Peroxidase- and nitrite-dependent metabolism of the anthracycline anticancer agents daunorubicin and doxorubicin.

Oxidation of the anticancer anthracyclines doxorubicin (DXR) and daunorubicin (DNR) by lactoperoxidase(LPO)/H(2)O(2) and horseradish peroxidase(HRP)/H(2)O(2) systems in the presence and absence of nitrite (NO(2)(-)) has been investigated using spectrophotometric and EPR techniques. We report that LPO/H(2)O(2)/NO(2)(-) causes rapid and irreversible loss of anthracyclines' absorption bands, suggesting oxidative degradation of their chromophores. Both the initial rate and the extent of oxidation are dependent on both NO(2)(-) concentration and pH. The initial rate decreases when the pH is changed from 7 to 5, and the reaction virtually stops at pH 5. Oxidation of a model hydroquinone compound, 2,5-di-tert-butylhydroquinone, by LPO/H(2)O(2) is also dependent on NO(2)(-); however, in contrast to DNR and DXR, this oxidation is most efficient at pH 5, indicating that LPO/H(2)O(2)/NO(2)(-) is capable of efficiently oxidizing simple hydroquinones even in the neutral form. Oxidation of anthracyclines by HRP/H(2)O(2)/NO(2)(-) is substantially less efficient relative to that by LPO/H(2)O(2)/NO(2)(-) at either pH 5 or pH 7, most likely due to the lower rate of NO(2)(-) metabolism by HRP/H(2)O(2). EPR measurements show that interaction of anthracyclines and 2,5-di-tert-butylhydroquinone with LPO/H(2)O(2)/NO(2)(-) generates the corresponding semiquinone radicals presumably via one-electron oxidation of their hydroquinone moieties. The possible role of the (*)NO(2) radical, a putative LPO metabolite of NO(2)(-), in oxidation of these compounds is discussed. Because in vivo the anthracyclines may co-localize with peroxidases, H(2)O(2), and NO(2)(-) in tissues, their oxidation via the proposed mechanism is likely. These observations reveal a novel, peroxidase- and nitrite-dependent mechanism for the oxidative transformation of the anticancer anthracyclines, which may be pertinent to their biological activities in vivo.

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.

Lactoferrin binds CpG-containing oligonucleotides and inhibits their immunostimulatory effects on human B cells.

Unmethylated CpG dinucleotide motifs in bacterial DNA, as well as oligodeoxynucleotides (ODN) containing these motifs, are potent stimuli for many host immunological responses. These CpG motifs may enhance host responses to bacterial infection and are being examined as immune activators for therapeutic applications in cancer, allergy/asthma, and infectious diseases. However, little attention has been given to processes that down-modulate this response. The iron-binding protein lactoferrin is present at mucosal surfaces and at sites of infection. Since lactoferrin is known to bind DNA, we tested the hypothesis that lactoferrin will bind CpG-containing ODN and modulate their biological activity. Physiological concentrations of lactoferrin (regardless of iron content) rapidly bound CpG ODN. The related iron-binding protein transferrin lacked this capacity. ODN binding by lactoferrin did not require the presence of CpG motifs and was calcium independent. The process was inhibited by high salt, and the highly cationic N-terminal sequence of lactoferrin (lactoferricin B) was equivalent to lactoferrin in its ODN-binding ability, suggesting that ODN binding by lactoferrin occurs via charge-charge interaction. Heparin and bacterial LPS, known to bind to the lactoferricin component of lactoferrin, also inhibited ODN binding. Lactoferrin and lactoferricin B, but not transferrin, inhibited CpG ODN stimulation of CD86 expression in the human Ramos B cell line and decreased cellular uptake of ODN, a process required for CpG bioactivity. Lactoferrin binding of CpG-containing ODN may serve to modulate and terminate host response to these potent immunostimulatory molecules at mucosal surfaces and sites of bacterial infection.

Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi.

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2-during phagocytosis of opsonized promastigotes. Addition of the O2- scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2- enhances macrophage leishmanicidal activity. The importance of NO. produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO. in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor N(G)-monomethyl-L-arginine inhibited IFN-gamma-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-gamma. These data suggest that O2- and NO. each contribute to intracellular killing of L. chagasi in human and murine macrophages.

Adequacy of fellowship training: results of a survey of recently graduated fellows.

The adequacy of fellowship training in the field of infectious diseases was assessed by means of a survey of recently graduated fellows. Surveys were mailed to all individuals who had passed the American Board of Internal Medicine's board certification examination in infectious diseases since 1992. A total of 666 completed surveys were returned by the deadline (response rate, 36%). Although most recent graduates thought that training in the standard components of clinical infectious diseases was adequate, only 50% thought that training in infection control was adequate. Fewer than 1 in 3 believed that they had received adequate training in the business aspects of infectious diseases practice. The adequacy and duration of research training were linked to ultimate career choice. These results form the basis for the Infectious Diseases Society of America's new initiatives to assist with more-diversified and relevant fellowship training.

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.

Gallium disrupts iron metabolism of mycobacteria residing within human macrophages.

Mycobacterium tuberculosis and M. avium complex (MAC) enter and multiply within monocytes and macrophages in phagosomes. In vitro growth studies using standard culture media indicate that siderophore-mediated iron (Fe) acquisition plays a critical role in the growth and metabolism of both M. tuberculosis and MAC. However, the applicability of such studies to conditions within the macrophage phagosome is unclear, due in part to the absence of experimental means to inhibit such a process. Based on the ability of gallium (Ga(3+)) to concentrate within mononuclear phagocytes and on evidence that Ga disrupts cellular Fe-dependent metabolic pathways by substituting for Fe(3+) and failing to undergo redox cycling, we hypothesized that Ga could disrupt Fe acquisition and Fe-dependent metabolic pathways of mycobacteria. We find that Ga(NO(3))(3) and Ga-transferrin produce an Fe-reversible concentration-dependent growth inhibition of M. tuberculosis strains and MAC grown extracellularly and within human macrophages. Ga is bactericidal for M. tuberculosis growing extracellularly and within macrophages. Finally, we provide evidence that exogenously added Fe is acquired by intraphagosomal M. tuberculosis and that Ga inhibits this Fe acquisition. Thus, Ga(NO(3))(3) disruption of mycobacterial Fe metabolism may serve as an experimental means to study the mechanism of Fe acquisition by intracellular mycobacteria and the role of Fe in intracellular survival. Furthermore, given the inability of biological systems to discriminate between Ga and Fe, this approach could have broad applicability to the study of Fe metabolism of other intracellular pathogens.

Inducible resistance to oxidant stress in the protozoan Leishmania chagasi.

Leishmania sp. protozoa are introduced into a mammalian skin by a sandfly vector, whereupon they encounter increased temperature and toxic oxidants generated during phagocytosis. We studied the effects of 37 degrees C "heat shock" or sublethal menadione, which generates superoxide and hydrogen peroxide, on Leishmania chagasi virulence. Both heat and menadione caused parasites to become more resistant to H(2)O(2)-mediated toxicity. Peroxide resistance was also induced as promastigotes developed in culture from logarithmic to their virulent stationary phase form. Peroxide resistance was not associated with an increase in reduced thiols (trypanothione and glutathione) or increased activity of ornithine decarboxylase, which is rate-limiting in trypanothione synthesis. Membrane lipophosphoglycan increased in size as parasites developed to stationary phase but not after environmental exposures. Instead, parasites underwent a heat shock response upon exposure to heat or sublethal menadione, detected by increased levels of HSP70. Transfection of promastigotes with L. chagasi HSP70 caused a heat-inducible increase in resistance to peroxide, implying it is involved in antioxidant defense. We conclude that leishmania have redundant mechanisms for resisting toxic oxidants. Some are induced during developmental change and others are induced in response to environmental stress.

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.

The Pseudomonas aeruginosa secretory product pyocyanin inactivates alpha1 protease inhibitor: implications for the pathogenesis of cystic fibrosis lung disease.

Alpha1 Protease inhibitor (alpha1PI) modulates serine protease activity in the lung. Reactive oxygen species inactivate alpha1PI, and this process has been implicated in the pathogenesis of a variety of forms of lung injury. An imbalance of protease-antiprotease activity is also detected in the airways of patients with cystic fibrosis-associated lung disease who are infected with Pseudomonas aeruginosa. P. aeruginosa secretes pyocyanin, which, through its ability to redox cycle, induces cells to generate reactive oxygen species. We tested the hypothesis that redox cycling of pyocyanin could lead to inactivation of alpha1PI. When alpha1PI was exposed to NADH and pyocyanin, a combination that results in superoxide production, alpha1PI lost its ability to form an inhibitory complex with both porcine pancreatic elastase (PPE) and trypsin. Similarly, addition of pyocyanin to cultures of human airway epithelial cells to which alpha1PI was also added resulted in a loss of the ability of alpha1PI to form a complex with PPE or trypsin. Neither superoxide dismutase, catalase, nor dimethylthiourea nor depletion of the media of O2 to prevent formation of reactive oxygen species blocked pyocyanin-mediated inactivation of alpha1PI. These data raise the possibility that a direct interaction between reduced pyocyanin and alpha1PI is involved in the process. Consistent with this possibility, pretreatment of alpha1PI with the reducing agent beta-mercaptoethanol also inhibited binding of trypsin to alpha1PI. These data suggest that pyocyanin could contribute to lung injury in the P. aeruginosa-infected airway of cystic fibrosis patients by decreasing the ability of alpha1PI to control the local activity of serine proteases.

Electron paramagnetic resonance detection of free tyrosyl radical generated by myeloperoxidase, lactoperoxidase, and horseradish peroxidase.

Phagocytes secrete the heme protein myeloperoxidase, which is present and active in human atherosclerotic tissue. These cells also generate hydrogen peroxide (H2O2), thereby allowing myeloperoxidase to generate a range of oxidizing intermediates and stable end products. When this system acts on L-tyrosine in vitro, it forms o, o'-dityrosine, which is enriched in atherosclerotic lesions. Myeloperoxidase, therefore, may oxidize artery wall proteins in vivo, cross-linking their L-tyrosine residues. In these studies, we used electron paramagnetic resonance (EPR) spectroscopy to identify an oxidizing intermediate in this reaction pathway and in parallel reactions catalyzed by horseradish peroxidase and lactoperoxidase. Using an EPR flow system to rapidly mix and examine solutions containing horseradish peroxidase, H2O2, and L-tyrosine, we detected free tyrosyl radical (a2,6H = 6.3 G, a3,5H = 1.6 G, and abetaH = 15. 0 G). We then used spin trapping techniques with 2-methyl-2-nitrosopropane (MNP) to further identify this intermediate. The resulting three-line spectrum (aN = 15.6 G) was consistent with an MNP/tyrosyl radical spin adduct. Additional MNP spin trapping studies with ring-labeled L-[13C6]tyrosine yielded a characteristic eight-line EPR spectrum (aN = 15.6 G, a13C (2) = 8.0 G, a13C (1) = 7.1 G, a13C (1) = 1.3 G), indicating that the MNP adduct resulted from trapping a carbon-centered radical located on the aromatic ring of L-tyrosine. This same eight-line spectrum was observed when human myeloperoxidase or bovine lactoperoxidase was substituted for horseradish peroxidase. Furthermore, a partially immobilized MNP/tyrosyl radical spin adduct was detected when we exposed a synthetic polypeptide composed of glutamate and L-tyrosine residues to the myeloperoxidase-H2O2-L-tyrosine system. The broadened EPR signal resulting from this MNP/polypeptide adduct was greatly narrowed by proteolytic digestion with Pronase, confirming that the initial spin-trapped radical was protein-bound. Collectively, these results indicate that peroxidases use H2O2 to convert L-tyrosine to free tyrosyl radical. They also support the idea that free tyrosyl radical initiates cross-linking of L-tyrosine residues in proteins. We suggest that this pathway may play an important role in protein and lipid oxidation at sites of inflammation and in atherosclerotic lesions.

Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.

Evidence for the existence of a surface receptor for ferriclactoferrin and ferrictransferrin associated with the plasma membrane of the protozoan parasite Leishmania donovani.

Previous work has demonstrated the ability of the promastigote form of the protozoan parasite Leishmania chagasi to utilize iron chelated to lactoferrin and transferrin for growth and metabolism. We have obtained evidence suggesting that the promastigote form of the parasite possesses specific binding sites for lactoferrin and transferrin. Lactoferrin binding appears to be: 1) independent of whether or not the protein contains iron; 2) not inhibited by transferrin; and 3) independent of whether the organism is in log or stationary phase of growth. Transferrin binding is: 1) markedly greater if the protein is iron loaded; 2) inhibited by the presence of lactoferrin; and 3) independent of whether the organism is in log or stationary growth phase. Preliminary ligand blot analyses are consistent with the presence of a protein or proteins which bind lactoferrin and/or transferrin. The relationship to these binding sites to those described in other protozoan species requires further investigation.

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.

Endogenous superoxide dismutase levels regulate iron-dependent hydroxyl radical formation in Escherichia coli exposed to hydrogen peroxide.

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O2.- and H2O2. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H2O2 but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (.OH) as a consequence of the reaction of H2O2 with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H2O2 in the presence of an alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)-ethanol spin-trapping system, the 4-POBN-.CH(CH3)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating .OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H2O2 in a similar manner, the magnitude of .OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of .OH spin trapped. Exogenous SOD failed to inhibit .OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in .OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in these mutants lacking SOD.

Iron acquisition by parasitic protozoa.

The efficient uptake of iron by microorganisms is essential for their survival. Mammalian hosts possess elaborate means of sequestering their iron stores to protect themselves against invading pathogens. In this review, Mary Wilson and Bradley Britigan summarize mechanisms by which bacteria and protozoa effectively scavenge iron from their hosts during infection, as well as the potential and proven effects of these mechanisms on microbial virulence.

Lack of Fc-epsilon receptors on murine eosinophils: implications for the functional significance of elevated IgE and eosinophils in parasitic infections.

Chronic infection with Schistosoma mansoni induces in humans and mice a Th2-dominant immune response in which eosinophils and IgE are conspicuously elevated. Human eosinophils express IgE receptors that participate in an IgE-dependent eosinophil-mediated ADCC reaction against Schistosomula larvae in vitro. To investigate the expression of IgE receptors on murine eosinophils, they were purified (>95% pure by Giemsa-stained cytospin preparations) from liver granulomas of Schistosoma-infected mice. Flow cytometric analysis showed the absence of the low-affinity IgE receptor Fc-epsilon RII (CD23) and Mac-2 and the absence of binding of murine IgE. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of granuloma eosinophil mRNA did not detect transcripts for Fc-epsilon RII or the alpha-chain of the high-affinity IgE receptor Fc-epsilon RI, but did detect transcripts that encode Mac-2 and the low-affinity IgG receptors Fc-gamma RIIb2, Fc-gamma RIII, and the FcR-associated gamma-chain. In vitro stimulation of granuloma eosinophils with interleukin-4 (IL-4) did not induce IgE binding, surface expression of Mac-2, or the transcription of Fc-epsilon receptors (Fc-epsilon RI, Fc-epsilon RII/CD23). To investigate normal murine eosinophils, we cultured normal mouse bone marrow cells with recombinant IL-3, recombinant IL-5, and recombinant granulocyte-macrophage colony-stimulating factor, conditions that promote eosinophil differentiation. Flow cytometric analysis of bone marrow-derived eosinophils failed to detect IgE binding or cell surface expression of Fc-epsilon RII and Mac-2, and RT-PCR analysis of fluorescence-activated cell sorted bone marrow-derived eosinophils failed to detect transcripts that encode Fc-epsilon RI or Fc-epsilon RII. These findings show that, in contrast to human eosinophils, murine eosinophils do not express cell surface receptors that bind IgE. However, because IgG receptors (Fc-gamma RIIb2, Fc-gamma RII) were present on eosinophils purified from granulomas, we investigated whether they might be involved in eosinophil activation. We found that an oxidative burst in eosinophils could be triggered through their IgG receptors.