Institute of Infectious Diseases and Public Health, University of Ancona, Piazza Cappelli 1, 60121 Ancona, Italy
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Abstract |
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Introduction |
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The ion transport system (ITS) is the target of amiloride, a substituted pyrazinoyl guanidine therapeutically useful as a potassium-sparing diuretic, and its analogue bearing substituents either on the 5-amino nitrogen or on a terminal guanidine nitrogen atom.6 Amiloride analogues such as 5-(N,N-dimethyl)amiloride (DMA), 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and, particularly, 5-(N-methyl-N-isobutyl)amiloride (MIBA) are selective inhibitors of Na+/H+ antiport. These agents are able to affect the mechanisms that regulate the intracellular pH and therefore cause intracellular acidification. Moreover, benzamil, a Na-benzyl derivative of amiloride, is a selective and potent blocker of Na+/H+ and Na+/Ca2+ channels.7 The ability of these drugs to suppress the Na+/H+ antiport in tumour cells and their efficiency in causing cell killing have yet to be evaluated. Little information is available about their toxicity, pharmacokinetics and mechanisms of interaction with other molecules, either in vitro or in vivo.
Cationic peptides and amiloride analogues act primarily on the structure or function of biological membranes. Therefore one can speculate about a possible synergic interaction between these two different groups of molecules. In the present study we have investigated the in vitro activity of four cationic peptides, tested alone and in combination with amiloride and its analogues, on the growth of C. parvum in the A549 cell line.
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Materials and methods |
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Oocysts of C. parvum were isolated from stools of six different patients with AIDS. Stool specimens were stored at 4°C in 2.5% (w/v) potassium dichromate (Sigma-Aldrich, Milan, Italy) for up to 4 months until processing.
Drugs
Cecropin P1, magainin II, indolicidin and ranalexin (all from Sigma-Aldrich) were solubilized in phosphate-buffered saline (PBS, pH 7.2) (BioWhittaker, Walkersville, USA), yielding 1 mM stock solutions. Amiloride, DMA, EIPA, MIBA and benzamil (all from Sigma-Aldrich) were dissolved in 2% dimethylsulphoxide (DMSO) and then brought to the final concentration of 1 mM in distilled water.
Parasite preparation
These procedures have been described in detail previously.8 Briefly, stools were homogenized in physiological saline and filtered through a metal sieve to remove coarse debris. Fatty material was removed by ether sedimentation: stools were separated into 9 mL aliquots and ether (1 mL) was mixed with each aliquot, which was then centrifuged (200g for 20 min). The supernatant with the fatty plug was discarded. Oocysts were successively purified and concentrated by flotation in Sheather's sugar solution (500 g sucrose and 6.5 g phenol in 320 mL of distilled water). The upper layer was removed and collected. Contaminating bacteria were eliminated by three washes in sterile distilled water followed by two washes in 0.05% sodium hypochlorite and finally by incubation in PBS containing penicillin G (2 MU/L), streptomycin (2000 mg/L) and amphotericin B (10 mg/L) for 4 h at 37°C. Excystation of sporozoites was achieved by incubating oocysts in PBS containing 0.25% trypsin and 0.75% sodium taurocholate for 60 min at 37°C. Free sporozoites were pelleted by centrifugation (200g for 20 min) and resuspended in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker). Finally, the six isolates were pooled, counted in a haemocytometer and aliquoted for culture.
Cell cultures
Cells (BioWhittaker) were maintained in 25 cm2 tissue culture flasks. The medium consisted of DMEM with 10% fetal calf serum (BioWhittaker), 1% l-glutamine (BioWhittaker), 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) (Sigma-Aldrich), penicillin G (100 mU/L), streptomycin (100 mg/L) and amphotericin B (0.5 mg/L). Cells were lifted from the surface of flasks using a solution of 0.25% trypsin and 0.53 mM EDTA in PBS and quantified with a haemocytometer. Forty-eight hours before parasite inoculation, A549 cells were plated into 35 mm diameter tissue culture plates at a concentration of 105 viable cells in a total volume of 5 mL. Viability was assessed by trypan blue exclusion. Infection of the cell monolayer was initiated by adding 105 pooled sporozoites in 50 µL of medium. After incubation for 4 h at 37°C in 5% CO2 to allow attachment and penetration of sporozoites, the monolayers were washed with DMEM to remove non-invasive sporozoites, residual oocysts and non-adherent epithelial cells, and 5 mL of new growth medium with or without antimicrobial agents was added. Infected cell cultures were kept at 37°C in 5% CO2 throughout the study.
In vitro studies
Cecropin P1, magainin II, indolicidin and ranalexin were examined singly at concentrations of 0.5, 5 and 50 µM. In experiments to test drug interactions, the three above-mentioned concentrations of each cationic peptide were tested in combination with each ITS inhibitor at concentrations of 5 and 50 µM. Antibiotic-free plates were used as controls. Experiments were performed in triplicate. C. parvum pooled sporozoites were added at a concentration of 105 sporozoites per plate. The monolayers were incubated for 72 h at 37°C in 5% CO2. Following four washes in PBS to remove free oocysts and non-adherent epithelial cells, 5 mL of new growth medium was added and the monolayers were observed under Nomarski interference contrast optics at x1000 magnification.9 Parasite growth was assessed at 48 h after infection in 50 random fields. Only meronts and gamonts were enumerated, to avoid counting non-viable, but adherent, sporozoites or merozoites.
Cytotoxicity assay
The cytotoxicities of drugs and their combinations were determined by the CellTiter 96 AQ cell proliferation assay (Promega Corp., Lyon, France). Controls for each cytotoxicity assay included (i) uninfected cells incubated in DMEM, (ii) infected cells incubated in DMEM and (iii) cells exposed to a freezethaw lysate containing 104 oocyst equivalents in DMEM. The cytotoxicity levels were indicated as no (05%), mild (625%), moderate (2650%) and severe (51100%) cytotoxicity.
Analysis of results
The anti-cryptosporidial activity of each compound and combination was evaluated by comparing the parasite count from plates with antimicrobial-supplemented medium with that from control plates without antimicrobials. The number of parasites was calculated as the mean of the number of organisms observed in three monolayers exposed to the same concentration of drug, by microscopic examination of 50 random fields from each monolayer. Each drug concentration was defined as inhibitory if it caused a significant decrease in parasite count when compared with control plates. The significance of differences was evaluated by Student's t test. To assess the efficacy of drug combinations, the significance of differences between results obtained by testing the peptides in combination with amiloride and its analogues were compared with those of control plates by Student's t test. A P value of 0.05 was considered significant.
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Results and discussion |
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In control plates without drugs the average number of parasites in 50 random fields was 36.8 (range 29.748.1). A high preponderance of meronts over microgamonts was observed. Macrogamonts were not seen at 48 h after infection. A significant inhibitory effect on parasite growth was noted for each peptide at the concentrations of 5 and 50 µM (Table). All peptides were similarly effective, although ranalexin exhibited the highest activity with Meront and gamont growth suppressed by >40% at 50 µM. Cecropin P1, magainin II, indolicidin and ranalexin at concentrations of 5 µM produced a decrease in parasite counts of 11.8, 13.7, 12.2 and 14.7%, respectively. The same peptides at concentrations of 50 µM produced a decrease in parasite counts of 30.6, 33.2, 38.5 and 42.1%, respectively. No peptide was able to inhibit parasite growth completely. Amiloride, DMA, EIPA, MIBA and benzamil had no significant inhibitory effects on C. parvum (data not shown).
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Cationic peptides and ITS inhibitors may be able to perturb biological membrane function, combining the effect of two or more different mechanisms. The membrane-active peptides are interesting compounds, but further research is needed to characterize in more detail the mode of action, the in vivo toxicity, the pharmacokinetics and the mechanisms of interaction with other molecules. The fact that cationic peptides are found in mammals and in insects hints that antimicrobial peptides may be a universal means for defence against infections. Our data suggest that by varying the amino acid sequence it may be possible to develop synthetic peptides with high antimicrobial activity combined with low toxicity for the host: with thousands of theoretical variants of small amino acid peptides, there is ample scope for designing improved synthetic peptides.
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Notes |
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References |
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Received 28 January 1999; returned 6 August 1999; revised 1 September 1999; accepted 27 November 1999