이 작품은 건강 (NIH) AI091759과 Crohn의와 미국의 윌리엄과 셀비 모델이란 가족 재단 연구 수상 (MN까지)의 대장염 재단의 국립 연구소에 의해 지원되었다. Schistosome에 감염된 생쥐는 NIAID Schistosome 리소스 센터 (NIAID 계약 N01 A130026)가 제공되었다.

1. Schistosoma mansoni 달걀을 정화 <ol><li> Schistosoma 수명주기 8 전염성 무대 schistosome cercariae와 스위스 웹스터 쥐를 감염. 양자 택일로, NIAID Schistosomiasis 리소스 센터 (에서 schistosome에 감염된 생쥐를 얻을 <a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/~~V</a> ). 그 시간을 포인트로, 계란은 간장에 존재하며 아래 설명에 따라 (그림을 참조하십시오. 1) 복구할 수, 감염 다음 6-7주 위해 생쥐를 남겨주세요. </li><li> CO 2와 쥐를 안락사시켜야하고, 70 % 에탄올로 젖었어. 뒷면에 마우스를 놓습니다. 피부와 기본 복막 버려야하는 무딘 집게와 가위를 사용하십시오. ribcage 및 격막 아래에있는 소비세 간,. </li><li> 잘게 간은를 말하다 및 collagenase / dispase (0.5 밀리그램 / ML), 페니실린 (100 U / ML)과 PBS의 스트렙토 마이신 (100 μg / ml) 쇼핑 구성되어 소화 혼합물의 간 당 20 ML을 추가합니다. mixt 넣고50 ML 팔콘 튜브의 우레와 37 ° C 흔드는에서 하룻밤 사이에 알을 품다. </li><li> 400 XG / 3 분 / 20 ° C.에 혼합물을 원심 분리기 부드럽게 과잉 액체를 부어하고 PBS로 튜브를 입력하십시오. 다시 원심 분리기와 PBS에 세차를 반복하십시오. resuspend 펠렛 25 ML PBS에 마지막으로 원심 분리에 따라. </li><li> 좋은 체 (미세 체를 통해 혼합물을 강요하는 10 ML의 주사기를 사용하여 혼합물을 통과하여 다음 기준 부엌 금속 스트레이너 (유리 비커에를 통해 매쉬 간 조각 50 ML 주사기 플런저를 사용)를 통해 resuspended 펠릿 스트레인 두 번째 유리 비커에). </li><li> 50 ML 팔콘 튜브에 무한 섞어 넣고. 400 XG / 5에서 원심 분 / 20 ° C. 펠렛을 없애지 않고 뜨는을 자세히 쏟아야. 3 ML PBS에서 Resuspend 펠릿. </li><li> 오버레이는 20 % Percoll에 부드럽게 섞어, 8 ML Percoll과 32 ML 0.25M 자당 (FW = 342.3 그래서 8.56 g sucrose/100 ML H 2 0)의 40 ML 기울기. </li><li> 10 분 / 800 XG / 20 ° C를 원심 분리기 를 피펫D 간 세포의 젤라틴 상위 계층을 버리고. 트랜스퍼 피펫과 하단에 계란 펠릿를 제거하고 15 ML 팔콘 튜브에 넣어. </li><li> 10 ML PBS / 1 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산) / 1 MM EGTA 사용하는 원심 분리기 설정에서 워시 펠릿 배 : 3 분 / 30 XG / 20 ° C. 500 μL PBS에 Resuspend. </li><li> 10 분 / 800 XG / 20 ° C. : 새 25 % Percoll, 원심 설정을 사용하여 10 ML 기울기 (2.5 ML Percoll과 7.5 ML 0.25M 자당)에서 오버레이 믹스 </li><li> 원심 설정을 사용하여 10 ML PBS로 3 배 씻으 : 3 분 / 30 XG / 20 ° C. </li><li> 지난 10 ML 세척에는 달걀을 계산하여 100 μL를 제거합니다. 계란 빠르게 정착, 팔콘 튜브가 샘플을 제거하기 전에 잘 혼합되어야하므로. 900 μL PBS로 100 μL 시료를 희석. 믹스 100 μL은 해부 현미경 (1시 10분 희석)를 사용하여 현미경 슬라이드에 방울로 간주되어야합니다. 넓은 구멍 피펫 팁을 사용해야합니다. </li><li> 50,000 계란 / PBS에 ML에서 계란 Resuspend. 계란은 -80 ° C에 몇 개월 동안 저장되고 한 twic 해동 수사용을위한 전자. 사용하기 전에, 달걀 (그림을 참조하십시오. 1) 아직도 건재한지 현미경으로 검사한다. </li></ol> 2. S로 Intraperitoneal Sensitization mansoni 달걀 : 주 영 <ol><li> 5 ML의 snapcap 관에서 5000 eggs/100 μL PBS에서 계란을 준비합니다. 주사의 경우 필요한 50 % 이상 더 많은 알을 예상됩니다. </li><li> 23 게이지 바늘 3/4inch와 마우스 당 계란 서스펜션의 100 μL로 한 ML의 주사기를로드합니다. 달걀은 사용하기 바로 전에 계란과 함께, 그래서 부하 바늘을 해결. </li><li> 전에 각 주사에 알을 섞어 및 마우스 당 정지 intraperitoneally 100 μL를 삽입할 앞뒤 락 주사기. </li></ol> 3. S.와 레트로 - 궤도 정맥 도전 mansoni 달걀 : 주 14 <ol><li> 5000 eggs/100 μL에서 위의 계란을 준비합니다. </li><li> xylazine (10 밀리그램 / ㎏)와 케타민 (120 밀리그램 / ㎏)과 (4 %) isoflurane, 또는 intraperitoneally와 전문 챔버의 생쥐를 마취. anest 심도hesia는 노상 강도의 곤란과 더 물리적인 응답을 확보하지 의해 결정됩니다. </li><li> 달걀 한 ML의 주사기 23 게이지 바늘 3/4inch의로드에 따라 각도로 아래로 향하게 베벨과 90 ° 각도로 바늘을 구부하는 포셉를 사용합니다. 아무런 거품이 존재하지 있는지 확인합니다. </li><li> 한쪽에 마우스를 놓습니다. 눈이 내다, 그리고 코에 45 ° 각도로 안구 뒤쪽 혈관에 100 μL를 주입 않도록 피부를 철회. </li><li> 바늘을 철회하고 출혈을 조절하기위한 가벼운 압력을 적용합니다. </li></ol> 이러한 절차들은 모두 세심하게 실시해야하며 그들이 마취에서 회복하기 전까지 마우스는 모니터링되어야한다. 이러한 프로토콜은 펜실베니아 기관 동물 관리 및 사용위원회 (IACUC)의 대학에 의해 승인되었다. 4. 실험 하베스트 : 주 22 <ol><li> CO 2와 함께 쥐를 안락사. </li><li> 뒷면의 70 % 에탄올, 장소와 젖은 생쥐. </li><li> 무딘 그렇게해야 사용EPS는 마우스 복부를 파악하고 피부에 작은 절개를합니다. 조심스럽게 가위로 피부를 제거하고 흉곽 및 복막을 폭로. </li><li> 복막을 열 컷. 파스퇴르 피펫으로 혈액을 복구하는 복부 대동맥을 절단. 얼음을 저장합니다. </li><li> 다이어프램을 폭로 간이 아래로 이동합니다. 피임기구를 잘라 미세 가위를 사용합니다. 조심스럽게 폐 조직을 노출하는 갈빗대 오픈 했네요. </li><li> 미세 집게로 폐를 드레인 parathymic 림프절을 복구할 수 있습니다. 이러한 thymus 9의 양쪽에, 흉곽 아래에 자리잡고있다. 1mL 멸균 미디어 얼음 저장소 (DMEM은 Invitrogen에서 사용 가능한 모든 10% 열 inactivated 소태아 혈청, 100 U / ML 페니실린, 100 μg / ML 스트렙토 마이신, 2 밀리미터 L-글루타민, 50 μm의 2 - 메르 캅 토 에탄올과 보충). </li><li> BAL을 복구하고, histologic 분석을 위해 폐가 팽창하기 in​​tratracheal 삽관법이 수행되어야합니다. 측면으로 침샘을 이동하여 호흡 관을 폭로하고, placi호흡 관 밑에 NG 벌금 포셉. 미세 가위를 사용하여, 호흡 관의 중앙에 구멍을 절단하고, 폐 속에 튜브를 삽입합니다. 수술용 실로 묶는로 튜브를 고정합니다. </li><li> 튜빙에 PBS-가득한 한 ML의 주사기를 첨부하고 1 ML PBS로 폐를 부풀려. 조심스럽게 주사기로 BAL 세차를 검색합니다. 얼음을 저장합니다. </li><li> 튜빙에 PBS-가득한 한 ML의 주사기에 4 % paraformaldehyde (PFA)를 첨부하고 폐를 부풀려. </li><li> 튜브를 제거하고, PFA의 누출 방지하기 위해 수술 스레드를 조입니다. </li><li> 조심스럽게 5 ML 4퍼센트 PFA로 50 ML 팔콘 튜브의 폐 조직과 장소를 해부. </li></ol> 5. 복구된 조직의 준비 <ol><li> 세럼 : 응고 혈액을 위해 30 분 ~ 2 H 위해 얼음 같은 스토리지, 혈청은 (13,000 XG / 10 분 / 4 ℃) 원심 분리에 의해 혈액에서 발견됩니다. 혈청은 -20 ° C에서 저장되고 시토킨 ELISAs 또는 S. 사용할 수 있습니다 mansoni 계란 항원에 특정한 IgG isotype ELISAs <sup&gt; 6, 10. </li><li> 림프절 : 단일 세포 현탁액 준비와 셀 카운트는 면역 반응의 정도를 평가하는 데 사용됩니다. Th2 세포 분극을 검토하기 위해 세포는 S로 다시 자극하고 72 시간 mansoni의 달걀 항원. Intracellar 시토킨 염색법은 유동세포계측법 검사를받을 수 있으며, supernatants은 Th2 크린 시토킨의 엘리사 (IL-4, IL-5, IL-13) 6에 대한 복구 및 -20 ° C에 저장할 수 있습니다. </li><li> BAL : 세포 (400 XG / 5 분 / 4 ℃) pelleted 있습니다. BAL 액은 엘리사에 의해 Th2 크린 시토킨의 분석을위한 복구 및 -20 ° C에 저장됩니다. BAL 세포 카운트는기도 염증에 대한 판독으로 사용됩니다. 10,000-100,000 세포 (500 RPM / 5 분 / 4 ℃)의 Cytocentrifuge의 준비는 Diffquik의 염색법 뒤에 것은 염증의 열거는 4, 6에 침투해 수 있습니다. 또는 BAL 세포 유동세포계측법 6 시까지 심사를 할 수 있습니다. </li><li> 폐 조직 : 폐 조직이 stor입니다4에 하룻밤 에드 ° C. 고정 조직은 파라핀 - 임베디드, sectioned 및 histological 및 immunofluorescence 분석을 위해 슬라이드에 마운트됩니다. </li></ol> 6. 대표 결과 이 프로토콜 세부 정보는 (i) 생체내 사용에 대한 Schistosoma mansoni 달걀 (그림 1), (2) 감광성 이러한 계란과 생쥐에 도전 투석을 그리고 (iii) 폐 염증 반응의 시험 기관을 복구 준비에 필요한 모든 단계. 생체내 모델이 reproducibly 적정량 Th2 폐 염증 반응을 조절합니다. 이것은 같은 BAL 셀 카운트 및 cytocentrifuge의 준비 (그림 2)에 의해 시각기도 염증과 eosinophilia에 의해 표시됩니다. H &amp; E-스테인드 폐 부분은 달걀 유발 granulomas (그림 3A)에 대한 검사 및 폐 섹션 immunofluorescent 염색법은 또한 활성화 macrophages와 같은 Resis 같은 Th2 시토킨 유도된 유전자의 시각화을 허용하실 수 있습니다주석과 같은 분자 (RELM) α (그림 3B). 항원에 특정한 CD4 Th2 시토킨 반응은 IL-5 ELISAs을 면밀히 검토, IL-4 및 항원 자극 parathymic 림프절 세포의 IL-13 (그림 4). 있습니다 <img alt="그림 1" src="/files/ftp_upload/3905/3905fig1.jpg" /> 그림 1 Schistosoma mansoni 계란 분사 모델 대표 높은 S.mansoni - 달걀 부담의 특징 granulomatous 간은 사진, 그리고 S.mansoni 달걀의가 표시됩니다 -.. 평균 크기 (L) (W) 50 μm의 작품 120 μm의. <img alt="그림 2" src="/files/ftp_upload/3905/3905fig2.jpg" /> 그림 2. S. mansoni 달걀 주입은기도 염증을 조절합니다. A. 나이브 (N) 또는 S.mansoni (SM) 달걀 주입 생쥐에서 BAL 세포 숫자 (x10 5). * P &lt;0.05. BAL 세포의 바실러스 Cytocentrifuge 준비. 맥, 대식 세포, EO초, EOSINOPHIL, 림프, 림프구. 바, 10 μm의. <img alt="그림 3" src="/files/ftp_upload/3905/3905fig3.jpg" /> 그림 3. 폐 granulomas 및 S.mansoni 달걀을 둘러싸고 또는 활성 macrophages의 시각화. A. SM 계란 (검은색 화살표)를 둘러싼 폐 granuloma는 H &amp; E-스테인드 폐 부분의 시각이었다. RELMα위한 B. Immunofluorescence의 염색법 (녹색), mannose 수용체 (적색) 및 DAPI (파란색) autofluorescent SM 계란 (검은색 화살표)를 둘러싼 granuloma에 또는 활성 macrophages (흰색 화살표)를 보여준다. 바, 50 μm의. <img alt="그림 4" src="/files/ftp_upload/3905/3905fig4.jpg" /> 그림 4. S.mansoni 계란 항원에 특정한 Th2 시토킨 반응. 달걀 주입 나이브 (N) 또는 SM 생쥐에서 parathymic 림프절 세포를 배수가 SM과 자극했다 &lt;/ 그들 IL-4, IL-5 및 IL-13에 대한 supernatants의 엘리사 다음에 72 시간&gt; 계란 항원. 규모, NG / ML. * P &lt;0.05.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis:+report+of+a+WHO+expert+committee.">Prevention and control of schistosomiasis and soil-transmitted helminthiasis: report of a WHO expert committee.</a> WHO Technical Report Series 912. , (2002).</li><li>Pearce, E. J., MacDonald, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+schistosomiasis.">The immunobiology of schistosomiasis.</a> Nat. Rev. Immunol. 2, 499-511 (2002).</li><li>Sandler, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+profiles+during+acute+pathogen-induced+pulmonary+inflammation+reveal+divergent+roles+for+Th1+and+th2+responses+in+tissue+repair.">Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and th2 responses in tissue repair.</a> J. Immunol. 171, 3655-3667 (2003).</li><li>Perrigoue, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-31-IL-31R+interactions+negatively+regulate+type+2+inflammation+in+the+lung.">IL-31-IL-31R interactions negatively regulate type 2 inflammation in the lung.</a> J. Exp. Med. 204, 481-487 (2007).</li><li>Sandor, M., Weinstock, J. V., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulomas+in+schistosome+and+mycobacterial+infections:+a+model+of+local+immune+responses.">Granulomas in schistosome and mycobacterial infections: a model of local immune responses.</a> Trends Immunol. 24, 44-52 (2003).</li><li>Nair, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatively+activated+macrophage-derived+RELM-{alpha}+is+a+negative+regulator+of+type+2+inflammation+in+the+lung.">Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung.</a> J. Exp. Med. 206, 937-952 (2009).</li><li>Dalton, 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=A+method+for+the+isolation+of+schistosome+eggs+and+miracidia+free+of+contaminating+host+tissues.">A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.</a> Parasitology. 115, 29-32 (1997).</li><li>Lewis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+laboratory+maintenance+of+Schistosoma+mansoni,+with+observations+on+three+schistosome/snail+host+combinations.">Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations.</a> J. Parasitol. 72, 813-829 (1986).</li><li>Tilney, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+lymphatic+drainage+in+the+adult+laboratory+rat.">Patterns of lymphatic drainage in the adult laboratory rat.</a> J. Anat. 109 (Pt. 3), 369-383 (1971).</li><li>Lewis, F., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Current protocols in immunology. Chapter 19, Unit 19 (2001).</li><li>Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2+cells+are+required+for+the+Schistosoma+mansoni+egg-induced+granulomatous+response.">Th2 cells are required for the Schistosoma mansoni egg-induced granulomatous response.</a> J. Immunol. 160, 1850-1856 (1998).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrotic+disease+and+the+T(H)1/T(H)2+paradigm.">Fibrotic disease and the T(H)1/T(H)2 paradigm.</a> Nat. Rev. Immunol. 4, 583-594 (2004).</li><li>Cheever, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+hepatic+fibrosis+and+granuloma+size+among+mouse+strains+infected+with+Schistosoma+mansoni.">Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni.</a> Am. J. Trop. Med. Hyg. 37, 85-97 (1987).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

여기에 2를 입력 폐 염증을 유발해서 Schistosoma mansoni 달걀을 고용 모델이 설명되어 있습니다. 이 모델의 특징 특징은 강력한 Th2 면역 반응,기도 염증과 폐의 granuloma 형성을 포함합니다. 이러한 매개 변수가 CD4 Th2 세포 반응 11 의존 때문에, 이것은 Th2 세포 반응에 대한 단백질의 특정 또는 혈통 고유의 삭제 효과를 조사하기위한 유용한 모델입니다. 여기에 설치 했어요는 항원에 특정?...

<table border="1"><tbody><tr><td> 시약 </td><td> 크기 / 설명 </td><td> 회사 </td><td> # 카탈로그 </td></tr><tr><td> Collagenase / Dispase </td><td> 500 MG </td><td> 로슈 응용 과학 </td><td> 11 097 113 001 </td></tr><tr><td> 비교 Quik Hema 3 청바지 </td><td> 짐 </td><td> 피셔 과학 (주) LLC </td><td> 122-911 </td></tr><tr><td> DMEM </td><td> 액체 </td><td> Gibco </td><td> 11,965 </td></tr><tr><td> 팔콘 50mL </td><td> 짐 </td><td> 매 </td><td> 352,070 </td></tr><tr><td> Intramedic PE 관 </td><td> 0.58 mm </td><td> Becton 디킨슨과 (주) </td><td> 427,410 </td></tr><tr><td> Isoflurane </td><td> 250 ML </td><td> 원장 동물 건강 </td><td> 52600405 </td></tr><tr><td> 케타민을 </td><td> 100 MG / ML </td><td> 포트 닷지, 동물 건강 </TD&gt; <td> N / A </td></tr><tr><td> Mannose 수용체 항체 </td><td> 비오틴 </td><td> AbD Serotec </td><td> MCA2235B </td></tr><tr><td> 메탈릭 스트레이너 </td><td> 표준 부엌 스트레이너 </td><td> 목표 </td><td> Laurentiis </td></tr><tr><td> Paraformaldehyde </td><td> 16% </td><td> 전자 Microsc. 과학 </td><td> 15,710 </td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> 100x </td><td> Gibco </td><td> 15,070 </td></tr><tr><td> Percoll </td><td> 1 리터 </td><td> GE 헬스케어 Biosciences </td><td> 17 0,891 01 </td></tr><tr><td> RELMα 항체 </td><td> 50 μg </td><td> Peprotech </td><td> 500-P214 </td></tr><tr><td> 외과 스레드 </td><td> 2.0 USP 100 yds </td><td> 외과 특산품 주식 회사 </td><td> SP118 </td></tr><tr><td> 주사기 바늘 </td><td> 23g, 4분의 3 인치 </td><td> BD Vacutainer 연구소. 메드. </td><td>305,143 </td></tr><tr><td> 미국 표준 시험 체 </td><td> 150 μm의, 0.0059 &quot; </td><td> WS 타일러 주식 회사 </td><td> ASTME-11, 사양. 번호 100 </td></tr><tr><td> Xylazine </td><td> 20 MG / ML </td><td> 집사 </td><td> N / A </td></tr></tbody></table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

Schistosoma의 기생충은 전세계 약 2억명 1 감염 혈액 flukes입니다. Schistosoma, 간 섬유증 및 splenomegaly 포함한 심각한 병리,과 만성 감염에 오히려 기생충 자체가 2보다 기생충 달걀에 대한 면역 반응 때문에 발생합니다. 기생충 알은 IL-4의 생산, IL-5 및 IL-13, macrophages의 대안 활성화 및 호산구의 채용 특징으로 Th2 반응을 유도. 여기서는 기생충 특정 폐에 Th2 시토킨 반응 및 배수 림프절, 계란을 둘러싼 폐 granulomas의 형성과기도 염증을 검사하기위한 모델로 Schistosoma mansoni 달걀의 주입을 설명합니다. intraperitoneal sensitization과 정맥 도전, S. 따라 mansoni 달걀은 그들이 폐 실질 조직 내에 갇혀있다 폐의 동맥을 통해 폐로 이송된다lymphocytes, 호산구 및 또는 활성화 macrophages 3-6로 구성되어 granulomas에 의해. 야생마 - 폐포 공간에서 granuloma 형성, 염증과 연관된, 배수 림프절과 CD4의 확장은 T 세포 활성을 관찰할 수있다. 여기서 우리가 세부 사항, 감염된 간은 (7에서 변경)에서 Schistosoma mansoni 달걀을 분리 쥐를 sensitizing과 도전, 그리고 분석을위한 기관 (야생마 - 폐포 세척 (BAL), 폐 및 배수 림프절)를 복구하는 프로토콜. 우리는 또한 대표 histologic 및 추가 immunologic 분석을위한 immunologic 데이터 및 제안을 포함합니다. 전반적으로,이 방법은 생체내 모델에서이 기생충 감염, fibrotic 질환, 알레르기 염증과 천식을 포함한 Th2 염증 질환의 연구에 광범위하게 적용하는 폐 안에 기생충 유발 immunologic 응답을 조사합니다. 일반의 연구에 대해이 모델의 장점폐에서 전자이 염증은 폐에 유력한 Th2 염증 반응의 재현성 및 배수 림프절, 계란을 둘러싼 granulomas의 histologic 심사에 의한 염증의 평가의 용이성, 그리고 기생충의 장기 보관의 가능성을 포함 계란.

Watch this Scientific Journal Video about Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation at JoVE.com

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

Schistosoma mansoni 달걀은 T 도우미 유형 2 (Th2) 면역 반응, 기생충 감염의 특징, 천식과 알레르기 염증의 강력한 stimulators 있습니다. 이 프로토콜은 S.을 활용 CD4 Th2 시토킨 유도된 염증성 계란 주위에 폐 granuloma 형성을 특징으로 폐에 반응, eosinophilia와 대식 세포 대안 활성화를 생성하는 mansoni 계란 분사.

에서 계란을 사용하여<em&gt; Schistosoma mansoni</em&gt; 등<em&gt; 생체내에서</em기생충 유도된 폐 염증의&gt; 모델

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

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Immunology and Infection

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

13.7K 조회수.  University of Pennsylvania. Schistosoma mansoni 달걀은 T 도우미 유형 2 (Th2) 면역 반응, 기생충 감염의 특징, 천식과 알레르기 염증의 강력한 stimulators 있습니다. 이 프로토콜은 S.을 활용 CD4 Th2 시토킨 유도된 염증성 계란 주위에 폐 granuloma 형성을 특징으로 폐에 반응, eosinophilia와 대식 세포 대안 활성화를 생성하는 mansoni 계란 분사.

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

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 변화와 체외에서</em의> 양성 Schistosoma mansoni Schistosomules

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

연구

면역학 및 감염병학

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

이식 - 대 - 호스트 질병의 유도 및<em&gt; 생체 내</emMHC 검색 손쥐 모델을 사용&gt; T 셀 모니터링

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

생체 내 이미징 방법에 급성 및 만성 염증을 구별하는

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

<em&gt; 생체 내</em&gt; 급성 뇌 조각의 희소 돌기 아교 세포 지시 T 세포 공격의 모델

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

인간 백신 닭 계란에 인플루엔자 바이러스 전파

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

마우스 비장에서 불변 자연 살해 T 세포를 분리하고 확장을위한 최적화 방법

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

새로운 박테리아 특정의 광학 검사 Confocal 레이저 Endomicroscopy에 의해 전 비보 인간의 폐 조직에 프로브

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

세포 독성의 평가 인간의 정밀 컷 폐 분할 영역에서 물질의 Immunomodulatory 효과

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

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.

절연 및 Extracellular 소포 특성 성인 Schistosoma 나무 에서

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