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Recent studies suggesting the involvement of singlet oxygen in the pathogenesis of multiple diseases have attracted renewed attention to lipid oxidation mediated by singlet oxygen. Although the rate constants for singlet oxygen quenching by antioxidants have been measured extensively, the inhibition of lipid oxidation mediated by singlet oxygen has received relatively less attention, partly because a convenient method for measuring the rate of lipid oxidation is not available. The objective of this study was to develop a convenient method to measure plasma lipid oxidation mediated by singlet oxygen which may be applied to a rapid assessment of the antioxidant capacity to inhibit this oxidation using a conventional microplate reader. Singlet oxygen was produced from naphthalene endoperoxide, and lipid hydroperoxide production was followed by using diphenyl-1-pyrenylphosphine (DPPP). Non-fluorescent DPPP reacts stoichiometrically with lipid hydroperoxides to give highly fluorescent DPPP oxide. It was found that plasma oxidation by singlet oxygen increased the fluorescence intensity of DPPP oxide, which was suppressed by antioxidants. Fucoxanthin suppressed the oxidation more efficiently than β-carotene and α-tocopherol, while ascorbic acid and Trolox were not effective. The present method may be useful for monitoring lipid oxidation and also for rapid screening of the capacity of dietary antioxidants and natural products to inhibit lipid oxidation in a biologically relevant system.

Multiple chemical sensitivity (MCS) is a chronic acquired disorder characterized by non-specific symptoms in multiple organ systems associated with exposure to odorous chemicals. We previously observed significant activations in the prefrontal cortex (PFC) during olfactory stimulation using several different odorants in patients with MCS by near-infrared spectroscopy (NIRS) imaging. We also observed that the patients with MCS did not adequately distinguish non-odorant in the late stage of the repeated olfactory stimulation test. The sensory recovery of the olfactory system in the patients with MCS may process odors differently from healthy subjects after olfactory stimulation. We examined the recovery process of regional cerebral blood flow (rCBF) after olfactory stimulation in patients with MCS. NIRS imaging was performed in 6 patients with MCS and in 6 controls. The olfactory stimulation test was continuously repeated 10 times. The study also included a subjective assessment of the physical and psychological status and of the perception of irritating and hedonic odors. After olfactory stimulation, significant activations were observed in the PFC of patients with MCS on both the right and left sides compared with controls. The activations were specifically strong in the orbitofrontal cortex (OFC). Compared with controls, autonomic perception and feelings identification were poorer in patients with MCS. OFC is associated with stimuli response and the representation of preferences. These results suggest that a past strong exposure to hazardous chemicals activates the PFC during olfactory stimuli in patients with MCS, and a strong activation in the OFC remains after the stimuli.

The technological development in endoscopy is directed toward improved accuracy of the diagnoses of novel diseases. The capsule endoscope and balloon-assisted endoscope are examples of such technological development. By these novel technologies, the small intestine can be examined in more detail. Therefore, an increasing number of novel diseases have been discovered, requiring the establishment of diagnosis and treatment strategies for these unknown diseases. In particular, obscure gastrointestinal bleeding, Crohn's disease, and nonsteroidal anti-inflammatory drug-induced enteropathy are of great interest to endoscopists. The capsule endoscope is the best method for screening the small intestine; however, the development of supporting methods such as the patency capsule is eagerly desired.

Esophageal visceral hypersensitivity has been proposed to be a pathogenesis of heartburn in nonerosive reflux disease (NERD), but its further mechanisms are unclear. Recently, it has been suggested that nociceptors and neuropeptides control sensory and pain mechanisms. Therefore, the objective of the present study was to estimate expression of acid-sensitive nociceptors such as transient receptor potential vanilloid 1 (TRPV1) and acid-sensing ion channel 3, protease-activated receptor 2 (PAR2), neuropeptides such as substance P and calcitonin-gene-related peptide, and their receptors such as neurokinin 1 receptor (NK1R) and receptor activity-modifying protein 1 in the esophageal mucosa of NERD patients. Biopsy samples were taken from NERD patients and healthy control subjects without heartburn. The expression level of nociceptors, neuropeptides, and their receptors were assessed by real-time RT-PCR and enzyme immunoassay. Localization of substance P and CGRP in the esophageal mucosa was determined by immunohistochemical staining. Expression of mRNA for TRPV1 and PAR2 was significantly elevated in the esophageal mucosa of NERD patients. Substance P protein level and its receptor NK1R mRNA also increased in NERD patients. A positive correlation between the substance P protein level and reflux symptoms was observed. Immunohistochemical study revealed the presence of substance P-positive nerves in the lamina propria of the esophagus. These findings suggest that visceral hypersensitivity in NERD patients is involved in neurogenic inflammation showing the increase in both substance P release and NK1R expression, which may be associated with the activation of TRPV1 and PAR2.

Flexible spectral imaging color enhancement (FICE), or image-enhanced endoscopy, can enhance visualization of surface and vascular patterns of colorectal polyps. Resolution of FICE has recently been improved. We evaluated diagnostic accuracy for neoplastic and non-neoplastic colorectal polyp differentiation with detection of surface patterns by FICE without magnification. Retrospective analysis of 151 colorectal polyps evaluated by FICE without magnification was performed. Neoplastic surface patterns were defined as tubular and oval pit. We aimed to determine sensitivity, specificity, positive and negative predictive values (PPV and NPV), and accuracy in correlating diagnosis by FICE without magnification with histology. Moreover, findings were compared to those of white-light endoscopy (WL) and chromoendoscopy (CHR). Of the 151 colorectal polyps, 95 were identified as neoplastic and 56 were identified as non-neoplastic. FICE without magnification had a sensitivity of 89.4%, specificity of 89.2%, PPV of 93.4%, NPV of 83.3%, and accuracy of 89.4%. The accuracy of FICE value was higher than that of WL (sensitivity of 74.7%, specificity of 73.2%, PPV of 82.5%, NPV of 63.0%, and accuracy of 74.1%) and was worse than that of CHR (sensitivity of 96.8%, specificity of 89.2%, PPV of 93.9%, NPV of 96.1%, and accuracy of 94.7%). Imaging evaluation was validated by inter-/intra-observer measurements, demonstrating consistent results. The detection of surface patterns by FICE without magnification is useful for differential diagnosis of colorectal polyps. We believe that FICE without magnification is more convenient and easier method than CHR.

Endogenous hydrogen sulfide (H(2)S) is increasingly being recognized as an important gaseous physiological mediator. Accumulating evidence shows the functions of H(2)S in various models of disease, but rarely in colitis. In this study, we investigated the role of endogenous H(2)S in a dextran sodium sulfate (DSS)-induced colitis model.Acute colitis was induced using 8% DSS in male BALB/c mice. The mRNA expression of cystathionine γ-lyase (CSE), the primary synthetase of H(2)S in the gastrointestinal tract, and cystathionine-β-synthetase (CBS) was measured by real-time RT-PCR. The amount of H(2)S in the colonic mucosa was measured by gas chromatography. Colitis severity was evaluated clinically, histologically, and biochemically under the condition of co-treatment with DL-propargylglycine (PAG), an irreversible CSE inhibitor, and sodium sulfide (Na(2)S), an H(2)S donor.The mRNA expression levels of CSE and CBS, and the H(2)S content in the colonic mucosa were increased with time after DSS administration. The disease activity index, which was determined by weight loss, stool consistency, and intestinal bleeding, increased after DSS administration. PAG significantly enhanced the increase in the disease activity index scores. PAG also significantly increased tissue-associated myeloperoxidase activity and thiobarbituric acid-reactive substances in the inflamed mucosa. Moreover, Na(2)S counteracted these effects of PAG.Taken together, the results indicated that the inhibition of endogenous H(2)S generation caused the deterioration of DSS-induced colitis. We conclude that physiological H(2)S might act as an anti-inflammatory molecule in colitis.

Recent development of proteomic array technology, including protein profiling coupling ProteinChip array with surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF/MS), provides a potentially powerful tool for discovery of new biomarkers by comparison of its profiles according to patient phenotypes. We used this approach to identify the host factors associated with treatment response in patients with chronic hepatitis C (CHC) receiving a 48-wk course of pegylated interferon (PEG-IFN) alpha 2b plus ribavirin (RBV). Protein profiles of pretreatment serum samples from 32 patients with genotype 1b and high viral load were conducted by SELDI-TOF/MS by using the three different ProteinChip arrays (CM10, Q10, IMAC30). Proteins showed significantly different peak intensities between sustained virological responders (SVRs), and non-SVRs were identified by chromatography, SDS-PAGE, TOF/MS and tandem mass spectrometry (MS/MS) assay. Eleven peak intensities were significantly different between SVRs and non-SVRs. The three SVR-increased peaks could be identified as two apolipoprotein (Apo) fragments and albumin and, among the eight non–SVR-increased proteins, four peaks identified as two iron-related and two fibrogenesis-related protein fragments, respectively. Multivariate analysis showed that the serum ferritin and three peak intensity values (Apo A1, hemopexin and transferrin) were independent variables associated with SVRs, and the area under the receiver operating characteristic (ROC) curves for SVR prediction by using the Apo A1/hemopexin and hemopexin/transferrin were 0.964 and 0.936. In conclusion, pretreatment serum protein profiling by SELDI-TOF/MS is variable for identification of response-related host factors, which are useful for treatment efficacy prediction in CHC receiving PEG-IFN plus RBV. Our data also may help us understand the mechanism for treatment resistance and development of more effective antiviral therapy targeted toward the modulation of lipogenesis or iron homeostasis in CHC patients.

Autofluorescence (AF) imaging, which can potentially differentiate tissue types based on differences in fluorescence emission, may be useful in the diagnosis and treatment of colorectal malignancies. This study was designed to assess the potential usefulness of AF colonoscopy for evaluating superficial colorectal neoplastic lesions. A total of 49 colorectal lesions in 43 patients were investigated. All superficial colorectal neoplastic lesions were identified with white light (WL) colonoscopy. Each detected lesion was investigated by WL colonoscopy, AF colonoscopy, and chromoendoscopy using 0.2% indigo carmine dye. Three endoscopists, blinded to each patient's history, evaluated the still images (as obtained with these three methods) in random order and evaluated their influence on the assessment of lesion visualization. All the lesions were biopsied or resected endoscopically, with the pathological results used as the gold standard. For visualization of the surface appearance and differences in color of the lesions compared with the surrounding mucosa, AF colonoscopy was superior to WL colonoscopy (p

To protect the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs is one of the critical issues in the field of gastroenterology. Polaprezinc (PZ), a gastric muco-protecting agent, has been widely used for the treatment of gastric ulcer and gastritis for its unique effects, such as its strong reactive oxygen species (ROS)-quenching effect. The aim of this study was to clarify the mechanism by which indomethacin-induced small intestinal mucosal injury occurs, by using a rat intestinal epithelial cell line (RIE-1). In addition, the protective role of PZ and the possible mechanism of its effect on indomethacin-induced small intestinal injury were investigated.Cell death was evaluated by methyl thiazolyl tetrazolium (MTT) assay and a double-staining method with Hoechst33342 dye and propidium iodide. Indomethacin-induced ROS production was evaluated by detecting the oxidation of a redox-sensitive fluorogenic probe, RedoxSensor, and the oxidation of cysteine residues of proteins (protein S oxidation). The activation of cytochrome c, smac/DIABLO, and caspase-3 was assessed by western blotting. In some experiments, PZ or its components, L: -carnosine and zinc, were used.We found that indomethacin caused apoptosis in RIE-1 cells in a dose- and time-dependent manner. Indomethacin also induced ROS production and an increase in the protein S oxidation of RIE-1. Pretreatment of RIE-1 with PZ or zinc sulfate, but not L: -carnosine, significantly reduced the indomethacin-induced apoptosis. PZ prevented ROS production and the increase in protein S-oxidation. PZ inhibited indomethacin-induced cytochrome c and smac/DIABLO release and subsequent caspase-3 activation.The protective effect of PZ on indomethacin-induced small intestinal injury may be dependent on its ROS-quenching effect.

Endoscopic submucosal dissection (ESD) has been reported to be effective for the en bloc resection of large colorectal tumors. Our study investigated whether ESD was suitable for elderly people with large colorectal tumors in terms of its invasiveness.We studied 119 colorectal tumors that were treated with ESD at Kyoto Prefectural University of Medicine or Nara City Hospital between 2006 and 2009. We classified each patient as either elderly, i.e., more than 75 years old, or non-elderly, i.e., less than 75 years old. Thirty-two of the cases were classified as elderly. Performance status, tumor size, operation time, rate of en bloc resection, histopathological diagnosis, complications, and hospital stay after ESD were analyzed retrospectively in both groups.In the elderly group, the average tumor size was 32.6 mm; the average operation time, 96 min; the rate of en bloc resection, 81.2%; the rate of perforation, 3.1%; and hospital stay after ESD, 5.1 days. Histopathological diagnosis for 16 tumors was adenoma; for 13, carcinoma with invasion into the mucosa; and for three, carcinoma with invasion into the submucosa. There were no statistical differences between the two groups in any of these data. The case with perforation was treated conservatively without urgent surgery in the elderly group.ESD for colorectal tumors resulted in favorable rates of en bloc resection in elderly people. Perforation occurred in elderly people, but these patients were cured with conservative treatment. ESD is a safe and minimally invasive treatment for elderly people with colorectal tumors.

CORM-released CO has been shown to be beneficial in resolution of acute inflammation. The acute phase of intestinal ischemia-reperfusion (I/R) injury is characterized by oxidative stress-related inflammation and leukocyte recruitment. In this study, we assessed the effects and potential mechanisms of CORM-2-released CO in modulation of inflammatory response in the small intestine following I/R-challenge. To this end mice (C57Bl/6) small intestine were challenged with ischemia by occluding superior mesenteric artery (SMA) for 45 min. CORM-2 (8 mg/kg; i.v.) was administered immediately before SMA occlusion. Sham operated mice were injected with vehicle (0.25% DMSO). Inflammatory response in the small intestine (jejunum) was assessed 4 h following reperfusion by measuring tissue levels of TNF-alpha protein (ELISA), adhesion molecules E-selectin and ICAM-1 (Western blot), NF-kappaB activation (EMSA), along with PMN tissue accumulation (MPO assay) and leukocyte rolling/adhesion in the microcirculation of jejunum (intravital microscopy). The obtained results indicate that tissue levels of TNF-alpha, E-selectin and ICAM-1 protein expression, activation of NF-kappaB, and subsequent accumulation of PMN were elevated in I/R-challenged jejunum. The above changes were significantly attenuated in CORM-2-treated mice. Taken together these findings indicate that CORM-2-released CO confers anti-inflammatory effects by interfering with NF-kappaB activation and subsequent up-regulation of vascular pro-adhesive phenotype in I/R-challenged small intestine.

We present the case of a 62-year-old Japanese man whose histological diagnosis was adenoendocrine cell carcinoma of the gallbladder at autopsy, but whose antemortem diagnosis was squamous cell carcinoma. The patient was admitted to hospital with complaints of occasional vomiting and abdominal pain. Abdominal computed tomography revealed a large tumor on the gallbladder involving the adjacent liver, colon, and duodenum, with multiple metastases in the greater omentum and paraportal lymph nodes. The serum level of squamous cell carcinoma antigen (SCCA) was high, whereas that of carbohydrate antigen (CA) 19-9, as well as that of carcinoembryonic antigen (CEA) was within the normal range. Due to these clinical features, we first suspected advanced squamous cell carcinoma of the gallbladder. After two cycles of gemcitabine monotherapy, the tumor had become enlarged and the regimen was changed to a combination of docetaxel and cisplatin. Though tumor regression was achieved and his serum SCCA level normalized after 3 months, the patient rejected additional chemotherapy and died 8 months after the diagnosis. The histopathological findings made by autopsy demonstrated the tumor to be an adenoendocrine cell carcinoma without squamous carcinoma cells. The case is interesting in that the clinical features were similar to those of squamous cell carcinoma of the gallbladder.

Nonsteroidal antiinflammatory drug (NSAID)-induced enteropathy is clinically very important, but the pathological mechanisms remain unclear. Mast cells have been reported to play an important role in the pathogenesis of indomethacin-induced small intestinal injury. In this study, we investigated the role of mast cells in indomethacin-induced small intestinal injury using mast cell deficiency (Ws/Ws) rat.Ws/Ws rats and control (W+/W+) rats were given indomethacin (15 mg/kg) subcutaneously, and the intestinal mucosal damage was estimated after 24 h.The area (mm2) of macroscopic visible lesions, the concentrations of thiobarbituric acid-reactive substances (TBARS) as an index of lipid peroxidation, myeloperoxidase (MPO) activity as an index of neutrophil accumulation, and the content of cytokine-induced neutrophil chemoattractant-1 (CINC-1) were significantly increased in indomethacin-treated groups compared with the sham groups. The development of intestinal lesions in response to indomethacin was prevented in Ws/Ws rats compared with W+/W+ rats, together with significant suppression of the increased levels of TBARS, MPO activities, and CINC-1 levels.These results indicate that mast cells are involved in the pathogenesis of the intestinal mucosal damage induced by indomethacin.

We examined the effect of sildenafil, an inhibitor of phosphodiesterase subtype 5, that catalyzes hydrolysis of 3′,5′-cyclic guanosine monophosphate (cGMP), on indomethacin-induced small-intestinal ulceration in rats and investigated the mechanism of this action, especially in relation to endogenous nitric oxide (NO). Animals without fasting were given indomethacin (10 mg/kg) s.c. and then killed 24 h later. Indomethacin produced hemorrhagic lesions in the small intestine, accompanied by a promotion of enterobacterial invasion and the expression of inducible NO synthase (iNOS) as well as myeloperoxidase (MPO) activity in the mucosa. Sildenafil (3–20 mg/kg), given p.o. 30 min before indomethacin, dose-dependently reduced the severity of these lesions, with concomitant suppression of the increase in MPO activity, iNOS expression and bacterial invasion. These effects were attenuated by the prior administration of the nonselective NOS inhibitor, N G-nitro-l-arginine methyl ester, in an l-arginine-reversible manner. Indomethacin also decreased the secretion of mucus and fluid (enteropooling) and enhanced intestinal motility, but these responses were all prevented by the prior administration of sildenafil. Likewise, pretreatment of the animals with NOR-3, a NO donor, also reversed the functional changes caused by indomethacin, followed by suppression of bacterial invasion and iNOS expression, and prevented the development of intestinal lesions. These results suggest that sildenafil prevents indomethacin-induced small-intestinal ulceration in rats, via a NO/cGMP-dependent mechanism, and this effect is functionally associated with an increase in the secretion of mucus/fluid and a decrease of hypermotility, resulting in the suppression of bacterial invasion and iNOS expression following indomethacin treatment.

Ménétrier's disease has been reported to be associated with Helicobacter pylori infection. The aim of this study was to investigate the genetic characteristics of various virulence factors and cytokine expression profiles in Helicobacter pylori isolated from patients with Ménétrier's disease. The genotyping of virulence factors was accomplished by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Induction of various cytokines in MKN45 cells or gastric fibroblasts by Helicobacter pylori stimulus was measured by real-time reverse transcription-PCR. We found that the Helicobacter pylori strain isolated from a patient with Ménétrier's disease was different from other strains in the MseI-RFLP pattern of the ureC gene. Helicobacter pylori isolated from a patient with Ménétrier's disease showed the highest hepatocyte growth factor (HGF) and TNF-alpha mRNA expressions from gastric fibroblasts, and the highest TNF-alpha expression from MKN45 cells. The results in this study suggest that the difference in cytokine production, depending on the difference in bacteria components, plays an important role in the development of Ménétrier's disease.

In Japanese healthy CYP2C19 extensive metabolizers, rabeprazole 10 mg shows a faster onset of action and stronger inhibition of acid secretion than does omeprazole 20 mg on the first 3 days of administration. We evaluated gastric ulcer improvement after 1 week's treatment with rabeprazole or omeprazole in relation to CYP2C19 polymorphism. A 6-mm rubber disc was placed temporarily at the side of the ulcer for measurement of the ulcer area. The improvement ratios of ulcer area in homozygous extensive metabolizers (homoEMs), heterozygous extensive metabolizers (heteroEMs) and poor metabolizers (PMs) treated with rabeprazole 10 mg were 60.8, 65.0 and 55.3%, respectively, and these values are not significantly different. Corresponding values with omeprazole 20 mg were 46.3, 61.7 and 63.2%, respectively, and the value of homoEMs was significantly smaller than that of heteroEMs. The improvement ratios with rabeprazole in homoEMs and heteroEMs were significantly greater than that with omeprazole in homoEMs.

Interleukin (IL)-8 has been reported to participate in neutrophil infiltration in Helicobacter pylori (H. pylori)-induced gastritis in humans. In this study, we investigated the anti-inflammatory actions beyond the suppression of acid secretion by proton pump inhibitors (PPI), such as omeprazole and lansoprazole, on IL-8 production by gastric epithelial cells (MKN45) and human umbilical vein endothelial cells (HUVEC) and on the transendothelial migration of polymorphonuclear neutrophils (PMN).MKN45 and HUVEC were stimulated with H. pylori water extract (HPE) and IL-1beta, respectively, and nuclear factor kappa B (NFkappaB) activation and subsequent IL-8 production was assessed in the absence or presence of PPI. We also assessed the effect of PPI on IL-8-induced PMN transendothelial migration and on the alteration of cytoplasmic calcium concentration in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN.HPE and IL-1beta induced a significant increase in IL-8 production by MKN45 and HUVEC, respectively, along with NFkappaB activation, which was significantly inhibited by PPI. PPI also inhibited the IL-8-induced transendothelial migration of PMN and the fMLP-induced cytosolic calcium increase in PMN.PPI attenuate PMN-dependent gastric mucosal inflammation partly by interfering with NFkappaB activation in vascular endothelial cells and gastric epithelial cells, and partly by modulating the calcium concentration of PMN.

Reflux of the duodenal contents with gastric acid has been reported to contribute to the development of esophageal mucosal damage and inflammation. Recent studies show that pancreatic trypsin can stimulate the production of inflammatory mediators, including chemokines and prostaglandins from human esophageal epithelial cells in vitro. The aim of the present study was to investigate the role of pancreatic trypsin in the pathogenesis of chronic esophageal inflammation induced by gastroduodenal reflux in rats.Esophagogastroduodenal anastomosis was carried out in male Wistar rats by anastomosing the jejunum to the gastroesophageal junction under diethyl ether inhalation anesthesia. The animals undergoing surgery were treated with the control diet, rabeprazole sodium, nizatidine, ecabet sodium, camostat mesilate (CMM), ONO-1714, a specific inducible nitric oxide synthase (iNOS) inhibitor, or meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor. Esophageal injury was evaluated by macroscopic and microscopic findings, and mRNA expression for CINC-1, COX-2, and iNOS was determined by real-time polymerase chain reaction (PCR). Trypsin activity within the esophageal lumen was measured 2 weeks after surgery, and the expression of protease-activated receptor (PAR)-1 and -2 was confirmed by reverse transcription (RT)-PCR.At 8 weeks after surgery, gastroduodenal reflux induced esophageal erosions and ulcer formation as well as marked thickening of the esophageal wall. Histological study showed an increase of thickness of the esophageal mucosa, hyperplasia of the epidermis and basal cells, ulcer formation, and marked infiltration of inflammatory cells. The macroscopic ulcer score and histological ulcer length were significantly reduced by treatment with rabeprazole or CMM but not by nizatidine or ecabet sodium, compared with each control. Rabeprazole, nizatidine, or ecabet sodium did not affect the severity of mucosal hyperplastic scores or histological parameters in esophagitis. In contrast, the CMM group showed a significant decrease in the mucosal hyperplastic and inflammatory scores. The enhanced expression of CINC-1, COX-2, and iNOS mRNA in the control group was also markedly inhibited in the CMM-treated group. ONO-1714 or meloxicam treatment significantly reduced the macroscopic scores of ulcer and hyperplasia. The trypsin activity in the esophageal lumen was significantly increased in the control diet group, and this increase was significantly inhibited in the CMM-treated group. The expression of PAR-1 and -2 mRNA was confirmed in rat esophageal epithelium.With this model, we have demonstrated that CMM significantly reduces inflammation and hyperplasia in the esophageal mucosa. These results indicate that trypsin, which is primarily inhibited by CMM, plays an important role in the mucosal damage induced by gastroduodenal reflux and that it can be a therapeutic target in patients with gastroduodenal reflux esophagitis.

Although several studies have demonstrated that airway exposure to diesel exhaust particles (DEP) induces lung inflammation, the signaling pathways involved in the pathogenesis remain unclear. Toll-like receptors (TLRs) are generally accepted to be pathogen recognition receptors in mammalians. In the present study, we investigated the role of TLR-4 in DEP-induced lung inflammation and cytokine expression in the lung in TLR-4 point mutant (C3H/HeJ) mice and corresponding control (C3H/HeN) mice. Both the types of mice were randomized into four experimental groups that received vehicle or DEP (12 mg/kg body weight) by intratracheal instillation (n = 8-10 in each group). Cellular profile of bronchoalveolar lavage (BAL) fluid, expressions of cytokines and chemokines in the lung, and circulatory fibrinogen levels were evaluated 24 h after the instillation.DEP challenge revealed a significant increase in the numbers of total cells and neutrophils in the BAL fluid as compared to vehicle challenge, however, the numbers were less in C3H/HeJ mice than in C3H/HeN mice. DEP exposure significantly induced the lung expression of interleukin (IL)-1beta, keratinocyte chemoattractant (KC), and macrophage inflammatory protein (MIP)-1alpha when compared to vehicle challenge in both genotypes of mice. In the presence of DEP, the level of MIP-1alpha was significantly lower in C3H/HeJ mice than in C3H/HeN mice, however, the levels of IL-1beta, KC, and fibrinogen showed opposite findings. These results suggest that TLR-4 is one of recognition receptors against DEP in the airways.

Rebamipide, a gastro-protective drug, was developed in Japan for the treatment of peptic ulcer disease. It was proven superior to the former same category drug cetraxate in a randomized, controlled, double-blind, comparative clinical study in 1989. Rebamipide's mechanisms of actions are different from anti-secretory drugs; it accelerates and improves the quality of ulcer healing and reduces ulcer recurrence rate. Numerous studies have been conducted to explain the mechanisms responsible for these actions, 37 papers were published by 1998. Major properties of rebamipide include: stimulation of prostaglandin and mucus glycoprotein synthesis, inhibition of reactive oxygen species, inflammatory cytokines and chemokines, and inhibition of neutrophils activation. Since 1998, 107 papers were published, clarifying further effects of rebamipide on cyclooxygenase-2, prostaglandin E receptors, growth factors (i.e., HGF, EGF, and VEGF), heat-shock proteins, nitric oxide, adhesion molecules, neutrophils, and Helicobacter pylori- and NSAID-related pathology. Moreover, inhibitory action of rebamipide on gastric cancer growth has also been shown. In this issue we reviewed recent advances in understanding of rebamipide's mechanism of action and its newest clinical applications.

Rebamipide, a gastromucosal protective drug, suppresses indomethacin-induced gastropathy in humans and rodents. Effects of rebamipide on gene expression in indomethacin-treated gastric mucosal cells (RGM1) were investigated using high-density oligonucleotide arrays. Indomethacin induced apoptosis in RGM1 cells in a dose-dependent manner. Rebamipide pretreatment significantly reduced indomethacin-induced apoptosis. We used gene expression profiling on high-density oligonucleotide probe arrays to characterize the transcriptional response of RGM1 cells to indomethacin treatment for 6 hr. Of the 8,799 probes examined, 717 (8.1%) were induced (400 probes) or repressed (317 probes) at least 1.5-fold. Among the 158 genes that were induced by indomethacin at least 2.0-fold, four genes that were down-regulated by rebamipide at least 2.0-fold are listed: growth arrest and DNA-damage-inducible 45 alpha (GADD 45 alpha), golgi SNAP receptor complex member 1, iodothyronine deiodinases, and transcription factor 8. Real time-PCR confirmed GADD 45 alpha expression and its inhibition by rebamipide. Inhibition of apoptosis-related genes is possibly important for the cytoprotective effect of rebamipide against indomethacin-induced gastric mucosal cell injury.

Gastric ischemia-reperfusion is a relatively common condition leading to mucosal injury and may affect mucosal repair via modulating the gene expression of growth factors. Therefore, precise understanding of the molecular mechanism of ischemia or ischemia-reperfusion may lead to the discovery of new mucosal protective drugs. DNA microarray analysis followed by powerful data analysis has the potential to uncover previously undescribed genes involved in gastric injury and lead to an increased understanding of gastric mucosal cytoprotection. We introduced the laser-assisted microdissection to obtain cell-specific RNA from gastric mucosa in vivo and obtained sufficient amounts of cRNA for GeneChip analysis. This comprehensive approach enabled the simultaneous analysis of many genes, including transcriptional factors, as well as the generation of novel hypothesis on the mechanism of action of gastro-protective agents.

Proinflammatory factors, including neutrophil-derived oxygen free radicals and inflammatory cytokines, have recently been implicated in the pathogenesis of reflux esophagitis. Rebamipide has been widely used as an anti-ulcer agent. The aim of the present study was to assess the protective effect of rebamipide against acute reflux esophagitis in rats. Esophagitis was induced in rats by ligation at the limiting ridge and the lower portion of the duodenum. Vehicle or rebamipide were given as a single dose intraduodenally. Lesion index (LI), thiobarbituric acid-reactive substances (TBA-RS), myeloperoxidase (MPO) activity, mRNA and protein of tumor necrosis factor (TNF)-alpha and cytokine-induced neutrophil chemoattractant (CINC)-1 in the esophageal mucosa were markedly increased; pretreatment with rebamipide, however, significantly reduced both macroscopic and microscopic injuries and increases in inflammatory mediators. The results of this study indicate that rebamipide protects against the occurrence of esophagitis and has highly promising potential as a new therapeutic agent for reflux esophagitis.

Although several studies have reported that diesel exhaust particles (DEP) affect cardiorespiratory health in animals and humans, the effect of DEP on animal models with spontaneous allergic disorders has been far less intensively studied. The Nc/Nga mouse is known to be a typical animal model for human atopic dermatitis (AD). In the present study, we investigated the effects of repeated pulmonary exposure to DEP on airway inflammation and cytokine expression in NC/Nga mice. The animals were randomized into two experimental groups that received vehicle or DEP by intratracheal instillation weekly for six weeks. Cellular profiles of bronchoalveolar lavage (BAL) fluid and expressions of cytokines and chemokines in both the BAL fluid and lung tissues were evaluated 24 h after the last instillation. The DEP challenge produced an increase in the numbers of total cells, neutrophils, and mononuclear cells in BAL fluid as compared to the vehicle challenge (P0.01). DEP exposure significantly induced the lung expressions of interleukin (IL)-4, keratinocyte chemoattractant (KC), and macrophage inflammatory protein (MIP)-1alpha when compared to the vehicle challenge. These results indicate that intratracheal exposure to DEP induces the recruitment of inflammatory cells, at least partially, through the local expression of IL-4 and chemokines in NC/Nga mice.

The role of resistin in human biology remains uncertain. We measured serum resistin levels in Japanese patients with (n=111) and without (n=98) type 2 diabetes mellitus and investigated the significance of this hormone in the pathophysiology of diabetes. The levels of serum adiponectin and leptin were also measured. Resistin levels were increased significantly in patients with type 2 diabetes compared with non-diabetic subjects (24.7+/-2.6 vs. 15.0+/-1.2 ng/ml, p=0.0013). However, there was no correlation in either patient group between serum resistin levels and markers of insulin resistance, obesity or hyperlipidaemia. These results were in direct contrast to the data of leptin or adiponectin, both of which were closely related to these clinical markers of diabetes. Multivariate regression analysis on the combined data of the two groups demonstrated that the presence of diabetes and HDL cholesterol levels were significant predictors of serum resistin levels (diabetes: beta=0.159, p=0.035; HDL: beta=-0.172, p=0.039). No correlation was observed between C-reactive protein and resistin adjusted for BMI. Taken together, these findings demonstrate that serum resistin levels are increased in patients with type 2 diabetes, but this increase is not linked to markers of insulin resistance or adiposity. Further studies are necessary to elucidate the significance of serum resistin concentration in human pathophysiology.

The aim of the present study was to investigate the antioxidative effects of water-soluble vitamin E derivative, 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), on ischemia-reperfusion (I/R) -induced gastric mucosal injury in rats. Gastric ischemia was induced by applying a small clamp to the celiac artery and reoxygenation was produced by removal of the clamp. The area of gastric mucosal erosion, the concentration of thiobarbituric acid-reactive substances, and the myeloperoxidase activity in gastric mucosa significantly increased in I/R groups compared with those of sham-operated groups. These increases were significantly inhibited by pretreatment with TMG. The contents of both mucosal TNF-alpha and CINC-2beta in I/R groups were also increased compared with the levels of those in sham-operated groups. These increases of the inflammatory cytokines were significantly inhibited by the treatment with TMG. It is concluded that TMG inhibited lipid peroxidation and reduced development of the gastric mucosal inflammation induced by I/R in rats.

It has recently been reported that not only endogenous nitric oxide (NO) but also carbon monoxide (CO) produced by heme oxygenase (HO) have many physiological functions. The objective of the present study was to determine whether endogenous NO or CO is involved in the experimental pulmonary or liver metastasis of colon cancer in mice. Intravenous or intrasplenic injection of colon 26 cells from a mouse colon adenocarcinoma cell line resulted in multiple pulmonary or liver metastases. NG-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthase (NOS), or zinc deuteroporphyrin 2, 4-bis glycol (ZnDPBG), a competitive inhibitor of HO, was administered to the mice only on the day of tumor inoculation. We assessed the number of tumor cells 24 h later and the outcome of metastases of the target organ. In the pulmonary metastasis model, L-NAME increased both the number of tumor cells 24 h later and outcome of metastases 18 days later, but did not have a significant effect on liver metastasis. On the other hand, metastasis to the liver, but not that to the lung, increased following administration of ZnDPBG. These results suggest that the activities of NOS and HO could influence experimental metastasis in an organ-specific manner.

Our aim was to understand the mechanism of immunological changes associated with the use of an adsorptive-type extracorporeal device (Adacolumn) that has been developed for selective adsorption of granulocytes and monocytes/macrophages from peripheral blood of patients with active ulcerative colitis. The column is filled with carriers (G-1 beads) that have a diameter of 2 mm and are made of cellulose diacetate. In peripheral blood treated with the G-1 beads or peripheral blood from patients with active ulcerative colitis following granulocyte and monocyte adsorption apheresis, a significant suppression of proinflammatory cytokines (tissue necrosis factor-alpha, interleukin-1beta, interleukin-6, and interleukin-8) production by leukocytes, neutrophil chemotaxis, down-regulation of leukocyte adhesion molecule (L-selectin) and neutrophil adhesion to interleukin-1beta-activated endothelial cells were observed. Furthermore, after granulocyte adsorption therapy, the number of CD10-negative premature granulocytes increased, indicating increased turnover of these cells in the circulation. Our observations suggest that selective granulocyte and monocyte adsorption is associated with modified peripheral blood leukocyte function favorable to patients with ulcerative colitis and possibly other autoimmune disorders which reflect leukocyte hyperactivity.

In active rheumatoid arthritis, large numbers of granulocytes and macrophages are found in the inflamed joints. These leucocytes can promote inflammation and tissue injury by releasing inflammatory cytokines, proteinases and oxygen derivatives. To see if granulocyte and monocyte (GM) depletion produces anti-inflammatory effect, GM adsorption apheresis was performed in rabbits with immune arthritis by using a column (Adacolumn) filled with cellulose diacetate beads (G-1 beads) as adsorptive carriers which selectively adsorb CD11b positive GMs. Injection of ovalbumin into the knee joints of ovalbumin-sensitized rabbits caused a marked increase in peripheral blood leucocytes, joint swelling, increased granulocyte adhesion to G-1 beads and elevated TNF-α production by peripheral blood mononuclear cells (PBMC). When rabbits received a 60 min adsorption apheresis, there was suppression of CD11b positive leucocyte infiltration into the joint and reduced joint swelling (P < 0.01) compared with controls. Additionally, there was a significant (p < 0.01) suppression of TNF-α production by PBMC in the post column blood. These results suggest that GM depletion may serve as a non-pharmacological strategy to modify inflammatory disorders.

We previously showed that the cell surface-expressed Mr 70,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3+, CD4-, CD8-, T-cell receptor (TCR)alphabeta-, natural killer recetor-P1- T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the Vdelta6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR gammadelta chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR Vdelta6 family are likely to contribute to host resistance to tumor cells.

We examined the roles of lipid peroxidation, neutrophil accumulation, and inflammatory cytokines in the protective effect of polaprezinc against aspirin-induced gastric mucosal injury in rats. The intragastric administration of acidified aspirin induced hyperemia and hemorrhagic erosions in rat stomachs. The increase in the total gastric erosive area after aspirin administration was significantly inhibited in a dose-dependent manner by treatment with polaprezinc. The increases in thiobarbituric acid-reactive substances and tissue-associated myeloperoxidase activity 3 hr after aspirin administration were significantly inhibited by pretreatment with polaprezinc. The gastric concentration of TNF-alpha increased after aspirin administration, and the increase was also inhibited in a dose-dependent manner by treatment with polaprezinc. The peak expression of TNF-alpha mRNA 1 hr after aspirin administration was inhibited by 30 mg/kg of polaprezinc. Based on these data, the beneficial effects of polaprezinc on aspirin-induced gastric mucosal injury may be attributed to its antioxidative and antiinflammatory properties.

We investigated the effect of vitamin E on aspirin-induced gastric mucosal injury in rats. Twenty-eight male Sprague-Dawley rats were divided into four groups and were fed for 20 weeks with a diet containing

In Helicobacter pylori-associated gastricmucosal injury, interleukin (IL)-8, a potent leukocytechemoattractant, is produced by epithelial cellsinfected by H. pylori and directs neutrophils to thegastric mucosa. According to previous studies, the IL-8production requires direct contact between the bacteriaand epithelial cells. The aims of the present study wereto determine whether an H. pylori water extract (HPE) induces IL-8 production by gastricepithelial cells and to characterize IL-8-inducingsubstances in HPE. Extracts were prepared from astandard strain and from strains obtained from patientswith gastric ulcers. After addition of HPE to MKN 45cells, a gastric cancer cell line, IL-8 in supernatantsand IL-8 mRNA were measured by immunoassay and reversetranscription-polymerase chain reaction, respectively. For characterization, active fractions obtainedby gel filtration of standard-strain HPE were treated byheating or trypsinization. To study the signal pathwayleading to IL-8 production, inhibitors for protein kinase A (PKA), protein kinase C (PKC),or protein tyrosine kinase (PTK) were incubated withMKN45 cells before HPE stimulation. HPE from thestandard strain and one of these clinical strainsinduced IL-8 production. Lipopolysaccharide or cagA inthe strains showed no correlation with IL-8concentration. Standard-strain HPE induced IL-8 mRNAexpression in MKN 45 cells. Gel filtration localizedactivity to a low-molecular-weight fraction of about 7kDa, which was resistant to heat and trypsin digestion.PKC inhibitors significantly blocked HPE-induced IL-8production by MKN 45 cells; however, the PKA inhibitor or PTK inhibitors showed a partial inhibitoryeffect. HPE contains a nonprotein substance of lowmolecular weight that is responsible for IL-8 inductionin gastric epithelial cells. This induction is mainly dependent on the activation of PKC butpartially also dependent on PKA or PTK.

The role of active oxygen species and lipidperoxidation in the pathogenesis of duodenal ulcersinduced by mepirizole was investigated in rats. Oraladministration of mepirizole (200 mg/kg) resulted in ulcer lesions in the proximal duodenum.Thiobarbituric acid-reactive substances (TBA-reactivesubstances), an indicator of lipid peroxidation, alsosignificantly increased in the duodenal mucosa.Myeloperoxidase (MPO) activity in the duodenal mucosa, a signof polymorphonuclear leukocyte (PMN) accumulation,significantly increased. Combination treatment withpolyethylene glycol-modified Serratia Mn-SOD andcatalase significantly decreased the size of the ulcersand TBA-reactive substances in the duodenal mucosa.Allopurinol, a xanthine oxidase inhibitor, also reducedthe size of duodenal ulcers. Both the size of the ulcers and the increase in TBA-reactivesubstances in the duodenal mucosa were significantlylower in PMN-depleted rats. Mepirizole increased thesurface expression of adhesion molecule CD18 on PMNs in vitro. These results suggest that lipidperoxidation, mediated by active oxygen speciesgenerated from xanthine oxidase and PMNs, plays animportant role in the pathogenesis of duodenal ulcersinduced by mepirizole.

We examined the role of gastric mucosal blood flow, lipid peroxidation, and neutrophil accumulation mediated by platelet-activating factor in the protective effect of rebamipide against gastric mucosal injury in rats. The intravenous injection of platelet-activating factor induced hyperemia and hemorrhagic erosions in rat stomachs. Rebamipide did not affect the decrease in the gastric mucosal blood flow induced by platelet-activating factor. The increase in gastric injury score after platelet-activating factor injection and the increase in thiobarbituric acid-reactive substances were significantly inhibited by the administration of rebamipide. The gastric injury score was closely correlated with the accumulation of lipid peroxides. Tissue-associated myeloperoxidase activity in the gastric mucosa significantly increased after platelet activating factor injection; this increase was not influenced by rebamipide treatment. The protective effect of rebamipide against the platelet-activiting factor-induced gastric mucosal injury may be due to direct inhibition of lipid peroxidation or scavenging of oxygen radicals that initiate lipid peroxidation.

The purpose of this study was to investigate the effect of regular meals on the daily profile of blood oxidative stress markers in type 2 diabetic patients with postprandial hyperglycaemia. %CoQ10, calculated as the ratio of ubiquinone-10 (oxidised form of coenzyme Q10) to ubiquinol-10 (reduced form), was used as a sensitive marker of oxidative stress. Blood samples were collected from patients before and 2 h after breakfast, lunch and supper, and at 10 p.m. Patients were selected for the study if their blood glucose levels were7 mmol/l before breakfast andor =11.1 mmol/l on at least one occasion after breakfast. %CoQ10 levels after breakfast and throughout the day were significantly higher than those before breakfast (p=0.006-0.04). In contrast to the wave-like changes in plasma glucose levels, %CoQ10 levels increased after breakfast and remained at high levels throughout the day. These results indicated that diabetic patients with postprandial hyperglycaemia were exposed to meal-induced periods of oxidative stress during the day. Postprandial hyperglycaemia therefore has the potential to increase the risk of atherosclerotic cardiovascular disease through induction of oxidative stress.

The objectives of this study were to determine the roles of neutrophil-endothelial cell interactions and oxygen-derived free radicals in the pathogenesis of aspirin-induced gastric mucosal injury in rats. Oral administration of acidified aspirin (200 mg/kg) resulted in linear hemorrhagic erosions and an increase in myeloperoxidase activity, an index of neutrophil infiltration, in the gastric mucosa. Aspirin-induced gastric damage and the increase in myeloperoxidase activity were significantly inhibited by the injection of anti-CD11a, anti-CD11b, anti-intercellular adhesion molecule-1 monoclonal antibodies, and the combination of superoxide dismutase and catalase, which are scavengers of active oxygen species. These results suggest that neutrophil-endothelial adhesive interactions, which occur via CD11a/ CD18- and CD11b/CD18-dependent interactions with intercellular adhesion molecule-1, and oxygen-derived free radicals produced by neutrophils are implicated in the production of aspirin-induced gastric mucosal injury.

Background Genome-wide maps of linkage disequilibrium (LD) and haplotypes have been created for different populations. Substantial sharing of the boundaries and haplotypes among populations was observed, but haplotype variations have also been reported across populations. Conflicting observations on the extent and distribution of haplotypes require careful examination. The mechanisms that shape haplotypes have not been fully explored, although the effect of sample size has been implicated. We present a close examination of the effect of sample size on haplotype blocks using an original computational simulation. Results A region spanning 19.31 Mb on chromosome 20q was genotyped for 1,147 SNPs in 725 Japanese subjects. One region of 445 kb exhibiting a single strong LD value (average |D'|; 0.94) was selected for the analysis of sample size effect on haplotype structure. Three different block definitions (recombination-based, LD-based, and diversity-based) were exploited to create simulations for block identification with θ value from real genotyping data. As a result, it was quite difficult to estimate a haplotype block for data with less than 200 samples. Attainment of a reliable haplotype structure with 50 samples was not possible, although the simulation was repeated 10,000 times. Conclusion These analyses underscored the difficulties of estimating haplotype blocks. To acquire a reliable result, it would be necessary to increase sample size more than 725 and to repeat the simulation 3,000 times. Even in one genomic region showing a high LD value, the haplotype block might be fragile. We emphasize the importance of applying careful confidence measures when using the estimated haplotype structure in biomedical research.

Effects of treatment with free radical scavengers in the healing process of acetic acid-induced gastric ulcer on the ulcer aggravation induced by indomethacin were investigated. Gastric ulcers were produced on the anterior wall of the stomach of male Sprague-Dawley rats by submucosal injection of 20% acetic acid. To investigate the role of oxygen radicals, rats with gastric ulcer were treated with scavengers for six weeks and then treated with indomethacin (1 mg/kg/day). While superoxide dismutase (10,000 units/kg/day) did not affect the ulcer area after indomethacin treatment, allopurinol (50 mg/kg/day) slightly inhibited the increase in ulcer area. Dimethyl sulfoxide (1% solution, ad libitum) produced a significant decrease in size of the ulcer after indomethacin treatment. Increased lipid peroxides in the gastric mucosa after indomethacin treatment decreased significantly in the rats of the dimethyl sulfoxide and allopurinol groups. These results indicate that lipid peroxidation mediated by oxygen radicals plays an important role in the mechanism of ulcer aggravation induced by indomethacin.