The quality of cold-stored livers declines with the extension of ischemic time, increasing the risk of primary dys or nonfunction. A new concept to rescue preserved marginal liver grafts by gentle oxygenated warming-up prior to blood reperfusion was investigated. Porcine livers were preserved by cold storage (CS) in modified HTK-solution for 18 h. Some grafts were subsequently subjected to 90 min of controlled oxygenated rewarming (COR) by machine perfusion with gradual increase of perfusate temperature up to 208C or simple oxygenated machine perfusion in hypothermia (HMP) or subnormothermia (SNP). Graft viability was assessed thereafter by 4 h of normothermic blood reperfusion ex vivo. Endischemic tissue energetics were significantly improved by COR or SNP and to a notably lesser extent by HMP. COR significantly reduced cellular enzyme loss, gene expression and perfusate activities of TNF-alpha, radical mediated lipid peroxidation (LPO) and increase of portal vascular perfusion resistance upon reperfusion, while HMP or SNP were less protective. Only COR resulted in significantly more bile production than after CS. Histological injury score and caspase 3-activation were significantly lower after COR than after CS. Oxygenated rewarming prior to reperfusion seems to be a promising technique to improve subsequent organ recovery upon reperfusion of long preserved liver grafts.

Type I interferon (IFN) is a critical mediator of autoimmune diseases such as systemic lupus erythematosus (SLE) and Aicardi-Goutières Syndrome (AGS). The recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of type I IFN in response to cytosolic DNA and is potentially linked to SLE and AGS. Suppressive oligodeoxynucleotides (ODN) containing repetitive TTAGGG motifs present in mammalian telomeres have proven useful in the treatment of autoimmune diseases including SLE. In this study, we demonstrate that the suppressive ODN A151 effectively inhibits activation of cGAS in response to cytosolic DNA, thereby inhibiting type I IFN production by human monocytes. In addition, A151 abrogated cGAS activation in response to endogenous accumulation of DNA using TREX1-deficient monocytes. We demonstrate that A151 prevents cGAS activation in a manner that is competitive with DNA. This suppressive activity of A151 was dependent on both telomeric sequence and phosphorothioate backbone. To our knowledge this report presents the first cGAS inhibitor capable of blocking self-DNA. Collectively, these findings might lead to the development of new therapeutics against IFN-driven pathologies due to cGAS activation.

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PP of 90 min shortly before transplantation seems to be an efficient mechanism to reduce proinflammatory endothelial phenotype and improve functional outcome of kidney grafts even after preceding static storage.

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the pathogenesis of IFN-driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self-DNA and has been linked to TLR9-independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS-STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS-STING pathway and that targeting this pathway could be of therapeutic interest.Keywords: Cytosolic sensor r Dendritic cells r DNA r Innate immunity r Interferon r Toll-likereceptors Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionInnate immune cells utilize pattern recognition receptors (PRRs) to monitor the extracellular, endosomal, and cytosolic Eur. J. Immunol. 2016. 46: 1615-1621 I IFNs and various pro-inflammatory cytokines [3]. pDCs act in the host defense against viruses and bacteria by recognizing single-stranded RNA and unmethylated DNA sequences (CpG motifs) via the endosomal toll-like receptors (TLRs) TLR7 and TLR9, respectively [4,5]. This feature and the promising results in the therapy of viral infections and certain cancers with type I IFNs have placed pDCs in the focus of clinical interest [6]. Furthermore, in autoimmune disorders such as systemic lupus erythematodes (SLE) where DNA/RNA containing immune complexes trigger the inflammatory response, pDCs have been proposed as a main source of aberrant type I IFN production and a major driver of autoimmune progression [7]. Although TLR9 was shown to be critical to sense microbial DNA within the endosomal compartment, there is less known about the exact mechanism by which self-nucleic acids are recognized and whether TLR9 represents the only DNA sensor in pDCs. IFN-driven autoimmunity has been linked to TLR9-independent sensing of self-DNA [8][9][10], suggesting that innate sensing of DNA in pDCs is not limited to endosomes and might also occur via cytosolic receptors. Much progress has recently been made in understanding how nucleic acids are recognized by the discovery of the cytoplasmic DNA-sensor cyclic GMP-AMP synthase (cGAS) [11]. Upon DNA binding, cGAS activates the stimulator of interferon genes (STING) adaptor via the production of cyclic GMP-AMP (cGAMP) as a second messenger [12...

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Sheffield S10 2JF 1 Azathioprine metabolite concentrations were studied in 54 kidney transplant recipients. Thirty-seven of these patients were studied over a 6 month period to investigate the intrapatient variation in metabolite concentrations. All patients had stable functioning grafts and normal peripheral white blood cell counts. 2 The metabolites measured were plasma 6-mercaptopurine and red blood cell 6-thioguanine nucleotide. 3 There was no correlation between azathioprine dose and plasma 6-mercaptopurine concentration but there was a significant correlation between dose and red blood cell 6-thioguanine nucleotide concentration (r, = 0.41, P < 0.005). The individual transplant recipient showed little variation in metabolite concentrations over several months. 4 Using this group as a control we studied metabolite concentrations in patients with kidney transplants who developed leucopenia. Our preliminary findings indicate that elevated red cell 6-thioguanine nucleotide concentrations, above the control range, can be associated with bone marrow depression.

The effect of adding pulsatility to gaseous oxygen persufflation during liver preservation was studied in an isolated rat liver model. Livers from male Wistar rats were retrieved 30 min after cardiac arrest of the donor and subjected to 18 h of cold storage. Some grafts were subjected to nonpulsatile or pulsatile gaseous oxygen persufflation. Graft viability was assessed thereafter upon warm reperfusion in vitro (n = 5 per group). Pulsatile persufflation significantly improved parenchymal integrity (enzyme release, bile flow) upon reperfusion, with respect to nonpulsatile persufflation or cold storage (CS) (e.g., max. release of alanine aminotransferase: 44 ± 10 vs. 178 ± 29 vs. 345 ± 100 U/L; pulsatile vs. nonpulsatile persufflation vs. CS).The effect was associated with the prevention of the ischemic decline in gene and protein expression of the vasoprotective Krüppel-like factor 2, increased perfusate levels of the endogenous vasodilator nitric oxide, and reduced portal vascular resistance upon reperfusion, while nonpulsatile persufflation was less effective (e.g., vascular resistance: 1235 ± 108 vs. 1607 ± 155 vs. 2215 ± 208 Pa s/mL; pulsatile vs. nonpulsatile persufflation vs. CS). In conclusion, pulsatile mechanostimulation of the hepatovasculature seems a genuine protective mechanism, affecting early graft recovery upon reperfusion.