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/β–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.

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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.

We systematically evaluated the effects of test temperature and storage temperature on platelet aggregation using flow cytometry and impedance aggregometry. Aliquots of citrated whole blood from 27 healthy adult male volunteers were stored at 37 degrees C and 22 degrees C. Aliquots were subjected to impedance aggregometry in response to collagen, adenosine diphosphate, ristocetin, and arachidonic acid performed at 22 degrees C, 34 degrees C, 37 degrees C, and 40 degrees C. The expression of activated fibrinogen receptor was determined on adenosine diphosphate-activated platelets at 22 degrees C and 37 degrees C by whole blood flow cytometry using PAC-1 for fluorescent staining. Aggregation induced by collagen, ristocetin, and arachidonic acid was not significantly different at the test temperatures of 34 degrees C and 37 degrees C but was significantly impaired at 22 degrees C. In contrast, adenosine diphosphate-induced aggregation was significantly increased at both 34 degrees C and 22 degrees C. Hyperthermia exclusively impaired collagen-induced aggregation. Storage temperature of 22 degrees C exclusively enhanced adenosine diphosphate- and collagen-induced aggregation compared with storage at 37 degrees C. The binding of PAC-1 was enhanced at test temperatures below 37 degrees C. Prewarming the antibody above 22 degrees C significantly decreased binding. Our results suggest that mild hypothermic test conditions have no relevant effect, whereas profound hypothermia induces defects in adhesion, thromboxane generation, and activation. The pathomechanism for the increased response to adenosine diphosphate at mild and profound hypothermia remains unclear. Storage temperature considerably affects the aggregation response to the agonists adenosine diphosphate and collagen but not to arachidonic acid and ristocetin. Flow cytometry using the temperature-labile antibody PAC-1 fails to assess temperature effects on platelet aggregability.

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.

The mechanisms causing temperature-dependent bleeding, especially in hypothermic patients, warrant clarification. Therefore the aim of this study was to investigate platelet aggregation at the clinically important temperature range of 30-34 degrees C. After obtaining informed consent citrated whole blood was drawn from 12 healthy adult male volunteers, who had not taken any medication in the previous 14 days. After venipuncture blood samples were incubated at 37 degrees C until platelet testing. Platelet aggregation was performed in whole blood using the impedance aggregometer Multiplate at five different test temperatures between 30 degrees C and 34 degrees C. Aggregation responses at 37 degrees C served as controls. At temperatures of mild and moderate hypothermia (30-34 degrees C), overall platelet aggregation was increased compared to 37 degrees C. Increases were recorded in response to collagen, thrombin receptor activating peptide and ristocetin between 31 degrees C and 34 degrees C and in response to adenosine diphosphate between 30 degrees C and 34 degrees C. Overall platelet aggregation is increased at mild and moderate hypothermia down to 30 degrees C. These results indicate that bleeding complications reported in mildly hypothermic patients are not due to hypothermia-induced platelet inhibition. The pathomechanism of the overall increased platelet aggregation between 30 degrees C and 34 degrees C requires further detailed study.

The orphan receptor CLEC‐1 is part of a subfamily of C‐type lectin‐like receptors, which is encoded in the human natural killer gene complex and comprises several pattern recognition receptors important for innate immune functions. As information on human CLEC‐1 is still very limited, we aimed to further characterize this receptor. Similar to another subfamily member, LOX‐1, expression of CLEC‐1 mRNA was detected in myeloid cells as well as in endothelial cells. CLEC‐1 protein displayed N‐linked glycosylation and formed dimers. However, in contrast to other members of the subfamily, expression levels were upregulated by transforming growth factor (TGF)‐β, but not significantly affected by proinflammatory stimuli. It is intriguing that human CLEC‐1 could only be detected intracellularly with a staining pattern resembling endoplasmic reticulum proteins. Neither TGF‐β nor inflammatory stimuli could promote significant translocation to the cell surface. These findings are in accordance with a primarily intracellular localization and function of human CLEC‐1.