Promotion of liver regeneration by natural killer cells in a murine model is dependent on extracellular adenosine triphosphate phosphohydrolysis

Nucleotides, such as adenosine triphosphate (ATP), are released by cellular injury, bind to purinergic receptors expressed on hepatic parenchymal and nonparenchymal cells, and modulate cellular crosstalk. Liver resection and resulting cellular stress initiate such purinergic signaling responses between hepatocytes and innate immune cells, which regulate and ultimately drive liver regeneration. We studied a murine model of partial hepatectomy using immunodeficient mice to determine the effects of natural killer (NK) cell-mediated purinergic signaling on liver regeneration. We noted first that liver NK cells undergo phenotypic changes post-partial hepatectomy (PH) in vivo, including increased cytotoxicity and more immature phenotype manifested by alterations in the expression of CD107a, CD27, CD11b, and CD16. Hepatocellular proliferation is significantly decreased in Rag2/common gamma-null mice (lacking T, B, and NK cells) when compared to wildtype and Rag1-null mice (lacking T and B cells but retaining NK cells). Extracellular ATP levels are elevated post-PH and NK cell cytotoxicity is substantively increased in vivo in response to hydrolysis of extracellular ATP levels by apyrase (soluble NTPDase). Moreover, liver regeneration is significantly increased by the scavenging of extracellular ATP in wildtype mice and in Rag2/common gamma-null mice after adoptive transfer of NK cells. Blockade of NKG2D-dependent interactions significantly decreased hepatocellular proliferation. In vitro, NK cell cytotoxicity is inhibited by extracellular ATP in a manner dependent on P2Y1, P2Y2, and P2X3 receptor activation. Conclusion: We propose that hepatic NK cells are activated and cytotoxic post-PH and support hepatocellular proliferation. NK cell cytotoxicity is, however, attenuated by hepatic release of extracellular ATP by way of the activation of specific P2 receptors. Clearance of extracellular ATP elevates NK cell cytotoxicity and boosts liver regeneration.

Protein cross-linking mediated by tissue transglutaminase correlates with the maturation of extracellular matrices during lung development

At birth, the mammalian lung is still immature. The alveoli are not yet formed and the interairspace walls contain two capillary layers which are separated by an interstitial core. After alveolarization (first 2 postnatal weeks in rats) the alveolar septa mature: their capillary layers merge, the amount of connective tissue decreases, and the mature lung parenchyma is formed (second and third week). During the first 3 wk of life the role of tissue transglutaminase (tTG) was studied in rat lung by immunostaining of cryostat and paraffin sections, by Northern and Western blotting, and by a quantitative determination of gamma-glutamyl-epsilon-lysine. While enzyme activity and intracellular tTG were already present before term, the enzyme product (gamma-glutamyl-epsilon-lysine-crosslink) and extracellular tTG appeared between postnatal days 10 and 19 in the lung parenchyma. In large blood vessels and large airways, which mature earlier than the parenchyma, both the enzyme product and extracellular tTG had already appeared at the end of the first postnatal week. We conclude that tTG is expressed and externalized into the extracellular matrix of lung shortly before maturation of an organ area. Because tTG covalently and irreversibly crosslinks extracellular matrix proteins, we hypothesize that it may prevent or delay further remodeling of basement membranes and may stabilize other extracellular components, such as microfibrils.

Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting

Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETA″), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETA″, simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETA″ was much better tolerated than Ec1-ETA″; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy.

A novel blood-sparing agent in cardiac surgery? First in-patient experience with the synthetic serine protease inhibitor MDCO-2010: a phase II, randomized, double-blind, placebo-controlled study in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass

BACKGROUND Antifibrinolytics have been used for 2 decades to reduce bleeding in cardiac surgery. MDCO-2010 is a novel, synthetic, serine protease inhibitor. We describe the first experience with this drug in patients. METHODS In this phase II, double-blind, placebo-controlled study, 32 patients undergoing isolated primary coronary artery bypass grafting with cardiopulmonary bypass were randomly assigned to 1 of 5 increasing dosage groups of MDCO-2010. The primary aim was to evaluate pharmacokinetics (PK) with assessment of plasmatic concentrations of the drug, short-term safety, and tolerance of MDCO-2010. Secondary end points were influence on coagulation, chest tube drainage, and transfusion requirements. RESULTS PK analysis showed linear dosage-proportional correlation between MDCO-2010 infusion rate and PK parameters. Blood loss was significantly reduced in the 3 highest dosage groups compared with control (P = 0.002, 0.004 and 0.011, respectively). The incidence of allogeneic blood product transfusions was lower with MDCO-2010 4/24 (17%) vs 4/8 (50%) in the control group. MDCO-2010 exhibited dosage-dependent antifibrinolytic effects through suppression of D-dimer generation and inhibition of tissue plasminogen activator-induced lysis in ROTEM analysis as well as anticoagulant effects demonstrated by prolongation of activated clotting time and activated partial thromboplastin time. No systematic differences in markers of end organ function were observed among treatment groups. Three patients in the MDCO-2010 groups experienced serious adverse events. One patient experienced intraoperative thrombosis of venous grafts considered possibly related to the study drug. No reexploration for mediastinal bleeding was required, and there were no deaths. CONCLUSIONS This first-in-patient study demonstrated dosage-proportional PK for MDCO-2010 and reduction of chest tube drainage and transfusions in patients undergoing primary coronary artery bypass grafting. Antifibrinolytic and anticoagulant effects were demonstrated using various markers of coagulation. MDCO-2010 was well tolerated and showed an acceptable initial safety profile. Larger multi-institutional studies are warranted to further investigate the safety and efficacy of this compound.

Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Ca v1.2 channels

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

Impaired DNase1-mediated degradation of neutrophil extracellular traps is associated with acute thrombotic microangiopathies.

BACKGROUND Acute thrombotic microangiopathies (TMAs) are characterized by excessive microvascular thrombosis and are associated with markers of neutrophil extracellular traps (NETs) in plasma. NETs are composed of DNA fibers and promote thrombus formation through the activation of platelets and clotting factors. OBJECTIVE The efficient removal of NETs may be required to prevent excessive thrombosis such as in TMAs. To test this hypothesis, we investigated whether TMAs are associated with a defect in the degradation of NETs. APPROACH AND RESULTS We show that NETs generated in vitro were efficiently degraded by plasma from healthy donors. However, NETs remained stable after exposure to plasma from TMA patients. The inability to degrade NETs was linked to a reduced DNase activity in TMA plasma. Plasma DNase1 was required for efficient NET-degradation and TMA plasma showed decreased levels of this enzyme. Supplementation of TMA plasma with recombinant human DNase1 restored NET-degradation activity. CONCLUSIONS Our data indicates that DNase1-mediated degradation of NETs is impaired in patients with TMAs. The role of plasma DNases in thrombosis is, as of yet, poorly understood. Reduced plasma DNase1 activity may cause the persistence of pro-thrombotic NETs and thus promote microvascular thrombosis in TMA patients. This article is protected by copyright. All rights reserved.

Cancer risk and use of protease inhibitor or nonnucleoside reverse transcriptase inhibitor-based combination antiretroviral therapy: the d: a: d study.

BACKGROUND The association between combination antiretroviral therapy (cART) and cancer risk, especially regimens containing protease inhibitors (PIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs), is unclear. METHODS Participants were followed from the latest of D:A:D study entry or January 1, 2004, until the earliest of a first cancer diagnosis, February 1, 2012, death, or 6 months after the last visit. Multivariable Poisson regression models assessed associations between cumulative (per year) use of either any cART or PI/NNRTI, and the incidence of any cancer, non-AIDS-defining cancers (NADC), AIDS-defining cancers (ADC), and the most frequently occurring ADC (Kaposi sarcoma, non-Hodgkin lymphoma) and NADC (lung, invasive anal, head/neck cancers, and Hodgkin lymphoma). RESULTS A total of 41,762 persons contributed 241,556 person-years (PY). A total of 1832 cancers were diagnosed [incidence rate: 0.76/100 PY (95% confidence interval: 0.72 to 0.79)], 718 ADC [0.30/100 PY (0.28-0.32)], and 1114 NADC [0.46/100 PY (0.43-0.49)]. Longer exposure to cART was associated with a lower ADC risk [adjusted rate ratio: 0.88/year (0.85-0.92)] but a higher NADC risk [1.02/year (1.00-1.03)]. Both PI and NNRTI use were associated with a lower ADC risk [PI: 0.96/year (0.92-1.00); NNRTI: 0.86/year (0.81-0.91)]. PI use was associated with a higher NADC risk [1.03/year (1.01-1.05)]. Although this was largely driven by an association with anal cancer [1.08/year (1.04-1.13)], the association remained after excluding anal cancers from the end point [1.02/year (1.01-1.04)]. No association was seen between NNRTI use and NADC [1.00/year (0.98-1.02)]. CONCLUSIONS Cumulative use of PIs may be associated with a higher risk of anal cancer and possibly other NADC. Further investigation of biological mechanisms is warranted.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro.

Pestivirus N(pro) is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N(pro) blocks the host׳s interferon response by inducing degradation of interferon regulatory factor-3. N(pro׳)s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N(pro)-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N(pro) proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N(pro׳)s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N(pro) does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus.