2. The Intestinal Epithelial Barrier
3. Intracellular Regulators of Paracellular Permeability
4. Epithelial Restitution and Healing
5. Clinical Implications of Impaired Intestinal Permeability in IBD
6. Assessing Barrier Function in Clinical Practice Today
7. Biomarkers of Intestinal Barrier Function
Conflicts of Interest
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|Inhibit cell proliferation||TGF-β|
|Promote epithelial restitution via TGF-β dependent pathway||Epidermal growth factor (EGF)|
|Promote epithelial restitution via TGF-β independent pathway||Trefoil peptides|
|Keratinocyte growth factor (KGF)|
|Decrease epithelial restitution velocity||IL-13 |
|Promote epithelial proliferation||Epidermal growth factor (EGF)|
|Induce cell apoptosis||TNF-α|
|Prevent cell apoptosis||Prostaglandin E2|
|Technique||General Principle||Test Site||Test Method||Limitations|
|Lactulose/mannitol||Oligosaccharides of different sizes||Small intestine||Urine||Time-consuming.|
Metabolised in the colon so limited application in assessing the large intestine (e.g., ulcerative colitis (UC)).
Does not show permeation of bacterial components.
Mannitol is contraindicated with blood transfusions.
Does not show permeation of bacterial components.
|Multi-sugar test||Sucrose, lactulose, sucralose, erythritol, rhamnase||Whole intestine||Urine||Time-consuming.|
Does not show permeation of bacterial components.
|51Cr-EDTA||51Cr-EDTA crosses the intestinal barrier via the paracellular route and has similar physiological properties to oliogosaccharides.||Whole intestine||Urine||Invasive and complex detection method.|
Not readily available.
Impractical in clinical setting.
Does not show permeation of bacterial components.
|PEG4000/400||Polyethylene glycol, an inert molecule of different sizes.||Whole intestine||Urine||Time-consuming.|
The exact route of PEG is not well defined , thus implications in interpreting results.
Does not show permeation of bacterial components.
|Gadolinium-based MRI contrast agent ||Gadolinium (500–1000 Da)||Whole intestine||24-h urine collection||Lack of evidence in human studies.|
More expensive and may have higher toxicity than conventional sugars.
Partial hepatobiliary elimination.
Contraindicated in renal impairment.
|Ussing chambers||Ion transport across the intestinal epithelium tissue sample is measured using a short circuit current.||Site-specific||Biopsy||Invasive and complex detection method.|
Lack of correlation between Ussing chamber and other permeability assays.
|Confocal laser endomicroscopy||Intravenously-administered fluorescent contrast is seen to leak through the small intestinal mucosa under real time endoscopy.||Terminal ileum, colon, duodenum||Endoscopy||Invasive.|
Time-consuming (average of 46.5 minutes ).
Validated measurement scores include the Watson grade (semi-quantitative ) and confocal leak score (quantitative) .
Requires special training of the endoscopist.
Does not show permeation of bacterial components.
|Biomarkers of Intestinal Permeability|
|Claudin-3 ||Epithelial tight junction protein||NA||Urine||Limited data and lack of randomised trials.|
|Lipopolysaccharide (LPS) assay||Show endotoxemia from bacterial translocation due to barrier function failure.||Colon||Blood (portal venous)||Technical limitation in detecting low levels of LPS in the peripheral blood.|
Requires careful standardization of the measurement.
Evidence of use in Inflammatory Bowel Disease (IBD).
|Circulating endotoxin core antibodies||An indirect measure of translocation of bacterial products by quantifying immunoglobulins (IgG, IgM and IgA) against the inner core of endotoxin for acute phase of intestinal barrier damage and function .||Colon||Blood||Only study done on post-operative patients, not patients with chronic gastrointestinal disease.|
Evidence for use in IBD.
|Plasma d-lactate||d-lactate is produced by the gut bacteria and translocated across the intestinal mucosa with barrier dysfunction.||Colon||Blood||False positive test with bacterial over growth.|
Limited use in critically ill patients (e.g., ischemic colonic injury, acute necrotizing pancreatitis).
|Faecal butyrate concentrations||Butyrate is a barrier enhancing substance, modifying claudin-1 and -2 to preserve intestinal barrier function and preventing bacterial translocation.||Colon||Faeces||Poorly established. |
The test relies on the principle that butyrate as a single major component of the barrier function rather than a complex and interactive entity.
|Bacteria-derived haemolysin||Toxin that impair the intestinal barrier.||Colon||Poorly established. |
Results are attributed to only haemolysin-producing bacteria.
|Assessment of fatty liver disease||Inflammation and fatty liver disease result from translocation of bacteria and its products into the portal system.||Whole intestine||Imaging||Poor specificity.|
|Gassler et al., 2001 ||Surgical specimen||10 ulcerative colitis(UC)|
10 Crohn’s disease(CD)
10 sporadic colon cancer
|Reverse transcription |
Quantitative PCR and sequencing reaction
Immunofluorescence staining and immunoblotting Immunohistochemistry
Western blot and densitometric analysis
|In actively inflamed Inflammatory Bowel Disease(IBD) tissue: desmosome protein expressions (desmoplakin-1, desmoglein-2 and desmocllin-2) decreased with severity of inflammation in IBD tissue (p < 0.05); Adherens junction(AJ) proteins such as E-cadherin and α-catenin were highly reduced; APC, p 120, plakophilin-2, β-catenin and plakoglobin were decreased and correlated with degree of inflammation in UC; plakophilin-2 and plakoglobin, but not β-catenin or APC proteins were reduced in actively inflamed CD; Tight junction(TJ) strands were discontinuous with reduced ZO-1 and occludin expression. |
In inactive IBD tissue: AJ-associated proteins were affected, but not desmosomes and TJs. Therefore, these alterations are not a primary occurrence in IBD.
|Kucharzik et al., 2001 ||Colonic biopsy||11 active UC|
9 active CD
29 control (normal colorectal mucosa or surgical resection of colon cancer)
|Global downregulation of occludin in IBD compared to controls. |
In epithelial cells adjacent to transmigrating polynorphonuclear leukocytes(PMNs), expressions of other TJ and AJ proteins were also downregulated (i.e.,zonulin-1 (ZO-1), claudin-1, junction adhesion molecule(JAM), beta-catenin, and E-cadherin).
|Blair et al., 2006 ||Biopsy||5 UC |
6 control (adenocarcinoma)
|Quantitative immunofluorescence microscopy||Epithelial MLCK expression mildly upregulated in inactive IBD and further upregulated in active disease (increase in Myosine Light Chain Kinase(MLCK) expression correlate with histological disease activity). |
MLCK phosphorylation is also significantly increased in active, but not inactive IBD.
|Zeissig et al., 2007 ||Sigmoid colon biopsy||23 active CD|
15 inactive CD
|Ussing chamber |
Freeze fracture electron microscopy
|Occludin (p < 0.05), claudin-5 (p < 0.05) and -8 (p < 0.001) were downregulated and re-distributed in active CD compared to controls but not in inactive state. Claudin-2 was strongly upregulated and inducible by Tumor Necrosis Factor- α (TNF-α). Other claudins were unchanged (-1, -4. -7) or not detectable in sigmoid colon (claudin-11, -12, -14, -15, and -16). There were reduced and discontinuous TJ strands. Focal epithelial lesions (e.g., microerosions) did not contribute to barrier dysfunction in CD. However, epithelial apoptosis was increased in active but not inactive CD.|
|Vetrano et al., 2008 ||Tissue specimen||11 control|
|Western blot |
Immunofluorescence staining for anti-JAM-A, E-cadherin and ZO-1 and confocal fluorescence microscopy
|Loss of JAM-A expression in actively inflamed IBD (p < 0.01) but not in uninvolved mucosa of IBD. |
Western blot showed significantly lower JAM-A levels in inflamed mucosa of IBD (p < 0.05) compared to the controls.
|Oshima et al., 2008 ||Rectum biopsy||5 active UC|
|Antibody staining (for claudin-1, 2, 3, 4, and 7)|
|Expression of claudin-4 and -7 were decreased; claudin-2 was elevated and claudin-1 and -3 remained unchanged, compared to the control patients.|
|Thuijls et al., 2010 ||Colonic biopsy (only from IBD group) Urine samples||10 healthy|
10 IBD remission (5 CD, 5 UC)
10 active IBD (4 CD, 6UC)
|Immunostaining of claudin-3 |
Western blot for urinary claudin-3
|Less staining of claudin-3 was observed in tissue samples of active IBD compared to controls and IBD patients in clinical remission. This correlated with urinary claudin-3 levels (p < 0.001).|
|Poritz et al., 2011 ||Mucosa sample||UC|
|Western blot||Decrease in occludin and an increase in claudin-1, thus significant increase in claudin-1: occludin (C:O) ratio in diseased UC colon compared to non-diseased UC colon (p < 0.001) and normal colon tissue (p < 0.01). |
In CD, C:O ratio elevated in all CD tissue, irrespective of disease status.
|Vetrano et al., 2011 ||Colon biopsy||16 healthy |
12 active CD
13 active UC
|EPCR (endothelial cell PC receptor) and PC (protein C) expression in inflamed tissue samples from UC and CD was significantly lower compared to healthy individuals (p < 0.001). |
EPCR, PC and PAP-1 (Protease-activated receptor-1) were expressed by epithelial cells of both healthy and IBD but the expression was decreased in IBD epithelial cells by 47% and 30%.
Downregulation of mRNA for EPCR, PC as well as PAR-1 in active IBD.
|Das et al., 2012 ||Colonic biopsy||11 active CD |
10 active UC
10 untreated colonic tuberculosis
6 IBS as control
Transmission electron microscopy
|Claudin-2 upregulated in all disease groups (p = 0.002). Claudin-2 was expressed the full length of ICJ in IBD group while it was localised to the upper one-third in cTB and control groups. Claudin-4 expression was lower in disease compared to controls groups (p < 0.01). ZO-1 expression was reduced and focal in all disease group while it was diffused in control. Occludin expression were not significantly deviated in disease groups versus the control. |
Pentalaminar structure of TJ destroyed in IBD patients.
|Petit et al., 2012 ||Colon samples from IBD patients||24 IBD patients|
Control (colonic diverticulitis)
|PrPc was concentrated at cell-cell junction and largely co-localised with beta-catenin in controls. This was disorganised in the junctions of IBD mucosa, accompanied by an increase in intracellular signal. |
However, the mRNA and protein level of PrPc was not significantly deviated compared to the controls.
|Goswami et al., 2014 ||Duodenal biopsy||24 Celiac disease|
28 active CD
15 functional dyspepsia as controls
Transmission electron microscopy
|Overexpression of claudin-2 (p = 0.001 at villi and p = 0.007 at crypts) that did not reverse with six months of treatment. |
Occludin was significantly overexpressed (p < 0.001) compared to controls that did not decrease with treatment. ZO-1 was reduced in mucosal crypts (p = 0.004) that did not alter with treatment, however, western blotting did not find consistent results. No change in JAM-1 protein.
Altered ultrastructure of TJs such as pentalaminarstructure and TJ dilatation.
|Rodriguez-Feo et al., 2015 ||Tissue biopsy||15 inflamed CD |
6 non-inflamed CD (control)
|Immunohistochemistry, confocal microscopy, real-time PCR, Western blotting||IBD patient samples showed significant reduction of RTN-4B/NOGO-B expression in inflamed mucosa compared to non-inflamed mucosa which show patchy staining pattern mostly at surface epithelium.|
|Gu et al., 2017 ||Colon biopsy||40 IBD in remission (assessed at 6, 12, 24 months after baseline colonoscopy)||Quantitative real-time PCR |
|Baseline expression of platelet endothelial cell adhesion molecule (PECAM-1) (2.4 fold elevation, p = 0.02), ICAM-3 (1.9fold elevation, p = 0.03) and VCAM-1 (1.4fold elevation, p = 0.02) were significantly higher in patients who flared than those who did not. Elevation in PECAM-1 and ICAM-3 were significant as early as six months.|
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