Oxygen therapy often rescues and reduces the mortality resulting from acute respiratory distress syndrome, chronic obstructive pulmonary diseases, exposure to toxic fumes, and drowning (1). However, prolonged exposure to supra-physiological concentrations of oxygen, referred to as hyperoxia, causes extensive damage to the alveolar-capillary barrier resulting in increased permeability and decreased lung function (2). Although the molecular mechanisms of hyperoxia-induced lung injury and cell death are complex, recent studies suggest that the generation of excessive reactive oxygen species (ROS), 1 loss of antioxidant defense pathways, cytokine-mediated inflammation, and modulation of signal transduction may regulate pulmonary edema and apoptosis/necrosis of endothelial and epithelial cells (3). The vascular endothelium has long been recognized to generate superoxide (O 2 . ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical ( ⅐ OH), and nitric oxide (NO) via enzymatic and nonenzymatic reactions. In endothelial cells (ECs), in addition to the mitochondrial electron transport, other potential enzymatic pathways of ROS production include cyclooxygenase/lipoxygenase, cytochrome P450, xanthine oxidase, NADPH oxidase, NO synthase, and peroxidase. In the lung, the vascular NADPH oxidase seems to play an important role in excessive production of O 2 . in atherosclerosis, ischemic lung, pulmonary hypertension, and ventilator-associated lung injury (4 -9). NADPH oxidase catalyzes the one-electron reduction of molecular oxygen to O 2 . by using NADPH or NADH as an electron donor (9). Activated NADPH oxidase is a multimeric protein complex consisting of at least three cytosolic subunits of p47 phox , p67 phox , and p40 phox ; a regulatory small molecular weight G-protein of either Rac1 or Rac2 and a membraneassociated cytochrome b 558 reductase made up of p22 phox and gp91 phox . We and others (10, 11) have shown that most of the subcomponents of phagocytic NADPH oxidase are expressed in vascular ECs. ECs exhibit a low output in of O 2. production under basal conditions, and stimulation by TNF-␣, pulsatile stretch, hypoxia reoxygenation, and phorbol ester enhanced the
Nystatin forms two types of channels in sterol-containing planar bilayer membranes. One type is formed when it is added to only one side of the membrane; the other is formed when it is added to both sides of the membrane. The relative permeability of these channels to nonelectrolytes (urea and glycerol) is identical. The sensitivity of membranes to the one-sided action of nystatin is critically dependent on their thickness; in particular, membranes made from monoglycerides with more than 18 carbon atoms in their acyl chain are insensitive to nystatin's one-sided action. These data are consistent with a model in which the two types of channels formed by nystatin have essentially identical structures, except that the channel formed by its two-sided action is twice the length of that formed by its one-sided action, because it is a tail-to-tail dimer of the latter.
Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.
Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxiainduced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47 phox , a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47 phox to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47phox . In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxiainduced activation of NADPH oxidase and ROS generation in human lung endothelial cells. production that is dependent on NADPH oxidase activation and independent of the mitochondrial electron transport or xanthine/xanthine oxidase systems (10). The mechanisms of NADPH oxidase activation are complex. In phagocytes, activation of NADPH oxidase requires serine phosphorylation of the cytosolic p47 phox , p67 phox , and p40 phox components, assembly of the phosphorylated subunits with Rac2, and translocation to the phagosomes for association with cytochrome b 558. Here, one-electron reduction of molecular O 2 to O 2 . occurs with NADPH as the electron donor (7). In leukocytes, formyl-Met-Leu-Phe-OH or phorbol ester stimulates phosphorylation of p47 phox at multiple serine residues through reactions involving several protein kinases such as protein kinase C, protein kinase A, and mitogen-activated protein kinases (14 -17). In HPAECs, tumor necrosis factor-␣-medi-*This work was supported by National Institutes of Health Grants RO1 HL 69909 (to V. N.), PO1 HL 58064 (to V. N. and J. G. N. G.) and DE13079-01 and AI061042 (to L. H. R.). The costs of publication...
Upon activation with various noncytokine stimuli, polymorphonuclear leukocytes (PMNs) mobilize intracellular sialidase to the plasma membrane, where the sialidase releases sialic acid from the cell surface. This desialylation enhances PMN adherence, spreading, deformability, and motility, functions critical to diapedesis. We now have examined the role of sialidase activity in PMN adhesion to and migration across the endothelium in vivo. A polyclonal antibody prepared against Clostridium perfringens neuraminidase 1) detected surface expression of sialidase on human PMNs stimulated with IL-8 in vitro and on murine PMNs stimulated in vivo, but not on that of unstimulated cells, 2) recognized proteins in human PMN lysates and granule preparations that were not detected by preimmune antibody, 3) inhibited bacterial neuraminidase and human PMN sialidase activities in vitro, and 4) inhibited both pulmonary leukostasis in mice systemically infused with cobra venom factor and intrapulmonary transendothelial migration of PMNs into the bronchoalveolar compartment of mice intranasally challenged with interleukin-8. We conclude that the chemokine interleukin-8, like other PMN agonists, induces the translocation of sialidase to the PMN surface and that surface expression of this sialidase is a prerequisite to PMN recruitment in vivo. The ability of antibodies raised against a prokaryotic neuraminidase to recognize eukaryotic sialidase extends the concept of the neuraminidase superfamily to mammalian enzymes. Inhibition of mobilized endogenous sialidase may provide a novel strategy for limiting the inflammatory response.
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