Masahiro Iizuka

Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas

PTEN is a tumor suppressor gene mutated in many human cancers, and its expression is reduced or absent in almost half of hepatoma patients. We used the Cre-loxP system to generate a hepatocyte-specific null mutation of Pten in mice (AlbCrePten(flox/flox) mice). AlbCrePten(flox/flox) mice showed massive hepatomegaly and steatohepatitis with triglyceride accumulation, a phenotype similar to human nonalcoholic steatohepatitis. Adipocyte-specific genes were induced in mutant hepatocytes, implying adipogenic-like transformation of these cells. Genes involved in lipogenesis and beta-oxidation were also induced, possibly as a result of elevated levels of the transactivating factors PPARgamma and SREBP1c. Importantly, the loss of Pten function in the liver led to tumorigenesis, with 47% of AlbCrePten(flox/flox) livers developing liver cell adenomas by 44 weeks of age. By 74-78 weeks of age, 100% of AlbCrePten(flox/flox) livers showed adenomas and 66% had hepatocellular carcinomas. AlbCrePten(flox/flox) mice also showed insulin hypersensitivity. In vitro, AlbCrePten(flox/flox) hepatocytes were hyperproliferative and showed increased hyperoxidation with abnormal activation of protein kinase B and MAPK. Pten is thus an important regulator of lipogenesis, glucose metabolism, hepatocyte homeostasis, and tumorigenesis in the liver.

Wound healing of intestinal epithelial cells.

The intestinal epithelial cells (IECs) form a selective permeability barrier separating luminal content from underlying tissues. Upon injury, the intestinal epithelium undergoes a wound healing process. Intestinal wound healing is dependent on the balance of three cellular events; restitution, proliferation, and differentiation of epithelial cells adjacent to the wounded area. Previous studies have shown that various regulatory peptides, including growth factors and cytokines, modulate intestinal epithelial wound healing. Recent studies have revealed that novel factors, which include toll-like receptors (TLRs), regulatory peptides, particular dietary factors, and some gastroprotective agents, also modulate intestinal epithelial wound repair. Among these factors, the activation of TLRs by commensal bacteria is suggested to play an essential role in the maintenance of gut homeostasis. Recent studies suggest that mutations and dysregulation of TLRs could be major contributing factors in the predisposition and perpetuation of inflammatory bowel disease. Additionally, studies have shown that specific signaling pathways are involved in IEC wound repair. In this review, we summarize the function of IECs, the process of intestinal epithelial wound healing, and the functions and mechanisms of the various factors that contribute to gut homeostasis and intestinal epithelial wound healing.

No Mycobacterium paratuberculosis detected in intestinal tissue, including Peyer's patches and lymph follicles, of Crohn's disease.

Abstract: To clarify the etiologic significance of Mycobacterium paratuberculosis in Crohns disease, we investigated whether M. paratuberculosis was detected in intestinal tissues, including Peyers patches, where M. paratuberculosis invades, and colonic lymph follicles, where early lesions appear. Fifty-one samples of intestinal tissues, either therapeutically resected or biopsied, including 34 specimens from 30 patients with Crohns disease, were studied. Four Peyers patches and eight lymph follicles were included in the intestinal tissue samples of Crohns disease. They were visualized by acetic acid fixation. DNA extracted from intestinal tissues by proteinase K treatment was used for nested polymerase chain reaction (PCR) for detection of IS900, which is specific for M. paratuberculosis. PCR products were analyzed by agarose gel electrophoresis and subsequent Southern blot analysis. Our amplification system could detect 7.5 fg of M. paratuberculosis DNA. None of the tissue samples showed positive IS900 amplification, whereas they all showed amplification of the positive control human leukocyte antigen (HLA)-DQA DNA. Spiked experiments of tissue samples with M. paratuberculosis demonstrated that inhibitors of IS900 amplification were not present in the samples. Our study does not support the etiologic significance of M. paratuberculosis in Crohns disease.

Induction of major histocompatibility complex class II antigens on human colonic epithelium by interferon-gamma, tumor necrosis factor-alpha, and interleukin-2

Abstract: Class II antigens are strongly expressed on the inflamed colonic epithelium in inflammatory bowel disease. However, the mechanism of this epithelial class II antigen induction is not fully understood. Increased activities of interferon (IFN)γ, tumor necrosis factor (TNF)α, and interleukin (IL)2 have been shown in the inflamed mucosa of inflammatory bowel disease. Therefore, we studied whether these cytokines could induce class II antigens on the human colonic epithelium. By an organ culture technique, 284 normal colonic biopsy specimens obtained from 49 individuals were cultured in media containing different concentrations of cytokines with/without anti-IFNγ R antibody. Colonic epithelial class II antigens were identified by the indirect immunoperoxidase staining method with anti-human leukocyte antigen (HLA)-DR, DP, and DQ monoclonal antibodies. IFNγ, TNFα, and IL2 induced epithelial class II antigens in 16 of 16 cases (100%), 2 of 16 cases (12.5%), and 6 of 17 cases (35%), respectively. Epithelial class II antigen expression in response to TNFα was induced via IFNγ but not via IL2. This is the first demonstration that: (i) the induction of class II antigens on the colonic epithelium in response to TNFα is mediated via IFNγ, and (ii) that IL2 induces class II antigens.BACKGROUND The aim was to investigate whether intragastric pH, meal-stimulated gastrin release, or demographic factors predict the outcome of Helicobacter pylori treatment. METHODS Thirty-six patients with H. pylori infection were investigated with 24-h intragastric pH registration and meal-stimulated gastrin release before and during treatment with 20 mg omeprazole twice daily and 750 mg amoxicillin twice daily for 14 days. The influence of age, sex, smoking, ethnic origin, pH, and gastrin on treatment outcome were analysed with logistic regression. RESULTS Eradication of H. pylori was achieved in 18 of 34 (53%) patients. The univariate analysis showed that age, ethnic origin, more than 84.2% of the time with pH above 4, and continuous periods longer than 156 min with intragastric pH above 6 were significantly associated with successful treatment of H. pylori. In the multivariate analysis only the two pH variables were found to be independent factors for predicting treatment outcome. CONCLUSION The outcome of H. pylori treatment with omeprazole and amoxicillin may depend on several factors, such as age, ethnic origin, and a pronounced acid suppression. However, the only factor of independent importance in this study was prolonged and profound acid inhibition.

The Distribution of G (VP7) and P (VP4) Serotypes among Human Rotaviruses Recovered from Japanese Children with Diarrhea

Both the G (VP7) and P (VP4) serotypes of human rotaviruses collected over a 10‐year period from Japanese children with diarrhea were determined by recently‐developed polymerase chain reaction‐based typing assays. The combination of G1 and P8 was found in 65.2% and the combination of G2 and P4 was found in 15.2%. For the rest of the specimens, only a few other combinations occurred and their relative frequencies were less than 10%. The viruses carrying P9 were always associated with G3 as is the prototype strain AU‐1.

Ecabet sodium prevents the delay of wound repair in intestinal epithelial cells induced by hydrogen peroxide

BackgroundRecent studies showed that ecabet sodium (ES), a gastro-protective agent, also had a therapeutic effect on inflammation in ulcerative colitis. The aim of this study was to clarify the function of ES in wound repair in intestinal epithelial cells (IECs).MethodsThe activation of signal proteins (ERK1/2 mitogen-activated protein kinase MAPK, and IκB-α) in IEC-6 cells after stimulation with 2.5 mg/ml of ES was assessed by Western blot. The induction of transforming growth factor (TGF)-β1, TGF-α, and cyclooxygenase-2 (COX-2) mRNAs after the stimulation of IEC-6 cells with ES was assessed by reverse transcription ploymerase chain reaction (RT-PCR). IEC-6 cells were wounded and cultured for 24 h with various concentrations of ES in the absence or presence of 20 µM H2O2. Epithelial migration or proliferation was assessed by counting migrated or bromodeoxyuridine (BrdU)-positive cells observed across the wound border. We also assessed apoptotic epithelial cells after the culture.ResultsES clearly activated ERK1/2 MAPK and slightly activated IκB-α. ES also enhanced the expression of TGF-α and COX-2 mRNAs. This enhancement was suppressed by a MAPK/Erk kinase (MEK) inhibitor. ES did not enhance epithelial migration in the absence of H2O2. In contrast, ES significantly decreased the number of apoptotic cells and prevented the reduction of epithelial migration (51.1%; P < 0.01) and proliferation (56%; P < 0.01) induced by H2O2. The function of ES was suppressed by a cyclooxygenase-2 (COX-2) inhibitor and by the MEK inhibitor, and partly suppressed by a nuclear factor (NF)-κB inhibitor.ConclusionsES prevents the delay of wound repair in IEC-6 cells induced by H2O2, probably through the activation of ERK1/2 MAPK and the induction of COX-2.

Immunohistochemical analysis of the distribution of measles related antigen in the intestinal mucosa in inflammatory bowel disease.

BACKGROUND Measles virus is implicated in the aetiology of Crohns disease. This measles hypothesis is mainly supported by immunohistochemical findings that the measles related antigen is present in the intestine of patients with Crohns disease. Recently we isolated this antigen from the intestine of a patient with Crohns disease using a molecular cloning technique and produced the monoclonal antibody against it (designated 4F12). AIM To discover whether the measles related antigen is uniquely present in Crohns disease. SUBJECTS/METHODS Colonic mucosa samples from 20 patients with Crohns disease, 20 with ulcerative colitis, 11 with non-inflammatory bowel disease (IBD) colitis, and nine controls were immunohistochemically stained with the anti-measles monoclonal antibody 4F12. The numbers of positive cells, the ratio of positive cells to nucleated cells, and the staining intensity of the positive cells were compared. Furthermore, the distribution of the measles antigen in other human organs was examined. RESULTS Both the number of positive cells and the ratio of positive cells to nucleated cells were significantly increased in Crohns disease, ulcerative colitis, and non-IBD colitis compared with controls (p<0.05) but were similar among the three disease groups. The staining intensity of the positive cells was also similar among the three disease groups. Small numbers of positive cells were observed in the oesophagus, stomach, duodenum, jejunum, and lung. CONCLUSIONS The presence of the measles related antigen in the colonic mucosa was not unique to Crohns disease. These results, together with the observation that such a measles related antigen was derived from host protein, do not support the hypothesis that measles virus causes Crohns disease.

Listeria monocytogenes in Crohn's Disease

BACKGROUND In an immunohistochemical study a higher rate of reactivity of intestinal tissues to the antibody against Listeria monocytogenes was reported in Crohns disease as compared with controls. METHODS Seventy-six intestinal tissues, either therapeutically resected or biopsied, from 31 patients with Crohns disease, 20 with ulcerative colitis, and 21 with non-inflammatory bowel disease were studied. DNA extracted from intestinal tissues by proteinase K treatment was used for nested polymerase chain reaction (PCR), using two sets of primers. PCR products were analyzed with agarose gel electrophoresis and subsequent Southern blot analysis. RESULTS Our amplification system could detect 9 pg of L. monocytogenes DNA. L. monocytogenes was detected in only one sample, that from a patient with ulcerative colitis. CONCLUSIONS Our study does not support the etiologic significance of L. monocytogenes in Crohns disease.

Systemic and local evidence of increased Fas-mediated apoptosis in ulcerative colitis

Abstract. Background and aims: Recent studies suggest that Fas-mediated apoptosis is involved in the pathogenesis of inflammatory bowel disease (IBD). This study was conducted to clarify whether soluble forms of Fas (sFas) and Fas ligand (sFasL) are concerned with inflammation in IBD. Methods and patients: Concentration of serum sFas and sFasL was measured by enzyme-linked immunosorbent assay in 10 patients with ulcerative colitis (UC), 10 with Crohns disease (CD) in both active and remission stages, and 20 controls. Expression of Fas and sFas in colonic mucosa was examined by western blot. Distribution of Fas and FasL in colonic mucosa was examined by immunohistochemistry in 20 UC, 20 CD, and 10 non-IBD colitis patients and in 10 controls. Apoptotic cells were examined by TUNEL. Results: Concentration of systemic sFas was significantly lower in active UC than controls. The number of FasL-containing cells was significantly higher in active UC than in remission UC, non-IBD colitis, and controls. Apoptotic cells were increased in active UC. Conclusions: Our results demonstrate that systemic and local Fas-mediated apoptosis is promoted in UC, which might be involved in the pathogenesis in UC.

Metastatic Crohn's disease involving the penis

Metastatic Crohns disease is a rare complication in Crohns disease and there have been only several cases of metastatic Crohns disease involving the penis. We report one such case. A 22-year-old male student developed anal pain and alternative constipation and diarrhea in December, 1985, followed by diarrhea and lower abdominal pain in January, 1986. He was diagnosed as having Crohns disease of ileocolitis type. He was admitted to our hospital in July, 1987 because of exacerbation of Crohns disease. He had anal tags. Soon after admission, two red swollen lesions with central ulcer and erosions were demonstrated at the eversion of the foreskin adjacent to coronal sulcus. Histology of the lesions revealed granulomas with epithelioid cells and giant cells. The lesion responded to a topical steroid. Eight cases of metastatic Crohns disease involving the penis are briefly reviewed.