BackgroundUnmethylated cytosine-guanine (CpG) motif-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists of Toll-like receptor 9 (TLR9). ODNs with a phosphorothioate (PTO) backbone have been studied as TLR9 agonists since natural ODNs with a phosphodiester (PD) backbone are easily degraded by a serum nuclease, which makes them problematic for therapeutic applications. However, ODNs with a PTO backbone have been shown to have undesirable side effects. Thus, our goal was to develop nuclease-resistant, PD ODNs that are effective as human TLR9 (hTLR9) agonists.ResultsThe sequence of ODN2006, a CpG ODN that acts as an hTLR9 agonist, was used as the basic CpG ODN material. The 3'-end modification of ODN2006 with a PD backbone (PD-ODN2006) improved its potential as an hTLR9 agonist because of increased resistance to nucleolytic degradation. Moreover, 3'-end modification with oligonucleotides showed higher induction than modification with biotin, FITC, and amino groups. Further, enhancement of hTLR9 activity was found to be dependent on the number of CpG core motifs (GTCGTT) in the PD ODN containing the 3'-end oligonucleotides. In particular, ODN sequences consisting of two to three linked ODN2006 sequences with a PD backbone (e.g., PD-ODN2006-2006 and PD-ODN2006-2006-2006) acted as effective agonists of hTLR9 even at lower concentrations.ConclusionsThis study showed that PD-ODN2006-2006 and PD-ODN-2006-2006-2006 can be used as potentially safe agonists for hTLR9 activation instead of CpG ODNs with a PTO backbone. We propose these CpG ODNs consisting of only a PD backbone as a novel class of CpG ODN.

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Optimization of the extracellular environment is very important for hepatocyte function in vitro. We expressed new chimeric proteins of the collagen-binding domain (CBD) with cell attachment site (CAS) of fibronectin to enhance hepatocyte function, and the CBD-CAS proteins were immobilized on collagen-coated plates. We hypothesized that the high density of CAS would increase activity of the integrin-dependent intracellular signaling pathway, thus inducing hepatocyte function. Expression of albumin in the human hepatocyte cell line HepG2 was assessed on CBD-CAS-immobilized dishes. The results indicated that the CBD-CAS-immobilized plates induced albumin expression. Immobilized CBD-CAS induced activation of focal adhesion kinase and integrin-ligand clustering on the cell membrane. These results suggest that immobilized CBD-CAS improves the function of HepG2 cells. This system could therefore be applied to drug metabolism assay in the development of new drugs.

Hepatocytes are widely used in pharmaceutical drug discovery tests, but their hepatic functions decrease rapidly during in vitro culture. Many culture systems have been devised to address this problem. We here report that a three-dimensional (3D) collagen-based scaffold coated with simplified recombinant fibronectin (FN) enhanced the function of a hepatocyte cell line. The developed culture system uses a honeycomb collagen sponge coated with collagen-binding domain (CBD)-cell attachment site (CAS), a chimeric protein comprising the CBD and CAS of FN. The function of HepG2 cells grown on honeycomb collagen sponge coated with CBD-CAS was investigated by determining the messenger RNA (mRNA) expression levels of several genes. The mRNA expression level of albumin increased 3.25 times in cells grown on CBD-CAS-coated honeycomb collagen sponge for 3 days; the expression level of CCAAT/enhancer binding protein (C/EBPα) increased 40-fold after 1 d and up to 150-fold after 3 d. These results suggested that CBD-CAS-coated honeycomb collagen sponge could improve the functions of hepatocytes by inducing C/EBPα expression. The activation of cytochrome P450 (CYP) enzymes in HepG2 cells grown on CBD-CAS-coated honeycomb collagen sponge was measured at the mRNA level and was found to increase between two and six times compared to cells grown without the CBD-CAS coating, showing that this culture system induced CYP gene expression and thus may be useful in drug metabolism assays.