Annotine is a lycopodane-type alkaloid isolated from the Icelandic dub moss Lycopodium annotinum ssp.
Annotine is a lycopodium alkaloid with a lycopodane-type skeleton (Fig.
annotine. As DCs are the most potent professional antigen-presenting cells and can have a major effects on T cell responses they are attractive targets for clinical therapies targeting autoimmune diseases and chronic infections (Miles et al.
Annotine was isolated from the Icelandic club moss Lycopodium annotinum ssp.
The annotine was dissolved in 0.002% DMSO at concentrations of 1,10 and 100 [micro]g/ml and added to the imDCs at the same time as IL-1[beta], TNF-[alpha] and LPS.
For co-culture of DCs and T cells, allogeneic [CD4.sup.+] T cells were isolated from buffy coat using CD4 MicroBeads and 2 x [10.sup.5] cells/well co-cultured with 2 x [10.sup.4] cells/well DCs previously matured in the absence (C-mDCs) or presence (A-mDCs) of annotine at 100 [micro]g/ml.
Results are presented as secretion index (SI), which is the concentration of the cytokine in culture medium from cells stimulated in the presence of annotine divided by the concentration of the cytokine in culture medium from cells stimulated in the absence of annotine.
Annotine at 100 [micro]g/ml did not affect viability of mDCs as assessed by trypan blue staining and counting live cells (data not shown).
Annotine did not affect expression of surface molecules on mDCs cultured alone
Maturing DCs in the presence of annotine (100 [micro]g/ml) did not affect their expression levels (MFI) or the percentage of cells expressing CD 14, CD86 or HLA-DR (data not shown).
Annotine decreased mDC secretion of IL-6, IL-10, IL-12p40 and IL-23 but increased the ratio ofIL-12p40/lL-10
DCs matured in the presence of the highest concentration of annotine had a tendency toward secreting less IL-12p40 (P = 0.06) than DCs matured in the absence of annotine (Fig.