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PB125® is a phytochemical composition providing potent Nrf2 activation as well as a number of direct actions that do not involve Nrf2. Nrf2 is a transcription actor that helps maintain metabolic balance by providing redox-sensitive expression of numerous genes controlling normal day-to-day metabolic pathways. When ordinary metabolism is upset by extraordinary events such as injury, pathogenic infection, air or water pollution, ingestion of toxins, or simply by the slow but incessant changes brought about by aging and genetic variations, Nrf2 may also be called into action by the redox changes resulting from these events, whether acute or chronic. A complicating factor in all of this is that Nrf2 levels decline with aging, leaving the elderly less able to maintain proper redox balance. The dysregulated gene expression that results can cause or exacerbate a wide variety of pathological conditions, including susceptibility to viral infections. This review examines the characteristics desirable in Nrf2 activators that have therapeutic potential, as well as some of the patterns of dysregulated gene expression commonly observed during pulmonary infections and the normalizing effects possible by judicious use of phytochemicals to increase the activation level of available Nrf2., Graphical abstract Image 1

Helianthemum nummularium is a European shrub growing at high altitude where it copes with a high level of stress. It was found to be overexpressed in ungulates diets compared to more abundant surrounding plants. These elements combined with the fact that H. nummularium from the Alps has never been investigated prompted us to study the phytochemical composition of its aerial parts. The analysis of the polar extract allowed for the isolation of eight compounds: p-hydroxybenzoic acid, tiliroside, kaempferol, astragalin, quercetin, plantainoside B, quercetin-3-O-glucoside, and quercetin-3-O-glucuronide. We investigated the effect of the polar extract and isolated compounds on nuclear factor erythroid 2-related factor 2 transcription factor, which regulates the expression of a wide variety of cytoprotective genes. We found that the ethanolic extract activates the expression of nuclear factor erythroid 2-related factor 2 in a dose-dependent manner, whereas the pure compounds were much less active. The activation of the nuclear factor erythroid 2-related factor 2 pathway by the plant extract could pave the way for studies to promote healthy aging through protection of cells against oxidative stress. Moreover, the isolated compounds could be investigated alone or in combination in the perspective of making the link between the ungulateʼs preference for this plant and possible use of it for self-medication.

There has been a long history of human usage of the biologically-active phytochemicals in

Chronic HIV infection in the era of anti-retroviral therapy is associated with dramatically increased risk of developing severe cardio pulmonary disease. Common to these diseases is increased oxidative burden and chronic inflammation despite low viremia and restoration of CD4+ T-cell levels. Soluble viral factors are heavily implicated in these disease processes, including the HIV Transactivator of Transcription (Tat). Tat is produced in high levels during infection and secreted from infected cells into circulation where it is internalized by bystander cells and is known to regulate inflammatory pathways and elicit a pro-oxidant environment. We have examined the effects of Tat on the anti-oxidant regulatory network driven by the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in primary human pulmonary arterial endothelial cells, which are heavily involved in pathogenesis of HIV associated lung diseases including pulmonary arterial hypertension and COPD. Co-expression of Tat and a luciferase reporter construct driven by the Nrf2 activated anti-oxidant response element (ARE) demonstrated markedly reduced Nrf2/ARE activity, even when stimulated by the potent Nrf2 activating compound PB125. Additionally, Heme-oxygenase-1 (HO-1) transcription was potently repressed by Tat in a cell line as well as primary endothelial cells, and treatment with PB125 failed to restore transcriptional activity. Other anti-oxidant Nrf2 genes examined included NADPH Dehydrogenase Quinone 1 (NQO1) and Sulfiredoxin-1 (SRXN1). NQO1 was repressed basally by Tat, while SRXN1 transcription was refractory to activation by PB125 in the presence of Tat. Lastly, we demonstrated that Tat expressing cells have increased indicators of oxidative stress including elevated production of reactive oxygen species, measured by electron paramagnetic resonance spectroscopy, and increased levels of nitrotyrosine content. These observations suggest a novel mechanism by which HIV Tat increases oxidative burden by dysregulation of the Nrf2/ARE pathway.

Chronic HIV infection causes redox stress and increases the risk of acute and chronic lung injury, even when individuals are adherent to antiretroviral therapy. HIV-1 transgene expression in rats inhibits nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which regulates antioxidant defenses and alveolar epithelial cell (AEC) barrier function, but the mechanism is unknown. In this study, we present novel evidence that these pathological effects of HIV are mediated by microRNA-144 (miR-144). HIV-1 transgene expression in vivo increases the expression of miR-144 in the alveolar epithelium, and this can be replicated by direct exposure of naïve primary AECs to either Tat or gp120 ex vivo. Further, treating naïve primary AECs with a miR-144 mimic decreased the expression and activity of Nrf2 and inhibited their barrier formation. In contrast, treatment with a miR-144 antagomir increased the expression and activity of Nrf2 and improved barrier function in primary AECs isolated from HIV-1 transgenic rats. Importantly, either delivering the miR-144 antagomir intratracheally, or directly activating Nrf2 by dietary treatment with PB123, increased Nrf2 expression and barrier formation in HIV-1 transgenic rat AECs. This study provides new experimental evidence that HIV-induced inhibition of Nrf2 and consequent AEC barrier dysfunction are mediated via miR-144, and that these pathophysiological effects can be mitigated in vivo by either directly antagonizing miR-144 or activating Nrf2. Our findings suggest that targeting the inhibition of Nrf2 in individuals living with HIV could enhance their lung health and decrease the lung-specific morbidity and mortality that persists despite antiretroviral therapy.

Musculoskeletal dysfunction is an age-related syndrome associated with impaired mitochondrial function and proteostasis. However, few interventions have tested targeting two drivers of musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that stimulates transcription of cytoprotective genes and improves mitochondrial function. We hypothesized daily treatment with a Nrf2 activator in Hartley guinea pigs, a model of age-related musculoskeletal dysfunction, attenuates the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis, preserving musculoskeletal function. We treated 2-month- and 5-month-old male and female Hartley guinea pigs for 3 and 10 months, respectively, with the phytochemical Nrf2 activator PB125 (Nrf2a). Longitudinal assessments of voluntary mobility were measured using Any-Maze™ open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30 days of treatment. Mitochondrial oxygen consumption in permeabilized soleus muscles was measured using ex vivo high resolution respirometry. In both sexes, Nrf2a 1) increased electron transfer system capacity; 2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and 3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial, and cytosolic subfractions of the soleus. These improvements were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, these results demonstrate that treatment with an oral Nrf2 activator contributes to maintenance of skeletal muscle mitochondrial function and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these improvements are also accompanied by slowed progression of other aspects of musculoskeletal decline.

Plasma pharmacy is a subset of the broader field of plasma medicine. Although not strictly defined, the term aqueous plasma pharmacy (APP) is used to refer to the generation and distribution of reactive plasma-generated species in an aqueous solution followed by subsequent administration for therapeutic benefits. APP attempts to harness the therapeutic effects of plasma-generated oxidant species within aqueous solution in various applications, such as disinfectant solutions, cell proliferation related to wound healing, and cancer treatment. The subsequent use of plasma-generated solutions in the APP approach facilitates the delivery of reactive plasma species to internal locations within the body. Although significant efforts in the field of plasma medicine have concentrated on employing direct plasma plume exposure to cells or tissues, here we focus specifically on plasma discharge in aqueous solution to render the solution biologically active for subsequent application. Methods of plasma discharge in solution are reviewed, along with aqueous plasma chemistry and the applications for APP. The future of the field also is discussed regarding necessary research efforts that will enable commercialization for clinical deployment.

The growing need for scalable systems that can inactivate microbiological contaminants and recycle water in industrial operations has led to the development of a variety of new advanced oxidation process (AOP) technologies. In this paper, we report on the capability and techno-economics of a new AOP method to generate aqueous plasma species for inhibition of microbiological contaminants. The test microorganisms in this work were Acidithiobacillus ferrooxidans (a motile, Gram-negative bacterium that oxidizes sulfides to sulfates and ferrous iron to ferric iron, used as a model biofouling organism) and Legionella gratiana (a Gram-negative bacteria used as a surrogate of the human pathogen Legionella pneumophila, which can be a dangerous contaminant in cooling water systems). The cultured bacteria were dispersed in water and treated within a non-thermal plasma treatment system for varied exposure times, and then the bactericidal effects were measured. The results demonstrated plasma inhibition of A. ...

The solubility of the vitamin E-related compound α-tocopheryl succinate in supercritical carbon dioxide was measured at pressures ranging from (15.0 to 30.0) MPa and temperatures of (40 and 50) °C using a simple microsampling type apparatus with a 100.5 μL sample loop to remove aliquots and collect them in ethanol for off line analysis. α-Tocopheryl succinate concentrations in the collected samples were measured using HPLC-MS/MS analysis. The solubility of α-tocopheryl succinate in supercritical carbon dioxide ranged from mole fractions of 0.28 × 10(-5) at 15.0 MPa and 50 °C to 2.56 × 10(-5) at 30.0 MPa and 50 °C.

Intrapulmonary administration of bacterial lipopolysaccharide (LPS) induces a well-characterized lung inflammatory response involving alveolar macrophage activation, proinflammatory cytokine elaboration, and neutrophil influx. Vitamin E, a lipophilic antioxidant consisting of a family that includes tocopherols and tocotrienols, has previously been shown to have a variety of anti-inflammatory effects, raising interest in its possible uses in disease prevention or therapy. Because aerosol delivery is a specific and rapid way to administer agents to the lungs, the authors undertook to determine whether inhaled vitamin E aerosols would have an anti-inflammatory effect in the lungs. Using a rat model of acute lung inflammation caused by intratracheally administered LPS (10 microg Pseudomonas aeruginosa LPS), the authors examined the effect of aerosol-administered vitamin E, in this case alpha-tocopherol, on several indices of lung inflammation which are increased by LPS treatment. It was found that inhaled alpha-tocopherol aerosol, but not inhaled alpha-tocopherol acetate aerosol, decreased tumor necrosis factor alpha (TNFalpha) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA levels in lung tissue, TNFalpha and CINC-1 immunoreactive protein levels in lung lavage, and the number of neutrophils recoverable by lung lavage from rats given LPS intratracheally. These results contribute to the increasing body of work describing immunomodulatory functions of alpha-tocopherol, and support the idea that direct aerosol administration of alpha-tocopherol may play a beneficial role in strategies to control inflammatory lung illnesses.

For unknown reasons, serum ferritin levels increase in patients at risk for and with acute lung injury (ALI). To improve understanding of the relationship between serum ferritin alterations and the development of ALI, we investigated the effect of iron deficiency on the serum ferritin response of rats subjected to hemorrhage. We found that rats fed an iron-deficient diet for 6 weeks had decreased hemoglobin, hematocrit, liver total iron, liver total iron-binding capacity, and liver ferritin concentrations but the same serum ferritin concentrations as rats fed a control diet. Following hemorrhage, serum ferritin concentrations increased rapidly and progressively in rats fed a control diet. Along with increases in serum ferritin concentrations, control diet rats subjected to hemorrhage also had increased lung lavage leukocyte numbers, lung myeloperoxidase activities (lung inflammation), and lung lavage protein concentrations (lung leak) compared to control diet fed rats subjected to sham treatment. By comparison, the serum ferritin concentrations, lung inflammation, and lung leak of hemorrhaged rats fed an iron-deficient diet were decreased compared to hemorrhaged rats fed a control diet. These findings indicate that serum ferritin concentrations increase and acute lung injury develops following hemorrhage in rats fed a control, but not an iron-deficient, diet. A relatively brief exposure to an iron-deficient diet reduces hemorrhage-induced ALI.

Interleukin-1 (IL-1) levels and neutrophils are increased in the lung-lavage fluid of patients with acute lung injury (ALI), and instilling IL-1 intratracheally into rats causes rapid lung-neutrophil influx and neutrophil-dependent lung leakage. IL-1 insufflation also increases platelet-activating factor (PAF) activity in rat lung, and PAF is increased in the lung-lavage fluid of ALI patients. To assess the direct effects of PAF on the lung, we administered PAF intratracheally in rats. We found that rats given PAF (5 μg) intratracheally had increased lung nuclear factor-κB activation, myeloperoxidase activity, numbers of lavage neutrophils, lavage neutrophil nitroblue tetrazolium reduction, and leakage compared with sham-treated rats administered saline solution intratracheally. Electron microscopic examination also indicated that lungs from rats given PAF intratracheally had increased neutrophil infiltration, cell damage, and neutrophil-endothelial cell interface cerium chloride staining — a marker of hydrogen peroxide production — compared with sham-treated rats. Simultaneous treatment with a PAF receptor-antagonist, WEB 2086, decreased the aforementioned changes observed after intratracheal administration of PAF. (J Lab Clin Med 2002;140:312-9)

Inhalation therapy for infectious lung diseases, such as tuberculosis, is currently being explored, with microspheres being used to target alveolar macrophages. One method of drug encapsulation into polymeric microspheres to form hydrophobic ion-paired (HIP) complexes, and then coprecipitate the complex and polymer using supercritical fluid methodology. For the potent antituberculosis drug, isoniazid (isonicotinic acid hydrazide, INH), to be used in this fashion, it was modified into an ionizable form suitable for HIP. The charged prodrug, sodium isoniazid methanesulfonate (Na-INHMS), was then ion paired with hydrophobic cations, such as alkyltrimethylammonium or tetraalkylammonium. The logarithms of the apparent partition coefficients (log P') of various HIP complexes of INHMS display a roughly linear relationship with the numbers of carbon atoms in the organic counterions. The water solubility of the tetraheptylammonium-INHMS complex is about 220-fold lower than that of Na-INHMS, while the solubility in dichloromethane exceeds 10 mg/mL, which is sufficient for microencapsulation of the drug into poly(lactide) microspheres. The actual logarithm of the dichloromethane/water partition coefficient (log P) for tetraheptylammonium-INHMS is 1.55, compared to a value of - 1.8 for the sodium salt of INHMS. The dissolution kinetics of the tetraheptylammonium-INHMS complex in 0.9% aqueous solutions of NaCl was also investigated. Dissolution of tetraheptylammonium-INHMS exhibited a first-order time constant of about 0.28 min(-1), followed by a slower reverse ion exchange process to form Na-INHMS. The half-life of this HIP complex is on the order of 30 min, making the enhanced transport of the drug across biological barriers possible. This work represents the first use of a prodrug approach to introduce functionality that would allow HIP complex formation for a neutral molecule.

Iron, through its participation in reactions that generate reactive oxygen species, may contribute to the oxidative lung injury observed in patients with acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). A number of investigators have shown that the endogenous iron storage protein ferritin increases in the blood of patients with and at-risk for ALI and ARDS, but the significance of these increases are not known. In the present investigation, we measured lung tissue levels of thiobarbituric acid reactive substances (TBARS) and lung leak in isolated rat lungs perfused with xanthine oxidase (XO) and purine, an enzymatic system which generates reactive oxygen species. We found that adding ferritin (100 ng/mL) or desferrioxamine (DFO, 10 mM), an iron chelator, to the vascular perfusate solution decreased oxidant-induced leak in isolated rat lungs perfused with XO and purine. Addition of ferritin or DFO also decreased TBARS in isolated rat lungs perfused with XO and purine; neither ferritin nor DFO, however, decreased XO activity in vitro. Our results suggest that oxidative lung leak may be altered by the availability of reactive iron and that ferritin may contribute to protection against oxidative lung injury.

Acute lung injury (ALI) is characterized by increased lung levels of proinflammatory cytokines, inflammation, oxidative stress, edema, and impaired gas exchange. Notably, ALI patients also exhibit pulmonary surfactant abnormalities, including increased levels of phospholipids in their lung lavages. In the present study, to assess early alterations of the lung surfactant system in ALI, we induced inflammation and acute lung injury in rats by administering interleukin-1α (IL-1) intratracheally. Five h after IL-1 instillation, we examined lung tissue ultrastructure by electron microscopy using both routine staining methods and cerium chloride staining to localize hydrogen peroxide (H2O2) histologically. We also measured lung lavage phospholipid levels, lung tissue γ-glutamyl transpeptidase (GGT) activities (a marker of oxidative stress), and arterial blood oxygen tensions. We observed that lungs of rats given IL-1 intratracheally had increased neutrophil accumulation, increased H2O2 production, and increased alveolar type II (ATII) pneumocyte ultrastructural abnormalities compared to rats given saline intratracheally. Intratracheal instillation of IL-1 also increased phospholipid levels in the bronchoalveolar lavage (BAL), possibly as a consequence of the abnormal discharge of lamellar bodies into the alveolar lumen. In addition, IL-1-insufflated rats had increased lung GGT levels and impaired blood oxygenation compared to saline-insufflated rats. Treatment with mepacrine decreased lung neutrophil accumulation, ultrastructural lung abnormalities, lung lavage phospholipid levels, lung tissue GGT levels, and blood oxygenation impairment in rats given IL-1 intratracheally, suggesting a possible relationship between these events. Our results indicate that IL-1-induced acute lung injury in rats is marked by neutrophil-dependent oxidative stress, ATII cell defects, abnormal discharge of lamellar body phospholipids, and impaired blood oxygenation.

Background: Pretreatment with interleukin-1 (1L-1) induces resistance to lung injury from hyperoxia exposure and to cardiac dysfunction after ischemiareperfusion in animal models. In contrast, IL-1 pretreatment did not produce tolerance to ischemiareperfusion injury and did not seem to alter antioxidant enzyme activities in the kidney. Recently, we determined that mitochondria scavenge superoxide anion via a nonenzymatic mechanism and that this newly identified intracellular antioxidant function was inducible in the lung. Based on these observations, we hypothesized that organ differences after IL-1 pretreatment between the lung and the heart, which become tolerant, and the kidney, which does not become tolerant, were a consequence of differential responses in mitochondrial superoxide scavenging. Methods: Rats were given IL-1 a (50 ng intratracheally, 36 hrs before assay) and lung and kidney mitochondria were isolated. Mitochondrial scavenging of superoxide anion was then determined by using an assay that we developed and published previously. We then tested the effects of IL-1 pretreatment on

Because plasminogen activators (PA) may participate in the inflammatory process associated with the acute respiratory distress syndrome (ARDS), we measured the effect of tissue plasminogen activator (tPA) on inflammation and acute lung leak caused by intratracheal instillation of IL-1alpha (50 ng) into male (300-400 g) Sprague-Dawley rats. Lung leak, lung myeloperoxidase (MPO) activity, and lung lavage neutrophil counts were increased in rats given IL-1 intratracheally compared to control rats that were given saline intratracheally. Giving tPA (12 mg/kg) intraperitoneally increased lung tPA concentration and reduced acute lung leak in rats given IL-1 intratracheally (p.01; lung leak index for sham-treated rats: 0.040 + 0.001, n=6; IL-1: 0.10 + 0.01, n=10; tPA + IL-1: 0.050 + 0.002, n=6). In contrast, administering tPA did not change IL-1-induced increases in lung lavage neutrophils or lung MPO activity (sham: 0.003 x 106 + 0.001 x 10(6) cells; IL-1: 2.9 x 10(6) + 0.4 x 10(6) cells; tPA + IL-1: 2.7 x 10(6) + 0.4 x 10(6) cells; and sham: 0.6 + 0.2 U/g lung; IL-1: 11.2 + 2.9 U/g lung, tPA + IL-1: 11.1 + 1.6 U/g lung, respectively). Our results suggest that intraperitoneal tPA administration increases lung tissue tPA levels and decreases acute lung leak without reducing lung neutrophil infiltration in rats given IL-1alpha intratracheally. This work suggests that tPA may suppress neutrophil activation in vivo and accordingly have anti-inflammatory effects.

Hybertson, Brooks M., Roger P. Kitlowski, Eric K. Jepson, and John E. Repine. Supercritical fluid-aerosolized vitamin E pretreatment decreases leak in isolated oxidant-perfused rat lungs. J. Appl. Physiol. 84(1): 263–268, 1998.—We hypothesized that direct pulmonary administration of supercritical fluid-aerosolized (SFA) vitamin E would decrease acute oxidative lung injury. We previously reported that rapid expansion of supercritical CO2formed respirable particles of vitamin E and that administering SFA vitamin E to rats increased lung vitamin E levels and decreased neutrophil-mediated lung leak. In the present investigation, we found that pretreatment with SFA vitamin E protected isolated rat lungs against the oxidant-induced lung leak caused by perfusion with xanthine oxidase (XO) and purine, an enzyme system that generates superoxide anion ([Formula: see text]) and hydrogen peroxide. SFA vitamin E droplets were 0.7–3 μm in diameter, and inhalation of the airborne droplets for 30 min deposited ∼55 μg of vitamin E in rat lungs. Isolated rat lungs perfused with XO (0.02 U/ml) and purine (10 mM) gained more weight (1.75 ± 0.12 g, n = 8), retained more Ficoll (11.5 ± 1.2 mg/left lung, n = 7), and accumulated more Ficoll in their lung lavages (700 ± 146 μg/ml, n = 8) than control lungs [0.25 ± 0.06 g ( n = 10), 6.2 ± 1.2 mg/left lung ( n = 9), and 141 ± 31 μg/ml ( n = 8), respectively, P < 0.05]. In contrast, isolated lungs from rats that were pretreated with SFA vitamin E had decreased ( P < 0.05) weight gains (0.32 ± 0.06 g, n = 7), Ficoll retentions (3.3 ± 1.1 mg/left lung, n = 7), and lung lavage Ficoll concentrations (91 ± 26 μg/ml, n = 6) after perfusion with XO and purine compared with isolated lungs from control rats perfused with XO and purine. This protective effect was not observed in rat lungs given sham treatments (CO2alone or vitamin E acetate aerosolized with supercritical CO2). Our results suggest that direct pulmonary supplementation of vitamin E decreases susceptibility to vascular leakage caused by XO-derived oxidants.

Interleukin-1 (IL-1) and neutrophils are increased in lungs of patients with the acute respiratory distress syndrome (ARDS). We found that rats given IL-1 intratracheally rapidly developed lung neutrophil accumulation and a neutrophil-dependent acute edematous lung leak. Lung leak was associated with increased lung lavage cytokine-induced chemoattractant (CINC) levels and increased oxidative stress that was manifested by increased exhaled H2O2 levels and increased lung oxidized glutathione levels. IL-1-induced lung leak was decreased by treatment with superoxide dismutase (SOD), dimethylsulfoxide (DMSO), supercritical fluid-aerosolized vitamin E, interleukin-1-receptor antagonist (IL-1ra), or liposome-associated PGE1 (Lip-PGE1). Importantly, Lip-PGE1 treatment also reduced ventilator dependence in a small clinical study of ARDS patients. Another series of investigations revealed that IL-1 pretreatment could prevent lung leak in rats given IL-1 intratracheally. These findings point to the possible dual effects of IL-1 with respect to the development of acute lung injury.

We determined that lisofylline, a potent inhibitor of oleate- and linoleate-containing phosphatidic acid formation (half-maximal inhibitory concentration = 40 nM), prevented oxidant-mediated capillary leak in isolated rat lungs given interleukin-8 (IL-8) intratracheally and perfused with human neutrophils. Lung leak was prevented by lung, but not neutrophil, lisofylline pretreatment. Furthermore, although lisofylline inhibited IL-8-stimulated neutrophil production of phosphatidic acid in vitro, it did not prevent IL-8-stimulated neutrophil adherence, chemotaxis, or intracellular calcium mobilization or N-formyl-Met-Leu-Phe (fMLP)-stimulated oxidant production in vitro. Lisofylline also prevented acute capillary leak in isolated rat lungs perfused only with the oxidant generator purine-xanthine oxidase but did not scavenge[Formula: see text] or H2O2in vitro. Finally, lisofylline-mediated protection against lung leak in both models was associated with alterations in lung membrane free fatty acid acyl composition (as reflected by the decreased ratio [linoleate + oleate]/[palmitate]). We conclude that lisofylline prevented both neutrophil-dependent and neutrophil-independent oxidant-induced capillary leak in isolated rat lungs and that protection appears to be mediated by blocking intrinsic lung linoleoyl phosphatidic acid metabolism. We speculate that lisofylline, in addition to our previously reported effects on cytokine signaling by intrapulmonary mononuclear cells, alters intrinsic pulmonary capillary membrane composition and renders this barrier less vulnerable to oxidative damage.

Interleukin-1 (IL-1) is increased in lung lavages obtained from patients with acute respiratory distress syndrome, and administering IL-1 intratracheally to rats causes an acute, neutrophil-dependent, oxidative lung leak. We found that rats given IL-1 intratracheally had increased lung lavage fluid tumor necrosis factor (TNF) levels, and that rats treated with TNF binding protein (TNFbp) intravenously did not develop the increased lung leak that occurs after administration of IL-1 intratracheally. In contrast, rats given IL-1 intratracheally and TNFbp intravenously had the same elevations in lung lavage neutrophil accumulation and lung lavage cytokine-induced neutrophil chemoattractant levels as rats given IL-1 intratracheally. Our results show that TNFbp decreases neutrophil-mediated lung leak, but not lung neutrophil accumulation, after administration of IL-1 intratracheally in rats.

Hybertson, Brooks M., Stuart L. Bursten, Jonathan A. Leff, Young M. Lee, Eric K. Jepson, Chris R. Dewitt, John Zagorski, Hyun G. Cho, and John E. Repine. Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally. J. Appl. Physiol.82(1): 226–232, 1997.—Interleukin-1 (IL-1) is increased in lung lavages from patients with the acute respiratory distress syndrome, and administering IL-1 intratracheally causes neutrophil accumulation and a neutrophil-dependent oxidative leak in lungs of rats. In the present study, we found that rats pretreated intraperitoneally with lisofylline [( R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species, did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1. However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbers of lung lavage neutrophils as rats given IL-1 intratracheally. Lungs of rats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment with LSF. Our results reveal that LSF decreases lung leak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

We investigated the effects of N-acetylcysteine (NAC) pretreatment on paraquat-induced lung inflammation and leak. We found that administering a single intravenous dose (60 mg/kg) of paraquat rapidly (2 h) increased lung leak, lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, and lung myeloperoxidase (MPO) activity in rats. Rats pretreated with NAC (150 mg/kg, intraperitoneally) had increased lung tissue glutathione (GSH + GSSG) levels compared to saline-pretreated rats. In addition, rats pretreated with NAC and then given paraquat 2.5 h later had decreased lung leak compared to saline-pretreated rats given paraquat. In contrast, NAC pretreated rats given paraquat had the same lung lavage CINC levels and lung tissue MPO activity as saline-pretreated rats given paraquat. Our results indicate that paraquat causes an oxidative injury which may be decreased by the GSH-increasing or other properties of NAC.

We found that ventilation with nitric oxide (NO, 50 ppm) significantly (P < 0.05) reduced capillary leak (as reflected by weight gain and Ficoll retention) in isolated rat lungs perfused for 60 min with N-formyl-methionyl-leucyl-phenylalanine (fMLP; 10(-7) M) and human neutrophils (1,300/microliters). Perfusion with previously heated neutrophils (48 degrees C for 10 min, which inactivates NADPH oxidase) did not cause weight gain or Ficoll retention, indicating that neutrophil-derived oxidants mediated lung leak. Although perfusion with fMLP and neutrophils increased mean pulmonary artery pressures (PAP) from 7 to 11.7 +/- 0.5 mmHg at 10 min, lungs perfused with fMLP and neutrophils in which PAP was maintained at 7 mmHg by reducing perfusion flow rates also developed significant (P < 0.05) weight gain and Ficoll retention. Furthermore, inhaled NO did not reduce (P > 0.05) PAP at 10 min and only modestly reduced PAP at 30 and 60 min of perfusion. Our results suggest that oxidative endothelial damage, and not increased hydrostatic pressure, was the primary cause of the capillary leak, and that the protection provided by inhaled NO was not solely a consequence of vasodilation. We conclude that inhaled NO prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs, and speculate that inhaled NO has anti-inflammatory properties in addition to its ability to cause pulmonary vasodilation.

A flow injection method is described for the determination of low levels of nitrite ion in cell culture solutions. Nitrite-containing solutions are injected into a flowing stream containing phosphoric acid and iodide ion, which reduces nitrite to nitric oxide. The solution is directed through a microporous tube out of which nitric oxide gas diffuses for detection by chemiluminescence. The method has been applied to analysis of nitrite generated by cultured bovine pulmonary artery endothelial cells and human neutrophils.

For the past 40 years or so, oxidative stress has been increasingly recognized as a contributing factor in aging and in various forms of pathophysiology generally associated with aging. Our view of oxidative stress has been largely "superoxide-centric", as we focused on the pathological sources of this oxygen-derived free radical and the types of molecular havoc it can wreak, as well as on the protection provided by the antioxidant enzymes, especially the superoxide dismutases, catalases, and glutathione peroxidases. In the last decade our view of oxidative stress has broadened considerably, and it is now often seen as an imbalance that has its origins in our genes, and the ways in which gene expression is regulated. At the center of this new focus is the transcription factor called nuclear factor (erythroid-derived 2)-like 2, or Nrf2. Nrf2 is referred to as the "master regulator" of the antioxidant response, modulating the expression of hundreds of genes, including not only the familiar antioxidant enzymes, but large numbers of genes that control seemingly disparate processes such as immune and inflammatory responses, tissue remodeling and fibrosis, carcinogenesis and metastasis, and even cognitive dysfunction and addictive behavior. Thus, the dysregulation of Nrf2-regulated genes provides a logical explanation for the connections, both direct and indirect, between observable oxidative stress and perhaps 200 human diseases involving these various physiological processes, each reflecting a network involving many gene products. The evolutionary self-association of these many genes under the common control of Nrf2 suggests that the immune and inflammatory systems may present the largest demand for increased antioxidant protection, apart from constitutive oxidative stress resulting from mitochondrial oxygen consumption for metabolic purposes. Gene expression microarray data on human primary vascular endothelial cells and on the SK-N-MC human neuroblastoma-derived cell line have been obtained in response to the dietary supplement Protandim, a potent composition of highly synergistic phytochemical Nrf2 activators. Pathway analysis of results shows significant modulation by Protandim of pathways involving not only antioxidant enzymes, but of those related to colon cancer, cardiovascular disease, and Alzheimer disease.

Better methods for delivery of pharmacologic agents directly to and via the lung are needed. We found that a solution of α-tocopherol (vitamin E) dissolved in supercritical carbon dioxide ...

Palladium was pyrolytically deposited by a supercritical fluid transport-chemical deposition method using the metal β-diketonate complex bis-(2,2,7-trimethyl-3,5-octanedionato)palladium(II). Deposition at a rate of 95 nm/min occurred at a substrate temperature of 740 °C. X-ray diffraction and electron microprobe analyses of the reflective films that were deposited on fused silica substrates showed that they contained polycrystalline palladium. The films were electrically conductive, but conductivity was substantially lower than that for bulk palladium. X-ray diffraction of the films deposited on (100) Si substrates showed that palladium silicide was formed.

Nitrogen dioxide was used to oxidize nonsuperconducting, powdered YBa2Cu3O6.0 to form a material that exhibits the Meissner effect at liquid nitrogen temperature. Nitrogen dioxide treatment at 200 °C for 70 h results in a mixed-phase material containing superconducting YBa2Cu3O6.9 that exhibits onset of superconductivity at 91.5 K and a midpoint at 88 K. During a 70-h exposure of YBa2Cu3O6 to NO2 at 200 °C, a mixture of oxidation and degradation products is formed, including YBa2Cu3O6.9 (61% by weight) and Ba(NO3)2 (23% by weight). Heating the NO2-treated product to 900 °C results in the evolution of NO, NO2, and O2; cooling the sample and annealing at 400 °C in an O2 stream generates the superconducting oxide.

The solubility of the sesquiterpene alcohol patchoulol in supercritical carbon dioxide was measured at pressures (P) ranging from 10.0 MPa to 25.0 MPa and temperatures (T) of 40.0 and 50.0 °C using a simple microsampling-type apparatus with a 100.5 μL sample loop to remove aliquots for off-line analysis. The system was first validated using vanillin with off-line spectrophotometric analysis, then utilized for patchoulol measurements with off-line GC−MS analysis. The measured solubility of patchoulol in supercritical CO2 ranged from mole fractions of 0.43 × 10-3 at 10.0 MPa and 50.0 °C to mole fractions of 9.4 × 10-3 at 25.0 MPa and 40.0 °C.

To evaluate if pulmonary delivery of microparticles loaded with a prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), can target alveolar macrophages (AM) and reduce metabolism of INH, an HPLC-MS/MS assay with automated online extraction for quantification of INH and its metabolite acetylisoniazid (AcINH) in plasma and AMs was developed and validated. Reproducibility in rat plasma and homogenate of a rat AM cell line, NR8383, for INH and AcINH showed excellent precision and accuracy with calibration curves exhibiting linearity within a range of 1-250ng/ml of INH and 0.05-50ng/ml of AcINH (r(2)>0.99). The validated methods were successfully applied to pharmacokinetic study of INHMS-loaded microparticles in rats, demonstrating efficient targeting of AMs and reduction of INH metabolism.

Microparticles prepared by the precipitation with a compressed antisolvent (PCA) process were evaluated for their potential in targeting an ionizable prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), for sustained delivery of INH to alveolar macrophages (AMs). The charged prodrug was ion-paired with two different hydrophobic cations (tetrapentylammonium (TPA)- and tetraheptylammonium (THA)-bromide), and loaded separately into the poly(l-lactide) (PLA) microparticles. The drug/polymer particles were spherical in shape and between 1 and 3 mum in diameter. The choice of hydrophobic cations did not affect drug incorporation efficiencies or the release kinetics of INH from the microparticles. Using a sensitive liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay developed for INH, high level of INH was detected in NR8383, a rat AM cell line, following exposure of these cells to drug-loaded microparticles. To confirm the microparticles can target AMs in vivo, we compared the INH levels in lavaged bronchoalveolar macrophages by LC-MS/MS after the Sprague-Dawley rats were administered either INHMS in PLA microparticles by intra-tracheal instillation or INH solution by gavage or intra-tracheal instillation. As expected, only microparticles provided sustained and targeted delivery of INH to AMs. Most importantly, this method of delivery led to substantial reduction in the blood levels of acetylisoniazid (AcINH), a major and potential toxic metabolite of INH.

This study was conducted to assess the feasibility of a pulmonary formulation of tissue plasminogen activator (tPA) for nebulization into the airway by measuring protein stability, biologic activity, particle size, and estimating human lung distribution.Formulations were derived by varying the surfactant and protein concentrations. Protein stability and recovery of each nebulized tPA formulation were assessed by ultraviolet spectroscopy. Formulations that met protein stability feasibility criteria were assessed for biologic and fibrinolytic activities. Biologic activity was determined by their ability to inhibit superoxide anion production by human neutrophils. Fibrinolytic activity was assessed by the cleavage of plasminogen to plasmin. Aerodynamic properties were assessed using a cascade impactor, and an estimation of human airway deposition was made via a human lung replica.Twenty-seven tPA formulations were initially assessed, 15 of which met protein stability criteria. Subsequently, three of these formulations maintained biologic and fibrinolytic activities. These formulations exhibited particle sizes of 2.4-3.1 microm, and had respirable dosesor =65%. A formulation of 1mg mL(-1) tPA and 0.1% Tween 80 exhibited a 45% deposition in the lower airways of a human lung replica.A suitable pulmonary tPA formulation was identified that, following nebulization, maintained protein stability as well as biologic and fibrinolytic activities, and resulted in an optimal respirable dose and human airway deposition. This formulation may be applicable in the treatment of lung diseases, such as acute respiratory distress syndrome by permitting targeted pulmonary delivery of a therapeutic protein to the lungs.

Ferritin regulates iron levels and, for unknown reasons, serum ferritin concentrations are increased in patients at risk for and with acute lung injury (ALI) and multiple organ failure. Uncomplexed iron could exacerbate the toxicity of the increased oxidative stress that occurs in patients with ALI and multiple organ failure and thereby contribute to disease. In the present investigation, the authors found that serum and lung lavage ferritin concentrations increased in hemorrhaged rats that develop ALI as manifested by increased lung inflammation (increased lung lavage leukocyte counts and lung myeloperoxidase activities) and increased lung leak (increased lung lavage protein concentrations). Treatment with mepacrine, a phospholipase A2 inhibitor, attenuated the increases in serum and lung lavage ferritin concentrations, lung inflammation, and lung leak that occur in rats subjected to hemorrhage. The findings show that serum and lung ferritin levels increase and may play a role in the development of acute lung injury caused by hemorrhage.

For unknown reasons, serum ferritin levels increase in patients at risk for and with acute lung injury (ALI). To improve understanding of the relationship between serum ferritin alterations and the development of ALI, we investigated the effect of iron deficiency on the serum ferritin response of rats subjected to hemorrhage. We found that rats fed an iron-deficient diet for 6 weeks had decreased hemoglobin, hematocrit, liver total iron, liver total iron-binding capacity, and liver ferritin concentrations but the same serum ferritin concentrations as rats fed a control diet. Following hemorrhage, serum ferritin concentrations increased rapidly and progressively in rats fed a control diet. Along with increases in serum ferritin concentrations, control diet rats subjected to hemorrhage also had increased lung lavage leukocyte numbers, lung myeloperoxidase activities (lung inflammation), and lung lavage protein concentrations (lung leak) compared to control diet fed rats subjected to sham treatment. By comparison, the serum ferritin concentrations, lung inflammation, and lung leak of hemorrhaged rats fed an iron-deficient diet were decreased compared to hemorrhaged rats fed a control diet. These findings indicate that serum ferritin concentrations increase and acute lung injury develops following hemorrhage in rats fed a control, but not an iron-deficient, diet. A relatively brief exposure to an iron-deficient diet reduces hemorrhage-induced ALI.

Interleukin-1 (IL-1) is increased in lung lavages obtained from patients with acute lung injury (ALI) and administering recombinant human IL-1alpha (rhIL-1alpha) (50 ng) intratracheally causes an acute, neutrophil-dependent, oxidative lung leak in rats that closely resembles human ALI. In the present work, the authors tested the hypothesis that transforming growth factor beta (TGFbeta) contributes to the lung inflammation and injury that develops in rats given IL-1 intratracheally. They found that intravenous administration of a monoclonal antibody to TGFbeta (1.D.11.16, 0.5 mg/kg) attenuated lung injury responses, specifically lung leak index, lung lavage protein concentrations, and blood oxygenation abnormalities, that are observed 5 hours after intratracheal instillation of IL-1 in rats, but did not decrease indices of lung inflammation, specifically myeloperoxidase (MPO) activity in lung tissue, neutrophil counts in lung lavage, and cytokine-induced neutrophil chemoattractant (CINC) levels in lung lavage, in rats given IL-1 intratracheally. The results suggest that TGFbeta contributes to lung leak, but not lung inflammation, following intratracheal administration of IL-1 in rats.

Iron, through its participation in reactions that generate reactive oxygen species, may contribute to the oxidative lung injury observed in patients with acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). A number of investigators have shown that the endogenous iron storage protein ferritin increases in the blood of patients with and at-risk for ALI and ARDS, but the significance of these increases are not known. In the present investigation, we measured lung tissue levels of thiobarbituric acid reactive substances (TBARS) and lung leak in isolated rat lungs perfused with xanthine oxidase (XO) and purine, an enzymatic system which generates reactive oxygen species. We found that adding ferritin (100 ng/mL) or desferrioxamine (DFO, 10 mM), an iron chelator, to the vascular perfusate solution decreased oxidant-induced leak in isolated rat lungs perfused with XO and purine. Addition of ferritin or DFO also decreased TBARS in isolated rat lungs perfused with XO and purine; neither ferritin nor DFO, however, decreased XO activity in vitro. Our results suggest that oxidative lung leak may be altered by the availability of reactive iron and that ferritin may contribute to protection against oxidative lung injury.

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.

Publisher Summary This chapter focuses on the oxyradicals and acute lung injury. Oxyradicals can injure tissues in numerous ways. Key cellular components are directly susceptible to oxidant attack. Lipids, proteins, and DNA molecules are important targets. Oxidants damage proteins by several mechanisms: These include (1) direct attack causing conformational change in amino acids, (2) denaturation of proteins, and (3) oxidation, which renders proteins more susceptible to hydrolysis. Oxyradicals can also cause cellular damage indirectly. One example is by potentiating the deleterious effects of elastase released by activated neutrophils. Elastase is a serine protease contained in the azurophilic granules of neutrophils. The beneficial effect of oxyradicals now encompasses their role as mediators, as well as their bactericidal contributions. This system includes both intracellular and extracellular components, which reduce formation of oxyradicals, increase the metabolism of oxyradicals to less toxic forms, localize the reactions of oxyradicals to less vital cellular locations and/or repair oxidized targets.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2; encoded in humans by the NFE2L2 gene) is a transcription factor that regulates the gene expression of a wide variety of cytoprotec- tive phase II detoxification and antioxidant enzymes through a promoter sequence known as the antioxidant-responsive element (ARE). The ARE is a promoter element found in many cyto- protective genes; therefore, Nrf2 plays a pivotal role in the ARE-driven cellular defense system against environmental stresses. Agents that target the ARE/Nrf2 pathway have been tested in a wide variety of disorders, with at least one new Nrf2-activating drug now approved by the US Food and Drug Administration. Examination of in vitro and in vivo experimental results, and taking into account recent human clinical trial results, has led to an opinion that Nrf2-activating strategies - which can include drugs, foods, dietary supplements, and exercise - are likely best targeted at disease prevention, disease recurrence prevention, or slowing of disease progression in early stage illnesses; they may also be useful as an interventional strategy. However, this rubric may be viewed even more conservatively in the pathophysiology of cancer. The activation of the Nrf2 pathway has been widely accepted as offering chemoprevention benefit, but it may be unhelpful or even harmful in the setting of established cancers. For example, Nrf2 activation might interfere with chemotherapies or radiotherapies or otherwise give tumor cells additional growth and survival advantages, unless they already possess mutations that fully activate their Nrf2 pathway constitutively. With all this in mind, the ARE/Nrf2 pathway remains of great interest as a possible target for the pharmacological control of degenerative and immunological diseases, both by activation and by inhibition, and its regulation remains a promising biological target for the development of new therapies.

We hypothesized that phospholipase A2 (PLA2) metabolites contribute to the acute, neutrophil-dependent, edematous lung leak that develops after administration of interleukin-1 (IL-1) intratracheally to rats and tested this premise by using mepacrine to inhibit PLA2 activity in vivo. We found that lung PLA2 activity, lung lavage phospholipid content, lung leak index, lung weight gain, and lung lavage protein concentrations were increased in rats given IL-1 intratracheally compared with sham-treated control rats. By comparison, lungs of mecaprine and IL-1-treated rats had decreased PLA2 activity, lavage phospholipid content, leak, weight gain, and lavage protein increases compared with rats given IL-1 intratracheally. Mepacrine treatment also decreased lung neutrophil accumulation, but not lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, in rats given IL-1 intratracheally. In parallel experiments, mepacrine treatment reduced the adhesion of human neutrophils to IL-1-treated human umbilical vein endothelial cells in vitro. Our results indicate that PLA2 activity participates in the lung neutrophil retention and pulmonary vascular leak that develops in rats given IL-1 intratracheally.

Terada, Lance S., Brooks M. Hybertson, Kevin G. Connelly, David Weill, Dale Piermattei, and John E. Repine. XO increases neutrophil adherence to endothelial cells by a dual ICAM-1 and P-selectin-mediated mechanism. J. Appl. Physiol. 82(3): 866–873, 1997.—Circulating xanthine oxidase (XO) can modify adhesive interactions between neutrophils and the vascular endothelium, although the mechanisms underlying this effect are not clear. We found that treatment with XO of bovine pulmonary artery endothelial cells (EC), but not neutrophils or plasma, increased adherence, suggesting that XO had its primary effect on EC. The mechanism by which XO increased neutrophil adherence to EC involved binding of XO to EC and production of H2O2. XO also increased platelet-activating factor production by EC by a H2O2-dependent mechanism. Similarly, the platelet-activating factor-receptor antagonist WEB-2086 completely blocked XO-mediated neutrophil EC adherence. In addition, neutrophil adherence was dependent on the β2-integrin Mac-1 (CD11b/CD18) but not on leukocyte functional antigen-1 (CD11a/CD18). Treatment of EC with XO for 30 min did not alter intercellular adhesion molecule-1 surface expression but increased expression of P-selectin and release of von Willibrand factor. Antibodies against P-selectin (CD62) did not affect XO-mediated neutrophil adherence under static conditions but decreased both rolling and firm adhesive interactions under conditions of shear. We conclude that extracellular XO associates with the endothelium and promotes neutrophil-endothelial cell interactions through dual intercellular adhesion molecule-1 and P-selectin ligation, by a mechanism that involves platelet-activating factor and H2O2as intermediates.

The acute respiratory distress syndrome (ARDS) is a highly fatal form of acute lung injury (ALI) for which no effective therapy is currently available. Despite the fact that ARDS was first described more than 25 years ago, its etiology remains essentially unknown. Furthermore, because of the diverse variety of predisposing insults for the development of ARDS, the existence of complex pathogenetic pathways appears likely. A unifying, and perhaps a common hypothesized mechanism is that pro-inflammatory mediators, neutrophils, and reactive oxygen species contribute to the development of ARDS. Some of the evidence for this premise can be summarized as follows: ARDS patients have increased numbers of neutrophils in their lungs, and these neutrophils are potent sources of oxygen radicals [1, 2]; ARDS patients have elevated levels of the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-α (TNF) in their bronchoalveolar lavage (BAL) fluid and have alveolar macrophages that release increased amounts of IL-1 [3–5]; ARDS patients have increased lung lavage levels of factors such as IL-8 that can attract and stimulate neutrophils [4–10]; Neutrophils recovered from the blood or lungs of ARDS patients have altered functional activity, suggesting that they have been exposed to neutrophil activating factors [11, 12]; Stimulated neutrophils damage isolated perfused lung and lung endothelial cell monolayers [13, 14]; ARDS patients have increased plasma levels of xanthine oxidase, which can generate oxygen radicals [15]; ARDS patients have an increased burden of endogenous oxidants as evidenced by increased concentrations of hydrogen peroxide in their exhaled breaths [16, 17], increased levels of lipid peroxidation products in their blood [18], and increased levels of oxidized antiproteases in their lung lavage fluid [19]; and ARDS patients have decreased levels of the antioxidants glutathione and vitamin E in their lung lavage fluid and plasma [18, 20].

Although local tumor necrosis factor-alpha (TNF) release by alveolar macrophages has been postulated to contribute to the development of acute respiratory distress syndrome (ARDS), the effects of instilling TNF intratracheally on the development of acute lung leak are not known. Our goal was to determine the effect of intratracheally administered TNF on the development of acute lung leak in rats. We found that rats given TNF (500 ng) 5 hours previously intratracheally had increased (p0.05) lung lavage cytokine induced neutrophil chemoattractant (CINC) concentrations, lung lavage neutrophils, lung myeloperoxidase (MPO) activity, and lung leak compared to saline-treated control rats. However, all of the responses following TNF instillation were much lower than the responses to interleukin-1 alpha (IL-1) instillation. For example, instilling 50 ng of IL-1 caused 6.4 times the increases in lung lavage CINC concentrations, 15.5 times the increase in lung lavage neutrophils, 3.6 times the increase in lung MPO activity and 3.8 times the increase in lung leak caused by giving 500 ng of TNF intratracheally. Co-treatment with TNF-binding protein decreased both lung MPO and lung leak increases in rats given TNF intratracheally. These observations suggest that locally elevated levels of TNF may induce lung neutrophil recruitment and acute lung leak but that IL-1 is a much more potent agent than TNF in causing lung neutrophil accumulation and lung leak.