Apoptosis, oxidative metabolism and interleukin-8 production in human neutrophils exposed to azithromycin: effects of Streptococcus pneumoniae

Crystal C. Kocha, David J. Estebana,{dagger}, Alex C. China, Merle E. Olsonb,c, Ronald R. Readb,c, Howard Ceria,b,c, Douglas W. Morcka,c and Andre G. Bureta,c,*

a Departments of Biological Sciences and b Microbiology and Infectious Diseases, and c Biofilm Research Group, The University of Calgary, Calgary, Alberta, Canada, T2N 1N4


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pathogen virulence factors and the host inflammatory response cause tissue injury associated with respiratory tract infections. The azalide azithromycin has demonstrated efficacy in the treatment of these infections. It has been demonstrated previously that induction of polymorphonuclear leucocyte (PMN) apoptosis is associated with minimization of tissue damage and inflammation in the lung. We hypothesized that, in addition to its antibacterial effects, azithromycin may promote apoptosis. The aim of the study was to determine the effects of azithromycin on PMN apoptosis, oxidative function and interleukin-8 (IL-8) production in the presence or absence of Streptococcus pneumoniae, in comparison with penicillin, erythromycin, dexamethasone or phosphate-buffered saline. Human circulating PMNs were assessed for apoptosis (by annexin V labelling and ELISA), oxidative function (by nitroblue tetrazolium reduction) and IL-8 production (by ELISA). Azithromycin significantly induced PMN apoptosis in the absence of S. pneumoniae after 1 h (10.27% ± 1.48%, compared with 2.19% ± 0.42% in controls) to levels similar to those after 3 h induction with tumour necrosis factor-{alpha} (8.73% ± 1.86%). This effect was abolished in the presence of S. pneumoniae. Apoptosis in PMNs exposed to the other drugs was not significantly different from that in controls. Azithromycin did not affect PMN oxidative metabolism or IL-8 production. In summary, azithromycin-induced PMN apoptosis may be detected in the absence of any effect on PMN function, and the pro-apoptotic properties of azithromycin are inhibited in the presence of S. pneumoniae.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The pathogenesis of bacterial infection in the respiratory tract is a result of the combination of bacterial virulence factors and the host inflammatory response.1,2 Eukaryotic cells die by necrosis or apoptosis. When polymorphonuclear neutrophils (PMN) die by necrosis in inflamed tissues, the cells swell and their plasma membrane ruptures. Cytotoxic compounds such as elastase, hypochlorous acid and oxygen radicals, released by neutrophils recruited to sites of acute infection, can damage surrounding tissues and induce necrosis in neighbouring neutrophils.3,4 Neutrophils injured in this fashion promote the release of more cytotoxic compounds and pro-inflammatory mediators, which amplify and perpetuate inflammation.5 In contrast, when neutrophils die by apoptosis (also called programmed cell death), they lose the ability to release granule enzymes. The plasma membrane remains intact, the cytoplasm and the nucleus become condensed and DNA is cleaved into mono- and oligonucleosomes (which are commonly used as indicators of apoptosis).6,7 Apoptotic cells are phagocytosed by macrophages, stimulated by various surfacesignalling molecules, including phosphatidylserine exposed on the outer leaflet of the cell membrane.8,9 Unlike the phagocytosis of necrotic neutrophils, ingestion of apoptotic neutrophils fails to trigger the release of pro-inflammatory mediators by macrophages.8,10,11 Therefore, preservation of membrane integrity and clearance of apoptotic cells from the site of infection minimize inflammation and host tissue damage.

The antimicrobial activity of the azalide azithromycin appears to be enhanced by the ability of this compound to reach high concentrations in phagocytes.12 Azithromycin has a high affinity for neutrophils, which facilitates its delivery to the site of infection.13 A number of other studies have suggested that macrolides have anti-inflammatory effects, partly because they modulate the production of pro-inflammatory cytokines.14,15 Moreover, recent findings indicate that the anti-inflammatory benefits of tilmicosin, a macrolide used in the treatment of bovine pneumonic pasteurellosis, are associated with its induction of neutrophil apoptosis and concomitant inhibition of local leukotriene B4 (LTB4) release.2

While the clinical effectiveness of azithromycin may result mainly from its favourable pharmacodynamics, it may also promote apoptosis. The aims of this study were (i) to determine the effects of azithromycin on human neutrophil apoptosis, in the presence or absence of a common respiratory pathogen, Streptococcus pneumoniae, and (ii) to assess whether such effects may be associated with altered functional properties of neutrophils, i.e. oxidative metabolism and interleukin-8 (IL-8) synthesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Drugs

Azithromycin (Pfizer Canada, Montreal, Quebec, Canada), penicillin G (Sigma Diagnostics, St Louis, MO, USA) and erythromycin (Sigma) were dissolved in phosphate-buffered saline (PBS) (pH 7.1) to give a final concentration of 0.05 mg/L in each test. Dexamethasone (Schering Canada, Pointe-Claire, Quebec, Canada) was diluted in PBS (pH 7.1) to give a final concentration of 0.02 mg/L in each test.

Bacteria

S. pneumoniae 14259, a human clinical isolate obtained from Dr R. R. Read, Foothills Hospital, Calgary, Alberta, Canada, was grown on Columbia blood agar (CBA) plates at 37°C, suspended in PBS (pH 7.1) and diluted to 1.5 x 108 bacteria/mL using a 0.5 McFarland standard (Dalynn Laboratory Products, Calgary, Alberta, Canada); it was then serially diluted to determine the concentration of bacteria , and used either live or after sonication (for 4 h on ice; 85% duty cycle, output control setting 4; W-350 Sonifier, Branson Ultrasonics Corporation, Danbury, CT, USA).

Neutrophils

Blood was collected by venipuncture from healthy human volunteers and collected in sodium heparin Vacutainers (Becton Dickinson, Franklin Lakes, NJ, USA). Neutrophils were purified by density centrifugation using Histopaque 1119/1077 (Sigma) (800g, 30 min). The neutrophil layer was removed, washed in Hanks' balanced salt solution (HBSS) (Gibco BRL, Life Technologies, Grand Island, NY, USA), and centrifuged (250g, 10 min). Contaminating erythrocytes were lysed by resuspending the pellet in sterile distilled water for 30 s, and osmolarity was restored by adding 2 x HBSS. After washing in 1 x HBSS, purified cells were resuspended in either HEPES-buffered RPMI 1640 medium (Sigma) supplemented with 0.05 mg/L l- glutamine or Ca2+/Mg2+-free PBS (pH 7.1) (for nitroblue tetrazolium (NBT) assay), to a final concentration of 106 cells/mL. Cells were counted in a haemocytometer and viability was assessed by trypan blue (0.1%) exclusion. PMN purity was assessed by staining with Diff Quick (Baxter Healthcare, Miami, FL, USA) and direct microscopy differential leucocyte counts. Neutrophils were incubated (37°C, 5% CO2) for various times with PBS (pH 7.1) (controls), or with azithromycin, penicillin, erythromycin, dexamethasone or tumour necrosis factor-{alpha} (TNF-{alpha}) (R&D Systems, Minneapolis, MN, USA), with or without live S. pneumoniae or a lysate from S. pneumoniae at a 10:1 bacteria:cells ratio for all assays, excepting a 4:1 ratio for annexin V labelling.

Cfu counts

S. pneumoniae was incubated with PMNs and drugs (37°C, 5% CO2) at a ratio of 10:1 for 2 and 6 h, and plated on CBA plates after serial dilutions. Colonies were counted after 24 h incubation at 37°C.

Annexin V and propidium iodide labelling

Neutrophil apoptosis and necrosis were assessed by double staining with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide using an annexin V Fluos kit (Boehringer, Mannheim, Germany) as previously described.4 Briefly, after 1 h incubation (37°C, 5% CO2) with or without drugs and with or without bacterial lysate, cells were centrifuged (Biofuge A; Heraeus Sepatech, Germany) (200g, 5 min), then washed in PBS (pH 7.1). Cells were centrifuged again, resuspended in FITC-conjugated annexin V and propidium iodide staining solution and incubated for 15 min in the dark at room temperature. Samples were then centrifuged (200g, 5 min), resuspended in PBS (pH 7.1) and observed by fluorescence microscopy (at 450 and 535 nm) (Aristoplan; Leitz, Germany) at 400x magnification and the percentages of apoptotic and necrotic cells were determined.

As a positive control for the annexin V/propidium iodide labelling system, PMNs were incubated with 5 ng/mL (approximately 100 U/mL) recombinant human TNF-{alpha} in PBS (pH 7.1) with 0.1% bovine serum albumin (BSA) for 1, 2 or 3 h. TNF-{alpha} at this concentration is known to induce neutrophil apoptosis in a time-dependent manner.16

Cell death ELISA

Neutrophils (105 cells/mL) were incubated (37°C, 5% CO2) for 6 h with or without drugs and with or without live bacteria and frozen at –70°C. Samples were assayed for apoptosis with a cell death ELISA (Boehringer), which detects the histone region of mono- and oligonucleosomes formed during apoptosis. Plates were read at 405 nm (THERMOmax microplate reader; Molecular Devices, Menlo Park, CA, USA) at intervals for 58 min, starting 1 min after addition of substrate. Results were expressed as the ratio of the sample absorbance to the mean absorbance of the control, as previously.2

Nitroblue tetrazolium assay

Oxidative function of PMNs incubated (37°C, 5% CO2) for 1 h with or without drugs and with or without bacterial lysate was assessed by NBT reduction. Cells were incubated (37°C, 5% CO2) in chamber slides (Nalge Nunc International, Naperville, IL, USA) for 20 min with NBT (Sigma) in the presence or absence of 2 mg/L phorbol 12-myristate 13-acetate (PMA) (Sigma). Cells were fixed with methanol and counterstained with safranin (1%). The percentage of oxidatively active cells with blue formazan crystals in their cytoplasm was counted under oil immersion (1000x) with a light microscope (Carl Zeiss, North York, Ontario, Canada).

Interleukin-8 assay

PMNs (2 x 107 cells/mL) were incubated for 2 h with drugs (37°C, 5% CO2), then washed in PBS (pH 7.1) and incubated (24 h, 37°C, 5% CO2) with or without S. pneumoniae lysate. Cells were then centrifuged (at 800g for 10 min) and the supernatant was collected. IL-8 production was assessed by a Quantikine human IL-8 sandwich enzyme immunoassay (R&D Systems) according to the manufacturer's instructions.

Statistical analysis

Results were expressed as mean ± s.e.m. and compared by one-way analysis of variance (ANOVA) followed by Tukey's test for multiple comparison. P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Neutrophils

Viability of purified PMNs incubated in PBS (pH 7.1) or RPMI 1640 was >95%, and cell purity was >98%.

Cfu counts

To verify that the pharmacological compounds used in these experiments maintained their antibacterial properties in the presence of PMNs, S. pneumoniae cfu were counted 2 and 6 h after incubation with the various drugs. After 6 h, but not after 2 h, incubation with PMNs, azithromycin, penicillin and erythromycin significantly decreased S. pneumoniae numbers compared with controls (Figure 1Go). After 6 h, bacterial numbers in preparations containing azithromycin were significantly lower than those exposed to other antibiotics. Incubation with PMNs and dexamethasone did not affect bacterial numbers.



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Figure 1. S. pneumoniae recovery (log10cfu) after 2 h and 6 h incubation of PMNs with azithromycin ({blacksquare}), penicillin (•), erythromycin (x) and dexamethasone ({blacktriangleup}). Antibiotic treatments are significantly different from control ({bigcirc}) and dexamethasone at both times (n = 5). *, P < 0.05 compared with control. {triangleup}, P < 0.05 for azithromycin compared with all groups.

 
Neutrophil apoptosis

PMN apoptosis was first assessed by annexin V/propidium iodide fluorescent labelling. As this method detects early apoptotic events, i.e. the translocation of phosphatidylserine on to the outer plasma membrane leaflet, experiments were carried out after 1 h of incubation. This system allowed apoptotic cells (those labelled with FITC–annexin V only) to be distinguished from necrotic cells (those labelled with both FITC–annexin V and propidium iodide). TNF-{alpha} induced PMN apoptosis in a time-dependent fashion (Figure 2Go). In the absence of S. pneumoniae lysate, azithromycin induced PMN apoptosis at levels similar to those seen in cells incubated with TNF{alpha} for 3 h (Figure 3aGo). Cells incubated with penicillin, erythromycin or dexamethasone were not different from controls. Addition of bacterial lysate inhibited the induction of apoptosis by azithromycin (Figure 3bGo).



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Figure 2. Apoptosis in PMNs treated with 5 ng/ml TNF{alpha} for 1, 2 or 3 h (n = 4), determined by annexin V/propidium iodide labelling. The bar labelled ‘Control’ represents the mean of the control values (n = 6) from 1, 2, and 3 h. *P < 0.05 compared with control.

 


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Figure 3. Apoptosis in neutrophils determined by annexin V/propidium iodide labelling after 1 h incubation with azithromycin (AZI), penicillin (PEN), erythromycin (ERY) or dexamethasone (DEX), (a) without or (b) with S. pneumoniae lysate (4 cfu:1 PMN). The TNF{alpha} sample is the 3 h incubation value from Figure 2Go. *P < 0.05 compared with control. **P < 0.01 compared with control and other drugs (n >= 5).

 
In an attempt to explain this inhibition of apoptosis by bacterial lysate, cell necrosis was assessed. As shown in Figure 4Go, S. pneumoniae did not affect PMN necrosis. Neutrophil counts in samples incubated with azithromycin (6.06 x 106 ± 4.81 x 105 cells/mL), S. pneumoniae alone (6.88 x 106 ± 7.04 x 105 cells/mL) or azithromycin and S. pneumoniae (7.15 x 106 ± 3.31 x 105 cells/mL) were not significantly different from those in cells exposed to saline alone (7.43 x 106 ± 4.61 x 105 cells/mL).



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Figure 4. PMN necrosis in samples incubated with azithromycin (AZI), penicillin (PEN), erythromycin (ERY) or dexamethasone (DEX) and with (shaded bars) or without (white bars) S. pneumoniae lysate determined by annexin V/propidium iodide labelling. There was no significant difference between any samples (n >= 5).

 
To confirm the above findings, PMN apoptosis was also assessed with a system that detects late-stage apoptosis, i.e. the formation of oligonucleosomes. In order to reflect this delay, apoptosis was measured after 6 h of incubation with drugs. Induction of neutrophil apoptosis by azithromycin was confirmed by ELISA in the absence of S. pneumoniae (Figure 5aGo). The colorimetric reaction was rapid enough to be detected after 1 min of incubation with reagent. As was seen with annexin V labelling, S. pneumoniae abolished this effect (Figure 5bGo). Incubation with penicillin, erythromycin or dexamethasone did not affect PMN apoptosis in the presence or absence of S. pneumoniae.



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Figure 5. Apoptosis detected by cell death ELISA after 6 h incubation (a) without S. pneumoniae and (b) with live S. pneumoniae. Absorbance readings were plotted as a ratio of the sample absorbance to the control, which was set at 1. Azithromycin ({blacksquare}), penicillin (•), erythromycin (x), dexamethasone ({blacktriangleup}), and control ({bigcirc}). *P < 0.05 compared with control and dexamethasone (n >= 5).

 
Oxidative function and IL-8 production

As the results presented above showed that azithromycin induces apoptosis, additional studies determined the effects of the drugs on oxidative metabolism and IL-8 synthesis, two important parameters of PMN integrity in the context of their antibacterial function. None of the drugs affected oxidative function in resting or stimulated neutrophil populations after 1 h of incubation (Figure 6aGo), a time at which azithromycin-induced PMN apoptosis was already detectable (Figure 3aGo). Similarly, the drugs did not affect oxidative function when incubated with bacterial lysate (Figure 6bGo).



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Figure 6. Oxidative activity determined by the percentage of resting (white bars) and PMA-stimulated (black bars) PMNs able to reduce NBT. Samples were incubated for 1 h with azithromycin (AZI), penicillin (PEN), erythromycin (ERY) or dexamethasone (DEX), either without (a) or with (b) S. pneumoniae lysate. In (a) and (b), all stimulated groups are significantly different from all resting groups. There was no significant difference between drug-treated and control samples within any group (n = 5).

 
IL-8 production by activated PMNs plays a central role in the recruitment of additional neutrophils to the site of infection, and as such represents a significant functional parameter in the context of the present study. Exposure of neutrophils to S. pneumoniae lysate promoted synthesis of IL-8 (Figure 7Go). IL-8 secretion was not affected by any of the antibiotics in the presence or in the absence of S. pneumoniae lysate compared with controls (Figure 7Go). In contrast, dexamethasone significantly inhibited S. pneumoniae lysate-induced IL-8 synthesis.



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Figure 7. IL-8 production by PMNs incubated for 24 h with (black bars) or without (white bars) S. pneumoniae lysate following a 2 h incubation with azithromycin (AZI), penicillin (PEN), erythromycin (ERY) or dexamethasone (DEX). There was no significant difference between antibiotic-treated and control samples within groups treated with or without lysate (n >= 4). *P < 0.05 compared with sham-treated control.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study investigated the effects of azithromycin on human circulatory PMNs. The results indicate that this azalide induces apoptosis in human PMNs, and that azithromycin-induced neutrophil apoptosis may be detected in the absence of any effect on oxidative metabolism or IL-8 production in vitro. Incubation of neutrophils with S. pneumoniae inhibited the induction of apoptosis by azithromycin.

The present study shows that after 1 h of exposure to azithromycin, apoptosis can be detected in human neutrophils. Recent findings have demonstrated that induction of bovine neutrophil apoptosis by the veterinary macrolide tilmicosin may contribute to its anti-inflammatory benefits in vivo.2 This hypothesis is consistent with the observation that physiological removal of apoptotic neutrophils in the inflamed lung in vivo promotes the resolution of inflammation.10,11 Furthermore, accumulation and necrosis of neutrophils in tissues contribute to the propagation of inflammation in a number of inflammatory diseases including infectious pneumonia, whereas apoptotic neutrophils may be eliminated without spilling pro-inflammatory products in situ.17,18 The clinical significance of azithromycin-induced apoptosis thus warrants further investigation.

Annexin V labelling detects the exposure of phosphatidylserine on the outer leaflet of the cell membrane, an early event in cell death.4,19 Cells that are labelled by annexin V, but not by propidium iodide, are apoptotic. This observation, consistent with previous studies, was validated in the present experimental setting by using TNF-{alpha} as a positive control.16 Human neutrophils exposed to azithromycin for 1 h in vitro were committed to apoptotic death, while erythromycin, penicillin and dexamethasone did not induce apoptosis. In addition, formation of mono- and oligonucleosomes, a late event in apoptotic cell death, was detected by ELISA after 6 h of incubation with azithromycin. Other studies suggest that erythromycin (as well as roxithromycin, clarithromycin and midecamycin) may also induce apoptosis in neutrophils in vitro.20 The fact that erythromycin-induced apoptosis was not observed in the present study may be explained by the different incubation times and antibiotic concentrations; previous studies assessed induction of neutrophil apoptosis after 24 h of incubation, and used a minimum concentration of 1 mg/L.17 Therefore, our findings suggest that azithromycin induces neutrophil apoptosis more quickly and at lower drug concentrations than erythromycin. Additional studies will help assess whether this is is a result of the well established higher affinity for neutrophils of azithromycin versus erythromycin.12 The anti-inflammatory glucocorticoid dexamethasone has been reported to inhibit neutrophil apoptosis,21 but this effect was not seen in this study. Again, as inhibition of apoptosis in human PMNs was observed previously after 24 h exposure to dexamethasone, the 1 or 6 h incubations used in the present study may not have been sufficient to inhibit apoptosis significantly.21 Nevertheless, the present study demonstrates that azithromycin induces apoptosis in human neutrophils to a greater degree than other drugs. As apoptosis limits the ability of neutrophils to damage surrounding tissues and prevents further amplification of inflammation, additional studies are needed to determine whether this mechanism may contribute to the clinical efficacy of this azalide in the treatment of infectious pneumonia.

The results suggest that the pro-apoptotic effect of azithromycin is abolished when neutrophils are incubated with S. pneumoniae lysate. Levels of necrosis were not different between samples with or without lysate, indicating that the low level of apoptosis was not a result of an increase in necrosis. The mechanisms whereby S. pneumoniae products may reverse or inhibit the apoptotic signal remain unclear. Lipopolysaccharide of Gram-negative bacteria has been shown to inhibit TNF-{alpha}-induced apoptosis.22 It remains to be shown whether Gram-positive bacteria have components that also inhibit apoptosis, and whether this effect may contribute to the difficulty in treating such infections. Further studies are also needed to assess whether this inhibition occurs in the presence of other bacterial species, including Haemophilus influenzae, a respiratory tract pathogen against which azithromycin is very effective.12 Interestingly, a recent report speculated that macrolides may significantly reduce the inflammatory injury associated with the presence of H. influenzae in the lower respiratory tract, via unknown mechanisms.23

Previous studies have suggested that macrolides have anti-inflammatory effects, but the mechanisms underlying this benefit remain unclear.14 A number of experiments have focused on the effects of macrolides on cytokine production.15,2327 Although still the subject of current debate, findings from these studies indicate that erythromycin, roxithromycin and clarithromycin may reduce the production of pro-inflammatory IL-1, IL-6, IL-8 and TNF. The results presented herein indicate that after 2 h of exposure to azithromycin, the capability of a given neutrophil population to release IL-8 remains unchanged, despite the concurrent commitment of some of these cells to undergo programmed cell death. In addition, erythromycin A derivatives, including azithromycin, roxithromycin and clarithromycin, may inhibit the oxidative response of neutrophils in a time- and concentration-dependent fashion.2832 Again, findings from the present study indicate that 1 h of exposure to azithromycin is insufficient to reduce oxidative metabolism in human neutrophils, despite detectable levels of apoptosis in these cells. Taken together, these observations suggest that the effects of azithromycin on both IL-8 production and oxidative metabolism are time- and concentration-dependent.

In summary, azithromycin induces apoptosis in human neutrophils more effectively than erythromycin or penicillin. The oxidative function of resting or stimulated neutrophils and the production of IL-8 may remain unchanged, indicating that these functions are not affected in the early stages of azithromycin-induced apoptosis. The pro-apoptotic properties of azithromycin are inhibited by S. pneumoniae. Future studies will assess whether the induction of neutrophil apoptosis by azithromycin contributes to the clinical efficacy of this azalide in the treatment of lower respiratory tract infections, and whether inhibition of neutrophil apoptosis by S. pneumoniae contributes to the tissue injury caused by this pathogen.


    Acknowledgments
 
This work was supported by Natural Sciences and Engineering Research Council of Canada, the Alberta Heritage Foundation for Medical Research, and Pfizer Canada. A. G. B. is funded by the Margaret Gunn Endowment for Animal Health Research.


    Notes
 
{dagger} Present address. Saint Louis University, St Louis, MO 63108, USA. Back

* Corresponding author. Department of Biological Sciences, The University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4; Tel: +1-403-220-2817; Fax: +1-403-289-9311; E-mail: aburet{at}acs.ucalgary.ca Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 2 September 1999; returned 5 January 2000; revised 21 January 2000; accepted 22 February 2000