Ryohei Hayashi

Is It Possible to Discriminate Between Neoplastic and Nonneoplastic Lesions in Ulcerative Colitis by Magnifying Colonoscopy

Background:Colitis-associated cancer/dysplasia is an intestinal tract condition that can affect the life expectancy of patients with ulcerative colitis. It is often difficult to detect neoplastic lesions. This study evaluated whether any endoscopic features are effective for distinguishing colitis-associated cancer/dysplasia from nonneoplastic lesions in patients with ulcerative colitis. Methods:The study involved 52 patients with 61 lesions treated at Hiroshima University Hospital between September 1999 and May 2012: 10 patients with 11 dysplastic lesions, 5 patients with 5 intramucosal carcinomas, 3 patients with 3 submucosal carcinomas, and 34 patients with 42 nonneoplastic lesions. All patients had undergone targeted biopsy. Endoscopic findings were compared between patients with biopsy-determined neoplasia and those with biopsy-determined nonneoplasia. Multivariate regression analysis was performed to identify magnifying chromocolonoscopy features predictive of neoplasia. Results:No significant difference was found in conventional endoscopy features between the neoplastic and nonneoplastic lesions. Under magnifying chromocolonoscopy, the pit density of the neoplastic lesions was found to be significantly greater than that of the nonneoplastic lesions (89% [17/19] versus 60% [25/42], respectively). Pit margins were more frequently irregular in the neoplastic lesions than in the nonneoplastic lesions (63% [12/19] versus 33% [14/42], respectively). Conclusions:In differentiating between colitis-associated neoplastic and nonneoplastic lesions, focus should be on the high residual density of pits and irregular pit margins observed under magnifying chromocolonoscopy.

Atonal homolog 1 protein stabilized by tumor necrosis factor α induces high malignant potential in colon cancer cell line

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colitis‐associated colorectal cancer (CAC). CAC cells often develop chemoresistance, resulting in a poorer prognosis than that of sporadic colorectal cancer (CRC). The mechanism by which CAC enhances malignant potential remains unknown. We have previously reported that the proteasomal degradation of the transcription factor Atonal homolog 1 (Atoh1) protein results in the non‐mucinous form of CRC. It also remains unknown whether Atoh1 protein is expressed in CAC. Therefore, in the present study, we investigated whether Atoh1 protein stabilizes in CAC. Consequently, the treatment with TNF‐α stabilized Atoh1 protein through the inactivation of GSK‐3β via Akt, resulting in the mucinous form of CRC cell lines. Atoh1 protein also enriched cancer stem cells with upregulated Lgr5 expression and cells in G0/G1 cell cycle phase, resulting in both the chemoresistance to 5‐fluorouracil and oxaliplatin and the promotion of cell migration. Immunofluorescence of the human mucinous CAC specimens showed the accumulation of NF‐κB p65 at nuclei with the expression of Atoh1 in mucinous cancer. In conclusion, the inflammation associated with carcinogenesis may preserve the differentiation system of intestinal epithelial cell (IEC), resulting in the acquisition of both the mucinous phenotype and high malignant potential associated with the enrichment of cancer stem cell.

Long-term Inflammation Transforms Intestinal Epithelial Cells of Colonic Organoids

Background and Aims Patients with ulcerative colitis [UC] are at an increased risk of developing colitis-associated cancer [CAC], suggesting that continuous inflammation in the colon promotes the transformation of colonic epithelial cells. However, the mechanisms underlying cell transformation in UC remain unknown. We therefore aimed to investigate the effect of long-term inflammation on intestinal epithelial cells [IECs] using organoid culture. Methods IECs were isolated from mouse colon, and were cultured according to a method for a three-dimensional [3D] organoid culture. To mimic chronic inflammation, a mixture of cytokines and bacterial components were added to the medium for over a year. Cell signal intensity was assessed by 3D immunofluorescence. Cell transformation was assessed by microarray with gene set enrichment analysis. Results Stimulation with cytokines resulted in a significant induction of target genes for the nuclear factor [NF]-κB pathway in colonic organoids. Following 60 weeks of continuous stimulation, cell differentiation was suppressed. Continuous stimulation also resulted in significant amplification of NF-κB signalling. Amplified NF-κB signalling by long-term stimulation remained in colonic organoids even 11 weeks after the removal of all cytokines. Some genes were specifically upregulated only in colonic organoids after the removal all cytokines following long-term stimulation. Conclusions Colonic organoids stimulated with cytokines for a prolonged period were established as in vitro model to assess long-term epithelial responses to inflammatory cytokines. Chronic inflammation led to sustained NF-κB signalling activation in colonic organoids, resulting in cell transformation that might be related to the carcinogenesis of CAC in UC.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

BACKGROUND AND AIMS The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. METHODS Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. RESULTS We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. CONCLUSIONS The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

Clinical usefulness of classification by transabdominal ultrasonography for detection of small-bowel stricture.

Abstract Objective. To assess the clinical usefulness of transabdominal ultrasonography (TUS) for detection of small-bowel stricture. Patients and methods. Subjects were 796 patients undergoing double-balloon endoscopy (DBE), December 2003–October 2011. All underwent TUS prior to DBE. The TUS findings were classified by type as intestinal narrowing and distension at the oral side (Type A); extensive bowel wall thickening (Type B); focal bowel wall thickening (Type C) or no abnormality detected (Type D). We compared TUS findings against DBE findings with respect to small-bowel stricture, defined as failure of the enteroscope to pass through the small bowel. Results. Small-bowel stricture was detected by DBE in 11.3% (90/796) of patients. Strictures resulted from Crohns disease (n = 36), intestinal tuberculosis (n = 24), malignant lymphoma (n = 9), ischemic enteritis (n = 6), NSAID ulcer (n = 5), radiation enteritis (n = 2), surgical anastomosis (n = 2) and other abnormalities (n = 6). Stricture was detected by TUS in 93.3% (84/90) of patients, and each such stricture fell into one of the three types of TUS abnormality. The remaining 6 strictures were detected only by DBE. DBE-identified strictures corresponded to TUS findings as follows: 100% (43/43) to Type A, 59.1% (29/49) to Type B, 14.8% (12/81) to Type C and 1% (6/623) to Type D. Correspondence between stricture and the Type A classification (vs. Types B, C and D) was significantly high, as was correspondence between stricture and Type B (vs. Types C and D). Conclusions. TUS was shown to be useful for detecting small-bowel stricture. We recommend performing TUS first when a small-bowel stricture is suspected.

Choline Deficiency Causes Colonic Type II Natural Killer T (NKT) Cell Loss and Alleviates Murine Colitis under Type I NKT Cell Deficiency

Serum levels of choline and its derivatives are lower in patients with inflammatory bowel disease (IBD) than in healthy individuals. However, the effect of choline deficiency on the severity of colitis has not been investigated. In the present study, we investigated the role of choline deficiency in dextran sulfate sodium (DSS)-induced colitis in mice. Methionine-choline-deficient (MCD) diet lowered the levels of type II natural killer T (NKT) cells in the colonic lamina propria, peritoneal cavity, and mesenteric lymph nodes, and increased the levels of type II NKT cells in the livers of wild-type B6 mice compared with that in mice fed a control (CTR) diet. The gene expression pattern of the chemokine receptor CXCR6, which promotes NKT cell accumulation, varied between colon and liver in a manner dependent on the changes in the type II NKT cell levels. To examine the role of type II NKT cells in colitis under choline-deficient conditions, we assessed the severity of DSS-induced colitis in type I NKT cell-deficient (Jα18-/-) or type I and type II NKT cell-deficient (CD1d-/-) mice fed the MCD or CTR diets. The MCD diet led to amelioration of inflammation, decreases in interferon (IFN)-γ and interleukin (IL)-4 secretion, and a decrease in the number of IFN-γ and IL-4-producing NKT cells in Jα18-/- mice but not in CD1d-/- mice. Finally, adaptive transfer of lymphocytes with type II NKT cells exacerbated DSS-induced colitis in Jα18-/- mice with MCD diet. These results suggest that choline deficiency causes proinflammatory type II NKT cell loss and alleviates DSS-induced colitis. Thus, inflammation in DSS-induced colitis under choline deficiency is caused by type II NKT cell-dependent mechanisms, including decreased type II NKT cell and proinflammatory cytokine levels.

Successful Use of Adalimumab for Treating Pyoderma Gangrenosum with Ulcerative Colitis under Corticosteroid-tapering Conditions.

A 52-year-old woman with ulcerative colitis was admitted to our hospital for an ulcerative colitis flare-up under salazosulfapyridine therapy. The symptoms improved with high-dose corticosteroids. After prednisolone was tapered to 10 mg, the frequency of diarrhea increased. The diarrhea was accompanied by joint pain and a skin ulcer with abscess formation, which was diagnosed to be pyoderma gangrenosum. The patient was started on adalimumab. A positive response to the adalimumab therapy was observed after 2 weeks, during which time the ulcerative skin lesion healed completely, however, colonic mucosal healing was achieved at 2 months. Therefore, adalimumab appears to be an effective therapeutic option for patients with ulcerative colitis-associated pyoderma gangrenosum.

Reduced Human α-defensin 6 in Noninflamed Jejunal Tissue of Patients with Crohn’s Disease:

Background:Mucosal barrier dysfunction is considered a critical component of Crohn’s disease (CD) pathogenesis after the identification of susceptibility genes. However, the precise mechanism underlying mucosal barrier dysfunction has not yet been elucidated. We therefore aimed to elucidate the molecular mechanism underlying the expression of human &agr;-defensin 6 (HD6) in patients with CD. Methods:HD6 expression was induced by the transfection of an atonal homolog 1 (Atoh1) transgene and was assessed by reverse transcription polymerase chain reaction. The HD6 promoter region targeted by Atoh1 and &bgr;-catenin was determined by reporter analysis and chromatin immunoprecipitation assay. HD5/HD6/Atoh1/&bgr;-catenin expression in noninflamed jejunal samples collected by balloon endoscopy from 15 patients with CD and 9 non-inflammatory bowel disease patients were assessed by immunofluorescence. Results:Both promoter activity and gene expression of HD6 was significantly upregulated by the Atoh1 transgene in human colonic cancer cell line. We identified a TCF4 binding site and an E-box site, critical for the regulation of HD6 transcriptional activity by directly binding of Atoh1 in the 200-bp HD6 promoter region. The treatment with &bgr;-catenin inhibitor also decreases HD6 promoter activity and gene expression. Moreover, HD6 expression, but not HD5 expression, was found to be decreased in noninflamed jejunal regions from patients with CD. In HD6-negative crypts, nuclear accumulation of &bgr;-catenin was impaired. Conclusions:HD6 expression was found to be regulated by cooperation between Atoh1 and &bgr;-catenin within the HD6 promoter region. Downregulation of HD6 in noninflamed mucosa may contribute to mucosal barrier dysfunction of patients with CD.

Clinical usefulness of narrow band imaging magnifying colonoscopy for assessing ulcerative colitis-associated cancer/dysplasia

Background and study aims: Colitis-associated cancer/dysplasia (CC/D) can affect the life expectancy of patients with ulcerative colitis (UC). Although the utility of magnifying chromocolonoscopy has been shown, the use of optical magnification with narrow band imaging (NBI) for distinguishing CC/D from non-neoplastic lesions in patients with UC has not been reported. We evaluated whether endoscopic findings are distinguishing and thus assessed the clinical usefulness of NBI magnification for differentiating UC-associated lesions. Patients and methods: The study involved 27 patients diagnosed and treated at Hiroshima University Hospital between September 2005 and March 2015: a neoplasia group (16 lesions) and a non-neoplasia group (17 lesions). The neoplasias comprised 9 dysplastic lesions, 5 intramucosal carcinomas, and 2 submucosal carcinomas, and 17 non-neoplastic lesions. Targeted biopsy samples of suspicious lesions detected by conventional colonoscopy were classified pathologically as neoplastic or non-neoplastic, and NBI magnifying colonoscopy findings (i. e., the surface [unclear/regular/irregular/amorphous] and vascular [same as the background mucosa/regular/irregular/avascular] patterns) of the 2 lesion types were compared. Results: Irregular/amorphous surface patterns were significantly more common in neoplastic lesions than in non-neoplastic lesions (81 % [13/16] vs. 18 % [3/17], respectively, P < 0.001). Irregular/avascular vessel pattern tended to be more common in neoplastic lesions (75 % [12/16] vs. 41 % [7/17], respectively). The surface pattern correctly predicted 82 % of neoplastic lesions, and the vessel pattern correctly predicted 67 % of non-neoplastic lesions. The 2 endoscopic findings together correctly predicted 91 % of neoplastic lesions. Conclusion: Surface pattern, determined by magnifying colonoscopy with NBI, is useful for differenting between UC-associated neoplastic and non-neoplastic lesions.

Caudal type homeobox 2 expression induced by leukocytapheresis might be associated with mucosal healing in ulcerative colitis: CDX2 by LCAP for mucosal healing in UC

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with an intractable, recurrent course. Although the goal of UC therapy has recently been to target mucosal healing, the molecular mechanism of mucosal healing remains unknown. In this study, we aimed to elucidate the molecular dynamics related to the proliferation and differentiation of intestinal epithelial cells during cytapheresis therapy in a short duration.