scholarly journals Tu1847 – Urease-Positive Proteobacteria in Crohn's Disease Identified by Novel Ex-Vivo Mucosal Microbe Culture Combined with Metgenomic Sequencing (MC-MGS). the Enigma Study

2019 ◽  
Vol 156 (6) ◽  
pp. S-1146
Author(s):  
Erwin Berendsen ◽  
Emily C. Hoedt ◽  
Jing-Jie Teh ◽  
Jingwan Zhang ◽  
Fen Zhang ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo

ACTIVATED INTESTINAL MUSCLE CELLS PROMOTE PREADIPOCYTE MIGRATION: A NOVEL MECHANISM OF CREEPING FAT FORMATION IN CROHN’S DISEASE

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S34-S34
Author(s):  
Ren Mao ◽  
Genevieve Doyon ◽  
Ilyssa Gordon ◽  
Jiannan Li ◽  
Sinan Lin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Muscularis Propria ◽  
Dominant Factor ◽  
Stricture Formation ◽  
Intestinal Tissue ◽  
Mesenteric Fat

Abstract Background and Aims Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn’s disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. Methods Tissues from normal, ulcerative colitis, non-strictured and strictured Crohn’s disease intestinal specimens were obtained. Fresh and decellularized tissue, mesenteric fat explants, primary human adipocytes, pre-adipocytes, muscularis propria cells, and native extracellular matrix were used in multiple ex vivo and in vitro systems involving cell growth, differentiation and migration, proteomics, and integrin expression. Results Crohn’s disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by TGF-b1. The muscle cell-derived matrix triggered migration of pre-adipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5β1 on the pre-adipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularized intestinal tissue extracellular matrix. Conclusion Crohn’s disease creeping fat appears to result from the migration of pre-adipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.

scholarly journals Novel strain-level resolution of Crohn’s disease mucosa-associated microbiota via an ex vivo combination of microbe culture and metagenomic sequencing

2021 ◽  
Author(s):  
J. J. Teh ◽  
E. M. Berendsen ◽  
E. C. Hoedt ◽  
S. Kang ◽  
J. Zhang ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Taxonomic Composition ◽  
Strain Level ◽  
Patient Specific ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Functional Characterisation ◽  
Bacterial Lineages

AbstractThe mucosa-associated microbiota is widely recognized as a potential trigger for Crohn’s disease pathophysiology but remains largely uncharacterised beyond its taxonomic composition. Unlike stool microbiota, the functional characterisation of these communities using current DNA/RNA sequencing approaches remains constrained by the relatively small microbial density on tissue, and the overwhelming amount of human DNA recovered during sample preparation. Here, we have used a novel ex vivo approach that combines microbe culture from anaerobically preserved tissue with metagenome sequencing (MC-MGS) to reveal patient-specific and strain-level differences among these communities in post-operative Crohn’s disease patients. The 16 S rRNA gene amplicon profiles showed these cultures provide a representative and holistic representation of the mucosa-associated microbiota, and MC-MGS produced both high quality metagenome-assembled genomes of recovered novel bacterial lineages. The MC-MGS approach also produced a strain-level resolution of key Enterobacteriacea and their associated virulence factors and revealed that urease activity underpins a key and diverse metabolic guild in these communities, which was confirmed by culture-based studies with axenic cultures. Collectively, these findings using MC-MGS show that the Crohn’s disease mucosa-associated microbiota possesses taxonomic and functional attributes that are highly individualistic, borne at least in part by novel bacterial lineages not readily isolated or characterised from stool samples using current sequencing approaches.

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

scholarly journals CD4+ Tissue-resident Memory T Cells Expand and Are a Major Source of Mucosal Tumour Necrosis Factor α in Active Crohn’s Disease

2019 ◽  
Vol 13 (7) ◽  
pp. 905-915 ◽  
Author(s):  
Shrinivas Bishu ◽  
Mohammed El Zaatari ◽  
Atsushi Hayashi ◽  
Guoqing Hou ◽  
Nicole Bowers ◽  
...  
Keyword(s):  
T Cells ◽  
Crohn’S Disease ◽  
T Cell ◽  
Crohn's Disease ◽  
Tumour Necrosis Factor ◽  
Tumour Necrosis ◽  
Ex Vivo ◽  
Factor Α ◽  
And Control ◽  
Necrosis Factor

Abstract Background and Aims Tumour necrosis factor [TNF]α- and IL-17A-producing T cells are implicated in Crohn’s disease [CD]. Tissue-resident memory T [TRM] cells are tissue-restricted T cells that are regulated by PR zinc finger domain 1 [PRDM1], which has been implicated in pathogenic Th17 cell responses. TRM cells provide host defence but their role in CD is unknown. We thus examined CD4+ TRM cells in CD. Methods Colon samples were prospectively collected at endoscopy or surgery in CD and control subjects. Flow cytometry and ex vivo assays were performed to characterise CD4+ TRM cells. Results CD4+ TRM cells are the most abundant memory T cell population and are the major T cell source of mucosal TNFα in CD. CD4+ TRM cells are expanded in CD and more avidly produce IL-17A and TNFα relative to control cells. There was a unique population of TNFα+IL-17A+ CD4+ TRM cells in CD which are largely absent in controls. PRDM1 was highly expressed by CD4+ TRM cells but not by other effector T cells. Suppression of PRDM1 was associated with impaired induction of IL17A and TNFA by CD4+ TRM cells Conclusions CD4+ TRM cells are expanded in CD and are a major source of TNFα, suggesting that they are important in CD. PRDM1 is expressed by TRM cells and may regulate their function. Collectively, this argues for prospective studies tracking CD4+ TRM cells over the disease course.

Semi-Quantification of Ex-Vivo Sonoelastography in Mesenterial Lymph Nodes from Patients with Adenocarcinomas and Crohn's Disease

2011 ◽  
Vol 37 (8) ◽  
pp. S94
Author(s):  
R.F. Havre ◽  
S. Leh ◽  
O.H. Gilja ◽  
S. Ødegaard ◽  
J.E.R. Waage ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Lymph Nodes ◽  
Ex Vivo

scholarly journals Ex vivo model for a new bilateral antimesenteric V-modified side-to-side isoperistaltic anastomosis to prevent recurrence in ileocolic Crohn’s disease

2019 ◽  
Vol 101 (5) ◽  
pp. 313-317 ◽  
Author(s):  
V Celentano ◽  
F Luvisetto ◽  
S Toh
Keyword(s):  
Crohn’S Disease ◽  
Small Bowel ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
High Rate ◽  
Ex Vivo Model ◽  
Repeat Surgery ◽  
Handsewn Anastomosis ◽  
Surgical Recurrence ◽  
Mesenteric Border

Introduction The high rate of recurrence following ileocaecal resection for Crohn’s disease may lead to repeat surgery in 20–30% of patients at five years after surgery. Recurrence usually occurs at the anastomosis and the neoterminal ileum and the association of a strictureplasty to widen the bowel lumen in the regions immediately proximal (‘anastomotic inlet’) and distal (‘anastomotic outlet’) to the anastomosis may delay or reduce the risk of surgical recurrence. Materials and methods A side to side isoperistaltic anastomosis, with an associated V-modified strictureplasty on the anti-mesenteric border at the level of the anastomosis inlet and outlet has been designed. We produced a wet lab ex vivo model of the anastomosis and, to evaluate the different calibre of the anastomotic segments, we compared it with ex vivo models of three anastomotic configurations currently used in surgery for Crohn’s disease: i) side to side isoperistaltic anastomosis; ii) modified side-to-side isoperistaltic anastomosis with double Heineke–Mikulicz procedure (Sasaki anastomosis); iii) anti-mesenteric functional end-to-end handsewn anastomosis (Kono-S anastomosis). Results Differences were recorded at the level of the anastomosis inlet and outlet, with a larger volume estimated in the Sasaki anastomosis and in the V-modified anastomosis. The V-modified anastomosis had a larger volume compared with the Sasaki anastomosis for a longer segment of small bowel. Conclusions We have developed an experimental animal model for a new anastomotic technique which could be applied in surgery for Crohn’s disease following small-bowel or ileocolic resection.

Intestinal CD14+ Macrophages Protect CD4+ T Cells From Activation-induced Cell Death via Exosomal Membrane TNF in Crohn’s Disease

2020 ◽  
Vol 14 (11) ◽  
pp. 1619-1631 ◽  
Author(s):  
Huashan Liu ◽  
Zhenxing Liang ◽  
Fengwei Wang ◽  
Xiaobin Zheng ◽  
Ziwei Zeng ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Crohn’S Disease ◽  
T Cell ◽  
Crohn's Disease ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Cd4 T Cell ◽  
Activation Induced Cell Death ◽  
Sustained Activation

Abstract Background and aims Sustained activation of CD4+ T cells plays important roles in the pathogenesis of Crohn’s disease [CD]. Under physiologic conditions, activated T cells can be timely eliminated by a process termed activation-induced cell death [AICD], restraining T cell over-activation and preventing immunological destruction. We inquired whether defective AICD might explain CD4+ T cell over-activation in CD and investigated the underlying mechanisms. Methods CD14+ macrophages [Mφ] and CD4+ T cells were isolated from intestinal tissues or peripheral blood of controls and CD patients. An ex vivo evaluation system was employed to simulate AICD and cell apoptosis was measured by flow cytometry. Results CD4+ T cells from CD patients fail to undergo AICD in the ex vivo system. Specifically, proinflammatory type 1 helper T [Th1] and type 17 helper T [Th17] cells, rather than immunosuppressive regulatory T [Treg] cells evade AICD in CD. CD14+ Mφ in the intestinal inflammatory microenvironment of CD promote AICD resistance in CD4+ T cells via a cell-to-cell contact-independent manner. Mechanistically, CD14+ Mφ released exosomes express membrane tumour necrosis factor [TNF] which engages TNFR2 on CD4+ T cells and triggers NF-κB signalling, thereby causing AICD resistance. Importantly, clinically applicable anti-TNF antibodies effectively blocked exosomal membrane TNF-induced CD4+ T cell AICD resistance. Conclusions CD14+ Mφ participate in CD pathogenesis by inducing AICD resistance through release of exosomal membrane TNF to activate the TNFR2/NF-κB pathway in CD4+ T cells. These results present new insights into CD pathogenesis and extend mechanistic understanding of anti-TNF agents. Proposed model CD14+ Mφ in the intestinal microenvironment of CD patients maintain the sustained activation of CD4+ T cells through exosomal membrane TNF to induce apoptosis resistance via TNFR2/NF-κB signalling, which could be effectively blocked by clinically applicable anti-TNF agents.

scholarly journals Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn’s disease

2020 ◽  
Author(s):  
Rasa Elmentaite ◽  
Alexander Ross ◽  
Kylie R. James ◽  
Daniel Ortmann ◽  
Tomas Gomes ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Single Cell ◽  
Ex Vivo ◽  
Cell Types ◽  
Gut Development ◽  
Human Gut ◽  
Single Cell Sequencing ◽  
Computational Analyses

SummaryHuman gut development requires the orchestrated interaction of various differentiating cell types. Here we generate an in-depth single-cell map of the developing human intestine at 6–10 weeks post-conception, a period marked by crypt-villus formation. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells, which are distinct from LGR5-expressing cells. We use computational analyses to show that these cells contribute to differentiated cell subsets directly and indirectly via the generation of LGR5-expressing stem cells and receive signals from the surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNAseq profiles from paediatric Crohn’s disease epithelium alongside matched healthy controls to reveal disease associated changes in epithelial composition. Contrasting these with the fetal profiles reveals re-activation of fetal transcription factors in Crohn’s disease epithelium. Our study provides a unique resource, available at www.gutcellatlas.org, and underscores the importance of unravelling fetal development in understanding disease.

scholarly journals Validation and Optimization of an Ex Vivo Assay of Intestinal Mucosal Biopsies in Crohn’s Disease: Reflects Inflammation and Drug Effects

PLoS ONE ◽  
2016 ◽  
Vol 11 (5) ◽  
pp. e0155335 ◽  
Author(s):  
Kasper Vadstrup ◽  
Elisabeth Douglas Galsgaard ◽  
Jens Gerwien ◽  
Marianne Kajbæk Vester-Andersen ◽  
Julie Steen Pedersen ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Drug Effects ◽  
Ex Vivo Assay
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