Gene Ontology Analysis for Drug Targets of the Whole Genome Transcriptome of Human Vascular Endothelial Cells in Response to Proinflammatory IL-1

The innate immune system combats tissue injury and infection by activating the proinflammatory responses involving the humoral complement system, granulocytes, macrophages and vascular endothelial cells (VEC) (Newton and Dixit, 2012; Zhu et al., 2012). Macrophages mediate proinflammatory responses by releasing inflammatory cytokines such as IL-1β. Once secreted, IL-1β paracrinically acts on the VEC and massively change their functions. These perturbations include a change from the anticoagulant phenotype to a procoagulant state, enhanced expression of vasoactive substances, cell adhesion molecules as well as inflammatory mediators including chemoattractants, and endothelial barrier dysfunction causing microvascular leakage (Pober and Sessa, 2007). Although essential for the effective immune defense, uncontrolled or chronic inflammatory response causes tissue damage and loss of organ function (Lon et al., 2012)

Comparison of the Antimicrobial Activity of Deactivated Human Macrophages Challenged with Aspergillus fumigatus and Listeria monocytogenes

The anticonidial activity of human monocytes deactivated by cytokines interleukin (IL)-4 and IL-10 and the hormone dexamethasone was studied and compared with antilisterial activity. Dexamethasone had the largest effect on the anticonidial activity and suppressed germination-inhibiting activity and elimination of ingested spores by macrophages more than the cytokines did. Maximally active concentrations of IL-10 had a similar but significantly smaller deactivating effect. IL-4, in contrast to IL-10 and dexamethasone, did not reduce anticonidial activity. However, IL-4 and IL-10 were equally potent in deactivating human macrophages against Listeria monocytogenes, whereas dexamethasone was significantly less potent in the Listeria model. These observations indicate that all three mediators lessen antimicrobial activity but that this effect depends on the test organism studied and is apparently mediated through regulation of different antimicrobial systems operating against a particular microorganis

Differential Inhibition of Constitutive and Inducible Nitric Oxide Synthase in Vascular Endothelial Cells by Analogues of Tetrahydrobiopterin

In the vasculature, a physiologic production of nitric oxide (NO) is maintained by endothelial nitric oxide synthase (eNOS). Induction of inducible nitric oxide synthase (ÍNOS) under inflammatory conditions (e.g. septic shock) resulting in high levels of nitric oxide (NO) is believed to be partly responsible for the pathophysiologic changes in the vascular system that occur under inflammatory conditions (e.g. septic shock). Both NOS isoforms are dependent on the obligatory cofactor tetrahydrobiopterin (BH4). We investigated the selectivity and potency of the BH4 analogues 4-amino-BH4 and 5-methyl-BH4 in inhibiting eNOS and iNOS in a murine vascular endothelial cell (MVEC) model expressing either eNOS or iNOS under physiologic and inflammatory conditions, respectively. Exogenous BH4 and its precursor sepiapterin both enhanced physiologic eNOS activity in resting MVEC, while 4-amino-BH4 slightly inhibited eNOS. 5-methyl-BH4 did not have any effect on eNOS. BH4, sepi - apterin, and 5-methyi-BH4 had no effect on iNOS in inflammatory activated MVEC. In contrast, 4-amino-BH4 selectively inhibited iNOS with a potency comparable to the unselective NOS inhibitor Νω-monomethyl-L-argimne (L-NMMA). The present study demonstrates that 4-amino-BH4 selectively and potently inhibits iNOS in vascular endothelial cells, while its effect on eNOS is minimal. The selective inhibition of iNOS is a promising strategy for the treatment of inflammatory conditions with high output of NO. Further in vivo studies are required to determine whether inhibition of NO production by analogues of BH4 offers any advantage compared to inhibition by L-arginine analogue

Nitric Oxide Synthase Is Not a Constituent of the Antimicrobial Armature of Human Mononuclear Phagocytes

Nitric oxide synthase (NOS) has received immense interest as an antimicrobial and antitumoral effector system of mononuclear phagocytes from rodents. Because there is increasing doubt that an analogous system exists in human macrophages, NOS was reexamined in these cells. Under tightly controlled conditions, with murine macrophages as positive controls, human macrophages failed to secrete nitric oxide <0.1µmol/106 cells/24 h), even after activation with endotoxin, intcrferon-γ, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor- a, bacteria, or proliferating lymphocytes. The discrepancy between murine and human macrophages depended on neither the anatomic source (blood, peritoneum), the agent used for activation, nor the duration of activation. NOS activity was paralleled by metabolization of L-arginine to L-citrulline. Exogenous tetrahydrobiopterin, an essential cofactor of NOS not synthesized by human macrophages, did not support NOS activity in human macrophages. Also, no NOS activity was found in cellular subfractions of human macrophages. It appears that in humans, the inducible high-output NOS is not conserved as an antimicrobial system of macrophage

Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis

Schaer DJ, Schaer CA, Schoedon G, Imhof A, Kurrer MO. Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis. Objectives: Uncontrolled macrophage activation with hemophagocytosis is a distinctive feature of hemophagocytic syndromes (HPS). We examined whether lympho-histiocytic infiltration of the bone marrow and liver, as well as hemo-/erythrophagocytosis also occurs during sepsis and whether this process could account for the increased production of anti-inflammatory heme-oxygenase (HO-1) products observed during sepsis. Methods: Hemophagocytosis and expression of CD163, HO-1, ferritin as well as CD8 and granzyme-B were examined in post-mortem bone marrow samples from 28 patients with sepsis and from eight control patients. Results: Comparison of samples from non-septic patients with samples from patients with fatal sepsis revealed that the latter group displayed dense lympho-histiocytic bone marrow infiltration with CD163(+)/HO-1(+)/ferritin(+) macrophages as well as with CD8(+) and granzyme-B(+) T-cells. Hemophagocytosis with prominent phagocytosis of erythroid cells was readily apparent in septic patients, implying that this process is a likely stimulus for the up-regulation of macrophage HO-1 expression. Conclusions: Lympho-histiocytic activation with hemophagocytosis is a shared pathophysiologic mechanism in HPS and sepsis. Furthermore, the association of hemophagocytosis with an increase in HO-1 expression may indicate a novel role for this apparently futile process as a negative regulator of inflammation

Survival and graft function in a large animal lung transplant model after 30 h preservation and substitution of the nitric oxide pathway

Objective: Substitution of the nitric oxide- (NO-) pathway improves early graft function following lung transplantation. We previously demonstrated that 8-Br-cGMP (second messenger of NO) to the flush solution and tetrahydrobiopterin (BH4, coenzyme of NO synthase) given as additive during reperfusion improve post-transplant graft function. In the present study, the combined treatment with 8-Br-cGMP and BH4 was evaluated. Methods: Unilateral left lung transplantation was performed in weight matched outbred pigs (24-31kg). In group I, grafts were preserved for 30h (n=5). 8-Br-cGMP (1mg/kg) was added to the flush solution (Perfadex™, 1.5l, 1°C) and BH4 (10mg/kg/h) was given to the recipient for 5h after reperfusion. In group II, lungs were transplanted after a preservation time of 30h (n=3) and prostaglandin E1 (250g) was given into the pulmonary artery (PA) prior to flush. In all recipients 1h after reperfusion the contralateral right PA and bronchus were ligated to assess graft function only. Survival time after reperfusion, extravascular lung water index (EVLWI), hemodynamic variables, and gas exchange (PaO2) were assessed during a 12h observation period. Results: All recipients in group I survived the 12h assessment, whereas none of the group II animals survived more than 4h after reperfusion with a rapid increase of EVLWI up to 24.8±6.7ml/kg. In contrast, in group I EVLWI reached up to 8.9±1.5ml/kg and returned to nearly normal levels at 12h (6.1±0.8ml/kg). In two animals of group I the gas exchange deteriorated slightly. The other three animals showed normal arterial oxygenation over the entire observation time. Conclusion: Our data indicate that the combined substitution of the NO pathway during preservation and reperfusion reduces ischemia/reperfusion injury substantially and that this treatment even allows lung transplantation after 30h preservation in this mode

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Barrier function of Wnt5A-treated VEC in the presence or absence of WIF1 and sFRP1. a ECIS assisted measurements (Additional file 4: supplementary methods) showing resistance of HCAEC monolayers grown in 8W10E+ arrays treated with vehicle (black), Wnt5A (green), Wnt5A + WIF1 (yellow) and Wnt5A + sFRP1 (purple). Data shown are the resistance measurements conducted at 4000 Hz and are mean ± SEM of 2 wells from 1 out of three representative experiments. b Barrier function measurements indicating the significance of WIF1’s antagonistic effect on Wnt5A and are mean ± SEM of three independent experiments run with duplicate wells. *P < 0.05 vs non-treated, **P < 0.05 vs Wnt5A. (PDF 2125 kb

Deactivation of Macrophages with Interleukin-4 Is the Key to the Isolation of Tropheryma whippelii

Whipple's disease is a systemic illness caused by a specific agent. Despite recognition of bacteria in lesions, efforts to isolate the causative agent remained futile. A novel strategy was devised to culture Whipple bacilli in deactivated mononuclear phagocytes. Infected tissue was inoculated into human phagocytes deactivated with interleukin (IL)-4, IL-10, and dexamethasone. Within 8-10 days, diastase-resistant periodic acid-Schiff-positive inclusions appeared, corresponding to intact and degenerating bacteria shown to be Tropheryma whippelii by electron microscopy and molecular analyses. T. whippelii was passaged several times in deactivated monocytes and a monoblastic cell line. Time-kinetics growth studies and comparative polymerase chain reaction analysis documented multiplication of T. whippelii in deactivated macrophages. Complementary studies showed that IL-4 rendered phagocytes permissive for T. whippelii, a strong indication that host factors contribute to the pathogenesis of Whipple's disease. The propagation of T. whippelii will permit microbiologic, immunologic, seroepidemiologic, and therapeutic studies of this pathoge