Antioxidant enzyme expression in clinical isolates of Pseudomonas aeruginosa: identification of an atypical form of manganese superoxide dismutase.

Expression of superoxide dismutases (FeSOD and MnSOD) and catalases by laboratory strains of Pseudomonas aeruginosa is modulated by exogenous factors. Whether clinical isolates behave similarly and whether antioxidant enzyme expression influences P. aeruginosa virulence remain unclear. Fifty-seven P. aeruginosa blood culture isolates, plus seven pairs of blood and local-site isolates, were examined for FeSOD, MnSOD, and catalase production in vitro. Under iron-replete growth conditions FeSOD and catalase activities were maximized. MnSOD was not detected. FeSOD and catalase activity decreased under iron-limited growth conditions, whereas MnSOD activity appeared. SOD and catalase activity did not change with site of isolation or by patient. MnSOD could not be expressed by one isolate due to a missense mutation in sodA that produced a premature stop codon. Eleven percent of the isolates expressed a novel, rapidly migrating MnSOD that was associated with missense mutations in the normal stop codon of sodA. We conclude that clinical P. aeruginosa isolates vary little in FeSOD and catalase expression. Some strains produce a newly described MnSOD variant, whereas one is deficient in MnSOD production. The absence of MnSOD expression in a P. aeruginosa strain causing invasive human disease indicates that MnSOD is probably not essential for P. aeruginosa virulence.

Assessment of structural features of the pseudomonas siderophore pyochelin required for its ability to promote oxidant-mediated endothelial cell injury.

We previously showed that iron chelated to the Pseudomonas aeruginosa siderophore pyochelin enhances oxidant-mediated injury to pulmonary artery endothelial cells by catalyzing hydroxyl radical (HO(*)) formation. Therefore, we examined pyochelin structural/chemical features that may be important in this process. Five pyochelin analogues were examined for (i) capacity to accentuate oxidant-mediated endothelial cell injury, (ii) HO(*) catalytic ability, (iii) iron transfer to endothelial cells, and (iv) hydrophobicity. All compounds catalyzed similar HO(*) production, but only the hydrophobic ones containing a thiazolidine ring enhanced cell injury. Transfer of iron to endothelial cells did not correlate with cytotoxicity. Finally, binding of Fe(3+) by pyochelin led to Fe(2+) formation, perhaps explaining how Fe(3+)-pyochelin augments H(2)O(2)-mediated cell injury via HO(*) formation. The ability to bind iron in a catalytic form and the molecule's thiazolidine ring, which increases its hydrophobicity, are key to pyochelin's cytotoxicity. Reduction of Fe(3+) to Fe(2+) may also be important.

Biological effects of menadione photochemistry: effects of menadione on biological systems may not involve classical oxidant production.

Because cell-mediated reduction of menadione leads to the generation of reactive oxygen species (ROS), this quinone is widely used to investigate the effects of ROS on cellular functions. We report that A549 human lung epithelial cells exposed to menadione demonstrate a dose-dependent increase in both intracellular calcium ([Ca(2+)](i)) and ROS formation. The concentrations of menadione required to initiate these two events are markedly different, with ROS detection requiring higher levels of menadione. Modulators of antioxidant defences (e.g. buthionine sulphoximine, 3-amino-1,2,4-triazole) have little effect on the [Ca(2+)](i) response to menadione, suggesting that ROS formation does not account for menadione-dependent alterations in [Ca(2+)](i). Additional evidence suggests that menadione photochemistry may be responsible for the observed [Ca(2+)](i) effects. Specifically: (a) EPR studies with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) show that light exposure (maximum effect at 340 nm) stimulates menadione-dependent formation of the DMPO/(.)OH spin adduct that was not sensitive to antioxidant interventions; (b) DMPO inhibits menadione and light-dependent increases in [Ca(2+)](i); and (c) light (maximum effect at 340 nm) augments the deleterious effects of menadione on cell viability as determined by (51)Cr release. These photo effects do not appear to involve formation of singlet oxygen by menadione, but rather are the result of the oxidizing chemistry initiated by menadione in the triplet state. This work demonstrates that menadione species generated by photo-irradiation can exert biological effects on cellular functions and points to the potential importance of photochemistry in studies of menadione-mediated cell damage.

Iron acquisition from Pseudomonas aeruginosa siderophores by human phagocytes: an additional mechanism of host defense through iron sequestration?

Chelation of iron to iron-binding proteins is a strategy of host defense. Some pathogens counter this via the secretion of low-molecular-weight iron-chelating agents (siderophores). Human phagocytes possess a high-capacity mechanism for iron acquisition from low-molecular-weight iron chelates. Efficient acquisition and sequestration of iron bound to bacterial siderophores by host phagocytes could provide a secondary mechanism to limit microbial access to iron. In the present work we report that human neutrophils, macrophages, and myeloid cell lines can acquire iron from the two Pseudomonas aeruginosa siderophores. Analogous to iron acquisition from other low-molecular-weight chelates, iron acquisition from the siderophores is ATP independent, induced by multivalent cationic metals, and unaffected by inhibitors of endocytosis and pinocytosis. In vivo, this process could serve as an additional mechanism of host defense to limit iron availability to invading siderophore-producing microbes.

Despite structural similarities between gp91phox and FRE1, flavocytochrome b558 does not mediate iron uptake by myeloid cells.

Superoxide (O2-) generated by the phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is dependent on electron transfer by flavocytochrome b558 (flavocytochrome b), a transmembrane heterodimer that forms the redox center of the oxidase at the plasma or phagosomal membrane. The larger of its two subunits, gp91phox, is homologous to the yeast iron reductase subunit FRE1, and these two proteins share many structural and functional characteristics. Because FRE1 is required for iron uptake in yeast, we hypothesized that flavocytochrome b might serve a similar function in human phagocytes and thus provide a mechanism for the transferrin-independent iron acquisition observed in myeloid cells. To determine whether flavocytochrome b was required for iron uptake, we compared iron acquisition by polymorphonuclear neutrophils (PMNs) or Epstein-Barr virus (EBV)-transformed B lymphocytes derived from individuals with X-linked chronic granulomatous disease (CGD) with iron acquisition by normal cells. Our results indicate that all cells acquired iron to the same extent and that uptake could be significantly enhanced in the presence of the trivalent metal gallium. The gallium enhancement of iron uptake observed in PMNs or in EBV-transformed B lymphocytes derived from healthy individuals was mirrored by those derived from individuals deficient in flavocytochrome b. Furthermore, both normal and CGD-derived EBV-transformed B lymphocytes had similar iron reductase activity, suggesting that flavocytochrome b is not a biologically significant iron reductase. In contrast to previously suggested hypotheses, these results show conclusively that flavocytochrome b is not necessary for cellular iron acquisition, despite structural and functional similarities between yeast iron reductases and flavocytochrome b.

Pseudomonas pyocyanine alters calcium signaling in human airway epithelial cells.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes both acute and chronic lung disease. P. aeruginosa exerts many of its pathophysiological effects by secreting virulence factors, including pyocyanine, a redox-active compound that increases intracellular oxidant stress. Because oxidant stress has been shown to affect cytosolic Ca2+ concentration ([Ca2+]c) in other cell types, we studied the effect of pyocyanine on [Ca2+]c in human airway epithelial cells (A549 and HBE). At lower concentrations, pyocyanine inhibits inositol 1,4,5-trisphosphate formation and [Ca2+]c increases in response to G protein-coupled receptor agonists. Conversely, at higher concentrations, pyocyanine itself increases [Ca2+]c. The pyocyanine-dependent [Ca2+]c increase appears to be oxidant dependent and to result from increased inositol trisphosphate and release of Ca2+ from intracellular stores. Ca2+ plays a central role in epithelial cell function, including regulation of ion transport, mucus secretion, and ciliary beat frequency. By disrupting Ca2+ homeostasis, pyocyanine could interfere with these critical functions and contribute to the pathophysiological effects observed in Pseudomonas-associated lung disease.

Binding of iron and inhibition of iron-dependent oxidative cell injury by the "calcium chelator" 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA).

A role for increases in intracellular calcium (Ca2+) has been suggested in the pathophysiology of various forms of oxidant-mediated cell injury. In recent studies, we found that iron bound to the Pseudomonas aeruginosa siderophore, pyochelin, augments oxidant-mediated endothelial cell injury by catalyzing the formation of hydroxyl radical (HO.). To investigate the role of Ca2+ in this process, the effects of two Ca2+ chelating agents, Fura-2 and 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA), were assessed. BAPTA, but not Fura-2, was protective against H2O2/ferripyochelin-mediated injury. Subsequent data suggested that chelation of iron rather than Ca2+ by BAPTA was most likely responsible. Spectrophotometry demonstrated that both ferrous (Fe2+) and ferric (Fe3+) iron formed a complex with BAPTA. The affinity of BAPTA for the metals was Fe3+ > Ca2+ > Fe2+. BAPTA was found to decrease markedly iron-catalyzed production of HO. and/or ferryl species when analyzed by spin trapping. Although our results do not definitively prove that BAPTA protects endothelial cells from ferripyochelin-associated damage by chelating iron, these data indicate that caution must be exercised in utilizing protective effects of intracellular "Ca2+ chelating agents" as evidence for a role of alterations in cellular Ca2+ levels in experimental conditions in which iron-mediated oxidant production is also occurring.

Augmentation of oxidant injury to human pulmonary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin.

Pseudomonas aeruginosa causes acute and chronic infections of the human lung, with resultant tissue injury. We have previously shown that iron bound to pyochelin, a siderophore secreted by the organism to acquire iron, is an efficient catalyst for hydroxyl radical (HO.) formation and augments injury to pulmonary artery endothelial cells resulting from their exposure to superoxide (O2.) and/or H2O2. Sources for O2-. and H2O2 included phorbol myristate acetate (PMA)-stimulated neutrophils and pyocyanin. Pyocyanin, another P. aeruginosa secretory product, undergoes cell-mediated redox, thereby forming O2-. and H2O2. In P. aeruginosa lung infections, damage to airway epithelial cells is probably more extensive than that to endothelial cells. Therefore, we examined whether ferripyochelin also augments oxidant-mediated damage to airway epithelial cells. A549 cells, a human type II alveolar epithelial cell line, was exposed to H2O2, PMA-stimulated neutrophils, or pyocyanin, and injury was determined by release of 51Cr from prelabeled cells. Ferripyochelin significantly increased (> 10-fold) oxidant-mediated cell injury regardless of whether H2O2, neutrophils, or pyocyanin was employed. Apo-pyochelin was not effective, and ferripyochelin was not toxic by itself at the concentrations employed. Spin trapping with alpha-(4-pyrridyl-1-oxide)-N-t-butyl-nitrone-ethanol confirmed the generation of HO., and injury was decreased by a variety of antioxidants, including superoxide dismutase, catalase, and dimethylthiourea. These data are consistent with the hypothesis that the presence of ferripyochelin at sites of P. aeruginosa lung infection could contribute to tissue injury through its ability to promote HO.-mediated damage to airway epithelial cells.

Protease cleavage of iron-transferrin augments pyocyanin-mediated endothelial cell injury via promotion of hydroxyl radical formation.

Although a number of bacterium- and host-derived factors have been suggested to contribute to the pathogenesis of Pseudomonas aeruginosa-associated tissue injury, the mechanism remains unclear. We have previously shown that protease modification of iron (Fe)-transferrin generates new iron chelates capable of catalyzing hydroxyl radical (.OH) formation from superoxide and hydrogen peroxide. The latter two oxidants are generated during redox cycling of another P. aeruginosa secretory product, pyocyanin. The lung is a major site of P. aeruginosa infection, with damage to local endothelial cells contributing to the pathogenesis of such infections. Endothelial cells are highly susceptible to oxidant-mediated injury. Therefore, we examined whether pseudomonas elastase-cleaved Fe-transferrin and pyocyanin synergistically enhance pulmonary artery endothelial cell injury via .OH formation. By measuring 51Cr release from cultured endothelial cell monolayers, pseudomonas elastase-cleaved Fe-transferrin significantly augmented cell injury resulting from cellular exposure to sublethal concentrations of pyocyanin. This enhancement in injury was not protease specific, as similar results were obtained with pyocyanin in combination with trypsin- or porcine pancreatic elastase-cleaved Fe-transferrin. The association of iron with the transferrin appeared to be necessary in this process. Supporting the involvement of .OH generation via the Haber-Weiss reaction in augmenting cell injury, catalase, dimethyl thiourea, superoxide dismutase, deferoxamine, and dimethyl sulfoxide significantly inhibited cell injury resulting from exposure to pyocyanin and protease-cleaved Fe-transferrin. Furthermore, spin trapping demonstrated the production of .OH in this cellular system. We conclude that .OH formation resulting from the interaction of protease-cleaved Fe-transferrin and endothelial cell redox cycling of pyocyanin may contribute to P. aeruginosa-associated tissue injury via endothelial cell injury.

Bicarbonate and phosphate ions protect transferrin from myeloperoxidase-mediated damage.

Exposure to hypochlorous acid (HOCl), the main product of the reaction of neutrophil myeloperoxidase (MPO), H2O2, and Cl-, reportedly decreases apotransferrin's iron binding capacity. Optimal transferrin iron binding requires the coexistent binding of anions such as bicarbonate (HCO3-) near the protein's two iron binding sites. Recently, we found that if HCO3- was also present during HOCl exposure, apotransferrin retained its ability to inhibit iron-catalyzed hydroxyl radical generation. Therefore, we examined apotransferrin iron binding capacity after exposure to the MPO/H2O2/I- system in the presence and absence of several anions (HCO3-, H2PO4, SO4(2-), and ClO4-) known to bind to apotransferrin. Although the MPO system decreased apotransferrin iron uptake to only 46% of the untreated apotransferrin control, apotransferrin treated in the presence of 1 mM HCO3- or H2PO4- retained 84 and 74%, respectively, of its iron binding capacity. Similar results were seen when apotransferrin was treated with NaOCl. These results could not be explained on the basis of a loss of MPO activity or scavenging of HOCl. In contrast, SO4(2-) and ClO4- were unable to prevent the MPO-mediated loss of apotransferrin iron binding capacity. NaOCl had no effect on the ability of transferrin to bind any of these anions, as assessed by the anion-induced change in apotransferrin absorbance spectrum. HCO3- but not H2PO4-, SO4(2-), or ClO4- decreased MPO-mediated oxidation (iodination) of apotransferrin. Under some conditions H2PO4- actually increased apotransferrin iodination. HCO3- and H2PO4- may protect apotransferrin from MPO-mediated oxidative damage by preventing selective oxidation of one or both iron binding sites. This process may allow transferrin to retain its iron binding function during MPO exposure in vivo.

Pseudomonas siderophore pyochelin enhances neutrophil-mediated endothelial cell injury.

Pyochelin, a siderophore secreted by Pseudomonas aeruginosa, binds iron in a form which can catalyze the formation of hydroxyl radical (.OH) from neutrophil-derived superoxide (O2-.) and hydrogen peroxide (H2O2). Ferripyochelin induced a concentration-dependent increase in endothelial cell injury (51Cr release) resulting from exposure to H2O2, a xanthine/xanthine oxidase O2-./H2O2 generating system, or stimulated neutrophils. This process was dependent on the presence of iron. Formation of .OH was confirmed using spin trapping. Although a slight (13%) increase in neutrophil O2-. production in the presence of ferripyochelin was observed, this did not appear to account for the extent of endothelial cell injury observed. The antioxidants dimethylthiourea and catalase decreased endothelial cell injury, whereas dimethyl sulfoxide and superoxide dismutase were without effect. Fe-nitrilotriacetic acid and Fe-EDTA, which are also .OH catalysts, did not augment endothelial cell injury resulting from exposure to the above oxidant systems. In contrast to results with the endothelial cells, killing of P. aeruginosa by O2-./H2O2 derived from the reaction of xanthine and xanthine oxidase was not increased by ferripyochelin. These data are consistent with the possibility that the interaction of Pseudomonas- and phagocyte-derived secretory products could contribute to local tissue injury at sites of P. aeruginosa infection by causing the generation of .OH.

Interaction of lactoferrin and lipopolysaccharide (LPS): effects on the antioxidant property of lactoferrin and the ability of LPS to prime human neutrophils for enhanced superoxide formation.

Lactoferrin is a 77-kDa iron-binding protein to which a wide variety of divergent biologic functions have been ascribed. It has recently been reported that lactoferrin interacts with bacterial lipopolysaccharide (LPS) in such a fashion as to affect the binding of lactoferrin to myeloid cells. Two other potential interactions of LPS and lactoferrin were explored. Lactoferrin prevents hydroxyl radical formation by binding iron, even at low pH. Lactoferrin inhibited iron-catalyzed formation of hydroxyl radical in the presence of LPS at pH 7.4 and 4.5. Low concentrations of LPS can be used to "prime" neutrophils toward enhanced function, such as formation of stimulated superoxide anion. Lactoferrin inhibited LPS priming of neutrophils if LPS contamination of the protein (provided by commercial suppliers) was first reduced. Inhibition of LPS priming was observed whether apolactoferrin or iron-saturated lactoferrin was used. Similar inhibition of LPS priming was observed when neutrophils were incubated with other serum proteins (e.g., albumin, apotransferrin, or iron-saturated transferrin). These results show that LPS should not be expected to affect the free radical biology of lactoferrin, which is a crucial physiologic function of this protein. However, lactoferrin inhibits LPS priming, and this effect requires consideration in experimental models of inflammation.

Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury.

Pyocyanin, a secretory product of Pseudomonas aeruginosa, has the capacity to undergo redox cycling under aerobic conditions with resulting generation of superoxide and hydrogen peroxide. By using spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry (EPR), superoxide was detected during the aerobic reduction of pyocyanin by NADH or porcine endothelial cells. No evidence of hydroxyl radical formation was detected. Chromium oxalate eliminated the EPR spectrum of the superoxide-derived spin adduct resulting from endothelial cell exposure to pyocyanin, suggesting superoxide formation close to the endothelial cell plasma membrane. We have previously reported that iron bound to the P. aeruginosa siderophore pyochelin (ferripyochelin) catalyzes the formation of hydroxyl free radical from superoxide and hydrogen peroxide via the Haber-Weiss reaction. In the present study, spin trap evidence of hydroxyl radical formation was detected when NADH and pyocyanin were allowed to react in the presence of ferripyochelin. Similarly, endothelial cell exposure to pyocyanin and ferripyochelin also resulted in hydroxyl radical production which appeared to occur in close proximity to the cell surface. As assessed by 51Cr release, endothelial cells which were treated with pyocyanin or ferripyochelin alone demonstrated minimal injury. However, endothelial cell exposure to the combination of pyochelin and pyocyanin resulted in 55% specific 51Cr release. Injury was not observed with the substitution of iron-free pyochelin and was diminished by the presence of catalase or dimethyl thiourea. These data suggest the possibility that the P. aeruginosa secretory products pyocyanin and pyochelin may act synergistically via the generation of hydroxyl radical to damage local tissues at sites of pseudomonas infection.

[The circumstances of life and need for social measures of patients with prolonged psychiatric illness]. / Langtidssyge psykiatriske patienters levevilkår og behov for sociale foranstaltninger.

The circumstances of life and the need for social support were investigated in 142 patients with prolonged psychiatric illness who sought help consecutively in the psychiatric casualty department or who were referred to the psychiatric department of Odense Hospital during the period 11 Sep. 1989-31 Dec. 1989. 80% of these were psychotic and 20% were borderline psychotic. Approximately 90% were young adults (20-49 years). Schizophrenic men predominated. More than 40% of all the patients had some form of addiction as a contributory diagnosis and 71% of the patients with borderline psychoses were addicts. Approximately 3/4 of the patients lived alone in flats. The level of occupational training was low as compared with the remainder of the population. The majority received early pensions. The majority lived in social isolation from the remainder of the community. Only a single patient had no fixed abode but suitable forms of residence where social support could be given were insufficient. Similarly, the requirements for possible employment and leisure activities were great.

[Violent patients in a psychiatric outpatient department]. / Voldelige patienter på psykiatrisk skadestue.

During the period 23.11.1987-22.11.1989, a total of 7,906 patients attended the psychiatric outpatient department and observation section on 12,469 occasions. Sixty patients (0.8%) behaved violently on 85 occasions. None of these episodes of violence resulted in injuries to persons requiring medical attention. 90% of the violent patients were men and 70% were in the age group 20-39 years. Approximately 1/5 were psychotic and, of these, approximately half suffered from schizophrenia. Comparison with the non-violent patient revealed that the probability of violence was greater if the patient was a man under the influence of alcohol and who had the diagnoses of narcomania, drug abuse or personality disorders. Significantly fewer violent patients were admitted than non-violent patients. Over 80% of the violent patients had previously been admitted to psychiatric departments. Approximately 60% had behaved violently on previous consultations with the psychiatric outpatient department.

[Attempted suicide and depression]. / Selvmordsforsøg og depression.

The frequency of depressive illness was investigated in 195 patients who had been referred consecutively after attempted suicide during the period 15. February 1989-15, October 1989. A total of 130 of these patients were admitted to hospital while the remainder were treated in the psychiatric emergency room or admission department. Registration of depressive symptoms on admission revealed that 85% had depressed mood and other depressive symptoms. According to the criteria established by Feighner et al. 51% suffered from definite depressive disease on admission. According to Zung's Depression Scale, 60% were depressed. On the basis of observations during hospitalization, 25% suffered from depressive disease according to the criteria established by Feighner et al. 19% of these patients suffered from endogenic depression according to the Newcastle I scale which corresponds to 5% of all the hospitalized patients with attempted suicide. Approximately 10% were treated with antidepressives. Only 8% were discharged with the diagnoses of endogenic or reactive psychoses (ICD-8). It is concluded that depressive symptoms occur in the majority of patients with attempted suicide but that slight non-endogenic depressive states are most commonly concerned and that many of these improve rapidly during hospitalization without medicinal treatment. Restraint should be observed in prescription of antidepressive medicine to patients with attempted suicide until the diagnosis of depressive disease is verified.

Evidence from capping experiments for independence of the RT7 alloantigen and the leucocyte common antigen in the rat.

Experiments reported here demonstrate that the RT7 alloantigen and the L-C antigen are separate and distinct structures on the surface of rat lymphocytes. The distribution of the antigens in different rat strains, including the mutant WF/fz, clearly establish the RT7 antigenic system as a polymorphic diallelic system, whereas the recognized L-C antigenic determinant is monomorphic and present on the lymphocytes of all rat strains tested. These data were obtained using monoclonal antibodies to the antigens in indirect immunofluorescence experiments. The two antigens were shown to redistribute (cap) independently of one another on the surface of rat thymocytes. Cells that had been exposed to anti-L-C antibody and FITC-conjugated anti-Ig followed by anti-RT7.1 antibody and RITC-conjugated anti-Ig demonstrated FITC caps and RITC rings.