Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
hsuzuki@sc.itc.keio.ac.jp
CURRENT ACADEMIC POSITION: Associate Professor, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Fax: 81-3-5363-3967
EDUCATION 1983-1995 Keio University, Premedical Course, Tokyo, Japan. 1985-1989 Keio University School of Medicine, Tokyo, Japan (M.D. June, 1989) 1989-1993 Keio University, Postgraduate Course of Medicine, Tokyo, Japan 1994 Ph.D.; Thesis: Suzuki, H. "Paradoxical oxidative cell injury in hypoperfused rat liver. -Beneficial effects of prostaglandin E1-" 1993-1995 Postdoctoral Research Fellow, Institute for Biomedical Engineering, University of California at San Diego, La Jolla, California, U.S.A. 1995-2003 Instructor, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan 2003-2005 Visiting fellow, Yamagata Technopolis foundation BioRadical Institute 2003-present Assistant Professor (visiting), Department of Internal Medicine, Tokyo Dental College 2003-2006 Assistant Professor, Department of Internal Medicine, Keio University School of Medicine 2005-2005 Head, Department of Gastroenterology, Kitasato Institute Hospital 2006-present Associate Professor, Department of Internal Medicine, Keio University School of Medicine 2007-present Head of Outpatient Clinic, Division of Gastroenterology and Hepatology, Keio University Hospital
OTHER PROFESSIONAL EXPERIENCES: Associate Editor: “Journal of Gastroenterology” (Springer-Verlag) “Microvascular Communications and Review” (Elsevier)
Editorial Board: “Neurogastroenterology and Motility” (Blackwell) “Journal of Gastroenterology and Hepatology” (Blackwell) “World Journal of Gastroenterology” (Elsevier) “Journal of Clinical Biochemistry and Nutrition” (Nakanishi Printing Co.) “Helicobacter Research (in Japanese)” (Sentan Igaku Co.) “ GI Network (in Japanese)” (Medical Asahi Co.)
MEDICAL SPECIALIST AUTHORIZATION (Nintei-i)
1997: The Japanese Society of Internal Medicine (No. 11269; No. 11661) 1998: The Japanese Society of Gastroenterology (No. 23302) 1998: The Japanese Medical Association, Registered Industrial Physician (No. 9803377) 1999: The Japanese Society of Hepatology (No. 3157) 2000: The Japanese Society of Gastroenterological Endoscopy (No. 20000408) (2005-, Registered Instructor)
AWARDS AND HONORS 1997: Asian Union for Microcirculation (AUM) Young Investigator Award. 1999: Prize (Cultural Film) in the Educational Film, Video, Slide and CD-ROM Contest 1999, sponsored by the Japan Audio-visual Education Association. 2000: The 2nd prize of Van Leeuwenhoek distinctive Travel Award, 21st European Conference on Microcirculation. 2000: a commendation by the Minister of State for Science and Technology at the 41th Science and Technology Film/Video Festival of Japan 2001: Winner of the 6th Keio University Medical School Faculty and Alumni Grants 2002 : The Society for Free Radical Research Japan (SFRR-Japan) Award for young scientist 2002: The 1st Prize of the 3rd research forum of GI inflammation and PPI 2002: The 1st Prize of the 9th research forum of Gastric mucosal protection 2003: The Prize of the 14th research forum of Gastrointestinal Cell Function 2003: The 1st Prize of the Gastric Mucosal Bioregulation (GMB) Award 2004 : The 1st Prize of the 15th research forum of Gastrointestinal Cell Function 2004: Rokuzo Kobayashi Memorial Helicobacter Prize 2004: Award of Excellence for Outstanding Participation and Discussion, The 3rd Japan GRG/AGA Symposium 2004: Special Award, Hamanako International Symposium 2005: Award of Excellence for Outstanding Participation and Discussion, The 4th Japan GRG/AGA Symposium 2007: Kitajima Award from the Alumni Society for Keio University School of Medicine 2007: The 4th Pfizer Science and Research Symposium, Excellence Award
MEMBERSHIPS IN MEDICAL AND SCIENTIFIC SOCIETIES: International 1994-present The Microcirculatory Society 1994-present The Oxygen Society 1995-present The American Physiological Society (APS) (Member ID# 8660) 1996-present The American Association for the Study of Liver Diseases (AASLD) 2004-present The American Gastroenterological Association (AGA) 2006-present The American College of Gastroenterology
Domestic 1989-present. The Japanese Society of Internal Medicine 1990-present. The Japanese Society of Gastroenterology (2003-, Executive member) 1991-presnet. The Japanese Society of Gastroenterological Endoscopy (2006-, Executive member) 1989-present. The Japanses Society for Microcirculation (2001-, Executive member) 1990-present. The Japanese Society of Hepatology 1992-present. The Japanese Society of Autonomic Nervous System (1998-, Executive member) 1993-present. The Japanese Society of Experimental Ulcers (1998-, Executive member) 1994-present. The Japanese Society of Helicobacter Research (1999-, Executive member) 1993-present. Society for Free Radical Research, Japan (2001-, Executive member) 1996-present. The Japanese Society of Digestion and Absorption 2000-present The Japanese Society for Wound Healing 2001-present. The Japanese Gastroenterological Association (2009-, Executive member) 2002-present. The Japanese Society of Neurogastroenterology (2005-, Executive member)
Abstract: Background: Recently, the number of Helicobacter pylori isolates showing antibiotic resistance has been increasing. Rifabutin (RFB) is one of the possible candidates for H. pylori eradication. In the present study, the RFB minimum inhibitory concentrations (MICs) and the resistance-determining genes to RFB (rpoB) were examined to clarify the relationship between drug MICs, rpoB mutations, and past history of rifampicin (RFP) treatment. Methods: The MICs of RFB and rpoB mutations were examined for 48 strains with failure of H. pylori eradication in the University Hospital and 46 isolated from patients at a specialized hospital for chronic respiratory diseases without past H. pylori eradication. Past RFP treatment was also examined. Results: Eight of 94 strains showed high RFB MICs and 6 of the 8 strains showed rpoB point mutations. Although no strains showed high RFB MICs among 48 strains from the patients in the University Hospital, all 7 strains isolated from patients with past RFP treatment showed high RFB MICs (>/=0.12 mg/l). Conclusion: Although RFB might be a potential candidate component of a new H. pylori eradication regimen following the first- or second-line failure, it should be used after examining a past history of RFP treatment.
Abstract: VATER association represents a cluster of Vertebral, Anal, Tracheo-Esophageal, Radial and Renal malformations and caudal regression syndrome is an entity consisting of a spectrum of congenital anomalies of lower spine and hips associated with genitourinary and lower limb defects. The concurrence of various malformations may be explained by a common defect in blastogenesis, but direct evidence is yet to be accumulated. Here, by the use of autofluorescence and the teratogenic effect of Adriamycin, we demonstrated that Adriamycin administered to eggs of White Leghorns distributes to the caudal portion of the embryo and foregut epithelium, and induces caudal regression and tracheal and pulmonary agenesis. The induction of caudal regression syndrome-like anomaly was developmental stage- and dose- dependent. Embryos with caudal regression demonstrated tracheoesophageal anomalies, one of the defects included in VATER association. The stages at which anomalies were produced corresponded to that of human embryos between days 22 and 26 (Carnegie stages 10-11). In view of the anti-tumor activity of Adriamycin by intercalating to double-stranded DNA of undifferentiated cells undergoing rapid cell division, it is possible that Adriamycin had preferentially attacked cells in the caudal end where pronounced proliferation takes place during this narrow period of greatest susceptibility.
Abstract: MicroRNAs (miRNAs) are small noncoding RNAs that function as endogenous silencers of numerous target genes. Hundreds of human miRNAs have been identified in the human genome, and they are expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation. Links between miRNAs and human diseases are increasingly apparent, and aberrant expression of miRNAs may contribute to the development and progression of human malignancies. Recent studies have shown that some miRNAs play roles as tumor suppressors or oncogenes in gastrointestinal cancers. miRNA expression is regulated by different mechanisms including transcription factor binding, epigenetic alterations, and chromosomal abnormalities. miRNA expression profiling may be a powerful clinical tool for cancer diagnosis, and regulation of miRNA expression could be a novel strategy for the chemoprevention of human gastrointestinal cancers. In this article, the biological importance of miRNAs in human gastrointestinal cancers is summarized.
Abstract: Resistance of H. pylori has been demonstrated to the standard therapeutic antimicrobials (clarithromycin (CLR), metronidazole, amoxicillin (AMX) and tetracycline) [1], therefore, other treatment options must be urgently introduced. ...
Abstract: BACKGROUND/AIMS: To examine the influence of long-term treatment of H. pylori-infected patients with proton pump inhibitors (PPI) on the toxic oxidant production in the stomach. METHODOLOGY: Eight H. pylori-positive patients with gastric ulcer were enrolled, and tissue samples were obtained endoscopically from the antrum and corpus. The tissue contents of neutrophil myeloperoxidase (MPO) and oxygen-derived free radicals were quantified by ELISA and chemiluminescence (ChL) assay. The H. pylori density in the tissue specimens was scored by immunohistochemistry and the mucosal infiltration by neutrophils and monocytes was scored by histopathology. The effects of PPI on these parameters were evaluated by oral administration of lansoprazole (LPZ) at the dose of 30mg od for 8 weeks, followed by 15mg for 24 weeks. RESULTS: Eight-week treatment with LPZ significantly increased the corpus ChL by 400%, which remained unchanged at this level during the subsequent 24 weeks, but the antrum ChL decreased slightly following LPZ-treatment. No significant changes in the mucosal MPO activity or the histologically determined parameters of H. pylori density and neutrophil/monocyte infiltration were observed in either portion of the stomach. CONCLUSIONS: The mucosal oxidative stress level significantly increased in the corpus mucosa after long-term treatment with LPZ, which may be independent of inflammatory cell infiltration.
Abstract: Proton pump inhibitors (PPIs) are novel compounds that strongly inhibit the H(+)/K(+)-ATPase in the gastric parietal cells to cause profound suppression of acid secretion. Acid-generating ATPase, also known as vacuolar-type ATPase, is located in the lysozomes of leukocytes and osteoclasts and its activity is also reportedly influenced by treatment with PPIs. This concept is supported by the results of studies using autoradiography in which (3)H-Lansoprazole uptake sites were clearly detected in the cytoplasmic granules of neutrophils infiltrating the gastric mucosa. In vitro studies indicate that PPIs increase the intra-vacuolar pH in the lysosomes of purified neutrophils and attenuate the adherence of neutrophils to the vascular endothelium. In clinical practice, the acidic environment in the stomach plays a critical role in the development of gastritis induced by Helicobacter pylori (H. pylori). This is worthy of note, because persistent gastritis often results in atrophic and metaplastic changes in the gastric mucosa, which are believed to be preneoplastic abnormalities. In patients with H. pylori-infection, PPI therapy causes corpus-predominant gastritis, which is frequently found in the background mucosa in patients with gastric cancer. The efficacy and safety of long-term PPI-treatment have not been conclusive, thus we need to pay more attention to the additional pharmacological actions of PPIs.
Abstract: BACKGROUND AND AIM: This study was designed to investigate the efficacy of gatifloxacin (GAT)-based triple therapy as a third-line treatment for Helicobacter pylori (H. pylori) eradication, according to the assessment of the susceptibility to GAT and gyrA mutation. METHODS: Fourteen patients who had eradication failure following both clarithromycin-based triple therapy and metronidazole-based triple therapy, or who were infected with H. pylori isolates that were resistant to both clarithromycin and metronidazole after failure of clarithromycin-based triple therapy, were enrolled. These patients were randomly assigned to two groups: (i) rabeprazole and amoxicillin (RA) and (ii) rabeprazole, amoxicillin, and GAT for 7 days (RAG). The minimal inhibitory concentrations were determined by the agar dilution method. The gyrA gene was examined by sequencing. RESULTS: The eradication rate was 0% in the RA group and 75% in the RAG group. The eradication rate in the RAG group was 100% in patients infected with GAT-susceptible bacteria and/or bacteria without gyrA mutations, but was only 33.3% in those infected with GAT-resistant bacteria or bacteria with gyrA mutations. CONCLUSION: Although GAT may be a promising candidate for third-line therapy, its selection must be based on the results of drug susceptibility testing or gyrA analyses.
Abstract: BACKGROUND: Body mass index (BMI) and obesity are reportedly associated with symptoms of gastroesophageal reflux disease (GERD). The present study was designed to investigate the effect of metabolic disorders including obesity on the levels of functional gastroesophageal reflux by videoesophagography. METHODS: Twenty-one patients with GERD-associated symptoms were examined by videoesophagography. On their initial visit, all patients completed the Japanese version of the Carlsson-Dent self-administered questionnaire (QUEST). The findings of videoesophagography were evaluated by the X-ray severity scores for gastroesophageal reflux (XRSS), which were defined for the total diagnosis of functional gastroesophageal reflux. Correlation between XRSS scores and physical or metabolic markers was evaluated. RESULTS: The mean XRSS in the QUEST-positive group (4.7+/-0.6) was significantly higher than that in the QUEST-negative group (3.3+/-0.5, P<0.05). XRSS correlated positively with BMI (P<0.05) and waist circumference (P<0.05), but negatively with high-density lipoprotein-cholesterol (P<0.05), serum adiponectin (P<0.05) and active ghrelin (P<0.05). In the multivariate analysis, serum adiponectin level, BMI and triglyceride independently affected the XRSS. CONCLUSION: Videoesophagography is a useful diagnostic modality for the evaluation of patients with GERD symptoms. Functional gastroesophageal reflux is seen in obese patients, especially with decreased levels of adiponectin.
Abstract: BACKGROUND/AIMS: Polyarteritis nodosa (PN) has been classified into polyarteritis (PA) and microscopic polyarteritis (MA) histologically. To clarify of the characteristics of upper gastrointestinal bleeding lesions in PN, we investigated the patients of PN with rapidly progressive glomerulonephritis (RPGN) presenting with upper gastrointestinal bleeding. METHODOLOGY: The subjects of this study were 21 patients of PN with RPGN (PA: 11, MA: 10) who presented with upper gastrointestinal bleeding. The bleeding lesions and their locations were examined endoscopically in the study subjects, and the relationship of the bleeding to the severity of renal failure, the necessity of hemodialysis (HD), presence/ absence of H. pylori infection and the gender of the patients were analyzed. RESULTS: The bleeding lesions were endoscopically identified as esophageal ulcers in 2 cases, gastric ulcers in 15 cases and duodenal ulcers in 4 cases, respectively. In 10 of the 15 cases with gastric ulcers, the ulcer assumed the form of Dieulafoy's lesions affecting the gastric body, and the underlying disease was PA in all the 10 cases. In the remaining 5 cases of gastric ulcers and 2 cases of esophageal ulcer with underlying MA or 4 cases of duodenal ulcers, in whom assumed the bleeding form of oozing from the marginal zone of ulcers. In all of the 4 cases of duodenal ulcers, and the 1 case with underlying PA and the other cases with MA, no correlation was found between the onset of the upper gastrointestinal bleeding and the severity of renal failure or the necessity for HD, presence of H. pylori infection, or the gender of the patients. CONCLUSIONS: Dieulafoy's lesions are the most frequent sources of upper gastrointestinal bleeding in cases of PA.
Abstract: In the Krebs cycle of Helicobacter pylori, the absence of alpha-ketoglutarate dehydrogenase and succinyl CoA synthetase are shown. Instead, alpha-ketoglutarate is converted to succinyl CoA and succinate by alpha-ketoglutarate oxidoreductase (KOR) and CoA transferase (CoAT). In the present study, when H. pylori transformed to the coccoid form, a viable but non-culturable form of H. pylori with reduced metabolic activity, the KOR activity was enhanced while the CoAT activity was reduced. Direct inactivation of KOR could potently kill the bacteria without allowing conversion to the coccoid form, suggesting a novel treatment strategy for the eradication of H. pylori, especially in cases infected with multiple antibiotic-resistant strains.
Abstract: Proton pump inhibitors (PPIs) are now commonly used for the treatment of acid related diseases such as peptic ulcer and reflux esophagitis. Because of their ability to produce direct inhibition of the proton pump, PPIs provide more sustained increase of the gastric pH than H(2)-receptor (H(2)R) antagonists. Diverse reports have been published on gastric epithelial cell modality associated with PPI treatment both in animal models and clinical settings. The present review summarizes the recent accumulated evidence on gastric epithelial cell modality associated with PPI treatment, including the formation of gastric carcinoid tumors and fundic gland polyps, and the development of gastric mucosal atrophy. Long-term PPI treatment has been reported to cause enlargement of the parietal cells and enterochromaffin-like cells, and to decrease the number of chief cells without affecting A-like cell. Although the development of gastric carcinoid tumors after chronic PPI treatment has been reported in animal studies, no such occurrences have been demonstrated in humans. The effect of PPIs on the formation of fundic gland polyps and the development of atrophic gastritis should be investigated in future studies.
Abstract: Immune thrombocytopenia purpura (ITP) is a bleeding disorder in which platelet-specific autoantibodies cause a loss of platelets. In a subset of patients with ITP and infected with Helicobacter pylori, the number of platelets recovers after eradication of H. pylori. To examine the role of H. pylori infection in the pathogenesis of ITP, the response of 34 ITP patients to treatment with a standard H. pylori eradication regimen, irrespective of whether they were infected with H. pylori, was evaluated. Eradication of H. pylori was achieved in all H. pylori-positive patients, and a significant increase in platelets was observed in 61% of these patients. By contrast, none of the H. pylori-negative patients showed increased platelets. At baseline, monocytes from the H. pylori-positive patients exhibited an enhanced phagocytic capacity and low levels of the inhibitory Fcgamma receptor IIB (FcgammaRIIB). One week after starting the H. pylori eradication regimen, this activated monocyte phenotype was suppressed and improvements in autoimmune and platelet kinetic parameters followed. Modulation of monocyte FcgammaR balance was also found in association with H. pylori infection in individuals who did not have ITP and in mice. Our findings strongly suggest that the recovery in platelet numbers observed in ITP patients after H. pylori eradication is mediated through a change in FcgammaR balance toward the inhibitory FcgammaRIIB.
Abstract: OBJECTIVE: Harmonic flash echo imaging, which is an intermittent second harmonic imaging technique, has recently been developed for the evaluation of blood flow. The present study was designed to investigate human gastric blood flow during administration of non-steroidal anti-inflammatory drugs (NSAIDs) by harmonic flash echo imaging. MATERIAL AND METHODS: Eight healthy volunteers were enrolled in the study. After an overnight fast, the volunteers were requested to drink 400 ml water and remain in the sitting position. Seven milliliters (300 mg/ml) Levovist (SHU508A) was then injected intravenously at the rate of 1 ml/s, and intermittent harmonic scanning was carried out at 1-s intervals. A similar examination was carried out one hour after each subject took an oral tablet of diclofenac sodium (50 mg). To minimize the effect of variations in acoustic attenuation among patients, the ratio of the maximum amplitude in the gastric wall to that in the portal vein was expressed as the gastric perfusion index. RESULTS: Strong ultrasonographic contrast enhancement of the gastric wall and portal vein was observed. The area under the curve was significantly reduced in the images obtained after ingestion of the diclofenac sodium tablet. The gastric perfusion index was significantly reduced from 0.617+/-0.114 to 0.480+/-0.127 in the antrum and from 0.659+/-0.103 to 0.509+/-0.107 in the lower gastric corpus after ingestion of the diclofenac sodium tablet (p<0.05). CONCLUSIONS: A reduction in the human conscious gastric transmural blood flow following ingestion of an NSAID is revealed by harmonic flash echo imaging.
Abstract: BACKGROUND/AIMS: Sonic hedgehog (Shh) is a morphogen involved in many aspects of patterning of the gut during embryogenesis and in gastric fundic gland homeostasis in the adult. Shh expression is reportedly to be reduced in Helicobacterpylori-associated gastritis. The aim of this study was to assess the restoration of Shh expression after H. pylori eradication. METHODOLOGY: Twenty H. pylori-positive patients who underwent upper gastrointestinal endoscopy before and after the eradication were studied. Biopsy specimens were taken from the greater curvature of the middle third of the stomach body. The specimens were evaluated for the severity of acute and chronic inflammation and for that of mucosal atrophy, based on the updated Sydney system. Immunohistochemistry for Shh and H(+)-, K(+)-ATPase was also performed; the percentages of positively stained epithelial cells for the two molecules were expressed as the Shh index and H(+)-, K(+)-ATPase index, respectively. RESULTS: There was a significant decrease in the acute and chronic inflammation scores and also in the mucosal atrophy score following the eradication. The Shh and H(+)-, K(+)-ATPase index were significantly increased following the eradication. CONCLUSIONS: Suppressed Shh expression in the gastric mucosa by H. pylori infection was significantly restored following eradication of the infection.
Abstract: BACKGROUND AND AIM: Several scoring systems have been devised to identify patients with upper gastrointestinal (UGI) bleeding who are at a high risk of adverse outcomes. We retrospectively evaluated the accuracy of the Blatchford scoring system for assessing the need for clinical intervention in cases of UGI bleeding admitted to the emergency department (ED). METHODS: This was a retrospective study conducted on patients who underwent emergency GI endoscopy at the ED of our hospital. Those who needed blood transfusion, operative or endoscopic interventions to control the hemorrhage were classified into the 'high risk' group. RESULTS: Of the 93 enrolled patients, 70 (75.3%) were classified into the high risk group. The Blatchford score was significantly higher in the high risk group than in the low risk group. When a cut-off value of 2 was used, the sensitivity and specificity of the Blatchford scoring system were determined to be 100% and 13%, respectively. Thus, the Blatchford scoring system was deemed to be useful for distinguishing between the high risk group and the low risk group of patients with GI hemorrhage admitted to the ED. CONCLUSION: The Blatchford scoring system is accurate for identifying definitively low-risk patients of GI hemorrhage, even prior to the performance of emergency UGI endoscopy at the ED.
Abstract: BACKGROUND: Ghrelin, an appetite-promoting peptide secreted from the stomach, is reported to enhance preprandial acid output, possibly through stimulation of the cephalic phase. The present study was designed to clarify the dynamics of ghrelin in H(2) receptor (H(2)R)-null mice by genetic H(2)R knockout. METHODS: Fifteen-week- and 54-week-old H(2)R-null mice and their littermates were used. After evaluating the levels of food intake and body-weight increments, mice were killed, and the plasma and gastric active and total ghrelin levels were examined by radioimmunoassay, and gastric preproghrelin mRNA expression was examined by quantitative reverse transcriptase-polymerase chain reaction. Furthermore, each stomach specimen was evaluated by immunohistochemistry and transmission electron microscopy for ghrelin. RESULTS: The levels of food intake and body-weight gain of the H(2)R-null mice were higher than those of wild-type mice. The gastric pH of the 54-week-old H(2)R-null mice was lower than that of the 15-week-old mice. Gastric preproghrelin mRNA expression, plasma ghrelin level, and density of ghrelin-immunoreactive cells in the gastric mucosa of the H(2)R-null mice were significantly increased compared with those of the wild-type mice. Ghrelin-positive immunogold density seen in the electron micrograph was significantly reduced in A-like cells of the H(2)R-null mouse stomach. CONCLUSIONS: Ghrelin production and secretion from A-like cells in the gastric fundus are upregulated in H(2)R-null mice, a genetic H(2)R knockout model.
Abstract: BACKGROUND: It is helpful in clinical practice to predict the effects of eradication therapy on Helicobacter pylori. AIM: To develop a useful predictor of the response to metronidazole (MNZ)-containing second-line regimens by combining minimal inhibitory concentrations (MICs) of both amoxicillin (AMX) and MNZ, and the results of urea breath test (UBT) before the treatment. METHODS: We enrolled 107 patients who showed eradication failure following first-line triple therapy with a proton pump inhibitor, AMX and clarithromycin. The eradication resistance index was defined as: [pre-treatment UBT result ( per thousand)] x [AMX MIC (microg/ml)] x [MNZ MIC (microg/ml)]. Second-line eradication therapy with lansoprazole, AMX and MNZ was administered for 1 week. Eradication was confirmed by the UBT. RESULTS: The eradication resistance index in subjects showing eradication failure and those showing successful eradication was 9.72 +/- 6.63 and 1.25 +/- 2.31, respectively (p < 0.001). When a cutoff value of 3 was used, the eradication resistance index predicted the response to therapy with a specificity of 93.8%, sensitivity of 81.8%, and accuracy of 92.5%. CONCLUSIONS: The eradication resistance index is a more useful predictor of response to MNZ-containing regimens for second-line treatment than only pretreatment UBT results or MNZ resistance.
Abstract: Expression of sonic hedgehog (Shh), a morphogen for the gastric fundic glands, is reduced in the atrophic mucosa that develops in association with Helicobacter pylori infection, resulting in impaired differentiation of the fundic gland cells, increased expression of trefoil factor family 2 (TFF2) and the formation of spasmolytic polypeptide (SP)-expressing metaplasia (SPEM), a preneoplastic lesion. However, it is still unresolved whether H. pylori-induced inflammation and the resultant reduction in parietal cell number or reduced parietal cell function per se reduces Shh expression. The present study was designed to clarify the expression of Shh and TFF2 in the context of parietal cell dysfunction in the absence of inflammation, using histamine H(2) receptor-knockout (H(2)R-null) mice and an acid exposure model. Age-matched H(2)R-null mice and wild-type (WT) mice were used. The expression of Shh and TFF2 mRNA was quantified by quantitative RT-PCR. Immunohistochemistry was also performed to detect the expression of Shh, TFF2 and cell markers. To study the effects of acid exposure, HCl solution was administered to the animals. The H(2)R-null mice exhibited higher gastric pH, increased TFF2 expression and reduced Shh expression. Impaired mucous neck-to-zymogenic cell differentiation was observed in the H(2)R-null mice. Furthermore, Shh expression increased in the presence of gastric acid and showed a significant correlation with gastric surface pH. In conclusion, our results suggest that persistent parietal cell dysfunction alone (suppressed gastric acid secretion), in the absence of inflammation or parietal cell loss caused by H. pylori infection, may be sufficient to down-regulate Shh expression in TFF2-overexpressing preneoplastic lesions of the gastric fundus. Since exposure to acid restored fundic Shh expression, appropriate gastric acid secretion may play an important role in the morphogen dynamics involved in the maintenance of gastric fundic gland homeostasis.
Abstract: Comparative genomics is a promising approach for identifying regulatory elements governing the unique spatio-temporal expression patterns of morphogenetic genes. Conserved noncoding genomic sequences are candidate regulatory elements. Here we performed a survey for conserved noncoding elements (CNE) nested within the SALL1 gene; mutations in this gene result in the Townes-Brocks syndrome. A comparison of the genomic sequence between humans and chicken revealed five CNE. Genomic fragments corresponding to each CNE were inserted into reporter cassettes consisting of eGFP cDNA and a minimal promoter. These constructs were electroporated into chick embryos during gastrula, neurula, and pharyngula stages. Among the five CNE that were examined, one 443 bp CNE exhibited tissue-specific enhancer activity. At the neurula stage, the eGFP signal was visualized in the prosencephalon. At the pharyngula stage, the eGFP signal was confined within the anterior neural ridge, which represents one of the morphogenetic centers regulating the patterning of the anterior neural plate. This report identifies, for the first time, an enhancer element of SALL1.
Abstract: The important human gastric pathogen Helicobacter pylori is the subject of many studies, and as a consequence it is frequently being transported between national and international laboratories. Unfortunately, common bacterial growth and transport media contain serum- and animal tissue-derived materials, which carry the risk of spreading infectious diseases. We have therefore developed a growth and transport medium for H. pylori, designated 'Serum- and Animal Tissue-Free Medium' (SATFM), which does not contain serum- or animal tissue-derived components. SATFM supported growth of H. pylori isolates to similar levels as obtained with serum-supplemented Brucella medium, and SATFM with 0.5% agar supported transport and storage of H. pylori strains, as 4/4 reference strains and 11/11 clinical isolates survived for at least 3 days at room temperature in SATFM, with some strains (2/15) even surviving for up to 7 days. In conclusion, SATFM can be used both as transport and growth medium for H. pylori. The formulation of SATFM may allow its use in international transport of H. pylori, and may also allow certified use in immunization studies requiring growth of H. pylori and other bacterial pathogens.
Abstract: BACKGROUND: One week of Helicobacter pylori eradication therapy is insufficient for healing of gastric ulcers. We examined the efficacy of rebamipide in gastric ulcer healing following 1 week of eradication therapy in a randomized, double-blind, placebo-controlled trial. METHODS: Patients with H. pylori-positive gastric ulcer were enrolled and received 1 week of eradication therapy, followed by 100 mg of rebamipide or placebo for 7 weeks. The primary end point was the gastric ulcer healing rate. RESULTS: Of the 309 patients entered in the trial, 301 completed H. pylori eradication therapy; 154 patients took rebamipide, and 147 took placebo. The healing rate in the rebamipide group was higher than that in the placebo group in the per-protocol analysis-80.0% (104/130) versus 66.1% (82/124) [95% confidence interval (CI), 3.1-24.7; P = 0.013)-and in a full analysis-70.1% (108/154) versus 60.5% (89/147) (95% CI, -1.1 to 20.3; P = 0.080). CONCLUSIONS: Compared with placebo, rebamipide significantly promoted gastric ulcer healing following 1 week of eradication therapy.
Abstract: Helicobacter pylori strains with reduced susceptibility to fluoroquinolones have a mutation at either codon 87 Asn or 91 Asp of the gyrA gene. A rapid test based on an allele-specific PCR (AS-PCR) was designed to detect the gyrA mutations. Clinical H. pylori isolates were obtained from the stomachs of 51 patients with H. pylori infections who showed treatment failure. The MICs of gatifloxacin (GAT) were determined by the agar dilution method. Identical genotyping results were obtained with AS-PCR and conventional PCR. The gyrA mutations of H. pylori causing reduced susceptibility to fluoroquinolones could be detected successfully by this method. A significant association was observed between the presence of mutations, as detected by AS-PCR, and the resistance of the strains to GAT. Moreover, genotyping by AS-PCR took less than 3 to 4 h. The AS-PCR method for the detection of gyrA mutations in H. pylori is useful for easy identification of fluoroquinolone-resistant strains of H. pylori.
Abstract: The discovery of Helicobacter pylori has already changed the natural history of peptic ulcer disease, with most patients being cured at their first presentation. Similarly, the incidence of gastric cancer and other diseases related to H. pylori are likely to be greatly reduced in the near future. Isolation of the spiral intragastric bacterium H. pylori totally reversed the false dogma that the stomach was sterile, and it taught us that chronic infectious disease can still exist in modern society. Helicobacter pylori's unique location, persistence, and evasion of the immune system offer important insights into the pathophysiology of the gut. Also, the fact that it was overlooked for so long encourages us to think "outside the box" when investigating other diseases with obscure etiologies. We should consider such provocative scientific ideas as bridges to the future disease control.
Abstract: BACKGROUND: With the increase in the frequency of clarithromycin-resistant Helicobacter pylori (H. pylori), there is rising concern about the decline of the eradication rate of this infection following treatment. The Tokyo Hp Study Group examined the eradication rate in response to a second-line regimen consisting of proton pump inhibitor (PPI), amoxicillin, and metronidazole by conducting a multicenter study in the Tokyo Metropolitan Area. MATERIALS AND METHODS: Two hundred and twenty-eight patients with H. pylori infection, in whom the first-line therapy with a PPI, amoxicillin, and clarithromycin administered for 1 week had failed to eradicate the infection, were enrolled in this study. These cases were randomly assigned to one of the two second-line regimens containing metronidazole (PPI/AM500 or PPI/AM750) administered for 1 week. 13C-urea breath test was performed as a diagnostic method test for H. pylori infection not earlier than 8 weeks after the second-line therapy. RESULTS: Intention-to-treat (ITT) and per-protocol (PP) analyses revealed an eradication rate of 87.6 and 90.6%, respectively, following PPI/AM500 treatment, and 86.9 and 88.6%, respectively, following PPI/AM750 treatment. Neither analysis revealed any significant difference in the eradication rate between PPI/AM500 and PPI/AM750 (p = .876 and .621, respectively). According to ITT and PP analyses, the eradication rates following treatment with PPI/AM500 were 85.2 and 88.5% with the use of lansoprazole, 62.5 and 62.5% with the use of omeprazole, and 93.2 and 96.5% with the use of rabeprazole, respectively. There was a significant difference in the eradication rates between PPI (omeprazole)/AM500 and PPI (rabeprazole)/AM500. In the case of PPI/AM750, the corresponding eradication rates were 84.8 and 87.0% with the use of lansoprazole, 92.9 and 92.9% with the use of omeprazole, and 92.9 and 92.9% with the use of rabeprazole, respectively. There were no significant differences in the eradication rates obtained with the use of the three PPIs. CONCLUSIONS: Both PPI/AM500 and PPI/AM750 administered for 1 week appeared to be highly effective second-line regimens for the treatment of H. pylori infection in Japanese patients. From the viewpoint of adverse events, PPI/AM500 appeared to be safe compared with PPI/AM750.
Abstract: Helicobacter pylori is associated with gastric disorders in humans and some experimental animals, and possesses the luxS/type 2 autoinducer (AI-2) system. The effects of a specific luxS mutation on the characteristics of H. pylori were examined. On 0.3 % agar medium, motility of H. pylori HPKY08 (luxS : : cat) was significantly lower than that of wild-type H. pylori TK1402. The luxS-complemented strain HPKY21 exhibited motility comparable to that of H. pylori TK1402. It was shown that the luxS/AI-2 system plays an important role in H. pylori motility. The luxS mutant exhibited a reduced infection rate relative to the wild-type parent strain TK1402 in a Mongolian gerbil model. At 3 months after oral inoculation, lower numbers of H. pylori were detected by semi-quantitative real-time reverse transcription PCR (qRT-PCR) in luxS(-) mutant-infected gerbils than in TK1402-infected gerbils. Gastric inflammation and increased antibody titre for H. pylori were observed in TK1402-infected gerbils only.
Abstract: The possible involvement of Toll-like receptor (TLR) genome DNA in the prolongation and relapse of inflammatory intestinal diseases and alcoholic hepatic diseases has been reported. In this study, we examined the relationship of mutations of the TLR 2, 4, 6 and 9 genomic DNA to recurrent or intractable gastritis or gastric ulcers. The subjects were 32 patients, including 6 with H. pylori (Hp)-positive gastritis, 4 with Hp-negative gastritis, 10 with Hp-positive tractable gastric ulcer, 5 with Hp-positive recurrent gastric ulcer after Hp eradication, and 7 with Hp-negative easily recurrent gastric ulcer after Hp eradication. Gastric mucosal tissue and peripheral blood specimens were collected from each of the patients. DNA was extracted from the tissue and blood specimens and subjected to electrophoresis by the PCR method, using the oligonucleotide primers of TLR 2, 4, 6 and 9. The gastric mucosal tissue specimens were collected endoscopically from the sites of the lesions. Subsequently, the presence or absence of genomic DNA mutations in the blood and tissue specimens was examined using a DNA sequencer. TLR 2, 4, 6 or 9 DNA mutations were not observed in any of the gastric mucosal or peripheral blood specimens obtained from patients with tractable gastritis or gastric ulcer, or from those with intractable gastric ulcer who were Hp-positive or Hp-negative or had become Hp-negative after eradication therapy. These data suggest that mutations of the TLR 2, 4, 6 and 9 genome DNA may not be involved in the recurrence, delayed healing or intractability of gastritis and gastric ulcers.
Abstract: Ghrelin, a novel gastrointestinal peptide with 28 amino acids, is secreted from the A-like cells of the gastric fundus. This peptide hormone does not only promote the release of growth hormone, but also stimulates food intake, gastric motility and cardiac output. Increased plasma ghrelin level has been reported in patients with upper gastrointestinal (GI) disease or in their disease animal model, suggesting its important role in the pathogenesis of upper GI disease.
Abstract: Thirty-seven patients with idiopathic thrombocytopenic purpura (ITP) were treated with a standard Helicobacter pylori eradication regimen irrespective of H. pylori infection. Our results indicate that platelet recovery results from the disappearance of H. pylori itself, and is mediated, in part, through suppression of anti-platelet autoantibody production.
Abstract: Gene mutations are essential to carcinogenesis. If an evident difference is observed in gastric mucosal chromosomal structure aberrations between H. pylori (Hp)-negative and Hp-positive gastric cancer patients, it may be interpreted as suggesting the involvement of Hp in gene mutations. This study was undertaken to compare chromosomal structural aberrations between Hp-negative and Hp-positive gastric cancer patients and to evaluate the effects of Hp eradication on chromosomal structures in clinical cases. The subjects of this study were 40 patients with gastric cancer divided into four groups: Group A was composed of 12 patients with Hp-negative gastric cancer (well-differentiated gastric cancer in 5 cases and poorly-differentiated in 7 cases), Group B of 8 patients with Hp-negative gastric cancer following Hp eradication (well-differentiated in 4 case and poorly-differentiated in 4 cases), Group C of 13 patients with Hp-positive gastric cancer (well-differentiated in 7 cases and poorly-differentiated in 6 cases) and Group D of 7 patients with gastric cancer (well-differentiated in 5 cases and poorly-differentiated in 2 cases) undergoing Hp eradication at subtotal gastrectomy. In each of the groups A, B and C, the structural chromosomal aberration such as loss of heterozygosity (LOH) and microsatellite instability (MSI) was analyzed. In Group D, changes in structural chromosomal aberrations after Hp eradication as compared to the pre-eradication structures were also analyzed. LOH and MSI were examined by PCR, using DNA extracted from the cancer-affected and intact gastric mucosal tissue specimens from each patient. In A, B and C groups, structural chromosomal aberrations were noted, and these aberrations tended to be more marked in cases of poorly-differentiated gastric cancer in each group. In terms of structural chromosomal aberrations, there was no marked difference between Group A and either Group B or C. Hp eradication resulted in no change in chromosomal structure as compared to the pre-eradication structure in Group D. These results suggest the possibility that Hp eradication does not affect chromosomal structures and Hp is involved in gastric carcinogenesis as an additive environmental factor rather than as a factor acting at the gene level.
Abstract: Isolation of the gastric spiral bacterium Helicobacter pylori totally reversed the false dogma that the stomach was sterile. In addition to its causal role in peptic ulceration, the newly identified bacterium has now been implicated in other gastric and even extragastric diseases, including chronic atrophic gastritis, gastric MALT lymphoma, gastric cancer, functional dyspepsia, idiopathic thrombocytopenic purpura (ITP), iron deficiency anemia, chronic urticaria, ischemic heart disease, and others. The majority of the reports are anecdotal, epidemiologic, or eradication studies, but there are also relevant in vitro studies. ITP represents one disease showing a strong link with H pylori infection. There are also accumulating data on the role of H pylori infection in iron deficiency anemia and ischemic heart disease. In summary, the association between H pylori infection and other extragut diseases is still controversial but worthy of further investigation.
Abstract: Although placebo response rates in clinical trials for functional dyspepsia (FD) are more than 30%, a recent meta-analysis based on randomized controlled trials (RCTs) showed that antisecretory drugs were more or less superior to placebos. On the other hand, large-scale RCTs on the efficacy of treatment with prokinetics on FD are still needed. Indications for antibiotic eradication therapy for Helicobacter pylori-positive FD are still controversial, but there seems to be a small but significant therapeutic gain achieved with H. pylori eradication. Since preprandial and postprandial symptomatic disturbances are very important targets for FD treatment, ghrelin, a novel appetite-promoting gastrointestinal peptide that also promotes gastric motility or basal acid secretion can be expected to be a therapeutic target. In the recently published Rome III classification, FD is redefined for patients with symptoms thought to originate from the gastroduodenal region, specifically epigastric pain or burning, postprandial fullness, or early satiation, and it is divided into the subcategories postprandial distress syndrome and epigastric pain syndrome. These new criteria are of value in clinical practice, for epidemiological, pathophysiological, and clinical research, and for the development of new therapeutic strategies.
Abstract: The genomic DNA of toll-like receptor (TLR) 2, TLR4, radioprotective 105, TLR6, and TLR9 were examined for mutations in 48 patients with gastric cancer. Of these, 22 had well-differentiated and 20 had poorly-differentiated adenocarcinomas, the latter group including 10 with signet ring cell carcinomas. The remaining 6 had gastric adenomas. Ten healthy volunteers with no family history of malignant diseases served as controls. DNA was extracted from peripheral blood and subjected to electrophoresis using PCR oligonucleotide primers. The resultant gel was analyzed with a DNA sequencer. None of the healthy volunteers, patients with gastric adenomas or those with well-differentiated gastric adenocarcinomas showed mutations. However, 8 of the 20 with poorly-differentiated gastric adenocarcinoma showed heterozygosity at the 135th position of the amino acid sequence of TLR4, and a mutation from threonine to alanine was found at this site. Analysis of the entire available amino acid sequence of TLR4 revealed that this mutation occurred at a leucine-rich repeat corresponding to one of its extracellular components. This suggests a disturbance in the protein phosphorylation reaction of TLR4, and that this disturbance is related to the development of poorly-differentiated gastric adenocarcinomas.
Abstract: Musashi-1 is involved in maintenance of neural stem cells. In the rat stomach, we found some cells in the luminal compartment of the mucosa that stained positively for Musashi-1. These cells were distinct from other cells of epithelial lineage, except for parietal cells, and coexpressed HES5. The Musashi-1-positive cells exfoliated after damage, while Musashi-1 expression in neck cells were upregulated, and proliferating cells diminished before reappearing and increasing in number thereafter. We conclude that a subpopulation of parietal cells acts as a source of Musashi-1, which contributes to rapid re-epithelization by restoration of stem cells and regulation of cell differentiation.
Abstract: A high resistance rate (47.9%) to gatifloxacin (GAT; 8-methoxy fluoroquinolone) in Helicobacter pylori (H. pylori) strains from 48 Japanese patients is observed after unsuccessful H. pylori eradication. A significant association between MICs for GAT equal to or above 1 microg/ml and mutations of the gyrA gene of H. pylori was demonstrated.
Abstract: BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) for early gastric cancer (EGC) has improved the success rate of en-bloc resection. We report here on a new technique using an external grasping forceps. PATIENTS AND METHODS: A total of 25 patients with suitable EGCs over 10 mm in diameter located in the gastric body were enrolled. After submucosal injection followed by circumcision of the lesion with a needle-knife, an external grasping forceps was introduced with the help of a second grasping forceps and anchored at the distal margin of the lesion. With gentle oral traction applied with this forceps, the lesion was dissected endoscopically in retroversion from the aboral side. RESULTS: The mean lesion size was 15.0 mm (range 10 - 25 mm). Using the technique described, all lesions could be resected en bloc with free margins. The mean procedure time was 45 min (range 30 - 80 minutes). No significant bleeding requiring blood transfusion or perforation occurred. CONCLUSIONS: This technical modification may simplify and shorten the gastric ESD procedure, except for lesions in distal locations, without compromising the efficacy.
Abstract: Zinc is an essential nutrient with a wide range of functions and closely involved in a variety of enzymatic processes of importance in glucose, protein and lipid metabolism. Ghrelin is the endogenous ligand of the G protein coupled growth hormone secretagogue receptor. The regulatory mechanism that explain the biosynthesis and secretion of ghrelin in the gastrointestinal tract has not been clarified. This study was undertaken to examine the effect of zinc supplementation on the streptozotocin (STZ)-induced diabetic rats, which exhibits ghrelin production and secretion, and lipid metabolism on the gastrointestinal tract. The animals were divided into four groups. Group I: Non-diabetic untreated animals. Group II: Zinc-treated non-diabetic rats. Group III: STZ-induced diabetic untreated animals. Group IV: Zinc-treated diabetic animals. Zinc sulfate was given to some of the experimental animals by gavage at a dose of 100 mg/kg body weight every day for 60 days. In the zinc-treated diabetic group, the blood glucose levels decreased and body weight increased as compared to the diabetic untreated group. Zinc supplementation to STZ-diabetic rats revealed the protective effect of zinc on lipids parameters such as total lipid, cholesterol, HDL-cholesterol and atherogenic index. There is no statistically change in ghrelin-immunoreactive cells in gastrointestinal tissue. But, it has found that zinc supplementation caused a significant reduction in densities of ghrelin-producing cells of fundic mucosa of zinc-treated diabetic animals as compared to untreated, non-diabetic controls. Zinc supplementation may contribute to prevent some complications of diabetic rats, biochemically.
Abstract: Alcohol dehydrogenase (ADH) participates in the formation of retinoic acid from retinol in various organs including the gastric mucosa. However, its clinical significance still remains to be clarified. In this study, we identified the ADH isoforms responsible for the retinoic acid formation among various ADH isoforms and examined associations among the ADH activities, the retinoic acid formation level, and morphological changes in the human gastric mucosa. Human gastric samples were endoscopically obtained from 67 male subjects. Morphological changes were assessed by the Sydney system and activities of class I, III, and IV ADH isoforms were determined in each specimen. In 26 cases, levels of all-trans retinoic acid (ATRA) formation from all-trans retinol were examined. Among activities of the three ADH isoforms, class IV ADH activity was solely associated with the ATRA formation level. This association was found even when subjects' age and Helicobacter pylori infection status were adjusted. As the degrees of inflammation, atrophy, and intestinal metaplasia increased, the class IV ADH activity as well as the potential for the ATRA formation decreased. Class IV ADH is a major enzyme in the retinoic acid supply in the human gastric mucosa, and the reduction of its activity was associated with decreasing retinoic acid supply and progression of inflammation, atrophy, and intestinal metaplasia in the gastric mucosa. In that retinoic acid is a key molecule for maintaining normal morphology, the reduction of class IV ADH activity may be involved in the pathogenesis of these morphological changes in the human gastric mucosa.
Abstract: AIM: Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor, and it plays a role in stimulating the growth hormone secretion, food intake, body weight gain and gastric motility. Eradication of Helicobacter pylori (H pylori) was shown to be associated with increase of the body weight. On the other hand, H pylori infection evokes the release of gastric IL-1beta. The present study was designed to investigate the involvement of the gastric IL-1 signal in the ghrelin dynamics in H pylori-colonized mice. METHODS: Twelve-week-old female IL-1-receptor type 1-homozygous-knockout mice (IL-1R1(-/-)) and their wild-type littermates (WT) were orally inoculated with H pylori (Hp group), while other cohorts received oral inoculation of culture medium (Cont group). Thirteen weeks after the inoculation, the mice were examined. The plasma and stomach ghrelin levels and the gastric preproghrelin mRNA were measured. RESULTS: Although the WT mice with H pylori infection showed a significantly decreased body weight as compared with that of the animals without H pylori infection, H pylori infection did not influence the body weight of the IL-1R1-knockout (IL-1R1(-/-)) mice. In the H pylori-infected IL-1R1(-/-) mice, the total and active ghrelin levels in the plasma were significantly increased, and the gastric ghrelin level was decreased. No significant differences were noted in the gastric preproghrelin mRNA expression. CONCLUSION: Ghrelin secretion triggered by H pylori infection might be suppressed by IL-1beta, the release of which is also induced by the infection, resulting in the body weight loss of mice with H pylori infection.
Abstract: BACKGROUND/AIMS: It has previously been reported that human-beta defensin 2 (hBD2) has a physiological role as a proinflammatory mediator in gastric mucosal inflammation as well as an antimicrobial peptide for Helicobacter pylori (Hp). The present study was conducted to evaluate the possibility of hBD2 as a molecular marker of gastric mucosal inflammation. METHODOLOGY: The subjects were 40 A1 to S2 gastric ulcer patients, with or without Hp infection. Biopsy specimens of the mucosa were obtained endoscopically before and after the administration of lansoprazole (LPZ) (15mg/day) or famotidine (FAM) (40 mg/day or 20 mg/day), consecutively, and each set of samples was divided into two groups; one group was subjected to RT-PCR to assess the expression of hBD2, and the other was subjected to immunohistochemical analysis for evaluating the expression of CD68. RESULTS: The expression of hBD2 was observed through the stage of gastric ulcer, from A1 to S1, regardless of the presence or absence of Hp infection, both before and after LPZ or FAM administration, and its intensity of expression decreasing as the number of CD68-positive cells decreased. The number of CD68-positive cells deceased as the severity of the ulcer increased from stage A1 to S2, regardless of the presence/absence of Hp infection. CD68-positive cells could hardly be observed in stage S2 gastric ulcers, in which hBD2 expression was also only scarcely noted. CONCLUSIONS: These results suggested the possibility that hBD2 may be a molecular marker of gastric mucosal inflammation, irrespective of the presence/absence of Hp infection.
Abstract: Functional dyspepsia (FD) refers to a broad range of chronic upper abdominal symptoms associated with food intake. A definitive treatment for FD has not yet been established, and the effect of Helicobacter pylori (H. pylori) eradication still remains under debate. The Gastrointestinal Symptom Rating Scale (GSRS) is a specific questionnaire for patients with gastrointestinal symptoms. The present study examined the quality of life (QOL) of patients with H. pylori-positive FD following H. pylori eradication. Methods: Sixty-eight patients with FD who gave informed consent were recruited for the study. H. pylori infection was diagnosed by the culture and histological methods, and the H. pylori eradication consisted of a 7-day course of lansoprazole, amoxicillin and clarithromycin. The overall success of the treatment was confirmed by a 13C urea breath test (UBT) conducted 3 months after the eradication. The GSRS questionnaire was administered to the patient just before the start of the eradication therapy and at 3 months after the therapy, just before the UBT was performed. Results: In successfully eradicated patients, the total GSRS and the abdominal pain score significantly decreased. In particular, the abdominal pain score and indigestion score were significantly decreased after successful eradication in patients with ulcer-like FD or dysmotility-like FD. Conversely, in patients in whom the eradication was unsuccessful, neither the total GSRS nor any of the individual symptom scores showed any significant change. Conclusion: Successful H. pylori eradication improved the QOL of patients with FD, in particular H. pylori-positive patients with ulcer-like FD or dysmotility-like FD.
Abstract: Sonic hedgehog (Shh) is a morphogen involved in many aspects of patterning of the gut during embryogenesis and in gastric fundic gland homeostasis in the adult. Intestinal metaplastic change of the gastric epithelium is associated with the loss of Shh expression, and mice that lack Shh expression show intestinal transformation of the gastric mucosa. The present study was designed to investigate the alteration of Shh expression in the stomach of an experimental model of Helicobacter pylori (H. pylori) colonization. Male Mongolian gerbils were inoculated with H. pylori and examined 4 and 51 weeks later. The level of Shh mRNA expression was determined by quantitative RT-PCR and in situ hybridization. Shh protein expression was determined by immunoblotting and immunohistochemistry. Shh was expressed in the parietal cells, zymogenic cells, and mucous neck cells of the gastric fundic glands of gerbils. Prolonged colonization by H. pylori led to extension of the inflammation from the antrum to the corpus of the stomach, with loss of Shh expression. Loss of Shh expression correlated with loss of parietal cells, disturbed maturation of the mucous neck cell-zymogenic cell lineage, and increased cellular proliferation. Shh expression is significantly reduced in H. pylori-associated gastritis. These data show for the first time that H. pylori infection leads to down-regulation of the expression of a morphogen with an established role (Shh) in gastric epithelial differentiation.
Abstract: We report on a case of chronic atrophic gastritis in which the serological markers of gastric diseases were strictly monitored for 2 years after successful Helicobacter pylori (H. pylori) eradication. A 31-year-old man with upper abdominal pain was diagnosed as having H. pylori infection. Laboratory examination revealed low serum levels of pepsinogen (PG) I, low PG I/II ratio, and low plasma levels of ghrelin. Upper gastrointestinal endoscopy revealed severe corpus-dominant atrophic gastritis. H. pylori eradication therapy was performed. Successful eradication was confirmed three months later by the 13C urea breath test. Decreased serum PG II levels and an increased serum PG I/II ratio were detected a week after completion of the eradication therapy. The serum anti-H. pylori IgG titer decreased to less than 75% of the baseline level by 24 weeks after completion of the eradication therapy. On the other hand, the plasma levels of total and active ghrelin showed no marked changes after successful eradication therapy. This is the first report of long-term follow-up of changes of the plasma ghrelin levels after H. pylori eradication therapy, the observations suggesting that reduction of plasma ghrelin levels cannot be achieved merely by H. pylori eradication, without resolution of the gastric atrophy.
Abstract: The proton pump inhibitors (PPIs) are a relatively new class of agents used for the treatment of acid-related disorders, including peptic ulcer diseases, reflux oesophagitis and Zollinger-Ellison syndrome, and in enhancing antibiotic therapy in the eradication of Helicobacter pylori in patients with peptic ulcer disease. The PPIs are the most potent gastric acid-suppressing agents currently in clinical use. According to the recent basic study, PPIs may act not only as potent acid suppressants, but also as anti-inflammatory or pro-regenerative agents. On the other hand, in the clinical field, general practitioners still tend to prescribe PPIs for unlicensed indications, such as non-ulcer dyspepsia and nonspecific abdominal pain. This article reviews the novel pharmacological action other than acid secretion and the diverse clinical usage of PPIs, in order to seek possible extensions of the use of this unique agent.
Abstract: Ghrelin, produced and secreted by the A-like cells of the stomach, stimulates growth hormone secretion, gastric motility, and food intake. Cysteamine inhibits the release of somatostatin and induces the formation of duodenal ulcers in rats. The present study was conducted to investigate the dynamics of ghrelin secretion in rats treated with cysteamine. Male Wistar rats (7 wk old) were administered three doses of cysteamine (400 mg/kg) orally; at 50 h after the first dose, duodenal ulcers were induced, and the plasma level of somatostatin and gastric density of somatostatin-immunoreactive cells were significantly reduced. The plasma total and active ghrelin levels were significantly higher in the cysteamine-treated rats than in the control rats, whereas the gastric ghrelin levels, number of gastric ghrelin-immunoreactive cells, and preproghrelin mRNA expression levels were significantly lower. Even at the time points of 2 and 10 h after the first dose of cysteamine, at which time no significant ulcer formation or antral neutrophil accumulation was yet noted, a significant increase in the plasma ghrelin level and decrease in the gastric ghrelin level were observed. Furthermore, although lansoprazole treatment attenuated the duodenal ulceration induced by cysteamine, the increase in the plasma level of ghrelin could still be demonstrated. Because an inverse correlation was found between the plasma ghrelin and somatostatin levels, the inhibition of somatostatin secretion may be associated with the increased ghrelin secretion. In conclusion, an increase in the plasma ghrelin level precedes the formation of duodenal ulcers in rats treated with cysteamine.
Abstract: BACKGROUND AND AIM: Helicobacter pylori is known to be a major pathogenic factor in the development of gastritis, peptic ulcer disease and gastric cancer. Recently, chicken egg yolk immunoglobulin Y (IgY) has been recognized as an inexpensive antibody source for passive immunization against gastrointestinal infections. The present study was designed to investigate the effect of anti-urease IgY on H. pylori infection in Mongolian gerbils. METHODS: H. pylori-infected Mongolian gerbils were administered a diet containing anti-urease IgY, with or without famotidine (F). After 10 weeks, bacterial culture and measurement of the gastric mucosal myeloperoxidase (MPO) activity were performed. In a second experiment, another group of gerbils was started on a diet containing F + IgY a week prior to H. pylori inoculation. After 9 weeks, these animals were examined. RESULTS: In the H. pylori-infected gerbils, there were no significant differences in the level of H. pylori colonization among the different dietary and control groups. However, the MPO activity was significantly decreased in the H. pylori group administered the F + IgY diet compared with that in the H. pylori group administered the IgY, F, or control diet. Furthermore, in the gerbils administered the F + IgY diet prior to the bacterial inoculation, inhibition of H. pylori colonization and suppression of the elevated gastric mucosal MPO activity were observed. CONCLUSIONS: Oral administration of urease-specific IgY not only inhibited H. pylori disease activity in H. pylori-infected gerbils, but also prevented H. pylori colonization in those not yet infected. These encouraging results may pave the way for a novel therapeutic and prophylactic approach in the management of H. pylori-associated gastroduodenal disease.
Abstract: OBJECTIVE: Although curative treatment of Helicobacter pylori infection markedly reduces the relapse of peptic ulcers, the details of the ulcers that do recur is not well characterized. The aim of this study is to describe the recurrence rate and specific features of peptic ulcers after cure of H. pylori infection. METHODS: This was a multicenter study involving 4940 peptic ulcer patients who were H. pylori negative after successful eradication treatment and were followed for up to 48 months. The annual incidence of ulcer relapse in H. pylori-cured patients, background of patients with relapsed ulcers, time to relapse, ulcer size, and site of relapsed ulcers were investigated. RESULTS: Crude peptic ulcer recurrence rate was 3.02% (149/4940). The annual recurrence rates of gastric, duodenal and gastroduodenal ulcer were 2.3%, 1.6%, and 1.6%, respectively. Exclusion of patients who took NSAIDs led annual recurrence rates to 1.9%, 1.5% and 1.3%, respectively. The recurrence rate was significantly higher in gastric ulcer. Recurrence rates of patients who smoked, consumed alcohol, and used NSAIDs were significantly higher in those with gastric ulcer recurrence compared to duodenal ulcer recurrence (e.g. 125 of 149 [83.9%] relapsed ulcers recurred at the same or adjacent sites as the previous ulcers). CONCLUSIONS: Curative treatment of H. pylori infection is useful in preventing ulcer recurrence. Gastric ulcer is more likely to relapse than duodenal ulcer. Recurrent ulcer tended to recur at the site of the original ulcers.
Abstract: BACKGROUND: The gastric mucosal alcohol dehydrogenase activity was reported to be reduced in subjects with Helicobacter pylori-associated chronic gastritis. However, the role of H. pylori infection in the metabolism of ingested ethanol has not been fully elucidated. AIM: To clarify whether H. pylori infection exerts any influence on the metabolism of small amounts of ethanol in humans. SUBJECTS AND METHODS: 13C-ethanol (100 microL) diluted in 100 mL water was administered orally to 22 healthy volunteers before breakfast. Breath samples were then collected every 10 min for up to 60 min. The content of 13CO2 (per thousand) was analysed using a mass-spectrometric method. The excretion rate of labelled CO2 was analysed by calculation of the following mathematical parameters, i.e., Tmax, Cmax, and AUC60. RESULTS: In H. pylori-negative controls (n = 11), the Tmax, Cmax and AUC60 were 0.47 +/- 0.13, 19.1 +/- 6.0 and 13.7 +/- 4.5, respectively. The corresponding values were 0.53 +/- 0.16 (n.s.) and 12.7 +/- 4.8 (P < 0.05) and 9.0 +/- 3.6 (P < 0.05), respectively, in subjects with H. pylori infection (n = 11). Successful eradication of H. pylori was associated with significant increase of these parameters (Cmax)and AUC60, n = 6). CONCLUSIONS: The metabolism of small amounts of ethanol is attenuated in subjects with H. pylori infection.
Abstract: BACKGROUND/AIMS: Problems after Helicobacter pylori (Hp) eradication therapy include recurrence of Hp-negative peptic ulcers. We investigated the pathophysiological characteristics of Hp-negative recurrent ulcer scars, and performed proton pump inhibitor (PPI) maintenance therapy as a new therapy for prevention of recurrence in patients with Hp-negative recurrence after Hp eradication and investigated its usefulness. METHODOLOGY: The subjects were 21 patients with Hp-negative recurrent peptic ulcers after Hp eradication (gastric ulcer: 19, duodenal ulcer: 2) and 25 patients with non-recurrent ulcers (gastric ulcer: 20, duodenal ulcer: 5). The mucosa from the ulcer scar lesion was endoscopically obtained from patients, and HE staining, CD68 immunohistochemical staining, and investigation of the mucosal expression levels of TNF-alpha and IFN-gamma were performed by ELISA. Patients with recurrence after eradication were divided into two groups at the time of ulcer scar after the first treatment, and received maintenance therapy: the intermittent treatment group that received lansoprazole (LPZ), 30 mg/day, on two days on weekends (gastric ulcer: 9, duodenal ulcer: 1) and the ranitidine (RAN) 150 mg/day daily treatment group (gastric ulcer: 8, duodenal ulcer: 1). RESULTS: Infiltration of CD68-positive inflammatory cells was observed in the lamina propria mucosae over the epithelial layer in ulcer scars of the Hp-negative recurrent ulcer group compared with the non-recurrent ulcer group, and TNF-alpha and IFN-gamma significantly increased (27.22+/-6.23 pg/mg, 52.12+/-5.41 pg/mg vs. 4.23+/-2.14 pg/mg, 7.11+/-3.06 pg/mg, P<0.001). In the RAN maintenance therapy group, the ulcer recurred within 10 months in all patients, while the ulcer recurred in only one patient in the intermittent LPZ treatment group. CONCLUSIONS: These results suggested that the pathophysiological characteristic of Hp-negative recurrent ulcer scar lesions after eradication was infiltration of inflammatory cells, mainly monocytes/macrophages, in the lamina propria mucosae over the epithelial layer, and this may be a key factor in ulcer recurrence. Furthermore, intermittent PPI therapy using LPZ may be a useful maintenance therapy for prevention of recurrence in these cases.
Abstract: BACKGROUND/AIMS: Gastric mucosa of patients with chronic renal failure on regular hemodialysis is known to retain fundic glands relatively intact, but no evidence for regeneration of fundic glands by hemodialysis has been provided to date. This study was performed to investigate endoscopically and histopathologically if hemodialysis to treat renal failure would regenerate the background gastric mucosa and to elucidate factors associated with the mucosal regeneration. METHODOLOGY: First, the relationship between duration of hemodialysis and the degree of atrophy of the background gastric mucosa was investigated in patients with chronic renal failure treated and not treated by hemodialysis. Treated patients were further divided into long-term group treated for 4 years or longer and short-term group treated for shorter than 4 years. The degree of atrophy of gastric mucosa was evaluated by hematoxylin-eosin staining, PAS-alucian blue staining and immunohistochemical staining to detect Ki-67 expression using biopsy specimens obtained from gastric mucosa. The labeling index is the proportion of positively labeled cells with respect to the total number of cells. The proliferative index was calculated by multiplying the labeling index and the proliferative zone (length of the area between the uppermost and lowest labeled cells). Serum gastrin, glucagon and cholecystokinin were assayed as well as urine epidermal growth factor to elucidate factors associated with regeneration of gastric mucosa. Helicobacter pylori infection was examined by ELISA. RESULTS: In the long-term group, the degree of atrophy of gastric mucosa was endoscopically evaluated to be C1 type. In both of the two hemodialysis groups, endoscopically identified fundic gland region was histologically confirmed to be fundic glands by both hematoxylin-eosin staining and PAS-alucian blue staining. Epithelial cell proliferative index was significantly higher in long-term and short-term hemodialysis group than non-hemodialysis group (P=0.0001). No significant difference in serum gastrin, glucagon and cholecystokinin as well as urine epidermal growth factor was detectable among the three groups. Most patients of both hemodialysis groups were H. pylori-negative. CONCLUSIONS: A possibility of regeneration of the background gastric mucosa in proportion to the duration of hemodialysis was suggested on the basis of histopathological evidence. The observed regeneration of gastric mucosa was ascribable to elimination of factors associated with atrophy of gastric mucosa including H. pylori by hemodialysis.
Abstract: We have demonstrated anti-proliferation and anti-metastasis effects of both interferon-alpha and a histone deacetylase inhibitor, sodium butyrate, on human liver cancer cell lines. In this study, invasive ability of human liver cancer cell lines through the matrix-coated membrane was examined and inhibitory effect of interferon-alpha and sodium butyrate was investigated. Among six human liver cancer cell lines, HLE and HLF showed high invasive ability using the Matrigel invasion assay. This invasion ability was significantly inhibited by pretreatment of the cells with 1000 IU/ml of interferon-alpha or 2 mM of sodium butyrate. Gelatin zymography and the matrix metalloproteinase-2 and -9 activity assay showed that these two cell lines produce active- and pro-matrix metalloproteinase-2 and -9, and their activity was significantly reduced by pretreatment with both agents. Real-time quantitative reverse transcription-polymerase chain reaction showed decrease in matrix metalloproteinase-1 mRNA levels by pretreatment with both agents, but mRNA levels of tissue inhibitor of matrix metalloproteinase-1 and -2 were differently modulated by interferon-alpha and sodium butyrate. These results suggest that interferon-alpha and sodium butyrate reduce a chance of invasion and metastasis of human liver cancer cells by inhibiting matrix metalloproteinase activity, although its inhibitor is differently regulated.
Abstract: Cell proliferation and migration are important repair mechanisms in cell defect type mucosal injuries, such as peptic ulcers. To evaluate the level of cell restitution in vitro, we established a normalized assay system for analyzing the area of a tissue defect created in the center of a cultured cell layer. Although proton pump inhibitors are known to be potently effective in the treatment of peptic ulcers by inducing acid suppression, they are also effective in low-acid conditions, such as in gastric ulcers associated with severe atrophic gastritis of the corpus. The present study was designed to examine the pH-independent effect of lansoprazole (LPZ) on cell restitution in vitro. The mouse gastric mucosal cell line, GSM06, was cultured to confluence. A 4-fluoric ethylene-tipped aluminum stick was then used to produce a cell-free area in the center of the culture well. After measuring the area of the cell defect using a digital analyzer equipped with an inverted microscope, LPZ was added to each well; the area of the residual cell defect was then measured 6 and 24 hours after LPZ administration. To investigate the involvement of the p44/p42 mitogen-activated protein kinase (MAPK) and p38 MAPK in this process, PD98059 (a MEK inhibitor) or FR167653 (a p38 MAPK inhibitor) was added to the cell cultures. In a separate experiment, GSM06 cells were cultured to the subconfluent level, each test agent was added, and the cell number in each well was measured using an MTT assay 16 hours after the administration of the agents. Six hours after the addition of LPZ, a slight but significant increase in the cell restitution rate was observed in the LPZ-treated groups compared with that in the control group. After 24 hours, a further significant increase in the cell restitution rate was observed in the LPZ groups compared with that in the control group. While the addition of PD98059 significantly attenuated the cell restitution rate in the LPZ groups, the addition of FR167653 had no such effect. The total cell number in the subconfluent cell cultures was significantly increased in the LPZ-treated groups compared with that in the control group. In conclusion, LPZ promotes the healing of injured gastric mucosal cells following injury by enhancing cell proliferation and migration. Furthermore, the mechanism by which cell proliferation and migration is promoted by LPZ may involve the activation of p44/p42 MAPK.
Abstract: BACKGROUND: and aim: Although ghrelin, a novel growth hormone releasing peptide localised mainly in the gastric fundus, is reported not only to accelerate food passage and gastrointestinal motility but also to affect appetite and weight control, regulation of gastric ghrelin secretion under the conditions of gastric Helicobacter pylori infection is unknown. The present study was designed to investigate plasma and gastric ghrelin levels in Mongolian gerbils with H pylori colonisation of the gastric mucosa. METHODS: Gerbils orally inoculated with H pylori were examined after inoculation. To examine preproghrelin mRNA expression in the gastric mucosa, cDNA encoding the gerbil preproghrelin and glyceraldehyde-3-phosphate dehydrogenase homologue was isolated and a quantitative reverse transcription-polymerase chain reaction system was established. RESULTS: In gerbils showing H pylori colonisation (H pylori group), expression of preproghrelin mRNA and total ghrelin levels were significantly decreased 17 and 23 weeks later (p<0.01). Although the number of ghrelin immunoreactive cells decreased as the stomach weight increased, the gastric contents of total and active ghrelin in this group were the same as those in controls. Gastric myeloperoxidase activity showed a positive correlation with plasma ghrelin levels. On the other hand, at 17 weeks, plasma ghrelin levels were significantly increased in the H pylori group (p<0.05), suggesting a compensatory increase in secretion of the peptide at this time point. CONCLUSION: The present experimental study demonstrated that gastric and plasma ghrelin dynamics are altered in response to H pylori infection.
Abstract: BACKGROUND: Helicobacter pylori resistance to clarithromycin, probably due to the frequent use of this antibiotic for the treatment of other diseases, is the greatest obstacle against its eradication. AIM: To clarify the prevalence of clarithromycin-resistant H. pylori in patients with non-tuberculous mycobacterial lung disease receiving multiple antibiotic treatment, including clarithromycin. METHODS: We enrolled 88 patients with non-tuberculous mycobacterial lung disease; 29 underwent upper gastrointestinal endoscopy for the diagnosis of H. pylori infection prior to treatment, and 60 underwent it during treatment. The diagnosis of H. pylori infection was confirmed by histological examination, urease test and microaerobic bacterial culture. The minimum inhibitory concentration of clarithromycin was determined and the DNA was analysed for each of the isolated H. pylori strains. RESULTS: Patients during the treatment had a high prevalence rate of clarithromycin-resistant H. pylori (100%). Analysis of DNA of the clarithromycin-resistant H. pylori isolates revealed point mutations at A2142G or A2143G. Moreover, a linear correlation was found between the total cumulative dose of clarithromycin and the minimum inhibitory concentration. CONCLUSION: All patients with non-tuberculous mycobacterial lung disease being treated long-term with multiple antibiotics, including clarithromycin, harboured clarithromycin-resistant H. pylori in the stomach. Therefore, eradication of H. pylori before commencement of long-term therapy including clarithromycin should be recommended.
Abstract: BACKGROUND: Although Helicobacter pylori infection is now acknowledged as a major promoter of gastric cancer in humans, the carcinogenetic process of this effect has not been fully elucidated. Precancerous lesions such as intestinal metaplasia, enhanced proliferation of epithelial cells and elevated level of growth factors have been postulated to play a role. AIM: To analyse a relationship between gastric mucosal proliferation, mucosal content of hepatocyte growth factor and prevalence of intestinal metaplasia before and after successful H. pylori eradication therapy. METHODS: We evaluated 25 H. pylori-eradicated patients. At initial endoscopic examination, two biopsy tissue samples each were obtained from the antrum and great curvature of the corpus. Tissue content of hepatocyte growth factor and neutrophil myeloperoxidase were measured using an ELISA method, and histological assessment of intestinal metaplasia (haematoxylin and eosin) and proliferating cells (Ki-67 immunostaining) was performed. The patients were treated with a 1-week course of triple therapy. At 10 months after successful eradication, biochemical and histological assessments were repeated. RESULTS: Among all patients (n = 25), no intestinal metaplasia was detected in the corpus mucosa, but was observed in 10 patients (40%) in the antrum. This prevalence ratio was not changed after eradication. A slight decrease in HGF content was demonstrated in both sites, but the level of antral hepatocyte growth factor was significantly decreased in patients with intestinal metaplasia but not in those without. Proliferative index (Ki-67 positive cells/epithelium) was decreased after eradication therapy in both sites. An increase in proliferative index was observed in the antrum with intestinal metaplasia compared with that without, which significantly decreased after eradication therapy. CONCLUSIONS: H. pylori eradication therapy in the present study afforded an inhibitory effect on epithelial cell proliferation and on the mucosal content of HGF.
Abstract: The capacity of lymph nodes to eradicate cancer is a controversial issue. The purpose of this study was to determine the interplay between tumor growth and host resistance at early stages of lymph node metastasis. A metastasis model was made in the rat mesenteric lymph node, and migration of cancer cells was visualized in vivo. The lymph node was removed for histologic analysis and cytokine measurement. Migrant cancer cells were initially arrested in the marginal sinus. After an initial increase, the number of cancer cells in the marginal sinus declined until 48 hours after inoculation. Germinal centers and lymphoid cells in the medulla proliferated before 48 hours. ED3(+) macrophages incorporated apoptotic cancer cells, but significant cancer proliferation occurred after 4 days. Lymph nodes depleted of macrophages were massively invaded by cancer cells. Tumor necrosis factor alpha and interleukin (IL)-1beta in the nodes transiently increased after 1 hour and 3 hours, respectively, and were expressed in ED3(+) and ED2(+) macrophages, respectively. These changes were followed by a transient increase in IL-2. Interferon-gamma and IL-12 did not increase during the early stages of metastasis, but they decreased after 48 hours. In conclusion, the marginal sinus constitutes a mechanical barrier against cancer cell passage. Early pathological manifestations in the regional lymph node are consistent with those in cancer patients with improved survival. Parasinus macrophages play a role in the transient antimetastatic capability of the node, and cytokines secreted by these cells increased at the early stages of metastasis. Deterioration of cytokine induction may be responsible for subsequent cancer proliferation.
Abstract: BACKGROUND: The effects of bacterial eradication therapy cannot be fully explained simply by elimination of the target bacteria, if one considers the effects of eradication therapy in H. pylori-negative cases of low-grade malignancy MALTomas of the rectum. The present study was undertaken to examine the possibility and mechanism of direct induction of apoptosis of the tumor cells by the antibiotics used for bacterial eradication therapy. MATERIALS AND METHODS: A B cell lymphoma cell line (300-19) derived from BALB/c mice was co-cultured with amoxicillin or clarithromycin, and amoxicillin and clarithromycin at concentrations equal to or 1/10 x MIC of either drug. Cells co-cultured with 1/100 x MIC of the standard anti-tumor agents, adriamycin, vincristine and cyclophosphamide, served as positive controls. Cells cultured without any antibiotic or anti-tumor agent served as controls. In each group, the following analyses were performed: (i) the time-course of changes in the cellular morphology, (ii) the time-course of occurrence of DNA fragmentation, (iii) the appearance of apoptotic changes as evaluated by trypan blue staining, (iv) Bcl-2 expression as examined by immunoblotting; and (v) the expression of TNFR1, Fas, FasL and caspase-3, -8 and -9, as evaluated by immunoblotting. RESULTS: Cells treated with amoxicillin and clarithromycin showed the formation of apoptotic bodies, and degeneration and detachment of the cells in a dose-dependent manner. DNA fragmentation was induced in these cells to a degree similar to that seen in cells treated with the anti-tumor agents. Trypan blue staining also demonstrated apoptosis of the cells and loss of cell viability. Bcl-2 expression was seen only in the control group and FasL was never seen, while the expression of TNFR1, Fas and caspase-3, -8 and -9 was seen in the amoxicillin-treated group, clarithromycin-treated group, amoxicillin and clarithromycin-treated group and the positive control group. CONCLUSION: Antibiotics used for the eradication of H. pylori can also directly induce apoptosis in mouse B cell lymphoma cells, an action which involves the TNF system.
Abstract: Human beta-defensins (HBDs) recognized in the stomach include HBD1, which is the constitutional human beta-defensin (HBD), and HBD-2 and HBD-4, which are inducible HBDs. HBD-2 is an antimicrobial peptide that is involved in host defences against bacterial infections, such as Helicobacter pylori (H.pylori) in infection of the gastric mucosal epithelium. We examined the pathophysiological role of HBD-2, besides their roles as antimicrobial peptides. The materials used for the study consisted of gastric mucosal tissue specimens collected endoscopically from patients with conditions such as chronic gastritis associated with H. pylori infection, and gastric ulcers and gastritis due to non-steroidal anti-inflammatory drugs (NSAIDs) with or without H. pylori infection. We investigated the expression of HBD-2 and NF-kappaB by RT-PCR and immunoblotting, and the relation between the localization of HBD-2 and follicular dendritic cells (FDCs) by immunohistochemistry. Expression of HBD-2 was recognized in all the mucosal tissue specimens, irrespective of the presence or absence of H. pylori infection. All of the mucosal specimens expressing HBD-2 also revealed expression of NF-kappaB. In consecutive immunohisto-chemical staining, while expression of HBD-2 was observed in the gastric mucosal epithelium, FDCs were found to be localized in the lamina propria mucosae under the epithelial cell layer. These data suggested that in addition to being antimicrobial peptides, HBD-2 may also have a pathophysiological role as proinflammatory mediators, and that the HBD may act as proinflammatory mediators in concert with the dendritic cells (DC) by transmitting a signal from the mucosal surface to the lamina propria mucosae, which seems to be the original site of gastric mucosal damage.
Abstract: BACKGROUND/AIMS: Ghrelin, a novel growth-hormone-releasing peptide, has been reported to be localized mainly in the A-like cells in the gastric fundic mucosa. With the extension of gastric inflammation caused by H. pylori infection, gastric mucosal atrophy extends from the antrum to the corpus, which is the predominant site of localization of the ghrelin-producing A-like cells. The present study was designed to investigate the correlation between the plasma ghrelin levels and the extent of gastric mucosal atrophy in patients with chronic gastritis caused by H. pylori infection. METHODOLOGY: Sixty-nine patients with dyspeptic symptoms were enrolled for the study. Of these, 41 patients were confirmed to become negative for H. pylori after therapy to eradicate the infection. The other 28 patients were diagnosed as positive for H. pylori infection. Blood samples were collected from all the patients after 12 hours of fasting, before upper gastrointestinal endoscopy was performed. The plasma levels of total and active ghrelin, as well as the serum levels of pepsinogen I (PGI) and pepsinogen II (PGII) were measured by radioimmunoassay. Based on endoscopic assessment, the atrophic changes in the gastric mucosa were classified as open-type atrophy or closed-type atrophy. RESULTS: There were no significant differences in the plasma total and active ghrelin levels between H. pylori-positive and H. pylori-eradicated (negative) patients. The serum levels of PGI correlated well with the plasma levels of total ghrelin (p<0.01, r=0.38) and active ghrelin (p<0.05, r=0.29). The ratio of serum PGI to PGII level (PG I/II ratio) also correlated well with the plasma level of total ghrelin (p<0.05. r=0.31) and active ghrelin (p<0.05, r=0.27). The plasma levels of total as well as active ghrelin were significantly decreased in patients with low PG levels as compared with those in patients with high PG levels (PGI > 70 ng/mL or PGI/II >3.0). The plasma levels of total as well as active ghrelin were also significantly decreased in patients with endoscopically diagnosed open-type atrophy as compared with those in patients with endoscopically diagnosed closed-type atrophy (p < 0.01), especially in the H. pylori-eradicated cohorts. CONCLUSIONS: The plasma levels of ghrelin, which correlated well with the serum levels of PGI as well as the PGI/II ratio, decreased with increasing extent of gastric mucosal atrophy, suggesting that it could be a potentially useful non-invasive marker for chronic atrophic gastritis.
Abstract: A 47-year-old woman was admitted to our hospital with complaints of fever, upper abdominal pain, and back pain. The serum amylase, C-reactive protein (CRP), and IgG (especially IgG4) were elevated, and abdominal computed tomography (CT) revealed diffuse enlargement of the pancreas and pseudocysts. Endoscopic retrograde pancreatography (ERP) revealed diffuse irregular narrowing of the main pancreatic duct. Histopathological examination of the pancreatic tissue showed fibrotic change with lymphocytic infiltration. Based on these findings, we diagnosed this case as a case of autoimmune pancreatitis. This case also fully satisfied the diagnostic criteria for autoimmune pancreatitis established by the Japan Pancreas Society in 2002. Few reports have been published on cases of autoimmune pancreatitis complicated by the formation of pseudocysts in the pancreas. We, therefore, report this case here to emphasize that cases of autoimmune pancreatitis can be complicated by the development of pseudocysts.
Abstract: Recently, chicken egg yolk was recognized as an inexpensive antibody source, and the therapeutic usefulness of egg yolk immunoglobulin Y (IgY) in oral passive immunization has been investigated. Although multiple antibiotic treatments eradicate most Helicobacter pylori (H. pylori) infections, therapy fails in 10-15% of cases due to the development of drug resistance. Consequently, it is important that new, more broadly based therapies for the treatment of H. pylori infection should be identified. The present study evaluated the effect, on H. pylori infection, of IgY prepared from egg yolk of hens immunized with H. pylori urease (anti-HpU IgY). Seventeen asymptomatic volunteers diagnosed as H. pylori-positive by the 13C-urea breath test (UBT) were orally administered anti-HpU IgY for 4 weeks. Four weeks later, UBT values were significantly decreased although no case showed H. pylori eradication. An H. pylori-positive 53-year-old female gastritis patient administered anti-HpU IgY plus lansoprazole for 8 weeks showed a decrease in serum pepsinogen (PG) I and UBT values as well as an increase in the PG I/II ratio. In conclusion, anti-HpU IgY may mitigate H. pylori-associated gastritis and partially attenuate gastric urease activity. Furthermore, anti-HpU IgY combined with antacids appears to ameliorate gastric inflammation. These encouraging results may represent a novel approach to the management of H. pylori-associated gastroduodenal disease.
Abstract: BACKGROUND/AIMS: The perforin/granzyme and Fas/Fas ligand pathways are two known major pathways of cytotoxic T lymphocyte-mediated apoptosis. We designed a clinical study to examine whether cytotoxic T lymphocyte-mediated apoptosis associated with peptic ulcer formation may occur via either or both of these two pathways. METHODOLOGY: Mucosal biopsy specimens were obtained endoscopically from the marginal zone of active stage gastric and duodenal ulcers in patients with or without Helicobacter pylori infection. RT-PCR, immunoblotting and immunohistochemistry were used to study the samples for the expression of apoptotic cells, perforin, granzyme B, Fas, Fas ligand and caspase 3. RESULTS: Apoptotic cells (Tunnel positive cells) appeared in the marginal zone of gastric and duodenal ulcers with and without H. pylori infection. Perforin/granzyme B and caspase 3 were expressed consistently, however Fas ligand was not. Furthermore, the immunohistochemical findings demonstrated apoptotic changes of target cells caused by perforin/granzyme B. CONCLUSIONS: These results suggest that the main pathway of cytotoxic T lymphocyte-mediated apoptosis in peptic ulcer formation is the perforin/granzyme pathway irrespective of H. pylori infection.
Abstract: BACKGROUND AND AIM: Although rabeprazole (RPZ), a proton pump inhibitor, has been reported to have a bactericidal effect on Helicobacter pylori (H. pylori), no studies have been conducted regarding the effect of RPZ on gastric mucosal lesion formation caused by this bacterium. In the present study, we investigated the effect of RPZ on H. pylori-associated gastric mucosal lesion formation. METHODS: Sixty-two male Mongolian gerbils were inoculated with H. pylori (ATCC43504) (Hp group) and 60 gerbils with the culture media alone (control group). Some gerbils in the Hp group and in the control group were injected with RPZ (1 mg/kg/day, for 7 days) at the 5th week. Gerbils were evaluated at the 12th, 24th and 48th weeks. RESULTS: In the Hp group, all gerbils were persistently infected for 24 weeks, but 36% became negative for H. pylori at the 48th week. In the Hp + RPZ group, 18% of gerbils at the 12th week, 40% at the 24th week, and 80% at the 48th week, became negative for H. pylori. The level of neutrophil infiltration was significantly decreased in the Hp + RPZ group in comparison to the Hp group, possibly through the effects of RPZ on initial bacterial colonization and resultant inflammation. Even in the gerbils that became H. pylori-negative, the level of neutrophil infiltration was lower in the Hp + RPZ group than in the Hp group. RPZ treatment significantly increased the level of the reduced form of glutathione (GSH) at the 48th week. The elevated levels of the reduced form of GSH may have been reduced by an antioxidation process in the H. pylori-positive Hp + RPZ group. CONCLUSION: Administration of RPZ not only inhibited gastric H. pylori colonization, but also reduced gastric mucosal inflammation in gerbils, possibly through its antibacterial action as well as pharmacological recruitment of the reduced form of GSH.
Abstract: Helicobacter pylori (H. pylori) is a spiral shaped bacterium that resides in the stomach mucosa. Isolation of H. pylori from the stomach mucosa changed the erstwhile widely held belief that the stomach contains no bacteria and is actually sterile. Once H. pylori is safely ensconced in the mucus, it is able to neutralize the acid in the stomach by elaborating an enzyme called urease. Urease converts urea, of which there is an abundant supply in the stomach (derived from saliva and the gastric juice), into bicarbonate and ammonia, which are strong bases. These bases form a cloud of acid-neutralizing chemicals in the vicinity of the organisms, protecting them from the acid in the stomach. This urea hydrolysis reaction is utilized for the diagnosis of H. pylori infection in the urea breath test (UBT) and the rapid urease test (RUT). In Japan, both invasive tests, such as bacterial culture, histopathology and RUT, and non-invasive tests such as UBT and serology are conducted for the diagnosis of H. pylori infection. For confirming the results of eradication therapy, UBT is considered to be the most sensitive and specific. In order to treat H. pylori infection, a new one-week triple therapy regimen (lansoprazole or omeprazole + amoxicillin + clarithromycin) has been approved for use in patients with peptic ulcer disease in Japan. As for H. pylori eradication in the case of other diseases in which the bacterium has been implicated (e.g., chronic atrophic gastritis, gastric MALT lymphoma, gastric cancer, non-ulcer dyspepsia, chronic urticaria, idiopathic thrombocytopenic purpura (ITP)), further basic and clinical investigation is required.
Abstract: Ceramides have emerged as key participants in the signaling pathway of cytokines and apoptosis. We previously revealed that phorbol 12-myristate 13-acetate (PMA) induced experimental ulcers in rat gastric mucosa. In this study, we investigated the role of ceramide in ulcer formation and its relation to the activation of transcription factors and apoptosis. PMA was subserosally injected to rat glandular stomach. Fumonisin B1 (FB1), an inhibitor of ceramide synthase, was administered together with the PMA. The time course of ceramide content was quantified using thin layer chromatography and the number of apoptotic cells was determined by immunohistochemistry. The activation of transcription factor nuclear factor-kappaB (NF-kappaB) or activator protein-1 (AP-1) was evaluated using an electrophoretic mobility shift assay. The administration of FB1 attenuated PMA-induced gastric ulcer formation in a dose-dependent manner. Before the ulcers became obvious, the ceramide content (C18 and C24 ceramide) increased significantly in the gastric wall. The activation of NF-kappaB and AP-1 and an increase in the number of apoptotic cells were also observed. Both of these were significantly inhibited by the coadministration of FB1. However, NF-kappaB inhibitors attenuated gastric ulcer formation without affecting the ceramide content or the number of apoptotic cells. Ceramide formation in the stomach significantly contributes to PMA-induced tissue damage, possibly via the activation of transcription factors and an increase in apoptosis in the gastric mucosa. However, after the increase in ceramide levels, the NF-kappaB and apoptosis pathways may be separately involved in ulcer formation.
Abstract: Ghrelin is a novel gastrointestinal peptide that stimulates growth hormone secretion, food intake, and body weight gain. Increased ghrelin secretion has been reported in such negative energy states as starvation and low body weight. We investigated the dynamics of ghrelin in rats with streptozotocin-induced diabetes, because they present reduced body weight and hyperphagia. The plasma ghrelin levels and gastric preproghrelin mRNA expression levels of the diabetic rats increased significantly and their gastric ghrelin levels decreased significantly. Negative energy balance may enhance preproghrelin mRNA expression and ghrelin secretion into the bloodstream.
Abstract: Deregulated cell turnover in Helicobacter pylori (H. pylori)-colonized gastric mucosa has been suggested to be linked to the gastric carcinogenesis pathway. We previously reported attenuation of apoptosis and enhancement of cellular proliferation in the H. pylori-colonized gastric mucosa of Mongolian gerbils as compared to that in mice, which might reflect a specific link between H. pylori colonization and carcinogenesis in the Mongolian gerbils; the difference between the two strains could be attributable to differences in the host genetic background. Inducible-type nitric oxide synthase (iNOS) is thought to participate in not only the inflammatory response, but also in the regulation of gastric mucosal cell turnover in H. pylori-colonized gastric mucosa. Thus, the present study was designed to examine gastric leukocyte activation and epithelial cell apoptosis in the gastric mucosa following H. pylori inoculation in iNOS-knockout mice. METHODS: iNOS-knockout mice (iNOS(-/-)) and their iNOS(+/+) littermates were orally inoculated with the Sydney strain of H. pylori (SS1, 10(8) colony-forming units [CFU]). H. pylori infection was confirmed by microaerobic bacterial culture. The stomach of each mouse was evaluated 14 weeks and 30 weeks after the inoculation. Gastric mucosal accumulation of polymorphonuclear leukocytes (PMN) was assessed by determining the myeloperoxidase (MPO) activity and histological score based on the updated Sydney system. The level of apoptosis was determined by estimation of the cytoplasmic levels of mono- and oligonucleosomes and by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. RESULTS: The SS1-inoculated mice showed persistent H. pylori colonization for 12 weeks. While gastric mucosal PMN infiltration increased following SS1 inoculation in both iNOS(+/+) and iNOS(-/-)strains, enhanced DNA fragmentation was observed in only SS1-colonized iNOS(+/+) mice, and not in the iNOS(-/-) mice. In conclusion, although the recruitment of PMN in response to H. pylori was evoked even in the gastric mucosa of iNOS(-/-) mice, epithelial cell apoptosis induced by H. pylori was attenuated in this strain. These data suggest that iNOS may play an important role in promoting apoptosis in the H. pylori-infected inflamed gastric mucosa, and that persistent inflammation without apoptosis in iNOS(-/-) mice with H. pylori infection may be linked to preneoplastic transformation.
Abstract: The relationship between Helicobacter pylori colonization and the formation of stress-induced gastric mucosal injury remains unknown. Since ammonia (NH(3)) is known as one of the injurious factors in H. pylori-colonized gastric mucosa, the present study is designed to investigate the level of stress-induced gastric mucosal oxidative injury with or without intragastric NH(3) overloading. To apply emotional stress, the communication box paradigm was used in the mouse model. Mice (C57BL/6, male) were pretreated with distilled water (responder-H(2)O) or 0.01% NH(3) (responder-NH(3)) through a gastric tube once a day for a week. Emotional stress was then applied to the responder mice for 3 h per day for 3 d by watching and hearing the behavior of the sender mice subjected to electric shocks to the feet (2 mA, 10 s, 50 s interval). After the communication box protocol, the tissue MPO activity, the contents of TBA-reactive substances (TBARS), and the level of gastric mucosal HSP70 were examined. Responder-NH(3) mice developed more severe gastric lesions than the responder-H(2)O subjects. MPO activity and TBARS contents were enhanced significantly in the responder-NH(3) group compared with the responder-H(2)O subjects. Although the contents of HSP70 in the gastric mucosa increased in the responder-H(2)O group compared with the control-H(2)O animals, they were significantly attenuated in the responder-NH(3) mice. Excess intragastric NH(3) was able to enhance the formation of emotional stress-induced gastric mucosal lesions. This injury may be associated with the enhanced production of oxygen free radicals from accumulated neutrophils under the NH(3)-mediated cancellation of gastric mucosal cytoprotective HSP70.
Abstract: Although intestinal epithelial cells (IECs) are known as an important source for IL-6, it is not known whether mechanical forces affect IL-6 production. We investigated how transmural pressure modulates IL-6 synthesis and activation of transcription factors in IECs. Pressure was loaded onto IEC-18 cells by introducing compressed helium gas into the cell culture flask for 1-48 h. IL-6 release into the culture media was determined by cell proliferation bioassay using an IL-6-dependent mouse hybridoma cell line (7TD1). Exposure to pressure (80 mmHg) significantly enhanced IL-6 release into the culture media from IEC-18 cells at 12 h. Under control conditions, IL-6 secretion was directed to the basolateral side, but after exposure to pressure IL-6 secretion was increased in both the apical and basolateral sides. A nuclear factor kappa B (NF-kappaB) decoy reversed completely the pressure-induced increase of IL-6 secretion by IEC-18 cells. Pressure treatment enhanced IL-6 mRNA expression in IECs within 6 h. Pressure loading significantly enhanced the activation of both NF-kappaB and NF-IL-6 from 1h in the nuclear protein of IEC-18 cells as assessed by the electrophoretic mobility shift assay using FITC-conjugated specific primers. Increased phosphorylation of I-kappa B was also demonstrated in the cytosol of IEC cells within 1h by Western blot analysis. These results suggest a possible role for pressure loading in immune modulation of the intestinal mucosa by the stimulation of IL-6 release from intestinal epithelial cells.
Abstract: Although T cell involvement in Helicobactor pylori-induced gastritis is known, mechanism about T cell recruitment is not understood. In this study we examined how mucosal addressin cell adhesion -molecule-1 (MAdCAM-1) is involved in lymphocyte recruitment in murine chronic gastritis induced by H. pylori. C57 BL/6 mice were infected with Sydney strain (SS1). Six months after infection, the stomach was removed. The expression of adhesion molecules, MAdCAM-1, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and the cell surface antigens CD4, CD8, CD45R/B220 or beta7-integrin were determined by immunohistochemistry. A significant increase in CD4 lymphocytes was observed in the body portion of stomach in SS1-infected mice and most of these CD4 cells express beta7-integrin, a known counter ligand for MAdCAM-1 molecule. Strong MAdCAM-1 expression was observed adjacent to these cells in the lamina propria as well as in the submucosa of SS1-infected stomach. Quantitative analysis showed that the area of MAdCAM-1 expression well correlated with the infiltration of beta7-integrin positive lymphocytes. On the other hand, expression of ICAM-1 or VCAM-1 in the lamina propria was few even in the SS1-infected stomach. Increased expression of MAdCAM-1 was well correlated to the location of lymphocytes, which express CD4 and beta7-integrin. These results suggest the possibility that MAdCAM-1 may be largely involved in the lymphocyte recruitment in the gastritis mucosa with H. pylori.
Abstract: Since Marshall's discovery before 20 years, Helicobacter pylori (H. pylori) infection is reportedly to be associated with a variety of clinical outcomes including peptic ulcer disease and gastric cancer. The first step of the H. pylori colonization might be its adhesion to the surface epithelial cells, which evokes gastric inflammatory events initiated by neutrophil recruitment from the microcirculation. Mongolian gerbil is one of the suitable animal models for H. pylori infection, which exerts gastric ulcer and cancer with its bacterial infection. In H. pylori-colonized gerbils, extensive levels of microvascular leukocyte adhesion and migration into the parenchymal side and significant levels of inflammatory cell infiltration are encountered. Bacterial urease not only neutralizes gastric luminal acid, but also plays as an adhesion factor to the surface epithelium. Recently, such an adhesion to the epithelium is reported to be important for bacterial type IV secretory system, which intermediates Cag A injection into the epithelial cells. Then, multiple chemokine and cytokine networks are activated and mucosal inflammatory lesion formation would be completed. In the long-term colonization of H. pylori, gastric mucosal cell turnover would be modified due to persistent inflammation and then such deregulation of cell turnover might link to the precancerous lesion formation.
Abstract: BACKGROUND: Although oxygen-derived free radicals are known to play a role in cell injury and DNA alterations, the role of active oxidants in chronic pancreatitis has not been fully elucidated. Using WBN/Kob rats, which spontaneously develop chronic pancreatitis-like lesions, we investigated whether xanthine oxidase (XOD)-derived oxygen radicals are involved in pancreatic tissue injury. METHODS: WBN/Kob rats were fed a control or a tungsten diet. The latter depletes XOD activity. Histologic al changes, glutathione (GSH) content and XOD and superoxide dismutase (SOD) activities were determined in pancreatic tissue. Pancreatic 8-hydroxy-deoxyguanosine (8-OH-dG) levels and lithostathine mRNA were also examined. RESULTS: In WBN/Kob rats, parenchymal destruction and fibrosis developed at approximately 12 weeks of age and progressed with each month. The activity of XOD was significantly higher in the early period (8-12 weeks), whereas the levels of GSH and SOD decreased after 16 weeks. Levels of 8-OH-dG in WBN/Kob rats were significantly elevated at 16 weeks. Lithostathine mRNA levels started to increase at 8 weeks, but were suppressed at 16 weeks. The tungsten diet significantly attenuated the histological changes in WBN/Kob rats. The increase in pancreatic XOD activity and 8-OH-dG content in WBN/Kob rats was significantly inhibited by the tungsten diet and lithostathine mRNA levels remained high at 16 weeks. CONCLUSION: These results suggest that oxygen radicals generated by XOD play an important role in oxidative DNA damage and the development of chronic pancreatic injury.
Abstract: Evidence is increasing in hypertensive models for an inflammatory reaction in the microcirculation with abnormal leukocyte counts and adhesion to the endothelium, enhanced arteriolar tone, and microvascular and tissue apoptosis. The spontaneous form of hypertension (SHR) is accompanied by a glucocorticoid-dependent increase in circulating leukocyte count with elevated levels of activation and at the same time depressed leukocyte-endothelial interaction and endothelial P-selectin function. The SHR exhibits immune suppression with lymphocyte apoptosis in the thymus. Generation of reactive oxygen species (ROS) in and around microvascular endothelial cells may regulate signal transduction pathways responsible for controlling gene expression and protein modification and thereby cause an elevation of vascular tone and, in excess, may form an injury mechanism for cells and tissue. A series of enzyme systems such as xanthine oxidase, reduced nicotinamide adenine dinucleotide phosphate/reduced nicotinamide adenine dinucleotide oxidase, and cytochrome P450 monooxygenases in conjunction with suppression of ROS scavengers seem to be involved in the oxidative stress responses in hypertension. The increase in ROS generation contributes to vascular remodeling, apoptosis, and proliferation of vascular smooth muscle, whereas gaseous monoxides such as nitric oxide and carbon monoxide have the ability to modulate elevated vascular tone and proliferative cell responses. Such biological actions of gases not only regulate activation of soluble guanylate cyclase but could also be attributable to inhibition of cytochrome P450 monooxygenases. We examine here the molecular basis of signal transduction by ROS, NO, and CO and functional alterations in their sensor molecules. An inflammatory reaction may underlie the pathogenesis of hypertension and its associated lesion formation and organ dysfunction.
Abstract: AIM: To examine whether proton pump inhibitors modify the production of oxygen-derived free radicals and related cytokines in the human gastric mucosa infected with H. pylori. METHODS: Thirty-four H. pylori-positive peptic ulcer patients (23 gastric ulcer, 11 duodenal ulcer) were enrolled. Biopsy tissue samples were obtained endoscopically from the antrum and corpus. Tissue content of neutrophil myeloperoxidase (myeloperoxidase) and IL-8 was measured by ELISA. Mucosal production of oxygen-derived free radical was measured using luminol-dependent chemiluminescence (ChL). A proton pump inhibitor (either lansoprazole 30 mg, omeprazole 20 mg, or rabeprazole 10 mg) was administered daily by mouth to all patients for 8 weeks. Endoscopic examination was then repeated, and biochemical analysis was performed. RESULTS: Antral myeloperoxidase decreased significantly after proton pump inhibitor treatment (5.23 +/- 7.00-2.76 +/- 5.11 ng/mg, P < 0.02), but corpus myeloperoxidase was unchanged. IL-8 was also modified by proton pump inhibitors and these changes were parallel to those of myeloperoxidase. Corpus ChL was significantly increased from 88.5 +/- 69.8-159 +/- 172 counts/10 s/mg after proton pump inhibitor treatment, whereas antrum ChL was not altered. H. pylori infection rate was decreased in the antrum as well as the corpus. CONCLUSIONS: Proton pump inhibitor treatment stimulated oxygen-derived free radical production in the corpus mucosa.
Abstract: 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.
Abstract: BACKGROUND: Proton pump inhibitors have been reported to modify the level of Helicobacter pylori gastritis. AIM: To quantitatively investigate the effect of a proton pump inhibitor on the mucosal neutrophil reaction. METHODS: Forty-six H. pylori-infected patients (17 duodenal ulcer, 29 gastric ulcer) were enrolled. During endoscopic examination, biopsy samples were obtained from the antrum and the corpus. The tissue content of neutrophil myeloperoxidase was measured by enzyme-linked immunoabsorbent assay, and H. pylori infection was histologically assessed. A proton pump inhibitor was administered orally for 8 weeks. RESULTS: In the patients as a whole, antral myeloperoxidase decreased significantly after proton pump inhibitor treatment, but corpus myeloperoxidase remained largely unchanged. In duodenal ulcer patients, myeloperoxidase significantly decreased in the antrum, but increased in the corpus. In gastric ulcer patients, a significant reduction was observed in antral myeloperoxidase, but corpus myeloperoxidase remained unchanged. In the antral myeloperoxidase > corpus myeloperoxidase subgroup (n=24), antral myeloperoxidase significantly decreased, whereas corpus myeloperoxidase increased. No changes were observed at either site in the corpus myeloperoxidase > antral myeloperoxidase subgroup. Histology showed that the antral bacterial load of H. pylori decreased in all subgroups, but that it was mostly unchanged in the corpus. CONCLUSIONS: Proton pump inhibitor treatment stimulated the neutrophil reaction in the corpus mucosa of duodenal ulcer patients and of patients in whom antral neutrophil accumulation was more predominant than that of the corpus. This phenomenon may not be caused by increased bacterial density.
Abstract: BACKGROUND: We previously reported that attenuated epithelial apoptosis and enhanced proliferation in comparison with mice might link to the specific carcinogenesis in Mongolian gerbils and suggested that the difference in both strains might be due to a difference in genetic background. p53 is a well-known tumour suppressor gene, mutation of which is also known to be involved in gastric cancer formation. AIM: The present study was designed to examine the level of gastric epithelial apoptosis and proliferation in p53 heterozygous knockout mice (p53+/-) colonized with Helicobacter pylori (Sydney strain: SS1). METHODS: Female p53+/- mice and wild-type controls were orally inoculated with SS1 and the stomachs were examined 24 weeks later. DNA fragmentation was measured by levels of cytoplasmic mono- & oligo-nucleosomes as well as by the TUNEL method. Gastric mucosal proliferative activity was morphometrically evaluated from the PCNA-stained tissue specimens. Gastric mucosal myeloperoxidase (MPO) activity was measured to evaluate mucosal inflammation. RESULTS: DNA fragmentation and the number of TUNEL-positive cells, as well as PCNA-positive cell number increased significantly in both groups of H. pylori-infected mice, suggesting that levels of apoptosis and proliferation may be independent of a deficiency of one p53 allele. MPO activity in p53+/- mice and wild-type controls increased to the same level. CONCLUSION: Although H. pylori inoculation per se induces an increase in cell turnover in mice, heterozygous mutation of p53 did not significantly modify the balance in cell apoptosis and proliferation.
Abstract: Although the negative effect of excessive alcohol consumption on later stressful events has long been recognized, pathophysiological mechanisms are incompletely understood. We examined possible roles of oxygen radicals and glutathione content in mesenteric venules of chronically ethanol-fed rats exposed to ischemia-reperfusion. Changes in microvascular hemodynamics, such as red blood cell (RBC) velocity, leukocyte adherence, and albumin extravasation, were monitored in postcapillary venules by intravital fluorescence microscopy. Chronic ethanol feeding significantly exaggerated the magnitude of the decrease in RBC velocity, the increased number of adherent leukocytes, and increased albumin leakage elicited by 10 min of ischemia followed by 30 min of reperfusion. Oxidative stress in the endothelium of venules monitored by dihydrorhodamine 123 (DHR) fluorescence was more severe in rats fed ethanol chronically. Both superoxide dismutase and N-acetyl-L-cysteine, which is known to increase glutathione content, reduced the ischemia-reperfusion-induced decrease in RBC velocity, the number of adherent leukocytes, and the increase in albumin leakage, as well as oxidative activation of DHR. This suggests that the increased reperfusion-induced microvascular disturbances in the mesenteric venules of rats fed ethanol chronically are significantly correlated with excessive production of oxygen-derived free radicals and decreased glutathione synthesis.
Abstract: BACKGROUND: Human gastric mucosa contains three alcohol dehydrogenase (ADH) isozymes (classes I, III, and IV). Various factors such as Helicobacter pylori infection, sex, age, and the part of the stomach involved have been suggested to affect alcohol dehydrogenase activities, although these views are controversial. In this study, these unsettled issues were reexamined. METHODS: Activities of class I and IV ADHs were evaluated in the cytosolic fraction of human gastric mucosa samples by reduction of their preferred substrates, namely acetaldehyde and m-nitrobenzaldehyde, and activities of class III were evaluated by oxidation of its preferred substrate, formaldehyde. Then, effects of Helicobacter pylori infection, sex, age, and the part of the stomach involved were examined. RESULTS: Class I, III, and IV ADH activities were 17.5 +/- 8.4, 4.2 +/- 2.5, and 8.9 +/- 3.9 nmol of nicotinamide adenine dinucleotide oxidation per minute per milligram of protein, respectively, for the entire population. Helicobacter pylori infection significantly reduced class I and IV ADH activities but did not affect activity of class III. In the samples without Helicobacter pylori infection and severe gastritis, sex did not affect class I, III, or IV ADH activities. In the same series, class IV ADH activity significantly decreased with age (p = 0.006), whereas no correlation was found between age and ADH activity of class I and III ADHs. The level of class IV ADH activity was significantly higher in the upper body than in the lower regions, whereas no such heterogeneity was observed in class I and III ADH. CONCLUSIONS: Various factors affect human gastric ADH activities, such that careful interpretation of their significance is necessary.
Abstract: BACKGROUND/AIMS: Cytokine-induced neutrophil chemoattractant (CINC/gro), a member of interleukin-8 family, was found as a potent chemotactic factor for rat neutrophils. Although several chemokines have been shown to be potent regulators of T cell chemotaxis in vitro, the potential role of chemokines in T-cell migration in gut-associated lymphoid tissues has not been investigated in vivo. In the present study, the effects of CINC/gro on T-lymphocyte migration were examined in rat Peyer's patches. METHODS: T lymphocytes collected from intestinal lymph of rats were fluorescence-labeled and injected into the jugular vein. Peyer's patches of the recipient rats were observed with intravital fluorescence microscopy and the effects of CINC/gro infusion was investigated. Lymphocyte flux in mesenteric collecting lymphatics was also observed. RESULTS: In vivo infusion of CINC/gro significantly attenuated the initial lymphocyte interaction with postcapillary venules of Peyer's patches. However, once these lymphocytes adhered to venules, CINC/gro treatment significantly accelerated the transendothelial migration of T lymphocytes and they also significantly increased their subsequent flux in collecting lymphatics. CONCLUSION: There is a possibility that CINC/gro could modulate the characteristics of T lymphocyte homing in the inflammatory sites of gut.
Abstract: Intestinal mucosal immunity is modulated by cytokine release from intestinal cells, but little is known about the relation between nutrient absorption and cytokine release. In this study, we examined how exposure to fatty acids affects the production of growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) and interleukin (IL)-6 in rat intestinal epithelial cells (IEC). The long-chain fatty acids, oleic, linoleic and arachidonic acids, and the middle-chain fatty acid octanoic acid were administered to subconfluent cultures of IEC-6 cells alone, or in combination with IL-1beta and transforming growth factor (TGF)-beta. The GRO/CINC-1 and IL-6 concentrations in culture media were determined by sandwich enzyme immunoassay. In epithelial cells, GRO/CINC-1 and IL-6 mRNA expression were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and mitogen-activated protein kinase (MAPK) activities determined by immunoblotting. Administration of long-chain fatty acids significantly increased the GRO/CINC-1 and IL-6 secretion into culture media, and this secretion was markedly increased (P < 0.05) in the presence of IL-1beta or TGF-beta. Octanoic acid had no effect on GRO/CINC-1 or IL-6 production. Furthermore, treatment with long-chain fatty acids significantly enhanced the GRO/CINC-1 and IL-6 expression that was induced by IL-1beta or TGF-beta. MAPK activity was significantly enhanced by treatment with long-chain fatty acids. Inhibitors of phospholipase C, protein kinase C or MAPK significantly reduced the fatty acid-induced increase in GRO/CINC-1 secretion, whereas a calcium/calmodulin inhibitor did not attenuate the secretion. These results suggest that long-chain fatty acids enhance cytokine release under conditions of inflammatory stimulation in the intestinal mucosa.
Abstract: 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 (GRO/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, GRO/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.
Abstract: Intestinal mucosa is continuously exposed to mechanical forces. We examined whether pressure loading activates mitogen-activated protein kinase (MAPK) and expression of early immediate genes in intestinal epithelial cells. Pressure was applied to IEC18 cells by helium gas in a culture flask and pressure-induced cell proliferation was examined. The expression of early immediate genes, MAPK activity, and activation of nuclear factor activator protein-1 (AP-1) were also examined. Pressures significantly promoted cell proliferation with peak effect at 80 mm Hg. Pretreatment with either a protein kinase C inhibitor or tyrosine kinase inhibitors, but not calcium chelating agents significantly inhibited cell proliferation promoted by pressure. Early inductions of c-myc and c-fos proteins, increased activity of MAPK, and activation of AP-1 were observed by pressure loading. Our study showed that intestinal mucosal cell proliferation is promoted by mechanical pressure and various intracellular signaling pathways are involved in the process.
Abstract: 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 GROalpha, 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 G0 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.
Abstract: Apoptosis, a programmed cell death, was ignored, just like Helicobacter pylori, only to reappear recently. However, the number of current publications dealing with apoptosis or H. pylori has increased exponentially. Although gastric epithelial apoptosis is a programmed physiological event in the superficial aspect of the mucosa and is important for healthy cell turnover, H. pylori infection reportedly promotes such a cell death sequence. Because apoptosis regulates the cycle of cell turnover in balance with proliferation, dysregulation of apoptosis or proliferation evoked by H. pylori colonization would be linked to the gastric carcinogenesis. In other words, a reduced level of apoptosis could contribute to the generation of gastric cancer. Herein, we review apoptosis as well as its associated pathological events, such as hyperproliferation, in H. pylori-colonized gastric mucosa.
Abstract: Macrophages beneath the marginal sinus in the lymph nodes may play a role in defense against microorganism. The purpose of this study was to directly visualize the parasinus macrophages in the mesenteric lymph node. Fluorescent latex particles were injected into the appendix submucosa of rats. The mesenteric lymph node was epi-illuminated and observed with a fluorescent microscope. Fluorescent particles entered the marginal sinus of the mesenteric lymph node through the afferent lymphatic vessels, and distributed diffusely all over the marginal sinus. The particles became aggregated and interspersed 3 hr after injection, suggesting that particles were incorporated by phagocytes. The number of these particle-laden phagocytes increased up to 12 hr after injection, and then declined. Some phagocytes migrated rapidly within the marginal sinus. Morphology of these phagocytes in cell suspension was consistent with macrophages. In conclusion, we successfully visualized parasinus macrophages in vivo, which incorporated foreign bodies and migrated within the marginal sinus.
Abstract: We previously reported that in vitro hypoxic condition enhanced VEGF level and its receptor expression in hepatic cancer cell line, HepG2. Transcatheter hepatic arterial embolization (TAE) therapy is one of the vasculo-occlusive and hypoxic challenges to hepatocellular carcinoma (HCC). Therefore, we examined the level of VEGF in sera of patients with HCC who underwent TAE during the course of the treatment. Thirty-eight patients with HCC and hepatitis C virus-positive cirrhosis were studied. Peripheral blood samples were taken before and 1, 3 and 7 days after TAE with informed consent. The serum levels of VEGF as well as hepatocyte growth factor (HGF), another hepatic remodeling factor, were measured. The molar ratio (BTR) of serum branched chain amino acid (BCAA) to tyrosine (Tyr), the serum levels of AST, ALT and LDH were also examined. Although the level of AST, ALT and LDH reached the peak value within 1 day after TAE, VEGF level increased significantly 7 days later. On the other hand, there were no significant alterations in the levels of HGF and BTR during the course of TAE. Although the level of HGF was significantly correlated with the level of VEGF before TAE, this correlation was no more observed after TAE. These data collectively suggest that VEGF may be secreted in response to clinical hypoxic intervention, TAE, independent of HGF or altered amino acid metabolism. VEGF may play a role as a sensitive marker for tumor ischemia.
Abstract: 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 TBARS 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.
Abstract: The mechanisms that lead to organ injury in hypertension are incompletely understood. In particular, there is a lack of evidence that serves to link the elevation of arterial blood pressure with end organ damage. Experimental models of hypertension have a range of microvascular abnormalities in addition to a shift in blood pressure. There is evidence for an oxidative stress in microvascular endothelium derived from xanthine and NADPH oxidase. Furthermore, there exists an immune suppression accompanied by abnormally elevated circulating leukocyte counts, depression of selectin membrane adhesion to the endothelium and enhanced cell apoptosis. Many of the deficiencies in the spontaneously hypertensive rats can be corrected by adrenalectomy, suggesting a contribution of glucocorticoids to the abnormalities in this model. These observations suggest a significantly enhanced vascular oxidative stress which is accompanied by a frustrated inflammatory response due to a glucocorticoid dependent deficiency of leukocyte adhesion to vascular endothelium.
Abstract: The mechanisms contributing to organ injury in hypertension have been incompletely defined. The thymus gland of the spontaneously hypertensive rat (SHR) shows significant atrophy at the age of 15 wk compared with its normotensive control, the Wistar-Kyoto rat (WKY). The aim of the present study was to examine the thymus of SHR for evidence of DNA nicking as one of the mechanisms for thymic atrophy. SHR and WKY were subjected to adrenalectomy or sham surgery at 12 wk and studied at 15 wk. Adrenalectomy served to normalize the blood pressure in the SHR. DNA nicking was detected by in situ nick-end labeling (ISEL) of fixed tissue sections. Tissue sections were treated with proteolysis, and terminal deoxyribonucleotidyl transferase was used to incorporate biotinylated deoxynucleotides into DNA nick end in situ. Separately, DNA fragmentation was evaluated by measuring the level of released mono- and oligonucleosomes to the cytoplasm. A higher number of thymic ISEL-positive cells and a higher level of cytoplasmic mono- and oligonucleosomes were observed in SHR than in WKY. After adrenalectomy the enhanced level of ISEL and cytoplasmic mono- and oligonucleosomes in SHR was reduced to the level in WKY. Dexamethasone treatment (0.05 mg. kg-1. day-1) in WKY serves to decrease the thymus weight and significantly elevate the level of mono- and oligonucleosomes. Thus increased DNA fragmentation represents one of the mechanisms associated with thymic atrophy, a feature that reflects immune suppression in SHR.
Abstract: BACKGROUND: Spontaneously hypertensive rats (SHR) are a representative animal model for disturbance of the pituitary-adrenal axis, as well as disturbance of the autonomic nervous system. METHODS AND RESULTS: In this study, we showed that adrenalectomy in SHR-induced spontaneous gastric ulcer formation. We further investigated how abnormal adrenal secretion is related to the attenuation of gastric ulcerogenesis, in terms of leucocyte infiltration and nitric oxide (NO) formation. Bilateral adrenalectomy, as well as a sham-operation, were carried out at 12 weeks in hypertensive SHR and Wistar-Kyoto rats (WKY) and observations were made three weeks later. The number of myeloperoxidase (MPO) positive cells, NADPH diaphorase histochemistry and NO synthase (NOS) activity were determined in gastric specimens. Only in adrenalectomized, but not sham-operated SHR, WKY and adrenalectomized WKY, could gastric ulcers be observed. Although the number of cells positive for MPO was significantly lower in hypertensive SHR than those in WKY, such cells were increased after adrenalectomy in SHR. In contrast, adrenalectomized WKY developed no increase in MPO-positive numbers. The number of NADPH diaphorase-positive cells increased after adrenalectomy in both strains, the extent of which was much greater in SHR than in WKY. Although NOS activity in SHR was lower than that in WKY, it was significantly increased after adrenalectomy. CONCLUSIONS: Our data show that the development of a significant gastric ulceration may be associated with entrapment of activated leucocytes in the gastric mucosa, as well as with an excessive production of NO in adrenalectomized SHR. An enhanced adrenal glucocorticoid may be a key factor for protecting the gastric mucosa in SHR.
Abstract: The role of nitric oxide in ulcer formation remains unknown. Accordingly, we assessed local expression of inducible nitric oxide synthase (NOS) and nitration of tyrosine as an indicator of peroxynitrite formation in patients with Helicobacter pylori (HP)-associated gastric ulcers compared with HP-negative ulcers. Biopsy specimens were taken from the ulcer margin and from an area remote from the ulcer portion. Inducible NOS, nitrotyrosine, and macrophage immunoreactivity were assessed immunohistochemically using a labeled streptavidin-biotin method. In HP-positive gastric ulcers, inducible NOS and nitrotyrosine immunoreactivity was frequently observed at active ulcer margins, sometimes in surface epithelial cells as well as in the lamina propria. Occasionally, inducible NOS and nitrotyrosine reactivity were found in areas remote from the lesion in cases of HP-positive ulcer and HP-related gastritis. Macrophages accumulated significantly in the margin of HP-positive ulcers. In HP-negative gastric ulcers, inducible NOS and nitrotyrosine immunoreactivity also were frequent at the ulcer margin, but no significant immunoreactivity was observed at a distance. HP eradication caused significant attenuation in inducible NOS and macrophage immunoreactivity. In conclusion, nitric oxide and peroxynitrite formation is increased in HP-infected gastric mucosa, suggesting that HP promotes nitric oxide stress.
Abstract: 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 polaprezinc could attenuate the NH3Cl-induced DNA damage. Gastric cell lines, MKN45, were exposed to NH2Cl in Ca(2+)-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 0.001 mM 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.
Abstract: 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.
Abstract: In this study, the influence of omeprazole on the adhesive activity of neutrophils, provided by an extract of Helicobacter pylori, was determined. Human neutrophils were collected from peripheral blood and labelled with a fluorochrome. Helicobacter pylori (NCTC 11637) was cultured and its water extract was obtained by centrifugation of the bacterial suspension. Neutrophils were incubated with the extract in a plastic plate. Percentage adherence was calculated by measuring the fluorescence of floating and adherent cells. Rat mesenteric venule was prepared on an intravital microscope and the number of neutrophils which adhered to venular endothelium was counted. Neutrophil adherence to the plastic plate was increased by the presence of H. pylori extract. Pretreatment with omeprazole significantly decreased this adherence in a dose-dependent manner (10(-6)-10(-4)mol/L). Neutrophil adherence to the mesenteric venule was also increased by H. pylori extract and significantly inhibited by omeprazole. These results indicate that the neutrophil-endothelial adhesive interaction is inhibited by omeprazole, suggesting that omeprazole prevents neutrophil recruitment to the gastric mucosa associated with H. pylori infection.
Abstract: This study aimed to examine whether acetaminophen (AAP), an anti-inflammatory agent producing hepatocellular damages with its overdose, evokes hepatocellular dysfunction through mechanisms involving carbon monoxide (CO) generated by heme oxygenase (HO). In perfused rat livers, CO and bilirubin were determined in venous perfusate and bile samples as indices of heme degradation. Biliary excretion of transportally injected horseradish peroxidase was also determined to assess paracellular junctional permeability and vesicular transport across hepatocytes. AAP at 20 mmol/L induced a transient choleresis, followed by a reduction of bile output. Under these circumstances, the release of CO and bilirubin IXalpha, terminal products of the HO-mediated heme degradation, became 2. 5-fold greater than the control. The rate of CO production appeared stoichiometric to the degradation rate of microsomal cytochrome P-450. Mechanisms for the AAP-induced cholestasis involved an increase in the junctional permeability that coincided with a reduction of vesicular transport across hepatocytes. Clotrimazole, a cytochrome P-450 inhibitor, or zinc protoporphyrin IX, an HO inhibitor, but not copper protoporphyrin IX, which did not inhibit HO, attenuated these AAP-induced changes. Furthermore, administration of CO at concentrations comparable with those induced by AAP elicited a marked elevation of the paracellular junctional permeability concurrent with a reduction of transcellular vesicular transport, mimicking effects of the AAP administration. Thus, CO serves as a putative regulator of hepatocellular function that is overproduced through acute heme degradation during xenobiotic transformation.
Abstract: 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 1 (flt-1), in human umbilical venous endothelial cells (HUVEC). Hep G2 cells were cultured in 20% or 1% O2 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.
Abstract: 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.
Abstract: Helicobacter pylori colonized to gastric mucosa plays an important pathogenic role in gastric mucosal lesions. We previously reported that ethanol pretreatment promotes the extension of H. pylori-associated lesions. The present study was designed to examine the effect of rebamipide, a mucosal protective agent, on H. pylori-associated injury. Male Mongolian gerbils were orally inoculated with H. pylori; 30 min prior to inoculation, 40% ethanol was administered orally to these gerbils (Hp group). Controls were given 40% ethanol with culture medium (control group). Some gerbils in the Hp and control groups were fed rebamipide-containing diets, and the remaining gerbils received laboratory chow diets. H. pylori infection was evaluated by quantitative bacterial culture and histological examination. Although H. pylori was persistently detected and a remarkable mucosal leukocyte infiltration was observed in the Hp groups, the bacteria had disappeared naturally in 67% of the gerbils and mucosal damage was mitigated in the Hp + rebamipide group at four weeks after the inoculation. Collectively, rebamipide might play a role in inhibiting the level of H. pylori colonization and gastric lesion formation in Mongolian gerbils.
Abstract: Endotoxin is postulated to be an important aggravating factor for alcoholic liver disease. We have previously reported that rats fed ethanol are more vulnerable to endotoxin-induced liver damage, and hepatic microcirculatory disturbance plays an important role for this liver damage by observation with an intravital microscopy. In this study, we have investigated the role of adhesion molecules in endotoxin-induced microcirculatory disturbance in chronic ethanol-fed rats. Male Wistar rats were pair-fed with ethanol liquid diet (ethanol group) or an isocaloric control diet (control group) for 6 weeks. Leukocyte adherence to the hepatic sinusoid by stimulation with lipopolysaccharides (1 mg/kg of body weight) was observed by an inverted fluorescence microscopy equipped with a silicon-intensified target camera and was found to be enhanced in ethanol-fed rats. Tumor necrosis factor-alpha and GRO/CINC-1 (rat counterpart of interleukin-8) was increased in the blood in these animals. Subsequent expression of adhesion molecules, LFA-1 beta-chain on leukocytes were demonstrated by flow cytometry, which suggests a possible involvement of leukocyte adherence to the hepatic damage in ethanol-fed animals. Preadministration of anti-rat LFA-1 beta-chain monoclonal antibody effectively suppressed leukocyte adherence to the hepatic sinusoid. These results suggest that the enhanced sequestration of neutrophils to the liver with these adhesion molecules may play a significant role in the pathogenesis of alcoholic liver disease.
Abstract: Recent evidence in vivo indicates that spontaneously hypertensive rats (SHR) exhibit an increase in oxyradical production in and around microvascular endothelium. This study is aimed to examine whether xanthine oxidase plays a role in overproduction of oxidants and thereby may contribute to hypertensive states as a consequence of the increasing microvascular tone. The xanthine oxidase activity in SHR was inhibited by dietary supplement of tungsten (0.7 g/kg) that depletes molybdenum as a cofactor for the enzyme activity as well as by administration of (-)BOF4272 [(-)-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo(1,5-alpha)-1,3, 5-triazine-4-monohydrate], a synthetic inhibitor of the enzyme. The characteristic elevation of mean arterial pressure in SHR was normalized by the tungsten diet, whereas Wistar Koto (WKY) rats displayed no significant alteration in the pressure. Multifunctional intravital videomicroscopy in mesentery microvessels with hydroethidine, an oxidant-sensitive fluoroprobe, showed that SHR endothelium exhibited overproduction of oxyradicals that coincided with the elevated arteriolar tone as compared with WKY rats. The tungsten diet significantly repressed these changes toward the levels observed in WKY rats. The activity of oxyradical-producing form of xanthine oxidase in the mesenteric tissue of SHR was approximately 3-fold greater than that of WKY rats, and pretreatment with the tungsten diet eliminated detectable levels of the enzyme activity. The inhibitory effects of the tungsten diet on the increasing blood pressure and arteriolar tone in SHR were also reproducible by administration of (-)BOF4272. These results suggest that xanthine oxidase accounts for a putative source of oxyradical generation that is associated with an increasing arteriolar tone in this form of hypertension.
Abstract: The possession of the cytotoxin-associated gene (cagA) of Helicobacter pylori is thought to be highly associated with peptic ulcer disease. However, the pathogenic role of cagA is still unknown. We have emphasized the importance of the interrelationship between H. pylori-derived ammonia and oxygen radicals from infiltrated leucocytes. The aim of the present study was to explore the relationship between oxygen radical production and H. pylori strain diversity based on cagA possession. An endoscopic examination and gastric mucosal biopsy were performed in 30 H. pylori-infected patients with gastric ulcer. Myeloperoxidase (MPO) content and the luminol-dependent chemiluminescence value in the biopsied gastric specimens were measured as an index for leucocyte infiltration and oxygen radical production. From the precipitates of cultured bacterial isolates of biopsied specimens, bacterial DNA was purified and analysed by polymerase chain reaction to characterize the possession of cagA. While all patients had ureC-positive strains, 22 had cagA-positive and eight had cagA-negative strains. In patients with cagA-positive strains, MPO contents as well as chemiluminescence values in the gastric corpus were significantly higher than those in patients with cagA-negative strains. Gastric mucosal leucocyte recruitment and activation are suggested to be enhanced by cagA gene-positive H. pylori infection.
Abstract: Eradication therapy for Helicobacter pylori (H. pylori) has been established. However, the physiological factors influencing the success of treatment remain unclear. The aim of this study was to analyze these factors and to evaluate the efficacy of sofalcone on H. pylori eradication therapy. Forty-four H. pylori-infected and peptic ulcer patients were enrolled in this study. Twenty-seven patients were treated with lansoprazole (LPZ, 30 mg o.d. for 1-8 weeks) and amoxicillin (AMPC, 500 mg q.i.d, 1-2 weeks), followed by 8 weeks of treatment with famotidine (FAM, 20 mg o.d.). Moreover, sofalcone (SOF, 100 mg t.i.d) was administered to 17 patients throughout the therapeutic period. Endoscopic and serologic evaluations and the urea breath test (UBT) were performed before therapy. At the endoscopic examination, mucosal samples were biopsied and then tissue myeloperoxidase (MPO) content, an index of neutrophil infiltration was measured. Cure of H. pylori infection was determined 8 weeks after the cessation of LPZ. This eradication regimen afforded an overall cure rate of 63.0% (17/27) without SOF and 76.5% (13/17) with SOF. In the control group, treatment success was inversely associated with pre-UBT value (gastric urease activity), whereas this association was not observed in the SOF group. Furthermore, in the patients exhibiting a high preUBT value (>40%), a twofold higher eradication rate was obtained by the administration of SOF. In patients who were successfully eradicated, mucosal MPO level was slightly higher than those of unsuccessful cases, whereas there was no significant association with serum pepsinogen (PG I, PG II) concentration and its ratio (PG I/PG II). These results suggest that a low UBT value is a factor predicting treatment success. SOF administration may improve the eradication rate, especially in the high-UBT subgroup.
Abstract: C-X-C Chemokines play an important role for neutrophil extravasation through microvessels. Although the level of interleukin (IL)-8 is known to increase in the Helicobacter pylori-infected gastric mucosa, another C-X-C chemokine, GROalpha, has not been evaluated in the H. pylori-associated gastric mucosal injury. The present study was designed to investigate gastric contents of GROalpha in relation to those of IL-8 in the gastric mucosa of H. pylori-infected peptic ulcer patients. Thirty-eight patients with gastric ulcer and 41 with gastritis underwent endoscopy with informed consent and 49 were found to be H. pylori positive and 30 H. pylori negative. Biopsies from the gastric corpus were performed in each patient to examine the H. pylori colonization by bacterial culture, the rapid urease test and histological specimens as well as measurement of the contents of human GROalpha and IL-8. Helicobacter pylori infection was eradicated in 21 patients by triple therapy (lansoprazole 30 mg, amoxycillin 2.0 g, clarithromycin 600 mg; 2 weeks). The samples for GROalpha and IL-8 assay were homogenized in 0.02% aprotinin containing phosphate-buffered solution and the mucosal contents of GROalpha and IL-8 in the supernatants were quantified by sandwich enzyme immunoassay methods. The levels of GROalpha and IL-8 in H. pylori-positive gastric mucosa were significantly higher than those in the H. pylori-negative mucosa. There was a significant linear correlation between the levels of GROalpha and IL-8 (r = 0.798, P < 0.01). After the eradication of H. pylori by the triple therapy, the levels of GROalpha and IL-8 were significantly decreased. The GROalpha showed an increase in the H. pylori-positive gastric mucosa in a similar fashion as IL-8 contents, suggesting a pathogenetic role for GROalpha in H. pylori-associated gastric mucosal injury.
Abstract: We sought to clarify the role of nitric oxide (NO) generated from inducible NO synthase (iNOS) during healing of rat gastric ulcers. After gastric ulcers were induced by acetic acid, rats were treated with vehicle, N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG), and dexamethasone (Dex) by gastric intubation twice a day for 3 days to 1 week. L-NAME significantly delayed healing compared with vehicle. AG and Dex significantly reduced ulcer size 3 days after ulcer induction but did not further reduce ulcer size 1 week after induction. iNOS expression was present in inflammatory cells, some epithelial cells, and in vascular smooth muscle in the regenerating mucosa of the vehicle-treated group. However, the number of iNOS-positive inflammatory cells increased in the AG- and L-NAME-treated groups. AG and L-NAME significantly increased the number of inflammatory cells with endogenous peroxidase and significantly reduced the number of apoptotic inflammatory cells compared with vehicle. In conclusion, inhibition of iNOS increases the number of inflammatory cells in the ulcer margin and delays ulcer healing. These observations suggest that NO generated from iNOS not only participates in ulcer formation but also plays a beneficial role in ulcer healing, in part by the exclusion of iNOS-positive inflammatory cells from the regenerating mucosa.
Abstract: Although endotoxin exacerbates hepatic microcirculatory disturbance, little is known of the way in which it acts on the hepatic microcirculation. We measured endotoxin-induced changes in hepatic microcirculation and investigated the effect of endotoxin on hepatic microcirculation in rats. After male Wistar rats were anesthetized, a lobe of the liver was observed with an inverted intravital microscope. Erythrocytes (RBC) were labeled with fluorescein isothiocyanate (FITC) and injected. The flow velocity (FV) of FITC-RBC in sinusoids was measured with an off-line velocimeter. Portal pressure (PP) and mean arterial pressure (MAP) were measured with a catheter cannulated in the portal vein and the left carotid artery, respectively. After a small dose (1 mg/kg) of endotoxin had been administered intravenously, FV decreased and PP increased gradually after 30 min. MAP showed no significant change, except for an initial decrease. However, when 5 mg/kg of endotoxin was administered, FV and PP increased, with a peak at 10 min, which was not observed with the small dose. In the late phase, FV decreased and PP increased, as was seen with the small dose. Endotoxin increased serum aspartate aminotransferase and lactate dehydrogenase activities. These results suggest that endotoxin induces hepatic microcirculatory disturbance, which may cause liver injury.
Abstract: Neutrophil accumulation plays an important role in Helicobacter pylori-associated gastric mucosal injury. In this study, the mucosal content of myeloperoxidase (MPO), which is a measure of neutrophil accumulation and interleukin-8 (IL-8) was assayed and changes in MPO and IL-8 content were determined before and after H. pylori eradication therapy. Thirty-seven H. pylori-positive patients (11DU/26GU) underwent H. pylori eradication therapy with lansoprazole (30 mg/day, 6 weeks) and amoxicillin (2 g/day, 2 weeks), followed by famotidine (20 mg/day, 8 weeks). H. pylori-infection status was evaluated by routine endoscopic examinations (culture, CLO, histology). Immediately and 8 weeks after cessation of the anti-H. pylori therapy, these tests were repeated. Intragastric urease activity was estimated by delta 13CO2, which was obtained by the [13C]urea breath test (UBT). Mucosal samples were taken and tissue MPO and IL-8 contents were assayed by EIA and ELISA, respectively. Histologic examination was also performed. Among the 37 patients, 21 cases of H. pylori infection were eradicated (56.8%). Intragastric urease activity was dramatically reduced immediately after the anti-H. pylori therapy, whereas, it was re-elevated 8 weeks later in the relapsed cases. Antral MPO content was decreased in the eradicated and relapsed cases. MPO in the corpus was also decreased in the eradicated cases. Nevertheless, it was enhanced (3.5-fold) in the relapsed cases at 8 weeks after therapy. Changes in mucosal IL-8 content were similar to those of MPO. In eradicated cases, neutrophil infiltration is improved in both the antrum and corpus. However failure of eradication therapy results in the enhancement of neutrophil accumulation in the corpus. Further study is necessary to clarify the mechanism of neutrophil accumulation after therapy for H. pylori.
Abstract: Morphogenesis of the cardiovascular system is likely linked to its functional development. This report presents an approach to functional assessment of early cardiovascular development using parametric imaging based on videodensitometry. Trypan blue was injected into the sinus venosus of chick embryos at stage 20 (3.5 d of incubation). Images were recorded on videotape, digitized, and analyzed by a microcomputer. A sampling window was placed over the entire image, and a densogram (time-density curve) for each pixel in the window was obtained. Parameters extracted from the densogram and its derivative were plotted in form of: 1) maximal image, 2) peak derivative image, and 3) time to peak derivative image. This approach revealed isochronal lines that divide the aortic arch and dorsal aorta into several segments. Regional flow velocity at these segments was estimated by dividing the distance between isochronal lines by the time interval. Flow velocity at the mid-systolic phase at the dorsal aorta and at the fourth aortic arch was 35.9 and 45.0 mm/s, respectively. Shear rate at the vessel wall was estimated to be 2.7 times larger at the fourth aortic arch than at the dorsal aorta. The extensive remodeling experienced by the aortic arch system compared with the dorsal aorta could be related to increased shear rate on the walls of the aortic arches.
Abstract: BACKGROUND: Previously, we have reported that toxic oxidants produced by activated neutrophils play a pivotal role in the development of Helicobacter pylori-induced gastric mucosal damage. Microorganisms, however, are characterized by their ability to produce a variety of antioxidant enzymes. This study is designed to measure the oxygen radical scavenging enzymes, such as catalase and superoxide dismutase (SOD), and urease in the supernatant of H. pylori (NCTC11637) suspension. MATERIALS AND METHODS: H. pylori was inoculated on a sheep blood agar plate and harvested. Bacterial suspensions (10(9) cfu/ml phosphate buffer) were washed twice and incubated at 37 degrees C for 1, 2, 12, and 24 hours or were sonicated in ice. Their supernatants were obtained by centrifugation and filtration. SOD activity was measured spectrophotometrically by the cytochrome c method. Catalase activity was assayed by the fall in absorbance at 240 nm as H2O2 is degraded. Urease activity was determined by measuring the release of ammonia using Berthelot reaction. RESULTS: Activities of both SOD and catalase were detected in the supernatants of 1-hour microaerophilic incubation. Their activities were almost constant in 4-, 12-, and 24-hour microaerobic incubation or sonication. Urease activity was increased dramatically in proportion to the period of microaerobic incubation. CONCLUSION: Although H. pylori possesses antioxidant as a constitutive compartment, the magnitude of its secretion was below the detectable level. It is not likely that SOD and catalase play a significant role for scavenging the oxidants from injured gastric mucosa, such as infiltrated leukocytes.
Abstract: Colonization of Helicobacter pylori (Hp) to gastric mucosa plays an important role for the pathogenesis of gastric mucosal lesions. We previously reported the importance of monochloramine (NH2Cl), which was derived from the interaction between Hp-urease and infiltrated leukocytes, in the course of Hp-associated gastric mucosal injury. While the long-term infection of Hp in the gastric mucosa is known to be one of the virulent factors which closely link to the gastric carcinogenesis, the details of its pathogenetic mechanisms remain speculative. The present study is designed to examine whether a NH2Cl could damage the DNA of gastric cells. Rabbit gastric mucosal cells (RGMC) or KATO III cells were cultured and suspended. Cell suspensions were exposed to HOCl, NH3 or NH2Cl for 15 min to give a final concentration of 0.1 mM. The magnitude of a double strand break of DNA was quantified by measuring the remnant double strand stained by ethidium bromide (EB), and the fluorescence intensity of EB was analyzed by spectrophotometer. Separately, cell nuclei were stained by fluorescent dye (Hoechst No. 33258) in order to evaluate the levels of chromatin condensation evoked by DNA fragmentation. The number of cells with chromatin condensation was counted. During the entire experimental period, more than 85% of cells were persistently viable in all groups. NH2Cl significantly induced the DNA double strand break as well as chromatin condensation in RGMC and KATO III cells (P < 0.05). However, NH3 or HOCl did not induce the DNA double strand break as well as chromatin condensation in both cells. NH2Cl, but not its precursors (NH3 or HOCl), enhanced the levels of DNA injury, suggesting the possible involvement in the carcinogenesis of Hp-associated gastric mucosa.
Abstract: We previously reported that the response of the arterioles in spontaneously hypertensive rats (SHR) to histamine is blunted compared with that in normotensive control rats (Wistar-Kyoto rats [WKY]). The present study was designed to analyze the extent to which this blunted arteriolar response may be attributed in SHR to the concurrent elevation of circulating glucocorticoids through the use of adrenalectomy with and without dexamethasone supplementation. Mesenteric arterioles were observed by intravital microscopy under general anesthesia, and their lumen diameters were measured after histamine superfusion. The concentration-response curve with histamine was compared with that of an endothelium-independent vasodilator, sodium nitroprusside. At the end of each experiment, papaverine was applied topically to determine the maximal diameter for each vessel, from which a measure of arteriolar tone could be computed. The arteriolar tone in sham-operated SHR is set at a higher steady-state level than in sham-operated WKY. The concentration required for a 50% dilator response (EC50) of histamine in adrenalectomized SHR was restored to the level of WKY. Adrenalectomy did not significantly affect the EC50 of histamine in WKY. When adrenalectomized SHR received a supplement of dexamethasone, the arteriolar response was found to show the same refractory pattern to histamine as sham-operated SHR. In contrast, the EC50 of sodium nitroprusside in sham-operated and adrenalectomized SHR was similar to that in sham-operated WKY. Our results indicate that the impaired dilator response to histamine in SHR is related to an enhanced adrenal glucocorticoid secretion.
Abstract: Helicobacter pylori are believed to play an important role in the formation of gastric ulcer in a syndrome characterized by a high urease activity. On the other hand, the production of oxygen radicals and platelet activating factor (PAF) is enhanced in gastric ulcers. The present study is designed to investigate the relationship between the different aspects of gastric mucosal injury, urease activity, oxygen radical production, and PAF content in gastric specimens. Biopsy specimens taken from 35 gastric ulcer patients were studied. Urease activity was detected by a rapid urease test (CLO). Oxygen radical production was measured as a value of luminol-dependent chemiluminescence (ChL) and PAF content was determined by radioimmunoassay in the biopsy samples. The CLO-positive rate was significantly higher in the gastric ulcer group in comparison with that in controls. ChL values and PAF content were significantly increased in gastric ulcers, especially in CLO-positive specimens. The CLO-positive rate, ChL values, and PAF content were also found to be increased at a distant site beyond the ulcer lesions. During the course of macroscopic ulcer healing of CLO-positive cases, the CLO positive level and the ChL values were not significantly decreased, although PAF content was significantly lower. Enhanced oxygen radical and PAF production were observed not only in the ulcer region but also at a distant site from the ulcer in the urease-positive gastric mucosa. The persistent enhancement of ChL values during the healing stage of urease-positive gastric ulcers suggests its involvement in the recurrence of gastric ulcers.
Abstract: Although small intestine is frequently injured in endotoxin shock, the exact pathological sequence has not been fully understood. The major objective of this study is to elucidate the role of interleukin (IL)-1 in endotoxin-induced microcirculatory disturbance of rat small intestine. Mucosal and submucosal microvessels of the rat ileum were observed by intravital microscope with a high speed video camera system and the attenuating effect of E5090, an inhibitor of IL-1 generation, on endotoxin-induced intestinal microcirculatory disturbances was investigated. Endotoxin infusion produced significant mucosal damage, but before these morphological changes became significant, microvascular stasis in villi, decreased red blood cell velocity, and increased leukocyte adherence to venular walls were observed in intestinal microcirculatory beds 30 min after endotoxin administration. Intestinal IL-1alpha levels were also significantly increased at that time. Endotoxin treatment enhanced chemiluminescence activity from neurophils and rapidly mobilized CD18 on leukocytes. E5090, which suppressed the IL-1 production in intestinal mucosa, attenuated the microcirculatory disturbances induced by endotoxin, and significantly reduced the subsequent mucosal damage. E5090 also attenuated the increased chemiluminescence activity and CD18 expression on leukocytes. In conclusion, the production of IL-1alpha is enhanced in the intestinal mucosa during endotoxin infusion. IL-1 may be an important mediator of microcirculatory changes, including decreased red blood cell velocity and increased leukocyte sticking and its activation, leading to the mucosal damage.
Abstract: In alcoholic liver disease, endotoxin has been postulated to play an important role in its pathogenesis. Endotoxin is known to lead to impediment of hepatic microcirculation, including the adhesion of leukocytes to sinusoidal endothelial cells. In this study, the effect of chronic ethanol consumption on the leukocyte adhesion elicited by endotoxin was examined. Male Wistar rats were pair-fed with a liquid diet containing ethanol or an isocaloric control diet for 6 weeks. The liver of anesthetized rats were placed on the nonfluorescent cover-glass for observation by an intravital inverted microscope equipped with a silicon intensified target camera. The red blood cell (RBC) velocity in hepatic sinusoids was measured by an off-line temporal correlation velocimeter (Capiflow, Sweden) after intravenous injection of fluorescein isothiocyanate-labeled rat RBC. RBC velocity in sinusoids was more severely disturbed in ethanol fed rats than in controls. Leukocytes were stained by the intravenous injection of carboxyfluorescein succinimidyl ester for a fluorographic observation of leukocyte adhesion. After lipopolysaccharide injection, the number of adherent leukocytes was significantly greater in ethanol-fed rats than in controls. Plasma tumor necrosis factor-alpha levels were also higher in ethanol-fed rats than in controls. These results suggest that chronic ethanol consumption aggravates endotoxin induced leukocytes adhesion that may result in hepatic microcirculatory disturbances. Leukocyte adhesion to the sinusoidal wall may be associated with increased in tumor necrosis factor-alpha levels.
Abstract: The factors that predispose to the accelerated organ injury that accompanies the hypertensive syndrome have remained speculative and without a firm experimental basis. Indirect evidence has suggested that a key feature may be related to an enhanced oxygen radical production. The purpose of this study was to refine and use a technique to visualize evidence of spontaneous microvascular oxidative stress in vivo in the spontaneously hypertensive rat (SHR) compared with its normotensive control, the Wistar-Kyoto rat (WKY). We investigated the effects of adrenal glucocorticoids on the microvascular oxidative stress sequence. The mesentery was superfused with hydroethidine, a reduced, nonfluorescent precursor of ethidium bromide. In the presence of oxidative challenge, hydroethidine is transformed intracellularly into the fluorescent compound ethidium bromide, which binds to DNA and can be detected by virtue of its red fluorescence. The fluorescent light emission from freshly exteriorized and otherwise unstimulated mesentery microvessels was recorded by digital microscopy. The number of ethidium bromide-positive nuclei along the arteriolar and venular walls in SHR was found to be significantly increased above the level exhibited by WKY. The elevation in ethidium bromide fluorescence in SHR arterioles could be attenuated by a synthetic glucocorticoid inhibitor and in rats subjected to adrenalectomy. The administration of glucocorticoids after adrenalectomy by injection of dexamethasone restored the oxidative reaction in SHR arterioles. Treatment with dimethylthiourea and with a xanthine oxidase inhibitor attenuated the superoxide formation. Although a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester) enhanced the ethidium bromide staining in WKY, it did not affect that in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Leukocyte adhesion is a key factor in the pathogenesis of organ injury following a variety of stimuli. In this study we have addressed the role of leukocyte adhesion in hypertensives as a risk factor for organ injury. In the spontaneously hypertensive rat (SHR), the number of circulating leukocytes and their level of activation are significantly increased compared with its normotensive control, the Wistar-Kyoto rat (WKY). We have demonstrated that elevated levels of glucocorticoid in SHR suppress P-selectin-mediated leukocyte-endothelial interaction in the microcirculation. It is possible that the disturbance in leukocyte-endothelial interactions may result in an elevated number of leukocytes in the circulation. The aim of the present study was to investigate the contribution of the adrenal glands to the disturbance in leukocyte behavior in SHR by subjecting the animals to bilateral adrenalectomy and investigating the effect of hydrocortisone. In addition, we have studied by immunohistochemistry the expression of the endothelial adhesion molecule, P-selectin, in response to histamine in the mesenteric venules of normal and adrenalectomized SHR and WKY. The elevated blood pressure, above-normal leukocyte counts, and elevated number of activated neutrophils (nitroblue tetrazolium test) in SHR were blunted after adrenalectomy. The blunted histamine-induced leukocyte-endothelial interaction in the mesenteric venules of SHR was restored after adrenalectomy. Treatment with hydrocortisone significantly attenuated the elevated leukocyte adhesion in the adrenalectomized SHR as well as in WKY. The suppressed P-selectin expression in SHR mesentery was restored after adrenalectomy. In conclusion, the subnormal leukocyte-endothelial interaction in response to an inflammatory stimulation in SHR is abolished after adrenalectomy, suggesting a relationship between the altered leukocyte adhesiveness and the adrenal corticosteroids in hypertensives.
Abstract: Substance P (SP), one of the established neurotransmitters, evokes an immunoinflammatory response involving leukocyte adhesion to venular endothelium and the degranulation of mast cells. The pathogenetic relationship between these responses, however, remains unresolved. In this study, we propose to examine the changes associated with the activation of mast cells, as well as leukocyte adhesion to venular endothelium by in vivo observation of the rat mesentery. The use of an in vitro assay for intracellular Ca2+ mobilization and the degranulation of mast cells demonstrated the significant upper shift of concentration response to SP (10(-4)-10(-5) M). In vivo experiments on the mesenteric microcirculation also showed that SP induced a significant increase in the number of degranulated mast cells as well as in the number of leukocytes adherent to the venular wall. Tranilast, a mast cell stabilizer, as well as SP antagonist (CP-96,345) significantly attenuated the extent of mast cell degranulation and leukocyte adhesion elicited by SP. Although an immunoneutralization against CD18 by WT-3 significantly attenuated the leukocyte adhesion, it had no influence on the mast cell degranulation after SP superfusion. These separate in vivo observations show that SP induces leukocyte adhesion to the venular endothelium, possibly through the degranulation of mast cells.
Abstract: This study is designed to explore the effect of adrenal glucocorticoids on arteriolar behavior under steady state conditions in spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto (WKY) rats. Mesenteric arterioles were observed by intravital microscopy under general anesthesia and arteriolar diameters were measured. At the end of each experiment, papaverine was topically applied to determine maximum diameter of the vessels for which arteriolar tone was computed. The adrenal contribution to the atypical arteriolar tone in SHR was explored by subjecting them to bilateral adrenalectomy with or without glucocorticoid replacement therapy. Adrenalectomy led to a reduction in the elevated blood pressure in SHR to normotensive levels, which could be restored to its hypertensive level by treatment with dexamethasone. Adrenalectomy had so significant effect on blood pressure in WKY rats, although administration of dexamethasone but not aldosterone did lead to an elevation of the blood pressure in sham-operated WKY. Arteriolar tone in SHR was set at a higher steady state level than in WKY rats. In adrenalectomized SHR, tone fell to the levels observed in WKY rats. This decrease in arteriolar tone in the adrenalectomized SHR could in turn be restored to the hypertensive levels with dexamethasone but not aldosterone. Our results suggest that the enhancement of arteriolar tone, as well as the high blood pressure in SHR, may be related to the above normal levels of adrenal glucocorticoids present in the bloodstream.
Abstract: Recent evidence suggests that spontaneously hypertensive rats (SHR) exhibit an impaired response to inflammatory mediators. We designed this study to analyze the response of arterioles of SHR after stimulation with a proinflammatory agent, histamine, compared with the response of arterioles of normotensive Wistar-Kyoto (WKY) controls. We observed mesenteric arterioles by intravital microscopy in rats under general anesthesia and measured their lumen diameters after histamine superfusion. To compare the concentration-response curve with histamine, we also studied the effect of an endothelium-dependent vasodilator, acetylcholine, and an independent vasodilator, sodium nitroprusside. At the end of each experiment we applied papaverine topically to determine the maximal diameter for each vessel, from which we computed arteriolar tone. Arteriolar tone in SHR is set at a higher steady state level than in WKY. The concentration required for a 50% dilator response (EC50) of histamine in SHR was significantly higher than that in WKY. In SHR the arteriolar response showed the same refractory pattern to histamine as to acetylcholine. In contrast, the EC50 of sodium nitroprusside in SHR was similar to that in WKY. Our results indicate that SHR exhibit an impaired dilator response to histamine that is due to a blunted endothelium-dependent vasodilation.
Abstract: The present study was designed to elucidate whether molecular mechanisms for leukocyte adhesion to microvascular endothelium may differ between spontaneously hypertensive rats and Wistar Kyoto rats. Leukocyte rolling and adhesion were investigated while monitoring venular wall shear rates in the mesenteric microcirculation stimulated with histamine or tert-butyl hydroperoxide in the two strains. In Wistar Kyoto rats, 10 microM histamine as well as 500 microM tertbutyl hydroperoxide promoted a significant reduction of venular leukocyte rolling velocity and subsequent adhesion. These changes in leukocyte behavior were blocked by monoclonal antibodies against P-selectin (PB 1.3) and against sialyl Lewis X-like carbohydrates (2H5). However, spontaneously hypertensive rats exhibited a blunted response of the stimulus-elicited leukocyte rolling, which was associated with impairment of venular P-selectin expression as well as a decrease in the expression of sialyl Lewis X-like carbohydrates on circulating neutrophils. No significant differences were detected between the two strains not only in the surface CD11b/CD18 expression but also in the CD18-mediated adhesivity of neutrophils to intracellular adhesion molecule-1 transfectants in vitro. These results suggest that impairment of selectin-mediated leukocyte adhesion is an event responsible for disorders of inflammatory responses in spontaneously hypertensive rats.
Abstract: To gain insight into the mechanisms responsible for muscle dysfunction after ischemia-reperfusion, a rat spinotrapezius muscle preparation was developed which enabled sequential measurements of in vivo maximum tetanic force production and cell death assessed using digital microfluorographic determination of propidium iodide (PI) staining. After 60 min of no-flow ischemia, maximum tetanic force fell significantly during 90 min of reperfusion compared with control, nonischemic muscles. The most striking fall was evident within 30 min of reperfusion and occurred concomitant with an explosive increase in PI-positive myocyte nuclei. Treatment with the oxygen radical scavenger, dimethylthiourea, attenuated both the fall in force and increased PI staining. Indeed, the rise in PI-positive nuclei correlated closely (r= 0.728) with the reduction of maximum tetanic force developed following ischemia and reperfusion under all conditions. Superoxide dismutase also attenuated the rise in PI-positive nuclei. Assessment of mitochondrial inner membrane potential (deltapsi) using Rhodamine 123 fluorescence revealed that myocytes with the lowest initial mitochondrial membrane potential were subject to the greatest injury after 90 min of reperfusion (r= 0.828). These results support the hypothesis that myocyte injury, as visualized by PI-staining, reflects an impaired contractile function in fibers with a low oxidative potential which is likely mediated by oxygen radicals.
Abstract: This study was designed to investigate the effects of prostaglandin E1 on reductive stress and the subsequent oxidative cell injury in hypoperfused rat liver. The intralobular heterogeneity of hepatocellular redox state, mitochondrial dysfunction, and intracellular hydroperoxide formation were visually monitored by digital microfluorography of pyridine nucleotide autofluorescence, rhodamine 123, and dichlorofluorescein fluorescence, respectively. Under the 25% low flow perfusion, pyridine nucleotide autofluorescence increased time-dependently and reached a steady state at 10 min among the entire lobules. The decrease in mitochondrial membrane potential was > 20 mV in all portions of the lobules at 60 min. The onset of hydroperoxide formation was observed at 40 min in the marginally oxygenated proximal portion of anoxic pericentral regions and the oxidative impact reached a maximum level at 60 min. Sodium (-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazo [1,5-a]-1,3,5-triazine-4-olate monohydrate (BOF 4272), a novel xanthine oxidase inhibitor, suppressed the zone-specific oxidative changes without attenuating the increase in pyridine nucleotide autofluorescence and mitochondrial dysfunction. Pretreatment with prostaglandin E1 not only abrogated an early increase in pyridine nucleotide fluorescence and mitochondrial dysfunction induced by hypoperfusion but also diminished the subsequent midzonal oxidative injury. Since prostaglandin E1 has no oxyradical-scavenging action, the preventive effect of this reagent on the hypoxia-induced oxidative cell injury is attributable to the attenuation of mitochondrial dysfunction. These results suggest that, in low flow hypoxia, early reductive stress plays a key role in the initiation of xanthine oxidase-mediated midzonal oxidative changes, which may lead to subsequent centrilobular necrosis.
Abstract: The effects of rat CINC/gro, a member of the interleukin-8 family, on the endothelium-neutrophil interaction and transendothelial macromolecular leakage were studied in rat mesenteric microcirculation. Thirty minutes after superfusion with 10 nM CINC/gro, the number of neutrophils adherent to the venular endothelium and those migrated across the venules were significantly increased with a concomitant elevation of luminol-dependent chemiluminescence at the site of adhesion. Transendothelial macromolecular leakage as assessed by the relative length of venular wall stained with monastral blue B was also increased at 30 min after the start of CINC/gro superfusion. Pretreatments with a CD18-directed monoclonal antibody, WT-3 (1 mg/kg), significantly attenuated the increase in number of adherent and migrated neutrophils, the increase in luminol-dependent chemiluminescence, and the venular macromolecular leakage after the application of CINC/gro. These data suggest that CINC/gro is a novel stimulator that evokes not only locomotive but also secretagogue activation of neutrophils via a CD18-dependent mechanism in vivo.
Abstract: E. coli O157:H7 produces a cytotoxin active against Vero cells that has been termed verotoxin. In this study, we demonstrated that local intraarterial injection of verotoxin induced a decrease in blood flow and an increase in hemorrhagic lesions in rat small intestine. Significant increases in the area of hemorrhagic lesions were observed at 120 min after verotoxin injection. These lesions were produced by either verotoxin 1 or 2, but verotoxin 2 produced more extensive lesions. The temporal alteration of vasoactive substances in microcirculatory beds was determined after the administration of culture filtrate of E. coli O157:H7. Tissue-type plasminogen activator activity in regional plasma was significantly elevated as early as 30 min, suggesting that local fibrinolytic activation mediated by microvascular endothelium occurred. There was also early elevation of platelet-activating factor content in the ileal mucosa and its level remained significantly elevated thereafter. Intestinal blood flow, as determined by a laser Doppler flowmeter, started to decrease at about 45 min. The platelet-activating factor antagonist CV6209 was shown to attenuate the decrease in blood flow as well as the development of hemorrhagic lesions, demonstrating that platelet-activating factor is an important mediator for the microcirculatory damage. Accumulation of neutrophils demonstrated by myeloperoxidase activity in the intestinal mucosa and overproduction of oxygen-radicals from neutrophils of the mesenteric veins determined by the luminol-dependent chemiluminescence assay were observed at 60 min, corresponding with the decreased blood flow. Platelet-activating factor may be closely involved in the process of leukocyte accumulation and increased oxygen radical generation, because CV6209 also significantly attenuated these changes.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Hypertension is associated with a progressive organ injury whose etiology remains largely speculative. An increasing database shows that activated leukocytes, while affording an important immune protection, may be a contributing factor to several of the pathogenetic features of the hypertension syndrome. The purpose of this study was to determine the extent to which the glucocorticoid pathway may be involved in the atypical kinetics of leukocytes in spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) rats. The typical venular leukocyte adhesion induced by histamine application was significantly lower in SHR, and a comparison of normalized leukocyte rolling velocity (VWBC/VRBC) showed the values to be significantly higher in SHR relative to WKY controls. This abnormal trend in adherent leukocyte numbers and in VWBC/VRBC values could be counteracted when SHR were pretreated with RU 486, a synthetic glucocorticoid inhibitor, and restored to the levels observed in WKY rats. Anti-P-selectin monoclonal antibody (PB1.3) attenuated in SHR and WKY rats the increment of adherent leukocyte numbers as well as the decrement of VWBC/VRBC value that developed under combined histamine and RU 486 superfusion. Furthermore, an anti-intercellular adhesion molecule-1 monoclonal antibody (1A29) served to attenuate the increment of adherent leukocyte number induced by a combination of histamine and RU 486 superfusion in WKY rats and SHR. The results indicate that the deficient leukocyte-endothelial cell interaction in SHR can be circumvented by a glucocorticoid inhibitor.
Abstract: The study was designed to examine the changes of thymus in sulfhydryl blocker-induced colitis. We used N-ethylmaleimide (NEM) as sulfhydryl blockers. Fasted male Sprague-Dawley rats were given 3% NEM in 1% methyl cellulose into the colon. N-ethylmaleimide treatment caused severe diarrhoea with bleeding for the first 7 days. At autopsy, adhesions, colon dilatation, and single or multiple erosions and ulcers were observed. Time-course studies revealed that the lesions were most extensive and severe 3 or 7 days after the administration of NEM. Histological examination of colon on the 3rd day after NEM treatment demonstrated mucosal erosion, oedema and extensive infiltration of neutrophils. The mucosal lesions extended into the submucosa and muscle on the 7th day after NEM treatment. Immunohistochemical studies showed that T cells and macrophages were markedly increased in the lamina propria of colonic mucosa. After 3 weeks, the infiltration of chronic inflammatory cells was observed and regeneration of the mucosa was noticed. The thymus gland was significantly decreased in weight and size on the 3rd day after NEM treatment, but the weight loss of thymus gland was regained in 3 weeks. Transient atrophy of thymus gland was noticed in this colitis model. The phenotypes of thymocytes were not influenced by NEM treatment. It is concluded that the thymus abnormalities in human ulcerative colitis are not induced in this animal model and that other chronic models are necessary for the elucidation of the immunological abnormalities, including thymus abnormalities.
Abstract: This study was aimed to determine the mechanism by which endogenous nitric oxide suppression promotes leukocyte adhesion in vivo. The rat mesenteric microcirculation was superfused with NG-nitro-L-arginine methyl ester (L-NAME; 100 microM), and intracellular oxidant formation in several microcirculatory cellular components such as arteriolar and venular endothelium and mast cells was visually monitored by digital microfluorography assisted by carboxydichlorofluorescein (CDCF), a hydroperoxide-sensitive fluorogenic probe. Adherent leukocyte density was measured simultaneously. L-NAME induced a significant time-dependent increase in CDCF fluorescence in arteriolar and venular endothelium and mast cells followed by firm adhesion of leukocytes. L-NAME-induced CDCF elevation showed a different spatial distribution compared with that evoked by N-formylmethionyl-leucyl-phenylalanine, in which only local venular segments with adhering leukocytes exhibited CDCF fluorescence enhancement. The level of hydroperoxide formation in arterioles and venules evoked by 60-min L-NAME superfusion was equivalent to that induced by the superfusion of approximately 880 microM tert-butyl hydroperoxide for 10 min. Pretreatment with anti-intracellular adhesion molecule-1, anti-P-selectin, or anti-CD18 monoclonal antibody attenuated L-NAME-elicited venular leukocyte adhesion without abolishing CDCF fluorescence in situ. Pretreatment with desferioxamine (50 mg/kg iv; 1 h before L-NAME superfusion) significantly diminished the iron-catalyzed hydroperoxide formation in arterioles and venules, but not in interstitial mast cells, as well as subsequent venular leukocyte adhesion. These findings indicate that endogenous nitric oxide may modulate oxidative stress in mast cells, arteriolar and venular microvascular endothelium and thereby can play a crucial role in leukocyte recruitment in venules.
Abstract: The effect of rebamipide, a novel antiulcer compound, on Helicobacter pylori activated neutrophil dependent in vitro gastric epithelial cell injury was investigated. Luminol dependent chemiluminescence (ChL), which detects toxic oxidants from neutrophils exhibited a 12-fold increase when the bacterial suspension of H pylori was added to the isolated human neutrophils. This change was significantly attenuated by rebamipide at a concentration less than 1 mM, showing that rebamipide may inhibit oxidant production from H pylori elicited neutrophils. To assess whether rebamipide attenuates gastric mucosal injury, we tested its inhibitory action on H pylori induced gastric mucosal damage associated with neutrophils in vitro. Rabbit gastric mucosal cells were monolayered in culture wells and coincubated with human neutrophils and H pylori, and the cytotoxicity index was then calculated. Cultured gastric cells were significantly damaged when they were incubated with human neutrophils activated by H pylori. This cellular damage was attenuated by rebamipide in a dose-dependent manner. Furthermore, spectrophotometrical measurement showed that rebamipide (1 mM) inhibits urease activity by 21.7%. As monochloramine (an oxidant yielded by reaction of neutrophil derived chlorinated oxidant and ammonia) is proposed as an important toxic molecule in this model, the current findings suggest that the preventive effect of rebamipide on H pylori elicited neutrophil induced gastric mucosal injury may result from its inhibitory actions on the neutrophilic oxidative burst as well as H pylori derived urease activity.
Abstract: Intralobular distribution of Ito cells (fat-storing cells) in hepatic microcirculatory units was investigated through intravital fluorescence microscopy. By using a low-level ultraviolet epi-illumination and a sensitive silicon intensified target camera, the intrahepatic autofluorescence was visualized and digitally processed. In rats fed an ordinary diet, the ultraviolet-excited autofluorescence was composed of at least two different origins that could not be spectrophotometrically distinguished, namely, multiple patchy autofluorescence activities along the sinusoids and terminal hepatic venules and diffuse parenchymal autofluorescence. Multiple patchy activities showed a rapid photobleaching phenomenon under the continuous ultraviolet excitation. These fluorescent activities were completely eliminated by depleting the intrahepatic retinoid contents using a vitamin A-deficient diet for 4 weeks. Furthermore repeated administration of vitamin A significantly enhanced the patchy fluorescent activities. Electron microscopy revealed that these vitamin A fluorescent activities are colocalized with the fat droplets in Ito cells, providing evidence that Ito cells exist not only in perisinusoidal spaces but also in the perivascular spaces of the terminal hepatic and portal venules. The current intravital technique thus provides a new method to observe Ito cells in hepatic microcirculatory units in vivo.
Abstract: Intralobular oxygen radical formation was examined in cerulein-stimulated rat pancreatic acinar cells by digital imaging microscopic fluorography using a hydroperoxide-sensitive fluorescent probe, dichlorofluorescin (DCFH) diacetate. The isolated pancreatic acinar cells loaded with DCFH diacetate were microscopically observed, and the dichlorofluorescein (DCF) fluorescence yielded by DCFH oxidation via hydroperoxides was digitally processed. Within the initial 20 min after the application of cerulein (10 microM), intracellular oxidative stress was observed as indicated by the increase in DCF fluorescence intensity and reached its maximum at 60 min. DCF fluorescence intensity was then gradually decreased until 80 min, followed by a marked increase in propidium iodide (PI) fluorescence, suggesting irreversible cell death. Allopurinol (1 microM), a xanthine oxidase inhibitor, significantly attenuated the early increase of DCF fluorescence intensity as well as the late cell damage. Treatment with hyperbaric oxygen (PO2 300 mm Hg) also significantly attenuated both the increase of DCF fluorescence and the number of PI-positive cells. The results suggest that xanthine oxidase-mediated oxygen radicals may play an important role in cerulein-induced intracellular oxidative stress in pancreatic acinar cells of rats.
Abstract: The role of neutrophil and its chlorinated oxidant were investigated in Helicobacter pylori-induced gastric mucosal injury in vitro. Luminol-dependent chemiluminescence (ChL) was used to detect neutrophil-derived oxidants. ChL activity was significantly elevated when neutrophils were incubated in H. pylori, indicating that H. pylori actually elicits oxidative burst of neutrophils. To assess whether H. pylori-activated neutrophils exert the cytotoxicity for gastric mucosal cells, rabbit gastric mucosal cell was monolayered in culture wells and labeled with a fluorescence dye, 2',7'-bis(2-carboxyethyl)-5(6)carboxy-fluorescein, which is retained in the intracellular space as long as the cell membrane is intact. Labeled cells were coincubated with neutrophils and H. pylori. We inferred from the cytotoxicity index (specific %cytotoxicity), which was calculated from fluorometrical measurements of supernatant and lysate, that the mucosal cells were significantly damaged by H. pylori-activated neutrophils. This injury was largely attenuated by eliminating urea from the incubation mixture or by acetohydroxamic acid, a potent urease inhibitor. Additionally, the scavengers of neutrophil-derived oxidants, including taurine, methionine, and catalase, also attenuated this injury. Cultured mucosal cells that were exposed to the solution containing monochloramine (an oxidant yielded by reaction of hypochlorous acid and ammonia) were highly damaged compared with cells exposed to hypochlorous acid or hydrogen peroxide at physiological concentrations. These data suggest that H. pylori-activated neutrophils promote gastric mucosal cell injury and that monochloramine plays a unique and important role in this process.
Abstract: In this study, we have demonstrated that repeated electrical stimuli to the artery of the mesenteric pedicle can produce mesenteric microcirculatory disturbance by autonomic nervous irritation in rats. The parameters to demonstrate microcirculatory damage were observed and quantitatively analyzed using the intravital microscopy after electrical stimulation for 40 minutes. The blood flow of arterioles and venules in the mesentery showed ischemia-reperfusion pattern during the repeated electrical stimulations. The diameter of arterioles did not show significant change, while RBC velocity of arterioles was significantly decreased at 30 min after the irritation. The RBC velocity in venules was decreased from the early period to about 20%. But this values were not significantly dropped during the later observation period, suggesting the formation of short circuit flow by passing the collapsed capillary beds. The number of rolling WBC in the venules was notably increased at the time immediately after irritation, and thereafter the number of rolling WBC number was rather reduced. The number of sticking WBC in venules was time-dependently increased and reached its maximum at 30 min. When permeability of venular wall was determined by the injection of pontamine sky blue, significant increase in permeability was already shown immediately after irritation, suggesting that the integrity of microvascular wall was disturbed in this early period. The permeated area expanded thereafter in parallel with the increase in sticking WBC number. From these observations, it is suggested that endothelial cell damage and following leukocyte-endothelium interaction induced by autonomic nerve irritation appear to be an important factor in microcirculatory disturbances.
Abstract: BACKGROUND: Although it has been postulated that mitochondrial dysfunction and oxygen radicals may play a role in the mechanisms of hypoxic liver injury, little is known about the intralobular topographic relationship between a disorder in oxygen metabolism and cell death during hypoxia. EXPERIMENTAL DESIGN: Intralobular heterogeneity of mitochondrial dysfunction and its topographic relationship to cell death during 25% low-flow hypoxia was visually demonstrated in perfused rat hepatic microcirculatory units by dual-color digital microfluorography using rhodamine 123, a fluorochrome sensitive to the mitochondrial membrane potential, and propidium iodide, which labels the nuclei of nonviable cells with a concomitant enhancement of fluorescence. RESULTS: In the normoperfused liver from fasted rats, no significant depletion of the mitochondrial membrane potential values was observed in any portion of lobules during 100 minutes. In the liver treated with the 40-minute hypoxia, the mitochondrial membrane potential values within the entire lobule were significantly decreased. After a longer period of hypoxia, the pericentral fluorescence decreased to background levels, and the intralobular gradient showed a steep decline midway between the periportal and pericentral regions. Cell death was at first prominent in the midzone at 40 minutes, and then extended towards the pericentral regions, forming centrilobular necrosis at 100 minutes. Although allopurinol diminished the early midzonal cell death and retarded the development of centrilobular necrosis, it did not attenuate depression of the mitochondrial membrane potential. CONCLUSIONS: It is conceivable that mitochondrial dysfunction per se may not be related to the intralobular distribution of cell death during hypoxia. On the other hand, mitochondrial dysfunction in the centrilobular regions seems to be an important event leading to early midzonal oxidative tissue damage in that this change may involve ATP breakdown and the subsequent enhancement of xanthine oxidase-mediated oxidative cell injury in the midzonal regions, which are incompletely oxygenated.
Abstract: Rat cytokine-induced neutrophil chemoattractant (CINC) is a member of the IL-8 family, and its human counterpart is gro/MGSA but not IL-8. We ascertained that chemically synthesized CINC was comparable to native CINC/gro with regard to chemotactic activity for rat neutrophils and studied the effect of synthesized CINC/gro on circulating leukocytes in microvascular vessels of rat mesentery. Exposure of rat mesentery to 10(-8)M authentic CINC/gro induced neutrophil adherence to and extravasation from postcapillary venules (PCVs) but not from capillaries or arterioles. CINC/gro concentrations as low as 10(-10) M were effective in causing neutrophil adherence. Neutrophils adhered to thin PCVs (mean diameter, approximately 25 microns) after exposure to CINC/gro for 15 min. The mean diameters of the PCV with adherence of neutrophils after exposure to CINC/gro for 30 and 60 min were 37 and 43 microns, respectively. The diameters of PCV with extravasation of neutrophils also increased in a time-dependent manner. The starting position of adherence of neutrophils was approximately 25-50 microns away from the upper junction of two vessels and remained virtually unchanged during exposure to CINC/gro for 60 min. However, the distance from the start to the end of neutrophil adherence increased in a time-dependent manner. The effect of CINC/gro on adherence and extravasation of leukocytes was neutrophil specific since other leukocytes such as lymphocytes and monocytes were not identified among the adherent and extravasated leukocytes.
Abstract: Intralobular heterogeneity of oxidative stress and its topographic relationship with cell death during low-flow hypoxia were shown in perfused rat liver by digital microfluorography using dichlorofluorescin diacetate, a fluorochrome sensitive to intracellular hydroperoxide formation, and propidium iodide, which labels the nuclei of nonviable cells. The surface of the liver loaded with two precursors was microscopically visualized, and the fluorescence of dichlorofluorescein, a highly fluorescent molecule generated by hydroperoxide-mediated dichlorofluorescin oxidation, was digitally processed. Dichlorofluorescein fluorescence significantly increased in midzonal regions as early as 20 minutes after starting the 25% low-flow hypoxia. At 40 minutes the fluorograph showed multiple dotted patterns, and the intensity peaked at 60 minutes. The onset of cell death studied by propidium iodide was observed at 40 minutes, and its topographic distribution corresponded to the dichlorofluorescein-enhanced midzonal regions. Allopurinol diminished the early midzonal oxidative stress and retarded the onset of cell death. The current findings show that xanthine oxidase-dependent oxidative stress and the resultant cell death during low-flow hypoxia are spatially restricted in the intermediate zone between the periportal and pericentral regions.
Abstract: Spatial and temporal alterations in carbon tetrachloride-induced oxidative stress were studied in perfused hepatic microcirculatory units of the rat by digital imaging microscopic fluorography using dichlorofluorescin diacetate, a hydroperoxide-sensitive fluorogenic probe. The surface of the liver loaded with dichlorofluorescin was microscopically visualized, and the fluorescence of dichlorofluorescin (DCF), a highly fluorescent molecule generated by hydroperoxide-mediated oxidation of dichlorofluorescin, was digitally processed. After completing the experiments, fluorescein-labeled albumin was injected into the perfusate to confirm the state of sinusoidal perfusion and the topography of hepatic microangioarchitecture in the area studied. During transportal infusion of carbon tetrachloride, DCF fluorescence was observed predominantly in the pericentral area and was attenuated by pretreatment with SKF-525A, suggesting the involvement of cytochrome P-450. After peaking at the maximum level, the pericentral DCF fluorescence gradually decreased in parallel to the loss of viability, implicating the causal role of intracellular hydroperoxide formation in hepatocellular injury. In retrogradely perfused liver, in which intralobular O2 gradient was reversed, no significant activation of DCF fluorescence was observed among hepatic lobules. It is therefore conceivable that the zonal heterogeneity of carbon tetrachloride-induced lipid peroxidation may depend on at least two factors, sinusoidal oxygenation and the intralobular distribution of cytochrome P-450. Furthermore, development of the current technique will provide a useful method to investigate the microtopography of oxidative stress in organ microcirculatory units.
