JHN is supported by the Swiss National Foundation (SNSF 310030_146290).

The authors have nothing to disclose.

her modelde olduğu gibi, yukarıda tarif edilen antijen odaklı kolit modeli tekniği gerçekleştiren araştırmacı farkında olmalıdır birkaç sorunları ortaya çıkabilir. OT-II hosts / Kırmızı +, CD4 + T CD62L + hücreleri enjekte zaman araştırmacı periton boşluğuna iğneyi çok nazik ve dikkatli olmak gerekir. Aksi takdirde, ölüme yol açabilecek bir fare ya da bir hastalık oluşturmak olmaz hücre deri altına bağırsak yırtılabilir. <p class="jove...

bağırsak dış ortama maruz vücudun büyük bir yüzeydir. yerleşik mikropların geniş diziler bağırsak Mikrobiyota (veya mikroflora) oluşturmak için insan bağırsağa kolonize. Bu kadar 100 trilyon mikrobiyal hücrelerin oluştuğu tahmin ve biyoloji 1-3 bilinen en yoğun nüfuslu bakteriyel habitatların biri oluşturmaktadır. GIT bakteriler hayatta ve 4 çarpın bir bağırsak niş kolonize. Buna karşılık, Mikrobiyota genomu 1 kodlanmış almayan ek fonksiyonel özellikleri ile ev sahibi endows. Örneğin Mikrobiyota, epitel hücrelerinin çoğalmasını uyarır kendileri tarafından ev sahipliği üretemez vitamin üretir, metabolizmasını düzenler ve patojenlere 4-6 karşı korur. Bu yarar ilişkisi göz önüne alındığında, bazı yazarlar insanların bakteriyel ve insan genlerinin 7,8 bir karışımı olan &quot;süper organizmalar&quot; veya &quot;holobionts&quot; olduğunu ileri sürmüşlerdir. (Insan) ana bilgisayarda Mikrobiyota yararlı etkileri göz önüne alındığında, intestinal bağışıklık sistemi lümen varlıklarını etkinleştirmek değil, aynı zamanda luminal kenarında 9-11 istila patojenleri öldürmek için ortakçı mikropların tahammül gerekiyor. Bağırsak bağışıklık sistemi zararsız ve potansiyel olarak zararlı lümen mikroplar ayırt etmek mekanizmalar geliştirmiştir; Ancak bu mekanizmalar henüz tam olarak 12 anlaşılamamıştır. Bağırsak bütünlüğünü korumak tolerans ve bağışıklık 13 arasındaki dengeyi korumak için sıkı regüle bağışıklık homeostazı gerektirir. Immün homeostasisin bir dengesizlik enflamatuar bağırsak hastalığı (IBD), 3,14 gibi bağırsak hastalıklarının indüksiyonu katkıda bulunur. Crohn hastalığı (CD) ve ülseratif kolit (UC), iki büyük IBD tipi vardır. Bu hastalıkları olan hastalar genellikle rektal kanama, şiddetli ishal ve karın ağrısı 15,16 muzdarip. IBD tek sebebi halagenetik faktörler, çevresel etkilere ve düzensiz immün yanıtın bilinmeyen, ama bir arada hastalık gelişimi 15 anahtar olay olabilir. IBD için hayvan modelleri 50 yılı aşkın bir süredir kullanılmaktadır. Son birkaç yıl içinde yeni IBD model sistemler IBD 17,18 patogenezinde ilişkin çeşitli hipotezleri test etmek için geliştirilmiştir. Kronik kolit en iyi karakterize edilmiş bir model T-hücresi homeostazında 19,20 bozulmasını mukozada T-hücre transferi modelidir. 16,21 Bu model T ve (- / - ve SCID farelerde bu RAG gibi) B hücreleri eksikliği konaklara bağışıklığı yeterli fareden naif T hücreleri aktarımı kapsamaktadır. Bu modelde hastalığın gelişimi vücut ağırlığı ishal varlığı, düşük fiziksel aktivite ve zarar değerlendirerek 3-10 hafta boyunca izlenir. Bu yüzden israf sendromu 16 olarak adlandırılır. Sağlıklı farelere kıyasla nakledilen konak kolon doku thicke olduğunur, daha kısa ve 16 ağır. T hücre transferi modelini kullanarak, T hücre popülasyonları IBD 22 patogenezinde nasıl katkıda bulunabileceğini farklı anlamak mümkündür. T hücre transferi modeli antijene özgü bir şekilde hastalık sürecinde APC ve T hücreleri arasındaki etkileşim analiz etmez. Miyeloid hücreler ve lenfoid hücreler arasında bir etkileşim bağırsak iltihabı 23 gelişiminden sorumlu olabileceği gösterilmiştir. IBD pek çok açıdan açıklık olmasına rağmen, hastalık gelişmesine neden ilk olaylar hala net bir şekilde anlaşılması gerekir. Bu Mikrobiyota transfer kolit yokluğunda gösterilmiştir 24 tespit edilemez. Son zamanlarda, çeşitli teoriler İBH bakterilere 25 karşı bir bağışıklık tepkisinin bir sonucu olabileceğini düşündürmektedir. Yazarlar ayrıca ortakçı bakteri uzak bağırsakta inflamasyon uyarmak için gerekli olduğunu önerdi26. Germ bilgilerini (GF) hayvanlar intestinal immün sistemi genellikle 27,28 bozulmuş, fakat tam yetkin bağırsak bağışıklık sisteminin 29 geliştirilmesine özel patojen içermeyen bakteriler sonuç karışımı ile, bu farelerde bir kolonizasyon. Bu nedenle, Mikrobiyota yatkınlık veya bağırsak iltihabı 30,31 gelişmesine karşı koruyan bir mekanizma olarak ya patogenezde önemli bir unsur olarak gözükmektedir. Güncel teoriler İBH genetik olarak yatkın hastalarda 32, dysbiosis denilen mikrobiyal dengesizlik, bir sonucu olduğunu düşündürmektedir, ancak henüz belli değil dysbiosis neden veya hastalık 12 sonucu ise. IBD gelişiminde mikroorganizmaların rolü dikkate alındığında, in vitro deneyler, CD4 + T hücreleri, bağırsak bakterileri 33,34 ile doldurulan APC &#39;ler ile aktive edilebilir olduğunu göstermiştir. Ayrıca, antijenlerin gösterilmiştirBöyle E. gibi farklı kommensal bakteri türleri, E. coli, Bacteroides, Eubacterium ve Proteus, CD4 + T hücrelerini aktive 35 edebiliyoruz. Bu T hücrelerine bakteriyel antijenlerin sunumu IBD gelişimi için önemli olduğunu gösterir. Hastalık sürecinde mikroflorasının türetilen birden fazla antijene karmaşıklığını azaltmak için, bir E. coli suşu OVA antijen üreten oluşturuldu. - / - Transfer kolit RAG olarak OVA spesifik T hücreleri enjekte edilerek indüklendi hayvanların OVA eksprese eden E. ile kolonize E. coli. Bu model CX 3 CR1 + milletvekilleri, kolon lamina propria (CLP) 36 büyük bir hücre alt kümesi, aktarım sırasında CD4 + T hücreleri ile etkileşim olduğunu düşündüren son kanıtlara dayanmaktadır 37 kolit. Milletvekilleri kendi dendrit 36, 38,39 kullanılarak, bakteri gibi partikül antijeni için bağırsak lümen, örnek. Önceki çalışmalarMilletvekilleri ayrıca 40,41 lümen intestinal sokulan gibi OVA olarak çözünür antijenler, sürebilir gösterdi. CLP CX 3 CR1 + milletvekilleri bolluğu göz önüne alındığında, bu hücrelerin lümen bakterileri örnek ve CD4 T hücreleri ile etkileşim olması mümkündür. E. ile kolonize OVA&#39;ya özel CD4 + T hücreleri ile transplante farelerin Konfokal görüntüleme E. coli CFP OVA CX 3 CR1 + MP&#39;ler antijen tahrik kolit gelişimi sırasında OT-II, CD4 + T hücresi ile temas içinde olduğunu göstermektedir. Bu model, bağırsak APC &#39;sadece bağırsak lümeninde özel bir antijen-ifade eden bakteriler için spesifik T hücreleri arasındaki antijen sunumu işlemi etüdüne olanak sağlamıştır.

<table><tbody><tr><td>LB Broth, Miller (Luria-Bertani)</td><td>Difco</td><td>244620</td><td></td></tr><tr><td>Rotary Shake</td><td>Reiss Laborbedarf e. K.</td><td>Model 3020 GFL</td><td></td></tr><tr><td>2 mm gap couvettes&amp;nbsp;</td><td>Peqlab Biotechnologie GmbH</td><td>71-2020</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516-100ML</td><td></td></tr><tr><td>Gene Pulser Xcell system&amp;nbsp;</td><td>BioRad Laboratories GmbH</td><td>1652660</td><td></td></tr><tr><td>LB Agar, Miller (Luria-Bertani)</td><td>Difco</td><td>244510</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9393-5G</td><td></td></tr><tr><td>SOC Medium</td><td>Sigma-Aldrich</td><td>S1797-100ML</td><td></td></tr><tr><td>High Pure Plasmid Isolation Kit</td><td>Roche</td><td>11754777001</td><td></td></tr><tr><td>Agarose</td><td>Carl Roth GmbH &amp;amp; Co</td><td>3810.1</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884-100G</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T5941</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>537020&amp;nbsp;</td><td></td></tr><tr><td>Gel chamber&amp;nbsp;</td><td>PEQLAB Biotechnology GmbH</td><td>40-0708</td><td></td></tr><tr><td>Loading Dye</td><td>Thermo Fisher</td><td>R0611</td><td></td></tr><tr><td>GeneRuler 1 kb DNA Ladder&amp;nbsp;</td><td>Thermo Fisher</td><td>SM0312</td><td></td></tr><tr><td>Ethidium bromide solution</td><td>Carl Roth GmbH &amp;amp; Co. KG</td><td>2218.3</td><td></td></tr><tr><td>Photo-documentation system&amp;nbsp;</td><td>Decon Science Tech GmbH</td><td>DeVision G&amp;nbsp;</td><td></td></tr><tr><td>DNA sequencing&amp;nbsp;</td><td>MWG-Biotech GmbH</td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Biochrom</td><td>L182-50</td><td></td></tr><tr><td>Fluorescent microscope&amp;nbsp;</td><td>Zeiss</td><td>HBO 100</td><td></td></tr><tr><td>Mini-PROTEAN Tetra System</td><td>Bio-Rad Laboratories GmbH</td><td>1658005</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder&amp;nbsp;</td><td>Fermentas, St. Leon-Rot, Germany</td><td></td><td></td></tr><tr><td>IstanBlue Solution</td><td>Expedeon, Cambridgeshire, United Kingdom</td><td></td><td></td></tr><tr><td>Nitrocellulose membrane&amp;nbsp;</td><td>Macherey-Nagel GmbH &amp;amp; Co. KG</td><td>741280</td><td></td></tr><tr><td>Electro blotter&amp;nbsp;</td><td>Biometra GmbH</td><td>846-015-600</td><td></td></tr><tr><td>Bovine Serum Albumins (BSA)</td><td>Sigma-Aldrich</td><td>A6003-25G</td><td></td></tr><tr><td>Anti-Ovalbumin antibody&amp;nbsp;</td><td>Abcam</td><td>ab181688</td><td></td></tr><tr><td>Anti-rabbit IgG&amp;nbsp; HRP</td><td>Sigma-Aldrich</td><td>A0545&amp;nbsp;</td><td></td></tr><tr><td>Pierce ECL Plus Western Blotting Substrate</td><td>Pierce Biotechnology, Thermo Fischer Scientific Inc</td><td>32132</td><td></td></tr><tr><td>Forene</td><td>Abbott</td><td>2594.00.00</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10500-064</td><td></td></tr><tr><td>Falcon Cell Strainers</td><td>Fischer Scientific&amp;nbsp;</td><td>08-771-2</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>254134-5G</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>10708976001</td><td></td></tr><tr><td>CD4&lt;sup&gt;+&lt;/sup&gt; CD62 L&lt;sup&gt;+&lt;/sup&gt; T isolation kit&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-093-227&amp;nbsp;</td><td></td></tr><tr><td>MACS LS Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MACS MS Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>MidiMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>MiniMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Feeding Needle 20G</td><td>SouthPointe Surgical Supply, Inc</td><td>FN-7903</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Paraffin</td><td>Sigma-Aldrich</td><td>1496904</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>H9627</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma-Aldrich</td><td>230251&amp;nbsp;</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D9779&amp;nbsp;</td><td></td></tr><tr><td>Collagenase type VIII</td><td>Sigma-Aldrich</td><td>C-2139</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) medium</td><td>AppliChem</td><td>A2044, 9050</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>438456</td><td></td></tr><tr><td>Mouse BD Fc Block</td><td>BD Pharmingen</td><td>553141</td><td></td></tr><tr><td>FITC-conjugated mAb binding V&amp;szlig; 5.1, 5.2&amp;nbsp;</td><td>BD Pharmingen</td><td>553189</td><td></td></tr><tr><td>APC-conjugated mAb binding CD4 GK1.5&amp;nbsp;</td><td>eBioscience</td><td>17-0041-83</td><td></td></tr><tr><td>FACS Calibur&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FCS Express V3 software</td><td>DeNovo</td><td></td><td></td></tr><tr><td>Meta scanning confocal microscope&amp;nbsp;</td><td>Zeiss</td><td>LSM 710&amp;nbsp;</td><td></td></tr><tr><td>Zeiss Workstation</td><td>Zeiss</td><td>LSM 7</td><td></td></tr><tr><td>Zeiss ZEM software&amp;nbsp;</td><td>Zeiss</td><td>v4.2.0.121</td><td></td></tr><tr><td>Maxisorp immuno plates&amp;nbsp;</td><td>NUNC, Roskilde</td><td>442404</td><td></td></tr><tr><td>Streptavidin conjugated alkaline phosphatase</td><td>Jackson Immuno Research</td><td>016-050-084</td><td></td></tr><tr><td>Alkaline phosphatase substrate 4-Nitrophenyl phosphate disodium salt hexahydrate</td><td>Sigma-Aldrich</td><td>71768-5G</td><td></td></tr><tr><td>mAb R4-6A2</td><td>BD Biosciences</td><td>551216</td><td></td></tr><tr><td>mAb XMG1.2&amp;nbsp;</td><td>BD Biosciences</td><td>554410</td><td></td></tr><tr><td>TECAN microplate-ELISA reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>EasyWin software</td><td>Tecan</td><td></td><td></td></tr><tr><td>DNase I recombinant</td><td></td><td>4536282001</td><td></td></tr></tbody></table>

development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to t cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

Antijen tahrik kolit modeli, bir E. kurmak coli suşu güçlü yapısal promotör P hiper (Şekil 1A) kontrolü altında ifade edilir CFP gen tavuk ovalbümin proteininin füzyon yapısı için kodlama dizisine kaynaşmış olduğu bir plazmidi içeren inşa edilmiştir. Floresan mikroskopi gösterir Rekombinant E. E. coli pCFP OVA, fakat ana E. E. coli DH10B, CFP (Şekil 1B) ifa...

Watch this Scientific Journal Video about Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells at JoVE.com

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Bir Antijen odaklı Kolit Modelinin Geliştirilmesi Antijen T hücreleri için Hücreler sunulması ile Antijen Sunumu Eğitim için

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

development-an-antigen-driven-colitis-model-to-study-presentation

Research

JoVE Journal

Immunology and Infection

9.0K Görüntüleme.  University College Cork. In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Video: Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Azoxymethane (AOM) ve Dekstran Sülfat Sodyum (DSS) ile Modelleme Kolit-İlişkili Kanser

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Tıp

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Fare Barsak ayrı dendritik hücreler ve makrofajlar İzolasyonu ve Karakterizasyonu

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

İmmünoloji ve Enfeksiyon

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

<em&gt; Citrobacter rodentium</em&gt; Mouse Model: Bulaşıcı Kolit için çalışılması Patojen ve Host Katılımlar

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

İmmünofluoresans kullanarak TNBS'ye Bağlı Crohn Hastalığı olan Farelerin Kolonlarında Östrojen Reseptörlerinin Görüntülenmesi

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Biyokimya

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

DNBS / TNBS kolit Modelleri: Koliti içine Trendleri ve Diyet Yağ Etkileri sağlanması

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Efektör CD4 Evlatlık transferi ile Kemirgen Bağırsak Enflamasyon indüksiyonu<sup&gt; +</sup&gt; CD45RB<sup&gt; Yüksek</sup&gt; Bağışık yetmezliği olan farelere içine T Hücreleri

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Raportör fareler kullanılarak IL-22 eksprese eden lenfositlerin Görselleştirme

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Fare Distal kolostomi, C57BL/6 farelerde saptırma kolit roman modeli

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

<meta charset="utf8"></head><body>CD40 Agonistik Antikor ile İndüklenen Kolit Modelinde Hedef Gen Fonksiyonunun CRISPR/Cas9 Tabanlı Teknolojiler Kullanılarak İncelenmesi

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

CD40 Agonistik Antikor ile İndüklenen Kolit Modelinde Hedef Gen Fonksiyonunun CRISPR/Cas9 Tabanlı Teknolojiler Kullanılarak İncelenmesi

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

Bir Antijen odaklı Kolit Modelinin Geliştirilmesi Antijen T hücreleri için Hücreler sunulması ile Antijen Sunumu Eğitim için

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CD40 Agonistik Antikor ile İndüklenen Kolit Modelinde Hedef Gen Fonksiyonunun CRISPR/Cas9 Tabanlı Teknolojiler Kullanılarak İncelenmesi