Japanin modulates dendritic cell maturation, rather than simply inhibiting it.

<p>Dendritic cells were cultured in the presence or absence of Japanin (500 ng/ml) and LPS (100 ng/ml) for 18–20 hours. (A) CD40, CD83, CD86, CD274 and HLA-DR expression were then assessed by flow cytometry, and (B)the concentration of pro-inflammatory cytokines in the culture supernatant was measured by Luminex1. Modelled means ±95% confidence intervals using data from at least four experiments are shown, except where marked ‡ where above-scale readings in the LPS-only made it impossible to calculate meaningful confidence intervals; the graphs show the lowest possible mean value (taking an above-scale value to be equal to the maximum possible on-scale value). ** p<0.01, * p<0.05, p<0.1, NS p>0.05.</p

Japanin blocks differentiation of DC from monocytes.

<p>Monocytes were cultured with GM-CSF (1000 U/ml) and IL4 (500 U/ml) with or without Japanin (500 ng/ml). Before the culture, and again after 3 and 5 days of culture, CD1a and CD14 expression were assessed by flow cytometry, in order to monitor differentiation into CD1a⁺CD14^low dendritic cells. Data shown is from one experiment, representative of three independent experiments using cells from different donors.</p

Japanin-like proteins form a clade within hard tick lipocalins.

<p>(A) A phylogenetic tree derived from maximum-likelihood analysis of hard tick lipocalins (including Japanin and its homologues), as well as the soft tick lipocalins OmCI, monomine and Am182. Sequences were aligned using ClustalX and manually refined, then Mega5.1 was used to construct a phylogeny. The frequency with which associated taxa clustered together in the bootstrap test is shown. For reasons of clarity, only selected protein names and bootstrap frequency labels are shown, and the Japanin clade is shown in detail in (B), with the full tree supplied as supplementary data.</p

Sequence alignment of Japanin and its homologues.

<p>Alignments were generated with ClustalX and manually refined. Shading intensity indicates BLOSUM62 score. N-glycosylation sequences and conserved cysteine residues are boxed. Ra-FS-HBP2 (PDB ID 1QFT) is aligned as an example of a tick lipocalin with low sequence similarity to Japanin.</p

Pretreatment of dendritic cells with Japanin inhibits their upregulation of CD86 in response to LPS.

<p>Dendritic cells were incubated with japanin for 24 hours prior to the addition of LPS (100 ng/ml) for a further 18–20 hours. CD86 expression was then analysed by flow cytometry. (A) The results from a representative experiment using 500 ng/ml japanin. (B) Titration of Japanin concentration, showing a dose-dependent inhibition of CD86 upregulation. The range and mean of duplicate measurements from one representative experiment are shown. This experiment was performed four times, with dose-dependency demonstrated each time, but with EC₅₀ varying between donors.</p