| Project/Area Number |
12680638
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | University of Shizuoka |
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Principal Investigator |
IMAI Yasuyuki University of Shizuoka, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (80160034)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | O157 / Vero toxin / mucosal immunity / Carbohydrate recognition / IgA / pathogenic E. coli / EHEC / germinal center |
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| Research Abstract |
We investigated mucosal immune response against Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC) toward the end of development of IgA monoclonal antibodies. As an antigen, carbohydrate recognition subunits (Stx-1B) were expressed and the proteins were purified until homogeneity. The activity was confirmed by binding to glycolipid Gb_3 and to CD77 positive Burkitt's lymphoma cell lines. We prepared globotriose-conjugated poly-lysine, and their binding to immobilized Stx-1B was measured by an ELISA format. Antiserum from mice that had parenterally been immunized with the Stx-1B specifically interfered with the ligand binding. This result suggests the usefulness of the assay as a screening method of blocking antibodies. We also immunized mice through mucosal route using Stx-1B with cholera toxin as a mucosal adjuvant. We observed antigen specific serum IgG and IgA as well as specific secreted IgA in feces and intestinal wash. We also detected antigen specific IgA-producing cells from intestinal lamina propria using ELISPOT assay and the specific IgA in the culture supernatant of lamina propria lymphocytes from immunized mice. We then examined the ability of Stx-1B as a probe to detect biological ,ligands using an immunohistochemical approach. The Stx-1B specifically bound to renal tubules and collecting ducts in mouse kidney frozen sections. This is consistent with the result of human tissues. On the other hand, germinal centers developed in mouse lymph nodes and Peyer's patches were not stained by Stx-1B. The results suggested that, unlike human immune system, Stx would not impede germinal center functions (affinity maturation and class switch) of mouse immune system. This is consistent with the results that Stx-1Bspecific IgA was produced after mucosal immunization.
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