Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.     

The effect of synthetic iron chelators on bacterial growth in human serum

Abstract The effect of synthetic iron chelators of the 1-alkyl-3-hydroxy-2-methylpyrid-4-one class (the L1 series) and 1-hydroxypyrid-2-one (L4) on bacterial growth in human serum was compared with those of the plant iron chelators mimosine and maltol and of the microbial siderophore desferrioxamine. None of the synthetic chelators enhanced growth of 3 Gram-negative organisms ( Yersinia enterocolitica, Escherichia coli and Pseudomonas aeruginosa ); in some cases they were even inhibitory. L4 strongly stimulated growth of Staphylococcus epidermidis , but the L1 series had only a marginal effect. Maltol was mildly inhibitory to all 4 bacterial species, while mimosine enhanced the growth of S. epidermidis and Y. enterocolitica but had little effect on E. coli or P. aeruginosa . Desferrioxamine enhanced the growth chelators of synthetic or plant origin may carry less risk of increasing susceptibility to bacterial infection in patients undergiong chelation therapy for iron overload than does desferrioxamine, the drug currently in clinical use.     

The effect of synthetic iron chelators on bacterial growth in human serum

The effect of synthetic iron chelators of the 1-alkyl-3-hydroxy-2-methylpyrid-4-one class (the L1 series) and 1-hydroxypyrid-2-one (L4) on bacterial growth in human serum was compared with those of the plant iron chelators mimosine and maltol and of the microbial siderophore desferrioxamine. None of the synthetic chelators enhanced growth of 3 Gram-negative organisms (Yersinia enterocolitica, Escherichia coli and Pseudomonas aeruginosa); in some cases they were even inhibitory. L4 strongly stimulated growth of Staphylococcus epidermidis, but the L1 series had only a marginal effect. Maltol was mildly inhibitory to all 4 bacterial species, while mimosine enhanced the growth of S. epidermidis and Y. enterocolitica but had little effect on E. coli or P. aeruginosa. Desferrioxamine enhanced the growth of all except E. coli. These results suggest that the chelators of synthetic or plant origin may carry less risk of increasing susceptibility to bacterial infection in patients undergoing chelation therapy for iron overload than does desferrioxamine, the drug currently in clinical use.     

Effect of iron on fluconazole activity against Candida albicans in presence of human serum or monocyte-derived macrophages

Human serum, transferrin, and apotransferrin are known to profoundly inhibit the growth of Candida albicans by iron deprivation. On the other hand, iron overload (iron saturated transferrin) is a serious risk factor for candidiasis in newborn and in leukemic patients. We tested the efficacy of fluconazole and the previously demonstrated synergy of fluconazole and effector cells against C. albicans under iron overload conditions where efficacy might be diminished. We confirm that exogenous iron completely reversed the inhibitory effect of human serum and report that the efficacy of fluconazole against C. albicans was not significantly compromised in a 24 h assay system. Although exogenous iron inhibited fungistatic activity of monocyte-derived macrophages, it did not interfere with the synergistic candidacidal activity of fluconazole and monocyte-derived macrophages. In 72 h assays, where fluconazole had candidacidal activity, exogenous iron did not compromise efficacy of fluconazole, and fluconazole activity was often increased. These in vitro results suggest that effectiveness of fluconazole therapy would not be compromised in iron overload situations in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of ceruloplasmin in macrophage iron efflux during hypoxia

The reticuloendothelial system has a central role in erythropoiesis and iron homeostasis. An important function of reticuloendothelial macrophages is phagocytosis of senescent red blood cells. The iron liberated from heme is recycled for delivery to erythrocyte precursors for a new round of hemoglobin synthesis. The molecular mechanism by which recycled iron is released from macrophages remains unresolved. We have investigated the mechanism of macrophage iron efflux, focusing on the role of ceruloplasmin (Cp), a copper protein with a potent ferroxidase activity that converts Fe2+ to Fe3+ in the presence of molecular oxygen. As shown by others, Cp markedly increased iron binding to apotransferrin at acidic pH; however, the physiological significance of this finding is uncertain because little stimulation was observed at neutral pH. Introduction of a hypoxic atmosphere resulted in marked Cp-stimulated binding of iron to apotransferrin at physiological pH. The role of Cp in cellular iron release was examined in U937 monocytic cells induced to differentiate to the macrophage lineage. Cp added at its normal plasma concentration increased the rate of 55Fe release from U937 cells by about 250%. The stimulation was absolutely dependent on the presence of apotransferrin and hypoxia. Cp-stimulated iron release was confirmed in mouse peritoneal macrophages. Stimulation of iron release required an intracellular "labile iron pool" that was rapidly depleted in the presence of Cp and apotransferrin. Ferroxidase-mediated loading of iron into apotransferrin was critical for iron release because ferroxidase-deficient Cp was inactive and because holotransferrin could not substitute for apotransferrin. The extracellular iron concentration was critical as shown by inhibition of iron release by exogenous free iron, and marked enhancement of release by an iron chelator. Together these data show that Cp stimulates iron release from macrophages under hypoxic conditions by a ferroxidase-dependent mechanism, possibly involving generation of a negative iron gradient.     

Transferrin: a potential source of iron for oxygen free radical-mediated endothelial cell injury.

The ability of transferrin to potentiate oxygen free radical-mediated endothelial cell injury was assessed. 51Cr-labeled endothelial cells derived from rat pulmonary arteries (RPAECs) were incubated with hydrogen peroxide (H2O2) in the presence and absence of holosaturated human transferrin, and the effect of transferrin on H2O2-mediated endothelial cell toxicity was determined. Addition of holosaturated transferrin potentiated H2O2-mediated RPAEC cytotoxicity at concentrations of H2O2 greater than 10 microM, suggesting that transferrin may provide a source of iron for free radical-mediated endothelial cell injury. Free radical-mediated injury is dependent on non-protein-bound iron. The ability of RPAECs to facilitate the release of iron from transferrin was assessed. We determined that RPAECs facilitate the release of transferrin-derived iron by reduction of transferrin-bound ferric iron (Fe3+) to ferrous iron (Fe2+). The reduction and release of transferrin-derived Fe2+ were inhibited by apotransferrin and chloroquine, indicating a dependence on receptor-specific binding of transferrin to the RPAEC cell surface, with subsequent endocytosis, acidification, and reduction of transferrin-bound Fe3+ to Fe2+. The release of transferrin-derived Fe2+ was potentiated by diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD). In contrast, exogenous SOD did not alter iron release, suggesting that intracellular superoxide anion (O2-) may play an important role in mediating the reduction and release of transferrin-derived iron. Results of this study suggest that transferrin may provide a source of iron for oxygen free radical-mediated endothelial cell injury and identify a novel mechanism by which endothelial cells may mediate the reduction and release of transferrin-derived iron.     

Iron at the cell surface controls both DNA synthesis and plasma membrane redox system

Summary Addition of the impermeable iron II chelator bathophenanthroline disulfonate (BPS) to cultured Chinese hamster lung fibroblast (CCL 39 cells) inhibits DNA synthesis but not protein synthesis or cytoplasmic alkalinization, when cell growth is initiated with growth factors such as EGF plus insulin, thrombin, or ceruloplasmin. The BPS inhibition is reversed by addition of stoichiometric ferrous iron at stoichiometric concentration. BPS does not inhibit cell growth stimulated by fetal calf serum. The effect of the BPS differs from the inhibition of growth by hydroxyurea which acts on the ribonucleotide reductase. The BPS treatment leads to release of iron from the cells as determined by BPS iron II complex formation over 90 min. Cells treated with BPS just during starvation period cannot re-initiate DNA synthesis after mitogen stimulation even if BPS is removed from the medium and cells are previously washed. BPS treatment also inhibits transplasma membrane electron which is restored by incubation of cells with 10 M ferric ammonium citrate. Growth factor stimulation of DNA synthesis is restored by addition of 1 M ferrous ammonium sulfate or ferric ammonium citrate, or 0.1 M diferric transferrin. Copper, cobalt, nickel, zinc, gallium, aluminum, or apotransferrin cannot restore the activity. The BPS effect is consistent with removal of iron from a site on the cell surface which controls electron transport and DNA synthesis.Abbreviations BCS bathocuproine disulfonate - BPS bathophenan-throline disulfonate - CUP ceruloplasmin - FCS fetal calf serum - Fe₂Tf diferric transferrin - EGF epidermal growth factor - HU hydroxyurea - THR -thrombin     

Dependence of Staphylococcus epidermidis on non-transferrin-bound iron for growth

The ability of Staphylococcus epidermidis strains to grow in the presence of human transferrin and varying amounts of ferric iron was studied. At initial bacterial densities up to 10(4) cfu ml(-1), none of the three strains grew when transferrin iron saturation was below the full saturation point, whereas the bacteria grew consistently when transferrin was fully iron-saturated and there was non-transferrin-bound iron in the medium. Precultivation of the bacteria under iron-restricted conditions to induce siderophore production did not abolish the growth dependence on non-transferrin-bound iron. At initial bacterial densities of 10(6) cfu ml(-1), the bacteria proliferated consistently also in the presence of partially saturated transferrin. The results indicate that at low bacterial densities, S. epidermidis cannot utilise transferrin-bound iron for growth and that its proliferation is dependent on non-transferrin-bound iron.     

Expression of virulence factors by Staphylococcus aureus grown in serum

Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl₃ into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.     

Catecholamine inotropes as growth factors for Staphylococcus epidermidis and other coagulase-negative staphylococci

Drugs commonly used in intensive care settings were assayed for their ability to affect the growth of Staphylococcus epidermidis in a minimal salts medium containing 30% serum. Of 28 compounds tested, the inotropic catecholamines adrenaline, dobutamine, dopamine, isoprenaline and noradrenaline significantly stimulated bacterial growth. These drugs, but not structurally similar compounds lacking a dihydroxybenzoyl moiety (such as tyramine, phenylephrine and salbutamol), were able to remove iron from iron-saturated transferrin and to supply transferrin-bound 55Fe to S. epidermidis cells. Similar results were observed with a range of coagulase-negative staphylococci associated with line infections, but not with Staphylococcus aureus (including MRSA).     

Effects of ferrous iron and transferrin on cell proliferation of human diploid fibroblasts in serum-free culture

Summary Iron-free RITC 80-7 defined medium was used to examine effects of ferrous iron and transferrin on cell proliferation of human diploid fibroblasts. Both ferrous iron and holotransferrin stimulated cell proliferation in the medium, but apotransferrin did not. When 5 g/l human serum albumin (HSA) was added to the defined medium, excellent growth was obtained under hypoxic conditions, whereas a reduction of cellular growth during the culture periods was observed under aerobic conditions. When ferrous iron was added to the HSA medium alone, the reduction in growth increased in proportion to the concentrations, whereas the addition of transferrin prevented this reduction in a concentration-dependent manner. This suggests that the ferrous iron concentration in media causes a reduction in growth under aerobic conditions and transferrin prevents this reduction because it decreases the ferrous iron concentration. Further, serum albumin seems to be a source of iron in media.     

金诺芬对表皮葡萄球菌及其生物膜的影响

目的 研究金诺芬对表皮葡萄球菌及其生物膜的作用。方法 利用微量稀释法药敏试验检测金诺芬对表皮葡萄球菌浮游菌生长的影响,并利用结晶紫染色和激光共聚焦显微镜观察金诺芬对表皮葡萄球菌生物膜形成的影响。结果 金诺芬对表皮葡萄球菌的MIC和MBC分别为0.125~0.250 μg/mL和2.000~4.000 μg/mL。同时,4 μg/mL金诺芬还能显著抑制表皮葡萄球菌标准菌株和临床菌株生物膜的形成（P<0.05）。通过激光共聚焦显微镜观察发现金诺芬能有效抑制生物膜的形成,降低生物膜的聚集,并增加死亡细菌的比例。结论 金诺芬能显著抑制表皮葡萄球菌浮游菌的增殖和生物膜的形成。     

Iron transfer from ferritin to transferrin. Effect of serum factors

The transfer of iron from 59Fe-labelled human spleen ferritin to human apotransferrin occurs in the absence of either reducing or chelating agents. The reaction is first order with respect to ferritin and zero order to apotransferrin. The transfer is enhanced by low-Mr substances from human serum such as ascorbate, citrate, bicarbonate and lactate. A mixture of the four molecules at their normal physiological concentrations can increase the iron exchange to the same extent as that observed with an ultrafiltrate of serum. A pathway of intracellular iron mobilization is considered.     

Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Anticryptococcal activity of human serum and apotransferrin in RPMI 1640 was studied in vitro. The effects of varying concentrations of FeCl₃ on this activity was investigated. Possible synergy of serum and apotransferrin with fluconazole was also measured. The fungistatic activity of human serum, whether lyophilized, stored at 4 °C, fresh frozen or purchased from commercial sources vs. Cryptococcus neoformans was comparable. There was no significant loss of fungistatic activity after freezing and thawing the serum up to 10 times. The fungistatic activity of human serum was similar when tested in different tissue culture media with the exception of Medium 199. The addition of apotransferrin (2.0 or 0.2 mg/ml) to RPMI 1640 had an inhibitory effect on cryptococcal growth. This effect was reversed by 20 M of FeCl₃ at both apotransferrin concentrations. By contrast, addition of FeCl₃ to human serum and RPMI 1640 did not reverse inhibition of growth. Fluconazole synergized with the human serum preparations described, but not with pooled commercial serum, for fungicidal activity. Synergistic activity of fluconazole and human serum was not affected by the addition of FeCl₃. Apotransferrin did not show any synergistic fungicidal activity with fluconazole.     

A lipophilic iron chelator can replace transferrin as a stimulator of cell proliferation and differentiation

Of the different growth supplements used in chemically defined media, only transferrin is required for differentiation of tubules in the embryonic mouse metanephros. Since transferrin is an iron-carrying protein, we asked whether iron is crucial for tubulogenesis. Differentiation of metanephric tubules both in whole embryonic kidneys and in a transfilter system was studied. The tissues were grown in chemically defined media containing transferrin, apotransferrin, the metal-chelator complex ferric pyridoxal isonicotinoyl hydrazone (FePIH), and excesses of ferric ion. Although we found that apotransferrin was not as effective as iron-loaded transferrin in promoting proliferation in the differentiating kidneys, excess ferric ion at up to 100 microM, five times the normal serum concentration, could not promote differentiation or proliferation. However, iron coupled to the nonphysiological, lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone, to form FePIH, could sustain levels of cell proliferation and tubulogenesis similar to those attained by transferrin. Thus, the role of transferrin in cell proliferation during tubulogenesis is solely to provide iron. Since FePIH apparently bypasses the receptor-mediated route of iron intake, the use of FePIH as a tool for investigating cell proliferation and its regulation is suggested.     

Effects of spermine on transferrin and iron uptake by reticulocytes: II. Changes in intravesicular pH

An increase in extracellular spermine concentration brought about a progressive rise in intralysosomal pH in rabbit reticulocytes. Since intracellular release of iron from transferrin is believed to involve the protonation of the iron-transferrin complex, the rise in intralysomal pH could account for the inhibitory effect of spermine on iron uptake. The inhibition could be reversed if spermine was removed by washing. As a result of spermine treatment, more acid-labile N-terminal monoferric transferrin and less apotransferrin were released from the cell. These results are consistant with the protonation theory of iron release.     

Growth of Staphylococcus aureus with defective siderophore production in human peritoneal dialysate solution

In this study, we attempted to determine the effects of iron-availability and the activity of the bacterial iron-uptake system (IUS) on the growth of Staphylococcus aureus in human peritoneal dialysate (HPD) solution. A streptonigrin-resistant S. aureus (SRSA) strain, isolated from S. aureus ATCC 6538, exhibited defective siderophore production, thereby resulting in ineffective uptake of iron from low iron-saturated transferrin. The growth of both strains was stimulated in HPD solution supplemented with FeCl3 and holotransferrin, but growth was inhibited in HPD solution which had been supplemented with apotransferrin and dipyridyl. The SRSA strain grew less robustly than did its parental strain in both iron-supplemented HPD solution and regular HPD solution. These results indicate that iron-availability and siderophore-mediated IUS activity in particular, the ability to produce siderophores and thus capture iron from low iron-saturated transferrin play critical roles in the growth of S. aureus in HPD solution. Our results also indicated that the possibility of using iron chelators as therapeutic or preventive agents warrants further evaluation.     

Growth-stimulating effect of transferrin on a hybridoma cell line: Relation to transferrin iron-transporting function

The relation of the growth-stimulating capacity of transferrin to its iron-transporting function was investigated in mouse hybridoma PLV-01 cells cultivated in a chemically defined medium. The cells were precultivated in protein-free medium supplemented either with ferric citrate (cells with a high intracellular iron level) or with iron-saturated transferrin (cells with a low intracellular iron level). Iron uptake was monitored after the application of 59Fe-labeled ferric citrate or pig transferrin. Cultivation of the cells at the optimum growth-stimulating concentration (500 microM) of ferric citrate resulted in an intracellular iron level about 100-fold higher than that of cells cultivated at the optimum transferrin concentration (5 micrograms/ml). Replacement of pig transferrin with bovine transferrin resulted in similar intracellular iron levels, but the growth-stimulating effect of bovine transferrin was more than one order of magnitude lower. Cells with a high intracellular iron level grew equally well when cultivated with iron-saturated transferrin or with apotransferrin + deferoxamine (2 micrograms/ml). On the other hand, cells with a low intracellular iron level required iron-saturated transferrin for further growth and apotransferrin + deferoxamine was ineffective. The results suggest that transferrin can act as a cell growth factor only in the iron-saturated form. However, several findings of this work indicate that supplying cells with iron cannot be accepted as the full explanation of the transferrin growth-stimulating effect.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.