TRAIL(+) human plasmacytoid dendritic cells kill tumor cells in vitro: mechanisms of imiquimod- and IFN-α-mediated antitumor reactivity.

Dendritic cells (DCs) not only exhibit the unique capacity to evoke primary immune responses, but may also acquire TLR-triggered cytotoxic activity. We and others have previously shown that TLR7/8- and TLR9-stimulated plasmacytoid DCs (pDCs) isolated from human peripheral blood express the effector molecule TRAIL. The exact mechanisms through which pDCs acquire and elicit their cytotoxic activity are still not clear. We now show that in the absence of costimulators, TRAIL induction on pDCs occurs with agonists to intracellular TLRs only and is accompanied by a phenotypic as well as functional maturation, as evidenced by a comparatively superior MLR stimulatory capacity. pDCs acquired TRAIL in an IFN-α/ß-dependent fashion and, notably, TRAIL expression on pDCs could be induced by IFN-α stimulation alone. At a functional level, both TLR7/8- (imiquimod [IMQ]) and TLR9-stimulated (CpG2216) pDCs lysed Jurkat T cells in a TRAIL- and cell contact-dependent fashion. More importantly, IFN-α-activated pDCs acquired similar cytotoxic properties, independent of TLR stimulation and maturation. Both IMQ- and IFN-α-activated pDCs could also lyse certain melanoma cell lines in a TRAIL-dependent fashion. Interestingly, suboptimal doses of IMQ and IFN-α exhibited synergistic action, leading to optimal TRAIL expression and melanoma cell lysis by pDCs. Our data imply that tumor immunity in patients receiving adjuvant IMQ and/or IFN-α may involve the active participation of cytotoxic pDCs.

Infliximab induces downregulation of the IL-12/IL-23 axis in 6-sulfo-LacNac (slan)+ dendritic cells and macrophages.

BACKGROUND: The spectrum of TNF-α-producing cells in patients with psoriasis, as well as their fate during treatment with TNF-α antagonists, is not clearly defined. OBJECTIVE: We sought to analyze the effects of anti-TNF-α treatment on TNF-α(+) cells in the skin and blood of patients with psoriasis. METHODS: Lesional psoriatic skin was analyzed by means of immunohistologic staining and quantitative RT-PCR, and peripheral blood cells were phenotypically characterized by means of multicolor immunofluorescence labeling. RESULTS: By using a tyramide-based signal amplification system, TNF-α was detected in dermal CD45(+)HLA-DR(+) leukocytes consisting of CD11c(+) dendritic cells and CD163(+) macrophages. In peripheral blood we observed an increase in the TNF-α-producing myeloid subsets of CD14(-) 6-sulfo-LacNac(+) dendritic cells and CD14(+)CD16(+) "intermediate" monocytes compared with healthy control subjects. Strikingly, we did not find detectable levels of TNF-α in other cells, including keratinocytes or T cells, making these cell types unlikely targets of TNF-α blockers. Up to 48 hours after the intravenous administration of the TNF-α antagonist infliximab, we encountered no overt changes in numbers of TNF-α(+) cells or signs of apoptosis in lesional psoriatic skin. Yet we observed a rapid decrease in IL-12p40, IL-1ß, CCL20, and IL12RB1 mRNA levels. Consistently, TNF-α blockade during in vitro stimulation of 6-sulfo-LacNac DCs resulted in decreased production of IL-12 and IL-23 but not IL-6. In a mixed leukocyte reaction infliximab led to significantly decreased proliferation rates of T cells independent of the Fc antibody fragment. CONCLUSION: The decrease in tissue inflammation during anti-TNF-α therapy is not due to immediate killing of TNF-α-producing cells but rather results from a rapid downregulation of the pathogenic IL-12/IL-23-driven immune response.

The effect of ex vivo anticoagulants on whole blood platelet aggregation.

Pre- and intraoperative platelet function monitoring is increasingly recommended in order to detect risk factors for bleeding and to target coagulation management. The ideal anticoagulant for accurate platelet aggregometry remains controversial. The aim of this experimental trial was to compare platelet aggregability in whole blood stored in citrate, heparin and direct thrombin inhibitors. Whole blood was drawn from 11 healthy adult volunteers who had not taken any medication in the previous 14 days. Blood was stored in trisodium citrate, unfractionated heparin, melagatran, lepirudin and argatroban. Platelet aggregation was performed using the impedance aggregometer Multiplate (Dynabyte, Munich, Germany) with adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP), collagen, arachidonic acid and ristocetin as agonists. Samples were analysed immediately after blood sampling (baseline), as well as 30 and 120 min afterwards. At baseline there were no significant differences in aggregability between samples containing direct thrombin inhibitors and heparin. In contrast, aggregation in response to all agonists except for ristocetin was significantly impaired in citrated blood. During storage the response to arachidonic acid and collagen was maintained by direct thrombin inhibitors and heparin, whereas ADP-, TRAP- and ristocetin-induced aggregation varied considerably over time in all ex vivo anticoagulants tested. Pre-analytical procedures should be standardized because storage duration and anticoagulants significantly affect platelet aggregability in whole blood. For point-of-care monitoring with immediate analysis after blood withdrawal all tested direct thrombin inhibitors as well as unfractionated heparin can be used as anticoagulants whereas citrate is not recommended.

Garlic at dietary doses does not impair platelet function.

BACKGROUND: In vitro studies suggest that various bioactive constituents of Allium sativum (garlic) inhibit platelet function. The extent, however, to which dietary doses of garlic influence platelet function remains unknown. Therefore, we tested the effect of raw garlic on platelet function using point-of-care monitoring devices sensitive for cyclooxygenase I-inhibition and platelet adhesion. METHODS: Whole blood from 18 healthy volunteers was investigated before and 5 h after ingestion of the study medication consisting of Greek tsatsiki with 4.2 g raw garlic (verum), or Greek tsatsiki without garlic (placebo), in a randomized, crossover, observer-blinded, placebo-controlled study. The potential long-term effects of garlic were investigated in five volunteers after daily ingestion of 4.2 g of raw garlic over 1 wk. Platelet function was assessed with the Platelet Function Analyzer (PFA-100), impedance aggregometry (Multiplate), and thrombelastographic Platelet Mappingtrade mark. In vitro experiments were performed to prove the sensitivity of the assays to garlic-induced platelet inhibition. RESULTS: Baseline values of platelet function were within normal range in all volunteers. Platelet function was not impaired by single and repeated oral consumption of Greek tsatsiki containing raw garlic in any point-of-care monitoring test used. CONCLUSIONS: Platelet function is not impaired by single and repeated oral consumption of a dietary dose of garlic in healthy volunteers. Dishes containing socially acceptable doses of raw garlic are unlikely to increase the risk of perioperative bleeding. Further studies are warranted to determine the potential additive effects of platelet-inhibiting drugs combined with garlic and other herbs.