Norihisa Ishimura

Strategic compartmentalization of Toll-like receptor 4 in the mouse gut.

Pattern recognition receptors (PRRs), which include the Toll-like receptors (TLRs), are involved in the innate immune response to infection. TLR4 is a model for the TLR family and is the main LPS receptor. We wanted to determine the expression of TLR4 and compare it with that of TLR2 and CD14 along the gastrointestinal mucosa of normal and colitic BALB/c mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS). Mucosa from seven segments of the digestive tract (stomach, small intestine in three parts, and colon in three parts) was isolated by two different methods. Mucosal TLR4, CD14, TLR2, MyD88, and IL-1β mRNA were semiquantified by Northern blotting. TLR4 protein was determined by Western blotting. TLR4/MD-2 complex and CD14 were evaluated by immunohistochemistry. PRR genes were constitutively expressed and were especially stronger in colon. TLR4 and CD14 mRNA were increased in the distal colon, but TLR2 mRNA was expressed more strongly in the proximal colon, and MyD88 had a uniform expression throughout the gut. Accordingly, TLR4 and CD14 protein levels were higher in the distal colon. TLR4/MD-2 and CD14 were localized at crypt bottom epithelial cells. TLR4/MD2, but not CD14, was found in mucosal mononuclear cells. Finally, DSS-induced inflammation was localized in the distal colon. All genes studied were up-regulated during DSS-induced inflammation, but the normal colon-stressed gut distribution was preserved. Our findings demonstrate that TLR4, CD14, and TLR2 are expressed in a compartmentalized manner in the mouse gut and provide novel information about the in vivo localization of PRRs.

SEROLOGICALLY SILENT HEPATITIS B VIRUS COINFECTION IN PATIENTS WITH HEPATITIS C VIRUS-ASSOCIATED CHRONIC LIVER DISEASE: CLINICAL AND VIROLOGICAL SIGNIFICANCE

Frequent coinfection of surface antigen‐negative hepatitis B virus (silent HBV) in hepatitis C virus (HCV)‐associated chronic liver disease (CLD) has been reported. The clinical and virological significance of silent HBV infection was investigated in 65 patients with HCV‐associated CLD who subsequently received interferon (IFN) therapy. HBV DNA was detected in 34 (52.3%) patients by a nested polymerase chain reaction (PCR). Virologically, all of the 34 patients were found to have HBV with an eight‐nucleotide deletion in the core promoter. Coinfection of silent HBV was more frequent with HCV genotype 1b than in 2a (64.3% vs 28.6%, P < .01). With HCV genotype 1b, the serum RNA level was significantly higher (≥106 copies per milliliter vs ≤105 copies per milliliter) in patients with silent HBV than those without coinfection (P < .01). Clinically, silent HBV was associated with a higher level of serum alanine aminotransferase (158.5 ± 104.8 vs 121.8 ± 78.6 IU/l; mean ± SD) and a greater histological activity of hepatitis as evaluated by histological activity index score (9.4 ± 3.8 vs 8.6 ± 4.5; mean ± SD), although it was not statistically significant. Silent HBV was also associated with poor efficacy of IFN therapy (P < .01). The results suggest that silent HBV has some promoting effect for HCV replication, at least for HCV genotype 1b, and may affect the histological activity of hepatitis and IFN response in HCV‐associated CLD. J. Med. Virol. 58:201–207, 1999.

Essential Role of MD-2 in TLR4-Dependent Signaling during Helicobacter pylori-Associated Gastritis

TLR4, a member of pattern recognition receptors, is the main receptor of LPS. MD-2 physically associates with TLR4 on the cell surface and confers LPS responsiveness. Helicobacter pylori LPS is one of the major virulence factors for induction of gastritis. We demonstrated in this study the role of MD-2 in TLR4-dependent signaling in H. pylori-associated gastritis. Gastric biopsy samples collected from patients with and without H. pylori infection and four gastric cancer cell lines were used for this study. TLR-4 and MD-2 expression in biopsy specimens and the cell lines was examined by using RT-PCR. Localization of TLR-4 in histological sections was evaluated by immunohistochemistry. For in vitro functional assays, we established stable transfectants of AGS cells expressing TLR4 and MD-2. Cellular distribution of TLR4 was examined by flow cytometry. NF-κB activation and activation of IL-8 and MD-2 promoters were assessed by reporter gene assay. H. pylori infection up-regulated the TLR4 and MD-2 expression in gastric mucosa. TLR4 staining was observed predominantly in epithelial cells, located in both the cytoplasm and at the apical surface. MD-2 transfection in AGS cells markedly increased cell surface expression of TLR4 and augmented the activation of NF-κB and IL-8 promoter upon stimulation with H. pylori LPS. Live H. pylori also stimulated transcriptional activation of MD-2. This study revealed that MD-2 expression is elevated in gastric epithelial cells during H. pylori infection, suggesting that the TLR4/MD-2 system is a potent receptor complex involved in the response to H. pylori LPS in the stomach.

Co-infection by serologically-silent hepatitis B virus may contribute to poor interferon response in patients with chronic hepatitis C by down-regulation of type-I interferon receptor gene expression in the liver

Intrahepatic mRNA levels of type‐I interferon (IFN) receptor genes have been shown to correlate with the clinical efficacy of IFN therapy in patients with chronic hepatitis C. Recently, co‐infection by serologically‐silent hepatitis B virus (HBV) has been assumed to be associated with the poor IFN response in patients with chronic hepatitis C. The aim of this study was to investigate the relationship between the co‐infection of serologically‐silent HBV and type‐I IFN receptor gene expression in the liver of patients with chronic hepatitis C. The intrahepatic mRNA levels of IFNAR2, one of the two subunits of the type‐I IFN receptor, were quantified and compared with both the prevalence of HBV DNA and the hepatitis C virus (HCV) genotype in 45 patients with chronic hepatitis C, who were negative for hepatitis B surface antigen. Co‐infection, as evaluated by a nested polymerase chain reaction, was present in 22 patients (48.9%), with dominance of the HCV genotype 1b (65.2%) over genotype 2a (31.8%). Co‐infection was associated with lower IFNAR2 mRNA levels, higher levels of serum HCV RNA, and a poor IFN response, regardless of the HCV genotype. The findings suggest the possibility that co‐infection by serologically‐silent HBV is one of the factors that can lead to an unfavorable IFN response in chronic hepatitis C by down‐regulation of IFN receptor gene expression in the liver. J. Med. Virol. 63:220–227, 2001.

BRAF and K-ras gene mutations in human pancreatic cancers

We investigated the frequency of BRAF mutations in human pancreatic cancer specimens to determine its role in the development of pancreatic cancer. Nine pancreatic cancer samples without a K-ras codon 12 mutation and 19 with a K-ras mutation were included in the study. Analyses of the BRAF sequence revealed mutations in exon 15 (V599E) in two cases, both of which also exhibited a K-ras codon 12 mutation. No BRAF mutation was found in cases without a K-ras mutation. The BRAF V599E mutation was not found to be a major mutation in pancreatic cancers that had no K-ras codon 12 mutation.

Hepatitis B Virus with X Gene Mutation Is Associated with the Majority of Serologically "Silent" Non-B, Non-C Chronic Hepatitis

Hepatitis B virus (HBV) with X gene mutations has been a putative pathogen of chronic hepatitis without serological markers of known hepatitis viruses. The aim of this study was to reconfirm whether the HBV with the X gene mutation is associated with these serologically “silent” non‐B, non‐C (NBNC) chronic hepatitis, alcoholic liver disease (ALD) and autoimmune hepatitis (AIH). HBV DNA was amplified from serum and sequenced in 30 patients with NBNC chronic hepatitis in comparison with 20 patients with ALD and 5 patients with AIH. HBV DNA was identified in 21 patients (70%) in NBNC chronic hepatitis by nested polymerase chain reaction while only one patient (5%) in ALD and none in AIH showed HBV DNA. Eighteen (85.7%) of the 21 identified HBV DNAs had an identical 8‐nucleotide deletion mutation at the distal part of the X region. This mutation affected the core promoter and the enhancer II sequence of HBV DNA and created a translational stop codon which truncated the X protein by 20 amino acids from the C‐terminal end. All the HBV DNAs had a precore mutation at the 83rd nucleotide resulting in disruption of HBe antigen synthesis. These results indicate that HBV mutants are closely associated with the majority of serologically “silent” NBNC chronic hepatitis cases and the population of such mutant HBV DNAs is not uniform.

A20 is an early responding negative regulator of Toll-like receptor 5 signalling in intestinal epithelial cells during inflammation.

Several negative regulatory mechanisms control Toll‐like receptor (TLR)‐mediated inflammatory responses and restore immune system balance, including the zinc‐finger protein A20, a negative regulator of TLR signalling that inhibits nuclear factor kappa B (NF‐κB) activity. In the present study, we investigated TLR‐5‐mediated A20 expression and its role in intestinal epithelial cells (IECs) during inflammation. HCT‐15 and HT‐29 cells were stimulated with flagellin, then the expressions of A20, interleukin‐1 receptor‐associated kinase (IRAK‐M) and Tollip were evaluated using RNase protection assay. Furthermore, experimental colitis was induced in tlr4‐deficient CH3/HeJ mice by administration of dextran sodium sulphate (DSS), then flagellin was injected anally, and the colonic expression of A20 was examined by real‐time polymerase chain reaction (PCR) and immunohistochemistry. To confirm flagellin‐induced expression of A20, we employed an organ culture system. The role of A20 in flagellin‐induced tolerance induction was evaluated in vitro, using a gene knock‐down method targeting A20. A20 expression increased rapidly and peaked at 1 h after flagellin stimulation in cultured IECs, then declined gradually to the basal level. In vivo, anal injection of flagellin induced epithelial expression of A20 in injured colonic tissue, whereas flagellin did not cause a significant increase in A20 expression in non‐injured normal tissue, which was also confirmed in vitro using the organ culture system. Gene knock‐down using A20 siRNA did not influence tolerance induced by restimulation with flagellin. A20 is an early response negative regulator of TLR‐5 signalling in IECs that functions during intestinal inflammation. Our results provide new insights into the negative feedback regulation of TLR‐5 signalling that maintains the innate immune system in the gut.

Barrett’s esophagus in Japanese patients: its prevalence, form, and elongation

BackgroundBarrett’s esophagus is a well-known acquired condition resulting from gastroesophageal reflux disease (GERD). However, it is still unknown whether Barrett’s esophagus develops gradually over time in patients with GERD. To address this issue, we investigated the change in the prevalence and length of short-segment Barrett’s esophagus (SSBE) over time.MethodsFrom January 2005 to March 2007, we enrolled 5338 patients who received upper gastrointestinal endoscopy. Prevalence and length of endoscopically identified SSBE were evaluated within groups divided on the basis of 10-year age intervals. The factors possibly influencing SSBE length such as symptoms, antacid use, and endoscopic findings were also evaluated. Additionally, the length change in 236 patients with histologically confirmed Barrett’s esophagus was evaluated over a 2-year follow-up.ResultsOf the 5338 enrolled patients, 1997 had SSBE. The prevalence of endoscopically identified SSBE was significantly higher and its length was significantly longer in elderly patients. Multiple regression analysis showed that age, presence of reflux esophagitis, reflux symptoms, and hiatal hernia were positively correlated with SSBE length. Analysis of the 2-year follow-up study of histologically confirmed SSBE revealed significant extension of Barrett’s length in 28.0% of 236 patients. Presence of reflux symptoms and hiatal hernia were identified as positive predictors and proton pump inhibitor administration as a negative predictor of SSBE elongation.ConclusionsPositive predictors for the extension of SSBE were presence of hiatal hernia and reflux symptoms, but not age.

Crystal Violet Chromoendoscopy with Mucosal Pit Pattern Diagnosis is Useful for Surveillance of Short-Segment Barrett's Esophagus

BACKGROUND:Because of a rapid increase in the incidence of Barretts cancer, the appropriate surveillance method for Barretts esophagus is of interest. Methylene blue chromoendoscopy has been reported to be an effective and inexpensive method to improve biopsy surveillance of Barretts epithelium. However, the usefulness of this method in short-segment Barretts esophagus cases is still controversial.AIMS:This study was undertaken to evaluate the abilities of crystal violet and methylene blue chromoendoscopy to detect potentially dysplastic Barretts epithelium in cases with short-segment columnar-appearing epithelium of the esophago-gastric junction.PATIENTS AND METHODS:Four hundred patients with endoscopically suspected short-segment Barretts esophagus were enrolled and randomly assigned to receive chromoendoscopy with 0.05% crystal violet, 0.1% crystal violet, 0.5% methylene blue, or 1.0% methylene blue. During crystal violet and methylene blue chromoendoscopy, biopsy specimens were obtained from stained and unstained columnar-appearing epithelium of the esophago-gastric junction, and the detection rates of Barretts epithelium were evaluated. The value of pit pattern diagnosis was also evaluated as a possible way to detect dysplastic Barretts epithelium.RESULTS:Chromoendoscopy with 0.05% crystal violet detected histologically confirmed Barretts epithelium with the highest sensitivity (89.2%) and specificity (85.7%). Crystal violet clearly stained both dysplastic and nondysplastic Barretts epithelia and made the surface pit pattern easy to observe without using magnifying endoscopy.CONCLUSIONS:The combination of crystal violet chromoendoscopy and pit pattern diagnosis is considered to be useful for the surveillance of short-segment Barretts esophagus.

Effectiveness of interferon-alpha therapy in chronic hepatitis C is associated with the amount of interferon-alpha receptor mRNA in the liver

BACKGROUND/AIMS This study aimed to investigate the relationship between interferon-alpha receptor mRNA in the liver and the response to interferon therapy in chronic hepatitis C. METHODS Interferon-alpha receptor mRNA was quantified by reverse transcription polymerase chain reaction using liver biopsies from 40 patients, comprising 20 responders and 20 non-responders to subsequent interferon therapy. RESULTS The amount of interferon-alpha receptor mRNA was significantly larger in interferon-responders (0.72+/-0.12) than non-responders (0.26+/-0.08) (p<0.01). Regardless of the response to interferon, histological activity index scores and the amount of HCV-RNA showed significant inverse correlation to the amount of interferon-alpha receptor mRNA, whereas the HCV-RNA genotype was not associated with the amount of interferon-alpha receptor mRNA. Logistic analysis and multiple regression analysis showed that the amount of interferon-alpha receptor mRNA was significantly associated with the efficacy of interferon (p=0.0275), but not with fibrosis of the liver (p= 0.2726). CONCLUSIONS Our results suggest that the amount of interferon-alpha receptor mRNA is an important factor determining the response to interferon, and may be a new predictor of interferon response in chronic hepatitis C.