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Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S. maltophilia can be cultured from respiratory tract secretions. Identification of S. maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified as B. cepacia by clinical microbiology laboratories were in fact S. maltophilia. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S. maltophilia directly from sputum. The SS-PCR was developed and tested against a panel of 112 S. maltophilia isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S. maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify S. maltophilia in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identify S. maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum.

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The simultaneous production of nitric oxide and superoxide anion leads to the formation of peroxynitrite, a potent oxidant which may be an important mediator of cellular injury. Oxidation of dichlorofluorescin to the fluorescent dichlorofluorescein has been used as a marker for cellular oxidant production. The mechanisms of peroxynitrite-mediated oxidation of dichlorofluorescin to dichlorofluorescein were investigated. Chemically synthesized peroxynitrite (50-500 nM) induced the oxidation of dichlorofluorescin to dichlorofluorescein in a linear fashion. In addition, the simultaneous generation of nitric oxide and superoxide anion induced the oxidation of dichlorofluorescin to dichlorofluorescein, while nitric oxide (1-10 microM) alone under aerobic conditions did not. Peroxynitrite-mediated oxidation of dichlorofluorescin was not inhibited by the hydroxyl radical scavengers mannitol (100 mM) or dimethylsulfoxide (100 mM). Moreover, peroxynitrite-mediated oxidation of dichlorofluorescin was not dependent upon metal ion-catalyzed reactions. Furthermore, dichlorofluorescein formation was diminished at alkaline pH. These findings suggest that peroxynitrite-mediated dichlorofluorescein formation results directly from the protonation of peroxynitrite to form the conjugate peroxynitrous acid. L-cysteine was an efficient inhibitor (KI approximately 25 microM) of dichlorofluorescin oxidation through competitive oxidation of free sulfhydryls. Urate was a less efficient with a maximum inhibition of only 49%. These results demonstrate that dichlorofluorescin is efficiently oxidized by peroxynitrite. Therefore, under conditions where nitric oxide and superoxide are produced simultaneously, oxidation of dichlorofluorescin may be mediated by the formation of peroxynitrite.

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OBJECTIVES: Production of nitric oxide via the cytokine-mediated activation of myocardial inducible nitric oxide synthase decreases myocardial contractility. Whether myocardial dysfunction is mediated directly by nitric oxide or indirectly through the formation of secondary reaction products, such as peroxynitrite, has not been established. Peroxynitrite, but not nitric oxide, reacts with the phenolic ring of tyrosine to form the stable product 3-nitro-L-tyrosine. Demonstration of tissue nitrotyrosine residues, therefore, infers the presence of peroxynitrite or related nitrogen-centered oxidants. DESIGN: Retrospective analysis of human autopsy specimens. SETTING: University pathology and basic science laboratories. PATIENTS: Formalin-fixed, paraffin-embedded myocardial tissue samples were obtained from 11 patients with a diagnosis of sepsis, seven patients with a diagnosis of viral myocarditis, and five control patients without clinical or pathologic cardiac disease. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Specific antibodies to nitrotyrosine were utilized to detect nitrotyrosine residues in human autopsy specimens. Cardiac tissue obtained from patients with myocarditis or sepsis demonstrated intense nitrotyrosine immunoreactivity in the endocardium, myocardium, and coronary vascular endothelium and smooth muscle. In contrast, connective tissue elements were without appreciable immunohistochemical staining. Nitrotyrosine antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin, but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. In situ reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. Densitometric analysis of nitrotyrosine immunoreactivity demonstrated significantly higher values for specimens from myocarditis and sepsis patients when compared with control tissue specimens. CONCLUSION: These results demonstrate the formation of peroxynitrite within the myocardium during inflammatory disease states, suggesting a role for peroxynitrite in inflammation-associated myocardial dysfunction.

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Oxidant-mediated toxicity resulting from acute pulmonary inflammation has been demonstrated in acute lung injury. A potent biological oxidant, peroxynitrite, is formed by the near diffusion-limited reaction of nitric oxide with superoxide. In addition to having hydroxyl radical-like oxidative reactivity, peroxynitrite is capable of nitrating phenolic rings, including protein-associated tyrosine residues. Nitric oxide does not directly nitrate tyrosine residues, therefore, demonstration of tissue nitrotyrosine residues infers the action of peroxynitrite or related nitrogen-centered oxidants. Lung tissue was obtained from formalin-fixed, paraffin-embedded autopsy specimens, and specific polyclonal and monoclonal antibodies to nitrotyrosine were visualized by diaminobenzidene-peroxidase staining. Acute lung injury resulted in intense staining throughout the lung, including lung interstitium, alveolar epithelium, proteinaceous alveolar exudate, and inflammatory cells. In addition, staining of the vascular endothelium and subendothelial tissues was present in those patients with sepsis-induced acute lung injury. Antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. Reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. In control specimens with no overt pulmonary disease, there was only slight staining of the alveolar septum. These results demonstrate that nitrogen-derived oxidants are formed in human acute lung injury and suggest that peroxynitrite may be an important oxidant in inflammatory lung disease.

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Nitric oxide (NO.) is a free radical and will react efficiently with other radicals. The reaction between NO. and superoxide anion (O2.-) is a pivotal reaction by which NO. affects oxidant metabolism. This reaction may scavenge O2.- before further reactions can occur that lead to the biosynthesis of more potent oxidants such as hydroxyl radical. The product of the reaction between NO. and O2.-, however, is peroxynitrite anion, which is also a potent oxidant capable of participating in several oxidative reactions. Among these reactions are oxidation of sulfhydryl groups, oxidation of lipids, and nitration of tyrosine by noncatalyzed and catalyzed mechanisms. The conformation, and therefore specific reactivity, of peroxynitrite are dependent on pH. Based on an understanding of this concept, sulfhydryl oxidation should be the predominant oxidative reaction of peroxynitrite in biological systems. Some experimental data support this conclusion. There is increasing evidence from isolated cell systems that peroxynitrite is produced under the influence of inflammatory mediators. Most data from animal models suggest that increased NO. production in acute lung injury is detrimental. We have performed immunohistochemical evaluation of lung tissue from pediatric patients with acute lung injury using an antinitrotyrosine antibody and have found evidence of extensive nitrotyrosine formation. This observation suggests a significant effect of peroxynitrite on lung tissue in this disorder. NO. has a variety of nonoxidant effects that also may also have a role in acute lung injury. With the information currently available, one cannot conclude with certainty whether the net effect of increased NO. production in inflammatory disorders of the lung is beneficial or injurious. However, simultaneous increases in NO. and O2.- occurring during inflammation may lead to peroxynitrite formation and subsequent oxidative tissue injury.

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Nitric oxide reacts with superoxide to form peroxynitrite, a potential mediator of oxidant-induced cellular injury. The endothelium is a primary target of injury in many pathological states, including acute lung injury, sepsis, multiple organ failure syndrome, and atherosclerosis, where enhanced production of nitric oxide and superoxide occurs simultaneously. It was hypothesized that stimulation of endothelial cell nitric oxide production would result in formation of peroxynitrite. Immediate oxidant production was detected by luminol- and lucigenin-enhanced chemiluminescence from cultured bovine aortic endothelial cells exposed to bradykinin or to the calcium ionophore A23187. Luminol-enhanced chemiluminescence was efficiently inhibited by the nitric oxide synthase inhibitor nitro-L-arginine methyl ester and by superoxide dismutase, implying dependence on the presence of both nitric oxide and superoxide for oxidant production. Inhibition of luminol-enhanced chemiluminescence by nitro-L-arginine methyl ester was partially reversed by L-arginine, but not by D-arginine. Cysteine, methionine, and urate, known inhibitors of peroxynitrite-mediated oxidation, inhibited luminol-enhanced chemiluminescence, while the hydroxyl radical scavengers, mannitol and dimethylsulfoxide, and catalase did not. Bicarbonate increased luminol-enhanced chemiluminescence in a concentration-dependent manner. Superoxide production, detected by lucigenin-enhanced chemiluminescence, was slightly increased in the presence of nitro-L-arginine methyl ester, suggesting that endothelial cell-produced superoxide was partially metabolized by reaction with nitric oxide. These results are consistent with agonist-induced peroxynitrite production by endothelial cells and suggests that peroxynitrite may have an important role in oxidant-induced endothelial injury.

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Nitric oxide reacts with superoxide to form peroxynitrite, which may be an important mediator of free radical-induced cellular injury. Oxidation of dihydrorhodamine to fluorescent rhodamine is a marker of cellular oxidant production. We investigated the mechanisms of peroxynitrite-mediated formation of rhodamine from dihydrorhodamine. Peroxynitrite at low levels (0-1000 nM) induced a linear, concentration-dependent, oxidation of dihydrorhodamine. Hydroxyl radical scavengers mannitol and dimethylsulfoxide had minimal effect (< 10%) on rhodamine production. Peroxynitrite-mediated formation of rhodamine was not dependent on metal ion catalyzed reactions because studies were performed in metal ion-free buffer and rhodamine formation was not enhanced in the presence of Fe3+ ethylenediaminetetraacetic acid (EDTA). Thus, rhodamine formation appears to be mediated directly by peroxynitrite. Superoxide dismutase slightly enhanced rhodamine production. L-cysteine was an efficient inhibitor (KI approximately 25 microM) of dihydrorhodamine oxidation through competetive oxidation of free sulfhydryls. Urate was also an efficient inhibitor (KI approximately 2.5 microM), possibly by reduction of an intermediate dihydrorhodamine radical and recycling of dihydrorhodamine. Under anaerobic conditions, nitric oxide did not oxidize dihydrorhodamine and inhibited spontaneous oxidation of dihydrorhodamine. In the presence of oxygen, nitric oxide induces a relatively slow oxidation of dihydrorhodamine due to the formation of nitrogen dioxide. We conclude that dihydrorhodamine is a sensitive and efficient trap for peroxynitrite and may serve as a probe for peroxynitrite production.

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Airway inflammation is important in the development and progression of many pulmonary disorders, including asthma. We hypothesized that the hydrogen peroxide (H2O2) concentration in expired breath may be a marker of airway inflammation. Expired breath condensate was collected by cooling and the H2O2 concentration was measured fluorimetrically. Thirty-five samples were collected from 22 pediatric patients with asthma who were 7 to 18 yr of age and from 11 healthy, nonasthmatic controls. Asthmatic subjects were determined to be well or sick (acute disease of the upper or lower respiratory tract) by clinical examination. Pulmonary function tests were determined to be abnormal if there was a > 15% reduction in FEV1 or > 20% reduction in FEF25-75 compared with baseline values. Expired breath H2O2 was elevated in asthmatic subjects compared with controls (0.81 +/- 0.70 versus 0.25 +/- 0.27 mumol/L). The difference was primarily due to elevation of H2O2 in sick asthmatic subjects, whose expired breath H2O2 level of 1.5 +/- 0.5 (n = 10) was different from that of well asthmatics (0.54 +/- 0.56, n = 25). There was a high correlation between expired breath H2O2 and clinical status. Elevation of expired H2O2 occurred with either acute upper or lower respiratory tract disease. There was no statistically significant correlation between expired breath H2O2 level and pulmonary function test results. We conclude that elevation of H2O2 in the expired breath condensate is a simple, noninvasive method that can be used as a biochemical marker of airway inflammation.

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2',7'-Dichlorofluorescein and dihydrorhodamine 123 were evaluated as probes for detecting changes in intracellular H2O2 in cultured endothelial cells. Stable intracellular levels of these probes were established within 15 min of exposure to the probe in culture medium. With continued presence of the probe in the medium, intracellular levels were unchanged for 1 h. However, if medium without the probes was used after intracellular loading had occurred, there was a greater than 90% loss of intracellular dichlorofluorescin, dichlorofluorescein, and dihydrorhodamine 123 while intracellular rhodamine 123 decreased by only 15%. Exposure of endothelial cells to exogenous 100 microM H2O2 for 1 h increased intracellular rhodamine 123 by 83%, but there was a reproducible decrease of 53% in intracellular dichlorofluorescein. Exposure to 0.05 mM BCNU plus 10 mM aminotriazole for 2 h increased intracellular rhodamine 123 by 111%. In vitro studies of dihydrorhodamine 123 oxidation were similar to previous reports of dichlorofluorescin oxidation. Oxidation of dihydrorhodamine 123 does not occur with H2O2 alone, but is mediated by a variety of secondary H2O2-dependent intracellular reactions including H2O2-cytochrome c and H2O2-Fe2+. Our results suggest that detection of increased oxidation of these probes in endothelial cells is most useful as a marker of a change in general cellular oxidant production.

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Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.

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Endotoxic shock is associated with acute vascular endothelial injury resulting in edema. Tumor necrosis factor (TNF) and interleukin 1 (IL-1) are cytokines produced by endotoxin-stimulated mononuclear phagocytes that are potential mediators of endotoxic shock. In this study, we investigated the effects of TNF and IL-1 alpha on vascular endothelial cell permeability in vitro. The movement of radiolabeled macromolecules of different sizes (57Co-vitamin B12, 125I-cytochrome c, and 131I-albumin; 6.5-35A) across bovine aortic endothelial cell monolayers was measured after exposure to these cytokines. TNF induced a time- and dose-dependent increase in endothelial cell monolayer permeability that was enhanced in the presence of serum. The peak increase was noted after 12 h of incubation with less alteration of permeability with longer incubations. IL-1 alpha caused a similar time-dependent increase in endothelial cell monolayer permeability, but the peak effect of IL-1 alpha was seen after 24 h. Therefore the increased permeability seen with TNF cannot be explained by release of endogenous IL-1 alone. Neither TNF nor IL-1 alpha increased release of [14C]adenine, and the only effect on lactate dehydrogenase release was a small, but statistically significant, increase after 24 h of incubation. From these studies, we conclude that TNF and IL-1 alpha directly increase vascular endothelial cell permeability in vitro and speculate that these cytokines may be involved in the acute vascular endothelial injury associated with endotoxic shock.

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