNF aktif HBEC-5i bağlama, similar seviyesi (veriler gösterilmemiştir). 4 P. burada açıklanan protokol ile test edildi falciparum suşlarının: HB3, IT/FCR3, 3D7 ve DD2 (Tablo 1). Sadece son 5 tur seçim sonrasında bile HBEC-5i bağlamak mümkün değil kanıtladı. HB3 parazitler de İnsan Dermal ve Pulmoner mikrovasküler endotel hücreleri uygulamaları (HDMEC ve HPMEC) cytoadherence için seçildi. Burada anlatılan yöntemi kullanarak 4 tur seçim ardından, yüksek bağlayıcı nüfus HDMEC ve HPMEC (Şekil 4) hem de elde edildi. <img alt="Şekil 1" src="/files/ftp_upload/3122/3122fig1.jpg" /> Şekil 1, seçim sürecinde genel bakış . <img alt="Şekil 2" src="/files/ftp_upload/3122/3122fig2.jpg" /> Şekil 2. P. Gelişim falciparum, kırmızı kan hücreleri enfekte . Giemsa smear 1000X büyütme mikroskop altında görüntülendi. <img alt="Şekil 3" src="/files/ftp_upload/3122/3122fig3.jpg" /> Şekil 3 HBEC-5i için bağlayıcı parazitler tipik bir örnektir . (A) mavi katmanı, Giemsa ile glutaraldehit ve boyanmış HBEC-5i sabit 1000X büyütmede mikroskop altında görüntülendi. URBC ve bağlanmamış iRBC yıkanıp sonra çekilen fotoğraflar. Sol panelinde tek bir P. gösterir falciparum HB3 (seçilmemiş) parazit HBEC-5i bağlı. Sağ panelde HB3 HBEC parazitler 5 tur için seçilen olmuştu. (B) Veriler, her biri, iki nüsha halinde yapılan iki bağımsız deneyler ortalama temsil eder. Endotel hücre başına bağlı parazit sayısı oldu. <img alt="Tablo 1" src="/files/ftp_upload/3122/3122table1.jpg" /> Tablo 1. Plasmodium falciparum suşlarının Özet HB3 TNF-aktif HB seçildi HBEC-5i. Bağlanarak başarıyla seçildiEC-5i. 5 tur seçim ardından, DD2 gerginlik seçilmemiş-DD2 göre HBEC-5i bağlayıcı herhangi bir artış gösterdi. <img alt="Şekil 4" src="/files/ftp_upload/3122/3122fig4.jpg" /> Şekil 4 P. bağlanması HDMEC ve HPMEC için kültür ortamı biraz farklılık olsa da önce ve seçim 4 tur sonra dermal (HDMEC) ve akciğer (HPMEC) endotel hücreleri, falciparum HB3 parazitler. (bkz. tedarikçinin talimatlarına), seçimi için kullanılan protokol olduğu gibi aynı oldu HBEC-5i. Resimler 400X büyütmede alınır. <table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> DMEM-F12 Ham </td><td> Sigma </td><td> D6421 </td><td> DMEM tam orta </td></tr><tr><td> L-glutamin 200mm </td><td>Gibco </td><td> 25030 </td><td> DMEM ve RPMI tam orta </td></tr><tr><td> 100X Penisilin / streptomisin (10000 adet / ml ve 10mg/ml) </td><td> ScienCell </td><td> 0503 </td><td> DMEM tam orta </td></tr><tr><td> Fetal Bovine Serum ısı ile inaktive </td><td> ScienCell </td><td> 0025 </td><td> DMEM tam orta </td></tr><tr><td> Tripsin-EDTA (% 0.025 tripsin, 0.5mm EDTA) </td><td> ScienCell </td><td> 0103 </td><td></td></tr><tr><td> Endotelyal hücre büyüme ek </td><td> ScienCell </td><td> 1052 </td><td> DMEM tam orta </td></tr><tr><td> Doku kültürü tedavi edilen 60 mm X 15 mm Petri kapları </td><td> BD </td><td> 353002 </td><td></td></tr><tr><td> İnsan Fibronektin </td><td> Millipore </td><td> FC010 </td><td> 2 mg / cm 2 </td></tr><tr><td> TNF </td><td> Ar-GeD Systems </td><td> 210-TA </td><td> isteğe bağlı, 50 mg / ml </td></tr><tr><td> RPMI 1640 </td><td> Lonza </td><td> BE12-167F </td><td> RPMI tam orta </td></tr><tr><td> Gentamisin 50mg/ml </td><td> Lonza </td><td> 17-518Z </td><td> RPMI tam orta </td></tr><tr><td> HEPES 1M </td><td> Lonza </td><td> BE17-737E </td><td> RPMI tam orta </td></tr><tr><td> HDMEC </td><td> ScienCell </td><td> 2000 </td><td> Birincil hücre hattı </td></tr><tr><td> HPMEC </td><td> ScienCell </td><td> 3000 </td><td> Birincil hücre hattı </td></tr><tr><td> HBEC-5i </td><td></td><td></td><td> Francisco Candal (fcandal@cdc.gov) &#39;dan elde edilenler </td></tr></tbody></table> Tablo 2. Malzeme

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+potentiate+brain+endothelial+alterations+induced+by+Plasmodium+falciparum.">Platelets potentiate brain endothelial alterations induced by Plasmodium falciparum.</a> Infect. Immun. 74, 645-653 (2006).</li></ol>

Biz ifşa etmek başka bir şey var.

Serebral sıtma damgasını P. sekestrasyon beyin mikrovasküler 2, 3 içinde falciparum iRBC. Ancak, in vitro kültürlerde P. falciparum HBEC-5i, insan beyninin mikrovasküler endotel için bir model için sadece kötü cytoadhere. Burada fenotip bir daha &quot;gibi in vivo&quot;, HBEC-5i için bağlayıcı bir nüfus zenginleştirmek için basit bir tahlil geliştirdi. 4 P. Üç falciparum suşlarının başarıyla bu yöntem kullanılarak seçilmiştir. Dahası, HB3, bu protokol, çeşitli parazit ve endotel hücre türleri için kullanılabilir olduğunu belirten HDMEC ve HPMEC seçildi. Farklı hipotezler DD2 suşu ile bağlayıcı olmaması açıklayabilir. Bu parazit hattı knobless olduğu gerçeğini olma olasılığı en yüksek olan derin cytoadherence 13, 14 engellemektedir. Biz seçim yanlısı yanında kültür seçilmemiş parazitler önermektedir.Karşılaştırma için bir kontrol sağlamak için girilmesini sağlar. Bu parazit ligand adayları keşfetme umuduyla, bağlayıcı ve bağlayıcı olmayan parazitler transcriptome karşılaştırarak, örneğin, izin verecektir. TNF, serebral sıtma hastaları yüksek düzeyde bulunan bir sitokin ve göstermek için birçok yüzey proteinleri HBEC-5i (Claessens ve hazırlık al, ve 8, 15, 16) ifadesi neden olmuştur . Bu durumda, bağlı iRBC miktarı aktif HBEC-5i ile karşılaştırıldığında, normal HBEC-5i benzer oldu. Bu &quot;sekestrasyon modeli&quot; iRBC ve endotel hücre arasındaki moleküler etkileşim, olası anti-cytoadherence ilaçların etkisinin yanı sıra aynı zamanda çalışma için kullanılabilir. Bu durumda, &quot;8 kuyu odasına slaytlar&quot; (BD 354.628) veya &quot;CultureWell&quot; (Sigma C7735-20EA) gibi daha küçük kuyu, HBEC-5i kaplama öneririz.

selection of plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells

Çoğu insan sıtma ölüm, kan aşamalı Plasmodium falciparum parazitlerin neden olur . Serebral sıtma, hastalığın en çok yaşamı tehdit eden bir komplikasyon, 2-4 beyin mikrovasküler pigmentli trophozoite aşamada Plasmodium falciparum bulaşmış kırmızı kan hücreleri (iRBC) birikimi ile karakterize edilir . Bu mikrovasküler obstrüksiyon (sekestrasyon), asidoz, hipoksi ve zararlı inflamatuvar sitokinler (5) yol açar . İhtiyati Haciz, aynı zamanda insan vücudunda 2, 3 en mikrovasküler dokularda bulunur. IRBC kan damarı duvarlarının eklemek hangi mekanizma hala tam olarak anlaşılamamıştır. Ölümsüzleştirdi İnsan Beyin mikrovasküler endotel hücre hattı (HBEC-5i) kan-beyin bariyerini 6 in vitro model olarak kullanılmıştır. Ancak, Plasmodium falciparum iRBC yoğun sequestrat aksine sadece in vitro HBEC-5i kötü takmakserebral sıtma vakaları meydana gelir iyon. Bu nedenle, (zenginleştirmek) çeşitli P. seçmek için kaydırma tahlil geliştirdi HBEC-5i yapışma falciparum suşlarının yüksek bağlayıcı parazitler, in vivo olarak ortaya çıkar ne daha fazla temsilcisi nüfusu elde etmek için. Pigmentli trophozoite aşamada bir parazit kültürünün bir örnek (iRBC ve bulaşmamış RBC karışım) Petri kabı yetiştirilen HBEC-5i bir katman üzerinde yıkanır ve inkübe edilir. Inkübasyondan sonra, çanak nazikçe uRBC ve bağlanmamış iRBC ücretsiz yıkanır. Taze uRBC HBEC-5i ve inkübe gecede bağlı birkaç iRBC eklenir. Schizont sahne parazitler patlaması gibi, reinvade RBC ve bu halka sahne parazitler, ertesi gün hasat merozoites. Yeterince malzeme elde edilir (genellikle 2 ila 4 hafta) ve yeni bir tur seçim yapılabilir kadar Parazitler kültür. P. bağlı falciparum gerilme, 4 ila 7 tur seçim bir nüfusa almak için gereklien parazitler HBEC-5i bağlamak. Kademeli olarak bağlayıcı fenotip böylece HBEC-5i olağan seçim fenotip korumak için gerekli olan varyant yüzey antijeni gen ekspresyonu bir geçiş gösteren, bir kaç hafta sonra kaybolur. Özetle, biz bir seçim testi render P. geliştirdi falciparum parazitler &quot;serebral sıtma yapıştırıcı&quot; fenotip. 4 P. 3 seçmek için başardık HBEC-5i falciparum suşlarının. Bu testte de başarılı bir insan dermal ve pulmoner endotel hücreleri için bağlayıcı için parazitler seçmek için kullanılır olmuştur. Önemlisi, bu yöntem, beyin endotel bağlama aday parazit ligandlar belirlemek amacıyla doku-spesifik parazit nüfusu seçmek için kullanılabilir. Ayrıca, bu testte varsayılan anti-sekestrasyon ilaçlar 7 için ekran kullanılabilir.

Watch this Scientific Journal Video about Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells at JoVE.com

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Bir<em&gt; In vitro</em&gt; Serebral sıtma sekestrasyon model açıklanmıştır<sup&gt; 1</sup&gt;.<em&gt; Plasmodium falciparum</em&gt; Bulaşmış kırmızı kan hücreleri ölümsüzleştirdi insan beyin mikrovasküler endotel hücreleri için bağlayıcı için seçilir. Seçilen parazitler farklı bir fenotip göstermektedir. Seçim süreci çeşitli kullanılarak yapılabilir<em&gt; S. falciparum</em&gt; Suşları ve endotel hücre hatları.

Seçimi Plasmodium falciparum İnsan Beyninin endotel hücreleri Cytoadhesion için Parazitler

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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JoVE Journal

Immunology and Infection

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

18.9K Görüntüleme.  University of Edinburgh. Bir In vitro Serebral sıtma sekestrasyon model açıklanmıştır 1. Plasmodium falciparum Bulaşmış kırmızı kan hücreleri ölümsüzleştirdi insan beyin mikrovasküler endotel hücreleri için bağlayıcı için seçilir. Seçilen parazitler farklı bir fenotip göstermektedir. Seçim süreci çeşitli kullanılarak yapılabilir S. falciparum Suşları ve endotel hücre hatları.

Video: Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Bir<em&gt; In vitro</emOkumak&gt; Co-enfeksiyon Modeli<em&gt; Plasmodium falciparum</emİnsan İlköğretim Monosit kökenli Bağışıklık Hücrelerde&gt;-HIV-1 Etkileşimleri

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

İmmünoloji ve Enfeksiyon

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Trombosit-aracılı kesekleşme için Basit Protokolü<em&gt; Plasmodium falciparum</emBir kaynak Poor Setting&gt; ile enfekte Eritrosit

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Den Beyin sekestre Lökositler İzolasyonu ve Analizi<em&gt; Plasmodium berghei</em&gt; ANKA-enfekte Fare

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Ayrılması<em&gt; Plasmodium falciparum</emManyetik Yoluyla&gt; Geç Evre-enfekte Eritrositler

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

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 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

İnsan Thymus ile myeloid dendritik hücreleri ve epitel hücreleri İzolasyonu

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Yüksek Kapasiteli saflaştırılması<em&gt; Plasmodium falciparum</emOpsonizing Antikor Analizlerde kullanım için&gt; merozoitler

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

Yöntemler Küçük RNA&#39;ların ve Ribosomal doluluk Düzenleyici Rolü Araştırma<em&gt; Plasmodium falciparum</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Sürekli Akışta Birincil İnsan Endotel Hücrelerinin Pnömokok Enfeksiyonu

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Neisseria meningitidis İndüklenmiş Pluripotent Kök Hücre Kaynaklı Beyin Endotel Hücrelerinin Enfeksiyonu

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...