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In early stages of calcific aortic valve disease (CAVD), immune cells infiltrate into the valve leaflets and release cytokines such as interferon (IFN)-γ. IFN-γ has context-dependent direct effects, and also regulates other immune pathways. The purpose of this study was addressing the effects of IFN-γ on human aortic valve interstitial cells (AVICs), focusing on the pathogenic processes underlying CAVD. Strikingly, under normoxic conditions, IFN-γ induced hypoxia inducible factor (HIF)-1α expression, an effect strongly potentiated by the Toll-like receptor (TLR)-4 ligand lipopolysaccharide (LPS). Immunodetection studies confirmed the nuclear translocation of HIF-1α. Gene silencing showed that HIF-1α expression is dependent on signal transducer and activator of transcription (STAT)-1 expression. Consistent with HIF-1α induction, the secretion of the endothelial growth factor was detected by ELISA, and downregulation of the antiangiogenic factor chondromodulin-1 gene was observed by qPCR. Results also disclosed IFN-γ as a proinflammatory cytokine that cooperates with LPS to induce the expression of adhesion molecules, prostaglandin E 2 and interleukins. Moreover, IFN-γ induced an osteogenic phenotype and promoted in vitro calcification that were markedly potentiated by LPS. Pharmacological experiments disclosed the involvement of Janus Kinases (JAK)/STATs as well as ERK/HIF-1α routes on the induction of calcification. Notably, IFN-γ receptor 1 expression, as well as ERK/HIF-1α activation, and the subsequent responses were more robust in male AVICs. This is the first report uncovering an immune and non-hypoxic activation of HIF-1α via STAT1 in AVIC. The aforementioned results and the sex-differential responses may be potentially relevant to better understand CAVD pathogenesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Unlabelled: This study aimed to set up an experimental model of long bone atrophic nonunion and to explore the potential role of PTH-1-84 (PTH 1-84) and strontium ranelate (SrR). A model of atrophic nonunion was created in Sprague-Dawley rats at the femoral midshaft level. The animals were randomised into four groups. Group A1: control rodents, fracture without bone gap; Group A2: rodents with subtraction osteotomy (non-union model control) treated with saline; Group B: rodents with subtraction osteotomy treated with human-PTH (PTH 1-84); and Group C: rodents with subtraction osteotomy treated with strontium ranelate (SrR). The groups were followed for 12 weeks. X-rays were be obtained at weeks 1, 6 and 12. After sacrificing the animals, we proceeded to the biomechanical study and four point bending tests to evaluate the resistance of the callus and histological study. In second phase, the expression of genes related to osteoblast function was analysed by reverse transcription-quantitative PCR in rats subjected to substraction osteotomy and treated for 2 weeks. The animals were randomised into three groups: Group A2: rodents treated with saline; Group B: rodents treated with PTH 1-84 and Group C: rodents treated with SrR., Results: No significant histological differences were found between animals subjected to subtraction osteotomy and treated with either saline or PTH (p=0.628), but significant difference existed between animals receiving saline or SrR (p=0.005). There were no significant differences in X-ray score between the saline and PTH groups at either 6 or 12 weeks (p=0.33 and 0.36, respectively). On the other hand, better X-ray scores were found in the SrR group (p=0.047 and 0.006 in comparison with saline, at 6 and 12 weeks, respectively). In line with this, biomechanical tests revealed improved results in the SrR group. Gene expression analysis revealed a slightly decreased levels of DKK1, a Wnt pathway inhibitor, in rats treated with SrR., Conclusions: SrR increases has a beneficial effect in this atrophic non-union model in rats. This suggests that it might have a role may have important implications for the potential clinical role in the treatment of fracture nonunion., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Human brucellosis is characterized by the presence of both acute inflammatory episodes and chronic inflammation with granuloma formation. On this basis, the proinflammatory effects of smooth lipopolysaccharide of Brucella (S-LPS) were addressed and compared to those of LPS from Escherichia coli. For this purpose, the induction of cyclooxygenase-2 (COX-2), the production of the chemokine monocyte chemoattractant protein-1 (MCP-1), and the activation of the nuclear factor kappa B (NF-kappa B) were studied. S-LPS was found to induce both COX-2 expression and MCP-1 production; however, the potency of E. coli LPS exceeded that of Brucella S-LPS by some orders of magnitude. However, at concentrations above 1 microg/ml, all of the LPS produced comparable effects, including their ability to activate the NF-kappa B system. These observations help explain the inflammatory events associated with Brucella infection and the ability of Brucella to produce monocyte recruitment and granuloma formation.

The effect of secretory phospholipase A2 (sPLA2) on intracellular Ca2+ signaling in human astrocytoma cells was studied. sPLA2 increased cytosolic [Ca2+] ([Ca2+]c) in both Ca2+-containing and Ca2+-free medium, thus suggesting Ca2+ release from intracellular stores. The activation by sPLA2 of arachidonate release via cytosolic PLA2 (cPLA2) was also independent of extracellular Ca2+. As sPLA2 requires Ca2+ for activity, these results indicate that both Ca2+ mobilization and cPLA2 activation induced by sPLA2 are unrelated to phospholipase activity but dependent on signaling mechanisms. The sPLA2-induced [Ca2+]c peak was sensitive to Bordetella pertussis toxin and inhibited by caffeine, suggesting its mediation by inositol 1,4,5-trisphosphate (IP3). sPLA2 induced tyrosine phosphorylation and membrane targeting of phospholipase Cgamma-1 (PLCgamma-1). Moreover, the Ca2+ peak was sensitive to protein tyrosine kinase inhibitors. sPLA2 activates two signaling pathways: one leading to the activation of the MAP kinase/cPLA2 cascade and another leading to PLCgamma activation and Ca2+ release., (Copyright 1999 Academic Press.)

The production of nitric oxide (NO) by rat adherent peritoneal cells stimulated with preformed IgE/Dinitrophenyl-BSA (DNP-BSA) complexes and its dependence on the activation of the transcription factor NF-kappa B were studied. Stimulation with IgE/DNP-BSA complexes at equivalence induced both the production of NO and an increased expression of the inducible isoform of NO synthase (iNOS) protein. Both events were also elicited by a rabbit polyclonal F(ab')2 anti-CD23 cross-reacting with rat CD23, thus suggesting Fc epsilon RII/CD23 antigen as the IgE-binding structure involved in the triggering of the response and ruling out an interaction of the antibody via its Fc portion. Inhibition of redox-sensitive signaling mechanisms by the antioxidant pyrrolidine dithiocarbamate (PDTC) blocked NO production, iNOS expression, and NF-kappa B activation elicited by both IgE/DNP-BSA complexes and anti-CD23 F(ab')2, thus suggesting the involvement of NF-kappa B in the signaling pathway leading to the transcriptional activation of iNOS. These results show the existence in rat peritoneal macrophages of a signaling pathway triggered by CD23 engagement that promotes nuclear translocation of NF-kappa B and transcriptional activation of the inducible isoform of NO synthase.

Human neutrophils were homogenized and fractionated on a continuous sucrose gradient to assess the subcellular location of acetyl-CoA: lyso-PAF acetyltransferase and of newly synthesized PAF (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Acetyltransferase activity showed two subcellular locations in resting neutrophils. One of them cofractionated with plasma membrane and endoplasmic reticulum markers, whereas another major location corresponded to a region of the gradient enriched in tertiary granules. No PAF was detected in resting neutrophils, but PAF synthesis was induced by cell stimulation with ionophore A23187. Most of the newly synthesized PAF was found cell-associated, showing a bimodal subcellular distribution similar to that found for acetyltransferase activity in activated cells. PAF and acetyltransferase were located in a light membrane fraction, enriched in plasma membrane and endoplasmic reticulum, and in an ill-defined region of the gradient between the specific and azurophilic granules in A23187-stimulated cells. These data support the involvement of the acetyltransferase pathway in the synthesis of PAF induced by ionophore A23187, and demonstrate the synthesis and accumulation of newly synthesized PAF in a light membrane fraction as well as in an intracellular dense organelle upon neutrophil activation.