Perfluorochemical liquid-adenovirus suspensions enhance gene delivery to the distal lung.

WE COMPARED LUNG DELIVERY METHODS OF RECOMBINANT ADENOVIRUS (RAD): (1) rAd suspended in saline, (2) rAd suspended in saline followed by a pulse-chase of a perfluorochemical (PFC) liquid mixture, and (3) a PFC-rAd suspension. Cell uptake, distribution, and temporal expression of rAd were examined using A549 cells, a murine model using luciferase bioluminescence, and histological analyses. Relative to saline, a 4X increase in transduction efficiency was observed in A549 cells exposed to PFC-rAd for 2-4 h. rAd transgene expression was improved in alveolar epithelial cells, and the level and distribution of luciferase expression when delivered in PFC-rAd suspensions consistently peaked at 24 h. These results demonstrate that PFC-rAd suspensions improve distribution and enhance rAd-mediated gene expression which has important implications in improving lung function by gene therapy.

IL-10 modulates placental responses to TLR ligands.

PROBLEM: Intra-uterine infections increase production of pro-inflammatory cytokines. It is unclear whether different infectious agents determine the relative expression of pro-and anti-inflammatory cytokines. METHODS OF STUDY: We compared the placental inflammatory response induced by bacterial lipopolysaccharide (LPS, endotoxin from Gram-negative bacteria) with those induced by lipoteichoic acid (LTA, a cell wall component of Gram-positive bacteria). Placental explants from term delivery were treated with either LPS or LTA, in the presence or absence of IL-10, for 24 hrs. Cytokines, prostaglandin E(2) (PGE(2)) production and cyclo-oxygenase-2 (COX-2) expression were quantified. RESULTS: Both LTA and LPS significantly induced several cytokines with LPS eliciting more potent effects. IL-6 and IL-8 were induced to comparable levels in response to both LTA and LPS whereas monocyte chemotactic protein-1 (MCP-1) production was induced more by LTA, demonstrating a differential placental response to a specific toll-like receptor (TLR) ligand. IL-10 treatment significantly reduced most pro-inflammatory cytokines as well as PGE(2) induced by both LPS and LTA. Interestingly, IL-10 down-regulated LTA-mediated MCP1 induction, but not that mediated by LPS. Moreover, IL-10 was more effective in down-regulating PGE(2) after LPS- when compared with LTA stimulation. CONCLUSIONS: Our results demonstrate that placental exposure to LTA and LPS appear to trigger distinct cytokine responses that can be modulated by IL-10.

Superoxide dismutase attenuates hyperoxia-induced interleukin-8 induction via AP-1.

Exposure of lung epithelial cells to hyperoxia results in the generation of excess reactive oxygen species (ROS), cell damage, and production of proinflammatory cytokines (interleukin-8; IL-8). Although activation of the NF-kappaB and c-Jun N-terminal kinase (JNK)/activator protein (AP)-1 transcription pathways occurs in hyperoxia, it is unclear whether activation of the AP-1 pathway has a direct impact on IL-8 production and whether overexpression of superoxide dismutase (SOD) can mitigate these proinflammatory processes. A549 cells were exposed to 95% O(2), and ROS production, AP-1 activation, and IL-8 levels were determined. Experimental groups included cells transduced with a recombinant adenovirus encoding CuZnSOD or MnSOD (two- to threefold increased activity) or transfected with a JNK1 small interfering RNA (RNAi). Hyperoxia resulted in significant increases in ROS generation, AP-1 activation, and IL-8 production, which were significantly attenuated by overexpression of either MnSOD or CuZnSOD. JNK1 RNAi also moderated IL-8 induction. The data indicate that activation of JNK1/AP-1 and subsequent IL-8 induction in hyperoxia are mediated by intracellular ROS, with SOD having significant protective effects.

Pseudomonas aeruginosa induces localized immunosuppression during pneumonia.

Hospital-acquired bacterial pneumonia is a common and serious complication of modern medical care. Many aspects of such infections remain unclear, including the mechanisms by which invading pathogens resist clearance by the innate immune response and the tendency of the infections to be polymicrobial. Here, we used a mouse model of infection to show that Pseudomonas aeruginosa, a leading cause of hospital-acquired pneumonia, interferes with the ability of recruited phagocytic cells to eradicate bacteria from the lung. Early in infection, phagocytic cells, predominantly neutrophils, are recruited to the lungs but are incapacitated when they enter the airways by the P. aeruginosa toxin ExoU. The resulting paucity of functioning phagocytes allows P. aeruginosa to persist within the lungs and results in local immunosuppression that facilitates superinfection with less-pathogenic bacteria. Together, our results provide explanations for previous reports linking ExoU-secreting P. aeruginosa with more severe pulmonary infections and for the tendency of hospital-acquired pneumonia to be polymicrobial.

Antioxidants improve antibacterial function in hyperoxia-exposed macrophages.

Hyperoxia and pulmonary infections are well known to increase the risk of acute and chronic lung injury in newborn infants, but it is not clear whether hyperoxia directly increases the risk of pneumonia. The purpose of this study was to examine: (1) the effects of hyperoxia and antioxidant enzymes on inflammation and bacterial clearance in mononuclear cells and (2) developmental differences between adult and neonatal mononuclear cells in response to hyperoxia. Mouse macrophages were exposed to either room air or 95% O2 for 24 h and then incubated with Pseudomonas aeruginosa. After 1 h, bacterial adherence, phagocytosis, and macrophage inflammatory protein (MIP)-1alpha production were analyzed. Bacterial adherence increased 5.8-fold (p < 0.0001), phagocytosis decreased 60% (p < 0.05), and MIP-1alpha production increased 49% (p < 0.05) in response to hyperoxia. Overexpression of MnSOD or catalase significantly decreased bacterial adherence by 30.5%, but only MnSOD significantly improved bacterial phagocytosis and attenuated MIP-1alpha production. When monocytes from newborns and adults were exposed to hyperoxia, phagocytosis was impaired in both groups. However, adult monocytes were significantly more impaired than neonatal monocytes. Data indicate that hyperoxia significantly increases bacterial adherence while impairing function of mononuclear cells, with adult cells being more impaired than neonatal cells. MnSOD reduces bacterial adherence and inflammation and improves bacterial phagocytosis in mononuclear cells in response to hyperoxia, which should minimize the development of oxidant-induced lung injury as well as reducing nosocomial infections.

Superoxide dismutase improves oxygenation and reduces oxidation in neonatal pulmonary hypertension.

RATIONALE: Hyperoxic ventilation in the management of persistent pulmonary hypertension of the newborn (PPHN) can result in the formation of reactive oxygen species, such as superoxide anions, which can inactivate nitric oxide (NO) and cause vasoconstriction and oxidation. OBJECTIVE: To compare the effect of intratracheal recombinant human superoxide dismutase (rhSOD) and/or inhaled NO (iNO) on systemic oxygenation, contractility of pulmonary arteries (PAs), and lung reactive oxygen species (isoprostane, 3-nitrotyrosine) levels in neonatal lambs with PPHN. METHODS: Six newborn lambs with PPHN (induced by antenatal ductal ligation) were killed at birth. Twenty-six PPHN lambs were ventilated for 24 h with 100% O(2) alone (n = 6) or O(2) combined with rhSOD (5 mg/kg intratracheally) at birth (n = 4), rhSOD at 4 h of age (n = 5), iNO (20 ppm, n = 5), or rhSOD + iNO (n = 6). Contraction responses of fifth-generation PAs to norepinephrine and KCl, lung isoprostane levels, and 3-nitrotyrosine fluorescent intensity were measured. RESULTS: Systemic oxygenation was impaired in PPHN lambs and significantly improved (up to threefold) in both rhSOD groups with or without iNO. Oxygenation improved more rapidly with the combination of rhSOD + iNO compared with either intervention alone. Norepinephrine- and KCl-induced contractions and lung isoprostane levels were significantly increased by 100% O(2) compared with nonventilated newborn lambs with PPHN. Both rhSOD and iNO mitigated the increased PA contraction response and lung isoprostane levels. Intratracheal rhSOD decreased the enhanced lung 3-nitrotyrosine fluorescence observed with iNO therapy. CONCLUSION: Intratracheal rhSOD and/or iNO rapidly increase oxygenation and reduce both vasoconstriction and oxidation in newborn lambs with PPHN. This has important implications for clinical trials of rhSOD and iNO in newborn infants with PPHN.

Perfluorochemical liquids enhance delivery of superoxide dismutase to the lungs of juvenile rabbits.

Previous studies suggest acute lung injury (ALI) in premature newborns is associated with relative deficiency of antioxidant enzymes that may be ameliorated by recombinant human superoxide dismutase (rhSOD). Perfluorochemicals (PFCs) are distributed homogeneously and support gas exchange in diseased lungs. We investigated whether PFCs could provide an effective delivery system for rhSOD. Juvenile rabbits were lung-lavaged, treated with surfactant, and randomized: group I: fluorescently labeled rhSOD (5 mg/kg in 2 mL/kg saline); group II: fluorescently labeled rhSOD (5 mg/kg in 18 mL/kg PFC). Animals were ventilated with oxygen for 4 h; the lungs were harvested for analysis of SOD distribution and oxidative injury. Cardiopulmonary indices remained stable and similar between groups. Qualitative assessment (QA) showed a more homogeneous lung SOD distribution in group II and a better histologic profile. QA of lung SOD distribution showed significant increase in SOD concentrations in group II (7.37 +/- 1.54 microg/mg protein) compared with group I (1.65 +/- 0.23 microg/mg protein). Oxidative injury as assessed by normalized protein carbonyl was 149.1 +/- 26.8% SEM in group II compared with 200.5 +/- 7.3% SEM in group I. Plasma SOD was significantly higher in group II. Administration of rhSOD with or without PFCs does not compromise cardiovascular function or impede lung recovery after ALI. PFCs enhance rhSOD delivery to the lungs by 400% while decreasing lung oxidative damage by 25% compared with rhSOD alone. These data suggest that PFCs optimize lung rhSOD delivery and might enhance the beneficial effects of rhSOD in preventing acute and chronic lung injury.

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells.

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.

Effects of transgene expression of superoxide dismutase and glutathione peroxidase on pulmonary epithelial cell growth in hyperoxia.

Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50-300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O(2) for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells (P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD (P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.

Superoxide dismutase moderates basal and induced bacterial adherence and interleukin-8 expression in airway epithelial cells.

Bacterial infection of the tracheobronchial tree is a frequent, serious complication in patients receiving treatment with oxygen and mechanical ventilation, resulting in increased morbidity and mortality. Using human airway epithelial cell culture models, we examined the effect of hyperoxia on bacterial adherence and the expression of interleukin-8 (IL-8), an important mediator involved in the inflammatory process. A 24-h exposure to 95% O(2) increased Pseudomonas aeruginosa (PA) adherence 57% in A549 cells (P < 0.01) and 115% in 16HBE cells (P < 0.01) but had little effect on Staphylococcus aureus (SA) adherence. Exposure to hyperoxia, followed by a 1-h incubation with SA, further enhanced PA adherence (P < 0.01), suggesting that hyperoxia and SA colonization may enhance the susceptibility of lung epithelial cells to gram-negative infections. IL-8 expression was also increased in cells exposed to both hyperoxia and PA. Stable or transient overexpression of manganese superoxide dismutase reduced both basal and stimulated levels of PA adherence and IL-8 levels in response to exposure to either hyperoxia or PA. These data indicate that hyperoxia increases susceptibility to infection and that the pathways are mediated by reactive oxygen species. Therapeutic intervention strategies designed to prevent accumulation of intracellular reactive oxygen species may reduce opportunistic pulmonary infections.

In vitro activity and synergy of bismuth thiols and tobramycin against Burkholderia cepacia complex.

OBJECTIVES: To determine the susceptibility of Burkholderia multivorans and Burkholderia cenocepacia to bismuth-thiols (BTs), and to examine the synergistic effects of tobramycin and subinhibitory concentrations of BTs against these organisms. METHODS: The susceptibilities of 25 clinical isolates each of B. multivorans and B. cenocepacia to six BTs were measured by broth dilution in accordance with NCCLS protocols. Ten strains were selected to evaluate the antimicrobial interaction between BTs and tobramycin. Fractional inhibitory concentration (FIC) and fractional bactericidal concentration (FBC) indices were calculated to assess synergy. RESULTS: B. multivorans and B. cenocepacia showed a wide range of susceptibilities to BTs. Bismuth ethanedithiol (BisEDT) was one of the more potent BTs against these organisms (MIC50 7.8 microM), and was selected for synergy studies. Selected strains were highly resistant to tobramycin. The addition of subinhibitory concentrations of BisEDT (2 microM) reduced the MIC and MBC of tobramycin against all strains, achieving synergy in many instances. The FIC index was in the range 0.28-0.66 and the FBC in the range 0.12-0.85. Most strains became susceptible to tobramycin at clinically achievable concentrations in the presence of non-toxic BisEDT levels. CONCLUSIONS: Treatment with subinhibitory BisEDT and tobramycin reduces the MICs and MBCs for B. multivorans and B. cenocepacia. BTs may represent an important adjunctive therapy for resistant Burkholderia cepacia complex.

Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury.

Oxidant insults can lead to apoptotic and nonapoptotic cell death. Lung epithelial cells exposed to high levels of oxygen do not die via apoptosis, but through a much slower, morphologically distinct process involving cell and nuclear swelling. In contrast, H2O2 induces a rapid apoptotic cell death. We first assessed the effect of oxidant exposure on activator protein-1 (c-Jun and Fos) and c-Jun N-terminal kinase (JNK) regulation in MLE12 cells. Both oxidants induced c-Jun and Fos expression, albeit with a different pattern of regulation-hyperoxia (95% O2) induced a biphasic response, whereas H2O2 (500 microM) induced a sustained response. We then examined the role of JNK by Western blot, JNK activity assay, and a pull-down assay and observed an identical pattern of regulation. To assess whether JNK functions in a pro-death or pro-survival capacity, we generated stable cell lines that constitutively express a dominant-negative mutation of JNK resulting in significant inhibition of JNK activity. Inhibition of the JNK pathway in this manner prevented hyperoxic and H2O2-induced cell death. These results demonstrate that hyperoxic cell death is pathway-driven and that both modes of death involve the JNK signaling pathway.

Suppression of nuclear factor-kappa B activity by nitric oxide and hyperoxia in oxygen-resistant cells.

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.

Effects of variable concentrations of inhaled nitric oxide and oxygen on the lungs of newborn piglets.

We previously demonstrated that inhalation of high concentrations of nitric oxide (iNO) and oxygen for 48 hr causes significant lung injury in newborn piglets. To determine if these effects persist at lower concentrations, groups of newborn piglets were mechanically ventilated for 48 hr with (study 1) constant O(2) (90-100%) and decreasing iNO (100-2 ppm) or (study 2) constant iNO (50 ppm) and decreasing O(2) (95-30%). Bronchoalveolar lavage (BAL) fluid was assayed for surfactant function, and markers of lung inflammation and physiologic parameters were monitored. Neutrophil chemotactic activity (NCA), % neutrophils, and total protein (TP) concentrations decreased significantly in BAL fluid of study 1 piglets as iNO was reduced and inhaled oxygen fraction remained constant, indicating less pulmonary injury at low iNO levels. Low-dose iNO (2 ppm) did not have antiinflammatory effects. However, surfactant function was minimally affected by lowering iNO, and was abnormal in all groups. In contrast, in study 2, pulmonary inflammation and injury were lower when O(2) was decreased to 70% or less, with iNO constant at 50 ppm. Surfactant function normalized and oxygenation improved in study 2 piglets when the inhaled oxygen fraction was decreased and iNO remained constant. These data suggest that iNO- and O(2)-induced lung injury may be minimized by weaning O(2) or iNO, although better physiologic function may be obtained when iNO concentrations are constant and O(2) is reduced. This has important implications in the clinical management of critically ill newborns treated with O(2) and iNO for pulmonary disorders.

Distinct patterns of apoptosis in the lung during liquid ventilation compared with gas ventilation.

To determine whether liquid ventilation (LV) causes less cell injury and improves lung function compared with conventional gas ventilation (GV), we analyzed pulmonary physiological profiles, lung histology, and cell death in 110- and 120-day preterm lambs, which were randomized to receive either ventilation modality on FI(O(2)) = 1. LV lungs were well expanded with adequate pulmonary function, whereas GV animals exhibited marked atelectasis, poor pulmonary function, and increased mortality. Both ventilatory strategies induced marked lung cell apoptosis, but with distinct patterns of distribution. Although GV induced apoptosis of epithelium primarily in the lining and within the lumina of bronchioles, LV induced significant apoptosis much more homogeneously throughout lung parenchyma including alveoli and interstitial spaces. These studies suggest that although both forms of ventilation cause regional apoptosis, LV more effectively delivers oxygen and recruits the lung more homogeneously than GV.

Subinhibitory bismuth-thiols reduce virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common pathogen in mechanically ventilated patients and produces a wide array of virulence factors. Bismuth-thiols (BTs) are active in vitro against all bacterial lung pathogens, including P. aeruginosa. The objective of these studies was to examine the biochemical and morphologic effects of sublethal BT concentrations on P. aeruginosa and to evaluate virulence in cell culture. Bismuth-dimercaprol, at a fraction of the minimal inhibitory concentration, reduced alginate expression by 67% in P. aeruginosa, whereas subinhibitory bismuth-ethanedithiol (BisEDT) reduced alginate by 92% in P. syringae. BisEDT effects on lipopolysaccharide content and type III secreted cytoxins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subinhibitory BisEDT reduced cell-associated lipopolysaccharide, and inhibited processing of the secreted cytotoxic protein ExoU. BisEDT-induced outer membrane blebbing and aggregation of cytoplasmic material was noted in electron microscopy. Virulence of P. aeruginosa was assessed by adherence to epithelial cells and sensitivity to serum killing. BisEDT inhibited adherence of P. aeruginosa to 16HBE14o- cells by 28% and to a collagen matrix by 53%. BisEDT-treated bacteria were also 100-fold more sensitive to serum bactericidal activity. In summary, low BT concentrations affect P. aeruginosa in a variety of ways, the combination of which may help prevent or resolve respiratory tract infection.