Secreted phospholipases A2 in hereditary angioedema with C1-inhibitor deficiency

BackgroundHereditary angioedema (HAE) caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) is a disabling, potentially fatal condition characterized by recurrent episodes of swelling. We have recently found that patients with C1-INH-HAE have increased plasma levels of vascular endothelial growth factors and angiopoietins (Angs), which have been associated with vascular permeability in several diseases. Among these and other factors, blood endothelial cells and vascular permeability can be modulated by extracellular or secreted phospholipases A2 (sPLA2s).ObjectiveWe sought to investigate the enzymatic activity and biological functions of sPLA2 in patients with C1-INH-HAE.MethodssPLA2s enzymatic activity was evaluated in the plasma from 109 adult patients with C1-INH-HAE and 68 healthy donors in symptom-free period and attacks. Plasma level of group IIA sPLA2 (hGIIA) protein was measured in selected samples. The effect of C1-INH-HAE plasma on endothelial permeability was examined in vitro using a vascular permeability assay. The role of hGIIA was determined using highly specific sPLA2 indole inhibitors. The effect of recombinant hGIIA on C1-INH activity was examined in vitro by functional assay.ResultsPlasma sPLA2 activity and hGIIA levels are increased in symptom-free C1-INH-HAE patients compared with controls. sPLA2 activity negatively correlates with C1-INH protein level and function. C1-INH-HAE plasma increases endothelial permeability in vitro, and this effect is partially reverted by a specific hGIIA enzymatic inhibitor. Finally, recombinant hGIIA inhibits C1-INH activity in vitro.ConclusionsPLA2 enzymatic activity (likely attributable to hGIIA), which is increased in C1-INH-HAE patients, can promote vascular permeability and impairs C1-INH activity. Our results may pave the way for investigating the functions of sPLA2s (in particular, hGIIA) in the pathophysiology of C1-INH-HAE and may inform the development of new therapeutic targets

Cellular subtype expression and activation of CaMKII regulate the fate of atherosclerotic plaque

Abstract Background and aims Atherosclerosis is a degenerative process of the arterial wall implicating activation of macrophages and proliferation of vascular smooth muscle cells. Calcium-calmodulin dependent kinase type II (CaMKII) in vascular smooth muscle cells (VSMCs) regulates proliferation, while in macrophages, this kinase governs diapedesis, infiltration and release of extracellular matrix enzymes. We aimed at understanding the possible role of CaMKII in atherosclerosis plaques to regulate plaque evolution towards stability or instability. Methods Clinically defined stable and unstable plaques obtained from patients undergoing carotid end arteriectomy were processed for evaluation of CaMKs protein expression, activity and localization. Results The larger content of CaMKII was found in CD14 + myeloid cells that were more abundant in unstable rather than stable plaques. To test the biological effect of activated CD14 + myeloid cells, VSMCs were exposed to the conditioned medium (CM) of macrophages extracted from carotid plaques. CM induced attenuation of CaMKs expression and activity in VSMCs, leading to the reduction of VSMCs proliferation. This appears to be due to the CaMKII dependent release of cytokines. Conclusions These results indicate a pivotal role of CaMKs in atherosclerosis by regulating activated myeloid cells on VSMCs activity. CaMKII could represent a possible target for therapeutic strategies based on macrophages specific inhibition for the stabilization of arteriosclerotic lesions

Mechanical behavior of hybrid fiber-reinforced composites manufactured by pulse infusion

Epoxy/carbon fiber composites have been manufactured by Pulse Infusion. Pulse Infusion allows to control the pressure of the vacuum bag on the dry fiber reinforcement by using a proper designed pressure distributor that induces a pulsed transverse action and promotes the through thickness resin flow. The adopted one-component commercial epoxy resin has been preliminary modified by adding 0.05% (w/w) of multiwalled carbon nanotubes, in order to take advantage of carbon nanotubes at low concentration. Both neat and hybrid realized composite panels have been mechanically characterized by performing experimental tests to evaluate tensile, interlaminar, and fracture properties in order to investigate the effect of Pulse Infusion and carbon nanotubes on the mechanical and fracture behavior of composites. Results demonstrated an improvement of 36.2% for the interlaminar shear strength, of 35% for the fracture energy at the crack initiation and of 14% for the fracture toughness in mode II. © 2015 Society of Plastics Engineers

GM-CSF and IL-3 Modulate Human Monocyte TNF-α Production and Renewal in In Vitro Models of Trained Immunity

GM-CSF and IL-3 are hematopoietic cytokines that also modulate the effector functions of several immune cell subsets. In particular, GM-CSF and IL-3 exert a significant control on monocyte and macrophage effector functions, as assessed in experimental models of inflammatory and autoimmune diseases and also in human studies. Here, we sought to investigate the mechanisms and the extent to which GM-CSF and IL-3 modulate the pro-inflammatory, LPS-mediated, activation of human CD14(+) monocytes taking into account the new concept of trained immunity (i.e., the priming stimulus modulates the response to subsequent stimuli mainly by inducing chromatin remodeling and increased transcription at relevant genetic loci). We demonstrate that GM-CSF and IL-3 priming enhances TNF-α production upon subsequent LPS stimulation (short-term model of trained immunity) in a p38- and SIRT2-dependent manner without increasing TNF primary transcript levels (a more direct measure of transcription), thus supporting a posttranscriptional regulation of TNF-α in primed monocytes. GM-CSF and IL-3 priming followed by 6 days of resting also results in increased TNF-α production upon LPS stimulation (long-term model of trained immunity). In this case, however, GM-CSF and IL-3 priming induces a c-Myc-dependent monocyte renewal and increase in cell number that is in turn responsible for heightened TNF-α production. Overall, our results provide insights to understand the biology of monocytes in health and disease conditions in which the hematopoietic cytokines GM-CSF and IL-3 play a role and also extend our knowledge of the cellular and molecular mechanisms of trained immunity

Simplexide Induces CD1d-Dependent Cytokine and Chemokine Production from Human Monocytes

Monocytes are major effector cells of innate immunity and recognize several endogenous and exogenous molecules due to the expression of wide spectrum of receptors. Among them, the MHC class I-like molecule CD1d interacts with glycolipids and presents them to iNKT cells, mediating their activation. Simplexide belongs to a novel class of glycolipids isolated from marine sponges and is structurally distinct from other immunologically active glycolipids. In this study we have examined the effects of simplexide on cytokine and chemokine release from human monocytes. Simplexide induces a concentration- and time-dependent release of IL-6, CXCL8, TNF-α and IL-10 and increases the expression of IL6, CXCL8 and IL10 mRNA. Cytokine and chemokine release induced by simplexide from monocytes is dependent on CD1d since: i) a CD1d antagonist, 1,2-bis (diphenylphosphino) ethane [DPPE]- polyethylene glycolmonomethylether [PEG], specifically blocks simplexide-induced activation of monocytes; ii) CD1d knockdown inhibits monocyte activation by simplexide and iii) simplexide induces cytokine production from CD1d-transfected but not parental C1R cell line Finally, we have shown that simplexide also induces iNKT cell expansion in vitro. Our results demonstrate that simplexide, apart from activating iNKT cells, induces the production of cytokines and chemokines from human monocytes by direct interaction with CD1d

Current Water Conditions in Massachusetts (2011-01-13)

BACKGROUND: Neutrophil functions have long been regarded as limited to acute inflammation and the defense against microbes. The role(s) of neutrophils in cancer remain poorly understood. Neutrophils infiltrate tumors and are key effector cells in the orchestration of inflammatory responses. Thyroid cancer (TC) is the most recurrent endocrine malignant tumor and is responsible for 70% of deaths due to endocrine cancers. No studies are so far available on the role of neutrophils in TC. OBJECTIVE: Our purpose was to study the involvement of tumor-associated neutrophils in TC. METHODS: Highly purified human neutrophils (>99%) from healthy donors were stimulated in vitro with conditioned media derived from TC cell lines TPC1 and 8505c (TC-CMs). Neutrophil functions (e.g., chemotaxis, activation, plasticity, survival, gene expression, and protein release) were evaluated. RESULTS: TC-derived soluble factors promoted neutrophil chemotaxis and survival. Neutrophil chemotaxis toward a TC-CM was mediated, at least in part, by CXCL8/IL-8, and survival was mediated by granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, each TC-CM induced morphological changes and activation of neutrophils (e.g., CD11b and CD66b upregulation and CD62L shedding) and modified neutrophils' kinetic properties. Furthermore, each TC-CM induced production of reactive oxygen species, expression of proinflammatory and angiogenic mediators (CXCL8/IL-8, VEGF-A, and TNF-α), and a release of matrix metalloproteinase 9 (MMP-9). Moreover, in TC patients, tumor-associated neutrophils correlated with larger tumor size. CONCLUSIONS: TC cell lines produce soluble factors able to "educate" neutrophils toward an activated functional state. These data will advance the understanding of the molecular and cellular mechanisms of innate immunity in TC

Lipopolysaccharide-Elicited TSLPR Expression Enriches a Functionally Discrete Subset of Human CD14(+) CD1c(+) Monocytes

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor (TSLPR) complex, a heterodimer composed of TSLPR and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLPR complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is poorly defined. We demonstrate that TSLP enhances human CD14(+) monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14(+) CD16(-) monocytes, TSLPR(+) monocytes (TSLPR(+) mono), expresses TSLPR complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional, and transcriptomic analysis revealed specific features of TSLPR(+) mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e., GAS6, ALOX15B, FCGR2B, LAIR1). Strikingly, TSLPR(+) mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14(+) CD1c(+) cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared with healthy control subjects. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14(+) CD16(-) monocytes and prompt further ontogenetic and functional analysis of CD14(+) CD1c(+) and LPS-activated CD14(+) CD1c(+) TSLPR(+) mono

Human lung-resident macrophages express CB1 and CB2 receptors whose activation inhibits the release of angiogenic and lymphangiogenic factors

Macrophages are pivotal effector cells in immune responses and tissue remodeling by producing a wide spectrum of mediators, including angiogenic and lymphangiogenic factors. Activation of cannabinoid receptor types 1 and 2 has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo. We investigated whether human lung-resident macrophages express a complete endocannabinoid system by assessing their production of endocannabinoids and expression of cannabinoid receptors. Unstimulated human lung macrophage produce 2-arachidonoylglycerol, N-arachidonoyl-ethanolamine, N-palmitoyl-ethanolamine, and N-oleoyl-ethanolamine. On LPS stimulation, human lung macrophages selectively synthesize 2- arachidonoylglycerol in a calcium-dependent manner. Human lung macrophages express cannabinoid receptor types 1 and 2, and their activation induces ERK1/2 phosphorylation and reactive oxygen species generation. Cannabinoid receptor activation by the specific synthetic agonists ACEA and JWH-133 (but not the endogenous agonist 2-arachidonoylglycerol) markedly inhibits LPS-induced production of vascular endothelial growth factor-A, vascular endothelial growth factor-C, and angiopoietins and modestly affects IL-6 secretion. No significant modulation of TNF-α or IL-8/CXCL8 release was observed. The production of vascular endothelial growth factor-A by human monocyte-derived macrophages is not modulated by activation of cannabinoid receptor types 1 and 2. Given the prominent role of macrophage-assisted vascular remodeling in many tumors, we identified the expression of cannabinoid receptors in lung cancer-associated macrophages. Our results demonstrate that cannabinoid receptor activation selectively inhibits the release of angiogenic and lymphangiogenic factors from human lung macrophage but not from monocyte-derived macrophages. Activation of cannabinoid receptors on tissue-resident macrophages might be a novel strategy to modulate macrophage-assisted vascular remodeling in cancer and chronic inflammation

Effect of increasing concentrations of natural or synthetic simplexide on IL-6 (panel A) and CXCL8 (panel B) release from monocytes.

<p>Polystyrene plates were coated with indicated concentrations of glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addiction of cells. After 24 hours of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as ng of IL-6 or CXCL8 per mg of total proteins. Data are the mean ± SEM of six experiments. * p<0.05 <i>vs</i>. control.</p