Key Points• Nonanticoagulant heparin is shown to bind histones and provide cytoprotection in mouse models of sterile inflammation and sepsis.Extracellular histones are considered to be major mediators of death in sepsis. Although sepsis is a condition that may benefit from low-dose heparin administration, medical doctors need to take into consideration the potential bleeding risk in sepsis patients who are already at increased risk of bleeding due to a consumption coagulopathy. Here, we show that mechanisms that are independent of the anticoagulant properties of heparin may contribute to the observed beneficial effects of heparin in the treatment of sepsis patients. We show that nonanticoagulant heparin, purified from clinical grade heparin, binds histones and prevents histone-mediated cytotoxicity in vitro and reduces mortality from sterile inflammation and sepsis in mouse models without increasing the risk of bleeding. Our results demonstrate that administration of nonanticoagulant heparin is a novel and promising approach that may be further developed to treat patients suffering from sepsis. (Blood. 2014;123(7):1098-1101) IntroductionSepsis and septic shock are serious clinical problems with high mortality rates for which no adequate treatment currently exists. 1Neutrophils respond to infection with the formation of neutrophil extracellular traps (NETs), 2,3 intricate networks containing DNA as the major structural component and proteins like histones and neutrophil elastase, which have antimicrobial properties. Extracellular histones, however, also exhibit cytotoxic activity toward host cells, including the endothelium. 4,5 Histone release can thus trigger a feedback cascade, resulting in more cell death and additional release of histones.6 Consequently, extracellular histones are considered interesting therapeutic targets for sepsis treatment. 4 Histones are positively charged, and NET-mediated cytotoxicity can be reduced with polysialic acid, a negatively charged polymer. 5 We hypothesized that heparin, a negatively charged polysaccharide, blocks histone cytotoxicity and reduces mortality from sterile inflammation and sepsis. Low dose unfractionated heparin (UFH) has been tested in a clinical trial as a complementary treatment of sepsis. 7 The study rationale linked infection, inflammation, and coagulation in sepsis and sought to inhibit the coagulation part with low doses of heparin so as not to increase the risk of bleeding in a patient who is already at risk due to sepsis-associated consumption coagulopathy. 7,8 Nevertheless, although this study failed to demonstrate a significant benefit on 28-day mortality rate, we hypothesize that the minor beneficial effects of heparin observed might be attributed to a mechanism independent of the anticoagulant properties of heparin. We reasoned that removing the anticoagulant fraction from UFH would yield an antithrombin affinity-depleted heparin (AADH) that neutralizes histone-mediated cytotoxicity and effectively treats sepsis without increasing risk of bleeding....

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

GAS6 signaling through AXL receptor contributes to the progression of nonalcoholic steatohepatitis (NASH). Soluble AXL significantly increases both in NASH patients and mouse models. Experimental AXL inhibition by bemcentinib diminishes inflammation and fibrosis, supporting its therapeutic use in NASH.BACKGROUND AND AIMS: GAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development.METHODS: GAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl -/-, Mertk -/and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients. RESULTS:In primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD-and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibroticand inflammatory-related genes in HFDfed mice after bemcentinib administration. HFD-fed Mertk -/mice exhibited enhanced NASH, while Axl -/mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH.CONCLUSION: AXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, reduces experimental NASH, revealing AXL as an effective therapeutic target for clinical practice.

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