Institute of Infectious Diseases and Public Health, University of Ancona, Italy
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Abstract |
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Introduction |
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Lactoferrin is an iron-binding glycoprotein that is synthesized by cells of the mucosal epithelium and neutrophils and released in response to inflammatory stimuli.810 Near the amino terminus of lactoferrin, there is a strongly basic region that binds to a variety of anionic biological molecules, including lipid A and glycosaminoglycans, which are found on the surface of most cells and in most extracellular matrices.11,12
Several authors have reported that lactoferrins have antimicrobial activity in vitro, and lactoferrin treatment in vivo has been reported to reduce the incidence of Gram-negative bacteraemia.10,1316 Recent studies have demonstrated that lactoferrin also has activity against candida.1719 This activity is commonly attributed to its ability to bind and sequester environmental iron.20 The iron-free form of lactoferrin (apo-lactoferrin) is able to kill both Candida albicans and Candida krusei, by mechanisms related to alterations in cell surface permeability.21
It has been demonstrated recently that non-transferrin-bound iron is increased in the lower respiratory tracts of patients with P. carinii pneumonia and that iron chelators are effective as anti-P. carinii agents in experimental models.2225 There is also evidence that combinations of macrolides and sulphamethoxazole, clindamycin and primaquine, atovaquone and dihydrofolate reductase inhibitors, and trimethoprimsulphamethoxazole (TMPSMX) have increased activity against P. carinii in vitro in the rat model and in humans.2628
In the present study we investigated the in vitro activity of lactoferrins alone and in combination with other clinically used antimicrobial agents against P. carinii and compared their activity with that of TMPSMX.
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Materials and methods |
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Three clinical isolates of P. carinii were obtained from bronchoalveolar lavages of three immunocompromised patients who had not received prior anti-P. carinii therapy. Specimens were combined, homogenized in an equal volume of phosphate-buffered saline (PBS) by forced passage through a thin needle in order to disaggregate the clumps, and then filtered through sterile gauze. The homogenate was centrifuged (1800g for 15 min) and the pellet resuspended in physiological saline. Contaminating bacteria were partially eliminated by four washes in 10 mL of physiological saline followed by incubation in phosphate-buffered saline containing ampicillin (2000 mg/L) and streptomycin (2000 mg/L) for 4 h at 37°C. Organisms were pelleted by centrifugation (1800g for 20 min), resuspended in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker, Walkersville, MD, USA), stained with methenamine silver (to detect cystic forms) or Giemsa stain (to detect P. carinii nuclei) and finally aliquoted for culture. The final inoculum was 5 x 103 P. carinii organisms/mL. Viability was assayed by double staining with fluorescein diacetate (FDA) (SigmaAldrich Srl, Milan, Italy) and propidium iodide (SigmaAldrich).29 Briefly, the organisms were resuspended in 0.1 mL of PBS. After addition of 0.1 mL of FDA 40 mg/L and 0.03 mL of propidium iodide 20 mg/L and incubation at room temperature for 5 min, the incubation mixtures were further diluted with an equal volume of PBS and analysed by flow cytometry.
The human lung epithelial cell line A-549 (BioWhittaker) was maintained in 25 cm2 tissue culture flasks. Medium consisted of DMEM (BioWhittaker) containing 0.10 mg/L Fe(NO3)3 with 10% fetal calf serum (BioWhittaker), 1% l-glutamine (BioWhittaker), 20 mM N-2-hydroxyethylpiperazine-N-ethanesulphonic acid (HEPES) (Sigma Aldrich), penicillin G 100 U/mL, streptomycin 100 mg/L and amphotericin B 0.5 mg/L. Cells were detatched from the surface of flasks using 0.25% trypsin and 0.53 mM EDTA in PBS; then they were counted using a haemocytometer. Forty-eight hours before P. carinii inoculation, 5 x 103 A549 viable cells/well were inoculated into 96-well plates in a total volume of 0.2 mL. Viability was assessed by trypan blue exclusion. After 48 h, monolayers were washed with DMEM to remove nonadherent epithelial cells and infection was initiated by adding 0.2 mL of P. carinii inoculum (c.1 x 103 organisms) in new growth medium to an adherent layer of 5070% confluent cells. Infected cell cultures were kept at 37°C in 5% CO2 throughout the study.
Drugs
Compounds were all purchased from SigmaAldrich, except clarithromycin (Abbott, Rome, Italy). Iron-free human lactoferrin (lactoferrin H) and iron-free bovine lactoferrin (lactoferrin B) were dissolved in DMEM. Trimethoprim, pyrimethamine and clarithromycin were dissolved in methanol/acetone (1:1) at a concentration of 1 mg/mL. Sulphamethoxazole was dissolved in dimethylsulphoxide at 1 mg/mL. Minocycline was dissolved in distilled water at 1 mg/mL. Solutions of drugs were made fresh on the day of assay or stored at 80°C in the dark for short periods.
Susceptibility testing
Serial dilutions of each drug were prepared in DMEM. All drugs were tested at concentrations close to that which could be achieved clinically. Lactoferrins H and B were tested at concentrations of 1, 10 and 20 mg/L, clarithromycin and minocycline at 1, 2 and 4 mg/L and pyrimethamine at 0.1, 0.2 and 0.4 mg/L. TMPSMX used as reference drug combination was tested at concentrations of 0.8/4, 1.6/8 and 3.2/16 mg/L. Preliminary experiments indicated that the final concentrations of methanol, acetone and dimethylsulphoxide (0.1%) used in the dilution of drugs did not inhibit the growth of P. carinii. In experiments to test drug interactions, the final concentrations in the culture medium were 1, 10 and 20 mg/L for the lactoferrins, 1, 2 and 4 mg/L for clarithromycin and minocycline, and 0.1, 0.2 and 0.4 mg/L for pyrimethamine. Antibiotic-free plates were used as controls in the study. Experiments were performed in triplicate. P. carinii was added at a concentration of 102103 organisms per well. The monolayers were incubated at 37°C in 5% CO2. After 72 h, 0.2 mL of supernatant containing nonadherent organisms was removed from each triplicate well and centrifuged (1800g for 15 min); P. carinii vegetative and cystic forms (precystic forms and mature cysts) were quantified by counting P. carinii nuclei in duplicate Giemsa-stained 10 µL drops delivered on to 25 mm2 squares on microscope slides. The A549 cells together with adherent P. carinii were removed from the plates by trituration following incubation in 1 mL of Ca2+- and Mg2+-free Hanks' balanced salt solution containing 10 mM EDTA for 30 min at 37°C.6 The suspensions were centrifuged (1800g for 15 min) and detached organisms were quantified by Giemsa staining, as described above.
The cytotoxicity of the lactoferrins was determined by the CellTiter 96 AQ cell proliferation assay (Promega Corp., Lyon, France). Control for each cytotoxicity assay included (i) uninfected cells incubated in DMEM; (ii) infected cells incubated in DMEM; and (iii) cells exposed to a freeze thawed lysate containing 5 x 103 P. carinii equivalents in DMEM.
Analysis of results
The activity of each agent and combination was evaluated by counting viable parasites from plates with antimicrobial-supplemented medium and comparing it with the count from control plates without drugs (counts were total numbers of parasites in the cell layer and supernatant). The average number of P. carinii parasites per millilitre was calculated by counting 50 oil-immersion fields (x1000 magnification) of each of three slides.
The 50% and 90% inhibitory concentrations (IC50 and IC90, respectively) of the drug were defined as the concentrations required to produce 50% and 90% reduction, respectively, in the mean cyst or trophozoite counts compared with controls without drug after 72 h incubation in the presence of drugs.
The activity of each compound was also expressed by calculating the ratio of the cystic and trophic form numbers in cultures containing lactoferrin H or B at 20 mg/L, singly or in combination with other agents, to the number of cysts and trophozoites in control cultures after 72 h incubation.
Cytotoxicity was calculated from the following formula: ((mean optical density (OD) of uninfected cells mean OD of infected cells)/mean OD of uninfected cells) x 100. Values of 05%, 625%, 2650% and 51100% were considered to indicate no toxicity, mild toxicity, moderate toxicity and severe toxicity, respectively, for A549 cells.
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Results |
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Lactoferrins H and B had similar effects (Table I). Lactoferrin B tested alone was more effective than lactoferrin H, with a higher activity against trophozoites than cysts. Lactoferrin B suppressed the growth of cysts and trophozoites by >60% at 20 mg/L (IC50 20 mg/L). Lactoferrin H, at the same concentration, showed slightly less activity against cysts and trophozoites, producing >50% reduction in the mean cyst and nucleus counts (IC50 20 mg/L). Neither lactoferrin B nor lactoferrin H achieved a 90% inhibitory effect at the concentrations tested, so their IC90 is >20 mg/L.
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Ratios of the peak number of viable organisms in drug-treated cultures to the peak number in control cultures showed that lactoferrin B 20 mg/L showed the highest activity against both cysts and trophozoites (peak ratio 0.39 and 0.37, respectively). Lactoferrin H tested alone at 20 mg/L showed similar activity (cyst ratio, 0.42; trophozoite ratio, 0.41). Lactoferrin B 20 mg/L in combination with clarithromycin 4 mg/L had in vitro anti-P. carinii activity (cyst ratio, 0.06; trophozoite ratio, 0.02) comparable to that of TMPSMX (cyst ratio, 0.06; trophozoite ratio, 0.08). These results are summarized in Table I.
The cytotoxicity of lactoferrins alone (7.3% to 12.2% cytotoxicity) and in all combinations (5.8% to 8.3% cytotoxicity) was very low.
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Discussion |
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The antimicrobial activities of lactoferrins make it a promising compound for combination therapy. The drugs tested in combination with lactoferrin in this study have distinct modes of action. Pyrimethamine is a specific inhibitor of dihydrofolate reductase in many microorganisms; this enzyme is involved in folate synthesis. Pyrimethamine has only a static effect on parasite growth. The antimicrobial activity of macrolides and tetracyclines results from their ability to inhibit protein synthesis by binding to the transpeptidation site of the larger ribosomal subunit. Studies of pharmacokinetic interaction of clarithromycin and other antimicrobial agents have shown that this macrolide, by inhibiting the cytochrome P-450 pathway, may reduce the metabolism of these agents, resulting in increased serum and tissue concentrations.31
In our experiments, lactoferrin had good activity against P. carinii, and this activity was enhanced when lactoferrin was combined with other drugs. The most active combination tested was lactoferrin plus clarithromycin. This result is in line with other studies that reported anti-P. carinii activity when lactoferrins were combined with clarithromycin, pyrimethamine or minocycline.17,32 This interaction mechanism appears to be complex. As mentioned above, lactoferrin has multiple mechanisms of antimicrobial action, while clarithromycin and minocycline inhibit protein synthesis and pyrimethamine indirectly inhibits folate synthesis. The additive effect of these compounds may result from the cumulative inhibitory effect on different, essential metabolic pathways.
The cytotoxicity assay indicates that these combinations are safe. Our results showed that the combinations tested were active in inhibiting P. carinii at concentrations which appeared to be non-toxic for the human cell monolayer.
In conclusion, our results lend experimental support to the use of iron-chelating agents such as lactoferrin, alone or in combination with macrolides, tetracyclines or dihydrofolate reductase inhibitors in P. carinii pneumonia.
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Notes |
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References |
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Received 4 January 2000; returned 27 April 2000; revised 16 May 2000; accepted 12 June 2000