Toru Maekawa

Regenerating gene protein may mediate gastric mucosal proliferation induced by hypergastrinemia in rats.

BACKGROUND & AIMS Regenerating gene (Reg) has been isolated from rat regenerating pancreatic islets, and Reg protein is mitogenic to islet cells. We have recently shown that Reg gene and Reg protein are expressed in gastric enterochromaffin-like (ECL) cells. This study aimed to clarify whether gastrin enhances Reg protein production in ECL cells and whether Reg protein is mitogenic to gastric mucosal cells. METHODS Reg gene expression in response to acute and chronic hypergastrinemia was investigated in rats. Immunohistochemical studies, Northern blotting, and in situ hybridization were performed to investigate the expression of Reg protein and Reg gene. The direct effect of gastrin on Reg gene expression was investigated using isolated ECL cells, and the trophic effect of Reg protein on cultured gastric epithelial cells was assessed by [3H]thymidine uptake. RESULTS Both chronic hypergastrinemia and short-term gastrin administration stimulated Reg gene expression and Reg protein production in fundic mucosa. Reg gene expression was also augmented in isolated ECL cells after incubation with rat gastrin. Reg protein was mitogenic to cultured rat gastric epithelial cells. CONCLUSIONS Gastrin stimulates the production of Reg protein in gastric ECL cells, which may be involved in the gastrin-induced gastric mucosal cell growth.

Presence of Prostaglandin EP4 Receptor Gene Expression in a Rat Gastric Mucosal Cell Line

RGM-1 is an epithelial cell line established from gastric mucosa of adult Wistar rats. In this study, we characterized this newly established cell line by Northern blot analysis. We also investigated deoxyribonucleic acid (DNA) synthesis and hexosamine production in RGM-1 by PGE2. Northern blot analysis did not detect any transcript of proton pump, gastrin receptor, histidine decarboxylase, somatostatin and pepsinogen 1, indicating the absence of characteristics of parietal, ECL, D and chief cells in RGM-1 cells. However, this periodic acid-Schiff (PAS)-positive cell line expressed prostaglandin EP4 receptor mRNA but not EP1 and EP3 receptor mRNAs. [3H]-thymidine incorporation into DNA of the cells was not increased by PGE2. In contrast, PGE2 increased hexosamine content in RGM-1 cells. These results suggest that RGM-1 may be a useful model of gastric mucosal cells and that PGE2 plays a role on mucin synthesis in RGM-1 cells possibly via EP4 receptors.

Relationship between severity and symptoms of reflux oesophagitis in elderly patients in Japan

Since information concerning reflux oesophagitis in the elderly is limited, particularly in Japan, the severity and symptomatic profiles of reflux oesophagitis in elderly patients were investigated. One hundred and nineteen patients with reflux oesophagitis found among 2278 endoscopy cases between 1993 and 1996 were investigated in this study. The patients were divided into two groups, elderly and non‐elderly. The severity of reflux oesophagitis was estimated by the Los Angeles classification. The presence or absence of typical symptoms (heartburn and regurgitation) was determined by interview. Reflux oesophagitis was not only more frequently found in the elderly group, but was more severe than in the non‐elderly. Although the degree of manifestation of typical symptoms was similar between the elderly and the non‐elderly with high‐grade oesophagitis, the elderly patients with mild reflux oesophagitis were less symptomatic than the non‐elderly. Mild reflux oesophagitis in the elderly may be missed due to its rarity of typical reflux symptoms and a substantial number of elderly persons might have subclinical reflux oesophagitis.

Expression of Protooncogene c-kit and Its Ligand Stem Cell Factor (SCF) in Gastric Carcinoma Cell Lines

We examined 13 human gastric carcinoma celllines for the expression of both c-kit and stem cellfactor (SCF). Expression of mRNAs was detected by bothNorthern blot analysis and reversetranscriptase-polymerase chain reaction (RT-PCR), and expression oftranslated proteins was detected by western blotting.Using RT-PCR we confirmed the expression of c-kit infive (ECC12, TMK1, MKN7, GCIY, and HGC27) cell lines. Northern blot analysis showed coexpression ofboth c-kit and SCF in ECC12 and expression of SCF infive other (MKN74, MKN1 OKAJIMA, KATOIII, and TMK1) celllines. SCF stimulated both tyrosine phosphorylation of c-kit and growth of ECC12, whereas it didnot stimulate those of GCIY. The sizes of c-kittranscript and protein in GCIY were slightly smallerthan those of the reported ones, suggesting the presence of a biologically inactive truncated form ofc-kit in GCIY. The present study suggests that c-kit/SCFsystem might play an important role in thecarcinogenesis and tumor growth of ECC12 and that thetruncated form of c-kit in GCIY might not be associatedwith malignant transformation.

Rat gastric mucosal cells express ICAM-1 and proinflammatory cytokines during indomethacin-induced mucosal injury

Adhesion molecules and cytokines are known to be involved in the formation of acute gastric mucosal injury. However, it is not clear whether the gastric mucosal cells express these molecules and modulate the inflammation. To clarify whether gastric mucosal cells express intercellular adhesion molecule-1 (ICAM-1) and proinflammatory cytokines (tumor necrosis factor-alpha (TNF-alpha), interleukin-1-alpha (IL-1-alpha), and cytokine-induced neutrophil chemoattractant-2-beta (CINC-2-beta)) in the formation of gastric mucosal injury, we have used rat indomethacin-induced gastric mucosal lesions as an in vivo model. The gene expression of all cytokines and ICAM-1 increases at the early stages of indomethacin-induced gastritis (TNF-alpha and IL-1-alpha gene expression began to increase earlier than that of ICAM-1 and CINC-2-beta) and can mainly be detected in the gastric epithelial layer. To further identify the source of those molecules, the epithelial cells were separated into seven fractions according to their sizes by a counterflow elution. ICAM-1 and CINC-2-beta gene expressions are particularly enhanced in the middle-sized cell fractions that are rich in gastric mucous-producing cells. The effect of TNF-alpha or IL-1-alpha on the gene expression of ICAM-1 and cytokines was examined by using RGM-1 cells as a model for gastric mucosal cells. RGM-1 cells show an augmented ICAM-1 and proinflammatory cytokine expression in response to TNF-alpha or IL-1-alpha stimulation. Moreover, immunohistochemical staining also reveals an increase in ICAM-1 and CINC protein production in RGM-1 cells in response to TNF-alpha stimulation. We conclude that gastric mucosal cells express various cytokines and an adhesion molecule during the formation of acute gastric mucosal injury and that they may modulate the inflammation.

Growth inhibitory effects of somatostatin on human leukemia cell lines mediated by somatostatin receptor subtype 1

Reverse transcription polymerase chain reaction analysis revealed that only somatostatin receptor (SSTR) 1 mRNA was expressed in Ball-1 B-, Jurkat T-, and HL60 leukemia cell lines. In contrast, human normal mononuclear cells expressed the mRNA of all five subtypes of SSTR, although the expression level of SSTR1 was the highest. A binding study, revealed that [125I]-somatostatin bound specifically to HL60 cells and this binding was inhibited concentration-dependently by unlabeled somatostatin (SS). A [3H]thymidine incorporation study showed that SS significantly and concentration-dependently inhibited HL60 and BALL-1 leukemia cell growth. Furthermore, this inhibition of leukemia cell growth was associated with reduces c-fos gene expression. These data indicate that leukemia cells express SSTR1 and SS reduce c-fos gene expression with resultant suppression of leukemia cell growth, possibly mediated by the SSTRI.

Helicobacter pylori induces proinflammatory cytokines and major histocompatibility complex class II antigen in mouse gastric epithelial cells

Although Helicobacter pylori has been reported to stimulate the release of various cytokines from gastric tissue, it remains unknown whether normal and nontumorous gastric epithelial cells produce these cytokines. Therefore, in this study, we used a normal mouse gastric surface mucous cell line (GSM06) to determine whether gastric epithelial cells produce proinflammatory cytokines in response to H. pylori. The expression of MHC class II antigen was also examined, to investigate whether gastric epithelial cells participate in the immune response to H. pylori. In the study, GSM06 cells were incubated with H. pylori or its lipopolysaccharide (LPS). Proinflammatory cytokines were detected by Northern and Western blot analysis. The expression of MHC class II antigen was examined by fluorescence activated cell sorter (FACS) analysis. Genetic expression of proinflammatory cytokines such as interleukin-1alpha, tumor necrosis factor-alpha, and cytokine-induced neutrophil chemoattractant-2beta was enhanced by both intact and sonicated H. pylori, but not by H. pylori LPS. The expression of MHC class II antigen was induced by H. pylori more strongly than by interferon-gamma. We conclude that H. pylori induces the expression of proinflammatory cytokines and MHC class II antigen in gastric epithelial cells. Gastric epithelial cells may act as antigen-presenting cells and participate in the immune response to H. pylori infection.

Augmentation of Water-Immersion Stress- Induced Gastric Mucosal Lesions in BALB/c Mice Infected with Helicobacter felis

Background and Aims: Inoculation of Helicobacter felis into the murine stomach has been reported to induce chronic gastric inflammation and may be a model of Helicobacter pylori-induced chronic gastritis. In this study, to characterize H. felis-induced gastritis, the gastric production of interleukin-1β (IL-1β) and hepatocyte growth factor (HGF) was measured in mice. Methods: Gastric mucosal lesions were induced in H. felis-infected BALB/c mice by water-immersion stress. The severity score of gastric erosions per stomach was measured as the sum of the length of erosions. Gene expression of IL-1β and HGF were analyzed by Northern blot analysis and production of HGF was examined using the enzyme immunoassay method. Results: Water-immersion stress induced gastric mucosal lesions accompanied by increased expression of IL-1β mRNA. H. felis infection evoked enhanced expression of IL-1β and HGF genes. When H. felis-infected mice were stressed by water immersion, the mucosal lesions were more severe than those in non-infected mice. Moreover, IL-1β gene expression as well as HGF production was further increased. Conclusions: Although H. felis inoculation did not cause gastric mucosal erosions by itself, it augmented the stress-induced erosions.

Geranylgeranylacetone, an anti‐ulcer drug, stimulates hexosamine production in a rat gastric mucosal cell line through binding to a specific cytosolic protein

An anti‐ulcer drug, geranylgeranylacetone (GGA), stimulates hexosamine production in a rat gastric mucosal cell line (RGM‐1). The aim of this study was to elucidate the mechanism of this action. The role of protein kinase A, inositol phospholipid turnover and tyrosine kinase in the stimulatory action of GGA on hexosamine production in RGM‐1 was determined by observing cAMP production, [3H]‐inositol phosphate turnover and western blotting of tyrosine phosphorylation, respectively. Any trophic effect of GGA on RGM‐1 was also checked by [3H]‐thymidine incorporation. Our experiments showed that GGA has no effect on cAMP production, inositol phospholipid turnover, tyrosine phosphorylation or DNA synthesis in RGM‐1. Finally, a [14C]‐GGA competitive receptor binding assay was performed on RGM‐1 and we found that [14C]‐GGA specifically bound to RGM‐1 cytosolic protein. Although retinoic acid (RA), another polyisoprenoid compound significantly stimulated hexosamine production in RGM‐1, we confirmed that the [14C]‐GGA binding site in RGM‐1 is different from the RA binding site. In summary, GGA stimulates hexosamine production in RGM‐1 and this action is probably mediated through its binding to a specific cytosolic protein in RGM‐1.

Immunological and molecular analysis of B lymphocytes in low-grade MALT lymphoma of the stomach. Are there any useful markers for predicting outcome after Helicobacter pylori eradication?

Background:Background: Although Helicobacter pylori eradication is effective in treating low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma, the condition in some patients deteriorates even after the eradication. Therefore, it is important to predict the disease outcome before starting H. pylori eradication. We investigated the usefulness of flow cytometry, quantifying CD19- and CD20-positive B lymphocytes in MALT lymphoma tissue, for predicting the disease outcome after H. pylori eradication. Methods: Tissue specimens from 14 patients with H. pylori-positive low-grade gastric MALT lymphoma were examined by histology, Southern blotting, and flow cytometry before therapy. Serum levels of soluble interleukin (IL)-2 receptor were also measured. The relationship between the data and the prognosis after H. pylori eradication was analyzed. Results: Remission occurred in 10 of the 14 patients. The condition in the 4 remaining patients deteriorated even after H. pylori eradication. The percentages of CD19- and CD20-positive cells in MALT lymphoma tissue from the patients in remission were both significantly lower than those in the tissue from patients not in remission. Indeed, 4 of the 5 patients in whom both CD19- and CD20-positive cells accounted for more than 50% of the total number of lymphocytes had gastrectomy, whereas all patients in whom both CD19- and CD20-positive cells accounted for less than 50% of the total number of lymphocytes achieved remission. Although immunoglobulin gene rearrangement was present in all patients operated on, there were also 6 patients whose MALT lymphoma was ameliorated in spite of the presence of gene rearrangement. The serum level of soluble IL-2 receptor was in the normal range in all patients tested. Conclusions:Analysis of mature B-cell markers in MALT lymphoma tissue is more useful than the examination of immunoglobulin gene rearrangement or serum levels of soluble IL-2 receptor in predicting the outcome of low-grade gastric MALT lymphoma after H. pylori eradication.