Published papers

Background & Aims: Acute pancreatitis is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with recruitment of leukocytes. We investigated the pathologic roles of innate immune cells, especially macrophages, in cerulein and L-arginineinduced acute pancreatitis in mice. Methods: Acute pancreatitis was induced by sequential peritoneal administration of cerulein to mice. We determined serum concentrations of amylase and lipase, pancreatic pathology, and features of infiltrating mononuclear cells. We performed parabiosis surgery to assess the hemodynamics of pancreatic macrophages. Results: Almost all types of immune cells, except for CD11b highCD11c - cells, were detected in the pancreas of healthy mice. However, activated CD11b highCD11c - cells, including Gr-1 low macrophages and Gr-1 high cells (granulocytes and myeloid-derived suppressor cells), were detected in damaged pancreas after cerulein administration. CCL2 -/- mice given cerulein injections developed significantly less severe pancreatitis, with less infiltration of CD11b highCD11c -Gr-1 low macrophages, but comparable infiltration of myeloid-derived suppressor cells, compared with cerulein-injected wild-type mice. Parabiosis and bone marrow analyses of these mice revealed that the CD11b highCD11c -Gr-1 low macrophages had moved out of the bone marrow. Furthermore, mice with macrophage-specific deletion of suppressor of cytokine signaling 3 given injections of cerulein developed less severe pancreatitis and Gr-1 low macrophage produced less tumor necrosis factor-α than wild-type mice given cerulein, although the absolute number of CD11b highCD11c -Gr-1 low macrophages was comparable between strains. Induction of acute pancreatitis by L-arginine required induction of macrophage migration by CCL2, via the receptor CCR2. Conclusions: Cerulein induction of pancreatitis in mice involves migration of CD11b highCD11c -Gr-1 low macrophage from the bone marrow (mediated by CCL2 via CCR2) and suppressor of cytokine signaling 3dependent activation of macrophage. These findings might lead to new therapeutic strategies for acute pancreatitis. © 2012 AGA Institute.

We previously reported that IL-7 -/-RAG -/- mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4 + T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4 + T cells. To further investigate these roles of NK cells, RAG -/- and IL-7 -/-RAG -/-mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (T EM) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44 +CD62L - T EM and unique CD44 -CD62L - T cell subsets were observed in the T cell-reconstituted RAG -/- recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44 +CD62L - T EM subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG -/- and IL-7 -/-RAG -/- recipient mice through targeting of colitogenic CD4 +CD44 +CD62L - T EM and, possibly, of the newly observed CD4 +CD44 -CD62L - subset present at the early stage of T cell development. Copyright © 2012 by The American Association of Immunologists, Inc.

Background & Aims: Antigen-presenting cells (APCs) are involved in the induction of liver inflammation. We investigated the roles of specific APCs in the pathogenesis of acute liver injury in mice. Methods: We used concanavalin A (con A) or carbon tetrachloride to induce acute liver inflammation in mice and studied the roles of macrophages that express CCR9. Results: After injection of con A, we detected CCR9 +CD11b +CD11c macrophages that express tumor necrosis factor (TNF)-α in livers of mice, whereas CCR9 +SiglecH +CD11bCD11c low plasmacytoid DCs (pDCs), which are abundant in normal livers, disappeared. The CCR9 + macrophages were also detected in the livers of RAG-2 -/- mice, which lack lymphocytes and natural killer T cells, after injection of con A. Under inflammatory conditions, CCR9 + macrophages induced naive CD4 + T cells to become interferon gammaproducing Th1 cells in vivo and in vitro. CCR9 -/- mice injected with con A did not develop hepatitis unless they also received CCR9 + macrophages from mice that received con A; more CCR9 + macrophages accumulated in their inflamed livers than CCR9 + pDCs, CCR9 pDCs, or CCR9 macrophages isolated from mice that had received injections of con A. Levels of CCL25 messenger RNA increased in livers after injection of con A; neutralizing antibodies against CCL25 reduced the induction of hepatitis by con A by blocking the migration of CCR9 + macrophages and their production of TNF-α. Peripheral blood samples from patients with acute hepatitis had greater numbers of TNF-αproducing CCR9 +CD14 +CD16 high monocytes than controls. Conclusions: CCR9 + macrophages contribute to the induction of acute liver inflammation in mouse models of hepatitis. © 2012 AGA Institute.

Background and objective: Capsule endoscopy (CE) is a comfortable for the patients; however, CE review is time-consuming. The aim of this study was (1) to evaluate the effectiveness of the CE software in reducing the CE reading time and the number of false negatives by beginners, and (2) to determine the learning curve for reading CE images. Methods: Capsule endoscopic images were captured by Pillcam SB (Given Imaging Ltd, Tokyo, Japan), and analyzed using the proprietary RAPID5 software. Comparison of CE reading using different software modes: manual mode, automatic mode, and QuickView (QV) mode. Three trainee endoscopists participated as CE readers. Each participant watched CE videos in which positive findings had been predefined by trained endoscopists. Each participant read the same CE record by using one of three different software modes. These were blinded on clinical history of patients. CE reading time was recorded, and the number of false negatives was counted. Each trainee endoscopist read a total of 45CE videos, in five steps divided into nine videos per step. Results: There was no significant reader associated difference between the results for the different modes. The QV software did miss some positive findings. Therefore, the total number of instances of FN by the software plus the reader in the QV mode was significantly higher than the others. The reading times in the automatic mode and the QV mode were significantly shorter than that in the manual mode. After the second step, the number of instances of false negatives significantly decreased. Conclusions: CE software is useful for reducing the reading time. Experience of approximately 20CE readings can be considered as the first step to becoming an expert. © 2011 Elsevier Masson SAS.

Some studies have indicated that dietary cholesterol has a role in the progression of liver fibrosis. We investigated the mechanisms by which dietary cholesterol might contribute to hepatic fibrogenesis. C57BL/6 mice were fed a high-cholesterol diet or a control diet for 4 weeks; liver fibrosis then was induced by bile-duct ligation or carbon tetrachloride administration. Hepatic stellate cells (HSCs) were isolated from mice fed high-cholesterol diets or from NiemannPick type C1-deficient mice, which accumulate intracellular free cholesterol. After bile-duct ligation or carbon tetrachloride administration, mice fed high-cholesterol diets had significant increases in liver fibrosis and activation of HSCs compared with mice fed control diets. There were no significant differences in the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation, between mice fed high-cholesterol or control diets. Levels of free cholesterol were much higher in HSCs from mice fed high-cholesterol diets than those fed control diets. In cultured HSCs, accumulation of free cholesterol in HSCs increased levels of Toll-like receptor 4 (TLR4), leading to down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor (a pseudoreceptor for transforming growth factor [TGF]β); the HSCs became sensitized to TGFβ-induced activation. Liver fibrosis was not aggravated by the high-cholesterol diet in C3H/HeJ mice, which express a mutant form of TLR4; HSCs that express mutant TLR4 were not activated by accumulation of free cholesterol. Dietary cholesterol aggravates liver fibrosis because free cholesterol accumulates in HSCs, leading to increased TLR4 signaling, down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor, and sensitization of HSC to TGFβ. This pathway might be targeted by antifibrotic therapies. © 2012 AGA Institute.

Background: Inflammatory bowel disease (IBD) is a chronic intestinal disorder that is associated with a limited number of clinical biomarkers. In order to facilitate the diagnosis of IBD and assess its disease activity, we investigated the potential of novel multivariate indexes using statistical modeling of plasma amino acid concentrations (aminogram). Methodology and Principal Findings: We measured fasting plasma aminograms in 387 IBD patients (Crohn's disease (CD), n = 165; ulcerative colitis (UC), n = 222) and 210 healthy controls. Based on Fisher linear classifiers, multivariate indexes were developed from the aminogram in discovery samples (CD, n = 102; UC, n = 102; age and sex-matched healthy controls, n = 102) and internally validated. The indexes were used to discriminate between CD or UC patients and healthy controls, as well as between patients with active disease and those in remission. We assessed index performances using the area under the curve of the receiver operating characteristic (ROC AUC). We observed significant alterations to the plasma aminogram, including histidine and tryptophan. The multivariate indexes established from plasma aminograms were able to distinguish CD or UC patients from healthy controls with ROC AUCs of 0.940 (95% confidence interval (CI): 0.898-0.983) and 0.894 (95%CI: 0.853-0.935), respectively in validation samples (CD, n = 63; UC, n = 120; healthy controls, n = 108). In addition, other indexes appeared to be a measure of disease activity. These indexes distinguished active CD or UC patients from each remission patients with ROC AUCs of 0.894 (95%CI: 0.853-0.935) and 0.849 (95%CI: 0.770-0.928), and correlated with clinical disease activity indexes for CD (r s = 0.592, 95%CI: 0.385-0.742, p<0.001) or UC (r s = 0.598, 95%CI: 0.452-0.713, p<0.001), respectively. Conclusions and Significance: In this study, we demonstrated that established multivariate indexes composed of plasma amino acid profiles can serve as novel, non-invasive, objective biomarkers for the diagnosis and monitoring of IBD, providing us with new insights into the pathophysiology of the disease. © 2012 Hisamatsu et al.

Background: Accurate evaluation of liver fibrosis in patients with chronic liver damage is required to determine the appropriate treatment. Various approaches, including laboratory tests and transient elastography, have been used to evaluate liver fibrosis. Recently, transient elastography with acoustic radiation force impulse (ARFI) has been developed and applied with conventional ultrasonography. The aim of this study was to evaluate the clinical utility of transient elastography with ARFI and to compare the results with this method and those of the Fibroscan® procedure. Methods: One hundred and thirty-one patients with liver damage, who underwent liver biopsy at our department, were enrolled prospectively in this study. Elastography with ARFI (applied with ACUSON S2000®), and Fibroscan® was performed at the same time as liver biopsy. These measurements were compared with histological findings in liver biopsy specimens, and measurement accuracy was evaluated by receiver-operating characteristic analysis. Results: Elastography values with both procedures were significantly correlated with the stages of liver fibrosis and there was little difference in the results obtained using the 2 procedures. The accuracy of differential diagnosis between no fibrosis at F0 and more than F1 stage was insufficient with ARFI, but this procedure was sufficient for diagnosing advanced fibrosis. The accuracy of ARFI was almost equivalent to that of the Fibroscan® method. Moreover, both ARFI and Fibroscan® values increased in proportion to the severity of hepatic inflammation when fibrosis stage is low, but not in proportion to the severity of steatosis. Conclusions: Transient elastography with ARFI is simple, non-invasive and useful for diagnosing the stage of fibrosis in chronic liver disease. The utility of ARFI was almost equivalent to that of the Fibroscan® method. © 2011 Springer.

Background: Routine diagnosis of the histopathological activity of ulcerative colitis (UC) requires multiple biopsy samples, and an endocytoscopy system (ECS) provides real-time ultra-magnifying microscopic imaging in vivo. Methods: We have established an ECS score (ECSS) to determine a histopathological activity index of UC. Fifty-five UC patients (mean age 40.7 years; 67% men) were enrolled. A super-magnifying ECS with magnification 450× was used, and sample biopsies were obtained. Matts' histopathological grade was determined, to evaluate disease severity, by two pathologists, with consensus. The ECSS of UC was independently determined by at least two investigators, with consensus. In total, 76 pairs of ECSS and Matts' histopathological grades were independently acquired. To validate the ECSS, inter-observer agreement between three endoscopists, with consensus, and another endoscopist, was calculated as the kappa value. We also evaluated the correlation between the ECSS and Matts' histopathological grade, and between the conventional Matts' endoscopic grade and Matts' histopathological grade. Results: The ECSS of UC intestinal mucosa, i.e., the sum of the indices for shape (0-3) and distance between crypts (0-2), and the visibility of superficial microvessels (0-1), showed a strong correlation with Matts' histopathological grades (ρ = 0.713, P < 0.001); as well, there was a strong correlation between the conventional Matts' endoscopic grade and Matts' histopathological grade (ρ = 0.694, P < 0.001). Furthermore, the ECSS showed high reproducibility (κ = 0.79, 95% confidence interval [CI] 0.71-0.87). Conclusions: Our novel ECSS has good predictive value for the histopathological activity of UC. © 2011 Springer.

Background & Aims: The egress of memory T cells from peripheral tissues, such as lung and skin, into the draining lymph nodes requires their expression of CC chemokine receptor 7 (CCR7). In the intestine, resident memory T cells in the intestinal lamina propria (LP) do not express CCR7, indicating that they are tissue bound and do not exit the intestine. Methods: We developed a cell transfer system, using rectal administration of lymphocytes to C57BL/6 mice. Lymphotoxin α-deficient mice were crossed with RAG-2 -/- (recombination-activating gene-2) mice to generate lymphotoxin α-deficient × RAG-2 -/- mice. Results: Severe combined immunodeficient (SCID) or RAG-2 -/- mice given rectal administration of splenic CD4 + T cells from normal mice developed colitis; the cells proliferated not only in the LP but also in spleen. SCID or RAG-2 -/- mice given rectal administrations of CD4 + T cells that expressed green fluorescent protein (GFP +CD4 + T cells) localized to the LP within 6 hours but were not found in the spleen until 24 hours after administration. Immunohistochemical and electron microscopic analyses detected CD4 + T cells in the intraepithelial space just 3 hours after intrarectal administration. However, neither CCR7 deficiency nor the sphingosine-1-phosphate receptor agonist Fingolimod impaired the egress of CD4 + T cells from LP to systemic circulation. Conclusions: CD4 + T cells not only penetrate from the luminal side of the intestine to the LP but also actively egress from the LP into the circulation. We developed a rectal administration system that might be used to further investigate cell trafficking in intestinal mucosa and to develop enema-based therapeutics for intestinal diseases. © 2011 AGA Institute.

Treatment with pegylated interferon alpha-2b (PEGIFN) plus ribavirin (RBV) is standard therapy for patients with chronic hepatitis C. Although the effectiveness, patients with high titres of group Ib hepatitis C virus (HCV) respond poorly compared to other genotypes. At present, we cannot predict the effect in an individual. Previous studies have used traditional statistical analysis by assuming a linear relationship between clinical features, but most phenomena in the clinical situation are not linearly related. The aim of this study is to predict the effect of PEG IFN plus RBV therapy on an individual patient level using an artificial neural network system (ANN). 156 patients with HCV group 1b from multiple centres were treated with PEGIFN (1.5 μg/kg) plus RBV (400-1000 mg) for 48 weeks. Data on the patients' demographics, laboratory tests, PEGIFN, and RBV doses, early viral responses (EVR), and sustained viral responses were collected. Clinical data were randomly divided into training data set and validation data set and analyzed using multiple logistic regression analysis (MLRs) and ANN to predict individual outcomes. The sensitivities of predictive expression were 0.45 for the MLRs models and 0.82 for the ANNs and specificities were 0.55 for the MLR and 0.88 for the ANN. Non-linear relation analysis showed that EVR, serum creatinine, initial dose of Ribavirin, gender and age were important predictive factors, suggesting non-linearly related to outcome. In conclusion, ANN was more accurate than MLRs in predicting the outcome of PEGIFN plus RBV therapy in patients with group 1b HCV. © 2011 Takayama et al.

Accurate evaluation of liver fibrosis in patients with chronic liver damage is required to determine the appropriate treatment. Various approaches, including laboratory tests and transient elastography, have been used to evaluate liver fibrosis. Recently, transient elastography with acoustic radiation force impulse (ARFI) has been developed and applied with conventional ultrasonography. The aim of this study was to evaluate the clinical utility of transient elastography with ARFI and to compare the results with this method and those of the Fibroscan(®) procedure. One hundred and thirty-one patients with liver damage, who underwent liver biopsy at our department, were enrolled prospectively in this study. Elastography with ARFI (applied with ACUSON S2000(®)), and Fibroscan(®) was performed at the same time as liver biopsy. These measurements were compared with histological findings in liver biopsy specimens, and measurement accuracy was evaluated by receiver-operating characteristic analysis. Elastography values with both procedures were significantly correlated with the stages of liver fibrosis and there was little difference in the results obtained using the 2 procedures. The accuracy of differential diagnosis between no fibrosis at F0 and more than F1 stage was insufficient with ARFI, but this procedure was sufficient for diagnosing advanced fibrosis. The accuracy of ARFI was almost equivalent to that of the Fibroscan(®) method. Moreover, both ARFI and Fibroscan(®) values increased in proportion to the severity of hepatic inflammation when fibrosis stage is low, but not in proportion to the severity of steatosis. Transient elastography with ARFI is simple, non-invasive and useful for diagnosing the stage of fibrosis in chronic liver disease. The utility of ARFI was almost equivalent to that of the Fibroscan(®) method.

Inflammatory bowel diseases (IBD) classified at the present time as ulcerative colitis (UC) and Crohn's disease (CD) are characterized by chronic relapsing inflammations of the gastrointestinal tract. In order to select the most appropriate treatment, patients must be assessed by determining the phenotype, disease extension, disease severity, and complicating cancer. Recent advances in endoscopic technology in addition to other modalities provide the possibility for the assessment of disease activity and the determination of the treatment for each IBD patient. Here we review the controversies in the use of diagnostic procedures for UC patients.

Routine diagnosis of the histopathological activity of ulcerative colitis (UC) requires multiple biopsy samples, and an endocytoscopy system (ECS) provides real-time ultra-magnifying microscopic imaging in vivo. We have established an ECS score (ECSS) to determine a histopathological activity index of UC. Fifty-five UC patients (mean age 40.7 years; 67% men) were enrolled. A super-magnifying ECS with magnification 450× was used, and sample biopsies were obtained. Matts' histopathological grade was determined, to evaluate disease severity, by two pathologists, with consensus. The ECSS of UC was independently determined by at least two investigators, with consensus. In total, 76 pairs of ECSS and Matts' histopathological grades were independently acquired. To validate the ECSS, inter-observer agreement between three endoscopists, with consensus, and another endoscopist, was calculated as the kappa value. We also evaluated the correlation between the ECSS and Matts' histopathological grade, and between the conventional Matts' endoscopic grade and Matts' histopathological grade. The ECSS of UC intestinal mucosa, i.e., the sum of the indices for shape (0-3) and distance between crypts (0-2), and the visibility of superficial microvessels (0-1), showed a strong correlation with Matts' histopathological grades (ρ = 0.713, P < 0.001); as well, there was a strong correlation between the conventional Matts' endoscopic grade and Matts' histopathological grade (ρ = 0.694, P < 0.001). Furthermore, the ECSS showed high reproducibility (κ = 0.79, 95% confidence interval [CI] 0.71-0.87). Our novel ECSS has good predictive value for the histopathological activity of UC.

Background & Aims: Although T-helper (Th) 17 and Th1 cells are involved in pathogenesis of intestinal inflammation, their developmental pathways and sufficiency to promote disease are not known; nor are the roles of CD4 +CD25 + regulatory T (T R) cells in their development. Methods: We performed adoptive transfer experiments to investigate the induction and suppression of colitis using nave CD4 +CD45RB high T cells and/or CD4 +CD25 + T R cells that were obtained from retinoid-related orphan receptor gamma t (RORγt) gfp/ + or Ly5.1/Ly5.2 congenic mice. Results: We observed 3 types of colitogenic CD4 + Th1 cells (interleukin [IL]-17A -interferon [IFN]-γ +): RORγt classical Th1 cells that differentiated directly from nave T cells; RORγt + Th1-like cells; and RORγt alternative Th1 cells that were terminally differentiated from RORγt + cells via Th17 (IL-17A +IFN-γ), Th17/Th1 (IL-17A +IFN- γ +), or Th1-like (IL-17AIFN-γ +) cells. In this pathway, CD4 +CD25 + T R cells suppress the development of not only classical Th1 cells, but also alternative Th1 cells at the transition of Th17/Th1 into alternative Th1 cells, resulting in accumulation of Th17 and Th17/Th1 cells in mice in which the development of colitis was suppressed. Furthermore, T R cells regulated the established balance of Th17 and Th1 cells under colitic conditions to yield a high ratio of Th17 and Th17/Th1 cells to Th1 cells in noncolitic conditions. Conclusions: Th17 and Th17/Th1 cells become colitogenic alternative Th1 cells via Th17, Th17/Th1, and Th1-like cells, independently of classical Th1 cells. T R cells suppress this pathway, resulting in accumulation of Th17 and Th17/Th1 cells. © 2011 AGA Institute.

Background and Aim: Colon capsule endoscopy has already been used for colon visualization and detection of polyps but its applicability to inflammatory bowel disease is still unconfirmed. The aim of this study was to assess the feasibility of evaluating the severity of mucosal inflammation in patients with ulcerative colitis (UC) using a second-generation colon capsule endoscope (CCE-2). Methods: Forty patients with histological confirmed diagnosis of UC were enrolled. Low-volume (2L) polyethylene glycol solution with prokinetics (mosapride citrate and metoclopramide) regimen was used for the bowel preparation. In Phase 1, consisting of 10 patients, to confirm appropriate CCE-2 bowel preparation for UC. In Phase 2, consisting of 30 patients, CCE-2 was performed with a fixed bowel preparation regimen. CCE-2 findings were recorded for 8h starting from capsule ingestion and conventional colonoscopy was subsequently performed on the same day. CCE-2 procedure completion rate and the colon cleansing level with a 4-point grading scale (poor, fair, good, and excellent) were evaluated in Phase 2. Correlations between Matts endoscopic scores as judged by CCE-2 and conventional colonoscopy were calculated. Results: CCE-2 procedure was completed within 8h in 69% of the patients. The proportion of patients with good or excellent cleansing level was below 50%. However, Matts endoscopic scores determined by CCE-2 showed a strong correlation with scores obtained by conventional colonoscopy (average ρ=0.797). Conclusions: Although modifications in bowel preparation are needed, CCE-2 might be feasible for assessing the severity of mucosal inflammation in patients with UC. © 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

It is not too much to say that infliximab has revolutionized the treatment of Crohn's disease. However, there is the problem of 'secondary failure' wherein the effect may diminish during treatment. This is a much-discussed topic. For example, not all patients with secondary failure can maintain remission with dose intensification to 10 mg/kg. It is also important to make the appropriate drug selection and to clarify the optimal timing of dose intensification for achieving long-term maintenance of remission, including discussion of the first priority use of adalimumab.