NOEsy and fluorescence spectroscopy reveal that conversion of conformationally flexible (phenylalkyl)pyridines into their corresponding N-methyl-pyridinium iodides results in intramolecular pi-stacking.
Unmethylated CpG sequences (CpG DNA) can induce Th1 response and thus become a potential immunotherapeutic agent. A key step in the treatment is to transport CpG DNA to its receptor TLR9 located in the endocytosis pathway of target immune cells. For the effective transport, we prepared a novel complex from a β-1,3-glucan schizophyllan (SPG) and CpG DNA, and administered the complex to murine peritoneal macrophages that had been previously activated by thioglycollate and expressed a major β-1,3-glucan receptor Dectin-1 on the cellular surface. Flow cytometric analysis and microscopic observation showed that the complex was taken up by the macrophage through Dectin-1 mediated pathway. Indeed, ELISA demonstrated that IL-12 production was increased sigmoidally with increasing SPG/CpG DNA ratio in the complexation, and reached the maximum at the SPG-rich composition. In the present work, we describe a new approach to deliver CpG DNA to immune cells by use of a β-1,3-glucan/DNA complex.
Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy.
A cationic polysaccharide bearing a beta-1,3-glucan main-chain structure (CUR-N(+)) forms a complex with a hetero-sequence oligonucleotide, that is, a CpG ODN, and facilitates the transportation of the resultant complex into a murine macrophage-like cell J774.A1, which induces an efficient secretion of a cytokine (IL-12) as compared with that induced by conventional carriers such as poly(ethyleneimine) (PEI) and poly(L-lysine) (PLL).
Unmethylated CpG sequences (CpG ODN) stimulate Toll-like receptor 9 (TLR9) to activate innate immunity. We made DNA duplexes from poly(dT)320 and CpG ODN with (dA)40 attached at the 3' end. Circular dichroism and gel electrophoresis indicated that the CpG parts turned outward from the duplex. When we changed the CpG ODN/poly(dT) molar ratio, the amount of IL-12 secreted from J774A.1 cells (murine macrophage-like) reached the maximum at the compositions with two to four CpG portions in one duplex, while the maximum loading was eight CpG ODNs per one poly(dT)320. When the residual free dT parts were hybridized with its control GpC ODN with (dA)40 tail or just (dA)40, the maximum disappeared and the secretion increased with increasing the CpG molar ratio. These results indicated that there is a particular DNA higher-order structure to activate TLR9 more efficiently than single CpG ODN.
CG sequences (CpG ODNs) can activate innate immune and thus be potentially useful for immune stimulants in therapeutic use. By hybridizing a CpG ODN having a (dA)40 tail with poly(dT), we prepared a DNA duplex that multiple CpG ODNs are hanging from the duplex. IL-12 cytokine secretion reached the maximum at the composition that two to four CpG ODNs in one duplex. These phenomena can be rationalized by an allosteric or cluster effect, and the present clustered CpG system is the simplest and most elucidating tool to investigate the cluster effect of CpG ODNs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.