Expression of Eotaxin in Gastric Epithelial Cells Stimulated with Helicobacter pylori Vacuolating Cytotoxin

Helicobacter pylori-infected gastric mucosa is characterized by infiltration of various inflammatory cells such as neutrophils and eosinophils. Although several mechanisms for neutrophil infiltration are well known, there has been little known the role of eotaxin, which is a potent chemoattractant for eosinophils, on the inflammatory process of H. pylori infection. The present study was to investigate the mechanisms of eotaxin expression in gastric epithelial cells stimulated with H. pylori vacuolating cytotoxin (VacA). Stimulation with VacA purified from VacA+ H. pylori slightly increased eotaxin expression in MKN-45 gastric epithelial cells. In contrast, the combined stimulation with VacA and IL-4 synergistically increased the eotaxin expression as determined by quantitative RT-PCR and ELISA. In MKN-45 cells transfected with an eotaxin promoter-luciferase reporter plasmid, costimulation with VacA and IL-4 induced more luciferase activity than either VacA or IL-4 alone did. However, such up-regulation was significantly decreased in the cells transfected with luciferase reporter plasmid bearing an eotaxin promoter which has a mutation at STAT6 binding site. These results suggest that the up-regulation of eotaxin in VacA-stimulated gastric epithelial cells may be synergistically facilitated by IL-4 via a STAT6-dependent mechanism.

Expression of Eotaxin in Gastric Epithelial Cells Stimulated with Helicobacter pylori Vacuolating Cytotoxin

Helicobacter pylori-infected gastric mucosa is characterized by infiltration of various inflammatory cells such as neutrophils and eosinophils. Although several mechanisms for neutrophil infiltration are well known, there has been little known the role of eotaxin, which is a potent chemoattractant for eosinophils, on the inflammatory process of H. pylori infection. The present study was to investigate the mechanisms of eotaxin expression in gastric epithelial cells stimulated with H. pylori vacuolating cytotoxin (VacA). Stimulation with VacA purified from VacA+ H. pylori slightly increased eotaxin expression in MKN-45 gastric epithelial cells. In contrast, the combined stimulation with VacA and IL-4 synergistically increased the eotaxin expression as determined by quantitative RT-PCR and ELISA. In MKN-45 cells transfected with an eotaxin promoter-luciferase reporter plasmid, costimulation with VacA and IL-4 induced more luciferase activity than either VacA or IL-4 alone did. However, such up-regulation was significantly decreased in the cells transfected with luciferase reporter plasmid bearing an eotaxin promoter which has a mutation at STAT6 binding site. These results suggest that the up-regulation of eotaxin in VacA-stimulated gastric epithelial cells may be synergistically facilitated by IL-4 via a STAT6-dependent mechanism.

The Role of Mitogen-activated Protein Kinase in Enteritis Induced by Bacteroides fragilis Enterotoxin

A 20 kDa heat-labile toxin (BFT) produced by enterotoxigenic Bacteroides fragilis (B. fragilis) is associated with diarrhea and mucosal inflammation. Although intestinal epithelial cells are known to activate mitogen-activated protein kinase (MAPK) in response to bacterial infection, there has been little understanding on the association between MAPK activation and BFT-induced enteritis. This study was performed to investigate the role of MAPK in enteritis induced by BFT. In human colon epithelial cells, BFT increased IL-8 secretion in a dose-dependent manner. BFT activated the three main MAPK cascades, including extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). BFT stimulation also activated AP-1 activation signals. Overexpression of dominant-negative plasmid of the c-Jun decreased the activated AP-1 signals and the up-regulated IL-8 expression induced by BFT stimulation. In addition, SB203580 and ERK inhibitor U0126 significantly reduced IL-8 secretion in colon epithelial cells stimulated with BFT. Furthermore, SB203580 significantly prevented BFT-induced severity of enteritis and fluid secretion in mouse ileum. These results suggest that MAPK activation may be required for IL-8 transcription in intestinal epithelial cells exposed to BFT and that the activated MAPK can mediate intestinal inflammation and mucosal damage induced by BFT.

The Role of Activation of MAP Kinase in Human Intestinal Epithelial Cells Stimulated with Bacteroides fragilis Enterotoxin

A ~20 kDa heat-labile toxin (BFT) produced by enterotoxigenic B. fragilis induces chemokine responses that are associated with mucosal inflammation. In the present study, we assessed whether the activation of mitogen-activated protein kinase (MAPK) affects the levels of IL-8 and MCP-1 produced by BFT stimulation in human epithelial HT-29 cells. Human intestinal epithelial HT-29 cell lines were incubated with purified BFT. MAPK and AP-1 in HT-29 cells were measured by Western blot and luciferase assay, respectively. The expression of chemokines such as IL-8 and MCP-1 were determined by quantitative RT-PCR, ELISA, and luciferase assay. BFT stimulation activated MAPK such as ERK1/2 and p38 in HT-29 cells. Treatment with MAPK inhibitors attenuated BFT-induced expression of IL-8 and MCP-1. Transfection with mutant genes for Ras or c-Jun did not only suppressed AP-1 reporter genes, but also inhibited BFT-induced expression of IL-8 and MCP-1 reporter genes. These results suggest that Ras and MAPK cascade may act as the upstream signaling for the activation of AP-1, which induce chemokine expression in BFT-stimulated intestinal epithelial cells.

Expression of Cyclooxygenase-2 in Human Intestinal Epithelial Cells Stimulated with Bacteroides fragilis Enterotoxin

A ~20 kDa heat-labile toxin (BFT) produced by enterotoxigenic B. fragilis induces chemokine responses that may be associated with mucosal inflammation. To determine whether epithelial cells can contribute to BFT-induced inflammation, we assessed the expression of cyclooxygenase (COX)-2 in BFT-stimulated human intestinal epithelial cells. Human intestinal epithelial cell lines, HT-29 and Caco-2, were incubated with purified BFT. COX-2 mRNA and protein expression were measured by quantitative RT-PCR and Western blot analysis, respectively. BFT increased expression of COX-2, not that of COX-1, in human intestinal epithelial cell lines. Up-regulated COX-2 expression was paralleled by increased prostaglandin E2 (PGE2) production measured by the radioimmunoassay. PGE2 was predominantly secreted from the basolateral surface of BFT-treated epithelial cells, whereas lactate dehydrogenase was released predominantly from the apical surface. Moreover, the COX-2 expression and PGE2 production were significantly suppressed when NF-kappaB activity was inhibited. The basolateral secretion of PGE2 by up-regulated COX-2 in the BFT-stimulated colon epithelial cells seems to contribute to the inflammatory response in the underlying intestinal mucosa.

Nuclear Factor - kappa B Activation and Signal Transduction Pathway in Human Intestinal Epithelial Cells Stimulated with Bacteroides fragilis Enterotoxin

No abstract available.

Cytokine gene expression of peritoneal tissues in response to mixed infection of Bacteroides fragilis and Escherichia coli

Bacteroides fragilis and Escherichia coli, normal colonic inhabitants, are the most frequently isolated bacteria in infected tissues, particularly in intraabdominal abscesses. This study was designed to determine whether enteric bacteria may alter the B. fragilis-induced expression of proinflammatory cytokines in mouse peritoneal tissue (MPT). After C57BL/6 mice were inoculated with abscess-forming mixture containing B. fragilis in the presence or absence of E. coli, RNA was extracted from MPT. Expression of interleukin (IL)-1alpha and tumor necrosis factor (TNF)alpha mRNA was assessed using RT-PCR and standard RNA. Each cytokine protein was also measured by ELISA. The co-inoculation of E. coli into mouse peritoneal cavity advanced the onset of abscess development by B. fragilis infection. When mouse was co-infected with E. coli and B. fragilis intraperitoneally, there was a synergistic increase in the expression of IL-1alpha and TNFalpha mRNA in MPT and this was paralleled by increased cytokine protein secretion. Mixed inoculation of heat-killed E. coli and B. fragilis did not cause a synergistic increase in those cytokine mRNA expression. These results suggest that enteric bacteria may significantly affect proinflammatory cytokine signal produced by host peritoneal cavity in response to B. fragilis infection.

Basolaterial Secretion of CXC Chemokines by Human Intestinal Epithelial Cells in Response to Bacteroides fragilis Enterotoxin Via NF-kB Pathway

No Abstract Available.

Basolaterial Secretion of CXC Chemokines by Human Intestinal Epithelial Cells in Response to Bacteroides fragilis Enterotoxin Via NF-kB Pathway

No Abstract Available.

Prevalence of Virulence Factors in Helicobacter pylori Isolated from Koreans and Proinflammatory Cytokine Gene Expression in Human Gastric Epithelial Cells Induced by H. pylori with Virulence Factors / 대한내과학회지

OBJECTIVES: CagA or cytotoxin-positive H. pylori may be associated with gastroduodenal diseases. However, controversies about this association also exist. Moreover, there could be geographic differences in the prevalence of virulence factors such as cagA or cytotoxin. In H. pylori infection, the gastric mucosa shows acute and chronic inflammation. However, the pathogenesis of such as an inflammation by H. pylori is not well elucidated. We performed this study 1) to determine prevalence of the genes of virulence factor such as cagA and cytotoxin in H. pylori, 2) to assess the correlation of their presence with clinical findings, and 3) to test whether the vacuolating cytotoxin of H. pylori could evoke proinflammatory cytokine gene expression in gastric epithelial cells. METHODS: 1) The prevalence of the cagA, vacA and adhesin genes in H. pylori strains isolated from Koreans was determined by PCR analysis. 2) H. pylori was cultured in Brucella broth containing 10% fetal bovine serum for 3 days using a shaker in a microaerophilic condition. Cytotoxin assay was performed by determining whether addition of the concentrated culture supernatants is able to cause vacuolization of HeLa cells. 3) After human gastric epithelial cells, Hs746T and AGS were incubated with the culture supernatants containing vacuolating cytotoxin, each RNAs were extracted from the gastric epithelial cells. And then various cytokine gene expression were assessed using RT-PCR. The expressed cytokine transcripts were quantified by RT-PCR and standard synthetic RNA. Among cytokines, IL-8 proteins were also measured by ELISA. RESULTS: 1) More than 95% of H. pylori isolates from Korean adults possessed cagA, vacA and adhesin genes. And 80.6% of H. pylori strains have expressed vacuolating cytotoxicity against HeLa cells within 24 hours. 2) There was no correlation between the virulence factors of H. pylori strains and clinical findings. 3) Cytotoxin-positive culture supernatants also caused vacuolization in gastric epithelial cells, both Hs746T and AGS. 4) Expression of mRNA for proinflammatory cytokines such as IL-1alpha: IL-8, MCP-1 and GM-CSF was much more upregulated by vacuolating cytotoxin-positive culture supernatants than cytotoxin-negative ones in both Hs746T and AGS cells. Number of molecules of the expressed IL-8 transcripts was parallel to the amounts of IL-8 protein secreted from gastric epithelial cells. CONCLUSION: These results suggest that virulence factors of H. pylori may not be factors determining disease entitiy in Korean patients infected with H. pylori. In addition, vacuolating cytotoxin secreted from H. pylori could give rise to vacuolization in gastric epithelial cells as well as induce proinflammatory cytokines from the cells.

Expression of Monocyte Chemotactic Protein-1 and Macrophage Inflammatory Protein-1alpha in Intraabdominal Abscess Induced by Bacteroides fragilis Infection

OBJECTIVE: Bacteroides fragilis is the most frequently isolated anaerobes in tissue of intraabdominal infection, particularly in intraabdominal sepsis or abscess. In acute experimental model with an intraabdominal infection, the response to B. fragilis is characterized by infiltration of neutrophils and monocytes. To understand the pathogenesis of B. fragilis infection, it is important to explore the mechanism for inflammatory signals such as chemokines induced by this bacteria. The goal of this study was to determine whether peritoneal monocytes or fibroblasts surrounding with intraabdominal abscess induced by B. fragilis could express chemokines such as monocyte chemotactic protein-1 (MCP-1) or macrophage inflammatory protein-1a (MIP-1a). METHODS: 1) After C57BL/6 mice were intraperitoneally inoculated with abscess-inducing agents containing B. fragilis, RNA was extracted from the intraperitoneal tissues of the mice using Ottawa sand and the guanidinium thiocyanate-phenol-chloroform method in 3 days later. 2) After C57BL/6 mouse peritoneal monocytes were infected with B. fragilis for 1, 4 and 9 hours, cellular RNA was extracted from the cells. 3) Fibroblasts isolated from intraabdominal abscess nodules induced by B. fragilis infection were growth in tissue culture for 3 to 4 weeks. After the fibroblasts were stimulated with IL-1alpha (0.1-10 ng/ml) or TNFalpha (0.1-10 ng/ml) for 24 hours, total cellular RNA was extracted. MCP-1 or MIP-1alpha mRNA expression was assessed using RTPCR. MCP-1 or MIP-1alpha proteins in cluture supernatants or tissue extracts were also measured by ELISA. RESULTS: 1) MCP-1 or MIP-1alpha mRNA was highly expressed in peritoneal tissue of C57BL/6 mice bearing with intraabdominal abscess induced by B. fragilis. 2) Expression of MCP-1 mRNA increased at 9 hours in mouse peritoneal monocytes infected with B. fragilis. MIP-1alpha mRNA was initially expressed and perisisted in the monocytes infected with B. fragilis for 9 hours. MCP-1 or MIP-1alpha proteins was also parallel to the expression of those chemokines. 3) The fibroblasts isolated from intraabdominal abscess nodules by B. fragilis infection constitutively expressed MCP-1, MIP-1alpha, production in the fibroblasts was significantly upregulated in response to proinflammatory cytokines produced in the monocytes, including IL-1alpha and TNFalpha, but MCP-1 production were not. The normal fibroblasts from uninfected mice didnot show significant production of MCP-1 or MIP-1a in response to IL-1a or TNFalpha. CONCLUSION: These results suggest that peritoneal monocytes and fibroblasts surrounding with abscesses induced by B. fragilis produce MCP-1 or MIP-1a. Furthermore, it could be extrapolated that those effects may play a role in the formation of intraabdominal abscess nodules.

A Case Of Di Guglielmo Syndrome

We have experienced a case of Di Guglielmo Syndrome in a 15 years old girl who had the cheif complaints of dizziness, gereral malaise and fine pustules around the nose. It is a systemic hemopathy characterized by abnormal proliferation of defective erythroid and myeloid cells and is a rare disease in childhood. The peripheral blood showed many rubriblasts, myeloblasts, metamyelocytes and bone marrow also showed atypical prorubricytes and bizzar multinucleated rubriblasts. Brief review of related literatures was made.