GCaMP6F Fluorescence in Ex Vivo Intestinal Preparation v1

2021 ◽  
Author(s):  
Bryan Yoo ◽  
Jessica Griffiths ◽  
Sarkis Mazmanian
Keyword(s):  
Ex Vivo ◽  
Intestinal Tissue ◽  
Intestinal Preparation

Protocol for GCaMP6F imaging in ex vivo intestinal tissue used in Yoo et al 2021

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 Imaging Fluorophore-Labelled Intestinal Tissue via Fluorescence Endoscope Capsule

Proceedings ◽  
2018 ◽  
Vol 2 (13) ◽  
pp. 766 ◽  
Author(s):  
James Beeley ◽  
Gianluca Melino ◽  
Mohammed Al-Rawahani ◽  
Mihnea Turcanu ◽  
Fraser Stewart ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Small Intestine ◽  
Fluorescence Imaging ◽  
Small Area ◽  
Ex Vivo ◽  
Surrounding Tissue ◽  
Capsule Endoscope ◽  
Human Intestine ◽  
Intestinal Tissue ◽  
White Light Imaging

The authors have developed a wireless fluorescence imaging capsule endoscope, potentially capable of detecting early signs of disease in the human intestine which can be missed by white-light imaging (WLI) capsule endoscopy (Figure 1). Intestinal fluorescence imaging exploits variations in tissue autofluorescence between healthy and diseased areas in response to illumination, or application of fluorescent labels which preferentially bind to diseased sites. To validate the capsule’s capability to image fluorescently-labelled tissue, a small area of a sample of ex vivo porcine small intestine was sonicated with 6 nm CdZnMg fluorescent quantum dots, and the labelled area clearly differentiated from surrounding tissue by the fluorescence imaging capsule.

A higher throughput and physiologically relevant two-compartmental human ex vivo intestinal tissue system for studying gastrointestinal processes

2019 ◽  
Vol 137 ◽  
pp. 104989 ◽  
Author(s):  
Lianne J. Stevens ◽  
Marola M.H. van Lipzig ◽  
Steven L.A. Erpelinck ◽  
Apollo Pronk ◽  
Joost van Gorp ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Intestinal Tissue ◽  
Tissue System

scholarly journals P51 A combined experimental approach to assess intestinal drug absorption in early childhood

2019 ◽  
Vol 104 (6) ◽  
pp. e38.1-e38
Author(s):  
M Kiss ◽  
J Nicolaï ◽  
JJMW van den Heuvel ◽  
R Mbasu ◽  
SM Botden ◽  
...  
Keyword(s):  
Drug Absorption ◽  
Drug Exposure ◽  
Ex Vivo ◽  
Ussing Chamber ◽  
Intestinal Transport ◽  
List Type ◽  
Intestinal Tissue ◽  
Metabolizing Enzymes ◽  
Intestinal Drug Absorption ◽  
Age Related

BackgroundDrug transporters and metabolizing enzymes located in the epithelial lining of the intestine limit or enhance systemic drug exposure. During child development, the abundance and activity of these transporters and enzymes - determining how fast and how much of a drug is being absorbed into the circulation - changes from birth to adolescence. As most drugs given to children are taken by mouth, the aim of this project is to study the abundance and activity of transporters and metabolizing enzymes, involved in the intestinal absorption of drugs.MethodsThe ex vivo Ussing chamber with pediatric small intestinal tissue is applied to evaluate intestinal drug absorption and metabolism.1 Transport and metabolism of a selection of drug molecules is assessed across these tissues by sampling the donor and receiver compartment at different intervals and sample analysis by LC-MS/MS. Viability, functionality and integrity of the tissues are monitored using electrophysiological parameters (dP, R, I). Ussing chamber experiments are combined with a targeted proteomics approach to quantify drug transporter and metabolizing enzyme abundance in these tissues.ResultsAn Using chamber method has been successfully set up using both adult and pediatric intestinal tissue. To date samples from three children of different ages have been evaluated and show promising results. Tissue from the same patients has been stored for proteomics analysis.ConclusionThe Ussing method presents an innovative, feasible approach to study active intestinal transport in children. Further studies are now underway to elucidate age-related variation in intestinal transport and metabolism.ReferenceSjöberg Å, Lutz M, Tannergren C, Wingolf C, Borde A, Ungell AL:Comprehensive study on regional human intestinal permeability and prediction of fraction absorbed of drugs using the Ussing chamber technique. Eur J Pharm Sci 2013 Jan 23;48(1–2):166–80.Disclosure(s)The collaboration project is financed by the Ministry of Economic Affairs by means of the PPP Allowance made available by the Top Sector Life Sciences & Health to stimulate public-private partnerships. Johan Nicolaï, Richard Mbasu and Anna-Lena Ungell are employees of UCB Biopharma SPRL.

scholarly journals Feruloyl esterase activity is influenced by bile, probiotic intestinal adhesion and milk fat

2016 ◽  
Vol 7 (4) ◽  
pp. 597-607 ◽  
Author(s):  
M.C. Abeijón Mukdsi ◽  
E. Argañaraz Martínez ◽  
A. Perez Chaia ◽  
R.B. Medina
Keyword(s):  
Intestinal Epithelium ◽  
Esterase Activity ◽  
Milk Fat ◽  
Ex Vivo ◽  
Positive Control ◽  
Feruloyl Esterase ◽  
Intestinal Tissue ◽  
Whole Cells ◽  
Methyl Ferulate

Cinnamoyl esterases (CE) are microbial and mammalian intestinal enzymes able to release antioxidant hydroxycinnamic acids from their non-digestible ester-linked forms naturally present in vegetable foods. Previous findings showed that oral administration of Lactobacillus fermentum CRL1446 increased intestinal CE activity and improved oxidative status in mice. The aim of this work was to evaluate the in vitro CE activity of L. fermentum CRL1446 and the effect of bile on this activity, as well as strain resistance to simulated gastrointestinal tract (GIT) conditions and its ability to adhere to intestinal epithelium and influence its basal CE activity. L. fermentum CRL1446 and L. fermentum ATCC14932 (positive control for CE activity) were able to hydrolyse different synthetic hydroxycinnamates, with higher specificity toward methyl ferulate (3,853.73 and 899.19 U/g, respectively). Feruloyl esterase (FE) activity was mainly intracellular in L. fermentum CRL1446 and cell-surface associated in L. fermentum ATCC14932. Both strains tolerated simulated GIT conditions and were able to adhere ex vivo to intestinal epithelium. Pre-incubation of L. fermentum strains with bile increased FE activity in both whole cells and supernatants (~2-fold), compared to controls, suggesting that cells were permeabilised by bile, allowing more substrate to enter the cell and/or leakage of FE enzymes. Three-fold higher FE activities were detected in intestinal tissue fragments with adhered L. fermentum CRL1446 cells compared to control fragments (without bacteria), indicating that this strain provides exogenous FE activity and could stimulate esterase activity in the intestinal mucosa. Finally, we found that milk fat had a negative effect on FE activity of intestinal tissue, in absence or presence of adhered L. fermentum. These results help explaining the increase in intestinal FE activity previously observed in mice fed with L. fermentum CRL1446, and support the potential use of this strain for the development of new functional foods directed to oxidative stress-related ailments.

scholarly journals Events associated with susceptibility to invasive Salmonella enterica serovar Typhi in BALB/c mice previously infected with Plasmodium berghei ANKA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasmin Cabral Moreira ◽  
Maele Jordão ◽  
Oscar Tadeu Ferreira da Costa ◽  
Elizangela Farias ◽  
Alysson Guimaraes Costa ◽  
...  
Keyword(s):  
Plasmodium Berghei ◽  
Ex Vivo ◽  
High Volume ◽  
Cytokine Profile ◽  
Mononuclear Leukocytes ◽  
Femoral Bone ◽  
Intestinal Tissue ◽  
Salmonella Enterica Serovar Typhi ◽  
Cytokine Levels ◽  
Femoral Bone Marrow

AbstractNumerous mechanisms have been proposed to explain why patients with malaria are more susceptible to bloodstream invasions by Salmonella spp., however there are still several unknown critical factors regarding the pathogenesis of coinfection. From a coinfection model, in which an S. enterica serovar Typhi (S_Typhi) was chosen to challenge mice that had been infected 24 h earlier with Plasmodium berghei ANKA (P.b_ANKA), we evaluated the influence of malaria on cytokine levels, the functional activity of femoral bone marrow-derived macrophages and neutrophils, and intestinal permeability. The cytokine profile over eight days of coinfection showed exacerbation in the cytokines MCP-1, IFNγ and TNFα in relation to the increase seen in animals with malaria. The cytokine profile was associated with a considerably reduced neutrophil and macrophage count and a prominent dysfunction, especially in ex vivo neutrophils in coinfected mice, though without bacterial modulation that could influence the invasion capacity of ex vivo S_Typhi obtained from liver macerate in non-phagocyte cells. Finally, irregularities in the integrity of intestinal tissue evidenced ruptures in the enterocyte layer, a presence of mononuclear leukocytes in the enterocyte layer, an increase of goblet cells in the enterocyte layer and a high volume of leukocyte infiltrate in the sub-mucosa were greatly increased in coinfected animals. Increases of mononuclear leukocytes in the enterocyte layer and volume of leukocyte infiltrate in the sub-mucosa were also seen in monoinfected animals with P. berghei ANKA. Our findings suggest malaria causes a disarrangement of intestinal homeostasis, exacerbation of proinflammatory cytokines and dysfunction in neutrophils that render the host susceptible to bacteremia by Salmonella spp.

scholarly journals Human Intestinal Tissue Explant Exposure to Silver Nanoparticles Reveals Sex Dependent Alterations in Inflammatory Responses and Epithelial Cell Permeability

2020 ◽  
Vol 22 (1) ◽  
pp. 9
Author(s):  
Kuppan Gokulan ◽  
Katherine Williams ◽  
Sarah Orr ◽  
Sangeeta Khare
Keyword(s):  
Silver Nanoparticles ◽  
Ex Vivo ◽  
Consumer Products ◽  
Cell Permeability ◽  
Gi Tract ◽  
Ex Vivo Model ◽  
Intestinal Tissue ◽  
Male And Female ◽  
20 Nm ◽  
Female Subjects

Consumer products manufactured with antimicrobial silver nanoparticles (AgNPs) may affect the gastrointestinal (GI) system. The human GI-tract is complex and there are physiological and anatomical differences between human and animal models that limit comparisons between species. Thus, assessment of AgNP toxicity on the human GI-tract may require tools that allow for the examination of subtle changes in inflammatory markers and indicators of epithelial perturbation. Fresh tissues were excised from the GI-tract of human male and female subjects to evaluate the effects of AgNPs on the GI-system. The purpose of this study was to perform an assessment on the ability of the ex vivo model to evaluate changes in levels of pro-/anti-inflammatory cytokines/chemokines and mRNA expression of intestinal permeability related genes induced by AgNPs in ileal tissues. The ex vivo model preserved the structural and biological functions of the in-situ organ. Analysis of cytokine expression data indicated that intestinal tissue of male and female subjects responded differently to AgNP treatment, with male samples showing significantly elevated Granulocyte-macrophage colony-stimulating factor (GM-CSF) after treatment with 10 nm and 20 nm AgNPs for 2 h and significantly elevated RANTES after treatment with 20 nm AgNPs for 24 h. In contrast, tissues of female showed no significant effects of AgNP treatment at 2 h and significantly decreased RANTES (20 nm), TNF-α (10 nm), and IFN-γ (10 nm) at 24 h. Smaller size AgNPs (10 nm) perturbed more permeability-related genes in samples of male subjects, than in samples from female subjects. In contrast, exposure to 20 nm AgNPs resulted in upregulation of a greater number of genes in female-derived samples (36 genes) than in male-derived samples (8 genes). The ex vivo tissue model can distinguish sex dependent effects of AgNP and could serve as a translational non-animal model to assess the impacts of xenobiotics on human intestinal mucosa.

scholarly journals Efficient Aflatoxin B1 Sequestration by Yeast Cell Wall Extract and Hydrated Sodium Calcium Aluminosilicate Evaluated Using a Multimodal In-Vitro and Ex-Vivo Methodology

Toxins ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 24
Author(s):  
Alexandros Yiannikouris ◽  
Juha Apajalahti ◽  
Hannele Kettunen ◽  
Suvi Ojanperä ◽  
Andrew N. W. Bell ◽  
...  
Keyword(s):  
Cell Wall ◽  
Yeast Cell ◽  
Aflatoxin B1 ◽  
In Vitro Model ◽  
Ex Vivo ◽  
Intestinal Tissue ◽  
Sodium Calcium ◽  
Calcium Aluminosilicate ◽  
Vitro Model

In this work, adsorption of the carcinogenic mycotoxin aflatoxin B1 (AFB1) by two sequestrants—a yeast cell wall-based adsorbent (YCW) and a hydrated sodium calcium aluminosilicate (HSCAS)—was studied across four laboratory models: (1) an in vitro model from a reference method was employed to quantify the sorption capabilities of both sequestrants under buffer conditions at two pH values using liquid chromatography with fluorescence detection (LC-FLD); (2) in a second in vitro model, the influence of the upper gastrointestinal environment on the mycotoxin sorption capacity of the same two sequestrants was studied using a chronic AFB1 level commonly encountered in the field (10 µg/L and in the presence of feed); (3) the third model used a novel ex vivo approach to measure the absorption of 3H-labelled AFB1 in the intestinal tissue and the ability of the sequestrants to offset this process; and (4) a second previously developed ex vivo model readapted to AFB1 was used to measure the transfer of 3H-labelled AFB1 through live intestinal tissue, and the influence of sequestrants on its bioavailability by means of an Ussing chamber system. Despite some sorption effects caused by the feed itself studied in the second model, both in vitro models established that the adsorption capacity of both YCW and HSCAS is promoted at a low acidic pH. Ex vivo Models 3 and 4 showed that the same tested material formed a protective barrier on the epithelial mucosa and that they significantly reduced the transfer of AFB1 through live intestinal tissue. The results indicate that, by reducing the transmembrane transfer rate and reducing over 60% of the concentration of free AFB1, both products are able to significantly limit the bioavailability of AFB1. Moreover, there were limited differences between YCW and HSCAS in their sorption capacities. The inclusion of YCW in the dietary ration could have a positive influence in reducing AFB1′s physiological bioavailability.

scholarly journals Human Intestinal Tissue Explant Exposure to Silver Nanoparticles Reveals Sex Dependent Alterations in Inflammatory Responses and Epithelial Cell Permeability

2020 ◽  
Author(s):  
Sangeeta Khare ◽  
Kuppan Gokulan ◽  
Katherine Williams ◽  
Sarah Beth Orr
Keyword(s):  
Silver Nanoparticles ◽  
Ex Vivo ◽  
Consumer Products ◽  
Cell Permeability ◽  
Gi Tract ◽  
Ex Vivo Model ◽  
Intestinal Tissue ◽  
Male And Female ◽  
20 Nm ◽  
Female Subjects

Abstract Background: Consumer products manufactured with antimicrobial silver nanoparticles (AgNPs) may affect the gastrointestinal (GI) system. The human GI-tract is complex and there are physiological and anatomical differences between human and animal models that limit comparisons between species. Additionally, induction of overt toxicity in animal and in vitro models by AgNP exposure can require doses that are well above what is feasible from environmental exposure or from the use of consumer products. This suggests that a practical assessment of potential AgNP effects on the human GI-tract may require tools that allow for the examination of subtle changes in inflammatory markers and indicators of epithelial perturbation. Methods: We utilize fresh tissue excised from the GI-tract of human male and female subjects to evaluate the effects of AgNPs on the GI-system. The purpose of this study was to perform an assessment on the ability of the ex vivo model to evaluate changes in levels of pro-/anti-inflammatory cytokines/chemokines and mRNA expression of intestinal permeability related genes induced by AgNPs in ileal tissues. Results: The ex vivo model preserved the structural and biological functions of the in-situ organ. Analysis of cytokine expression data indicated that intestinal tissue of male and female subjects responded differently to AgNP treatment, with male samples showing significantly elevated Granulocyte-macrophage colony-stimulating factor (GM-CSF) after treatment with 10-nm and 20-nm AgNPs for 2 hours and significantly elevated RANTES after treatment with 20-nm AgNPs for 24 hours. In contrast, tissues of female patients showed no significant effects of AgNP treatment at 2-hours and significantly decreased RANTES (20 nm), TNF-α (10 nm), and IFN-g (10 nm) at 24-hours. Smaller size AgNPs (10 nm) perturbed more permeability-related genes in samples of male subjects (38 up-regulated, 18 down-regulated), than samples of female subjects (23 up-regulated, 15 down-regulated). In contrast, exposure to 20-nm AgNPs resulted in upregulation of a greater number of genes in female-derived samples (36 genes) than in male-derived samples (8 genes). Conclusion: Results of this study suggest that the ex vivo tissue model deserves further evaluation as a tool to assess the impacts of nanomaterials and other xenobiotic compounds on human intestinal mucosa.

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