Several recent reports have suggested that c-di-GMP can stimulate a variety of signaling pathways in mammalian cells in vivo. One early report demonstrated that 50 µM of exogenous c-di-GMP inhibited growth of human colon cancer cells (Karaolis et al., 2005a) without toxicity against normal kidney cells. Two additional reports have indicated that c-di-GMP can function as an adjuvant. One group demonstrated a highly significant (>200-fold; P < 0.001) induction of anti-ClfA IgG2a titers when mice were vaccinated with ClfA and c-di-GMP as compared with vaccination with ClfA alone (Karaolis et al., 2007a) . Another group found a similar induction of anti--Gal titers when c-di-GMP was used as an adjuvant (Ebensen et al., 2007) . Both groups also found increased T cell responses in mice injected with c-di-GMP. Karoalis et al. (2007a) also showed that c-di-GMP has immunostimulatory effects in vivo and in vitro on innate cell populations, Toll-like receptor (TLR) 3, 7, 8, and 9 are transmembrane nucleic acid receptors that reside in intracellular compartments, where they detect endocytosed or autophagocytosed nucleic acids Medzhitov, 2007) . TLR7, 8, and 9 all require the signaling adaptor MyD88 to initiate downstream signaling, whereas TLR3 requires the signaling adaptor TRIF. Thus, MyD88 / Trif / double-knockout cells are deficient in signaling through all known TLRs that sense nucleic acids (Hoebe et al., 2003; Yamamoto et al., 2003) .