우리는 HBEC - 5i 전지 시스코 Candal, CDC 기술 이전 사무소, 아틀란타 조지아 감사합니다. 이 작품은 Wellcome 트러스트 (AC 및 JAR에 기본 의생명 과학 수석 원정대에 사년 박사 학생이기, 부여 번호 084226)에 의해 재정 지원되었다.

일반적인 권고 사항 인간 두뇌의 microvascular 내피 세포 (HBEC - 5i) 문화는 이전에 다음 ID로 8, 6 설명되었습니다. P.가 falciparum의 기생충은 9로 교양되었습니다. HBEC - 5i와 P. 모두 falciparum 기생충 문화는 항상 무균 상태에서 보관해야합니다. 모든 시약은 37 ° C.에 미리 예열한다 우리는 정기적으로 PCR (Minevera Biolabs, 다음과 같은 제조 업체의 지침)에 의해 mycoplasma 오염 10 확인하는 것이 좋습니다. 프로토콜은 그림 1에 요약됩니다. 1. 내피 세포 일상 문화 <ol><li> 필요한 시약을 준비합니다. </li></ol><table border="1"><tbody><tr><td> 준비 중 </td><td> 시약 </td><td> 양 </td></tr><tr><td> &quot;불완전한 DMEM&quot; </td><td> DMEM - F12 햄 </td><td> 500ml </td></tr><tr><td> 및nbsp; </td><td> L - 글루타민 200mM </td><td> 5ml </td></tr><tr><td></td><td> 100X 페니실린은 / 스트렙토 마이신 </td><td> 5 ML </td></tr><tr><td></td><td> NaOH 1M </td><td> 1.3 ML (7.4로 산도를 조절) </td></tr><tr><td> &quot;완료 DMEM&quot; </td><td> &quot;불완전한 DMEM&quot; </td><td> 450ml </td></tr><tr><td></td><td> Foetal 소 혈청 열 inactivated </td><td> 50ml </td></tr><tr><td></td><td> 내피 세포 형성제 </td><td> 5 ML </td></tr></tbody></table> <ol start="2"><li> 문화 5 % CO 2와 37 ° C 배양기에서 10ml DMEM 완전한 매체와 통풍 25cm 2 플라스크에 HBEC - 5i. </li><li> 통로 세포들이 합류된다. 흡입에 의해 기존의 미디어를 제거하고 두 번 DMEM 불완전 매체 또는 조직 문화 학년 PBS (CA 2 + 및 MG 2 + 무료) 37 미리 예열 ° C.를 사용하여 세척 </li><li> 사전 예열 시도의 1ml 추가플라스크의 전체를 커버하고 37 ~ 2 분 부화 psin - EDTA (에틸렌 다이아 민 테트라 초산) (0.025 % 트립신, 0.5mM EDTA (에틸렌 다이아 민 테트라 초산)), 소용돌이 ° C. </li><li> 세포의 최소 90 %가 분리되었다는 거꾸로 현미경으로 확인합니다. 필요한 경우, 부드럽게 점착 성의 세포를 이동시키다하는 술병의 바닥을 물릴. 15ml 원뿔 튜브에 트립신 및 전송 세포를 차단하는 DMEM 완료 매체의 10ml를 추가합니다. 실온 (RT)에서 300g에 4 분 원심 분리기. </li><li> 뜨는을 취소하고 DMEM 전체 매체의 10ml로 펠렛을 resuspend. 철저하게 세포를 resuspend에 대한 해결책을 피펫 아래. </li><li> 새로운 문화 플라스크에 세포 현탁액 1 또는 2 ML을 추가하고 문화를 유지하기 위해 새로운 매체의 8ml를 추가합니다. 거꾸로 현미경으로 매일 문화의 성장을 평가하고 노란색 변하기 전에 매 2 또는 삼일 매체를 변경합니다. </li></ol> 2. 선택에 대한 내피 세포를 준비 <ol><li> 전에 selectio의 하루 이틀<html> N 하나 (혹은 이상) 60mm 페트리 접시에 무균 PBS (2 μg / cm 2)에 희석 fibronectin을 추가합니다. 37 5-20 분 요리를 품어 ° C, 그럼 한달 동안 4 ° C에서 저장하고 한번 다시 사용할 수있는 fibronectin 솔루션을 제거합니다. </li><li> 통로 세포는 섹션 1.3에서 설명한. 1.5. 가정 백퍼센트 합류 세포 분리 및 10ml DMEM 전체 매체 (섹션 1.6., confluency이 낮은 경우 매체의 동등한 볼륨 resuspend, confluency는 80 % 였다면 예 8ml와 resuspend)에 resuspended 있었다 정지의 1.5ml 추가 fibronectin 코팅된 배양 접시와 DMEM 전체 매체의 다른 1.5ml로 세포. </li><li> 인큐베이터에있는 씨앗을 페트리 접시를 놓습니다. 참고 : 이상적으로, confluency 이틀 후에 선택의 시간을 90 % 정도됩니다. </li><li> 선택 사항입니다. HBEC - 5i 활성화하려면, 사전 선택에 50μg/ml 24 시간 최종 농도에서 TNF (Tumour의 괴사 팩터) 시토킨를 추가합니다. </li></ol>e_title &quot;&gt; 3. Plasmodium falciparum 루틴 문화 <ol><li> 필요한 시약을 (여기 아래 표 참조) 준비합니다. 백혈구 고갈 필터 (일반 말라리아 기생충의 culturing에 대한 출판 11 &quot;말라리아 연구의 방법&quot;참조)을 통해 통과하여 전체 혈액 (그룹 O +)를 분리하여 인간의 적혈구 (RBC)를 준비합니다. 5 분 400g에 centrifuging 10 ML RPMI 불완전한 그들을 resuspending하여 두 번 RBC 씻으십시오. 4 세탁 RBC 계속 ° C를 불완전 매체 50 %의 혈소판에서. </li></ol><table border="1"><tbody><tr><td> 준비 중 </td><td> 시약 </td><td> 양 </td></tr><tr><td> &quot;불완전한 RPMI&quot; </td><td> RPMI 1640 (소 포함) </td><td> 500ml </td></tr><tr><td></td><td> Hepes 1M </td><td> 12.5ml </td></tr><tr><td></td><td> 포도당 20% </td><td> 5ml </td></tr><tr><td></TD&gt; <td> L - 글루타민 200mM </td><td> 5ml </td></tr><tr><td></td><td> Gentamycin 50mg/ml </td><td> 250μl </td></tr><tr><td></td><td> NaOH 1M </td><td> 0.7 ML (7.2 산도를 조절) </td></tr><tr><td> &quot;RPMI 완료&quot; </td><td> &quot;불완전한 RPMI&quot; </td><td> 450ml </td></tr><tr><td></td><td> 인간 (비 면역) 혈청 풀링 </td><td> 50ml </td></tr></tbody></table> <ol start="2"><li> 문화 P. 37 2 %의 혈소판과 부화에 RPMI 완료 매체 ° C 3퍼센트 CO 2, 1 % O 2, 96% N 2.와 falciparum에 감염된 RBC 기생충 (그림 2)의 개발 단계를 평가하기 위해 Giemsa의 얼룩 11 매일합니다. </li><li> 정기적으로 (약 일주일에 한 번) sorbitol 치료 12 문화를 동기화할 수 있습니다. 일 이전에 선택 분석, 5 % parasitaemia 이상에서 반지 무대 문화에 대한 목표 (이상 최소 10 %). </li></ol> 4. P.의 선택 내피 세포에 cytoadhesion위한 falciparum <ol><li> HBEC - 5i 문화 (이상 90%) 50~100%에서 confluency되어야하는 동안 분석의 날, 기생충 문화 색소 trophozoite 단계 (그림 2) (이상 10 % parasitaemia)에 있어야합니다. 기생충 문화의 30μl 포장 셀 볼륨 HBEC - 5i 페트리 접시마다 필요합니다. </li><li> centrifuging (5 분 500g) 기생충 문화의 1.5ml로 두 번 기생충 씻으십시오. 뜨는 갓 만든, 예열, DMEM 불완전 매체 10ml로 resuspend 폐기하십시오. 세척에게 두 번째 시간을 반복합니다. 1 % BSA와 함께 불완전한 DMEM의 1.5ml와 30μl 모여 셀 볼륨을 Resuspend. </li><li> 두 매체 aspirating 및 불완전 DMEM의 3ml를 추가 HBEC - 5i 코팅 페트리 접시를 씻으십시오. </li><li> 37 HBEC - 5i 요리와 부화 기생충의 솔루션 ° 75min C를 추가합니다. 부화 기간 동안 (30 및 60 분 후) 두 번 기생충 Resuspend부드럽게 네 개의 방향뿐만 아니라, 시계 방향과 반 시계 방향으로 접시를 락을하여. </li><li> 부화 후, 3 따뜻한 DMEM 불완전 매체의 ML, 부드러운 흔들어 추가하는 플라스틱 파스퇴르 피펫을 사용하여 매체를 aspirating하여 접시에게 5 번 씻는다. 많은 요리가 사용되는 경우, 그러한 37 물이 담겨있는 대형 플라스크 ° C.로 따뜻한 표면에 그들을 계속 </li><li> 거꾸로 현미경 접시를 확인합니다. 많은 감염되지 않은 RBC 아직도 볼 수있다면, 위에서 설명한대로 더 세척 해. 오염의 위험을 피하기 위해 매우 조심스럽게 페트리 접시를 취급하고 있습니다. </li><li> 접시에서 매체를 제거하고 신선한 uRBC의 40μl 포장 셀 볼륨 따뜻한 RPMI 전체 매체의 3ml를 추가합니다. </li><li> 밀폐 챔버 잠복기에있는 접시, 3 분 가스를 놓고 37 ° C 야간에 인큐베이터에있는 챔버를 놓습니다. </li><li> 그날 이후, 섹션 4.4에서 설명한대로 유사한 방식으로, RPMI 불완전 매체로 세탁하여 기생충을 수확. 하지만 더 적극적으로. 키P 모든 매체 15ml 원뿔 튜브 (resuspended RBC를 포함)의 사용. 모든 RBC는 접시에서 제거된 것을 거꾸로 현미경으로 확인합니다. </li><li> 펠렛 기​​생충, 5ml RPMI 완료 매체와 장소 정상 culturing에 대한 플라스크에 혼합물에 resuspend 뜨는을 버리고. </li></ol> 5. 대표 결과 선택하지 않은 기생충 HBEC - 5i (그림 3A)에 바인딩의 낮은 수준을 보여줍니다. 따라서, 선택의 첫 라운드 이후 거의 기생충이 수확되고 그것은 선택의 두 번째 라운드에 대한 충분한 parasitaemia에 도달 culturing의 달 소요될 수 있습니다. 선택 각 라운드마다, 점점 더 많은 기생충은 내피 세포에 바인딩하고 선택은 짧은 시간 간격으로 반복 수 있습니다. 선택 4-5 라운드 후, 높은 바인딩 기생충의 인구는 (그림 3) 얻을 수 있습니다. 우리 손에, HBEC - 5i 또는 TNF 활성화 HBEC - 5i에 HB3 - HBEC의 바인딩은 시뮬레이션을했습니다ilar 수준 (데이터가 표시되지 않음). 프로토콜은 여기에서 설명한 4 P. 함께 테스트되었습니다 falciparum의 변종 : HB3, IT/FCR3, 3D7 및 Dd2 (표 1). 오직 후자도 선택 5 라운드 후 HBEC - 5i에 바인딩할 수 없습니다이었다. HB3 기생충은 또한 인간의 피부와 폐 Microvascular 내피 세포 (HDMEC 및 HPMEC)에 cytoadherence이 선택되었습니다. 방법을 사용하여 선택 4 라운드가 여기에 설명된 후, 높은 바인딩 인구는 HDMEC 및 HPMEC (그림 4) 모두에 얻은 것입니다. <img alt="그림 1" src="/files/ftp_upload/3122/3122fig1.jpg" /> 그림 1. 선정 과정의 개요. <img alt="그림 2" src="/files/ftp_upload/3122/3122fig2.jpg" /> P.의 그림 2. 개발 단계 falciparum은 적혈구를 감염. Giemsa의 얼룩은 1000X 확대 현미경 시각. <img alt="그림 3" src="/files/ftp_upload/3122/3122fig3.jpg" /> 그림 3. HBEC - 5i에 바인딩 기생충의 전형. (A) 파란색 레이어는 Giemsa와 글루 타 알데히드와 스테인드와 HBEC - 5i 고정 1000X 확대 현미경 시각. uRBC 및 언바운드 iRBC가 씻겨 나서 사진을 촬영했다. 왼쪽 패널은 단일 P.을 보여줍니다 falciparum HB3 (선택되지 않은) 기생충은 HBEC - 5i에 바인딩. 오른쪽 패널에서 HB3 - HBEC 기생충은 5 라운드를 위해 선택했다. (B) 데이터는 두 개의 독립적인 실험, 중복으로 수행 각각의 평균을 나타냅니다. 내피 세포 당 행 기생충의 수를 계산했다. <img alt="표 1" src="/files/ftp_upload/3122/3122table1.jpg" /> 표 1. 성공적으로 HB3 또한 TNF - 활성 HB에서 선택한 것을 HBEC - 5i. 참고로 바인딩 선정되었다 Plasmodium falciparum의 종자의 요약EC - 5i. 선택 5 회진 후, Dd2 긴장이 선택되지 않은 - Dd2에 비해 HBEC - 5i에 바인딩에 증가했다 없습니다. <img alt="그림 4" src="/files/ftp_upload/3122/3122fig4.jpg" /> 그림 4. P.의 바인딩 HDMEC 및 HPMEC에 대한 문화 매체 약간 차이가 있지만 이전과 선택 4 라운드 후 피부 (HDMEC)와 폐 (HPMEC) 내피 세포에 falciparum HB3 기생충. (업체의 지침을 참조) 선택에 사용되는 프로토콜과 마찬가지로 동일했습니다 HBEC - 5i. 사진은 400X 배율에서 찍은. <table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> DMEM - F12 햄 </td><td> 시그마 </td><td> D6421 </td><td> DMEM 전체 매체에 </td></tr><tr><td> L - 글루타민 200mM </td><td>GIBCO </td><td> 25,030 </td><td> DMEM과 RPMI 전체 매체에 </td></tr><tr><td> 100X 페니실린은 / 스트렙토 마이신 (10,000 대 / ML 및 10mg/ml) </td><td> ScienCell </td><td> 0503 </td><td> DMEM 전체 매체에 </td></tr><tr><td> Foetal 소 혈청 열 inactivated </td><td> ScienCell </td><td> 0025 </td><td> DMEM 전체 매체에 </td></tr><tr><td> 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) (0.025 % 트립신, 0.5mM EDTA (에틸렌 다이아 민 테트라 초산)) </td><td> ScienCell </td><td> 0103 </td><td></td></tr><tr><td> 내피 세포 형성제 </td><td> ScienCell </td><td> 1052 </td><td> DMEM 전체 매체에 </td></tr><tr><td> 조직 문화는 60mm를 치료 X 15mm 배양 접시 </td><td> BD </td><td> 353,002 </td><td></td></tr><tr><td> 인간 Fibronectin </td><td> Millipore </td><td> FC010 </td><td> 2 μg / cm 2 사용 </td></tr><tr><td> TNF </td><td> R &amp;D 시스템 </td><td> 210 - TA </td><td> 옵션, 50 μg / ML에서 사용 </td></tr><tr><td> RPMI 1640 </td><td> Lonza </td><td> BE12 - 167F </td><td> RPMI 전체 매체에 </td></tr><tr><td> Gentamycin 50mg/ml </td><td> Lonza </td><td> 17 - 518Z </td><td> RPMI 전체 매체에 </td></tr><tr><td> Hepes 1M </td><td> Lonza </td><td> BE17 - 737E </td><td> RPMI 전체 매체에 </td></tr><tr><td> HDMEC </td><td> ScienCell </td><td> 2000 </td><td> 기본 셀 라인 </td></tr><tr><td> HPMEC </td><td> ScienCell </td><td> 3000 </td><td> 기본 셀 라인 </td></tr><tr><td> HBEC - 5i </td><td></td><td></td><td> 시스코 Candal (fcandal@cdc.gov)에서 획득 </td></tr></tbody></table> 표 2. 재료

<ol>
	<li>Scherf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+variation+in+malaria:+in+situ+switching,+relaxed+and+mutually+exclusive+transcription+of+var+genes+during+intra-erythrocytic+development+in+Plasmodium+falciparum.">Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum.</a> Embo. J. 17, 5418-5426 (1998).</li><li>MacPherson, G. G., Warrell, M. J., White, N. J., Looareesuwan, S., Warrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cerebral+malaria.+A+quantitative+ultrastructural+analysis+of+parasitized+erythrocyte+sequestration.">Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration.</a> Am. J. Pathol. 119, 385-401 (1985).</li><li>Seydel, K. B., Milner, D. A., Kamiza, S. B., Molyneux, M. E., Taylor, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+and+intensity+of+parasite+sequestration+in+comatose+Malawian+children.">The distribution and intensity of parasite sequestration in comatose Malawian children.</a> J. Infect. Dis. 194, 208-208 (2006).</li><li>Taylor, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+the+pathologies+of+cerebral+malaria+by+postmortem+parasite+counts.">Differentiating the pathologies of cerebral malaria by postmortem parasite counts.</a> Nat. Med. 10, 143-145 (2004).</li><li>van der Heyde, H. C., Nolan, J., Combes, V., Gramaglia, I., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unified+hypothesis+for+the+genesis+of+cerebral+malaria:+sequestration,+inflammation+and+hemostasis+leading+to+microcirculatory+dysfunction.">A unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction.</a> Trends. Parasitol. 22, 503-508 (2006).</li><li>Dorovini-Zis, K., Prameya, R., Bowman, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+characterization+of+microvascular+endothelial+cells+derived+from+human+brain.">Culture and characterization of microvascular endothelial cells derived from human brain.</a> Lab. Invest. 64, 425-436 (1991).</li><li>Rowe, J. A., Claessens, A., Corrigan, R. A., Arman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+Plasmodium+falciparum-infected+erythrocytes+to+human+cells:+molecular+mechanisms+and+therapeutic+implications.">Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications.</a> Expert reviews in molecular medicine. 11, e16-e16 (2009).</li><li>Xiao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum:+involvement+of+additional+receptors+in+the+cytoadherence+of+infected+erythrocytes+to+microvascular+endothelial+cells.">Plasmodium falciparum: involvement of additional receptors in the cytoadherence of infected erythrocytes to microvascular endothelial cells.</a> Exp. Parasitol. 84, 42-55 (1996).</li><li>Corrigan, R. A., Rowe, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+variation+in+early+innate+cytokine+induction+by+Plasmodium+falciparum.">Strain variation in early innate cytokine induction by Plasmodium falciparum.</a> Parasite. Immunol. 32, 512-527 (2010).</li><li>Rowe, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+mycoplasma+contamination+in+Plasmodium+falciparum+cultures+and+methods+for+its+detection+and+eradication.">Implications of mycoplasma contamination in Plasmodium falciparum cultures and methods for its detection and eradication.</a> Mol. Biochem. Parasitol. 92, 177-180 (1998).</li><li>Moll, K., Ljungstr&#246;m, I., Perlmann, H., Scherf, A., Wahlgren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Malaria Research. , (2009).</li><li>Lambros, C., Vanderberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Plasmodium+falciparum+erythrocytic+stages+in+culture.">Synchronization of Plasmodium falciparum erythrocytic stages in culture.</a> J. Parasitol. 65, 418-420 (1979).</li><li>Herricks, T., Antia, M., Rathod, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformability+limits+of+Plasmodium+falciparum-infected+red+blood+cells.">Deformability limits of Plasmodium falciparum-infected red blood cells.</a> Cell. Microbiol. , (2009).</li><li>Nakamura, K., Hasler, T., Morehead, K., Howard, R. J., Aikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum-infected+erythrocyte+receptor(s)+for+CD36+and+thrombospondin+are+restricted+to+knobs+on+the+erythrocyte+surface.">Plasmodium falciparum-infected erythrocyte receptor(s) for CD36 and thrombospondin are restricted to knobs on the erythrocyte surface.</a> J. Histochem. Cytochem. 40, 1419-1422 (1992).</li><li>Wassmer, S. C., Cianciolo, G. J., Combes, V., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LMP-420,+a+new+therapeutic+approach+for+cerebral+malaria.">LMP-420, a new therapeutic approach for cerebral malaria.</a> Med. Sci. (Paris). 22, 343-345 (2006).</li><li>Wassmer, S. C., Combes, V., Candal, F. J., Juhan-Vague, I., Grau, G. 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>

우리는 공개 아무것도 없어.

대뇌 말라리아의 특징은 P.의 격리입니다 두뇌 microvasculature 2, 3 시간 falciparum iRBC. 그러나, P.의 체외 배양에 HBEC - 5i, 인간 두뇌 microvascular의 내피를위한 모델 falciparum은 제대로 cytoadhere. 여기 표현형보다 &quot;처럼 - 생체내&quot;HBEC - 5i에 바인딩을위한 인구를 풍성하게하는 직접적인 분석을 개발했습니다. 4 P. 중 3 falciparum의 변종이 성공적으로이 방법을 사용하여 선택되었습니다. 또한, HB3 또한이 프로토콜은 다양한 기생충 및 내피 세포 유형에 사용할 수 나타내는 HDMEC 및 HPMEC에 선정되었습니다. 다른 가설은 Dd2 변형과 바인딩의 부족을 설명할 수 있습니다. 이 기생충 라인 knobless이라는 사실되는 대부분, 이것은 깊이 cytoadherence 13, 14을 막아. 우리는 선택 프로와 함께 culturing 선택되지 않은 기생충을 권장비교를위한 컨트롤을 제공하기 위해 세금. 이것은 기생충 리간드 후보를 발견의 희망과 함께, 바인딩 및 구속력이 기생충의 transcriptome를 비교 예를 들어, 수 있습니다. TNF는 대뇌 말라리아 환자의 높은 수준에서 찾을 시토킨이며 HBEC - 5i (Claessens 동부 표준시 준비에 알, 8, 15, 16)의 많은 표면 단백질의 표현을 유도하기 위해 공연을하고 있습니다. 이 경우, 행 iRBC의 양은 활성화 HBEC - 5i에 비해 일반 HBEC - 5i와 비슷한되었습니다. 이 &quot;격리 모델은&quot;또한 iRBC와 내피 세포 사이의 분자 상호 작용뿐만 아니라, putative 방지 cytoadherence 약물의 효과를 연구하는 데 사용할 수 있습니다. 이 경우, 우리는 &quot;8 자 챔버 슬라이드&quot;(BD 354628) 또는 &quot;CultureWell&quot;(시그마 C7735 - 20EA)와 같은 작은 우물에서 HBEC - 5i 도금 권장합니다.

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

대부분의 인간의 말라리아 사망자는 혈액 단계 Plasmodium falciparum의 기생충에 의해 발생합니다. 대뇌 말라리아, 질병의 가장 생명을 위협하는 합병증은 뇌 2-4 microvasculature에 색소 trophozoite 단계에서 Plasmodium falciparum 감염 적혈구 (iRBC)의 축적이 특징입니다. 이 microvessel 방해 (격리)는 산증, hypoxia 해로운 염증 크린 시토킨 (5 검토)로 연결됩니다. 격리는 또한 인체 2, 3 대부분의 microvascular 조직에서 발견된다. iRBC은 혈관 벽에 부착되는 메커니즘은 아직 제대로 이해됩니다. 불후의 인간 두뇌 microvascular 내피 세포 라인 (HBEC - 5i)은 혈액 뇌 장벽 6 체외 모델로 사용되고 있습니다. 그러나, Plasmodium falciparum iRBC은 빽빽한 sequestrat 달리 체외에서 HBEC - 5i에만 제대로 첨부대뇌 말라리아 케이스에서 발생 이온. 따라서 (풍부) 다양한 P.을 선택 패닝 분석을 개발 HBEC - 5i에 부착을위한 falciparum의 변종 높은 바인딩 기생충, 생체내에서 발생하는 것을 더 대리인의 인구를 얻기 위해서 인치 색소 trophozoite 단계에서 기생충 문화의 샘플 (iRBC과 감염되지 않은 RBC의 혼합물)는 페트리 접시에서 성장 HBEC - 5i의 레이어에 세탁하고 incubated입니다. 부화 후 요리가 부드럽게 uRBC와 언바운드 iRBC에서 무료로 세탁합니다. 신선한 uRBC는 HBEC - 5i와 incubated 하룻밤에 연결된 몇 iRBC에 추가됩니다. schizont 무대 기생충이 버스트로 reinvade RBC 이러한 링 단계 기생충은 다음날 수확 아르 merozoites. 충분한 자료 얻은 것입니다 (일반적으로 2-4주)과 선택의 새로운 원형을 수행할 수있을 때까지 기생충이 양식입니다. P.에 따라 falciparum 변형, 선택의 4-7 라운드는 인구를 위해 필요한대부분의 기생충은 HBEC - 5i에 바인딩 어디에. 바인딩 표현형은 점차적 따라서 HBEC - 5i에 대한 일반 선택이 표현형을 유지하기 위해 필요한, ​​변형 표면 항원 유전자 발현에 스위치를 나타내는 몇 주 후에 손실됩니다. 요약에서 우리는 선택 분석 렌더링에게 P.을 개발 falciparum은 기생충보다 &quot;대뇌 말라리아 접착제&quot;표현형합니다. 우리는 4 P. 3를 선택할 수 있었다 HBEC - 5i에 falciparum의 변종. 이 분석은 또한 성공적으로 인간의 피부와 폐의 내피 세포에 바인딩에 대한 기생충을 선택하는 데 사용되었습니다. 중요한 것은,이 방법은 뇌의 내피에 바인딩을위한 후보 기생충의 리간드를 확인하기 위해 조직을 특정 기생충의 인구를 선택하는 데 사용할 수 있습니다. 또한,이 분석은 putative 방지 격리 마약 7 화면으로 사용할 수 있습니다.

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

<em&gt; 체외에서</em대뇌 말라리아의 격리를 위해&gt; 모델이 설명되어 있습니다<sup&gt; 1</sup&gt;.<em&gt; Plasmodium falciparum</em&gt; 감염된 적혈구는 영원히 인간의 뇌 microvascular 내피 세포에 바인딩을 위해 선택됩니다. 선택한 기생충은 별개의 표현형을 보여줍니다. 선정 과정은 다양한 사용하여 적용할 수<em&gt; P. falciparum</em&gt; 변종과 내피 세포 라인.

선정 Plasmodium falciparum</em인간 두뇌 내피 세포 Cytoadhesion위한> 기생충

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

selection-plasmodium-falciparum-parasites-for-cytoadhesion-to-human

Research

JoVE Journal

Immunology and Infection

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

18.9K 조회수.  University of Edinburgh.  체외에서 모델이 설명되어 있습니다 1. Plasmodium falciparum 감염된 적혈구는 영원히 인간의 뇌 microvascular 내피 세포에 바인딩을 위해 선택됩니다. 선택한 기생충은 별개의 표현형을 보여줍니다. 선정 과정은 다양한 사용하여 적용할 수 P. falciparum 변종과 내피 세포 라인.

비디오: 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.

<em&gt; 체외에서</em공부하기&gt; 공동 감염 모델<em&gt; Plasmodium falciparum</em인간의 기본 단핵구 파생 면역 세포에서&gt; - HIV-1 상호 작용

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

연구

면역학 및 감염병학

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.

의 혈소판 응집 중재를위한 간단한 프로토콜<em&gt; 변형체 falciparum의</em자원별로 설정에서&gt; - 감염된 적혈구

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.

에서 뇌 - 분리 된 백혈구의 분리 및 분석<em&gt; Plasmodium berghei</em&gt; ANKA에 감염된 마우스

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.

의 분리<em&gt; Plasmodium falciparum</em자기 수단으로&gt; 늦은 단계에 감염된 적혈구

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.

정량<em&gt; 체외</em정적 조건에서 활성화 된 기본 인간의 미세 혈관 내피 세포에 호중구 부착을 측정하는&gt; 분석

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.

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

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.

높은 수익률 정화<em&gt; 변형체 falciparum의</emOpsonizing 항체 분석 실험에 사용하기 위해&gt; Merozoites

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.

방법은 작은 RNA를과의 리보솜 점유 규제 역할을 조사하기 위해<em&gt; 말라리아 원충</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.

일정한 흐름에 있는 1 차적인 인간 내피 세포의 폐렴구균 감염

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 유도만능줄기세포 유래 뇌내피세포의 감염

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

선정 Plasmodium falciparum 기생충