저자의 연구실에서 작업은 자연 과학 및 캐나다의 공학 연구 협의회, 건강 연구의 캐나다 연구소, 그리고 캐나다 연구 의자 프로그램에서 교부금에 의해 지원됩니다. JL은 퐁 드 물리 엉 쌍떼 두 퀘벡에서 지원금을 통한 Groupe D&#39; 에투드 데스 Protéines Membranaires (GEPROM)에서 화목에 의해 지원됩니다.

1. 예선 : 박테리아 변종과 상피 세포. 세포 및 박테리아의 조작은 층류 후드 아래 무균 수행됩니다. <ol><li> 갓 E.을 분리 콜리 변종 2787, 2787Δ 아이다 및 C600 글리세롤 주식의 Lysogeny 스프 (LB) 한천 플레이트 (1 % tryptone, 0.5 %의 나트륨 염화물, 0.5 % 효모 추출물, 1.5 %의 한천)에 37에서 성장 ° C. 검정의 다양성을 최소화하기 위해, 그것은 갓 도금 변종을 항상을 사용하고 ° C 봉인 페트리 접시에서만 몇 주 최대 4에서 긴장을 유지 좋습니다. </li><li> 높은 포도당 Dulbecco의 수정 이글 중간 (DMEM)에 문화 형 간염 - 2 세포 (ATCC CCL - 23) 10 % 열 inactivated 소 혈청 (열 불활 성화가 ° C 30 분 56 수행)와 보충. 일상 문화 중에 우리는 또한 10 U / ML 페니실린 및 10 μg / ML 스트렙토 마이신을 추가합니다. </li><li> 37 형 간염 - 2 세포를 성장 ° CO 2 5 %를 포함하는 분위기 세포 배양기에서 C. 75cm 2 flasks 그들이 합류에 도달 subcultured 때마다에서 재배하는 세포를 유지하기 위해 표준 세포 배양 절차를 사용하십시오. 우리는 그들이 30-40 구절에 도달할 때까지 우리의 교양 세포를 사용하고 그들을 폐기. </li><li> 75cm이 플라스크에있는 C 형 간염 - 2 세포가 거의 합류에 도달하기 때문에 준수 분석을위한 준비가되면 분석을 준비합니다. </li></ol> 2. DAY 1 : inoculum와 상피 세포를 준비 <ol><li> 8g / L NaCl, 0.2 g / L KCl, 0.2 g / L KH 2 PO 4, 0.21 g / L 나 2 HPO 4 : : 7H 따뜻한 Dulbecco의 인산이 (DPBS 호수 버퍼와 한번 술병에있는 C 형 간염 - 2 세포를 씻어 2 O) </li><li> 신선한 따뜻한 전체 매체를 추가하기 전에 5 분 EDTA (에틸렌 다이아 민 테트라 초산) - 세포는 0.05 % 트립신과 함께 incubated 있습니다. 신선한 매체가 추가되면 전지와 위아래로 pipetting하여 resuspended 있습니다. </li><li> 5 분 2,000 RPM의 세포 현탁액을 원심 분리기와 DPBS에서 알약을 resuspend 다시 원심 분리기. </li><li> 10 %의 혈청과 보충하지만, 2x10 5 셀 / ML의 농도에서 더 항생제를 포함하지 신선한 매체 세포를 Resuspend. </li><li> 시드 그 중 한명은 24 잘 접시의 중앙에 중복 웰스 (각 변형에 대해 하나) 세​​ 가지 세트의 세포 현탁액의 밀리리터와 세포 문화 인큐베이터에서 하룻밤 접시를 품어. 참고 : 혈청이 완전히 decomplemented해야하고 항생제가이 단계 이후 생략하는 것이 중요합니다. 이렇게하지 ​​않으면 감염 박테리아를 죽일 수 있습니다. </li><li> LB의 국물 5 ML (1 % tryptone, 0.5 %의 나트륨 염화물, 0.5 % 효모 추출물)의 각 세균의 변형 중 하나를 격리 콜로니 (2787, 2787Δ 아이다 및 C600)을 접종하고 37 야간 성장 ° 활발한 잡고 C (180 RPM ). </li></ol> 3. DAY 2 : 세포의 감염 <ol><li> 그들이 최소한 90 % 합류하고 오염되지 않은 것을 확인할 거꾸로 현미경 형 간염 - 2 세포를 검사합니다. </li><li> 각 잘하는 따뜻한 DPBS하고있는 세포를 씻어, 10 %의 혈청과 보충 신선한 중간 1 ML을 추가하지만 항생제를 포함하지 않습니다. 세포없이 3 웰스 신선한 매체도를 추가합니다. 이것은 각 변형에 대한 inoculum에서 세균의 총 수를 결정하는 데 사용됩니다. </li><li> 세균성 문화 600 NM (OD 0.600 nm의)에서 광학 밀도를 측정합니다. C 형 간염 - 2 세포를 포함하는 중복 웰스 중 하나를 설정 하나 잘 포함하지 않는 세포를 각 세균성 문화의 나누어지는를 추가합니다. 보통, 우리는 10 6 콜로니 형성 단위 (cfu)에 해당하는 숙박 문화의 볼륨을 사용합니다. (세포 박테리아)이 5시 1분의 감염의 다중성을 (뫄) 나타냅니다. 우리가 직접 우리의 야간 문화를 사용하지만, (예 : cytotoxins과 같은 예를 들어) 야간 문화에있는 분비 분자의 해로운 효과를 피하기 위해 박테리아를 원심과 DPBS에서 그들을 resuspend하기 위해 때로는 좋습니다 참고 : 뫄는 100:1과 1시 10분 사이 다를 수 있습니다. 높은 뫄 높은 다양성과 배경을 산출하고 박테리아가 접시의 플라스틱에 집중하는 경향이 있습니다. 뫄 로우어도 높은 다양성을 산출. 뫄이 선택되면, 그것은 일관되게이 뫄 유지하는 필수적입니다. </li><li> 37 3 시간 동안 세포 배양기 문화에 감염된 세포를 품어 ° C 5 % CO 2. 참고 : 직접 세포 접촉에있는 모든 박테리아를 가지고하기 위해 잠시 저속 (1-2 분 예 : 1000 XG)에서 번호판을 원심 수도 있습니다. 이것은 감염을 동기화의 추가 혜택을 가지고 있으며, 짧은 배양 시간 (적게 15-30로 분)을 수 있도록합니다. </li><li> 감염된 세포에서 매체를 제거하고 따뜻한 DPBS와 세포에게 3 회 씻는다. 이 단계에서 자기편 박테리아는 일반적으로 표준 현미경으로 볼 수 우리는 정기적으로이 검사를 수행할 수 있습니다. </li><li> 세포를 lyse하고 준수 BAC를 분리teria는 트리톤 X - 100 각 1 %의 100 μl를 추가할 잘 세포를 포함하는. 기타 세제는 (예 : 예를 들어 사포닌 등) 사용할 수 있습니다. </li><li> 실온에서 10 분에 대한 세포를 부화 후 LB 매체 900 μl를 추가합니다. </li><li> 부드럽게 pipetting 상하 반복하여 suspensions을 균질. inoculum를 포함하는 세포가없는 우물은 비슷하게 부드럽게 균질하고 있습니다. 참고 : 일부 박테리아는 우리가 0.05 % 트립신과 부화하여 상피 세포를 분리되는 경우, 세제에 너무 민감한 것 - ​​EDTA (에틸렌 다이아 민 테트라 초산)을 20 분에 대해 37 ° C. 준수 박테리아와 함께 세포도 피펫 팁있는 우물에서 까진 수 있습니다. </li><li> LB 한천 3 dilutions (보통 1:1,000, 1:10,000과 1:100,000 dilutions)에서 LB의 국물과 플레이트 100 μl를 사용 준수 박테리아 및 inoculum의 suspensions 일련 10 배 dilutions을 준비하고 37 밤새 품어 ° C . </li></ol> 4. 3 일 : cfu의 계산 및 데이터 프레 젠 테이션. <ol><li> 접시에 식민지를 카운트하고 dilutions의 각 시리즈를 평균하여 준수 박테리아와 inoculum의 cfu의 개수를 계산합니다. 10-300 식민지 사이에서만 플레이트는 계산해야합니다. </li></ol> 5. 대표 결과 : 다음 표는 준수 박테리아와 다른 일을 수행할 세 실험에서 inoculum의 계수 cfu의 전형적인 결과를 보여줍니다 : <img src="/files/ftp_upload/2783/2783table1.jpg" alt="표 1" /> 그림 1A와 같이 결과는 직접으로 cfu을 준수보고 수 있습니다. inoculum 크기 때문에 성장 속도의 차이 또는 pipetting 오류의 변종마다 다를 수 있기 때문에 준수 박테리아의 비율로 결​​과를보고하는 것이 좋습니다. 그림 1B와 같이 준수 세균의 비율은, inoculum의 cfu의 숫자로 준수 박테리아의 cfu의 수를 나누어 계산하실 수 있습니다. 그림 1B의 모든 컬럼을 비교하기 위해 반복 측정 ANOVA와 Bonferroni 사후 테스트를 수행함으로써, 우리는 상당한 아이다 2787과 2787Δ 차이 (P는 &lt;0.05),뿐만 아니라 2787과 C600 사이에있다는 것을 볼 수 없지만, 더 큰 차이가 있습니다 2787Δ 아이다과 C600 사이. <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="그림 1"/> 그림 1. 준수 세균 cfu (A) 또는 준수 박테리아의 비율 (B)로 검색 결과의 표현 관찰 준수의 비율은 종종 실험에 매우 의존 셋업 (그리고, 낮은 정도로, 실험자에). 특히 중요한 뫄하고 세척의 수가 너무 비율은 문자 그대로 해석해서는 안됩니다. inoculum의 박테리아는 때때로 세포와 우물에서 세균의 실제 총수와 비교하여 inoculum의 크기를 왜곡, 세포의 존재에있는 박테리아보다 훨씬 빠르게 커질 수 있습니다. 이 문제를 완화하기 위해, 그것은 어느 것을 권장합니다 : C 형 간염 - 2 세포가 추가 우물에 씨앗을 품고 및 감염 : (I)는 inoculum을 결정하는 세포와 우물을 사용합니다. 대신 폐기의 분석의 마지막에서 뜨는 및 세탁 10 % 트리톤 100 μl는 X는 - 100은 잘에 추가됩니다. 세포가 lyse하고 준수 및 비 준수 세균을 포함하고있는 lysate는 부드럽게 반복 상하 pipetting하여 균질이며. 또는 (ii) 분석의 끝에 감염된 세포의 supernatants를 수집뿐만 아니라 supernatants에서 DPBS의 세척 및 그 풀링 supernatants에 cfu의 수를 결정합니다. 이것은 비 준수 박테리아의 cfu의 개수를 반환합니다. 준수 세균의 비율은 다음 준수 및 비 준수 박테리아의 cfu의 숫자의 추가에 의해 준수 박테리아의 cfu의 수를 나누어 계산하실 수 있습니다. 분석의 견고을 설명하기 위해, 우리는 최근에 설립 tracheal 돼지 세포 라인, NPTr 8 세포와 incubated S4074과 함께 수행한 분석, Actinobacillus pleuropneumoniae 7 접착제 돼지의의 병원성 변형,이 프로토콜을 비교할 수 있습니다. 이외에도 세균과 세포의 성장에 필요한 매체의 차이에서, 유일한 차이점은 감염에 사용되는 박테리아가 고정 상 야간 문화 기하 급수적으로 증가하는 문화, 그리고 아니라는 점이다. A.로 pleuropneumoniae 그것은 달리 독소의 분비는 세포 죽음과 바이어스 결과가 발생할 것이며, 뫄 10시 1분의를 존중하고 감염 3 시간을 초과하지하는 것도 매우 중요합니다. A.의 또한이 스트레인 그것이 합류 세포를 사용하여 세포가 꺼져 sloughing 아니라는 것을 시각적으로 확인하는 것이 중요하므로 pleuropneumoniae는 쉽게 플라스틱을 준수합니다.

<ol>
	<li>Kline, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesins+in+host-microbe+interactions.">Bacterial adhesins in host-microbe interactions.</a> Cell Host Microbe. 5, 580-580 (2009).</li><li>Cossart, P., Boquet, P., Normark, S., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+microbiology+emerging.">Cellular microbiology emerging.</a> Science. 271, 315-315 (1996).</li><li>Elsinghorst, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasion+by+gentamicin+resistance.">Measurement of invasion by gentamicin resistance.</a> Methods Enzymol. 236, 405-405 (1994).</li><li>Pizarro-Cerda, J., Lecuit, M., Cossart, P., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cellular Microbiology. , 161-161 (2002).</li><li>Srivastava, A., Isberg, R. R., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cellular Microbiology. , 179-179 (2002).</li><li>Benz, I., Schmidt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+an+adhesin+(AIDA-I)+involved+in+diffuse+adherence+of+enteropathogenic+Escherichia+coli.">Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.</a> Infect Immun. 57, 1506-1506 (1989).</li><li>Charbonneau, M. E., Berthiaume, F., Mourez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+processing+is+not+essential+for+multiple+functions+of+the+Escherichia+coli+autotransporter+adhesin+involved+in+diffuse+adherence+(AIDA-I).">Proteolytic processing is not essential for multiple functions of the Escherichia coli autotransporter adhesin involved in diffuse adherence (AIDA-I).</a> J Bacteriol. 188, 8504-8504 (2006).</li><li>Jacques, M., Paradis, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesin-receptor+interactions+in+Pasteurellaceae.">Adhesin-receptor interactions in Pasteurellaceae.</a> FEMS Microbiol Rev. 22, 45-45 (1998).</li><li>Auger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+of+Actinobacillus+pleuropneumoniae+with+porcine+lung+and+tracheal+epithelial+cells.">Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells.</a> Infect Immun. 77, 1426-1426 (2009).</li><li>Jacques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipo-oligosaccharides+and+lipopolysaccharides+in+bacterial+adherence.">Role of lipo-oligosaccharides and lipopolysaccharides in bacterial adherence.</a> Trends Microbiol. 4, 408-408 (1996).</li><li>Nataro, J. P., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diarrheagenic+Escherichia+coli.">Diarrheagenic Escherichia coli.</a> Clin Microbiol Rev. 11, 142-142 (1998).</li><li>Jallat, C., Darfeuille-Michaud, A., Rich, C., Joly, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+clinical+isolates+of+diarrhoeogenic+Escherichia+coli:+diffusely+adhering+E.+coli+strains+with+multiple+adhesive+factors.">Survey of clinical isolates of diarrhoeogenic Escherichia coli: diffusely adhering E. coli strains with multiple adhesive factors.</a> Res Microbiol. 145, 621-621 (1994).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 프로토콜은 (gentamicin 보호 분석 3을 사용 예) 침략을 공부하기 위해 수정할 수있는 표준 준수 세균 분석을 설명합니다. 콜로니 형성 단위의 계산은 Giemsa의 얼룩 같은 현미경 표준 시각화 기법에 의존 접근과 비교하여, 부량 수 있습니다. 후자는 접착력의 질적 전망을 제공하지만 접착력의 다양한 패턴을 차별하고 mechanistical 통찰력에게 9 줄 수 있기 때문에 종종 유용하게 보완합...

<table border="1"><tbody><tr><th> 시약 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 높은 포도당 DMEM </td><td> GIBCO - Invitrogen </td><td> 12430-054 </td></tr><tr><td> DDPBS </td><td> GIBCO - Invitrogen </td><td> 14190-144 </td></tr><tr><td> 보빈 성장 세럼 </td><td> Hyclone </td><td> SH3054 </td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> GIBCO - Invitrogen </td><td> 15140-148 </td></tr><tr><td> 24 웰 플레이트 </td><td> 코닝 </td><td> 3337 </td></tr><tr><td> 트리톤 X - 100 </td><td> 피셔 사이 언티픽 </td><td> BP151 - 100 </td></tr></tbody></table>

in vitro assay of bacterial adhesion onto mammalian epithelial cells

감염을 원인 박테리아들은 숙주를 식민지해야합니다. 세균성 병원균은 세포 하나를 호스트하기 위해 첨부 파일을 홍보할 수 다양한 분자 또는 구조를 표현한다. 이러한 adhesins는 숙주 세포 표면 수용체 또는 박테리아와 호스트 사이의 다리 역할을 수용성 단백질과 상호 작용에 의존하고 있습니다. 접착력 전에 침략 및 / 또는 독소의 분비에 중요한 첫 번째 단계이며, 따라서 그것은 세균 pathogenesis에서 공부하는 주요 행사입니다. 또한, 준수 박테리아는 종종 정교하게 미세 조정 세포 반응, &#39;세포 미생물학&#39;2의 분야에 출산을 가지고있는 연구를 유도. 세균성 호스트 세포의 접착과 침략에 ​​대한 강력한 assays 따라서 세균 pathogenesis 연구의 핵심 역할을하고 오랫동안 많은 선구자 실험실 3,4에서 사용되었습니다. 이러한 assays 이제 세균 pathogenesis 작업 대부분의 실험실에서 연습하고 있습니다. 여기, 우리는 특정 adhesin의 기여를 설명하는 표준 준수 분석을 설명합니다. 우리는 대장균 스트레인 2787 5, 확산 준수 (아이다)에 관련된 autotransporter Adhesin을 표현하는 인간 병원성 변형을 사용합니다. 컨트롤로서, 우리는 아이다 유전자, 2787Δ 아이다 (F. 버타이오메와 M. Mourez, 발표되지 않은), 그리고 E. 상업 실험실 부담을 부족한 돌연변이 변형을 사용하여 대장균, C600 (뉴 잉글랜드 Biolabs). 박테리아는 일반적으로 사용되는 C 형 간염 - 2 인간 상피 세포주에서 세포을 준수하기 위해 남아 있습니다. 이 분석은 덜 광범위 6 이전에 설명되었습니다.

Watch this Scientific Journal Video about In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells at JoVE.com

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

이 프로토콜은 교양 세포에 준수 아르 단위를 형성하는 박테리아 식민지의 숫자를 계산에 구성된 간단한 세균 유착 분석이다. 분석은 강력한이며, adhesin 독립 공부하고, 다수의 유사 콘텐츠는 세균 pathogenesis 작업을 대부분의 실험실에 사용됩니다.

포유류의 상피 세포에 박테리아의 접착 시험 관내 분석

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

in-vitro-assay-of-bacterial-adhesion-onto-mammalian-epithelial-cells

Research

JoVE Journal

Immunology and Infection

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

25.2K 조회수.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. 이 프로토콜은 교양 세포에 준수 아르 단위를 형성하는 박테리아 식민지의 숫자를 계산에 구성된 간단한 세균 유착 분석이다. 분석은 강력한이며, adhesin 독립 공부하고, 다수의 유사 콘텐츠는 세균 pathogenesis 작업을 대부분의 실험실에 사용됩니다.

비디오: In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

마우스의 응용은 M 세포에 의해 박테리아 이해를 평가하는 Peyer의 패치 장 루프 분석 출혈도 잡았

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

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 ...

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 ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

장 상피 세포의 장내 세균의 침입<em&gt; 체외</em&gt; 크게 수직 확산 챔버 모델을 사용하여 향상

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

시험관 내 병원균 유도 호중구 트랜스 상피 이동을 평가하는 공동 문화 분석

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

인간 호중구 유량 용기 접착 분석

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T 림프구에있는 인테그린 활성화의 연구에 대한 정적 접착 분석

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

포유 동물 세포에 기능 세균 이펙터의 전기

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

생체 외 및 생체 내 모델에서 혈관 벽에서 흐름 조건에 세균 부착을 연구하기 위해

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

T 림프구 - 접착 분자 상호 작용의 연구에 대한 접착에서 전단 응력의 분석

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...