CRP Induces NETosis in Heart Failure Patients with or without Diabetes.

C-reactive protein (CRP) is recognized as a biomarker of chronic, low-grade inflammation associated with vascular disorders. Lately, the role of neutrophils and neutrophil extracellular traps (NETs) has been investigated as a potential source of chronic inflammation and cardiovascular complications. This study investigated NETs as a marker of inflammation in patients with symptomatic heart failure (HF) with or without type 2 diabetes (T2DM) and examined the correlation between NETs and CRP. We performed a noninterventional study including patients with HF with or without T2DM, T2DM, and a healthy control (HC) group. NETs and other inflammatory markers in serum were measured by ELISA. The release of NETs (NETosis) in vitro under various stimuli was measured by confocal microscopy. The levels of NETs in the serum of HF patients were significantly higher compared with HC (112%). Serum CRP concentrations were significantly increased in HF and HF plus T2DM patients compared with HC, and a positive correlation was observed between serum CRP and NETs levels. Neutrophils from HF and HF plus T2DM patients underwent in vitro NETs release faster than T2DM and HC without any stimuli. In vitro, serum collected from the HF and the HF plus T2DM group induced NETosis in healthy neutrophils significantly more when compared with HC and T2DM, which was prevented by depletion from CRP. We confirmed in vitro that CRP induces a concentration-dependent NETs synthesis. This study proposes a mechanism by which CRP increases the risk of future cardiovascular events and supports mounting evidences on the role of neutrophils in chronic low-grade inflammation associated with HF.

Synthesis of Human Neutrophil Extracellular Traps Contributes to Angiopoietin-Mediated In Vitro Proinflammatory and Proangiogenic Activities.

Neutrophil extracellular traps (NETs) are composed of nuclear DNA in a web-like structure extruded from neutrophils in response to either bacterial infection or inflammation. We previously reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of both angiopoietins (Ang1 and Ang2) to induce proinflammatory activities, such as synthesis and release of platelet-activating factor, upregulation of ß2 integrin complex (CD11/CD18), and neutrophil chemotaxis. In contrast, only Ang1 but not Ang2 is capable of promoting translational and transcriptional activities in neutrophils. In this article, we addressed whether Ang1 and/or Ang2 could modulate the release of NETs and if they contribute to angiopoietin-mediated proinflammatory activities. We observed that Ang1 and Ang2, alone or combined (10 nM, 3 h), increase NET synthesis and release by ≈2.5-fold as compared with PBS-treated neutrophils. The release of NETs is Tie2 dependent and requires downstream intracellular participation of PI3K, p38, and p42/44 MAPK pathways; reactive oxygen species production; intracellular calcium store depletion; and protein arginine deiminase 4 activation. These isolated NETs induced neutrophil and endothelial cell activation, leading to neutrophil adhesion onto human extracellular matrix and HUVEC and in vitro formation of capillary-like tubes by endothelial cells. Our study reports the capacity of Ang1 and Ang2 to promote the release of NETs and that these NETs contribute to angiopoietin-mediated in vitro proinflammatory and proangiogenic activities.

New Insights into the Pro-Inflammatory Activities of Ang1 on Neutrophils: Induction of MIP-1ß Synthesis and Release.

We reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of angiopoietins (Ang1 and Ang2) to induce pro-inflammatory activities, such as platelet-activating factor synthesis, ß2-integrin activation and neutrophil migration. Recently, we observed differential effects between both angiopoietins, namely, the capacity of Ang1, but not Ang2, to promote rapid interleukin-8 synthesis and release, as well as neutrophil viability. Herein, we addressed whether Ang1 and/or Ang2 could modulate the synthesis and release of macrophage inflammatory protein-1ß (MIP-1ß) by neutrophils. Neutrophils were isolated from blood of healthy volunteers; intracellular and extracellular MIP-1ß protein concentrations were assessed by ELISA. After 24 hours, the basal intracellular and extracellular MIP-1ß protein concentrations were ≈500 and 100 pg/106 neutrophils, respectively. Treatment with Ang1 (10 nM) increased neutrophil intracellular and extracellular MIP-1ß concentrations by 310 and 388% respectively. Pretreatment with PI3K (LY294002), p38 MAPK (SB203580) and MEK (U0126) inhibitors completely inhibited Ang1-mediated increase of MIP-1ß intracellular and extracellular protein levels. Pretreatment with NF-κB complex inhibitors, namely Bay11-7085 and IKK inhibitor VII or with a transcription inhibitor (actinomycin D) and protein synthesis inhibitor (cycloheximide), did also abrogate Ang1-mediated increase of MIP-1ß intracellular and extracellular protein levels. We validated by RT-qPCR analyses the effect of Ang1 on the induction of MIP-1ß mRNA levels. Our study is the first one to report Ang1 capacity to induce MIP-1ß gene expression, protein synthesis and release from neutrophils, and that these effects are mediated by PI3K, p38 MAPK and MEK activation and downstream NF-κB activation.

Angiopoietin-1 but not angiopoietin-2 promotes neutrophil viability: Role of interleukin-8 and platelet-activating factor.

We previously reported the expression of angiopoietin receptor Tie2 on human neutrophils. Both angiopoietins (Ang1 and Ang2) induce platelet activating factor (PAF) synthesis from endothelial cells (ECs) and neutrophils. Both angiopoietins can also modulate EC viability and since PAF can promote pro-survival activity on neutrophils, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Neutrophils were isolated from venous blood of healthy volunteers and neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers (annexin-V and propidium iodide (P.I.), respectively). Basal neutrophil viability from 0 to 24 h post-isolation decreased from 98% to ≈45%. Treatment with anti-apoptotic mediators such as interleukin-8 (IL-8; 25 nM) and PAF (100 nM) increased neutrophil basal viability by 34 and 26% (raising it from 43 to 58 and 55%) respectively. Treatment with Ang1 (0.001-50 nM) increased neutrophil viability by up to 41%, while Ang2 had no significant effect. Combination of IL-8 (25 nM) or PAF (100 nM) with Ang1 (10 nM) further increased neutrophil viability by 56 and 60% respectively. We also observed that Ang1, but not Ang2 can promote IL-8 release and that a pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 81% respectively. Pretreatment with a selective PAF receptor antagonist (BN 52021), did abrogate PAF pro-survival activity, without affecting Ang1-induced neutrophil viability. Our data are the first ones to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.

Angiopoietin-1 but not angiopoietin-2 induces IL-8 synthesis and release by human neutrophils.

We previously reported Tie2 receptor expression on human neutrophils, which promote chemotactic activities upon activation by both angiopoietins (Ang1 and Ang2). Moreover, we observed that neutrophil pretreatment with Ang1 or Ang2 enhances interleukin-8 (IL-8) chemotactic effect. Therefore, we assessed the capacity of Ang1 and/or Ang2 to modulate neutrophil IL-8 synthesis and release. Neutrophils isolated from healthy donors were stimulated in a time- (1-6 h) and concentration-(10(-10) -10(-8) M) dependent manner with both angiopoietins. IL-8 mRNA production was measured by RT-qPCR, whereas its protein synthesis and release from neutrophils was assessed by ELISA. Ang1 (10(-8) M) induced a significant and maximal increase of IL-8 mRNA (4.7-fold) within 1 h, and promoted maximal IL-8 protein synthesis (3.6-fold) and release (5.5-fold) within 2 h as compared to control PBS-treated neutrophils. Treatment with Ang2 alone did not modulate IL-8 synthesis or release, and its combination to Ang1 did not affect Ang1 activity. Neutrophil pretreatment with a protein synthesis inhibitor (CHX) increased IL-8 mRNA synthesis by 18-fold, and reduced Ang1-mediated IL-8 protein synthesis and release by 96% and 92%, respectively. Pretreatment with a transcription inhibitor (ActD) reduced IL-8 mRNA synthesis by 54% and IL-8 protein synthesis and release by 52% and 79%, respectively. Using specific kinase inhibitors, we observed that Ang1-driven IL-8 mRNA and protein synthesis is p42/44 MAPK-dependent and -independent from p38 MAPK and PI3K activity. Our study is the first to report the capacity of Ang1 (as opposed to Ang2) to promote neutrophil IL-8 synthesis and release through the activation of p42/44 MAPK pathway.

Deodorant effects of a supercritical hops extract: antibacterial activity against Corynebacterium xerosis and Staphylococcus epidermidis and efficacy testing of a hops/zinc ricinoleate stick in humans through the sensory evaluation of axillary deodorancy.

BACKGROUND: There is little scientific evidence to support the efficacy of natural deodorants and therefore, such products may be perceived as inefficacious. The evaluation of the in vitro antibacterial activity of a hop extract and the evaluation of the odor-reducing capacity of a hops/zinc ricinoleate-containing product by a sensory evaluation panel is employed to verify deodorant performance. AIMS: The goal of this study was to evaluate the in vitro antibacterial activity of a hop extract against Corynebacterium xerosis and Staphylococcus epidermidis and to verify in vivo deodorant performance of a hops/zinc ricinoleate-containing product. METHODS: The hops extract was evaluated on a culture of an armpit swab from six volunteers. Furthermore, the extract was submitted to a zone of inhibition test and an agar-dilution assay against two major odor-causing bacteria. The clinical evaluation of the finished product was carried out according to a standard method for substantiating deodorant efficacy using trained odor judges for the assessment of axillary malodor (ASTM method E 1207-87 Standard Practice for the Sensory Evaluation of Axillary Deodorancy). RESULTS: The supercritical hops extract showed good antibacterial activities in all three tests. Minimum inhibitory concentration values of 6.25 and 25 mug/mL against C. xerosis and S. aureus, respectively, were obtained in the agar-dilution assay. In the clinical underarm odor-reduction evaluation, the mean malodor score dropped from 6.28 (+/-0.70) to 1.80 (+/-0.71) after 8 h of application. There was still a noticeable effect at both 12 and 24 h after the application, with a score of 1.82 (+/-0.74) and 2.24 (+/-0.77), respectively. CONCLUSION: The hops extract has good in vitro antibacterial properties and, in combination with zinc ricinoleate in an appropriate base, delivers in vivo odor reduction. The clinical efficacy is likely due to a combination of the base ingredients and the antibacterial actives.

Best practices in compound management for preserving compound integrity and accurately providing samples for assays.

Preserving the integrity of the compound collection and providing high-quality materials for drug discovery in an efficient and cost-effective manner are 2 major challenges faced by compound management (CM) at Bristol-Myers Squibb (BMS). The demands on CM include delivering hundreds of thousands of compounds a year to a variety of operations. These operations range from single-compound requests to hit identification support and just-in-time assay plate provision for lead optimization. Support needs for these processes consist of the ability to rapidly provide compounds as solids or solutions in a variety of formats, establishing proper long- and short-term storage conditions and creating appropriate methods for handling concentrated, potent compounds for delivery to sensitive biological assays. A series of experiments evaluating the effects of processing compounds with volatile solvents, storage conditions that can induce freeze/thaw cycles, and the delivery of compounds were performed. This article presents the results of these experiments and how they affect compound integrity and the accuracy of compound management processes.