Explore the vast range of titles available.

We examined the functional role of 14-kD secretory group V phospholipase A(2) (gVPLA(2)) on the barrier function of pulmonary endothelial cells (ECs) after LPS activation in vitro. Expression of gVPLA(2) was elicited by 20 ng/ml LPS as demonstrated by increased (1) mRNA, (2) protein content, and (3) cell surface expression of gVPLA(2) within 4 hours. The effect of LPS on EC barrier function was measured by transendothelial monolayer electrical resistance (TER). LPS increased permeability across EC monolayers at 2-3 hours, and was sustained for 10 hours or more. Blockade of gVPLA(2) with mouse monoclonal 3G1 (MCL-3G1) monoclonal antibody directed against gVPLA(2) inhibited EC barrier dysfunction elicited by LPS in a time- and concentration-dependent manner; control IgG had no effect on TER. Like LPS, exogenous gVPLA(2) caused increased EC permeability in a time- and concentration-dependent manner; neither gIIaPLA(2), a close homolog of gVPLA(2), nor W31A, an inactive mutant of gVPLA(2), caused a decrease in EC TER. Immunofluorescence analysis revealed comparable F-actin stress fiber and intercellular gap formation for ECs treated with either gVPLA(2) or LPS. Treatment with gVPLA(2) disrupted vascular endothelial-cadherin junctional complexes on ECs. Coincubation of ECs with MCL-3G1 substantially attenuated the structural changes caused by gVPLA(2) or LPS. We demonstrate that (1) gVPLA(2) is constitutively expressed in ECs and is up-regulated after LPS activation, (2) endogenously secreted gVPLA(2) from ECs after LPS increases EC permeability through F-actin and junctional complex rearrangement, and (3) inhibition of endogenous gVPLA(2) from ECs is sufficient to block disruption of the EC barrier function after LPS in vitro.

Background Cytosolic gIVaPLA 2 is a critical enzyme in the generation of arachidonate metabolites and in induction of β 2 -integrin adhesion in granulocytes. We hypothesized that gIVaPLA 2 activation also is an essential downstream step for post adhesive migration of PMN in vitro . Methods Migration of PMNs caused by IL-8/CXCL8 was assessed using a transwell migration chamber. PMNs were pretreated with two structurally unrelated inhibitors of gIVaPLA 2 , arachidonyl trifluoromethylketone (TFMK) or pyrrophenone, prior to IL-8/CXCL8 exposure. The fraction of migrated PMNs present in the lower chamber was measured as total myeloperoxidase content. GIVaPLA 2 enzyme activity was analyzed using [ 14 C-PAPC] as specific substrate F-actin polymerization and cell structure were examined after rhodamine-phalloidin staining. Results IL-8/CXCL8-induced migration of PMNs was elicited in concentration- and time-dependent manner. Time-related phosphorylation and translocation of cytosolic gIVaPLA 2 to the nucleus was observed for PMNs stimulated with IL-8/CXCL8 in concentration sufficient to cause upstream phosphorylation of MAPKs (ERK-1/2 and p38) and Akt/PKB. Inhibition of gIVaPLA 2 corresponded to the magnitude of blockade of PMN migration. Neither AA nor LTB 4 secretion was elicited following IL-8/CXCL8 activation. In unstimulated PMNs, F-actin was located diffusely in the cytosol; however, a clear polarized morphology with F-actin-rich ruffles around the edges of the cell was observed after activation with IL-8/CXCL8. Inhibition of gIVaPLA 2 blocked change in cell shape and migration caused by IL-8/CXCL8 but did not cause F-actin polymerization or translocation of cytosolic F-actin to inner leaflet of the PMN membrane. Conclusion We demonstrate that IL-8/CXCL8 causes a) phosphorylation and translocation of cytosolic gIVaPLA 2 to the nucleus, b) change in cell shape, c) polymerization of F-actin, and d) chemoattractant/migration of PMN in vitro. Inhibition of gIVaPLA 2 blocks the deformability and subsequent migration of PMNs caused by IL-8/CXCL8. Our data suggest that activation of gIVaPLA 2 is an essential step in PMN migration in vitro .

Cytosolic gIVaPLA.sub.2 is a critical enzyme in the generation of arachidonate metabolites and in induction of [beta].sub.2 -integrin adhesion in granulocytes. We hypothesized that gIVaPLA.sub.2 activation also is an essential downstream step for post adhesive migration of PMN in vitro. Migration of PMNs caused by IL-8/CXCL8 was assessed using a transwell migration chamber. PMNs were pretreated with two structurally unrelated inhibitors of gIVaPLA.sub.2 , arachidonyl trifluoromethylketone (TFMK) or pyrrophenone, prior to IL-8/CXCL8 exposure. The fraction of migrated PMNs present in the lower chamber was measured as total myeloperoxidase content. GIVaPLA.sub.2 enzyme activity was analyzed using [.sup.14.sup.C-PAPC] as specific substrate F-actin polymerization and cell structure were examined after rhodamine-phalloidin staining. IL-8/CXCL8-induced migration of PMNs was elicited in concentration- and time-dependent manner. Time-related phosphorylation and translocation of cytosolic gIVaPLA.sub.2 to the nucleus was observed for PMNs stimulated with IL-8/CXCL8 in concentration sufficient to cause upstream phosphorylation of MAPKs (ERK-1/2 and p38) and Akt/PKB. Inhibition of gIVaPLA.sub.2 corresponded to the magnitude of blockade of PMN migration. Neither AA nor LTB.sub.4 secretion was elicited following IL-8/CXCL8 activation. In unstimulated PMNs, F-actin was located diffusely in the cytosol; however, a clear polarized morphology with F-actin-rich ruffles around the edges of the cell was observed after activation with IL-8/CXCL8. Inhibition of gIVaPLA.sub.2 blocked change in cell shape and migration caused by IL-8/CXCL8 but did not cause F-actin polymerization or translocation of cytosolic F-actin to inner leaflet of the PMN membrane. We demonstrate that IL-8/CXCL8 causes a) phosphorylation and translocation of cytosolic gIVaPLA.sub.2 to the nucleus, b) change in cell shape, c) polymerization of F-actin, and d) chemoattractant/migration of PMN in vitro. Inhibition of gIVaPLA.sub.2 blocks the deformability and subsequent migration of PMNs caused by IL-8/CXCL8. Our data suggest that activation of gIVaPLA.sub.2 is an essential step in PMN migration in vitro.

We examined the functional role of 14-kD secretory group V phospholipase A(2) (gVPLA(2)) on the barrier function of pulmonary endothelial cells (ECs) after LPS activation in vitro. Expression of gVPLA(2) was elicited by 20 ng/ml LPS as demonstrated by increased (1) mRNA, (2) protein content, and (3) cell surface expression of gVPLA(2) within 4 hours. The effect of LPS on EC barrier function was measured by transendothelial monolayer electrical resistance (TER). LPS increased permeability across EC monolayers at 2-3 hours, and was sustained for 10 hours or more. Blockade of gVPLA(2) with mouse monoclonal 3G1 (MCL-3G1) monoclonal antibody directed against gVPLA(2) inhibited EC barrier dysfunction elicited by LPS in a time- and concentration-dependent manner; control IgG had no effect on TER. Like LPS, exogenous gVPLA(2) caused increased EC permeability in a time- and concentration-dependent manner; neither gIIaPLA(2), a close homolog of gVPLA(2), nor W31A, an inactive mutant of gVPLA(2), caused a decrease in EC TER. Immunofluorescence analysis revealed comparable F-actin stress fiber and intercellular gap formation for ECs treated with either gVPLA(2) or LPS. Treatment with gVPLA(2) disrupted vascular endothelial-cadherin junctional complexes on ECs. Coincubation of ECs with MCL-3G1 substantially attenuated the structural changes caused by gVPLA(2) or LPS. We demonstrate that (1) gVPLA(2) is constitutively expressed in ECs and is up-regulated after LPS activation, (2) endogenously secreted gVPLA(2) from ECs after LPS increases EC permeability through F-actin and junctional complex rearrangement, and (3) inhibition of endogenous gVPLA(2) from ECs is sufficient to block disruption of the EC barrier function after LPS in vitro.

Human papillomavirus-negative (HPV-neg) oropharyngeal squamous cell carcinomas (OPSCCs) are associated with poorer overall survival (OS) compared with HPV-positive (HPV-pos) OPSCCs. The major obstacle in improving outcomes of HPV-neg patients is the lack of robust biomarkers and therapeutic targets. Herein, we investigated the role of centrosome amplification (CA) as a prognostic biomarker in HPV-neg OPSCCs. A quantitative evaluation of CA in clinical specimens of OPSCC revealed that (a) HPV-neg OPSCCs exhibit higher CA compared with HPV-pos OPSCCs, and (b) CA was associated with poor OS, even after adjusting for potentially confounding clinicopathologic variables. Contrastingly, CA was higher in HPV-pos cultured cell lines compared to HPV-neg ones. This divergence in CA phenotypes between clinical specimens and cultured cells can therefore be attributed to an inaccurate recapitulation of the in vivo tumor microenvironment in the cultured cell lines, namely a hypoxic environment. The exposure of HPV-neg OPSCC cultured cells to hypoxia or stabilizing HIF-1α genetically increased CA. Both the 26-gene hypoxia signature as well as the overexpression of HIF-1α positively correlated with increased CA in HPV-neg OPSCCs. In addition, we showed that HIF-1α upregulation is associated with the downregulation of miR-34a, increase in CA and expression of cyclin- D1. Our findings demonstrate that the evaluation of CA may aid in therapeutic decision-making, and CA can serve as a promising therapeutic target for HPV-neg OPSCC patients.