| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
1. Cloning of genes encoded Toll-like receptors (TLRs) and related molecules
The full-length cDNAs encoding TLR2, TLR4, CD14 and MD2 were cloned from murine spleen and human peripheral mononuclear cells. These genes were transfected to cells to produce transfectants.
2. Preparation of monoclonal antibodies against murine TLR2, TLR4 or MD2
To prepare monoclonal antibodies, rats were immunized with transfectants encoding TLR2, TLR4 or MD2. Immunized lymphocytes were fused with SP2/0 myeloma cells using polyethylene glycol Hybri-Max. After hybridization and HAT selection hybridoma were cloned in the usual way. However, we did not yet get good monoclonal antibodies.
3. Analysis of TLRs recognized bacterial glycolipids
Trehalose 6,6'-dimycolate (TDM, cord factor), obtained from Mycobacterium tuberculosis, has various biological activates including macrophage activation and granuloma formation. Since the activities of TDM have never been demonstrated in vitro due to its hydrophobicity, we newly developed an in vitro assay system. TDM, coated on wells of culture plates, activated peritoneal macrophages from TLR4-deficient mice. In contrast, activation of TLR2-knockout macrophages by TDM was much weaker than by LPS.Using glycolipids containing different mycolic acids, the structure-activity relationship showed the importance of mycolic acid chain length for macrophage activation. Furthermore, we showed that similar to LPS a glycosphingolipid (GSL), obtained from Sphingomonas paucimobilis was recognized by TLR4.
In this project we have demonstrated that mycobacterial TDM activates innate immunity via TLR2.
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