Mucosal Tissue Of Patients Research Articles

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Altered Mucosal Immune-Microbiota Interactions in Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is a condition caused by a constitutional pathogenic variant of the adenomatous polyposis coli gene that results in intestinal adenoma formation and colorectal cancer, necessitating pre-emptive colectomy. We sought to examine interaction between the mucosal immune system and commensal bacteria in FAP to test for immune dysfunction that might accelerate tumorigenesis. Colonic biopsies were obtained from macroscopically normal mucosal tissue from 14 healthy donors and 13 patients with FAP during endoscopy or from surgical specimens. Intraepithelial and lamina propria lymphocytes were phenotyped. Intraepithelial microbes were labeled with anti-IgA/IgG and analyzed by flow cytometry. Proportions of resident memory CD103-expressing CD8 + and γδ T-cell receptor + intraepithelial lymphocytes were dramatically reduced in both the left and right colon of patients with FAP compared with healthy controls. In lamina propria, T cells expressed less CD103, and CD4 + CD103 + cells expressed less CD73 ectonucleotidase. IgA coating of epithelia-associated bacteria, IgA + peripheral B cells, and CD4 T-cell memory responses to commensal bacteria were increased in FAP. Loss of resident memory T cells and γδ T cells in mucosal tissue of patients with FAP accompanies intestinal microbial dysbiosis previously reported in this precancerous state and suggests impaired cellular immunity and tumor surveillance. This may lead to barrier dysfunction, possible loss of regulatory T-cell function, and excess IgA antibody secretion. Our data are the first to implicate mucosal immune dysfunction as a contributing factor in this genetically driven disease and identify potentially critical pathways in the etiology of CRC.

Transcription factor paired related homeobox 1 (PRRX1) activates matrix metalloproteinases (MMP)13, which promotes the dextran sulfate sodium-induced inflammation and barrier dysfunction of NCM460 cells

ABSTRACT Paired related homeobox 1 (PRRX1) is a newly identified transcription factor that regulates the expression of various genes. We aimed to investigate the roles of PRRX1 and Matrix metalloproteinases (MMP)13 in dextran sulfate sodium (DSS)-induced inflammation and barrier dysfunction of NCM460 cells. PRRX1 expression in the mucosal tissues of patients with ulcerative colitis was analyzed using the GSE87466 microarray. PRRX1 and MMP13 expression was examined using Western blotting and RT-qPCR following the exposure of the NCM460 cells to DSS. The JASPAR database was used to predict the binding sites of PRRX1 to the MMP13 promoter, which was verified by luciferase reporter and chromatin immunoprecipitation assays. MMP13 expression was then detected following PRRX1 silencing or overexpression. The levels of inflammatory factors were determined using ELISA. Finally, the expression of intestinal barrier function-related proteins was evaluated using Western blotting and cellular permeability was detected by Transepithelial electrical resistance. PRRX1 was upregulated in the mucosal tissue samples of patients with UC. DSS induction upregulated PRRX1 and MMP13 expression. PRRX1 bound to the promoter of MMP13, which was further supported by the decreased expression of MMP13 observed following PRRX1 knockdown and its increased expression following PRRX1 overexpression. Furthermore, PRRX1 deletion decreased TNF-α, IL-1β and IL-6 levels in the DSS-challenged NCM460 cells, which were subjected to MMP13 overexpression. Moreover, PRRX1 silencing upregulated ZO-1, occludin and claudin-1 expression and elevated the TEER value, whereas MMP13 overexpression attenuated these effects. Collectively, PRRX1 activates MMP13, which in turn promotes the DSS-induced inflammation and barrier dysfunction of NCM460 cells.

MicroRNA profiles in five pairs of early gastric cancer tissues and adjacent non-cancerous tissues.

Approximately half of the world's gastric cancer cases and deaths occur in China. In addition, the incidence and mortality rates of gastric cancer in Gansu province in China are much higher than the average nationwide levels. The present study investigated microRNA (miRNA/miR) profiles in early gastric cancer (EGC) without specific symptoms. miRNA expression levels in five pairs of EGC tissues and adjacent non-cancerous mucosa tissues of patients from Gansu province in China were analyzed using a miRNA microarray. A total of 47 differentially expressed miRNAs (DEMs) were identified. Subsequently, mRNA expression profiles of three pairs of cancer tissues and adjacent non-cancerous tissues from 3 Asian patients with stage I or stage II gastric cancer (stage I/II; American Joint Committee on Cancer classification, Eighth Edition) were obtained from The Cancer Genome Atlas database, and differentially expressed genes (DEGs) were identified. The target genes of DEMs were filtered from the DEGs using the miRDB database and a miRNA-gene network was constructed. The functions of DEMs were evaluated using the tool for annotations of human miRNAs database, and via Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis and Gene Set Enrichment Analysis of the target genes. Finally, survival analyses of DEMs, which were in the miRNA-gene network, was performed. The results suggested that a number of miRNAs, including hsa-let-7a-5p, hsa-miR-27a-3p, hsa-miR-126-5p and hsa-miR-424-5p, may serve critical roles in EGC. The present study could provide a basis for the identification of EGC screening biomarkers. Furthermore, the present study may provide a basis for the exploration of the cause of the high incidence of gastric cancer in Gansu province from the perspective of miRNAs.

Genome-wide analysis of long noncoding RNA expression profile in nasal mucosa with allergic rhinitis

BackgroundLong noncoding RNAs (lncRNAs) are involved in a variety of human immune diseases. However, the expression profile and precise function of lncRNAs in allergic rhinitis (AR) remain unknown. In the present study, genome-wide analysis of lncRNA expression was performed in the nasal mucosa tissue and mRNA regulatory relationship was examined among patients with or without AR.MethodsMicroarray assays were performed and the differential expressions of lncRNAs or mRNA were verified through RT-PCR. The lncRNA functions were annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The potential regulatory relationships between lncRNAs and the co-expressed mRNAs were analyzed using Cytoscape software. The expressions of specific lncRNAs and mRNAs were examined using an in vitro cell model.ResultsA total of 57 lncRNAs and 127 mRNAs were dysregulated in the nasal mucosa tissue of patients with AR, compared to those of patients without AR (fold change > 2.0 and P < 0.05). GO and pathway analysis indicated that the lncRNA–co-expressed mRNAs were enriched in several biological processes and cellular signaling pathways related to AR, such as positive regulation of the integrin biosynthetic process, cell adhesion, and leukocyte transendothelial migration. Some lncRNAs regulated the co-expressed genes in a cis- and/or trans-regulatory manner. Furthermore, allergen exposure significantly increased the expression of lnc-CXCL12-4, CXCL12, and CXCR4 in BEAS-2B cells compared to untreated cells (P < 0.01).ConclusionThe results of the present study suggest that lncRNAs participate in the biological pathways related to AR. Leukocyte transepithelial migration may be a potential target for lncRNAs to regulate allergic inflammation and CXCL12/CXCR4 axis plays an important role in the inflammatory process of AR.

Characteristics of microbiota of colonic lavage fluid from patients with diarrhea-predominant irritable bowel syndrome

Objective To investigate the characteristics of the microbiota of colonic lavage fluid from patients with diarrhoea-predominant irritable bowel syndrome (D-IBS). Methods From August 2014 to March 2015, at the Department of Gastroenterology Shanxi Provincial People′s Hospital, 39 patients with D-IBS were enrolled and during the same period 39 patients with self-reported symptoms such as occasional abdominal discomfort or abdominal pain but were excluded from the diagnosis of irritable bowel syndrome (IBS) were selected as control group. The colonic mucosal tissues and colonic lavage fluid of two groups of patients were collected. DNA was extracted from tissues and colonic fluid. Ten dominant bacteria, including Bacteriodes prevotella, Bacteroidetes, Clostridium, Bifidobacterium, Dialister pneumosintes, Enteropathogenic Escherichia coli, Faecalibacterium prausnitzii, Firmicutes, Lactobacillus and Salmonella, were analyzed by real-time fluorescence quantitative polymerase chain reacticn (RT-PCR). T test and Spearman correlation analysis were performed for statistical analysis. Results The numbers of Bacteriodes prevotella in colonic lavage fluid and the descending colon mucosal tissues of patients in D-IBS group were 3.096±1.462 and 4.278±0.290, respectively, which were lower than those of control group (4.842±1.032 and 5.341±0.433, respectively), and the differences were statistically significant (t=6.093 and 12.657, both P<0.01). Correspondingly, the numbers of Bacteroidetes were 3.259±0.607 and 3.698±0.554, respectively, which were lower than those of control group (4.085±0.504 and 4.339±0.263, respectively), and the differences were statistically significant (t=6.538 and 6.528, both P<0.01). The numbers of Clostridium were 5.254±0.522 and 4.471±0.690, which were lower than those of control group (5.949±0.505 and 4.950±0.604, respectively), and the differences were statistically significant ( t=5.976, P<0.01; t=3.262, P=0.002). The number of Faecalibacterium prausnitzii from colonic lavage fluid of patients in D-IBS group was 2.238±1.845, which was lower than that of control group (3.222±1.450), and the difference was statistically significant (t=2.619, P=0.011). The Firmicutes (r=0.678, P=0.012), Lactobacillus (r=0.358, P=0.034), Bifidobacterium (r=0.418, P=0.042) and Faecalibacterium prausnitzii (r=0.568, P=0.022) from colonic mucosal tissues were all positively correlated with those from colonic lavage fluid. However, Salmonella (r=-0.638, P=0.012) and Enteropathogenic Escherichia coli (r=-0.343, P=0.025) were negatively correlated with those from mucosal tissues. Conclusions An imbalance of the microbiota in colonic lavage fluid is found in D-IBS patients. The numbers of Bacteriodes prevotella, Bacteroidetes, Clostridium and Faecalibacterium prausnitzii significantly decrease. Key words: Irritable bowel syndrome; Colonic lavage fluid; Microbiota

Expression of CXC Chemokine Receptors in Acute Ulcerative Colitis: Initial Study from an Animal Model.

Background:Ulcerative colitis (UC) is an inflammatory disease which is characterized by infiltration of inflammatory cells, crypt abscesses, distortion of the mucosal glands, and goblet cell depletion. The existence of neutrophil-rich inflammation in colon tissues of patients with UC is one of the most significant histological features of this disease. Nonetheless, the expression of CXCR chemokine receptors which appear as the main chemical mediators governing the migration of neutrophils into the mucosal tissue of patients with UC has not been well clarified.Materials and Methods:In this experimental study, the UC model was induced in Wistar rats by administration of 2 ml 4% acetic acid into the large colon through the rectum. Animals were anesthetized after 48 h; their colon tissue samples were isolated for macroscopic and histopathological examination. The expression of receptor1-7 of CXC chemokine was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technique.Results:Heavy infiltration of neutrophils, coagulative necrosis, and ulcers were observed in H and E staining, which pathologically proved the UC model. qRT-PCR results indicated that CXCR2 as one of the important ELR+ chemokine family receptors bears the highest expression in the UC group (32 fold) than the control group (P ≤ 0.05). In addition, other CXCRs of this group including CXCR1 did not possess any change (P > 0.05). In contrast, RLR negative chemokine family receptors did not show any changes with the normal group.Conclusion:The results showed that CXCR2 is the only receptor for CXCL family which was remarkably upregulated in experimental UC and that CXCR2 might play a significant role in the pathogenesis of UC.

Expressions of serum 25(OH)D, vitamin D receptor and vitamin D1-α hydroxylase in intestinal mucosa tissues of patients with inflammatory bowel disease

Objective To investigate the expression of serum 25(OH)D, vitamin D receptor (VDR) and vitamin D1-α hydroxylase (CYP27B1) in intestinal mucosa tissues of patients with inflammatory bowel disease (IBD). Methods From January 1st to December 31st in 2014, 105 patients with IBD were enrolled, among them there were 49 cases of ulcerative colitis (UC) and 56 cases of Crohn′s disease (CD); there were 20 cases in remission, 26 cases in mild active phase, 37 cases in moderate active phase and 22 cases in severe active phase; and 50 cases with lesions located in the left colon and 55 cases with lesions located in the right colon. At the same period, 45 healthy individuals were also recruited as controls, whom were suspected as IBD but at last proved healthy. The lactulose and mannitol absorption ratio (LMR), serum endotoxin, tumor necrosis factor-α (TNF-α) and 25(OH)D levels, and the expressions of VDR and CYP27B1 in the intestinal mucosa tissues were detected in all the subjects. T test, one-way analysis of variance and chi-square test were used for statistical analysis. Results The LMR, endotoxin and TNF-α levels of UC group were (63.2±13.9)%, (118.9±19.7) EU/mL, and (109.6±18.4) ng/L, respectively, which were higher than those of the healthy control group ((3.3±1.2)%, (34.2±5.6) EU/mL, and (0.6±0.3) ng/L); the level of 25(OH)D was (36.6±9.7) nmol/L, which was lower than that of healthy control group ((49.6±10.9) nmol/L), and all the differences were statistically significant (t=28.796, 29.284, 27.817 and 6.118, all P<0.05). LMR, endotoxin and TNF-α levels of CD group were (52.9±11.3)%, (96.4±10.6) EU/mL and (83.0±16.1) ng/L, respectively, which were higher than those of the healthy control group, 25(OH)D level was (44.4±9.4) nmol/L, which was lower than that of healthy control group, and all the differences were statistically significant (t=34.555, 39.716, 34.293 and 0.012, all P<0.05). The differences in LMR, endotoxin, TNF-α and 25(OH)D levels among healthy control group, remission group, mild active group, moderate active group and severe active group were statistically significant (F=286.731, 385.690, 657.830 and 18.932, all P<0.01) which was dependent on the disease activity. Compared with those of the healthy controls, the levels of LMR, endotoxin and TNF-α of the left colon group and the right colon group increased, and 25(OH)D levels decreased. The high expression rates of VDR in UC group and CD group were 36.7% (18/49) and 55.4% (31/56), respectively, which were both lower than that of healthy control group (80.0%(36/45)), and the differences were statistically significant (χ2=38.574 and 13.837, both P<0.05). The high expression rates of CYP27B1 of UC group and CD group were 26.5% (13/49) and 35.7% (20/56), respectively, which were both higher than that of healthy control group (22.2%(10/45)), and the differences were statistically significant (χ2=6.499 and 4.430, both P<0.05). The differences in the high expression rates of VDR and CYP27B1 among healthy control group, remission group, mild active group, moderate active group and severe active group were statistically significant (F=33.470 and 27.142, both P<0.01), which was dependent on the disease activity. Compared with that of the healthy control group, the high expression rates of VDR of the left colon group and the right colon group decreased, and the high expression rates of CYP27B1 increased. Conclusion There is vitamin D metabolic imbalance in IBD patients, as well as low serum 25(OH)D level, low rate of high VDR expression in colonic mucosa tissues and high rate of high CYP27B1. Key words: Inflammatory bowel diseases; Vitamin D; Receptors, calcitriol; Vitamin D1-α hydroxylase

T-Cell Depletion in the Colonic Mucosa of Patients With Idiopathic CD4+ Lymphopenia.

Idiopathic CD4(+) lymphopenia (ICL) is a rare syndrome characterized by low peripheral CD4(+) T-cell counts that can lead to serious opportunistic infections. The pathogenesis of ICL remains unclear, and whether effector sites are also lymphopenic is unknown. In this study, rectosigmoid mucosal biopsy specimens from patients with ICL and healthy controls were evaluated. Significant T-cell lymphopenia was observed in the mucosal tissue of patients with ICL by flow cytometry and immunohistochemistry, compared with healthy controls. Functional capacity of T cells, assessed by production of interferon γ and interleukin 17, was preserved in the mucosa of patients with ICL. In contrast to T lymphocytes, the frequency of myeloid cells (neutrophils and macrophages) was elevated in the colonic mucosa of patients with ICL. Despite the observed mucosal abnormalities, plasma levels of intestinal fatty acid binding protein, a marker of enterocyte turnover and other inflammatory biomarkers, including interleukin 6, C-reactive protein, and tumor necrosis factor, were not elevated in patients with ICL, compared with healthy controls, whereas soluble CD14 levels were minimally elevated. These data suggest that patients with ICL, despite gut mucosal lymphopenia and local tissue inflammation, have preserved enterocyte turnover and T-helper type 17 cells with minimal systemic inflammation. These observations highlight differences from patients with human immunodeficiency virus infection, with or without AIDS, and may partially explain their distinct clinical prognosis.

Expression and relationship of pendrin and MUC5AC in patients with chronic rhinosinusitis

To explore the expression of pendrin in sinonasal mucosal tissue of patients with chronic rhinosinusitis (CRS) and examine its relationship with mucin5AC (MUC5AC). Thirty mucosal tissue (located on anterior wall of maxillary sinus ostium and close to uncinate process) of patients with CRS with or without nasal polyps and 10 normal sinonasal mucosal tissue located at the same sites as CRS were collected. Histological changes of sinonasal mucosa were examined by hematoxylin and eosin (HE) staining. The expression of total mucins was evaluated by periodic acid Schiff staining (PAS). And the expressions of pendrin and MUC5AC in sinonasal mucosa were determined by immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The tissue samples demonstrated mucosal thickening, goblet cell hyperplasia and metaplasia, epithelial cilia partial deletion, glandular hyperplasia and a dense infiltration of inflammatory cells in CRS with or without nasal polyps. IHC revealed that positive pendrin was expressed dominantly at apical membrane of epithelial cells and sparsely in submucosal glandular cells, there was a positive expression rate of CRS with nasal polyps and CRS without nasal polyps; MUC5AC was expressed abundantly in goblet cells and pseudostratified ciliated columnar epithelium cells and weakly in submucosal glandular cells in CRS. There were significant increases in positive expression rate of total mucins production, pendrin and MUC5AC in patients of CRS with nasal polyps or without nasal polyps than those in control (86.7% and 80.0% vs 20.0%, 73.3% and 70.0% vs 10.0%, 83.3% and 76.7% vs 20.0%, all P<0.05); the relative expression levels of mRNA and protein expression of pendrin were significantly greater in patients of CRS with or without nasal polyps than those in controls (1.685±0.111 and 1.537±0.262 vs 1.012±0.167, 2.066±0.188 and 1.996±0.153 vs 1.000±0.169, all P<0.05). The relative expression levels of mRNA expression of MUC5AC were significantly greater in patients of CRS with or without nasal polyps than in controls (1.861±0.718 and 1.562±0.428 vs 1.004±0.094, P<0.05). And the expression level of pendrin mRNA was positively correlated with that of MUC5AC mRNA in patients with CRS (r=0.670, P=0.006 and r=0.713, P=0.003). An up-regulation of pendrin is correlated with an over-expression of MUC5AC in patients with CRS. And pendrin is an important cause of mucus hypersecretion in CRS.

Nerve Fiber Outgrowth Is Increased in the Intestinal Mucosa of Patients With Irritable Bowel Syndrome

Mediators released by the intestinal mucosa of patients with irritable bowel syndrome (IBS) affect the function of enteric and extrinsic sensory nerves, which can contribute to the development of symptoms. Little is known about the effects of mucosal mediators on intestinal neuroplasticity. We investigated how these mediators affect the phenotypes of colonic mucosa nerve fibers, neuron differentiation, and fiber outgrowth. We analyzed mucosal biopsy samples collected from 101 patients with IBS and 23 asymptomatic healthy individuals (controls). We measured levels of neuronal-specific enolase, growth-associated protein 43, nerve growth factor (NGF), and tyrosine kinase receptor A (NTRK1) by immunohistochemistry and enzyme-linked immunosorbent assay. Primary rat enteric neurons and human SH-SY5Y cells were incubated with supernatants from the mucosal biopsies and analyzed by morphometric and polymerase chain reaction analyses. Compared with mucosal tissues of controls, mucosa from patients with IBS had a significant increase in the area of lamina propria occupied by neuronal-specific enolase-positive (57.7% increase) and growth-associated protein 43-positive fibers (56.1% increase) and staining density of NGF (89.3% increase) (P < .05 for all). Levels of NGF protein were also increased in tissues from patients with IBS vs controls (18% increase; P= .16) along with levels of NTRK1 (64% increase; P < .05). Mucosal supernatants from tissues of patients with IBS induced higher levels of neuritogenesis in primary culture of enteric neurons, compared with controls, and more NGF-dependent neuronal sprouting in SH-SY5Y cells. Nerve fiber density and sprouting, as well as expression of NGF and NTRK1, are significantly increased in mucosal tissues of patients with IBS. Mucosal mediators participate to these neuroplastic changes.

Association between Virulence Factors and TRAF1/4-1BB/Bcl-xL Expression in Gastric Mucosa Infected with Helicobacter pylori.

Objective. CagA+/vacAs1+/vacAm1+ Helicobacter pylori upregulates the expression of tumor necrosis factor receptor–associated factor 1 (TRAF1), tumor necrosis factor receptor superfamily member 9 (4-1BB), and B-cell lymphoma-extra large (Bcl-xL) in human gastric epithelial cells. We investigated the correlation between cagA/vacAs1/vacAm1 and TRAF1/4-1BB/Bcl-xL expression in gastric mucosal tissue of patients with gastric disorders. Methods. We collected gastric mucosa samples from 35 chronic, nonatrophic gastritis (CG) patients, 41 atrophic gastritis patients, 44 intestinal metaplasia with atypical hyperplasia (IM) patients, and 28 gastric carcinoma (Ca) patients. The expression of TRAF1, 4-1BB, and Bcl-xL was determined using western blotting. The expression of cagA, vacAs1, and vacAm1 in H. pylori was examined with polymerase chain reaction. Results. The expression of TRAF1, 4-1BB, and Bcl-xL was significantly upregulated in IM and Ca patients (P < 0.05 compared with CG). There were more cases of cagA+/vacAs1+/vacAm1+ H. pylori infection in samples with elevated TRAF1, 4-1BB, or Bcl-xL expression (P < 0.05). Additionally, there were a remarkably large number of samples with upregulated TRAF1/4-1BB/Bcl-xL expression in cases of cagA+/vacAs1+/vacAm1+ H. pylori infection (44 cases, 67.7%; P < 0.05). Conclusions. The pathogenesis of IM and Ca may be promoted by cagA+/vacAs1+/vacAm1+ H. pylori, possibly via upregulated TRAF1, 4-1BB, and Bcl-xL in gastric mucosal tissue.

Phosphorylation of eIF2α Is Dispensable for Differentiation but Required at a Posttranscriptional Level for Paneth Cell Function and Intestinal Homeostasis in Mice

Recent studies link endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) to inflammatory bowel disease. Altered eIF2α phosphorylation (eIF2α-P), a regulatory hub of the UPR, was observed in mucosal tissue of patients with inflammatory bowel disease. In this study, we examined the mechanistic role of eIF2α-P in intestinal epithelial cell (IEC) function and intestinal homeostasis in mice. We generated mice with villin-Cre-mediated conditional expression of nonphosphorylatable Ser51Ala mutant eIF2α in IECs (AA mice). We analyzed AA mice under normal conditions and on challenge with oral infection of Salmonella Typhimurium or dextran sulfate sodium-induced colitis. Loss of eIF2α-P did not affect the normal proliferation or differentiation of IECs. However, AA mice expressed decreased secretory proteins including lysozyme, suggesting eIF2α-P is required for Paneth cell function. The ultrastructure of AA Paneth cells exhibited a reduced number of secretory granules, a fragmented ER, and distended mitochondria under normal conditions. UPR gene expression was defective in AA IECs. Translation of Paneth cell specific messenger RNAs encoding lysozyme and cryptidins was significantly defective leading to the observed granule-deficient phenotype, which was associated with reduced ribosomal recruitment of these messenger RNAs to the ER membrane. Consequently, AA mice were more susceptible to oral Salmonella infection and dextran sulfate sodium-induced colitis. We conclude eIF2α phosphorylation is required for the normal function of intestinal Paneth cells and mucosal homeostasis by activating UPR signaling and promoting messenger RNA recruitment to the ER membrane for translation.

Regulation of anergy-related ubiquitin E3 ligase, GRAIL, in murine models of colitis and patients with Crohn’s disease

Abrogating tolerance is a critical step in the pathogenesis of Crohn's disease (CD). T cell-anergy is one of the main mechanisms of tolerance and is regulated by the gene related to anergy in lymphocytes (GRAIL). This study investigated the expressions and regulation of GRAIL in CD and murine colitis models. Expressions of GRAIL mRNA and protein in CD4+ T cells were investigated in the peripheral blood and mucosal tissues of patients with CD, mice with dextran sodium salt (DSS)-induced colitis, and Il-10-deficient mice. MicroRNAs responsible for the regulation of GRAIL were examined by miRNA microarray. GRAIL-overexpressing T cells were intravenously injected in mice with DSS-induced colitis. The GRAIL expression was higher in the lamina propria (LP) CD4+ T cells of CD patients than of the control subjects, while it was lower in the peripheral blood CD4+ T cells of the CD patients than of the control subjects. The GRAIL mRNA expression was lower, but the GRAIL protein expression was higher in the LP of colitic mice than that of non-colitic mice. The miRNA microarray identified miR-290-5p as an miRNA that inhibits expression of the GRAIL protein and that is highly expressed in the LP of non-colitic mice. GRAIL-expressing T cells expressed regulatory T cell markers and showed suppressive effects in murine DSS-induced colitis. Our results show that expression of GRAIL is uniquely regulated by the specific miRNA in the intestinal mucosa, and suggest that GRAIL may associate with the pathophysiology of CD.

Identification of Intracellular Escherichia Coli (IEC), Associated With Crohn’s Disease, in Other Intestinal Pathologies

Several studies have confirmed the presence of adherent-invasive Escherichia coli (AIEC) in intestinal mucosa of patients with Crohn’s disease (CD). AIEC invades intestinal epithelial cells and survives inside macrophages. CD inflammatory disorder, similar to ulcerative colitis (UC), and other intestinal pathologies (OP) has been proposed to be the result of an exaggerated immune response to luminal antigens. The aim of this study was to determine the presence of IEC strains in patients with CD, UC and OP, and its association with clinical and endoscopic features. For IEC isolation, we evaluated intestinal biopsies from 92 patients: 27 CD, 28 UC, 18 OP and 19 controls. Bacteria were recovered from intestinal biopsies using gentamicin-protection assay. Association studies were performed according to IEC presence, clinical and endoscopic characteristics of patients. The presence of IEC in 12/27 CD patients was significantly higher than control subjects (1/19) (P = 0.0326). This finding was similar to that observed for UC (p = 0.0126) and OP (P = 0.0028), with 10/28 and 10/18 presented IEC, respectively. The identification of these bacteria was not associated with gender, age, extent, location, and severity phenotype (p > 0.05), however patients presenting IEC, 70% were male and 60% presented a colonic location. In the UC group with isolated bacteria, 50% of patients showed extensive IEC. In the OP group, 62.5% patients with IEC have been diagnosed with diverticulitis. CONCLUSION(S): In this study we show that mucosa tissue of patients with other intestinal disorders, such as ulcerative colitis and diverticulitis, also present IEC, suggesting a role of these bacteria in the inflammatory process. FINANCED BY FONDECYT GRANT 1120577

Expression of 5-LOX and NF-κB P65 in mucosal tissues of patients with ulcerative colitis and their relationship

Expression of 5-LOX and NF-κB P65 in mucosal tissues of patients with ulcerative colitis and their relationship

The expression ofprotease-activated receptors in colon mucosa tissue of patients with diarrhea - predominantirritable bowel syndrome and its significance

Objective To investigate the expression of protease-activatedreceptors (PARs) in colon mucosal tissue of patients with diarrhea-predominant irritablebowel syndrome (D-IBS). Methods Colon mucosa tissues were obtained from 28 D-IBS patientsand 18 normal controls by colonoscopy.The expression of PARs was detected using Westernblotting and real-time PCR. Data were analysed by SPSS 18. 0 softwave, and comparisonsbetween groups were done using Mann-Whitney U test.Results There was no difference inexpression of PAR1 between D-IBS patients and normal controls (P=0. 300). The expressionsof PAR2 and PAR4 were higher in D-IBS patients than those in normal controls (0.99±0.67vs 0.63±0.38, P=0.038 and 0.37±0. 14 vs 0.25±0. 11, P=0.013,respectively). The resultsof real-time PCR were in accordance with those of Western blotting.Conclusion Theincreased expressions of PAR2 and PAR4 in colon mucosa of D-IBS patients indicate thatthese two PAR receptors may be involved in the process of D-IBS.

Cellular sources of MMP-7, MMP-13 and MMP-28 in ulcerative colitis

Objective. Matrix metalloproteinases (MMPs) are considered the predominant proteases in the pathogenesis of mucosal ulcerations associated with inflammatory bowel disease (IBD). Whether the malignancy associated MMP-7 and MMP-13 or the recently cloned MMP-28 convey a certain meaning for intestinal homeostasis and pathogenesis of IBD is currently unknown. We therefore set off to analyze regulation patterns and cellular origins of these MMPs in mucosal tissues of patients with ulcerative colitis (UC). Material and methods. Biopsy samples of affected and healthy tissues were obtained from 35 Norwegian patients with UC. RNA was quantified by quantitative real-time polymerase chain reaction to study MMP gene expression in both pathological and healthy mucosal specimens. Cellular origins were determined by immunohistology using surrogate markers for inflammation, neovascularization, and epithelial structures. Protein expression of MMP-7 and MMP-13 was quantified using enzyme-linked immunosorbent assay. Results. MMP-7 and MMP-13 gene expression was significantly increased in UC affected colonic mucosa whereas MMP-28 showed a decreased expression in inflamed mucosa. Endothelial cells and infiltrating leukocytes were identified as the major cellular sources of MMP-7 and MMP-13 in UC. Enterocytes represented the major cellular source of MMP-28 in healthy and inflamed mucosa. Conclusions. MMP-7 and MMP-13 expression in inflammatory and endothelial cells indicate a role of these MMPs for both colitis associated neoangiogenesis and inflammatory changes. Decreased MMP-28 expression in UC is most likely the result of colitis associated epithelial destruction and loss of cryptal architecture.

Mycobacterium avium subsp. Paratuberculosis (MAP) as a modifying factor in Crohnʼs disease

Crohn's disease (CD) is a multifactorial syndrome with genetic and environmental contributions. Mycobacterium avium subspecies paratuberculosis (MAP) has been frequently isolated from mucosal tissues of patients with CD but the cellular immune response to this bacterium has been poorly described. Our aim was to examine the influence of MAP on T-cell proliferation and cytokine responses in patients with inflammatory bowel disease (IBD). Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph node cells (MLNCs) were obtained from IBD patients and non-IBD controls. PBMC T-cell proliferation in response to MAP was determined using CFSE labeling and flow cytometry. The specificity of cytokine responses to MAP was controlled by parallel exposure to Listeria monocytogenes (LM) or Salmonella typhimurium (ST). Coincubation of PBMCs with MAP induced significantly more T-cell proliferation (P < 0.0001) in PBMCs isolated from CD patients compared to PBMCs obtained from ulcerative colitis (UC) patients or healthy volunteers. In addition, PBMCs from CD patients secreted significantly higher (P < 0.05) levels of tumor necrosis factor-alpha (TNF-alpha; 2302 +/- 230 pg/mL) and interleukin (IL)-10 (299 +/- 48 pg/mL) in response to MAP compared to UC patients (TNF-alpha: 1219 +/- 411 pg/mL; IL-10: 125 +/- 19 pg/mL) and controls (TNF-alpha: 1447 +/- 173 pg/mL; IL-10: 127 +/- 12 pg/mL). No difference in cytokine responses was observed in response to LM or ST. MLNCs from both CD and UC patients secreted significantly more TNF-alpha and IL-8 in response to MAP compared to MLNCs from non-IBD control patients. Increased proliferation of T cells and an altered cytokine response suggest that prior exposure to MAP and engagement of the immune system is common in patients with CD. This does not imply causation but does support further examination of this bacterium as an environmental modifying factor.

Apoptosis in bowel mucosal tissues of patients with inflammatory bowel disease

Apoptosis in bowel mucosal tissues of patients with inflammatory bowel disease