Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon

Wallon, Conny; Braaf, Ylva; Wolving, Mats; Olaison, Gunnar; Söderholm, Johan D.

doi:10.1080/00365520510012235

Cited by 81 publications

(95 citation statements)

References 27 publications

(24 reference statements)

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“…On the other hand, the conductance was lower in surgical specimens compared to biopsies in both tissue types, Po0.001. This is in line with previous results from human colon 26 and probably refers to the thicker subepithelial layer in surgical specimens, giving higher total resistance recordings. However, it could also refer to differences in the mounting process with the influence of stretching the epithelium in surgical specimens and a higher risk of edge damage in biopsy chambers.…”

Section: Equal Permeability In Surgical Specimens and Biopsiessupporting

confidence: 82%

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Characterization of antigen and bacterial transport in the follicle-associated epithelium of human ileum

Keita¹,

Gullberg

Ericson

et al. 2006

Laboratory Investigation

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100

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The follicle-associated epithelium (FAE), covering Peyer's patches, provides a route of entry for antigens and microorganisms. Animal studies showed enhanced antigen and bacterial uptake in FAE, but no study on barrier function of human FAE has been reported. Our aim was to characterize the normal barrier properties of human FAE. Specimens of normal ileum were taken from 30 patients with noninflammatory colonic disease. Villus epithelium (VE) and FAE were identified and mounted in Ussing chambers. Permeability to 51 Cr-EDTA, transmucosal flux of the protein antigen, horseradish peroxidase (HRP), and transport of fluorescent Escherichia coli (chemically killed K-12 and live HB101) were measured. Uptake mechanisms were studied by confocal-and transmission electron microscopy, and by using pharmacological inhibitors in an in vitro coculture model of FAE and in human ileal FAE. HRP flux was substantially higher in FAE than in VE, and was reduced by an amiloride analog. Electron microscopy showed HRP-containing endosomes. Transport of E. coli K-12 and HB101 was also augmented in FAE and was confirmed by confocal microscopy. In vitro coculture experiments and electron microscopy revealed actin-dependent, mainly transcellular, uptake of E. coli K-12 into FAE. 51 Cr-EDTA permeability was equal in FAE and VE. Augmented HRP flux and bacterial uptake but similar paracellular permeability, suggest functional variations of transcellular transport in the FAE. We show for the first time that FAE of human ileum is functionally distinct from regular VE, rendering the FAE more prone to bacterial-epithelial cell interactions and delivery of antigens to the mucosal immune system.

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Section: Equal Permeability In Surgical Specimens and Biopsiessupporting

confidence: 82%

“…Our group previously showed that permeability was equal in surgical specimens and biopsies of human colon 26 and initially we wanted to confirm these findings in human ileal mucosa. After 40 min of equilibration, PD was stable in all tissues.…”

Section: Equal Permeability In Surgical Specimens and Biopsiesmentioning

confidence: 94%

Characterization of antigen and bacterial transport in the follicle-associated epithelium of human ileum

Keita¹,

Gullberg

Ericson

et al. 2006

Laboratory Investigation

Self Cite

100

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“…Modified 3ml Ussing chambers (Mussler Scientific Instruments, Aachen, Germany) were used to mount biopsies as described previously [14]. …”

Section: Ussing Chamber Experimentsmentioning

confidence: 99%

Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function

Plessis

Vanheel

Janssen

et al. 2013

Journal of Hepatology

149

123

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Background & Aims: Bacterial infections commonly occur in decompensated cirrhosis resulting from bacterial translocation from the intestine. We studied the role of intestinal macrophages and the epithelial barrier in cirrhosis. Methods: Forty-four patients with NASH/ASH cirrhosis (decom-pensated n = 29, compensated n = 15) and nineteen controls undergoing endoscopy were recruited. Serum was obtained and LPS and LBP levels determined. Intestinal macrophages were characterized by flow cytometry, immunohistochemistry, and nitric oxide (NO) production measured in supernatant of cultured duodenal samples. Quantitative RT-PCR was performed on duo-denal biopsies assessing 84 inflammatory genes. Protein levels of cytokines/chemokines were assessed in serum and superna-tant. The duodenal wall was assessed by electron microscopy, tight junction protein expression determined by RT-PCR, immu-nohistochemistry, and Western blot and, functional analysis performed by transepithelial resistance measurement and per-meability studies. Results: Increased plasma LPS, LBP levels and higher numbers of duodenal CD33 + /CD14 + /Trem-1 + macrophages, synthesizing iNOS and secreting NO were present in decompensated cirrhosis. Upregulation of IL-8, CCL2, CCL13 at the transcriptional level, and increased IL-8, and IL-6 were detected in supernatant and serum in cirrhosis. IL-6 and IL-8 co-localised with iNOS + and CD68 + , but not with CD11c + cells. Electron microscopy demon-strated an intact epithelial barrier. Increased Claudin-2 was detected by Western blot and immunohistochemistry, while decreased transepithelial resistance and increased duodenal per-meability were detected in decompensated cirrhosis. Conclusions: Our study shows the presence of activated CD14 +-Trem-1 + iNOS + intestinal macrophages, releasing IL-6, NO, and increased intestinal permeability in patients with cirrhosis, sug-gesting that these cells may produce factors capable of enhancing permeability to bacterial products.

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“…Similarly, the excised ileal human tissue maintained viability during the experiment as confirmed by a starting PD of at least À0.2 mV and a PD that remained at a level of 475% of initial value. 35,37 Thus, neither the bacteria nor the nanoparticles affected the integrity of the cell monolayers or the tissue.…”

Section: Cell Monolayer and Tissue Integritymentioning

confidence: 99%

“…Transmucosal potential difference (PD), short circuit current and electrical resistance were assessed using two pairs of electrodes (Ag/AgCl with agar-salt bridges and platinum electrodes, respectively) for assessment of tissue viability as previously described. 33,35 Before transport experiments chambers were equilibrated for 20 min to achieve steady-state conditions in PD. Live Y. pseudotuberculosis (inv þ or invÀ) were added to the mucosal side at a final concentration of 1 Â 10 8 CFU/ml.…”

Section: Confocal Microscopy Studiesmentioning

confidence: 99%

Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis

Ragnarsson

Schoultz

Gullberg

et al. 2008

Laboratory Investigation

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Crohn's disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epitheliumderived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv þ ) or lacking (invÀ) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of b1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv þ Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, Po0.01) and ileal mucosa (268 ± 47% of control; Po0.01), whereas invÀ bacteria had no effect on transcytosis. The transcytosis was concentration-, particle size-and temperature-dependent, and possibly mediated via macropinocytosis. Y. pseudotuberculosis also induced apical expression of b1-integrin on epithelial cells. A slight drop in TER was seen after exposure to inv þ Y. pseudotuberculosis, whereas mannitol flux and tight junction protein expression was unchanged. In summary, Y. pseudotuberculosis induced apical expression of b1-integrin and stimulated uptake of nanoparticles via invasindependent transcytosis in human intestinal epithelium. Our findings suggest that bacterial factors may initiate transcytosis of luminal exogenous particles across human ileal mucosa, thus presenting a novel mechanism of intestinal barrier dysfunction.

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Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon

Abstract: Endoscopic biopsies of human colon are viable in Ussing chambers and are reliable tools for studies of mucosal permeability to protein antigens. The technique offers a broad potential for studies of mucosal function in the pathophysiology of human gastrointestinal diseases.

Cited by 81 publications

References 27 publications

Characterization of antigen and bacterial transport in the follicle-associated epithelium of human ileum

Characterization of antigen and bacterial transport in the follicle-associated epithelium of human ileum

Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function

Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis

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