Inflammatory Bowel Disease Mucosa Research Articles

Increased catabolism and decreased unsaturation of ganglioside in patients with inflammatory bowel disease.

To investigate whether accelerated catabolism of ganglioside and decreased ganglioside content contribute to the etiology of pro-inflammatory intestinal disease. Intestinal mucosa from terminal ileum or colon was obtained from patients with ulcerative colitis or inflammatory Crohn's disease (n = 11) undergoing bowel resection and compared to control samples of normal intestine from patients with benign colon polyps (n = 6) and colorectal cancer (n = 12) in this observational case-control study. Gangliosides and phospholipids of intestinal mucosa were characterized by class and ceramide or fatty acid composition using liquid chromatography triple-quad mass spectrometry. Content and composition of ganglioside classes GM1, GM3, GD3, GD1a, GT1 and GT3 were compared among subject groups. Content and composition of phospholipid classes phosphatidylcholine (PC) and phosphatidylethanolamine were compared among subject groups. Unsaturation index of individual ganglioside and phospholipid classes was computed and compared among subject groups. Ganglioside catabolism enzymes beta-hexosaminidase A (HEXA) and sialidase-3 (NEU3) were measured in intestinal mucosa using western blot and compared among subject groups. Relative GM3 ganglioside content was 2-fold higher (P < 0.05) in intestine from patients with inflammatory bowel disease (IBD) compared to control intestine. The quantity of GM3 and ratio of GM3/GD3 was also higher in IBD intestine than control tissue (P < 0.05). Control intestine exhibited 3-fold higher (P < 0.01) relative GD1a ganglioside content than IBD intestine. GD3 and GD1a species of ganglioside containing three unsaturated bonds were present in control intestine, but were not detected in IBD intestine. The relative content of PC containing more than two unsaturated bonds was 30% lower in IBD intestine than control intestine (P < 0.05). The relative content of HEXA in IBD intestine was increased 1.7-fold (P < 0.05) and NEU3 was increased 8.3-fold (P < 0.01) compared to normal intestine. Intestinal mucosa in IBD is characterized by increased GM3 content, decreased GD1a, and a reduction in polyunsaturated fatty acid constituents in GD3, GD1a and PC. This study suggests a new paradigm by proposing that IBD occurs as a consequence of increased metabolism of specific gangliosides.

ID: 150: Cytokines in inflammatory bowel diseases

Cytokines play a crucial role in the pathogenesis of inflammatory bowel diseases (IBD). IBD comprise to different disorders: Crohn’s disease and ulcerative colitis. These diseases are characterized by chronic diarrhea, abdominal pain and weight loss and place a significant burden on the affected patients. Cytokines control multiple aspects of the inflammatory response in IBD by binding to their specific cognate receptors on the surface of intestinal target cells. Additionally, cytokines reaching the systemic circulation have been implicated in driving extraintestinal symptoms of IBD (e.g. arthritis). In the inflamed mucosa in IBD, the balance between pro-inflammatory and anti-inflammatory cytokines is altered and pro-inflammatory cytokines dominate the cytokine milieu. This situation impedes the resolution of inflammation and instead leads to disease perpetuation and tissue destruction in IBD. Based on these observations, it is no surprise that anti-cytokine agents have been considered for therapy of IBD. While anti-TNF agents are now used in clinical routine for IBD therapy, other new anti-cytokine agents such as anti-IL12/IL-23 antibodies are on the horizon. Moreover, multi-cytokine blockers such as JAK inhibitors have been recently used in pilot studies for IBD therapy. In this presentation, the role of cytokines in IBD and the current status of anti-cytokine therapy will be reviewed.

Oral mucosa lesions and oral symptoms in inflammatory bowel disease patients.

Inflammatory Bowel Disease is known for its extra intestinal manifestations, the oral cavity is no exception. The aim of this study was to evaluate the association between Inflammatory Bowel Disease and oral mucosa lesions and symptoms, and complementary to evaluate their possible relation with oral hygiene, smoking habits, drug therapy, duration and activity of the disease. Patients were selected from the Gastroenterology Clinic of a Portuguese tertiary referral hospital. This sample consisted of 113 patients previously diagnosed with ulcerative colitis or Crohn's disease along with a control group of 58 healthy individuals that were accompanying the study group patients to their appointments. Clinical interviews and clinical examinations were performed for data collection. The patients in the study group were more affected by oral symptoms (P=0.011), and showed a trend towards a higher incidence of oral mucosal lesions, even though statistical significance was not reached (8.8% versus 3.4% in the control group; P=0.159). Patients in active phase were the most affected. No differences were detected between Crohn's disease and ulcerative colitis, or concerning smoking habits. The corticosteroid and immunosuppressant therapy seemed to increase the incidence of oral symptoms (P=0.052). The oral mucosa lesions increased and the oral symptoms decreased over the course of the disease, however without statistical significance. Oral mucosa's lesions and oral symptoms were positively associated with Inflammatory Bowel Disease, mainly during disease activity periods and conceivably, associated with corticosteroid and immunosuppressant therapy.

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease

Objective. Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn’s disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. Material and methods. Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). Results. GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3′,5′-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. Conclusions. GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

Curative endoscopic submucosal dissection of large nonpolypoid superficial neoplasms in ulcerative colitis (with videos)

Endoscopic resection of superficial neoplasms in inflammatory bowel disease (IBD) is appropriate if a complete resection can be achieved. However, EMR is ineffective for large, nonpolypoid neoplasms in IBD due to submucosal fibrosis, and no data are available on the efficacy of endoscopic submucosal dissection (ESD). To assess ESD feasibility and efficacy for large, nonpolypoid neoplasms in patients with IBD. Prospective case series. Multicenter: Italian and Japanese centers. Consecutive patients with long-standing ulcerative colitis and a superficial nonpolypoid neoplasm, >20 mm within the colitic mucosa. Neoplasm characterization and delineation by chromoscopy and narrow-band imaging. ESD performed according to the standard technique. Feasibility, safety, curative resection rates. Nine patients with 10 neoplasms were included (7 and 3 in the Italian and Japanese centers, respectively). Neoplasms were laterally spreading tumors-non-granular in 5 cases, in the left side of the colon in 7, had median size of 33 mm, and were associated with scar in 5 cases. Margin delineation was difficult in 5 cases. Submucosal fibrosis was present in 9 cases. ESD was en bloc with negative margins (R0) in 8 cases, and curative in 7. No endoscopic invisible dysplasia or cancer was found during the follow-up (median 24 months, range 6-72 months) at the resection site and elsewhere within the colitic mucosa. Small series. ESD achieves curative resections in patients with IBD, but the procedure is difficult because of the high prevalence of submucosal fibrosis. Patients need to be accurately evaluated before resection and adhere to strict long-term follow-ups.

Mo1724 Interleukin-17A Homodimer Reduces Pro-Inflammatory Cytokine Production by Inflammatory Bowel Disease Mucosa Cultured Ex Vivo

Introduction Interleukin (IL)-17A, which is up-regulated in inflammatory bowel disease (IBD) mucosal lesions, and IL-17F are normally present as IL-17AA and IL-17FF homodimers and may occasionally form IL-17A/F heterodimers. The role of each IL-17 dimer in IBD is currently unknown. Therefore, we studied the effects of IL-17AA, IL-17FF and IL-17-A/F in ulcerative colitis (UC) and Crohn’s disease (CD) mucosa. Methods Inflamed colonic biopsies from 17 IBD patients (6 UC and 11 CD) were cultured ex vivo for 24 h with IL-17AA, IL-17FF or IL-17A/F (1 ng/ml). Mucosal myofibroblasts isolated from the inflamed colon of 4 CD and 4 UC patients were cultured for 24 h with tumour necrosis factor (TNF)-α 20 ng/ml or with increasing concentrations (1–100 ng/ml) of IL-17AA, IL-17FF or IL-17A/F. IL-6 and IL-8 were measured in culture supernatants by ELISA. Results IL-17AA, but not IL-17FF, significantly reduced both IL-6 and IL-8 production by inflamed IBD biopsies cultured ex vivo , whereas IL-17A/F decreased IL-8 release by IBD mucosa. No difference was observed between CD and UC. Neither IL-17AA, nor IL-17FF, nor IL-17A/F exerted any effect on IL-6 and IL-8 production by IBD myofibroblasts. As expected, TNF-α stimulation significantly increased IL-6 and IL-8 production by both CD and UC myofibroblasts in vitro . No difference was observed between CD and UC myofibroblasts. Conclusion IL-17AA exerts an anti-inflammatory action on inflamed IBD biopsies cultured ex vivo . The action of IL-17AA is not mediated by myofibroblasts, therefore further studies are underway to ascertain which cell type is the main target of IL-17AA in IBD mucosa. Disclosure of Interest None Declared.

760 Confocal Endomicroscopy Demonstrates Increased Intestinal Permeability and Gut Dysfunction in Patients With Acute and Chronic Pancreatitis

Introduction Increased intestinal permeability and endotoxaemia occur in acute pancreatitis (AP) but have yet to be confirmed in chronic pancreatitis (CP). Confocal endomicroscopy (CEM) is a novel technology that has identified epithelial gaps and leakage of intravenously administered fluorescein from small intestinal mucosa in inflammatory bowel diseases. We aimed to determine intestinal permeability changes in AP and CP using CEM, assessing associations with endotoxaemia, circulating cytokines and alterations in duodenal flora. Method 182 patients were recruited: 35 mild AP; 11 moderate AP; 8 severe AP (Determinant Based Classification); 47 CP (27 surgically managed, 20 conservatively managed); 33 chronic liver disease and 48 non-ulcer dyspepsia (from whom 14 healthy H. pylorinegative controls were identified). Blood was sampled and duodenal fluid aspirated for culture. CEM was performed in consenting patients and fluorescein leakage scored by two independent blinded investigators. Plasma endotoxin antibodies, serum cytokine concentrations and lactulose:mannitol ratios were measured; D-amino oxidase concentrations were assayed in AP patients undergoing CEM. Results Fluorescein leakage from epithelial villi at CEM and lactulose:mannitol ratios were significantly higher in AP patients (p = 0.0346 and p = 0.046 respectively) and surgically managed CP (p = 0.032, p = 0.030) compared to healthy controls. Patients with AP more frequently showed positive duodenal bacterial cultures than controls (p = 0.004); positive duodenal cultures were associated with fluorescein leakage (p = 0.035). Plasma IgM and IgG concentrations were decreased in AP compared to controls, most markedly in severe AP, and multiple cytokines showed significantly increased concentrations in severe AP. D-amino oxidase enzyme concentrations were significantly increased in those AP patients who showed fluorescein leakage at CEM (p = 0.035). Conclusion CEM identified increased intestinal permeability in AP and surgically managed CP, associated with endotoxaemia, increased serum inflammatory cytokines and D-amino oxidase. These changes were most marked in severe AP. The association of fluorescein leakage with duodenal bacterial proliferation in AP indicates major gut dysfunction that may account for bacterial translocation via epithelial gaps, contributing to AP severity. Disclosure of interest None Declared.

Mo1692 Vascular Endothelial Transient Receptor Potential Vanilloid 4 Links Colonic Inflammation in Dextran Sulfate Sodium-Induced Colitis Model Mice

BACKGROUND&AIMS: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel, and involved in physical sensing in various types of tissues. TRPV4 is expressed in sensory neuron and vascular endothelial cells in various organs. In gastrointestinal tracts, TRPV4 activation in epithelial cells increases in intracellular calcium concentrations and induces colitis. However, the localization and the role of the TRPV4 channel expressed in vascular endothelial cells in colitis are still poorly defined. In the present study, we investigated the alteration of TRPV4 expression in the colonic vascular endothelial cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: Eight week-old wild type (WT) and TRPV4 knock-out (TRPV4 KO) mice were used. Colitis was induced by 2% DSS solution given as drinking water for 7 days. The disease activity index (DAI) was evaluated during the treatment, while colon length, histological damage, and myeloperoxidase (MPO) activity were examined post-DSS treatment. Immunohistochemical analysis was performed in distal colon. TRPV4-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. CD31, CD105, CD11b, CD68, Ly6B.2, and VEGF receptor-2 were detected by indirect staining with their specific antibodies. Vascular leakage was investigated using Evans blue dye extrusion assay. RESULTS: TRPV4 KO mice protected against DSS-induced weight loss, neutrophil (MPO), colon shortening, diarrhea, occult-fecal blood, and histological damage. TRPV4 immunoreactivities were detected mostly in epithelial cells of normal distal colon. DSS treatment drastically increased TRPV4 expression in vascular endothelial cells of mucosa and submucosal layer which colocalized with CD31, CD105, and VEGF receptor-2, but not epithelial cells. Repeated intrarectal and intravenous administration of TRPV4 agonist GSK1016790A exacerbated the severity of DSS colitis. Increased vascular permeability was observed following single intravenous administration of GSK1016790A in DSS colitis and this effect was abolished with TRPV4 antagonist RN1734. CD11b, CD68, Ly6B.2 immunopositive cells were detected around TRPV4 positive vascular endothelial cells in mucosa and submucosal layer in DSS colitis. CONCLUSION: These findings indicate that the alteration of TRPV4 in vascular endothelial cells may contribute to progression of colonic inflammation via increasing vascular permeability.

Mo1693 Activation of Bone Marrow-Derived TRPV4-Expressing M2 Macrophages Promotes Intestinal Contraction

BACKGROUND&AIMS: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel, and involved in physical sensing in various types of tissues. TRPV4 is expressed in sensory neuron and vascular endothelial cells in various organs. In gastrointestinal tracts, TRPV4 activation in epithelial cells increases in intracellular calcium concentrations and induces colitis. However, the localization and the role of the TRPV4 channel expressed in vascular endothelial cells in colitis are still poorly defined. In the present study, we investigated the alteration of TRPV4 expression in the colonic vascular endothelial cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: Eight week-old wild type (WT) and TRPV4 knock-out (TRPV4 KO) mice were used. Colitis was induced by 2% DSS solution given as drinking water for 7 days. The disease activity index (DAI) was evaluated during the treatment, while colon length, histological damage, and myeloperoxidase (MPO) activity were examined post-DSS treatment. Immunohistochemical analysis was performed in distal colon. TRPV4-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. CD31, CD105, CD11b, CD68, Ly6B.2, and VEGF receptor-2 were detected by indirect staining with their specific antibodies. Vascular leakage was investigated using Evans blue dye extrusion assay. RESULTS: TRPV4 KO mice protected against DSS-induced weight loss, neutrophil (MPO), colon shortening, diarrhea, occult-fecal blood, and histological damage. TRPV4 immunoreactivities were detected mostly in epithelial cells of normal distal colon. DSS treatment drastically increased TRPV4 expression in vascular endothelial cells of mucosa and submucosal layer which colocalized with CD31, CD105, and VEGF receptor-2, but not epithelial cells. Repeated intrarectal and intravenous administration of TRPV4 agonist GSK1016790A exacerbated the severity of DSS colitis. Increased vascular permeability was observed following single intravenous administration of GSK1016790A in DSS colitis and this effect was abolished with TRPV4 antagonist RN1734. CD11b, CD68, Ly6B.2 immunopositive cells were detected around TRPV4 positive vascular endothelial cells in mucosa and submucosal layer in DSS colitis. CONCLUSION: These findings indicate that the alteration of TRPV4 in vascular endothelial cells may contribute to progression of colonic inflammation via increasing vascular permeability.

Complete mucosal healing defined by endoscopic Mayo subscore still demonstrates abnormalities by novel high definition colonoscopy and refined histological gradings.

A novel high definition colonoscopy imaging technique (i-Scan) can characterize, in detail, colonic mucosa in patients with ulcerative colitis, and may provide additional information about mucosal healing. The aim of this study was to create a more refined histological and endoscopic criteria based on this novel technique in order to redefine inflammatory activity and mucosal healing. A total of 78 patients with ulcerative colitis were assessed by high definition colonoscopy as well as by white light endoscopy (WLE). Mayo endoscopic subscores were assigned to patients according to WLE findings. Mucosal and vascular patterns on high definition colonoscopy were each graded from 1 - 4. A histological scoring system (ECAP system) was designed to reflect all histological changes in ulcerative colitis. The overall high definition imaging scores (mucosal and vascular patterns) were significantly correlated with Mayo endoscopic subscores (rs = 0.86, 95 % confidence interval [CI] 0.79 - 0.91; P < 0.0001). Of those with Mayo endoscopic subscore of 0, 30.4 % had an abnormal mucosal pattern and 73.9 % of them had an abnormal vascular pattern on high definition colonoscopy; a score of 6 or less had a sensitivity of 95.8 % (95 %CI 85.7 % - 99.3 %) and specificity of 75.9 % (95 %CI 56.5 % - 90.0 %) to detect mucosal healing as defined by Mayo endoscopy subscore of 0 or 1. Furthermore, mucosal and vascular pattern scores were also significantly correlated with most parameters of the proposed ECAP score. The subtle histological abnormalities underlying the apparently healed mucosa in ulcerative colitis could be detected using high definition colonoscopy and the refined ECAP histology scoring system. These techniques detect residual abnormalities in the majority of patients with seemingly complete mucosal healing by conventional Mayo criteria.

Reduced expression of aquaporins in human intestinal mucosa in early stage inflammatory bowel disease

ObjectivesThe aim of this study was to investigate the relationship between aquaporin (AQP) water channel expression and the pathological features of early untreated inflammatory bowel disease (IBD) in humans.MethodsPatients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days, were examined at the local hospital. Colonoscopy with biopsies was performed and blood samples were taken. Patients who did not meet the diagnostic criteria for IBD and who displayed no evidence of infection or other pathology in the gut were included as symptomatic non-IBD controls. AQP1, 3, 4, 5, 7, 8, and 9 messenger RNA (mRNA) levels were quantified in biopsies from the distal ileum and colon by quantitative real-time polymerase chain reaction. Protein expression of selected AQPs was assessed by confocal microscopy. Through multiple alignments of the deduced amino acid sequences, the putative three-dimensional structures of AQP1, 3, 7, and 8 were modeled.ResultsAQP1, 3, 7, and 8 mRNAs were detected in all parts of the intestinal mucosa. Notably, AQP1 and AQP3 mRNA levels were reduced in the ileum of patients with Crohn’s disease, and AQP7 and AQP8 mRNA levels were reduced in the ileum and the colon of patients with ulcerative colitis. Immunofluorescence confocal microscopy showed localization of AQP3, 7, and 8 at the mucosal epithelium, whereas the expression of AQP1 was mainly confined to the endothelial cells and erythrocytes. The reduction in the level of AQP3, 7, and 8 mRNA was confirmed by immunofluorescence, which also indicated a reduction of apical immunolabeling for AQP8 in the colonic surface epithelium and crypts of the IBD samples. This could indicate loss of epithelial polarity in IBD, leading to disrupted barrier function.ConclusionAQPs 1 and 8 and the aquaglyceroporins AQPs 3 and 7 are the AQPs predominantly expressed in the lower intestinal tract of humans. Their expression is significantly reduced in patients with IBD, and they are differentially expressed in specific bowel segments in patients with Crohn’s disease and ulcerative colitis. The data present a link between gut inflammation and water/solute homeostasis, suggesting that AQPs may play a significant role in IBD pathophysiology.

Relationship between dendritic cells, interleukin-23 and ulcerative colitis

Objective To compare the levels of S100+ DC,CD83 + DC and IL-23P19 between the patients with mucosa of ulcerative colitis ( UC ) and those with irritable bowel syndrome ( 0BS ).Methods Paraffin-embedded colon tissue from colonoscopic biopsy was detected in sixty UC and sixty IBS cases for the positivity of SI00 + DC,CD83 + DC and IL-23P19 using immunohistochemical SP method.Results There was no significant difference in the positive rates of S100 + DC and CD83 + DC between the two groups ( P ＞ 0.05 ),while significant difference in IL-23P19 level was found between the two groups( 85.0％ vs.70.0％ ; x2 =3.87,P ＜0.05 ).The infiltration densities of S100 + DC,CD83 + DC and expression of IL-23 P19 in the mild and severe UC patients were 24.43 ± 8.26/mm2,5.23 ± 1.66/mm2,95.03 ± 18.39/mm2,and 36.13 ± 11.36/mm2,10.13 ±3.29/mm2,133.38 ± 25.32/mm2,respectively.The difference between the mild and severe UC patients was significant ( P ＜ 0.05 ).No significant difference was found regarding the above three parameters between severity-stratified subgroups of the UC patients ( P ＞ 0.05 ).Conclusion In UC colon tissues,the infiltration density and activity of DC were increased.IL-23P19 level was elevated.The levels of S100 + DC,CD83 + DC and IL-23P19 were increased with the advancement of UC disease. Key words: Ulcerative colitis ; Irritable bowel syndrome; Dendritic cell; Interleukin-23 ; Immunohistochemistry

Transcriptional changes between uninflamed ulcerative colitis and familial adenomatous polyposis pouch mucosa can be attributed to an altered immune response.

A total proctocolectomy with ileal pouch-anal anastomosis (IPAA) is considered the surgery of choice for definitive management of familial adenomatous polyposis (FAP) and some patients with ulcerative colitis (UC). However, this surgical treatment is often associated with pouchitis, a long-term complication that occurs mostly in UC patients. The purpose of this study was to better define the molecular background of pouchitis. A microarray-based survey was performed using pouch mucosal samples collected from 28 and 8 patients undergoing surgery for UC and FAP, respectively. There were 4,770 genes that significantly differentiated uninflamed from inflamed mucosal samples, and their functional features were represented mostly by metabolic and cell proliferation pathways. In contrast, functional analyses of aberrantly expressed genes between UC and FAP samples, irrespective of mucosal inflammation status, revealed multiple pathways and terms that were linked to changes in immune response. Interestingly, the comparison of uninflamed UC and FAP samples identified a set of 29 altered probe sets, including an inflammation-related transcript encoding a Charcot-Leyden crystal (CLC) protein. The most distinct changes in gene expression profiles differentiating uninflamed UC and FAP pouch mucosal samples were attributed to the Gene Ontology category innate immune response. Our study confirmed that alterations in immune responses can be found between patients who underwent surgery for UC and FAP, independent of the pouch inflammation status. This observation may be important when managing IPAA patients.

Interleukin (IL)-17AA Reduces Pro-Inflammatory Cytokine Production By Inflammatory Bowel Disease Mucosa Cultured Ex Vivo

Interleukin (IL)-17AA Reduces Pro-Inflammatory Cytokine Production By Inflammatory Bowel Disease Mucosa Cultured Ex Vivo

Pathomorphology of colonic mucosa in patients with chronic post-parasitic colitis

Aim. To identify pathologic features of the colonic mucosa in patients with chronic post-parasitic colitis. Methods. Under the observation were 80 patients aged 38-42 years (38 men and 42 women) 1.5-2 years after undergoing parasitic diseases (amebiasis, giardiasis, diphyllobothriasis). In 19 patients due to the presence of dyspeptic phenomena colon mucosa was evaluated by morphometric parameters using grid of Avtandilov. The control group consisted of 6 patients with adaptive norm, in which by complex evaluation (bacteriology, parasitology, endoscopic, histologic) the pathology has not been revealed. Biopsies of colon mucosa were fixed in 10% neutral formalin solution and embedded in paraffin. Histological sections were stained with 1% aqueous solution of Alcian blue, Mayer’s hematoxylin and eosin; for morphometric analysis of 24 objective indicators - azure II-eosin by Romanovsky, with eosin methylene blue and thionine by Nicolas. Results. It was found that the mucosa in chronic post-parasitic colitis in all cases different from the normal colonic mucosa by registered parameters: the amebiasis - in 50% of cases, giardiasis - in 54.1%, with difillobotriosis - in 70.8%. In post-difillobotriosis colitis the number of fibroblasts in the lamina propria was reduced. Post-lyambliotic colitis characterized by hypertrophy of the surface epithelium and a high mitotic activity of the epithelium of intestinal glands. Conclusion. After undergoing parasitic diseases, in the colon mucosa preserved histological changes corresponding to chronic ulcerative colitis; post-amebiasis chronic colitis characterized by catarrhal-haemorrhagic inflammation, chronic post-lyambliotic - catarrhal-follicular, post-difillobotriosis - catarrhal-haemorrhagic inflammation with high activity of the pathological process and moderate atrophy of the intestinal glands.

Mechanisms of Immune Signaling in Colitis-Associated Cancer

The inflammatory bowel diseases ulcerative colitis and Crohn’s disease are associated with an increased risk for the development of colorectal cancer. During recent years, several immune signaling pathways have been linked to colitis-associated cancer (CAC), largely owing to the availability of suitable preclinical models. Among these, chronic intestinal inflammation has been shown to support tumor initiation through oxidative stress–induced mutations. A proinflammatory microenvironment that develops, possibly as a result of defective intestinal barrier function and host–microbial interactions, enables tumor promotion. Several molecular pathways such as tumor necrosis factor/nuclear factor-κB or interleukin 6/signal transducer and activator of transcription 3 signaling have been identified as important contributors to CAC development and could be promising therapeutic targets for the prevention and treatment of CAC.

Abstract 5348: Impaired self-renewal and colitis susceptibility of colonic mucosa in mice lacking AKR1B8

Abstract Background: Ulcerative colitis (UC) is a major clinically defined form of inflammatory bowel disease which affects individuals throughout a life and associates with an increased risk of developing colorectal cancer. Aldo-keto reductase 1B10 (AKR1B10), isolated by our laboratory, is a novel monomeric NADPH-dependent enzyme with reductive activity towards reactive carbonyls. AKR1B10 also acts as a novel regulator of de novo fatty acid synthesis and lipid metabolism via regulating acetyl-CoA carboxylase-α (ACCA) stability. AKR1B10 is primarily expressed in normal human intestines, but lost in colorectal cancer and predisposition conditions, such as UC and adenomatous polyps, suggesting its pathogenic role in the these diseases. Aldo-keto reductase 1B8 (AKR1B8) is the mouse ortholog of human AKR1B10, having the similar enzyme activity to α,β-unsaturated carbonyls and capability of regulating fatty acid synthesis and lipid metabolism in mice. Thereby, we generated an AKR1B8 knockout (-/-) mouse strain to understand the etiopathogenic role of AKR1B10/8 in the UC and associated colorectal cancer to Methods: AKR1B8 -/- mouse strain was produced by homozygous recombination. Dextran sulfate sodium (DSS) was utilized to induce colitis and associated colorectal tumors. In detail, AKR1B8 -/- mice were fed with 2% DSS in drink water for four cycles (e.g., 7 days DSS, 14 days water). Inflammatory activity and masses were recorded, and histopathology of chronic colitis, crypt architecture, and micro-neoplastic lesions was examined. Profiles of multiple cytokines and reactive oxidative species in colonic mucosa were estimated. Potential DNA mutations were identified using whole Exome sequencing. Results: Targeted disruption of AKR1B8 disturbed the self-renewal and injury recovery of colonic cryptic cells due to decrease in lipid synthesis from butyrate. AKR1B8 deficient mice are highly susceptible to colitis induced by DSS at 2% and to carbonyl and oxidative DNA damage, leading to colitis-associated tumorigenesis and malignant progression. Histopathological studies showed that AKR1B8 -/- mice suffered severe inflammatory lesions and high-grade dysplasia. Importantly, visible large masses were seen in AKR1B8 -/- mice, but not in wild type that underwent the same DSS treatment. Furthermore, 269 gene variants (in exons) were identified uniquely in AKR1B8 -/- colitis mucosa and tumors, not in WT colitis. Among them, 20 genes were mutated in colitis only and 28 genes in tumors alone. A total of 221 genes were mutated in both AKR1B8 -/- colitis mucosa and tumors. These gene mutants may represent the DNA damage that drives the malignant transition of colitis. Conclusions: AKR1B8 (AKR1B10 in humans) is a critical protein that regulates the dynamic homeostasis of self-renewal and barrier function of colonic epithelium. AKR1B8 deficiency in colonic epithelium is a critical etiopathogenic factor of UC and its tumorigenic transformation. Note: This abstract was not presented at the meeting. Citation Format: Yi Shen, Jun Ma, Ruilan Yan, Hongyan Ling, Xiaoning Li, Wancai Yang, John Gao, Yiwen Bu, Ahmed Alsalman, Cheifei Huang, Yu Cao, Shuangbai Zhou, Laxiang Wan, Yingchun He, Zhiqi Wang, Ali Khazaal, Mei Chris Huang, Duan-Fang Liao, Deliang Cao. Impaired self-renewal and colitis susceptibility of colonic mucosa in mice lacking AKR1B8. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5348. doi:10.1158/1538-7445.AM2014-5348

Toll-like receptor expression in crypt epithelial cells, putative stem cells and intestinal myofibroblasts isolated from controls and patients with inflammatory bowel disease.

The aim of our studies was to investigate the expression of Toll-like receptor (TLR)-2 and TLR-4 (and in some studies TLR-5) in myofibroblasts and small and large intestinal crypt epithelial cells from control patients and those affected by Crohn's disease and ulcerative colitis. Isolated and disaggregated crypt epithelial cells and monolayers of myofibroblasts were used for studies by reverse transcription–polymerase chain reaction (RT–PCR), real-time RT–PCR, flow cytometry, immunocytochemistry and Western blot analysis. Compared to control cells, crypt epithelial cells isolated from active ulcerative colitis and Crohn's disease colonic mucosal samples showed significantly higher expression of TLR-2 and TLR-4 transcripts and protein (on the cell surface). There was also enhanced expression of TLR-4 in crypt cells from ileal Crohn's disease. Expression of TLR-2 and TLR-4 transcripts in crypt epithelial cells isolated from inflamed mucosa of distal ulcerative colitis did not differ significantly from such cells obtained from the normal proximal colon. Crypt epithelial cells with side population characteristics (putative stem cells) also expressed transcripts and protein for TLR-2, TLR-4 and TLR-5. Colonic myofibroblast expression of these TLRs was much weaker than in crypt epithelial cells. In conclusion, enhanced TLR-2 and TLR-4 expression by crypt epithelial cells in active inflammatory bowel disease likely reflects greater ability to respond to microbial products. Results from our studies using mucosal samples from patients with distal ulcerative colitis suggest that the enhanced expression of these TLRs could be constitutive. TLR-2, TLR-4 and TLR-5 expression by stem cells imply ability to respond to distinct bacterial products.

CCAAT/enhancer binding protein α (C/EBPα)+ M2 macrophages contribute to fibrosis in IgG4-related disease?

IgG4-related disease (IgG4-RD) is a new disease entity characterized by type 2 helper T (Th2)-dominant inflammation and progressive fibrosis. We found the infiltration of strange cell populations in the fibrotic lesions of submandibular gland specimens obtained from 15 patients with IgG4-RD. These cells expressed CCAAT/enhancer binding protein a (C/EBPα). Many of the cell populations were identified with M2 macrophages. The degrees of infiltration of C/EBPα+M2 macrophages and the ratio of fibrotic lesions in the specimens were correlated (r2 = 0.83, p < 0.01). We also analyzed the expression of C/EBPα in other chronic inflammatory disorders: synovium in rheumatoid arthritis (RA), liver tissue in chronic viral hepatitis, and mucosa in ulcerative colitis. The specimens from RA and chronic viral hepatitis showed infiltration of C/EBPα+ cells, but there were few C/EBPα-positive cells in ulcerative colitis. Fibrosis is not a major issue in ulcerative colitis. In conclusion, we found the remarkable infiltration of C/EBPα+M2 macrophages in cases of chronic inflammation with fibrosis, including IgG4-RD. This primitive study also disclosed that most of C/EBPα+M2 macrophages localized in fibrotic lesions, and the degree of the infiltration and the ratio of fibrotic area were correlated.

Telomere length in non-neoplastic colonic mucosa in ulcerative colitis (UC) and its relationship to the severe clinical phenotypes.

Telomere shortening occurs with human aging in many organs and tissues and is accelerated by rapid cell turnover and oxidative injury. To clarify the clinical importance of telomere shortening in colonic mucosa in ulcerative colitis (UC), we measured average telomere length using quantitative real-time PCR in non-neoplastic colonic mucosa in UC patients and assessed its relationship to various clinical subtypes. Relative telomere length in genomic DNA was measured in colonic biopsies obtained from rectal inflammatory mucosa from 86 UC patients as well as paired non-inflammatory proximal colonic mucosae from 10 patients. Data were correlated with various clinical phenotypes. In paired samples, average relative telomere length of rectal inflammatory mucosa was shortened compared to normal appearing proximal colon in eight out of ten cases (p=0.01). Telomere length shortening was significantly associated with more severe Mayo endoscopic subscore (p<0.0001) and cases needing surgery due to toxic megacolon or cancer occurrence (p=0.043). When the severe clinical phenotype was defined as having at least one of following phenotypes, more than two times of hospitalization, highest Mayo endoscopic subscore, steroid dependent, refractory, or needing operation, average relative telomere length was significantly shortened in the same phenotypes than the others (p=0.003). Telomere shortening is associated with more severe clinical phenotypes of UC, reflecting severe inflammatory state in the colonic mucosa.