Table 3.

Studies showing differential expressions of proteins contributing to the intestinal barrier function in IBD tissue.

Reference	Sample	Sample Size	Technique	Findings
Gassler et al., 2001 [9]	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 [30]	Colonic biopsy	11 active UC 9 active CD 29 control (normal colorectal mucosa or surgical resection of colon cancer)	Immunofluorescence Immunohistochemistry Western blotting	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 [39]	Biopsy	5 UC 15 CD 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 [24]	Sigmoid colon biopsy	23 active CD 22 control 15 inactive CD 15 UC	Ussing chamber Freeze fracture electron microscopy Western blot Immunohistochemistry	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 [75]	Tissue specimen	11 control 13 CD 15 UC	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 [25]	Rectum biopsy	5 active UC 5 control	Antibody staining (for claudin-1, 2, 3, 4, and 7) Immunofluorescence microscopy Western blot Real-time PCR	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 [27]	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 [29]	Mucosa sample	UC CD Control	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 [73]	Colon biopsy	16 healthy 12 active CD 13 active UC	Immunohistochemistry Flow cytometry RT-PCR	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 [28]	Colonic biopsy	11 active CD 10 active UC 10 untreated colonic tuberculosis 6 IBS as control	Immunohistochemistry 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 [23]	Colon samples from IBD patients	24 IBD patients Control (colonic diverticulitis)	Immunohistochemistry Electron microscopy Immunoblotting	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 [10]	Duodenal biopsy	24 Celiac disease 28 active CD 15 functional dyspepsia as controls	Light microscopy Immunohistochemistry Western blot 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 [74]	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 [76]	Colon biopsy	40 IBD in remission (assessed at 6, 12, 24 months after baseline colonoscopy)	Quantitative real-time PCR Gene array	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.