Shuji Kanmura

Increased rate of death related to presence of viremia among hepatitis C virus antibody–positive subjects in a community-based cohort study†

The overall mortality of patients infected with hepatitis C virus (HCV) has not been fully elucidated. This study analyzed mortality in subjects positive for antibody to HCV (anti‐HCV) in a community‐based, prospective cohort study conducted in an HCV hyperendemic area of Japan. During a 10‐year period beginning in 1995, 1125 anti–HCV‐seropositive residents of Town C were enrolled into the study and were followed for mortality through 2005. Cause of death was assessed by death certificates. Subjects with detectable HCV core antigen (HCVcAg) or HCV RNA were considered as having hepatitis C viremia and were classified as HCV carriers; subjects who were negative for both HCVcAg and HCV RNA (i.e., viremia‐negative) were considered as having had a prior HCV infection and were classified as HCV noncarriers. Among the anti–HCV‐positive subjects included in the analysis, 758 (67.4%) were HCV carriers, and 367 were noncarriers. A total of 231 deaths occurred in these subjects over a mean follow‐up of 8.2 years: 176 deaths in the HCV carrier group and 55 in the noncarrier group. The overall mortality rate was higher in HCV carriers than in noncarriers, adjusted for age and sex (hazard ratio, 1.53; 95% confidence interval, 1.13‐2.07). Although liver‐related deaths occurred more frequently among the HCV carriers (hazard ratio, 5.94; 95% confidence interval, 2.58‐13.7), the rates of other causes of death did not differ between HCV carriers and noncarriers. Among HCV carriers, a higher level of HCVcAg (≥100 pg/mL) and persistently elevated alanine aminotransferase levels were important predictors of liver‐related mortality. Conclusion: The presence of viremia increases the rate of mortality, primarily due to liver‐related death, among anti–HCV‐seropositive persons in Japan. (HEPATOLOGY 2009.)

Human neutrophil peptides 1-3 are useful biomarkers in patients with active ulcerative colitis.

Background: A specific useful biomarker for diagnosing ulcerative colitis (UC) has not yet been described. This study employed proteomics to identify serum protein biomarkers for UC. Methods: Ninety‐four blood samples were isolated from patients and controls (including 48 UC, 22 Crohns disease [CD], 5 colorectal cancer, and 6 infectious colitis patients and 13 healthy subjects). Serum samples were analyzed using the SELDI‐TOF/MS ProteinChip system. After applying the samples to ProteinChip arrays, we assessed differences in the proteomes using Ciphergen ProteinChip software and identified candidate proteins, which were then characterized in immunoassays. Results: Preliminary analysis using the ProteinChip system revealed significant peak‐intensity differences for 27 serum proteins between 11 patients with UC and 7 healthy subjects. Among these proteins, 3 proteins (with mass/charge ratios of approximately 3400) were identified as human neutrophil peptides 1–3 (HNP 1–3). The presence of HNP 1–3 in the patient sera was confirmed using immunoassays. Enzyme‐linked immunosorbent assays demonstrated that the mean plasma concentration of HNP 1–3 was significantly higher in patients with active UC (n = 28) than in patients whose UC was in remission (n = 20) or patients with CD (n = 22), infectious colitis, or healthy subjects, and tended to be higher than in patients with colon cancer. In addition, the plasma concentration of HNP 1–3 in patients that responded to corticosteroids‐based therapy decreased after treatment, whereas it was not changed in nonresponders. Conclusions: HNP 1–3 is a novel biomarker that may be useful for diagnosing patients with active UC and predicting treatment outcomes.

Clinical proteomics for liver disease: a promising approach for discovery of novel biomarkers

Hepatocellular carcinoma (HCC) is the fifth most common cancer and advanced hepatic fibrosis is a major risk factor for HCC. Hepatic fibrosis including liver cirrhosis and HCC are mainly induced by persistent hepatitis B or C virus infection, with approximately 500 million people infected with hepatitis B or C virus worldwide. Furthermore, the number of patients with non-alcoholic fatty liver disease (NAFLD) has recently increased and NAFLD can progress to cirrhosis and HCC. These chronic liver diseases are major causes of morbidity and mortality, and the identification of non-invasive biomarkers is important for early diagnosis. Recent advancements in quantitative and large-scale proteomic methods could be used to optimize the clinical application of biomarkers. Early diagnosis of HCC and assessment of the stage of hepatic fibrosis or NAFLD can also contribute to more effective therapeutic interventions and an improve prognosis. Furthermore, advancements of proteomic techniques contribute not only to the discovery of clinically useful biomarkers, but also in clarifying the molecular mechanisms of disease pathogenesis by using body fluids, such as serum, and tissue samples and cultured cells. In this review, we report recent advances in quantitative proteomics and several findings focused on liver diseases, including HCC, NAFLD, hepatic fibrosis and hepatitis B or C virus infections.

Transgenic expression of human neutrophil peptide‐1 enhances hepatic fibrosis in mice fed a choline‐deficient, L‐amino acid–defined diet

Neutrophils infiltrate the livers of patients with nonalcoholic steatohepatitis (NASH). Human neutrophil peptides (HNPs) induce cytokine and chemokine production under inflammatory conditions, which may contribute to the progression of NASH. In this study, we focused on the effects of HNP‐1 on hepatic steatosis and fibrosis in a mouse model of NASH induced by a choline‐deficient, L‐amino acid–defined (CDAA) diet.

Glycoprotein Nonmetastatic Melanoma B (Gpnmb)-Positive Macrophages Contribute to the Balance between Fibrosis and Fibrolysis during the Repair of Acute Liver Injury in Mice.

Background and aims Glycoprotein nonmetastatic melanoma B (Gpnmb), a transmembrane glycoprotein that is expressed in macrophages, negatively regulates inflammation. We have reported that Gpnmb is strongly expressed in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet. However, the role of macrophage-expressed Gpnmb in liver injury is still unknown. This study aimed to clarify the characteristics of infiltrating macrophages that express Gpnmb, and the involvement of Gpnmb in the repair process in response to liver injury. Methods C57BL/6J, DBA/2J [DBA] and DBA/2J-Gpnmb+ [DBA-g+] mice were treated with a single intraperitoneal injection of carbon tetrachloride (CCl4) at a dose of 1.0 mL/kg body weight. Mice were sacrificed at predetermined time points, followed by measurement of serum alanine aminotransferase (ALT) levels and histological examination. Expression of Gpnmb, pro-/anti-inflammatory cytokines, and profibrotic/antifibrotic factors were examined by quantitative RT-PCR and/or Western blotting. Immunohistochemistry, fluorescent immunostaining and flow cytometry were used to determine the expression of Gpnmb, CD68, CD11b and α-SMA, phagocytic activity, and the presence of apoptotic bodies. We used quantitative RT-PCR and ELISA to examine TGF-β and MMP-13 expression and the concentrations and supernatants of isolated infiltrating hepatic macrophages transfected with siGpnmb. Results In C57BL/6J mice, serum ALT levels increased at two days after CCl4 injection and decreased at four days. Gpnmb expression in the liver was stimulated four days after CCl4 injection. Histological examination and flow cytometry showed that Gpnmb-positive cells were almost positive for CD68-positive macrophages, contained engulfed apoptotic bodies and exhibited enhanced phagocytic activity. Isolated infiltrating hepatic macrophages transfected with siGpnmb showed high MMP-13 secretion. There was no significant difference in the magnitude of CCl4-induced liver injury between DBA-g+ and DBA mice. However, hepatic MMP-13 expression, as well as α-SMA expression and collagen production, increased significantly in DBA-g+ compared with DBA mice. Conclusions Gpnmb-positive macrophages infiltrate the liver during the recovery phase of CCl4–induced acute liver injury and contribute to the balance between fibrosis and fibrolysis in the repair process following acute liver injury.

Human neutrophil peptides induce interleukin-8 in intestinal epithelial cells through the P2 receptor and ERK1/2 signaling pathways

Human neutrophil peptides (HNPs) are antimicrobial peptides produced predominantly by neutrophils. We have previously reported that HNP 1-3 levels are increased in the sera and plasma of patients with active ulcerative colitis. The increased expression of interleukin-8 (IL-8) has also been demonstrated in the colonic mucosa of patients with active ulcerative colitis. HNPs induce IL-8 in lung epithelial cells and monocytes through the P2Y6 signaling pathway. However, the association between HNPs and IL-8 in the intestinal mucosa has not yet been investigated. In the present study, we investigated the effects of HNP-1 on the production of IL-8 by human intestinal epithelial cells and the underlying signaling mechanisms. We observed a significant increase in IL-8 expression in the human colon carcinoma cell line, Caco-2, following treatment with HNP-1. The non-selective P2 receptor antagonists, suramin and pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), significantly blocked the HNP-1-induced expression of IL-8 in the Caco-2 cells. The P2Y6-specific antagonist, MRS2578, led to a significant but partial decrease in IL-8 expression, suggesting that P2 receptors in addition to P2Y6 are involved in the HNP-1-induced production of IL-8 by Caco-2 cells. In agreement with this finding, HNP-1 also significantly increased IL-8 production in the P2Y6-negative human colon cancer cell line, HT-29, and this increase was blocked by treatment with suramin and PPADS. HNP-1 significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in the HT-29 cells. However, the HNP-1-induced production of IL-8 was suppressed by the ERK1/2 inhibitor, U0126, but not by the p38 MAPK inhibitor, SB203580. In conclusion, our data demonstrate that HNP-1 induces IL-8 production not only through P2Y6, but also through additional P2 receptors via an ERK1/2-dependent mechanism in intestinal epithelial cells.

Human neutrophil peptide-1 aggravates dextran sulfate sodium-induced colitis.

Background: Human neutrophil peptide (HNP)‐1, HNP‐2, and HNP‐3 (HNP‐1–3) are useful biomarkers for ulcerative colitis (UC). The precise roles of these peptides in UC are poorly understood, however. The aim of this study was to determine whether HNP‐1 affects disease activity in mice with experimental colitis. Methods: Experimental colitis was induced in BALB/c or severe combined immunodeficiency (SCID) mice using dextran sulfate sodium (DSS). Mice were subsequently treated intraperitoneally with HNP‐1 (100 &mgr;g/day) or phosphate‐buffered saline (PBS) from day 4 to day 6. The severity of colitis was evaluated based on a disease activity index, histologic score, and cytokine expression. Results: Body weight and colon length significantly decreased and the disease activity index score, histologic score, and myeloperoxidase activity significantly increased in HNP‐1‐treated BALB/c mice compared with PBS‐treated mice. Interferon‐&ggr; and tumor necrosis factor‐&agr; levels in colon culture supernatants‐derived HNP‐1‐treated mice were also significantly higher, and interleukin (IL)‐1&bgr; levels tended to increase in response to HNP‐1. In addition, treating SCID mice with HNP‐1 aggravated DSS‐induced colitis and IL‐1&bgr; levels in colon culture supernatants from these mice were significantly higher than in cultures obtained from control mice. Furthermore, in both BALB/c and SCID mice increased recruitment of F4/80‐positive macrophages was observed in the inflamed colonic mucosa following HNP‐1 injections. Conclusions: High concentrations of HNP‐1 aggravate DSS‐induced colitis, including upregulated expression of such macrophage‐derived cytokines as IL‐1&bgr;. These results indicate that high concentrations of HNP‐1–3 in patients with UC may exacerbate disease activity via increased cytokine production. (Inflamm Bowel Dis 2011;)

Effect of Endoscopic Submucosal Dissection for Superficial Esophageal Neoplasms and Risk Factors for Postoperative Stricture

AbstractEndoscopic submucosal dissection (ESD) enables wider tumor resection compared with endoscopic mucosal resection and en bloc resection of superficial esophageal neoplasms. However, ESD may cause difficult-to-treat stricture of the esophagus, and therefore, prediction of and measures against postoperative esophageal stricture are critical. The aim of this study was to evaluate the effect of ESD on superficial esophageal neoplasms and identify risk factors associated with esophageal stricture after ESD.This study included 165 lesions in 120 patients with superficial esophageal neoplasms, including cancer and neoplasia, who underwent ESD from 2009 to 2013.The complete resection rate of superficial esophageal neoplasms by ESD was 90.9%. After ESD, 22 subjects (18.3%) had symptomatic esophageal stricture, 12 (10.0%) had aspiration pneumonia of grade 2, and 7 (5.8%) had mediastinal emphysema of grade 2. Comparison of the 22 subjects with stricture with the 98 subjects without stricture showed significant differences in the rate of resection of >75% of the esophageal circumference, rate of whole circumference resection, and the required time for resection. The tumor size and the size of the resected tissue sample also differed between the 2 groups. The groups did not differ in age, sex, alcohol intake, and smoking; location, macroscopic, and histological tumor findings; chest pain; or use of anticoagulants for comorbidities. In multivariate analysis, tumor size and whole circumference resection were independent risk factors for stricture. Furthermore, in 45 subjects with resection of >75% of the esophageal circumference, whole resection of the esophagus was the only independent risk factor for stricture.This study suggests that ESD has a strong therapeutic effect on superficial esophageal neoplasms; however, a greater extent of resection of the esophagus increases the risk of postoperative esophageal stricture. Preventive measures against development of postoperative stricture require further study.

Correlation of serum levels of complement C4a desArg with pathologically estimated severity of glomerular lesions and mesangial hypercellularity scores in patients with IgA nephropathy

The aim of the present study was to explore serum biomarkers for the pathology of IgA nephropathy using serum proteomics. The subjects were 57 patients with IgA nephropathy who were divided into two groups (group 1, n=25; group 2, n=32) and 14 healthy controls. Serum protein profiles were analyzed using the ProteinChip surface-enhanced laser desorption ionization (SELDI) system. Associations between signal intensities of proteins and histological findings in patients with IgA nephropathy were studied in group 1. Serum levels of a candidate biomarker protein (complement component C4a desArg) for IgA nephropathy were determined by enzyme linked-immunosorbent assay (ELISA) in group 2 and the relationships of these levels with histological findings were evaluated. There were significant differences in 93 protein signals between patients in group 1 and controls. Among these signals, 3 proteins at 8592, 8757 and 8806 m/z were significantly correlated with the severity of glomerular lesions. The protein at 8592 m/z was identified as C4a desArg and the signal intensity of 8592 m/z was strongly correlated with serum C4a levels, including C4a desArg, determined by ELISA. In addition, the serum levels of C4a (mainly C4a desArg) were significantly higher in patients in group 2 compared to controls and were correlated with the severity of glomerular lesions and with mesangial hypercellularity scores. In conclusion, the serum levels of complement C4a desArg are significantly higher in patients with IgA nephropathy compared to healthy controls and are significantly correlated with the severity of glomerular lesions and mesangial hypercellularity scores. Thus, serum C4a desArg is a potential biomarker for the severity of histological findings in patients with IgA nephropathy.

Impact of antibody to hepatitis B core antigen on the clinical course of hepatitis C virus carriers in a hyperendemic area in Japan: A community-based cohort study

Subjects positive for antibody to hepatitis B core antigen (HBcAb) and negative for hepatitis B surface antigen (HBsAg) are considered to have occult hepatitis B virus (HBV) infection. The aim of this study was to determine the impact of occult HBV infection on aggravation of the clinical course in hepatitis C virus (HCV) carriers.