瀬戸 孝一

Background: Several immunochromatographic serological test kits have been developed to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, but their relative performance and potential clinical utility is unclear. Methods: Three commercially available serological test kits were evaluated using 99 serum samples collected from 29 patients diagnosed with coronavirus disease 2019 (COVID-19) and 100 serum samples collected from 100 healthy volunteers in 2017 as negative controls. Results: The specificity of the IgM and IgG antibodies showed comparable results among the three immunochromatographic serological test kits. The specificity for IgM antibody was 98.0%, 98.0%, and 97.0%, and the specificity for IgG antibody was identical among the three kits (99.0%). The IgM antibody-positive rates of the three test kits for samples taken at the early stage of the disease (0-4 days after onset) were consistent with all three kits (18.2%); however, the IgM antibody-positive rates thereafter showed considerable differences among the kits, making it difficult to interpret the kinetics of IgM response against SARS-CoV-2. The IgG antibody-positive rates for samples taken after 13 days of onset were 100.0%, 97.6%, and 97.6%, respectively. Conclusion: There were large differences among the results of the three test kits. Only few cases showed positive results for IgM, suggesting that at least 2 of these kits used in this study were unsuitable for diagnosis of COVID-19. The IgG antibody was positive in almost all samples after 13 days of onset, suggesting that it may be useful for determining infections in the recent past.

Lymphatic metastasis is common in advanced-stage carcinoma and is associated with a poor prognosis. However, few effective treatments to inhibit it are available. Z-100 is an immunomodulatory extract of Mycobacterium tuberculosis strain Aoyama B that contains polysaccharides such as arabinomannan and mannan. Here, we investigated the inhibitory effect of Z-100 on spontaneous lymphatic metastasis. C57BL/6N mice injected subcutaneously with B16-BL6 melanoma cells in the right hind footpad were administered Z-100 subcutaneously in the right inguinal region on a daily basis. On day twenty-one after the injection, the right inguinal lymph nodes were excised, and the extent of metastasis, the number of immune cells, and the amount of granzyme B protein in the lymph nodes were examined. We also investigated the combined effect of Z-100 and irradiation in this model. Results showed that Z-100 reduced number of animals with metastasis, with respective metastasis rates of 85.7%, 42.9%, 7.1% and 0.0% in saline, 0.1 mg/kg Z-100, 1 mg/kg Z-100 and 10mg/kg Z-100 group. Further, mice that had been given Z-100 were found to have more immune cells and granzyme B protein in the lymph nodes than control mice. The combination of low dose Z-100 and irradiation also inhibited spontaneous lymph node metastases. These findings suggest that Z-100 may be beneficial in preventing lymphatic. metastasis by enhancing the immune response.

Aims: As activation and overexpression of the cholecystokinin-2 (CCK-2)/gastrin receptor can lead to carcinogenesis, it has been explored as a therapeutic target in pancreatic cancer. We demonstrated that Z-360, a CCK-2/gastrin receptor antagonist, combined with gemcitabine prolonged survival and reduced gemcitabine-induced vascular endothelial growth factor (VEGF) expression in a pancreatic carcinoma orthotopic xenograft mouse. In this study, we investigated the role of the CCK-2/gastrin signaling pathway on gemcitabine-induced VEGF expression in PANC-1 human pancreatic carcinoma cells. Main methods: In PANC-1 cells treated with Z-360, anti-gastrin IgG or kinase inhibitors, the gene expression levels were analyzed by quantitative real-time RT-PCR, and the protein levels of Akt and phosphorylated Akt (p-Akt) in cellular extracts were measured by ELISA. Key findings: Gemcitabine-induced expression of VEGF and hypoxia-inducible factor-1 alpha (HIF-1 alpha) were suppressed by the treatment with an anti-gastrin antibody. In addition, VEGF and HIF-1 alpha gene expression was inhibited by treatment with an inhibitor of phosphatidylinositol 3-kinase (PI3K), which is involved in the downstream signaling pathway of the CCK-2/gastrin receptor, and was also suppressed by treatment with Z-360. Moreover, although Akt phosphorylation was increased by treatment with gemcitabine, this elevation was partially, but significantly, inhibited by an exposure of Z-360. Significance: Gemcitabine might induce gene expression of VEGF via the PI3K/Akt signaling pathway in the downstream of the CCK-2/gastrin receptor. The suppression of the CCK-2/gastrin signaling pathway by treatment with Z-360 could be a useful approach for potentiating prolonged survival of pancreatic cancer patients receiving gemcitabine therapy. (C) 2011 Elsevier Inc. All rights reserved.

Z-360 is a novel cholecystokinin (CCK)-2/gastrin receptor antagonist that is being developed for the treatment of pancreatic adenocarcinoma in combination with gemcitabine. A previous study shows that the co-administration of Z-360 with gemcitabine significantly prolonged the survival of mice with orthotopically implanted human pancreatic adenocarcinoma cell lines. To clarify the therapeutic effects of Z-360 in combined with gemcitabine, we analyzed gene expression. When gemcitabine was administered, CCK-2/gastrin receptor expression was induced in an orthotropic xenograft model; the result indicating that Z-360 could act on gemcitabine-sensitive cells. Both in vitro and in vivo studies showed that gemcitabine increased the expression of vascular endothelial growth factor A (VEGFA), a prognostic factor for survival in pancreatic cancer, while Z-360 suppressed this induction of VEGFA gene expression. These results help to explain how Z-360 prolongs survival when used in combination with gemcitabine.

Z-360, a novel cholecystokinin(2) (CCK2) receptor antagonist, has been developed as a therapeutic drug for pancreatic cancer and showed pain relief action in phase Ib/IIa clinical trial. This study was attempted to elucidate the analgesic efficacy of Z-360 in mice. Oral administration of Z-360 (30-300 mg/kg) showed a dose-dependent inhibitory effect on the late phase of nociceptive responses to formalin. YF476, another CCK2 receptor antagonist, was without effects at 1 and 10 mg/kg. In contrast, the CCK1 receptor antagonist devazepide inhibited the nociceptive responses to formalin. In a mouse model of cancer pain, significant anti-allodynic effect of Z-360 was observed after single and repeated oral administration of 100 and 300 mg/kg doses. Anti-allodynic effect was also observed after repeated administration of devazepide. Combined single treatment with morphine and Z-360 caused an increase inhibition of pain-related responses in the pain models produced by formalin and cancer. Although Z-360 has lower affinity for CCK1 receptor than for CCK2 receptor, Z-360 exhibited an inhibitory effect on sulfated CCK-8-induced gallbladder emptying, a CCK1 receptor-mediated effect, at a dose of 100 mg/kg. These results suggest that Z-360 inhibits inflammatory and cancer pain probably through the blockade of CCK1 receptors. Z-360 is expected to become a useful drug for the pancreatic cancer with analgesic effects as well as the prolongation of survival.

Acotiamide hydrochloride (Z-338) is a member of new class prokinetic agents currently being developed for the treatment of functional dyspepsia (FD). DNA microarray analysis showed that acotiamide altered the expressions of stress-related genes such as gamma-aminobutyric acid (GABA) receptors, GABA transporters and neuromedin U (NmU) in the medulla oblongata or hypothalamus after administration of acotiamide. Therefore, effects of acotiamide on stress-related symptoms, delayed gastric emptying and feeding inhibition, in rats were examined. Acotiamide significantly improved both delayed gastric emptying and feeding inhibition in restraint stress-induced model, but did not affect both basal gastric emptying and feeding in intact rats, indicating that acotiamide exerted effects only on gastric emptying and feeding impaired by the stress. On the other hand, mosapride showed significant acceleration of gastric emptying in intact and restraint stress-induced model, and itopride showed no effect on restraint stress-induced delayed gastric emptying. In addition, gene expression of NmU increased by restraint stress was suppressed by administration of acotiamide, while acotiamide had no effect on delayed gastric emptying induced by an intracerebroventricular administration of NmU, suggesting that the suppressive effect of acotiamide on gene expression of NmU might be important to restore delayed gastric emptying or feeding inhibition induced by restraint stress. These findings suggest that acotiamide might play an important role in regulation of stress response. As stress is considered to be a major contributing factor in the development of FD, the observed effects may be relevant for symptom improvement in FD.

Expression of heart-type fatty acid binding protein is restricted mainly to the skeletal and cardiac muscles and further regulated by peroxisome proliferator-activated receptor alpha. The molecular basis for the muscle-restricted peroxisome proliferator-activated receptor a action on the fatty acid binding gene was analyzed using normal and the receptor-null mice and the cultured cells. Two possible peroxisome proliferator-response elements were found in the promoter region of the mouse gene. A gel shift assay showed that both elements were functional. However, neither the tandem repeats of the elements nor the cloned promoter sequence could be activated by peroxisome proliferator-activated receptor alpha, and its ligand in the reporter gene assay using cultured cells. The cloned promoter responded to the ligand only in the muscle when the reporter gene was introduced into the mouse muscle. Using a chimeric receptor with the activation domain of herpes virus VP16 protein and the tandem repeats of the elements with or without mutation, the upstream element was finally demonstrated to be potentially involved in the receptor-dependent transcriptional activation., These results suggest that the peroxisome proliferator-response element of the mouse gene is atypical and there is a muscle-specific mechanism to enhance the weak binding of the receptor to the response element to ensure the muscle-specific action of peroxisome proliferator-activated receptor a oil the heart-type fatty acid binding protein, gene promoter. (c) 2005 Elsevier Ltd. All rights reserved.

Background. Although several kinds of evidence suggest that glycosaminoglycans (GAGs) and proteoglycans (PGs) may contribute to the development of beta(2) -microglobulin-related (Abeta(2) m) amyloidosis, the precise roles of these molecules for the development of Abeta(2) m amyloidosis are poorly understood. Methods. We investigated the effects of GAGs and PGs on the depolymerization of Abeta(2) m amyloid fibrils at a neutral pH, as well as on the formation of the fibrils at an acidic pH in vitro, using fluorescence spectroscopy with thioflavin T and electron microscopy. Results. Depolymerization of Abeta(2) m amyloid fibrils at pH 7.5 at 37degreesC was inhibited dose-dependently by the presence of some GAGs (heparin, dermatan sulfate, or heparan sulfate) or PGs (biglycan, decorin, or keratan sulfate proteoglycan). Electron microscopy revealed that a significant amount of Abeta(2) m amyloid fibrils remained in the reaction mixture with some lateral aggregation. Second, when monomeric beta(2) m was incubated with aggrecan, biglycan, decorin, or heparin at pH 2.5 at 37degreesC for up to 21 days, the thioflavin T fluorescence increased depending on dose and time. Electron microscopy revealed the formation of rigid and straight fibrils similar to Abeta(2) m amyloid fibrils in beta(2) m incubated with biglycan for 21 days. Conclusion. These results suggest that some GAGs and PGs could enhance the deposition of Abeta(2) m amyloid fibrils in vivo, possibly by binding directly to the surface of the fibrils and stabilizing the conformation of beta(2) m in the fibrils, as well as by acting as a scaffold for the polymerization of beta(2) m into the fibrils.

We previously identified pyruvate dehydrogenase kinase 4 (PDK4) mRNA as a most rapidly induced mRNA by fibrates and suggested the possibility that the coupled induction of PDK4 and reduction of serum triglyceride and fatty acid levels can cause protein degradation in muscles. To investigate whether the drugs that are known to have a risk of rhabdomyolysis induce PDK4 mRNA in skeletal muscle, the effects of statins and new quinolon anti-bacterial drugs on the expression levels of the mRNA were examined using mice and cultured cells. Several statins and new quinolon anti-bacterial drugs solely induced PDK4 mRNA in the muscle as efficiently as fibrates and at least some combinations were synergistic. The present results suggest that induction of PDK4 mRNA is involved in the drug induced acute rhabdomyolysis when the muscle is restricted to use fatty acids as a major energy source.

Peroxisome proliferator ( PPAR) ligand Wy14,643 induces liver-fatty acid binding protein ( FABP) spontaneously and heart-FABP gradually, but not intestine-FABP mRNA expression in the mouse liver. These strict regulations have not been reproduced in cultured cell systems. W applied a DNA electroporation method to directly introduce reporter gene constructs into the livers of mice. This system reproduced the in vivo responses of the above three FABP gene promoters to the PPARalpha ligand but not that of a promoter containing the typical three PPAR binding sites in tandem. Deletion and mutation analyses of the mouse L-FABP gene suggested that, in addition to the binding site for PPARalpha, a far upstream sequence is required for PPAR-dependent transactivation in the liver. In contrast to the cultured cell systems, our in vivo DNA electroporation method showed that PPAR binding to the promoter is necessary but not sufficient for PPARalpha ligand-dependent transcriptional activation of the L-FABP gene in vivo.

Although gastric cancer formation with H. pylori in Mongolian gerbils was recently reported, the same inoculation procedure did not result in cancer formation in other animals such as mice. Disturbed regulation of apoptosis and cell proliferation are known to link the multistep process of carcinogenesis. The present study is designed to examine the level of gastric epithelial cell apoptosis in Mongolian gerbils colonized with the H. pylori (Sydney strain: SS1) in comparison with that in mice. Mice (C57BL/6) and Mongolian gerbils were orally inoculated with SS1 and the stomachs were examined 9 and 18 months later. MPO activity increased persistently in gerbils, but increased transiently in mice. While the levels of DNA fragmentation, caspase-3 activity, and the number of TUNEL-positive cells increased significantly in mice, such parameters were attenuated in gerbils. On the other hand, the number of PCNA-positive cells increased after SS1 inoculation only in Mongolian gerbils, suggesting the enhancement of cell turnover in H. pylori-colonized gerbils. In conclusion, the SS1-induced increase in gastric mucosal apoptosis observed in mice was attenuated significantly in Mongolian gerbils, suggesting the causative role for the higher incidence of gastric carcinogenesis in this animal.

Background: We previously demonstrated that Helicobacter pylori colonization evokes gastric mucosal inflammation and an extensive increase in lipid peroxides and glutathione in Mongolian gerbils. Zinc and its derivative, polaprezinc, have been reported to be potent antioxidants in gastric mucosa. Aim: To examine the effect of polaprezinc on gastric mucosal oxidative inflammation in H. pylori-colonized Mongolian gerbils. Methods: Sixty-eight male Mongolian gerbils were orally inoculated with H. pylori (ATCC43504, 5 x 10(8) CFUs/gerbil; H. pylori group) and 35 gerbils were inoculated with the culture media (control group). Twenty-two gerbils in the H. pylori and 13 gerbils in the control group were fed with diets containing polaprezinc (0.06%, 100 mg/kg, 10 times the usual clinical dose) (H. pylori + polaprezinc group, polaprezinc group). The remaining gerbils were fed a standard laboratory chow diet. Neutrophil infiltration, assessed histologically and by the activity of myeloperoxidase, the contents of CXC-chemokine (GR0/CINC-1-like protein) and the contents of thiobarbituric acid-reactive substances, was evaluated in each group 12 weeks after the inoculation. Separately, gastric mucosal leucocyte activation and capillary perfusion were also assessed using intravital microscopy 2, 4, 8 and 12 weeks after the inoculation. Results: In all H. pylori-inoculated animals, the bacterial infection persisted throughout the experimental period. Gastric mucosal lesion formation in the H pylori group was significantly inhibited in the H. pylori + polaprezinc group. Elevated levels of myeloperoxidase activity, GR0/ CINC-1 and thiobarbituric acid-reactive substances in the H. pylori group at 12 weeks were attenuated significantly by polaprezinc treatment. Enhanced levels of venular leucocyte activation observed in the H. pylori group were attenuated significantly in the H. pylori + polaprezinc group during both the early phase (2 weeks) and late phase (12 weeks). Conclusion: Polaprezinc inhibited H. pylori-associated gastric mucosal oxidative inflammation, including initial micro-vascular leucocyte activation, in Mongolian gerbils.

We examined the roles of lipid peroxidation, neutrophil accumulation, and inflammatory cytokines in the protective effect of polaprezine 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 L 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.

Polaprezinc, N-(3-aminopropionyl)-L-histidinatozinc, has been shown to stimulate the production of insulin-like growth factor-1 (IGF-1) in mesenchymal cells, the polypeptide playing a role in the gastric epithelial wound repair. The present study was performed to examine the effect of polaprezinc on the impaired healing of chronic gastric ulcers in adjuvant-induced arthritic rats, in relation to IGF-1. Arthritis was induced in male Dark Agouti (DA) rats by a single injection of Freund's complete adjuvant (FCA), and the gastric ulcers were induced by thermal cauterization (70°C for 30 sec) 7 days after FCA injection. Omeprazole (30 mg/kg) was administered p.o. once daily, while recombinant human IGF-1 (rhIGF-1) (30 μg/kg, s.c.) or polaprezinc (3-10 mg/kg, p.o.) was administered twice daily, starting from 3 days after ulceration for 14 days. The healing of gastric ulcers was significantly delayed in arthritic rats as compared to normal rats on day 10 and 17 following ulceration. The expression of IGF-1 mRNA was markedly increased in the ulcerated mucosa, but this response was apparently attenuated in arthritic rats. Repeated administration of polaprezinc accelerated the healing of gastric ulcers in both normal and arthritic rats, in a dose-dependent manner, and this effect was more pronounced in arthritic rats. Likewise, treatment with omeprazole also significantly promoted the healing of gastric ulcers in both normal and arthritic rats. On the other hand, rhIGF-1 significantly promoted the gastric ulcer healing in arthritic rats Without any effect on that in normal rats. These results suggest that the impaired healing of chronic gastric ulcers in arthritic rats is, at least partly, accounted for by less expression of IGF-1, and the polaprezinc improves the delayed healing of gastric ulcers in arthritic rats, probably through an increase in IGF-1 production.

We examined the influence of diabetes mellitus (DM) on the healing of HCl-induced gastric lesions and the healing promoting effect of polaprezinc [N-(3-aminopropionyl)-6-histiclinnto zinc] on these lesions. Studies were performed on rats injected intraperitoneally with streptozotocin (STZ, 70 mg/kg) five weeks prior to experiments. Diabetic rats had blood glucose levels (BGLs) higher than 350 mg/100 ml. Randomly chosen animals were treated subcutaneously with insulin (4 IU/day/rat) starting 1 week after STZ. Animals were given 1 ml of 0.6 N HCl by oral gavage (per os) following 18 hr of fasting; they were fed normally from 1 hr later and killed at various lime points after HCl administration. Polaprezinc (3-30 mg/kg) or its components ZnSO4/7H(2)O and L-carnosine were given orally, twice daily fur four days following HCl treatment. Gastric lesions induced by HCl healed macroscopically to quiescence within 10 days. DM and insulin did not affect the development of HCl-invoked gastric lesions, but the healing of such lesions was markedly impaired in animals with DM. Daily administration of insulin returned high BGLs to significantly lower ranges (190-208 mg/100 ml) and markedly antagonized the healing impairment. Polaprezinc (> 10 mg/kg) significantly reversed the delay observed in diabetic rats without any notable effects on BGLs or acid secretion, Similar trends were observed with 1 ZnSO4/7H(2)O or a mixture of ZnSQ4/7H(2)O and L-carnosine, but not by L-carnosine alone. The mucosal expression of insulin-like growth factor-1 (IGF-I) mRNA was significantly lower in diabetic rats, a dysregulation partially corrected by insulin and polaprezinc. In addition, the delayed healing in diabetic rats was also significantly promoted by the repeated subcutaneous administration of rhIGF-I (> 10 mu g/kg, twice daily) without any notable effect on BGLs or acid secretion. These results suggest that DM exerted a deleterious influence on the healing of acute gastric lesions in both insulin- and zinc-sensitive manner. The salutary effects of polaprezinc on the impaired healing of gastric lesion in STZ-diabetic animals may at least be partly explained by enhancement of mucosal IGF-I mRNA expression in the stomach.

Background: CXC chemokines such as interleukin (IL)-8 are neutrophil chemoattractants, the levels of which increase in Helicobacter pylori-infected gastric mucosa. Many investigators have focused on the chemotactic aspects of IL-8; however, CXC chemokines are also reported to have angiogenic activity and to serve as remodelling factors. Rat GRO/CINC-1 is a rodent counterpart of human GRO alpha, a member of the family of CXC chemokines. Gastric mucosa infected with H. pylori is in a state of hyperproliferation, with increases in the amounts of growth factors such as hepatocyte growth factor (HGF). Aim: To investigate whether rat GRO/CINC1 had growth-stimulating activity for gastric epithelial cells. Methods: The rat gastric epithelial cell line RGM-1 was incubated in serum-free medium for 12 h to adjust the cell cycle to the G(o) phase, and GRO/CINC-1 was then added for 24 h. The total cell number was determined by fluorogenic analysis after propidium iodide staining, and cell proliferation was assessed by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation, The activity of p42/p44 mitogen-activated protein kinase (MAPK) was measured 5-20 min after the start of GRO/CINC1 exposure. Results: Cultures treated with GRO/CINC-1 showed a significant increase in cell number and BrdU incorporation in a concentration-dependent fashion. The MAPK activity increased within 5 min after GRO/ CINC-1 application and returned to the control level at 20 min. Conclusion: The growth-stimulatory effect of GRO/CINC1 on rat gastric epithelial cells suggests a dual function of this chemokine: proinflammatory action and induction of epithelial proliferation.

Background: CXC chemokines such as interleukin (IL)-8 are neutrophil chemoattractants, the levels of which increase in Helicobacter pylori-infected gastric mucosa. Many investigators have focused on the chemotactic aspects of IL-8: however, CXC chemokines are also reported to have angiogenic activity and to serve as remodelling factors. Rat GRO/CINC-1 is a rodent counterpart of human GROα, a member of the family of CXC chemokines. Gastric mucosa infected with H. pylori is in a state of hyperproliferation, with increases in the amounts of growth factors such as hepatocyte growth factor (HGF). Aim: To investigate whether rat GRO/CINC-1 had growth-stimulating activity for gastric epithelial cells. Methods: The rat gastric epithelial cell line RGM-1 was incubated in serum-free medium for 12 h to adjust the cell cycle to the G(o) phase, and GRO/CINC-1 was then added for 24 h. The total cell number was determined by fluorogenic analysis after propidium iodide staining, and cell proliferation was assessed by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation. The activity of p42/p44 mitogen-activated protein kinase (MAPK) was measured 5-20 min after the start of GRO/CINC-1 exposure. Results: Cultures treated with GRO/CINC-1 showed a significant increase in cell number and BrdU incorporation in a concentration-dependent fashion. The MAPK activity increased within 5 min after GRO/CINC-1 application and returned to the control level at 20 min. Conclusion: The growth-stimulatory effect of GRO/CINC- 1 on rat gastric epithelial cells suggests a dual function of this chemokine: proinflammatory action and induction of epithelial proliferation.

We previously reported that NH2Cl induced extensive DNA fragmentation in gastric cells. Polaprezinc, a zinc-carnosine chelate compound, is reported to be a potent antioxidant in gastric mucosa. The present study was designed to examine whether polaprezine could attenuate the NH2Cl-induced DNA damage. Gastric cell lines, MKN45, were exposed to NH2Cl in Ca2+-containing Hanks' balanced salt solution. DNA fragmentation was evaluated by photometric enzyme immunoassay for in vitro determination of cytoplasmic mono- and oligonucleosomes. Polaprezinc, L-carnosine, and zinc sulfate (ZnSO4) were added to the cell incubation medium to evaluate the inhibitory effect on the formation of cytoplasmic mono- and oligonucleosomes. Separately, the bleaching level of beta-carotene with the addition of each test solution was evaluated to confirm the inhibitory effect against hypochlorous acid. Polaprezinc or L-carnosine, but not ZnSO4, at a concentration of 0.001 mM, significantly attenuated the increased levels of cytoplasmic mono- and oligonucleosomes evoked by 0.001mM NH2Cl. Polaprezinc and L-carnosine, but not ZnSO4, also inhibited NH2Cl-induced beta-carotene bleaching in the cell-free system. In conclusion, polaprezinc, especially its subportion L-carnosine, inhibited NH2Cl-evoked gastric epithelial DNA fragmentation, suggesting a role for this agent in preventing the progression of gastric epithelial injury induced by NH2Cl.

Background: Mongolian gerbils have been reported to be a suitable model for Helicobacter pylori-associated gastric mucosal injury, including gastric cancer. Although ethanol is known to be one of the harmful substances in the gastric mucosa, the relationship between ethanol and H. pylori infection remains unknown. The aim of the present study is to investigate the effect of ethanol treatment prior to H. pylori inoculation on associated gastric mucosal injury. Methods: Male Mongolian gerbils were used for the study. Helicobacter pylori was orally inoculated after 15 h fasting (Hp group). Thirty minutes prior to H. pylori inoculation, a group of gerbils was orally treated with 40% ethanol (20 mL/kg; E + Hp group). Another group of animals was treated either with H. pylori culture media alone (controls) or with 40% ethanol plus culture media (E group). Gerbils were killed 2, 4 or 12 weeks after H. pylori inoculation. Helicobacter pylori infection was confirmed by both histological examination and serological tests. Mucosal damage was evaluated histologically according to the modified Sydney system. Results: Although in the controls and E group no significant change to the gastric mucose was observed, persistent H. pylori infection was seen in the mucosa and mucosal leucocyte infiltration and severe epithelial damage was observed in the Hp and E + Hp groups after 4 weeks. The histological scores for polymorphonuclear cell infiltration and myeloperoxidase activity were higher in the E + Hp group at 4 weeks than in the Hp group (P < 0.05). Conclusions: Ethanol intake preceding H. pylori inoculation could promote the progression of gastric mucosal inflammation in Mongolian gerbils. (C) 1999 Blackwell Science Asia Pty Ltd.

The effect of polaprezinc (N-(3-aminopropionyl)-L-histidinato zinc), a novel antiulcer drug containing zinc, on cellular proliferation was studied using cultured cells. In human umbilical vein endothelial cells (HUVEC) or human foreskin fibroblast cells, bromodeoxyuridine (BrdU) uptake and the number of cells were increased by polaprezine under low serum conditions, but polaprezinc had no effect on guinea pig gastric mucosal epithelial cells. In addition, L camosine (a component of poraprezinc) had no effect on cultured HUVEC, while zinc sulfate, a representative zinc compound, increased BrdU uptake by about 2-fold at 10(-9) M. However, the action of zinc sulfate was weaker than that of polaprezinc. The insulin-like growth factor I (IGF-I) mRNA level was increased in HUVEC by polaprezine at: 10(-9) M similar to 3 x 10(-8) M concentrations, causing stimulation of BrdU uptake. When an anti-IGF I antibody was added to cultures, the effects of polaprezinc on BrdU uptake was suppressed. These results suggest: that although polaprezinc, a novel antiulcer agent, does not have proliferative effects on epithelial cells, it does promote the proliferation of non-parenchymal cells, and IGF-I is involved in this action. (C) 1999 Elsevier Science Inc.

Helicobacter pylori colonized gastric mucosa is manifest in a significant neutrophil infiltration with an extensive level of oxyradical formation. Mongolian gerbil is one of the excellent models for H. pylori-infection. The present study was designed to investigate pro- and antioxidant formation in the stomach of H. pylori-positive gerbils. Fourteen male Mongolian gerbils (MGS/Sea) were orally inoculated with H. pylori (ATCC43504) (Hp group) and 15 gerbils were inoculated with the culture media (Control). H. pylori infection was confirmed by the serum anti-H. pylori IgG test. Each gerbil was evaluated 6 or 12 weeks after the inoculation. Neutrophil infiltration was assessed by the tissue MPO activity. Mucosal oxidative stress was evaluated by thiobarbituric acid-reactive substances (TBARS), total glutathione contents, glutathione peroxidase (GSHPx) activity and Cu-, Zn-superoxide dismutase (SOD) activity. In Hp group, the H. pylori was persistently infected until 12 weeks. The level of MPO activity was significantly higher in Hp group at 6 and 12 weeks. Although the levels of TEARS and total glutathione were within the same range as controls at 6 weeks, they were significantly increased at 12 weeks. However, GSHPx activity was significantly increased at 6 weeks, but became the same range with the controls at 12 weeks. SOD activity showed no significant increase in Hp group at 6 and 12 weeks. In conclusion, H. pylori inoculation induced gastric mucosal neutrophil activation and pro-oxidant formation and also increased total glutathione contents, one of the mucosal antioxidants in gerbils. (C) 1999 Elsevier Science Inc.

Although vascular endothelial growth factor (VEGF) plays a role in the growth of hypervascular tumors, mechanisms for paracrine regulation of its receptor expression on vascular endothelial cells remain unknown. This study aimed to investigate whether VEGF released from hypoxia-exposed Hep G2 cells alters expression of the two distinct receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase I (flt-1), in human umbilical venous endothelial cells (HUVEC). Hep G2 cells were cultured in 20% or 1% O-2 for 16 h to examine induction of VEGF mRNA and its protein expression. Conditioned medium from Hep G2 cells (CM) was applied to HUVEC under normoxic conditions, and expression of mRNA for the VEGF receptors was determined by RT-PCR. In response to the hypoxic challenge, Hep G2 cells upregulated VEGF mRNA and the release of VEGF. Hypoxia-CM preferentially stimulated the mRNA expression of flt-1 but not that of KDR in HUVEC. When the VEGF release from hypoxia-exposed Hep G2 cells was blocked by its antisense oligodeoxynucleotide, the endothelial flt-1 mRNA upregulation elicited by the hypoxia-CM was still maintained. These results suggest that hypoxia-exposed Hep G2 cells not only produce VEGF but also evolve paracrine induction of flt-1 through VEGF-independent mechanisms.

Background: Although the detailed mechanism is unclear, zinc and its derivative, polaprezinc, have been reported to accelerate gastric ulcer healing in vivo. Aim: To investigate the detailed cellular mechanism of polaprezinc on gastric epithelial cells and fibroblasts with special attention to insulin-like growth factor I (IGF-I). Methods: Isolated rabbit gastric epithelial cells formed a complete monolayer, from which a circular artificial wound with constant size was made, The restoration process was monitored by measuring wound size up to 48 h, Either polaprezinc, IGF-I, fibroblast conditioned medium or neutralized medium conditioned by anti-IGF-I antibody was added at the time of wounding. The expression of mRNA of IGF-I, hepatocyte growth factor (HGF) and transforming growth factor cc (TGF-a) in fibroblasts with or without polaprezinc treatment was tested using reverse transcription polymerase chain reaction (RT-PCR), Gastric epithelial cell proliferation was also examined by bromodeoxyuridine (BrdU) staining. Results: IGF-I and fibroblast conditioned medium treatment accelerated gastric epithelial restoration which included cell migration and proliferation. However, polaprezine and neutralized conditioned medium treatment did not accelerate epithelial repair. RT-PCR for growth factor mRNA revealed the IGF-I mRNA expression in fibroblasts was increased after treatment with polaprezinc. Conclusion: Polaprezinc induced IGF-I production from mesenchymal cells, resulting in stimulation of epithelial cell restoration through a paracrine pathway. IGF-I may play an important role in gastric wound repair.

Monochloramine (NH2Cl) is known to be one of the virulence factors in Helicobacter pylori-associated gastric mucosal injury. The present study was designed to examine NH2Cl-evoked DNA fragmentation in the gastric epithelial cell line MKN45. NH2Cl was produced by mixing NH3 with sodium hypochlorite (NaClO). MKN45 cells were exposed to NH2Cl, NH3, or NaClO in Hanks' balanced salt solution. DNA cleavage was evaluated quantitatively by photometeric enzyme immunoassay for the in vitro determination of cytoplasmic mono- and oligonucleosomes. Damage to the plasma membrane was assessed by measuring the activity of lactate dehydrogenase in the supernatants. Separately, DNA ladder formation was performed to confirm the incidence of DNA fragmentation. NH2Cl (0.001-0.01 mM) significantly increased the cytoplasmic mono- and oligonucleosomes, suggesting. the incidence of DNA cleavage, The DNA ladder was clearly evoked by NH2Cl. NH2Cl induced a DNA fragmentation, one of the important aspects in apoptosis, in the gastric cell line MKN45.

The bacterial toxin VacA produced by H. pylori induces large vacuoles in several types of cultured cells such as HeLa cells or gastric cells. To determine the mechanism of vacuolation induced by this toxin we employed several inhibitors of membrane trafficking and endocytosis, The development of vacuolation induced by VacA in HeLa cells were prevented by bafilomycin A1 and low temperature conditions that inhibited vesicle transport or endocytosis, Formation of large vacuoles was also inhibited by an antibody against EGF receptor, which was previously shown to be internalized by endocytosis, but not by an anti-transferrin receptor antibody. Moreover, proteins of 58 and 37 kDa, corresponding to fragments of VacA, were recognized by an anti-H. pylori antibody after immunoprecipitation with anti-EGF receptor of cell extracts from HeLa cells treated with VacA, but not from untreated HeLa cells. We suggest that VacA may enter cells by endocytosis mediated by the EGF receptor. These are the first data indicating that the EGF receptor may be significant in the development of vacuolation caused by VacA. (C) 1998 Federation of European Biochemical Societies.