Kanada Doğal Bilimler ve Mühendislik Araştırma Kurumu, Kanada Sağlık Araştırma Enstitüsü ve Kanada Araştırma Sandalyeler programı hibe yazarların laboratuarlarda çalışmalar desteklenmektedir. JL Viyana de Recherche en Sante du Quebec fon yoluyla Groupe d&#39;Etüt des Protéines Membranaires (GEPROM) bir burs ile desteklenmektedir.

1. Ön: Bakteriyel suşlar ve epitel hücreleri. Hücre ve bakteri manipülasyonlar laminer akış kaputu altında aseptik yapılmaktadır. <ol><li> Taze E. izole coli suşlarının 2787, 2787Δ aida C600 gliserol stokları Lysogeny Broth (LB) agar plakları (% 1 Tripton,% 0.5 sodyum klorür,% 0.5 maya özü,% 1.5 agar) ve 37 büyümeye ° C Tayininde değişkenliği en aza indirmek için, taze kaplama suşlar her zaman kullanmak ve sadece birkaç hafta maksimum 4 ° C kapalı Petri kapları suşları tutmak için tavsiye edilir. </li><li> Yüksek glukoz Dulbecco&#39;nun Modifiye Eagle Orta (DMEM) Kültür HEp-2 hücreleri (ATCC CCL-23)% 10 ısı inaktive sığır serum (ısı inaktivasyonu 56 ° C de 30 dakika süreyle yapılır) ile desteklenmiştir. Rutin kültür sırasında biz de 10 U / ml penisilin ve 10 mg / ml streptomisin ekleyin. </li><li> 37 Hep-2 hücreleri büyütün ° C% 5 CO 2 içeren bir atmosfere sahip bir hücre inkübatör. 75 cm 2 matara ve izdiham ulaşmak pasajlandı her zaman yetiştirilen hücreler, korumak için standart hücre kültürü yöntemleri kullanın. Biz kültür hücreleri 30 ila 40 pasajlar ulaşana kadar kullanın ve daha sonra atın. </li><li> 75 cm 2 balonuna HEp-2 hücreleri neredeyse izdiham ulaşan ve bu nedenle bir bağlılık testi için hazır olduğunuzda bir test hazırlayın. </li></ol> 2. 1. GÜN: inokulum hazırlanması ve epitel hücreleri <ol><li> 8 g / L NaCl, 0,2 g / L KCl, 0.2 g / L KH 2 PO 4, 0.21 g / L Na 2 HPO 4:: 7H sıcak Dulbecco&#39;nun Kullanıcı fosfat (DPBS tuzlu tamponlu ile bir kez balondaki HEp-2 hücreleri yıkayın 2 O) </li><li> Taze sıcak tam orta eklemeden önce 5 dakika boyunca EDTA hücrelerin% 0.05 tripsin ile inkübe edilir. Taze orta eklendikten sonra, hücreler yukarı ve aşağı pipetleme tarafından yeniden süspanse. </li><li> 2.000 rpm&#39;de 5 dakika boyunca hücre süspansiyonu Santrifüj ve DPBS pelet tekrar süspansiyon haline getirin ve tekrar santrifüj. </li><li> % 10 serum ile desteklenebilir ama 2x10 5 hücre / ml &#39;lik bir konsantrasyon, antibiyotik içeren taze orta hücreleri yeniden süspanse edin. </li><li> Tohum 24 plaka merkezi yinelenen kuyuları (her zorlanma biri) üç set hücre süspansiyonu mililitre ve geceleme bir hücre kültürü inkübatörde plaka kuluçkaya yatmaktadır. Not: serum iyice decomplemented ve bu antibiyotikler bu aşamadan sonra ihmal edildiği önemlidir. Bunu yapmazsanız, enfeksiyona yol açan bakterileri öldürür. </li><li> 5 ml (% 1 Tripton,% 0.5 sodyum klorür,% 0.5 maya özü) LB suyu her bakteri suşu izole koloni (2787, 2787Δ aida ve C600) İnokülasyon ve 37 o gecede büyümek ° C (180 rpm iyice sallanarak .) </li></ol> 3. 2. GÜN: hücrelerin Enfeksiyon <ol><li> Ters bir mikroskop altında en az% 90 konfluent ve kontamine olduğunu tespit etmek için HEp-2 hücreleri kontrol edin. </li><li> Her iyi sıcak DPBS ve hücreler yıkanmış, 1 ml% 10 serum ile desteklenmiş taze orta fakat antibiyotik içeren. Taze orta hücreleri olmaksızın üç kuyu da ekleyebilirsiniz. Bu her bir suş için inokulum toplam bakteri sayısını belirlemek için kullanılır. </li><li> Bakteri kültürleri, 600 nm (OD 0,600 nm) optik yoğunluk ölçülür. Hep-2 hücreleri içeren yinelenen kuyuların bir dizi ve bir de içeren hücrelerin her bakteri kültürü bir kısım ekleyin. Genellikle, biz 10 6 koloni oluşturan birim (kob) karşılık gelen bir gecede kültür hacmi kullanın. (: Hücrelerinin bakteriler) Bu bir enfeksiyon 05:01 çokluğu (İçişleri Bakanlığı) temsil eder. Biz doğrudan bir gecede kültürler kullanmalarına rağmen, (örneğin cytotoxins olarak, örneğin) bakteri gece kültürlerde salgılanan moleküllerin zararlı etkilerini önlemek için santrifüj ve DPBS onları tekrar süspansiyon bazen tavsiye edilir Not: İçişleri Bakanlığı 100:1 ve 1:10 arasında değişebilir. Yüksek İçişleri Bakanlığı, yüksek değişkenliğini ve arka plan verim ve bakteri plaka plastik sopa eğiliminde olacaktır. İçişleri Alt yüksek değişkenlik verir. Bir İçişleri Bakanlığı seçildikten sonra, tutarlı olması ve bu İçişleri Bakanlığı tutmak zorunludur. </li><li> , 37 ° 3 saat enfekte hücreleri hücre kültürü inkübatörde inkübe ° C,% 5 CO 2. Not: Bu bakteri hücreleri ile doğrudan temas halinde getirmek amacıyla, kısa bir süre düşük hızda (örneğin 1.000 xg 1-2 dakika) plaka santrifüj da mümkündür. Bu enfeksiyon senkronize da yarar vardır ve kısa inkübasyon süreleri (15-30 dakika kadar az) sağlayan. </li><li> Enfekte hücrelerden orta çıkarın ve sıcak DPBS hücreleri 3 kere yıkayın. Bu aşamada, yapışkan bakteri genellikle standart bir mikroskop ile görülebilir ve biz rutin olarak bu onay gerçekleştirmek. </li><li> Olmuş akyuvarlar ve yapıştırılır bac ayırmak içinmalıdır, Triton X-100 her biri için% 1 100 ul eklemek de içeren hücreler. Diğer deterjanlar (örneğin saponin gibi) kullanılabilir. </li><li> Hücreleri oda sıcaklığında 10 dakika inkübe edin ve sonra 900 LB ul orta ekleyin. </li><li> Süspansiyonlar pipetleme yukarı ve aşağı tekrar hafifçe karıştırılır. Inokulum içeren hücreler olmadan kuyu benzer hafifçe homojenize edilir. Not: C. Bazı bakteriler, bu durumda% 0.05 tripsin ile inkübasyon epitel hücreleri ayırmak, deterjan çok hassas olabilir - EDTA 20 dakika süreyle 37 ° Ile birlikte uyulması bakteri hücreleri de bir pipet ile kuyulardan kazınmış olabilir. </li><li> 3 LB agar dilüsyonları 1:1,000, 1:10,000 ve 1:100,000 dilüsyonları (genellikle) LB suyu ve plaka 100 ul kullanarak yapıştırılır bakteri ve inokulum süspansiyonlar seri 10 kat dilüsyonlarının hazırlayın ve 37 ° gece inkübe ° C . </li></ol> 4. 3. GÜN: kob sayma ve veri sunumu. <ol><li> Plakalar kolonileri sayın ve her dizi dilüsyonlarının ortalamasını alarak, uyulması bakteri ve inokulum kob sayısını hesaplamak. Sadece plakaları ile 10-300 koloniler arasında sayılmalıdır. </li></ol> 5. Temsilcisi sonuçları: Aşağıdaki tabloda, uyulması bakteri ve 3 farklı günlerde yapılan deneyler inokulum sayımı kob tipik sonuçları göstermektedir: <img src="/files/ftp_upload/2783/2783table1.jpg" alt="Tablo 1" /> Şekil 1A gösterildiği gibi sonuçları, doğrudan kob yapıştırılır rapor edilebilir. Inokulum boyutları, büyüme oranlarındaki farklılıklar veya pipetleme hataları nedeniyle suşları arasında değişebilir bu yana, uyulması bakteri yüzdesi olarak sonuçlarını rapor için genellikle tavsiye edilir. Şekil 1B gösterildiği gibi yapıştırılır bakterilerin yüzde inokulum mikroorganizma sayısına göre uyulması bakteri mikroorganizma sayısına bölünmesiyle hesaplanır. Şekil 1B tüm sütunları karşılaştırmak için tekrarlanan ölçümler ANOVA ve Bonferroni post-test yaparak, aida 2787 ve 2787Δ arasında önemli farklılıklar (p &lt;0.05) yanı sıra 2787 ve C600 arasında olduğunu, ancak anlamlı bir fark 2787Δ Aida ve C600 arasında. <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="Şekil 1"/> Şekil 1 uyulması bakteri kob (A) veya yapıştırılır bakteri yüzdesi (B) gibi sonuçlar Temsilciliği Gözlenen bağlılığın yüzde genellikle deneysel çok bağımlı set-up (ve bir dereceye kadar, deneyci). Özellikle önemli olan İçişleri Bakanlığı ve yıkar sayısı yüzdesi anlamıyla yorumlanmalıdır olmamalıdır. Inokulum bakteriler bazen bakteri hücreleri ile kuyu gerçek toplam sayısı ile karşılaştırılması inokulum büyüklüğü halde, hücre varlığında bakteriler çok daha hızlı büyüyebilir. Bu sorunu hafifletmek için, ya da tavsiye edilir: HEp-2 hücreleri iyi ek bir numaralı seribaşı ve enfekte: (i) hücreleri ile inokulum belirlemek için kuyu kullanabilirsiniz. Testin sonunda, At yerine atarak süpernatant ve çamaşır 100 ul,% 10 Triton X-100 de eklenir. Olmuş akyuvarlar ve yapıştırılır ve non-uyulması bakteri içeren lizat yavaşça tekrarlanan yukarı ve aşağı pipetleme homojenize veya, (ii) testin sonunda süpernatantlar enfekte olmuş hücreleri toplamak gibi süpernatantlar DPBS yıkar ve bu havuzlu Süpernatantları mikroorganizma sayısını belirlemek. Bu kob olmayan uyulması bakteri sayısı verecektir. Uyulması bakteri yüzdesi sonra yapıştırılır ve non-uyulması bakteri mikroorganizma sayısının yanı sıra uyulması bakteri mikroorganizma sayısına bölünmesiyle hesaplanır. Testinin sağlamlığını göstermek için, kısa bir süre önce kurulan trakeal piglet hücre hattı, NPTr 8 hücreleri ile inkübe S4074 ile yapılan tahlil, Actinobacillus pleuropneumoniae 7 yapışkan bir domuzlara patojenik, bu protokolü karşılaştırın. Bakteri ve hücrelerin büyüme için gerekli ortam farklılıkları bir yana bırakırsak, tek fark, bakteri enfeksiyonu için kullanılan sabit bir fazlı gecede kültür katlanarak büyüyen bir kültür ve bu. A. pleuropneumoniae aksi toksinlerin salgılanması hücre ölümüne neden olacak ve sonuçlar önyargı, bir İçişleri Bakanlığı 10:01 saygı ve enfeksiyon 3 saati geçmemelidir de çok önemlidir. A. ek olarak, bu gerginlik konfluent hücreleri ve hücrelerin dökülmesi kapalı olmadığını görsel doğrulamak için önemli böylece pleuropneumoniae plastik kolayca yapışabilir.

<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>

Bu protokol işgali çalışma (gentamisin koruma tahlil 3 kullanarak) modifiye edilebilir bir standart bakteriyel aderans deneyi açıklar. Koloni oluşturan birim sayma, Giemsa boyama gibi yaklaşımlar, mikroskop altında standart görselleştirme teknikleri dayanarak ile karşılaştırıldığında, ölçme sağlar. İkincisi, sadece yapışma niteliksel bir görünüm verir ama yapışma çeşitli desenler farklılaştırmak ve mechanistical anlayışlar vermek 9 yılından bu yana sık sık ...

<table border="1"><tbody><tr><th> Reaktif </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Yüksek glikoz 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> Bovine Büyüme Serumu </td><td> Hyclone </td><td> SH3054 </td></tr><tr><td> Penisilin / Streptomisin </td><td> Gibco-Invitrogen </td><td> 15140-148 </td></tr><tr><td> 24 kuyucuğu </td><td> Corning </td><td> 3337 </td></tr><tr><td> Triton X-100 </td><td> Fisher Scientific </td><td> BP151-100 </td></tr></tbody></table>

in vitro assay of bacterial adhesion onto mammalian epithelial cells

Enfeksiyonlara neden, bakteri, ev sahibi kolonize gerekir. Bakteriyel patojenler, hücreleri 1 barındırmak için ek teşvik etmek için mümkün olan çeşitli moleküller veya yapıları ifade eder. Bu adhezinleri konak hücre yüzey reseptörleri veya bakteri ve ev sahibi arasında bir köprü olarak hareket çözünür proteinler ile etkileşim güveniyor. Yapışma işgali ve / veya toksin salgılanması önce kritik bir ilk adım, bu nedenle bakteriyel patogenezi incelenmesi gereken önemli bir olay. Ayrıca, uyulması bakteri genellikle zarif ince ayar hücresel yanıtları, &#39;cep mikrobiyoloji&#39; 2 alanına doğum yapmış olan çalışmalar neden. Bakteriyel konak hücrelerine yapışma ve istila için sağlam testler, bu nedenle bakteriyel patogenezi çalışmalarda kilit rol oynayan ve uzun birçok öncü laboratuarlar 3,4 kullanılmıştır . Bu testler bakteriyel patogenezi üzerinde çalışan laboratuvarlar tarafından uygulanmaktadır. Burada, belirli bir adhesin katkıları gösteren standart bir bağlılık tahlil açıklar. Biz, Escherichia coli suşu 2787 5, Diffüz bağlılık (AIDA) ile ilgilendim Autotransporter Adhesin ifade eden bir insan patojenik kullanın . Kontrol olarak, Aida gen, 2787Δ aida (F. Berthiaume ve M. Mourez, yayınlanmamış), ve E. ticari bir laboratuvar suşu yoksun bir mutant suşu coli, C600 (New England Biolabs). Bakteriler yaygın olarak kullanılan HEp-2 insan epitel hücre hattı hücrelerinde uymak bırakılır. Bu testte, 6 önce, daha az yoğun tarif edilmiştir .

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

Bu protokol, kültür hücreleri üzerine yapıştırılır birimler oluşturan bakteri kolonisi sayısının sayma oluşan basit bir bakteriyel yapışma testtir. Testin sağlam, adhesin bağımsız olarak okudu ve çeşitli varyasyonlar bakteriyel patogenezi üzerinde çalışan çoğu laboratuvarlarda kullanılır.

Bakteriyel Yapışma Memeli Epitel Hücreleri üzerine In Vitro Testi

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

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JoVE Journal

Immunology and Infection

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

25.3K Görüntüleme.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. Bu protokol, kültür hücreleri üzerine yapıştırılır birimler oluşturan bakteri kolonisi sayısının sayma oluşan basit bir bakteriyel yapışma testtir. Testin sağlam, adhesin bağımsız olarak okudu ve çeşitli varyasyonlar bakteriyel patogenezi üzerinde çalışan çoğu laboratuvarlarda kullanılır.

Video: 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.

Fare Uygulama M hücreleri tarafından Bakteriyel Alımı değerlendirin Peyer Patch Bağırsak Loop Testi bağlandı

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

İmmünoloji ve Enfeksiyon

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.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

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.

Nicel<em&gt; In vitro</emStatik Koşullarında Account İlköğretim İnsan mikrovasküler endotel hücreleri için Nötrofil yapışma Tedbir&gt; Testi

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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.

Bağırsak Epitel Hücreleri Of Enterik Bakteriyel Invasion<em&gt; In Vitro</em&gt; Dramatik bir Dikey Difüzyon Odası Modeli Geliştirilmiş mı

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.

Tüp bebek Patojen Kaynaklı Nötrofil Trans-epitel Göçünü Değerlendirmek için Coculture Tahlili

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.

İnsan nötrofil Akış odası Yapışma Deneyi

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 Lenfositlerindeki Integrin Aktivasyonunun Çalışması için statik Adezyon Deneyi

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.

Memeli Hücreleri içinde fonksiyonel Bakteriyel Efektör elektroporasyonu

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.

In Vitro ve İn Vivo Modeli Gemi Duvar Altında Akış Koşulları Bakteriyel Yapışma Eğitim için

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 Lenfosit-Yapışma Molekülü Etkileşimleri Çalışmaları Yapışma Altında Kesme Stres Testi

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