mRNA expression of inducible nitric oxide synthase, endothelial nitric oxide synthase and vascular endothelial growth factor in esophageal mucosa biopsy specimens from patients with reflux esophagitis.

BACKGROUND/AIMS: Nitric oxide (NO) production is elevated in the intestine and may contribute to intestinal injury during inflammation. However, how the expression of inducible NO synthase (iNOS) mRNA and endothelial NO synthase (eNOS) mRNA in the esophageal mucosa contribute to mucosal damage caused by reflux esophagitis remains unknown. Since vascular endothelial growth factor (VEGF) exerts its action on microcirculation, contributing to angiogenesis and inflammation, we examined the role of VEGF together with iNOS and eNOS on development of reflux esophagitis. METHODOLOGY: The mRNA expression levels of iNOS, eNOS and VEGF were measured in biopsy specimens from 25 patients with reflux esophagitis, using TaqMan PCR and reverse transcription PCR. RESULTS: The expression of iNOS mRNA in the esophageal mucosa increased parallel to the severity of the esophagitis. There were no significant differences between both eNOS and VEGF mRNA expression levels and the severity of the esophagitis. The existence of gastric mucosal atrophy, hiatus hernia, therapy and Helicobacter pylori infection did not affect the levels of mRNA expression. CONCLUSIONS: The accumulation of NO, produced by iNOS, was considered to be related to the exacerbation of reflux esophagitis. Therapeutic intervention that reduces NO production may thus be of use in preventing development of esophageal mucosal injury in patients with reflux esophagitis.

Pleural effusions following endoscopic injection sclerotherapy for cirrhotic patients with esophageal varices.

BACKGROUND/AIMS: Endoscopic injection sclerotherapy is in widespread use for patients with esophageal varices. It is well known that pleural effusions are among complications following endoscopic sclerotherapy. However, there are few studies regarding the proportion of patients developing pleural effusions after sclerotherapy. METHODOLOGY: Between August 1991 and September 1998, 575 endoscopic injection sclerotherapies were carried out in 128 patients. Chest radiographs were obtained prior to and 24 hours after all procedures. We also obtained other clinical data from all patients. RESULTS: In total, 17.7% of post-sclerotherapy patients were diagnosed as having small amounts of pleural effusions. Logistic regression revealed pleural effusions after sclerotherapy to be associated with ascites, chest pain for 24 hours, total volume of sclerosant and submucosal injection of more than 4mL of sclerosant. In parallel with injection of an increasing amount of submucosal sclerosant, the proportion of patients with pleural effusion increased. CONCLUSIONS: Pleural effusions were related to ascites, chest pain for 24 hours, total sclerosant volume and submucosal injection of sclerosant.

Coexpression of glucose transporter 1 and matrix metalloproteinase-2 in human cancers.

BACKGROUND: Cancer cells express higher levels of glucose transporter proteins (Gluts) than do normal cells. Glut-1 overexpression is associated with invasiveness. Because matrix metalloproteinase-2 (MMP-2) is also overexpressed in cancer cells and is associated with invasiveness, we tested the hypothesis that Glut-1 may regulate MMP-2 expression. METHODS: We transiently transfected Glut-1 complementary DNA (cDNA) or Glut-1 antisense oligonucleotides in the human rhabdomyosarcoma cell line RD and analyzed MMP-2 mRNA expression and cell invasiveness. Empty vector and sense oligonucleotides were used for controls. We analyzed MMP-2 promoter activity in transfectants with a luciferase reporter assay and with p53 and Ets-1 gel mobility shift assays. Eight human cancer cell lines and 80 human cancer specimens were analyzed for coexpression of Glut-1 and MMP-2 by western blot and immunohistochemical analyses, respectively. RESULTS: Overexpression of Glut-1 in RD cells increased MMP-2 expression 4.3-fold (95% confidence interval [CI] = 3.7-fold to 4.9-fold) and invasiveness 3.2-fold (95% CI = 2.6-fold to 3.8-fold) relative to control transfected cells. Conversely, suppression of Glut-1 expression by antisense oligonucleotides decreased MMP-2 expression by 71.5% (95% CI = 71.1% to 71.9%) and invasiveness by 53.0% (95% CI = 47.5% to 58.5%). Glut-1-mediated MMP-2 expression involved the binding of the transcription factor p53 but not Ets-1. All eight human cancer cell lines coexpressed Glut-1 and MMP-2 by western blotting, and 45 of 80 human tumor tissues coexpressed Glut-1 and MMP-2 by immunohistochemistry. CONCLUSIONS: MMP-2 expression and cell invasiveness are tightly associated with Glut-1 expression in human cancer cell lines. Because suppression of Glut-1 decreased MMP-2 expression and cancer cell invasion, Glut-1 could be a target for therapy of various cancers that overexpress Glut-1.

Dysadherin overexpression in pancreatic ductal adenocarcinoma reflects tumor aggressiveness: relationship to e-cadherin expression.

PURPOSE: The E-cadherin-mediated cell adhesion system is frequently inactivated by multiple mechanisms and is involved in tumor progression in many types of cancer. Recently, we reported the cloning and characterization of dysadherin and showed that it downregulated E-cadherin and promoted metastasis. The aim of this study was to investigate the clinical significance of dysadherin expression and the relationship between dysadherin expression and E-cadherin expression in pancreatic ductal adenocarcinoma. PATIENTS AND METHODS: We examined dysadherin and E-cadherin expression in 125 surgically resected pancreatic ductal adenocarcinoma patients using immunohistochemistry. RESULTS: Dysadherin was expressed at the cell membrane of cancer cells, but not in nontumor duct and acinar cells. Its expression was stronger in infiltrative and poorly differentiated nests compared with well-differentiated nests. Although the correlation between the expression of dysadherin and E-cadherin was not significant, a group of patients showed reduced E-cadherin expression with dysadherin overexpression. Increased dysadherin expression was significantly correlated with distant metastasis (P =.047), high tumor grade (P =.006), positive tumor margins (P =.024), and infiltrative type of growth pattern (P =.014). A survival advantage was observed in patients with 0% to 20% dysadherin-positive cells compared with patients with 51% to 100% dysadherin-positive cells, independent of tumor-node-metastasis classification, and World Health Organization tumor grade (P =.019). A combination of increased dysadherin expression and reduced E-cadherin expression (< 90%) further worsened the prognosis. CONCLUSION: In pancreatic ductal adenocarcinoma, dysadherin expression seems to reflect tumor aggressiveness and to be a positive marker of poor prognosis when considered both alone and in combination with downregulation of E-cadherin.

Phosphatidylinositol 3-kinase suppresses glucose-stimulated insulin secretion by affecting post-cytosolic [Ca(2+)] elevation signals.

The role of phosphatidylinositol (PI) 3-kinase in the regulation of pancreatic beta-cell function was investigated. PI 3-kinase activity in p85 alpha regulatory subunit-deficient (p85 alpha(-/-)) islets was decreased to approximately 20% of that in wild-type controls. Insulin content and mass of rough endoplasmic reticula were decreased in beta-cells from p85 alpha(-/-) mice with increased insulin sensitivity. However, p85 alpha(-/-) beta-cells exhibited a marked increase in the insulin secretory response to higher concentrations of glucose. When PI 3-kinase in wild-type islets was suppressed by wortmannin or LY294002, the secretion was also substantially potentiated. Wortmannin's potentiating effect was not due to augmentation in glucose metabolism or cytosolic [Ca(2+)] elevation. Results of p85 alpha(-/-) islets and wortmannin-treated wild-type islets stimulated with diazoxide and KCl showed that inhibition of PI 3-kinase activity exerted its effect on secretion, at least in part, distal to a cytosolic [Ca(2+)] elevation. These results suggest that PI 3-kinase activity normally plays a crucial role in the suppression of glucose-stimulated insulin secretion.

Clinicopathological significance of galectin-3 expression in ductal adenocarcinoma of the pancreas.

PURPOSE: Galectin-3, a member of the beta-galactoside-binding lectin family, has multiple biological functions including cell-cell interactions and cell-extracellular matrix adhesion, cellular proliferation, cellular differentiation, and apoptosis. The aim of this study was to determine the relationship of galectin-3 expression to clinicopathological findings and patient prognosis in ductal adenocarcinoma of the pancreas. EXPERIMENTAL DESIGN: We examined galectin-3 expression in 104 surgically resected pancreatic ductal adenocarcinoma cases with stages I through IV using immunohistochemistry and investigated the relationship of it to overall survival. RESULTS: Patients were divided into two groups: a low expression group, where <60% of tumor cells were positive; and a high expression group, where > or =60% of tumor cells were positive. Cases in the low expression group had a significant tendency to be at later stages, to have distant metastasis, and to have less differentiated tumors, compared with cases in the high expression group (P = 0.001 for stage, P = 0.001 for metastasis, and P = 0.006 for differentiation). Postoperative overall survival was worse in the low galectin-3 expression group than in the high galectin-3 expression group (P = 0.004). Multivariate analysis showed that the risk ratio of prognosis was 2.06 among patients in the low galectin-3 expression group compared with the high galectin-3 expression group (P = 0.006). CONCLUSIONS: Decreased expression of galectin-3 was associated with advanced stage, tumor de-differentiation, and metastasis in ductal adenocarcinoma of the pancreas. Galectin-3 expression might be a useful prognostic marker for survival in ductal adenocarcinoma of the pancreas.

Efficacy of a triple therapy with rabeprazole, amoxicillin, and faropenem as second-line treatment after failure of initial Helicobacter pylori eradication therapy.

BACKGROUND/AIMS: Triple therapy consisting of lansoprazole, amoxicillin, and clarithromycin (LAC regimen) is widely used to eradicate Helicobacter pylori in Japan. However, the need for appropriate treatment after failure of initial therapy to eradicate H. pylori has been increasing. We therefore assessed the efficacy of a combination of rabeprazole, amoxicillin, and faropenem for second-line eradication therapy. METHODOLOGY: The subjects were 116 patients positive for H. pylori infection. Patients initially received lansoprazole 60 mg/day, amoxicillin 1500 mg/day and clarithromycin 400 mg/day in two divided doses for 7 days. Patients in whom eradication treatment failed were given rabeprazole 20 mg/day and amoxicillin 1500 mg/day in two divided doses, and faropenem 600 mg/day in three divided doses (RAF regimen) for 7 consecutive days. H. pylori status was assessed by the 13C-urea breath test combined with rapid urease test or H. pylori culture method 8 weeks after completion of therapy. Susceptibility to clarithromycin was determined by the agar dilution method, and genetic polymorphism of CYP2C19 was analyzed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The initial H. pylori eradication rate with the LAC regimen was 76.4% (84/110). Assessment of the CYP2C19 genotypes of the patients in whom eradication therapy failed revealed that homozygous extensive metabolizers accounted for 70.0% (16/23) and heterozygous extensive metabolizers for 30.0% (7/23), with no poor metabolizers. The acquired resistance rate for clarithromycin was 52.0% (12/23). The success rate of re-eradication with the RAF regimen was 91.3% (21/23) with no serious adverse effects. CONCLUSIONS: Triple therapy comprising rabeprazole, amoxicillin, and faropenem is effective for second-line eradication treatment of H. pylori infection, regardless of the genetic polymorphism of CYP2C19 or the presence of resistance to clarithromycin.

Insulin-induced cell cycle progression is impaired in chinese hamster ovary cells overexpressing insulin receptor substrate-3.

To analyze the roles of insulin receptor substrate (IRS) proteins in insulin-stimulated cell cycle progression, we examined the functions of rat IRS-1 and IRS-3 in Chinese hamster ovary cells overexpressing the human insulin receptor. In this type of cell overexpressing IRS-1 or IRS-3, we showed that: 1) overexpression of IRS-3, but not IRS-1, suppressed the G1/S transition induced by insulin; 2) IRS-3 was more preferentially localized to the nucleus than IRS-1; 3) phosphorylation of glycogen synthase kinase 3 and MAPK/ERK was unaffected by IRS-3 overexpression, whereas that of protein kinase B was enhanced by either IRS; 4) overexpressed IRS-3 suppressed cyclin D1 expression in response to insulin; 5) among the signaling molecules regulating cyclin D1 expression, activation of the small G protein Ral was unchanged, whereas insulin-induced gene expression of c-myc, a critical component for growth control and cell cycle progression, was suppressed by overexpressed IRS-3; and 6) insulin-induced expression of p21, a cyclin-dependent kinase inhibitor, was decreased by overexpressed IRS-3. These findings imply that: 1) IRS-3 may play a unique role in mitogenesis by inhibiting insulin-stimulated cell cycle progression via a decrease in cyclin D1 and p21 expressions as well as suppression of c-myc mRNA induction in a manner independent of the activation of MAPK, protein kinase B, glycogen synthase kinase 3 and Ral; and 2) the interaction of IRS-3 with nuclear proteins may be involved in this process.

A novel therapy for acute hepatitis utilizing dehydroepiandrosterone in the murine model of hepatitis.

Dehydroepiandrosterone (DHEA), one of the major androgens secreted by the adrenal cortex, has been shown to have potential immunoreguratory properties. In this study, we examined the effect of DHEA in a mouse model of hepatitis. Mice were treated with DHEA and injected with concanavalin A (Con A) or lipopolysaccharide (LPS)/D-galactosamine (GalN). Cytokine expression was measured by quantitative RT-PCR and ELISA. Apoptosis was detected by the TUNEL method and by DNA fragmentation analysis. In the DHEA-treated mice, the serum levels of ALT and expression of inflammatory mediators were significantly decreased. The number of apoptotic cells was also much lower than that observed in control, untreated mouse liver tissue. There were fewer tumor necrosis factor-alpha (TNF-alpha)-induced apoptotic cells in H4IIE hepatoma cells treated with DHEA than in non-treated cells. DHEA decreased the expression levels of mRNA transcripts encoding TNF-alpha and iNOS. These results suggest that DHEA can reduce T-cell-mediated injury in the liver as manifest by inhibition of the expression of several inflammatory mediators and hepatocyte apoptosis. DHEA should, thus, be considered as a novel candidate for the therapy of liver injury.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) inhibit the apoptosis in human peripheral blood lymphocytes.

BACKGROUND: Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) are the major steroid hormones secreted by the adrenal gland. Administration of DHEA has been reported to have beneficial effects on aging, diabetes, and atherosclerosis. Apoptosis is a normal physiologic process that occurs during embryonic development as well as in the maintenance of tissue homeostasis. In this study, we examined the suppressive effect of DHEA(S) on staurosporine-induced apoptosis in human peripheral blood lymphocytes (PBL). METHODS: Apoptosis was induced in human PBL with staurosporine and measured by flow cytometry utilizing Annexin V and propidium iodide (PI) staining. The quantity of FITC+/PI- cells corresponded to early apoptosis, while that of FITC+/PI+ cells corresponded to late apoptosis or secondary necrosis. RESULTS: The fraction of staurosporine-induced early apoptosis but not that of secondary necrosis in PBL was reduced by the treatment with either DHEA or DHEAS. Furthermore, this apoptosis was neither associated with androgen receptor (AR) nor with estrogen receptor (ER). CONCLUSIONS: This is the first study showing that DHEA(S) inhibits apoptosis in human PBL through a mechanism independent of either ARs or ERs. DHEA(S) may be a promising chemopreventive drug for aging, diabetes, and atherosclerosis.

Prevention of diabetes, hepatic injury, and colon cancer with dehydroepiandrosterone.

The levels of dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) peak in human in their twenties, then decrease gradually with age. The physiological importance of DHEA was not clear until recent research reports showing that DHEA has beneficial effects on preventing diabetes, malignancy, inflammation, osteoporosis, and collagen disease. We summarize our results concerning diabetes, hepatitis, and colon cancer. In 1982, Coleman et al. [Diabetes 31 (1982) 830] reported that DHEA decreased hyperglycemia in diabetic db/db mice, which become insulin resistant. We measured hepatic gluconeogenic enzymes in an attempt to elucidate the mechanical mechanism of DHEA action. The activity and gene expression of hepatic gluconeogenic enzyme such as glucose-6-phosphatase (G6Pase) was increased in db/db mice despite hyperinsulinemia compared to control db/+m mice. DHEA, like troglitazone, decreased these levels in db/db mice. We also showed that DHEA improved the insulin resistance caused by aging or obesity using the glucose clamp technique in another animal model. In humans, the serum DHEA concentration was shown to be associated with hyperinsulinemia in diabetes. It also became clear that DHEA increased insulin secretion in old-aged db/db mice. DHEA increases not only insulin sensitivity due to the effects in the liver and muscle, but also insulin secretion. As an effect of DHEA on T-cell mediated hepatitis induced by concanavalin A (ConA), DHEA reduced hepatic injury by inhibiting several inflammatory mediators and apoptosis. As an effect of DHEA on carcinogenesis, DHEA would be a potential chemopreventative agent against colon cancer because it decreases the number of azoxymethane (AOM) induced aberrant crypt foci, which is a possible precursor to adenoma and cancer in a murine model.Thus, since DHEA has many beneficial effects experimentally, we should consider administration of DHEA in the future, and common mechanisms among these actions of DHEA should be elucidated in further studies.

Regulation of feeding behavior, gastric emptying, and sympathetic nerve activity to interscapular brown adipose tissue by galanin and enterostatin: the involvement of vagal-central nervous system interactions.

Galanin and enterostatin, which are distributed in both the central nervous system and the gastrointestinal tract, regulate the feeding behavior. In the first set of experiments, we investigated the effects of galanin and enterostatin, injected into the third ventricle, on food intake, gastric emptying, and the sympathetic activity of nerves innervating interscapular brown adipose tissue in rats. Galanin dose-dependently increased the intake of a high-fat diet after overnight starvation, but it did not affect low-fat diet intake. In contrast, enterostatin suppressed the intake of the high-fat diet, while intake of the low-fat diet was not affected. Galanin significantly and dose-dependently suppressed gastric emptying rate. However, gastric emptying showed no response to enterostatin. Galanin produced a dose-dependent suppression of sympathetic firing rate. In rats fed a high-fat diet, the injection of enterostatin showed a dose-dependent increase in firing rate. In contrast, animals fed a chow diet showed almost no response. In the second set of experiments, we investigated the role of the hepatic vagus nerve in modulating the peripheral response to enterostatin in rats. Intraperitoneal (i.p.) enterostatin reduced the intake of a high-fat diet. Immunohistochemical identification indicated that the Fos protein was present in the nucleus tractus solitarius, and parabrachial, paraventricular, and supraoptic nuclei after IP enterostatin. These responses to i.p. enterostatin were blocked by hepatic vagotomy. These results suggest that galanin and enterostatin coordinate to regulate feeding behavior, gastric emptying, and sympathetic activity to interscapular brown adipose tissue via central and peripheral sites of action, one of which was the interaction which was found to exist through the vagal system.

The comparative effects of single intravenous doses of omeprazole and famotidine on intragastric pH.

BACKGROUND: The ideal medication for the treatment of acid-related diseases, for example, hemorrhagic ulcers and stress-related gastric bleeding, should have a rapid onset of action to promote hemostasis and alleviate symptoms. The aim of our study was to investigate the inhibitory effects on gastric acid secretion after single intravenous administrations of omeprazole 20mg and famotidine 20 mg. METHODS: Ten healthy Helicobacter pylori-negative male subjects participated in this randomized, double-masked, two-way crossover study. Intragastric pH was monitored continuously for 4 h after a single intravenous administration of omeprazole 20 mg and after a single intravenous administration of famotidine 20 mg. The administration of the two agents was separated by a 7-day washout period. RESULTS: In all ten subjects, the length of time that intragastric pH remained over 3, during the 0- to 3- and 0- to 4-h study periods, was greater after famotidine treatment than after treatment with omeprazole, and famotidine increased the average pH during the 0 to 3- and 0 to 4-h study periods significantly more than omeprazole did. During the 4-h study period, famotidine provided a longer duration of pH of more than 2, 3, 3.5, 4, 5, 6, and 7, compared to omeprazole. CONCLUSIONS: In Helicobacter pylori-negative healthy male subjects, an intravenous dose of 20 mg famotidine increased intragastric pH more rapidly than intravenous omeprazole 20 mg.

Screening and follow-up of diabetic retinopathy using a new mosaic 9-field fundus photography system.

AIM: To evaluate the clinical usefulness of a newly developed fundus photographing system and assess its applicability to telemedicine. METHODS: Nine overlapping 45 degrees fundus photographs were taken by a new camera equipped with nine internal fixation targets to provide standardized 9-field photographs. The digitally stored images were either edited in 3x3 form or reconstructed as collage (9F) and compared to the ophthalmological examination (OP) and single-field non-mydriatic photography (SC). In telemedicine, 9-field images derived from 61 adolescent diabetics were sent to ophthalmologists over an analog phone line. RESULTS: The sensitivities of the examinations by 9F without and with mydriasis (78 and 82%) were equivalent to OP (84%) and superior to SC (64%). The diagnosis of severity by 9F was also comparable to those by OP, whereas SC tended to underestimate the severity. An average of 1 min 19 s was required to send one edited 9-field photography (average size 259+/-30 KB) over the Internet. Twelve of these eyes were diagnosed as diabetic retinopathy on a desktop monitor whereas SC gave only seven. CONCLUSION: This new 9-field fundus photography system can be appropriate for the screening and follow-up of diabetic retinopathy in adult and adolescent diabetic subjects, especially when applied to telemedicine over the Internet.

Chemoprevention of precursors to colon cancer by dehydroepiandrosterone (DHEA).

Although dehydroepiandrosterone (DHEA) is recognized as one of the major adrenal androgens, its precise physiological role in the human endocrine system remains to be elucidated. In particular, the effect of DHEA on carcinogenesis has not been fully characterized. We undertook this study to determine whether DHEA has a chemopreventative effect on the precursors of colon cancer in a murine model of azoxymethane (AOM)-induced aberrant crypt foci (ACF). The number of ACF was significantly decreased in mice treated with 0.4% (p < 0.001) and 0.8% DHEA (p < 0.001), but there were no significant differences between DHEA-treated and control mice in terms of the ACF size, 3-catenin expression or level of dysplasia. This is the first study of colon cancer carcinogenesis demonstrating that DHEA treatment can decrease the number of ACF without apparently modifying their malignant potential. These data strongly suggest that DHEA might be a potential chemopreventative agent against human colon cancer.

Dehydroepiandrosterone decreases elevated hepatic glucose production in C57BL/KsJ-db/db mice.

Dehydroepiandrosterone (DHEA) is known to improve hyperglycemia in diabetic db/db mice that are obese and insulin resistant. In a previous study, we reported that DHEA suppresses the elevated hepatic gluconeogenic glucose-6-phosphatase (G6Pase) activity and gene expression in C57BL/KsJ-db/db mice. In the present study, we evaluated the total amount of gluconeogenesis using NaH[(14)C]CO(3) and hepatic glucose production using fructose as a substrate in primary cultured hepatocytes. Despite hyperinsulinemia, the glucose production of db/db mice in the total body and hepatocytes was elevated as compared to their heterozygote littermate C57BL/KsJ-db/+m mice. Administration of DHEA significantly decreased the blood glucose level and increased the plasma insulin level in db/db mice. Administration of DHEA decreased the elevated total body and hepatic glucose production in db/db mice. In addition, the glucose production in the primary cultured hepatocytes of db/db mice was decreased significantly by the direct addition of DHEA or DHEA-S to the medium. These results suggest that administration of DHEA suppresses the elevated total body and hepatic glucose production in db/db mice, and this effect on the liver is considered to result from increased plasma insulin and DHEA or DHEA-S itself.

Lactoferrin inhibits hepatitis B virus infection in cultured human hepatocytes.

We recently reported that lactoferrin (LF), a milk protein belonging to the iron transporter family, inhibits hepatitis C virus (HCV) infection in cultured human hepatocytes (PH5CH8) and that the interaction of LF with HCV is responsible for this inhibitory effect. As PH5CH8 cells were found to be a human hepatocyte line susceptible to hepatitis B virus (HBV) infection, we therefore examined if LF could effectively prevent HBV infection in PH5CH8 cells. Preincubation of the cell with bovine LF (bLF) or human LF (hLF) was required to prevent HBV infection of cells, and preincubation of HBV with bLF or hLF had no inhibitory effect on HBV infection. We further found that bovine transferrin, casein, and lactoalbumin had no anti-HBV activity. Our findings suggest that the interaction of LF with cells was important for its inhibitory effect, and that LF may well be among the candidates for an anti-HBV reagent that could prove effective in the treatment of patients with chronic hepatitis.