Pgn bs

Example 9

To test if recombinant PGLYRP1 can activate a TREM-1 response, the BWZ/hTREM-1 reporter cell line was seeded into black 96 well plates and stimulated with recombinant human PGLYRP1 (Cat, no. 2590-PG-050, R&D Systems: Minneapolis, Minn.) in the presence or absence of 10 μg/ml PGN. TREM-1 activation was read out after 24 hours of culture using the BetaGlo reagent (Cat. no. E4720, Promega Madison, Wis., USA) and luminescence measured using a TopCount Luminescence counter from Perkin Elmer. As shown in FIG. 5A, stimulation of the TREM-1 reporter cell line with PGLYRP1 induced a dose-dependent activation of TREM-1 in the presence of PGN. Several different PGN were tested, including PGN-EC (from E. coli), PGN-SA (from S. aureus) and PGN-BS (from B. subtilis) (Invivogen, San Diego, Calif., USA), and were all able to fascilitate the PGLYRP1-induced TREM-1 response.

The response induced by recombinant PGLYRP1 could be inhibited by addition of a recombinant TREM-1-Fc-fusion protein (SEQ ID NO: 2) (FIG. 5B) or by addition of polyclonal anti-PGLYRP1 antibody (Cat. no. AF-2590, R&D Systems: Minneapolis, Minn., USA) confirming that PGLYRP1 is the TREM-1 ligand and that soluble TREM-1 and anti-PGLYRP1 are potentially useful as TREM-1 antagonists.

Example 9

To test if recombinant PGLYRP1 can activate a TREM-1 response, the BWZ/hTREM-1 reporter cell line was seeded into black 96 well plates and stimulated with recombinant human PGLYRP1 (Cat. no. 2590-PG-050, R&D Systems: Minneapolis, Minn.) in the presence or absence of 10 μg/ml PGN. TREM-1 activation was read out after 24 hours of culture using the BetaGlo reagent (Cat. no. E4720, Promega Madison, Wis., USA) and luminescence measured using a TopCount Luminescence counter from Perkin Elmer. As shown in FIG. 5A, stimulation of the TREM-1 reporter cell line with PGLYRP1 induced a dose-dependent activation of TREM-1 in the presence of PGN. Several different PGN were tested, including PGN-EC (from E. coli), PGN-SA (from S. aureus) and PGN-BS (from B. subtilis) (Invivogen, San Diego, Calif., USA), and were all able to fascilitate the PGLYRP1-induced TREM-1 response.

The response induced by recombinant PGLYRP1 could be inhibited by addition of a recombinant TREM-1-Fc-fusion protein (SEQ ID NO: 2) (FIG. 5B) or by addition of polyclonal anti-PGLYRP1 antibody (Cat. no. AF-2590, R&D Systems: Minneapolis, Minn., USA) confirming that PGLYRP1 is the TREM-1 ligand and that soluble TREM-1 and anti-PGLYRP1 are potentially useful as TREM-1 antagonists.