Soluble bioactive microbial mediators regulate proteasomal degradation and autophagy to protect against inflammation-induced stress.

Bifidobacterium breve and other Gram-positive gut commensal microbes protect the gastrointestinal epithelium against inflammation-induced stress. However, the mechanisms whereby these bacteria accomplish this protection are poorly understood. In this study, we examined soluble factors derived from Bifidobacterium breve and their impact on the two major protein degradation systems within intestinal epithelial cells, proteasomes and autophagy. Conditioned media from gastrointestinal Gram-positive, but not Gram-negative, bacteria activated autophagy and increased expression of the autophagy proteins Atg5 and Atg7 along with the stress response protein heat shock protein 27. Specific examination of media conditioned by the Gram-positive bacterium Bifidobacterium breve (Bb-CM) showed that this microbe produces small molecules (<3 kDa) that increase expression of the autophagy proteins Atg5 and Atg7, activate autophagy, and inhibit proteasomal enzyme activity. Upregulation of autophagy by Bb-CM was mediated through MAP kinase signaling. In vitro studies using C2BBe1 cells silenced for Atg7 and in vivo studies using mice conditionally deficient in intestinal epithelial cell Atg7 showed that Bb-CM-induced cytoprotection is dependent on autophagy. Therefore, this work demonstrates that Gram-positive bacteria modify protein degradation programs within intestinal epithelial cells to promote their survival during stress. It also reveals the therapeutic potential of soluble molecules produced by these microbes for prevention and treatment of gastrointestinal disease.

[Jejunal duplication in an adult presenting with hematochezia].

A 56-year-old man was admitted to our hospital with appetite loss, palpitations, orthostatic syncope, and hematochezia. Contrast-enhanced abdominal computed tomography (CT) revealed a proximal jejunal diverticulum with contrast extravasation. We immediately performed transoral double balloon enteroscopy (DBE) to treat the bleed in the jejunum, and this revealed a small ulcer with an exposed vessel at the opening of the jejunal diverticulum. Hemostasis was achieved endoscopically with argon plasma coagulation (APC) and hemoclips. During subsequent surgery, the diverticulum was found on the mesenteric side of the jejunum. We performed laparoscopy-assisted partial resection of the jejunum, and pathological examination showed that the diverticulum shared a common proper muscle layer with the jejunum and was covered by jejunal mucosa with no ectopic mucosa. Therefore, we diagnosed jejunal duplication. After hospital discharge, the patient had no recurrence of hematochezia or anemia. We report a rare case of jejunal duplication presenting with hematochezia, which was diagnosed as jejunal diverticular bleeding by CT and DBE before surgery. Pathological analysis confirmed jejunal duplication after surgery. We suggest that intestinal diverticular bleeding, as well as duplication of the gastrointestinal tract, should be considered as part of the differential diagnosis of obscure gastrointestinal bleeding.

Primary squamous cell carcinoma of the duodenum effectively treated with TS-1: a case report.

An 89-year-old man was admitted to our hospital for thorough investigation of refractory diabetes mellitus, which revealed primary squamous cell carcinoma of the duodenum. After two courses of chemotherapy, follow-up esophagoduodenogastroscopy and duodenal biopsy showed no evidence of tumor. No findings were suggestive of recurrence of the primary lesion 19 months after starting chemotherapy. This case suggests that chemotherapy including TS-1 may be effective for treating unresectable primary squamous cell carcinoma of the duodenum.

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.

AIM: 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. METHODS: A prospective cohort study was performed in 400 subjects who were positive for anti-HCV antibody and negative for HBsAg. Among these subjects, 263 were HCV core antigen positive or HCV RNA positive (HCV carriers). We examined whether the presence of HBcAb affected the clinical course in these HCV carriers from 1996-2005. RESULTS: The HBcAb positive rates were 53.6% and 52.6% in HCV carriers and HCV RNA negative subjects, respectively. There were no differences in the incidence of hepatocellular carcinoma (HCC) and cumulative mortality associated with liver-related death between HCV carriers who were positive and negative for HBcAb. In multivariate analysis, age (≥65 years) and alanine aminotransferase level (≥31 IU/L) emerged as independent risk factors for HCC development and liver-related death, but the HBcAb status was not a risk factor. In addition, increased serum hepatic fibrosis markers (measured from 2001-2004) were not associated with HBcAb status. CONCLUSION: In our cohort study, the presence of HBcAb had no impact on HCC development, liver-related death and hepatic fibrosis markers in HCV carriers. Thus, our results indicate that occult HBV infection has no impact on the clinical course in HCV carriers.

Safety and pharmacokinetics of recombinant human hepatocyte growth factor (rh-HGF) in patients with fulminant hepatitis: a phase I/II clinical trial, following preclinical studies to ensure safety.

BACKGROUND: Hepatocyte growth factor (HGF) stimulates hepatocyte proliferation, and also acts as an anti-apoptotic factor. Therefore, HGF is a potential therapeutic agent for treatment of fatal liver diseases. We performed a translational medicine protocol with recombinant human HGF (rh-HGF), including a phase I/II study of patients with fulminant hepatitis (FH) or late-onset hepatic failure (LOHF), in order to examine the safety, pharmacokinetics, and clinical efficacy of this molecule. METHODS: Potential adverse effects identified through preclinical safety tests with rh-HGF include a decrease in blood pressure (BP) and an increase in urinary excretion of albumin. Therefore, we further investigated the effect of rh-HGF on circulatory status and renal toxicity in preclinical animal studies. In a clinical trial, 20 patients with FH or LOHF were evaluated for participation in this clinical trial, and four patients were enrolled. Subjects received rh-HGF (0.6 mg/m2/day) intravenously for 12 to 14 days. RESULTS: We established an infusion method to avoid rapid BP reduction in miniature swine, and confirmed reversibility of renal toxicity in rats. Although administration of rh-HGF moderately decreased BP in the participating subjects, this BP reduction did not require cessation of rh-HGF or any vasopressor therapy; BP returned to resting levels after the completion of rh-HGF infusion. Repeated doses of rh-HGF did not induce renal toxicity, and severe adverse events were not observed. Two patients survived, however, there was no evidence that rh-HGF was effective for the treatment of FH or LOHF. CONCLUSIONS: Intravenous rh-HGF at a dose of 0.6 mg/m2 was well tolerated in patients with FH or LOHF; therefore, it is desirable to conduct further investigations to determine the efficacy of rh-HGF at an increased dose.

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

UNLABELLED: 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 (>or=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.

Endoscopic characterization of the small bowel in patients with portal hypertension evaluated by double balloon endoscopy.

BACKGROUND: The endoscopic abnormalities present in the small bowel (SB) of patients with portal hypertension (PH) are not well understood. This study sought to evaluate endoscopic findings of the SB in patients with PH by double balloon endoscopy (DBE). METHODS: We evaluated the endoscopic findings of SB in 15 patients with PH and 49 controls without liver disease or PH. A total of 24 and 90 procedures were performed for PH patients and control patients, respectively, through oral and/or anal approaches. RESULTS: Fourteen of the 15 patients exhibited villous abnormalities, including edema (73%), atrophy (40%), and reddening (47%) of villi. Vascular lesions, such as angiodysplasia-like abnormalities (67%), dilated/proliferated vessels (93%), and varices (7%), were observed in all patients with PH. Although they were associated with ascites, these abnormalities did not correlate with any laboratory findings. None of these abnormalities was observed in controls. Definitive or suspected bleeding sources were identified in 9 of 13 patients with both PH and obscure gastrointestinal bleeding (OGIB), which was similar to the incidence in controls with OGIB. Although the frequency of postprocedure fever (>37.5 degrees C) was higher in patients with PH in comparison to controls (29% vs. 2%, P < 0.01), endoscopic treatment under DBE was performed on 3 PH patients without serious complications. CONCLUSIONS: Endoscopic abnormalities of the SB may be prevalent in patients with PH. Although postprocedure fever of DBE may occur more commonly in patients with PH, DBE is useful as both a diagnostic and therapeutic tool to evaluate the SB.

Translational research on HGF: A phase I/II study of recombinant human HGF for the treatment of fulminant hepatic failure.

Hepatocyte growth factor (HGF) is a potential therapeutic agent for fatal liver diseases, including fulminant hepatic failure (FHF). After performing a number of preclinical tests with recombinant human HGF (rh-HGF), we started a phase I/II study in September 2005 of patients with FHF or late-onset hepatic failure (LOHF), to examine the safety and clinical efficacy of rh-HGF. We first administered rh-HGF (0.6 mg/m(2)/day) for 13 days to a 67-year-old Japanese man with FHF. All data from this patient were reviewed by the independent data monitoring committee, and the safety of rh-HGF was recognized. Finally, a clinical trial of rh-HGF was approved to be continued. As of August 2007, we have administered rh-HGF to four patients with FHF or LOHF. All patients showed a moderate decrease in systolic blood pressure during rh-HGF administration, while the urinary excretion of albumin did not increase in all cases. In the first and third patients, hepatic failure gradually progressed, and they died 66 and 29 days, respectively, after encephalopathy occurred. The second and fourth patients are presently still alive. In conclusion, we started a clinical trial that examined the effects of rh-HGF in patients with FHF or LOHF, and in the four patients with FHF or LOHF enrolled in this study, repeated doses of rh-HGF did not produce any severe side effects.

[A case study: interferon-beta-induced remission of ulcerative colitis in a patient with type C chronic hepatitis].

A 66-year-old man patient with chronic hepatitis (CH) C and complications from ulcerative colitis (UC) was treated with interferon-beta (IFN-beta). Endoscopically, the UC disease activity was moderate before IFN-beta treatment but was in remission eight week after treatment. However, a few months after stopping IFN treatment, endoscopy revealed that the UC disease activity had returned to moderate levels. This result shows that UC improved with IFN treatment.

Effect of hepatocyte growth factor on endogenous hepatocarcinogenesis in rats fed a choline-deficient L-amino acid-defined diet.

Hepatocyte growth factor (HGF) is a promising agent for the treatment of intractable liver disease, due to its mitogenic, anti-apoptotic, and anti-fibrotic effects. We investigated the effect of recombinant human HGF (rh-HGF) on the development of both hepatocellular carcinoma (HCC) and preneoplastic nodules in rats fed a choline-deficient L-amino acid-defined (CDAA) diet, an animal model of hepatocarcinogenesis resembling human development of HCC with cirrhosis. From weeks 13 to 48 of the CDAA diet, rh-HGF (0.1 or 0.5 mg/kg/day) was administered intravenously to rats in four-week cycles, with treatment for five consecutive days of each week for two weeks, followed by a two-week washout period. Treatment with rh-HGF significantly inhibited the development of preneoplastic nodules in a dose-dependent manner at 24 weeks. Although the numbers and areas of the preneoplastic nodules in rats treated with rh-HGF were equivalent to those in mock-treated rats by 60 weeks, the incidence of HCC was reduced by HGF treatment. Although one rat treated with low-dose rh-HGF exhibited a massive HCC, which occupied almost the whole liver, and lung metastases, HGF treatment did not increase the overall frequency of HCC. Administration of high-dose rh-HGF, however, induced an increase in the urinary excretion of albumin, leading to decreased serum albumin at 60 weeks. These results indicate that long-term administration of rh-HGF does not accelerate hepatocarcinogenesis in rats fed a CDAA diet. However, these findings do not completely exclude the potential of HGF-induced hepatocarcinogenesis; this issue must be resolved before rh-HGF can be used for patients with intractable liver diseases, especially those with cirrhosis.

Repeated intravenous injection of recombinant human hepatocyte growth factor ameliorates liver cirrhosis but causes albuminuria in rats.

Hepatocyte growth factor (HGF) is a promising agent for the treatment of liver cirrhosis because of its mitogenic and anti-fibrotic effects. We investigated the effect of recombinant human HGF (rh-HGF) on cirrhosis development; its pharmacokinetics and nephrotoxicity in rats with liver cirrhosis induced by 4-week treatment with dimethylnitrosamine (DMN). rh-HGF (0.3 mg/kg) was intravenously administered to rats once a day for 4 weeks in parallel with DMN treatment or twice a day for the last 2 weeks of DMN treatment. Repeated doses of rh-HGF increased the liver weight and serum albumin, and reduced serum ALT. The development of hepatic fibrosis was inhibited more efficiently by extended low-dose treatment with rh-HGF. In cirrhotic rats, serum levels of rh-HGF increased and clearance was decreased, leading to an increase in the area under the plasma-concentration time curve and a decrease in the steady-state volume of distribution. Repeated doses of rh-HGF led to increased urinary albumin excretion, but no rh-HGF-treated animals developed increased serum creatinine levels. Urinary albumin excretion returned to baseline after the cessation of rh-HGF. These results suggest that extended treatment with rh-HGF is required for the attenuation of cirrhosis, and repeated doses of rh-HGF cause adverse effects in extra-hepatic organs. These issues must be resolved before the widespread application of rh-HGF in the treatment of liver cirrhosis.

Antihypertensive therapy improves insulin resistance and serum levels of interleukin-6 and -10 in spontaneously hypertensive rats with steatohepatitis.

Metabolic syndrome based on insulin resistance (IR) and hypertension is a risk factor for advanced liver disease and cardiovascular disease in patients with nonalcoholic steatohepatitis (NASH). The present study investigated the effects of severe hypertension induced by a high­salt (HS) diet and antihypertensive therapy on the pathophysiological condition of spontaneously hypertensive rats (SHRs) with steatohepatitis. Steatohepatitis was induced using a choline-deficient, L-amino acid-defined diet (CDAA). Male SHRs (7­week­old) were randomly divided into five groups: Those receiving 6 weeks of standard chow with a normal salt concentration, followed by an additional 8 weeks of standard chow or CDAA with a normal salt concentration (control and CDAA groups, respectively); and those receiving 6 weeks of standard chow with HS, followed by CDAA with HS for an additional 8 weeks, with or without the antihypertensive agents, amlodipine (Aml) or hydralazine. In the CDAA and CDAA+HS groups, blood pressure was significantly correlated with serum levels of insulin, fasting blood glucose and homeostasis model assessment (HOMA)­IR. Antihypertensive therapy ameliorated the elevated glucose, insulin and HOMA­IR. Furthermore, the increased levels of serum interleukin (IL)­6 following the CDAA+HS diet were attenuated by antihypertensive therapy. The serum levels of IL­10 were increased by antihypertensive therapy, and the decrease in the proportion of splenic CD4+CD25+forkhead box P3+ T cells observed following the CDAA+HS diet tended to be restored by Aml. In conclusion, antihypertensive therapy improved glucose metabolism and imbalances in cytokine expression in the rat model of hypertension with steatohepatitis, suggesting that antihypertensive therapy acting through immunological factors may be beneficial for patients with metabolic syndrome-associated NASH.

Low concentrations of human neutrophil peptide ameliorate experimental murine colitis.

Human neutrophil peptides (HNPs) not only have antimicrobial properties, but also exert multiple immunomodulatory effects depending on the concentration used. We have previously demonstrated that the intraperitoneal administration of high-dose HNP-1 (100 µg/day) aggravates murine dextran sulfate sodium (DSS)-induced colitis, suggesting a potential pro-inflammatory role for HNPs at high concentrations. However, the role of low physiological concentrations of HNPs in the intestinal tract remains largely unknown. The aim of this study was to examine the effects of low concentrations of HNPs on intestinal inflammation. We first examined the effects of the mild transgenic overexpression of HNP-1 in DSS-induced colitis. HNP-1 transgenic mice have plasma HNP-1 levels similar to the physiological concentrations in human plasma. Compared to wild-type mice treated with DSS, HNP-1 transgenic mice treated with DSS had significantly lower clinical and histological scores, and lower colonic mRNA levels of pro-inflammatory cytokines, including interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. We then injected low-dose HNP-1 (5 µg/day) or phosphate-buffered saline (PBS) intraperitoneally into C57BL/6N and BALB/c mice administered DSS. The HNP-1-treated mice exhibited significantly milder colitis with reduced expression levels of pro-inflammatory cytokines compared with the PBS-treated mice. Finally, we examined the in vitro effects of HNP-1 on the expression of cytokines associated with macrophage activation. Low physiological concentrations of HNP-1 did not significantly affect the expression levels of IL-1ß, TNF-α, IL-6 or IL-10 in colonic lamina propria mononuclear cells activated with heat-killed Escherichia coli, suggesting that the anti-inflammatory effects of HNP-1 on murine colitis may not be exerted by direct action on intestinal macrophages. Collectively, our data demonstrated a biphasic dose-dependent effect of HNP-1 on DSS-induced colitis: an amelioration at low concentrations and an aggravation at high concentrations. Low concentrations of HNPs may contribute to the maintenance of intestinal homeostasis.

Hepatocyte growth factor facilitates the repair of large colonic ulcers in 2,4,6-trinitrobenzene sulfonic acid-induced colitis in rats.

BACKGROUND: Hepatocyte growth factor (HGF) modulates intestinal epithelial cell proliferation and migration, serving as a critical regulator of intestinal wound healing. The aim of this study was to clarify the effects of administration of recombinant human HGF on colonic mucosal damage in vivo. METHODS: Rats were given 7.5 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS) per rectum on day 0. On day 5, the degree of TNBS-induced colitis was evaluated endoscopically, and rats suffering from large ulcers (occupying more than two thirds of the luminal circumference) were treated with intravenous bolus injections of recombinant human HGF (1.0 mg/kg per day) or phosphate-buffered saline (PBS) for 5 days. RESULTS: Rats with TNBS-induced colitis given human HGF showed a significant reduction in colonic ulcer coverage and large intestinal shortening compared with those treated with PBS. Administration of recombinant human HGF also stimulated the proliferation of epithelial cells and reduced the inflammatory cell infiltrate. Finally, HGF treatment decreased the myeloperoxidase activity and tumor necrosis factor alpha levels in the TNBS-inflamed colon tissues. CONCLUSIONS: These results indicate that intravenous injection of HGF accelerates colonic mucosal repair and reduces infiltration of inflammatory cells in rats with TNBS-induced colitis and suggest that HGF has the potential to be a new therapeutic modality to promote intestinal mucosal repair in patients with inflammatory bowel disease.

Mucosal repair and growth factors: recombinant human hepatocyte growth factor as an innovative therapy for inflammatory bowel disease.

The repair of intestinal mucosal injuries is a tightly regulated process involving epithelial restitution, cell proliferation and maturation, and the dedifferentiation of epithelial cells. Deeper injuries also require additional repair mechanisms, including inflammatory processes, angiogenesis, and extracellular-matrix deposition. Once intestinal mucosal injury occurs, numerous growth factors and cytokines, including hepatocyte growth factor (HGF), keratinocyte growth factor, endothelial growth factor, epidermal growth factor, transforming growth factor-beta1, intestinal trefoil factor, interleukin (IL)-1, and IL-2, are induced in both the intestinal lumen and submucosa, and these factors cooperatively stimulate epithelial mucosal repair. HGF, a major agent promoting hepatocyte proliferation, also modulates intestinal epithelial cell proliferation and migration, leading to the acceleration of intestinal mucosal repair. Additionally, the proteolytic activation of HGF, which is mediated by HGF activator, is essential for the regeneration of injured intestinal mucosa. Recently, several studies have shown that the administration of recombinant human HGF or HGF gene therapy abrogates disease severity in several animal models of inflammatory bowel disease (IBD). Recombinant human HGF will soon be available for administration to patients with fulminant hepatic failure. Although additional preclinical biological studies are required, HGF has the potential to be an important new treatment modality promoting intestinal mucosal repair in patients with IBD.

Molecular mechanisms of hereditary persistence of alpha-fetoprotein (AFP) in two Japanese families A hepatocyte nuclear factor-1 site mutation leads to induction of the AFP gene expression in adult livers.

alpha-Fetoprotein (AFP) is produced abundantly in fetal liver but is hardly detectable in adults. In this study, we investigated two unrelated Japanese families with hereditary persistence of AFP. A g-->a substitution at nucleotide -119 (-119g-->a) in the hepatocyte nuclear factor (HNF)-1 binding site of the AFP promoter was identified in both families. The activity of the wild- or variant-type human AFP promoter was evaluated by in vitro and in vivo transfection experiments. This substitution in the AFP promoter significantly stimulated its transcriptional activity in human hepatoma cells, regardless of their prior AFP production. The variant-type AFP promoter was also active in adult mouse livers in vivo. Additionally, overexpression of HNF-1alpha stimulated the activity of both the wild- and variant-type AFP promoters in hepatoma cells. HNF-1alpha expression also activated both AFP promoters even in nonhepatoma cells, and this activation was suppressed by nuclear factor (NF)-I overexpression. These results indicate that an HNF-1 binding site mutation leads to induction of the AFP gene expression in adult liver, and suggest that competition between HNF-1 and NF-I in this region is involved in transcriptional regulation of the AFP gene during hepatic development.

The peroxisome proliferator-activated receptor-gamma agonist, pioglitazone, inhibits fat accumulation and fibrosis in the livers of rats fed a choline-deficient, l-amino acid-defined diet.

Administration of a choline-deficient, l-amino acid-defined (CDAA) diet to rats causes steatohepatitis, hepatic fibrosis, and hepatocellular carcinoma, a pathology similar to that observed in non-alcoholic steatohepatitis (NASH). The aim of this study was to evaluate if a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, pioglitazone (PGZ), could ameliorate CDAA diet-induced fatty liver and cirrhosis. Rats were fed a CDAA diet for 1 week and were given the CDAA diet for an additional week with or without PGZ (2-week model). Also, after administration of the CDAA diet for 12 weeks, rats were administered the CDAA diet for an additional 4 weeks with or without PGZ (16-week model). The CDAA diet, administered for either one or 12 weeks, induced fatty liver or cirrhosis with up-regulation of hepatic PPAR-gamma expression, respectively. In the 2-week model, rats treated with PGZ for 1 week demonstrated significantly lower hepatic triglyceride content and serum levels of tumor necrosis factor-alpha. In the 16-week model, treatment for 4 weeks with PGZ ameliorated hepatic fibrosis with a decrease in the expression of procollagen, alpha-smooth muscle actin, and transforming growth factor-beta1 in comparison to rats without PGZ. These results suggest that PPAR-gamma agonist is a potential therapeutic modality to treat NASH.

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.

Expression of glycoprotein nonmetastatic melanoma protein B in macrophages infiltrating injured mucosa is associated with the severity of experimental colitis in mice.

Glycoprotein nonmetastatic melanoma protein B (Gpnmb) is a transmembrane glycoprotein, which negatively regulates the inflammatory responses of macrophages. However, the role of Gpnmb in intestinal macrophages remains to be fully elucidated. The present study aimed to investigate the expression of Gpnmb and its effects on colonic mucosal injuries associated with dextran sulfate sodium (DSS)­induced colitis in BALB/c mice, DBA/2J (D2) mice lacking Gpnmb and Gpnmb­transgenic DBA/2J mice (D2­gpnmb+). The colonic expression of Gpnmb increased with the severity of DSS­induced colitis in BALB/c mice, and macrophages infiltrating the inflamed mucosa were found to express Gpnmb. The D2 mice lacking Gpnmb exhibited more severe DSS­induced colitis, which was accompanied by higher levels of pro­inflammatory cytokines, including interleukin (IL)­1ß and IL­6, compared with the D2­gpnmb+ mice. Following lipopolysaccharide stimulation, macrophages from the D2 mice expressed higher levels of pro­inflammatory cytokines and lower levels of IL­10, compared with the D2­gpnmb+mice. In addition, in the RAW264.7 murine macrophage cell line, knockdown of Gpnmb by small interfering RNA was associated with increased production of pro­inflammatory cytokines, which were potentially mediated by the extracellular signal­regulated kinase (ERK) and p38 signaling pathways. The results of the present study indicated that macrophages infiltrating injured mucosa express Gpnmb, and that Gpnmb­positive macrophages may ameliorate inflammation in the intestinal mucosa by decreasing pro­inflammatory cytokine production via the ERK and p38 signaling pathways.