Regulated IFN signalling preserves the stemness of intestinal stem cells by restricting differentiation into secretory-cell lineages.

Intestinal stem cells (ISCs) are located at the crypt base and fine-tune the balance of their self-renewal and differentiation1,2, but the physiological mechanism involved in regulating that balance remains unknown. Here we describe a transcriptional regulator that preserves the stemness of ISCs by restricting their differentiation into secretory-cell lineages. Interferon regulatory factor 2 (IRF2) negatively regulates interferon signalling3, and mice completely lacking Irf24 or with a selective Irf2 deletion in their intestinal epithelial cells have significantly fewer crypt Lgr5hi ISCs than control mice. Although the integrity of intestinal epithelial cells was unimpaired at steady state in Irf2-deficient mice, regeneration of their intestinal epithelia after 5-fluorouracil-induced damage was severely impaired. Similarly, extended treatment with low-dose poly(I:C) or chronic infection of lymphocytic choriomeningitis virus clone 13 (LCMV C13)5 caused a functional decline of ISCs in wild-type mice. In contrast, massive accumulations of immature Paneth cells were found at the crypt base of Irf2-/- as well as LCMV C13-infected wild-type mice, indicating that excess interferon signalling directs ISCs towards a secretory-cell fate. Collectively, our findings indicate that regulated interferon signalling preserves ISC stemness by restricting secretory-cell differentiation.

CHAC1 overexpression in human gastric parietal cells with Helicobacter pylori infection in the secretory canaliculi.

BACKGROUND: Cation transport regulator 1 (CHAC1), a newly discovered enzyme that degrades glutathione, is induced in Helicobacter pylori (H. pylori)-infected gastric epithelial cells in culture. The CHAC1-induced decrease in glutathione leads to an accumulation of reactive oxygen species and somatic mutations in TP53. We evaluated the possible correlation between H. pylori infection and CHAC1 expression in human gastric mucosa. MATERIALS AND METHODS: Both fresh-frozen and formalin-fixed paraffin-embedded tissue samples of gastric mucosa with or without H. pylori infection were obtained from 41 esophageal cancer patients that underwent esophago-gastrectomy. Fresh samples were used for real-time polymerase chain reaction for H. pylori DNA and CHAC1 mRNA, and formalin-fixed samples were used for immunohistochemistry with anti-CHAC1 and anti-H. pylori monoclonal antibodies. Double-enzyme or fluorescence immunohistochemistry and immuno-electron microscopy were used for further analysis. RESULTS: Significant CHAC1 overexpression was detected in H. pylori-infected parietal cells that expressed the human proton pump/H,K-ATPase α subunit, whereas a constitutively low level of CHAC1 mRNA expression was observed in the other samples regardless of the H. pylori infection status, reflecting the weak CHAC1 expression detected by immunohistochemistry in the fundic-gland areas. Immuno-electron microscopy revealed intact H. pylori cells in the secretory canaliculi of infected parietal cells. Some parietal cells exhibited positive nuclear signals for Ki67 in the neck zone of the gastric fundic-gland mucosa with H. pylori infection. CONCLUSION: Cation transport regulator 1 overexpression in H. pylori-infected parietal cells may cause the H. pylori-induced somatic mutations that contribute to the development of gastric cancer.

Helicobacter pylori induces somatic mutations in TP53 via overexpression of CHAC1 in infected gastric epithelial cells.

Infection with Helicobacter pylori is known to decrease the level of glutathione in gastric epithelial cells and increase the production of reactive oxygen species (ROS), which can lead to DNA damage and the development of gastric cancer. Cation transport regulator 1 (CHAC1) has γ-glutamylcyclotransferase activity that degrades glutathione. We found that cagA-positive H. pylori infection triggered CHAC1 overexpression in human gastric epithelial (AGS) cells leading to glutathione degradation and the accumulation of ROS. Nucleotide alterations in the TP53 tumour suppressor gene were induced in AGS cells overexpressing CHAC1, whereas no mutations were detected in cells overexpressing a catalytically inactive mutant of CHAC1. A high frequency of TP53 mutations occurred in H. pylori-infected AGS cells, but this was prevented in cells transfected with CHAC1 siRNA. These findings indicate that H. pylori-mediated CHAC1 overexpression degrades intracellular glutathione, allowing the accumulation of ROS which subsequently causes mutations that could contribute to the development of gastric cancer.

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 α-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 ß-catenin was determined by reporter analysis and chromatin immunoprecipitation assay. HD5/HD6/Atoh1/ß-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 ß-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 ß-catenin was impaired. CONCLUSIONS: HD6 expression was found to be regulated by cooperation between Atoh1 and ß-catenin within the HD6 promoter region. Downregulation of HD6 in noninflamed mucosa may contribute to mucosal barrier dysfunction of patients with CD.

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-3ß 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.

Clinical characteristics of Japanese oropharyngeal squamous cell carcinoma positive for human papillomavirus infection.

CONCLUSION: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is considered to be a distinct entity in Japan. The combination of both HPV-DNA sequencing analysis and immunohistochemistry (IHC) for p16(INK4A) is useful to discriminate the OPSCC patients with a better prognosis from other cases, especially in the advanced stage. Surgical treatment is recommended for HPV-negative advanced cancer. OBJECTIVE: The number of HPV-related OPSCCs has been increasing worldwide. However, the incidence and prognostic significance of this cancer in Japan have not yet been fully elucidated. METHODS: Seventy-seven Japanese patients with OPSCC were enrolled in this study. The prevalence of HPV-DNA was assessed by PCR and sequencing. The expression of p16(INK4A) and p53 was examined by IHC. The clinicopathological parameters and disease-specific survival were analyzed for HPV-positive and -negative patients. RESULTS: HPV-DNA was detected in 32 patients. Thirty-four patients were p16(INK4A)-positive by IHC. The patients who were positive for HPV infection were significantly younger. Furthermore, in the stage III or IV cases, the 3-year disease-specific survival of the HPV infection-positive group was significantly better than that of the HPV-negative group. Surgical treatment was demonstrated to lead to a good prognosis for the patients with HPV-negative advanced cancer.

γ-Glutamylcyclotransferase as a novel immunohistochemical biomarker for the malignancy of esophageal squamous tumors.

Recently, overexpression of γ-glutamylcyclotransferase (GGCT) has been reported in various cancer tissues suggesting that it has significant potential as a diagnostic marker. The aim of this study was to examine the suitability of GGCT for the detection of high-risk lesions at an early stage of esophageal squamous cell carcinoma (ESCC). A total of 200 lesions, including 120 invasive ESCC, 80 esophageal squamous intraepithelial neoplasia consisting of 40 low-grade intraepithelial neoplasia (LGIEN) and 40 high-grade intraepithelial neoplasia (HGIEN), as well as 20 confounding lesions, were examined by immunohistochemical (IHC) staining for GGCT. IHC staining for Ki-67 and p53 was also performed in esophageal squamous intraepithelial neoplasia to compare the diagnostic power of GGCT. Increased expression of GGCT was common in invasive ESCC and HGIEN (87.5% and 85.0%, respectively), but was much less frequently observed in LGIEN (17.5%). GGCT expression significantly correlated with the presence of lymph node metastasis and the degree of differentiation. In the differential diagnosis of LGIEN and HGIEN, GGCT possessed both high sensitivity and high specificity, while Ki-67 and p53 only possessed either high sensitivity or high specificity. Additionally, GGCT expression was higher in 7 out of 8 ESCC cell lines (KYSE series) than in a normal esophageal squamous cell line (Het-1A). The expression levels strongly correlated with enzymatic activity (r=.92; P<.001). These results indicate that overexpressed GGCT retains its enzymatic activity and can become a valuable biomarker for the diagnosis of HGIEN that is likely to progress to subepithelial invasion.

"Crawling-type" adenocarcinoma of the stomach: a distinct entity preceding poorly differentiated adenocarcinoma.

BACKGROUND: Gastric "crawling-type" adenocarcinoma (CTAC) is a neoplasm histologically comprising irregularly fused glands with low-grade cellular atypia that tends to spread laterally in the mucosa. It is necessary to elucidate the clinicopathological characteristics of CTAC. METHODS: We evaluated 25 CTACs-16 intramucosal (M-) and 9 submucosal invasive (SM-) cancers-clinicopathologically and immunohistochemically. RESULTS: CTAC was most frequently located in the lesser curvature of the middle-third of the stomach. Macroscopically, 21 lesions were superficial-depressed and 4 were superficial-flat type. Histologically, all CTACs had cystic dilated glands and 16 lesions had focal signet-ring cells. All invasive areas of the SM-CTACs were occupied by poorly differentiated adenocarcinoma with an infiltrative growth pattern and abundant stroma. Fifteen CTACs were surrounded by mucosa with partial or no intestinal metaplasia. In the intramucosal area, 24 lesions were mixed phenotype with mucin and brush border immunoexpression. SM-CTAC was frequent in lesions with an intramucosal poorly differentiated component (PDC) greater than 10 mm in size (P = 0.041), and lymph node metastasis (LNM) was frequent in lesions with a PDC greater than 20 mm (P = 0.039). The frequency of an expanded pattern (Ki-67-positive cells occupying > 50 % of the mucosa) was higher in SM-CTAC than in M-CTAC (P = 0.027). p53 overexpression was not detected in the intramucosal areas of any of the lesions. CONCLUSION: CTAC is a distinct subgroup of gastric adenocarcinoma in the early phase. A larger PDC and a Ki-67 expanded pattern were predictive of submucosal invasion or LNM.

Reduced expression of cytokeratin 4 and 13 is a valuable marker for histologic grading of esophageal squamous intraepithelial neoplasia.

Histologic evaluation of low-grade or high-grade intraepithelial neoplasia (LG-IN or HG-IN) of the esophagus is important for estimating the risk of progression to invasive carcinoma. Discrimination between LG-IN and HG-IN, or neoplasia and non-neoplastic lesion (NNL), however, is occasionally difficult. This study was designed to evaluate whether cytokeratin expression can be used for discrimination of these lesions. Esophageal Iodine-unstained lesions (n=154), less than 10 mm, were classified into HG-IN, LG-IN, and NNL. These lesions together with 154 foci of normal esophageal epithelium (NEE) were examined by immunohistochemistry for cytokeratins (CK4 and CK13), p53 overexpression, and the MIB-1 labeling index. The ratios of CK4- and CK13-positive staining were scored from 1 to 3. The CK4- and CK13-positive staining ratios were decreased in NNL (73% and 78%), LG-IN (55% and 69%), and HG-IN (33% and 48%), compared to NEE (91% and 95%). The differences between LG-IN and HG-IN, neoplasia and NNL, and among these three lesions and NEE were statistically significant (p < 0.005). The cytokeratin scores correlated with the MIB-1 labeling index (both: p < 0.0001), but not with p53 overexpression. CK4 and CK13 immunohistochemistry could be an objective method for evaluating the risk for progression to invasive carcinoma.

Longitudinal cell formation in the entire human small intestine is correlated with the localization of Hath1 and Klf4.

BACKGROUND: Double balloon endoscopy (DBE) enables the observation and collection of viable specimens from the entire intestine, thereby allowing more detailed investigation of how the structure and function of the human small intestine are regulated. The present study aimed to elucidate the regulation of cell formation in the human small intestine using biopsy specimens collected from an entire individual small intestine by DBE. METHODS: The expression and the localization of representative genes for the differentiation program were analyzed in the entire small intestine of 10 patients. The functional correlation between Hath1 and Klf4 was analyzed in an intestinal cell line by using a Tet-On system. RESULTS: In longitudinal cell formation in the small intestine, it was shown that goblet cells, but not Paneth cells, increased toward the ileum in each individual small intestine. Immunohistochemistry showed that Hath1-expressing cells migrated from the base of the crypt to the top of the villi in the terminal ileum, while Klf4-expressing cells migrated from the top of the villus, resulting in the colocalization of Hath1 and Klf4 in the terminal ileum. Coexpression of Hath1 and Klf4 upregulated the expression of phenotypic genes for goblet cells following the downregulation of those for Paneth cells. CONCLUSIONS: Using mapping biopsy by DBE, we have demonstrated, for the first time, the molecular basis of the villus structure in the entire human small intestine in vivo. The present study showed that longitudinal cell formation was regulated by the colocalization of Hath1 and Klf4 that converted Paneth cell differentiation into goblet cell differentiation.

Airway pressure release ventilation reduces the increase in bronchoalveolar lavage fluid high-mobility group box-1 levels and lung water in experimental acute respiratory distress syndrome induced by lung lavage.

BACKGROUND AND OBJECTIVE: Airway pressure release ventilation (APRV) may provide better alveolar recruitment at a lower peak airway pressure than conventional mechanical ventilation (CMV) and, therefore, decrease the risk of barotrauma in patients with acute lung injury and acute respiratory distress syndrome. The present study compared the effects of APRV with low tidal volume ventilation (LTV) and CMV on the ongoing response in lung injury induced by whole lung lavage. METHODS: Lung injury was induced by whole lung lavage. Twenty-one Japanese white rabbits were randomized to receive CMV (tidal volume 10 ml kg, positive end-expiratory pressure 3 cmH2O), LTV (tidal volume 6 ml kg, positive end-expiratory pressure 10 cmH2O), or APRV (Phigh 20 cmH2O, Plow 5 cmH2O). After 4 h of treatment, the lungs and heart were excised en bloc. The left lung was lavaged, and high-mobility group box-1 (HMGB1) levels were measured in the lavage. The right lung was analysed histologically and its wet-to-dry weight ratio was calculated. RESULTS: PaO2 was decreased after the induction of lung injury, but the values were significantly higher in the APRV and LTV groups after treatment than in the CMV group. Serum HMGB1 levels did not change before and after lung injury; however, bronchoalveolar lavage fluid HMGB1 levels were significantly increased at the end of the experiment (266.8 +/- 47.9 in the CMV group, 137.4 +/- 23.4 in the LTV group, and 91.2 +/- 5.4 ng ml in the APRV group). The bronchoalveolar lavage fluid HMGB1 levels after experiment were significantly lower in the APRV group than in the CMV and LTV groups (P < 0.0001 and P = 0.0391, respectively). Wet-to-dry weight ratios were also lowest in the APRV group. CONCLUSION: APRV reduces bronchoalveolar lavage fluid HMGB1 levels and lung water and it preserves oxygenation and systemic blood pressure in experimental acute respiratory distress syndrome. The results suggest that APRV could be as protective for acute respiratory distress syndrome as LTV with positive end-expiratory pressure.