Intradermally administered TLR4 agonist GLA-SE enhances the capacity of human skin DCs to activate T cells and promotes emigration of Langerhans cells.

The natural TLR4 agonist lipopolysaccharide (LPS) has notable adjuvant activity. However, it is not useful as a vaccine adjuvant due to its toxicity. Glucopyranosyl lipid A (GLA) is a synthetic derivative of the lipid A tail of LPS with limited cytotoxicity, but strong potential to induce immune responses in mice, guinea pigs, non-human primates, and humans. In this study we determined how this synthetic TLR4 agonist affects the function of different subsets of human skin dendritic cells (DCs). The effect of GLA in an aqueous formulation (GLA-AF) or in an oil-in-water emulsion (GLA-SE) was compared to that of LPS and TLR3 agonist poly(I:C) using a human skin explant model with intradermal injections for the administration of the agonists. Intradermal injection of GLA-SE or LPS, but not GLA-AF, enhanced the emigration of CD1a(high)/langerin(+) Langerhans cells (LCs), but not dermal DCs (DDCs). LCs and CD14(-) DDCs exhibited an enhanced mature phenotype following intradermal administration of either of the two GLA formulations tested, similar to DCs that emigrated from LPS-injected skin. However, only injection of GLA-SE resulted in a significant increase in the production of the wide range of cytokines that is observed with LPS. Moreover, DCs that emigrated from GLA-SE-injected skin induced stronger CD4(+) T-cell activation, as indicated by a more pronounced T-cell proliferation, than DCs from skin injected with GLA-AF or LPS. Altogether, our data show that GLA-SE has a notable potency to stimulate the function of skin DCs, indicating that GLA-SE may be a good candidate as adjuvant for vaccines administered via the intradermal route.

Protease inhibitors atazanavir, lopinavir and ritonavir are potent blockers, but poor substrates, of ABC transporters in a broad panel of ABC transporter-overexpressing cell lines.

OBJECTIVES: A possible mechanism for HIV therapy failure is the efflux of HIV drugs from viral target cells or certain body compartments by ATP-binding cassette (ABC) transporters, allowing ongoing viral replication. Here, we investigated the interaction between protease inhibitors (PIs) and ABC transporters. METHODS: To explore the potential blocking capacity of PIs, we exposed cells overexpressing multidrug resistance 1 P-glycoprotein (MDR1 P-gp), multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP) to established cytotoxic substrates with or without one of the PIs atazanavir, lopinavir or ritonavir. Furthermore, to assess whether PIs serve as substrates, cell growth-inhibitory effects of these PIs were evaluated on cells overexpressing 1 of 11 ABC transporters and their parental counterparts. RESULTS: Atazanavir, lopinavir and ritonavir were highly effective in reversing resistance against established substrates in cells overexpressing MDR1 P-gp and MRP1, and, to a lesser extent, BCRP. Concurrently, however, PIs appeared to be relatively poor substrates for ABC transporters. Only a moderate level of resistance to atazanavir was observed in cells overexpressing MRP6 and MRP9 [resistance factor (RF): 2.0-2.6]. Cells overexpressing MDR1 P-gp, MRP3, MRP4 and MRP5 displayed low levels of resistance to atazanavir (RF: 1.3-1.7); MRP7- and MRP9-overexpressing cells to lopinavir (RF: 1.4-1.5); and MRP9-overexpressing cells to ritonavir (RF: 1.4). CONCLUSIONS: PIs can act as potent blockers of MDR1 P-gp, MRP1 and BCRP, but they are poor substrates for 11 ABC transporters. Consequently, ABC transporters are unlikely to play a major role in PI failure, but still may contribute to drug-specific adverse events and drug-drug interactions.

Dendritic cells require multidrug resistance protein 1 (ABCC1) transporter activity for differentiation.

Dendritic cells (DC) express the ATP-binding cassette (ABC) transporters P-glycoprotein (ABCB1) and multidrug resistance protein 1 (MRP1; ABCC1). Functionally, both these transporters have been described to be required for efficient DC and T cell migration. In this study, we report that MRP1 activity is also crucial for differentiation of DC. Inhibition of MRP1, but not P-glycoprotein, transporter activity with specific antagonists during in vitro DC differentiation interfered with early DC development. Impaired interstitial and Langerhans DC differentiation was characterized by 1) morphological changes, reflected by dropped side scatter levels in flow cytometric analysis and 2) phenotypic changes illustrated by maintained expression of the monocytic marker CD14, lower expression levels of CD40, CD86, HLA-DR, and a significant decrease in the amount of cells expressing CD1a, CD1c, and Langerin. Defective DC differentiation also resulted in their reduced ability to stimulate allogeneic T cells. We identified the endogenous CD1 ligands sulfatide and monosialoganglioside GM1 as MRP1 substrates, but exogenous addition of these substrates could not restore the defects caused by blocking MRP1 activity during DC differentiation. Although leukotriene C(4) was reported to restore migration of murine Mrp1-deficient DC, the effects of MRP1 inhibition on DC differentiation appeared to be independent of the leukotriene pathway. Though MRP1 transporter activity is important for DC differentiation, the relevant MRP1 substrate, which is required for DC differentiation, remains to be identified. Altogether, MRP1 seems to fulfill an important physiological role in DC development and DC functions.