| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Recently, intestinal bacteria might be the elusive pathogenic factors in ulcerative colitis (UC). However, which mucosal bacteria cause inflammation of the intestinal mucosa has not been determined. In this study, we investigated bacterial penetration and the secretion of proinflammatory cytokines in human colon epithelial cells in response to various mucosal bacteria and probiotics. Human colonic epithelial cells (SW480, ATCC) were cocultured with bacteria isolated from UC (F.valitrnl, E.coli, B.vulgatus, C.clostridloforme), H.pylori (ATCC 43504, TN2GF4), and probiotics (Lactobacillus johnsonii, L.delbrueckii subsp, bulgaricus). Cell cultures were infected with bacteria and resuspended in a 1:1 mixture of DMED and Ham's F-12 medium containing 10% FBS at a bacterium/cell ratio of 10001. The co-cultures further incubated for 1 h and 3h to allow bacterial adhesion and invasion to cells, and the supernatants were collected for attachment and invasion assay. The IL-8, IL-6, IL-18 and MCP-1
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concentrations in the supernatants were determined with ELISA kits. The induction of m-RNAs was measured by comparative Ct methods using real time PCR. The attachment and invasion were also analyzed by electron microscopy. In attachment assays, all bacteria were recovered at 0.01-1.49×10^5 CFU/mL, however L. delbrueckii subsp. bulgaricus was not detected by scanning electron microscope. The cytokines were not detected in all strains except for F.varium. IL-18 concentrations after exposure to three F.varium strains from UC were (mean) 19.7-52.9 pg/mL. In invasion assays, all bacteria except for probiotics were recovered from SW480 cells at 0.03-2.9 X 10^4 CFU/mL. IL-8 concentrations after exposure to F.varizrm were 148-210 pg/mL and IL-18 concentrations after exposure to two F.varium strains from UC were 32.0-51.5 pg/mL. IL-8 concentrations after exposure to B.vulgatus. C.clostridioforine and H.pylori (TN2BF4) were 35, 33 and 47 pg/mL, respectively. IL-6 and MCP-1 concentrations after exposure to all bacteria strains were below 0.150 and 31.3 pg/mL. However, m-RNA of IL-6, MCP 1 and IL-8 were increased in all strains except for probiotics, and marked increased in F varlum. In transmission electron microscopy, F. varium and H pylori were detected in SW480 cells. Probiotics did not invade the colonic epithelial cells and therefore did not induce inflammatory cytokines secretion in the cells. On the other hand, some mucosal bacteria, especially Fn invaded colonic epithelial cells and induced inflammatory cytokines secretion. Less
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