Activated eosinophils elicit substance P release from cultured dorsal root ganglion neurons

Allan Garland, Jonathan Necheles, Steven R. White, Scott P. Neeley, Alan R. Leff, Shannon S. Carson, Linda E. Alger, Kyron McAllister, and Julian Solway

Section of Pulmonary and Critical Care Medicine, Department of Medicine, The University of Chicago, Chicago, Illinois 60637; and Division of Pulmonary and Critical Care Medicine, Department of Medicine, Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

This study was performed to test the hypothesis that activated eosinophils or their secretory products can directly stimulate sensory neurons to release their neuropeptides. Neurons derived from neonatal rat dorsal root ganglia (DRG), which synthesize and store sensory neuropeptides, were placed in primary cell culture and were exposed to eosinophils or their bioactive mediators. The resultant release of substance P (SP) was measured by enzyme-linked immunosorbent assay and was expressed as a percent (mean ± SE) of total neuronal SP content. Eosinophils were isolated from human volunteers with a history of allergic rhinitis and/or mild asthma and were activated by incubation with cytochalasin B (5 µg/ml) and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 µM). Activated eosinophils [6 × 106/ml, suspended in Hanks' buffered salt solution (HBSS)] applied to cultured DRG neurons for 30 min increased basal SP release 2.4-fold compared with HBSS-exposed neurons (activated eosinophils 11.10 ± 2.48% vs. HBSS 4.59 ± 0.99%; P = 0.002), whereas neither nonactivated eosinophils nor cytochalasin B and FMLP in HBSS influenced SP release. Additional cultured DRG neurons were exposed to soluble products made by eosinophils. Compared with SP release under control conditions (2.37 ± 0.34%), major basic protein (MBP) increased release in a concentration-related fashion (e.g., 3 µM MBP: 6.23 ± 0.67%, P = 0.006 vs. control), whereas neither eosinophil cationic protein (3 µM), eosinophil-derived neurotoxin (3 µM), leukotriene D4 (500 nM), platelet-activating factor (100 nM), nor H2O2 (100 µM) affected SP release. These studies demonstrate that activated eosinophils can stimulate cultured DRG neurons directly and suggest that MBP may be the responsible mediator.

sensory C fiber; asthma; airway hyperresponsiveness; airway inflammation

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

BOTH EOSINOPHILS and sensory C fiber neurons may be involved in the pathophysiology of asthma. Eosinophils are prevalent in the airways of asthmatics and can release various bioactive mediators that potentially contribute to the bronchoconstriction, bronchial hyperreactivity, and inflammation that characterize this condition (8, 29, 40, 52). Nociceptive, unmyelinated C fiber nerve endings are present throughout the airways and contain neuropeptides [such as substance P (SP), neurokinin A, and calcitonin gene-related peptide] that are released locally upon stimulation of these afferent fibers (45). These neuropeptides can mediate bronchoconstriction and bronchovascular hyperpermeability in animal models of asthma (20, 26, 31, 44), and they might be involved in human asthma as well (36, 48).

Several lines of evidence indicate that the products of C fibers can influence the function of eosinophils (6, 13, 23, 35, 56) in ways that promote eosinophil influx or bioactive mediator release. Conversely, physiological studies performed in vivo or in intact airway preparations suggest that allergic reactions and eosinophil products in particular can lead to the release of tachykinins from C fiber neurons (10, 11, 15, 32, 42, 50) as well. The present study was performed to gain insight into the mechanisms by which eosinophils stimulate sensory neuropeptide release. During eosinophilic airway inflammation, airway C fibers are exposed to multiple cell types and to the mechanical consequences of airway narrowing (which might lead to mechanical stimulation of these neurons). As such, eosinophil products might stimulate C fiber neuropeptide release directly through action on the neurons themselves or indirectly through action on an intermediate cell type that releases another mediator that stimulates C fiber neuropeptide release or that induces motion that stimulates the neuron mechanically. In this study, we test the hypothesis that activated eosinophils and their secretory products can stimulate C fibers directly using an in vitro system of isolated human eosinophils and primary cultures of neurons derived from rat dorsal root ganglia (DRG), which synthesize and release SP in response to typical C fiber stimuli. In this system, neurons can be exposed to cellular and/or biochemical stimuli in the absence of confounding mechanical influences and in almost complete isolation from the influence of other cell types.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

DRG cell cultures. DRG were harvested from 1- to 5-day-old Sprague-Dawley rats and were placed into primary culture as described previously (19, 54). Briefly, neonatal rats were decapitated, their spinal cords were removed, and DRG were isolated (~45-50/pup). DRG neurons were dissociated by enzymatic digestion (collagenase, dispase, and trypsin) and mechanical trituration and then were plated at a density of 5 ganglia/cm2 in 35-mm Primaria plastic culture wells coated with laminin and fibronectin. DRG neuron cultures were maintained at 37°C in 5% CO2 and were fed with 1.5 ml of fresh isotonic growth medium [consisting of Ham's F-12 (GIBCO-BRL) supplemented with 1.5% fetal bovine serum, 5% heat inactivated rat serum, 100 µg/l 7S murine nerve growth factor (NGF; Sigma Chemical), 5 g/l glucose, 10 ml/l minimum essential medium vitamin solution, 10 ml/l L-glutamine, 1 mM CaCl2, and 10 ml/l penicillin-streptomycin solution (GIBCO-BRL)] on the first, third, fifth, and sixth days after plating. During the second, third, and fifth days, the growth medium also contained 10 µM cytosine arabinoside (Sigma Chemical) to suppress overgrowth by ganglionic fibroblasts. After culture in this manner for 7 days, >75% of DRG neurons immunostain positively for SP and exhibit increases in intracellular calcium ion concentration upon capsaicin exposure (19).

Seven days after plating, DRG neuron cultures were exposed to experimental intervention. As described below, DRG neurons were bathed during experimental intervention in a neuropeptide assay buffer consisting of growth medium without sera, NGF, or penicillin-streptomycin but with addition of 0.2% fatty acid-free bovine serum albumin. Sera, NGF, and antibiotics were removed from assay buffer to minimize potential interference with experimental interventions or with subsequent SP assay by enzyme-linked immunosorbent assay (ELISA; see below). To inhibit enzymatic degradation of SP, the angiotensin-converting enzyme inhibitor enalaprilat (10 µM; Merck, Sharp & Dohme) and the neutral endopeptidase inhibitor phosphoramidon (3 µM; Sigma Chemical) were also added. The effect of human eosinophils on cultured DRG neurons was evaluated by coincubation in Hanks' buffered salt solution (HBSS) with added enalaprilat and phosphoramidon, rather than in assay buffer.

To perform an experimental intervention, culture wells were washed two times with assay buffer (AB) or HBSS and then were exposed for 30 min to 1.5 ml of AB or HBSS at 37°C in 5% CO2 containing the desired additive or eosinophils. This supernatant "exposure sample" (ES) was then collected from the culture plate, placed in 5% acetic acid, boiled for 10 min, and kept on ice for further processing. Neuropeptides remaining within neurons in the culture dish were liberated by adding 1.5 ml of boiling 5% acetic acid, removing the cells from the plastic with a cell scraper, boiling for 10 min, and then sonicating for 30 s. To remove cell debris and larger proteins, an equal volume of 0.1% trifluoroacetic acid was added to the mixture, which was then centrifuged at 10,000 g for 20 min at 4°C; the resulting supernatant [designated the "cell sample" (CS)] was saved for further processing.

Determination of SP release. SP release from cultured neurons was determined as a marker of neuropeptide storage granule secretion. Numerous studies have demonstrated that SP is stored and released with the other sensory neuropeptides, including neurokinin A and calcitonin gene-related peptide (reviewed in Ref. 45). The SP content of each sample was established by ELISA (17) after partial purification with C18 Sep-Pak (Waters, Division of Millipore, Milford, MA) as described previously (19). This method allowed for reproducible measurement of 10-10,000 fmol SP/culture well with cross-reactivity for neurokinin A of 0.1%. Data were expressed as the fractional release of SP, defined as the percent of total SP contained within the cultured DRG neurons that was released during the 30-min exposure, and were calculated as 100 × SPES/(SPES + SPCS), where SPES and SPCS are the SP contents of the corresponding ES and CS samples.

Isolation and activation of human eosinophils. Human eosinophils were isolated from volunteers with eosinophil counts of 211-510 eosinophils/µl blood. All subjects had a history of allergic rhinitis and/or mild asthma. None was taking oral or parenteral corticosteroids at the time of the study. Eosinophils were isolated from peripheral blood as previously described (34) using a modification of the negative immunomagnetic separation technique of Hansel et al. (21). Briefly, 60-120 ml of heparinized blood were diluted 1:1 with HBSS without calcium, layered onto Percoll (1.089 g/ml), and centrifuged at 1,000 g for 30 min at 4°C. Erythrocytes within the granulocyte layer were lysed by addition of hypotonic saline. The remaining granulocytes (1.5-2.0 × 108 cells) were incubated with 75-100 µl of anti-CD16 immunomagnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany), and then neutrophils were removed by passing the suspension through a magnetic separation column. After repeat hypotonic lysis of the few contaminating erythrocytes, the remaining granulocytes were washed and then resuspended in HBSS (6 × 106 eosinophils/ml). Eosinophils isolated in this way are normodense, >97% pure, and have >98% viability (30, 34). Activation of eosinophils was accomplished by incubation for 30 min with cytochalasin B (5 µg/ml; Sigma Chemical) and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 µM; Sigma Chemical; see Refs. 33 and 34).

Specific interventions on neuron cultures. Experiments were performed to evaluate whether activated eosinophils are capable of stimulating cultured DRG neurons to release tachykinins. Groups of cultured DRG neurons were exposed at 37°C for 30 min in 1.5 ml of solution to 1) HBSS alone (negative control), 2) HBSS, cytochalasin B, and FMLP, 3) nonactivated eosinophils (i.e., 6 × 106 eosinophils/ml in HBSS), or 4) activated eosinophils (i.e., 6 × 106 eosinophils/ml preexposed for 30 min in HBSS to cytochalasin B and FMLP).

In additional studies, individual inflammatory mediators thought to be released by activated eosinophils were tested for their effects on neuronal SP release. Groups of DRG neuron cultures were exposed, for 30 min at 37°C, to 1.5 ml of AB to which was added either no additive (negative control), 10 µM capsaicin (Sigma Chemical) as a positive stimulation control, 50 mM KCl as a second positive control stimulus, 1 µM eosinophil major basic protein (MBP), 3 µM MBP, 10 µM MBP, 3 µM eosinophil cationic protein (ECP), 3 µM eosinophil-derived neurotoxin (EDN), 100 µM H2O2, 500 nM leukotreine (LT) D4 (Cayman Chemical, Ann Arbor, MI), or 100 nM C16 platelet-activating factor (PAF; Sigma Chemical). We studied LTD4 rather than LTC4 because LTC4 is converted to LTD4 in the airways, and each stimulates the same cysteinyl LT receptor. MBP, ECP, and EDN were kindly supplied by Dr. Gerald Gleich of Mayo Clinic and were solubilized in sodium acetate buffer, pH 7.4. Concentrations of individual eosinophil mediators were chosen based on published studies that demonstrate substantial biological effects in other in vitro model systems (1, 2, 4, 15, 22, 24, 25, 27, 28, 37, 38, 41, 49, 55, 57, 60) and parallel or exceed values found in bronchoalveolar lavage fluid or sputum from human asthmatic subjects (12, 14, 18, 39, 47). The final concentration of acetate buffer in AB applied to cells was 10% in MBP, ECP, and EDN groups; to test for a possible confounding effect of this substance, an additional group of DRG neuron cultures was exposed to AB with 10% sodium acetate buffer. Last, to test for the potential contribution of cationic charge to their activation, a group of DRG neurons in culture was exposed to 1 µM poly-L-lysine (mean molecular weight 18,000; Sigma Chemical).

SP content of eosinophils. To ensure that results were not confounded by any preformed SP that may be released from eosinophils (5), two separate samples of 9 × 106 nonactivated eosinophils in 1.5 ml of HBSS were boiled for 10 min, sonicated, and assayed as above for SP content.

Data analysis. All data are expressed as means ± SE, and P values <0.05 were considered statistically significant. To test for an effect of activated eosinophils or of inflammatory mediators on SP release from cultured DRG neurons, a comparison was made among relevant groups using one-way analysis of variance (ANOVA) followed by Fisher's least significant difference test to compare between groups if overall significance was found by ANOVA. For evaluations of added mediators, the positive stimulation groups (i.e., capsaicin and KCl) were included in the analysis.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

SP content of DRG neurons and of eosinophils. Experimental interventions were performed on 142 culture wells of rat DRG neurons. The total (ES + CS) SP content was 1,405 ± 837 (SD) fmol/well, similar to previously reported values (19). The SP content of two separate samples of 9 × 106 nonactivated eosinophils was <= 1 fmol each. Thus the eosinophils employed in the current study did not contribute substantially to the SP detected in culture supernatants or cell extracts.

Influence of known C fiber stimulants on SP release from cultured DRG neurons. The influence of a 30-min coincubation of cultured DRG neurons with capsaicin or KCl is shown in Fig. 1. Each of these known C fiber neuron secretagogues significantly augmented release of SP from our cultured DRG neurons.


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Fig. 1.   Effect of positive control interventions on release of substance P from cultured dorsal root ganglia (DRG) cells (as % of total cellular content). Capsaicin- and KCl-exposed wells demonstrate substantial substance P release in response to these known C fiber stimulants. Values are means ± SE. Number of wells per group appears in parentheses above each column. LSD, least significant difference; CON, control.

Influence of activated eosinophils on SP release from cultured DRG neurons. The influence of a 30-min coincubation of cultured DRG neurons with human eosinophils on the release of SP from cultures is shown in Fig. 2. Compared with the control HBSS group, 6 × 106 activated eosinophils/ml caused a statistically significant 2.4-fold greater SP release, whereas neither nonactivated eosinophils nor the combination of activating substances (FMLP and cytochalasin B) in the absence of eosinophils augmented tachykinin release. Thus substances released from activated eosinophils must be responsible for the observed stimulation of cultured DRG neuron neuropeptide release.


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Fig. 2.   Effect of exposure to eosinophils on release of substance P from cultured DRG neurons (as % of total cellular content). Values are means ± SE. Number of wells per group appears in parentheses above each column. Compared with the no-stimulation control exposure [Hanks' buffered salt solution (HBSS)], incubation with activated eosinophils caused a significant augmentation of substance P release (P = 0.002, Fisher's test). In contrast, neither nonactivated eosinophils nor the substances used to active the eosinophils [1 µM N-formyl-methionyl-leucyl-phenylalanine (FMLP) + 5 µg/ml cytochalasin B] caused significant augmentation.

Influence of individual eosinophil mediators on SP release from cultured DRG neurons. Figure 3 shows the effect of a 30-min exposure to inflammatory mediators on cultured DRG neurons. Among the individual eosinophil inflammatory substances tested, only eosinophil MBP augmented SP release. Neither ECP, EDN, LTD4, PAF, nor H2O2 had any effect on the concentrations studied. The polycation poly-L-lysine also caused a significant increase in release of neuronal SP stores. Note that the acetate buffer in which the eosinophil proteins were solubilized did not affect basal release itself. Figure 4 demonstrates that eosinophil MBP elicits cultured DRG neuron tachykinin release in a concentration-dependent fashion in which 10 µM MBP increases SP release fourfold compared with the negative control condition.


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Fig. 3.   Effect of exposure to inflammatory mediators on release of substance P from cultured DRG neurons (as % of total cellular content). Values are means ± SE. Number of wells per group appears in parentheses above each column. Of the eosinophil-derived substances, only major basic protein (MBP) caused an increased release (P = 0.006 compared with control conditions of isotonic assay buffer, Fisher's test). Poly-L-lysine (PLL) also augmented release (P < 0.001). Sodium acetate buffer, the solvent for MBP, EDN, and ECP, had no effect on basal release. ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; PAF, platelet-activating factor.


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Fig. 4.   Concentration-response curve of the effect of eosinophil MBP on release of substance P from cultured DRG neurons (as % of total cellular content). Values are means ± SE. Number of wells per group appears in parentheses above each datum. Compared with control conditions of isotonic assay buffer containing MBP solvent (10% sodium acetate buffer), each concentration of MBP significantly augmented release (P < 0.05, Fisher's test). Substance P released by 10 µM MBP was also significantly higher than that at 1 µM (P < 0.05, Fisher's test).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
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This study demonstrates that inflammatory substances released from activated eosinophils directly stimulate cultured DRG neurons to secrete their preformed neuropeptides. Activated, but not quiescent, eosinophils stimulated SP release from cultured DRG neurons when the two cell types were coincubated for 30 min (Fig. 2). Of the individual mediators known to be released from activated eosinophils, only MBP mimicked this effect of activated eosinophils on DRG neurons (Figs. 3 and 4). As such, it is likely that MBP released from activated eosinophils mediate their influence on SP-containing neurons in our culture system. Previous studies demonstrated that airway allergen challenge (31, 32, 50) or exposure to MBP or polycationic proteins (10, 11) lead to endogenous tachykinin release in vivo or in situ. Our present study extends current knowledge by demonstrating that eosinophil products can act directly on sensory neurons to stimulate their neuropeptide release and by implicating MBP as a key mediator of this direct effect.

Of the likely bioactive mediators released from activated eosinophils, only MBP elicited concentration-dependent cultured DRG neuron tachykinin release (Figs. 3 and 4), whereas neither ECP, EDN, LTD4, PAF, nor oxygen radicals (as represented by H2O2) had any effect in the concentrations tested. Thus it appears that MBP may be largely responsible for the stimulatory effect of activated eosinophils on our cultured DRG neurons. Because the polycation poly-L-lysine also dramatically stimulated DRG neuron SP release (Fig. 3), it is conceivable that the mechanism of action of MBP (also a polycation at physiological pH) might depend primarily on its positive charge. However, two findings suggest that features of MBP other than its charge also play a role in its observed effect. First, the lot of poly-L-lysine that we used had an average of 86 cationic charges per molecule, whereas the positive charge of MBP is approximately +16 per molecule (7). Thus 1 µM poly-L-lysine has a cationic charge concentration that is only one-half that of 10 µM MBP, yet it induced greater SP release than did MBP (1 µM poly-L-lysine 16.92 ± 2.00%; 10 µM MBP 8.09 ± 0.81%). Second, if charge per se is a potent stimulus for SP release from C fibers, then ECP with its average cationic charge of 14.5 per molecule (7) should be almost as potent a SP secretagogue as MBP. Yet, our studies (Fig. 3) clearly demonstrate that this is not so. Thus it seems likely that some as yet undefined property of MBP beyond its charge alone contributes to its ability to stimulate cultured DRG neurons. Although our study did not identify this property, MBP recently has been shown to cause selective M2 receptor inhibition, thus augmenting neural secretion in parasympathetic nerves (16). We also have shown that MBP causes airway contraction by a neurally mediated mechanism that also is related to the production of constrictor prostaglandins in guinea pigs (46). Perhaps cultured DRG neurons are stimulated by MBP, in part, through a mechanism related to its actions in these systems.

An important advantage of our DRG neuronal culture system is that it facilitates evaluation of the direct influence of activated eosinophils and their bioactive mediators on DRG neurons in the virtual absence of potentially confounding factors related to mechanical stimulation of sensory nerves or to intermediary factors elaborated by other resident or migratory cells within intact allergen-inflamed airways. However, it is true that some ganglionic fibroblasts do persist within our neuronal cultures, even though cytosine arabinoside is used to minimize their numbers. Thus it is conceivable that some unidentified factor elaborated from these few fibroblasts might have contributed to the apparently direct effects of activated eosinophils and/or MBP on cultured DRG neurons.

There are other potential limitations of our study. First, our cultured DRG neurons differ physically from the C fiber neurons innervating in an intact airway in several respects. Neuronal axons are transected during harvest; thus, only DRG neuron cell bodies are placed in primary cell culture, although axons do regrow partially within 7 days (19). In vivo, DRG neurons exhibit plasticity of neuropeptide expression after axonal transection (43, 59), with a greater proportion of DRG cells expressing neuropeptide Y, galanin, and vasoactive intestinal peptide and fewer DRG neurons expressing SP. Application of NGF at the injury site prevents SP loss (59), however. In our experiments, the entire neuronal cell body is exposed to the stimulus, whereas in intact airways only the nerve terminals would be exposed. Furthermore, neuronal receptor expression might change as a consequence of cell culture. Together, these certain and potential physical differences between intact C fiber neurons and our cultured DRG neurons dictate against direct extension of results from our in vitro model system to cell-cell interactions in inflamed human airways in vivo.

Second, because we tested for individual effects on cultured DRG neurons of only a subset of the bioactive substances produced by eosinophils (52), it remains possible that other eosinophil-derived products contribute to the effects of activated eosinophils. In addition, it is well established that various compounds can sensitize or inhibit C fiber activation to other stimulants (15, 51, 53, 58), but we tested only individual mediators on our cultured DRG neurons. Thus it is plausible that two or more eosinophil products might act in a more complex fashion to influence C fiber degranulation in intact airways.

Third, we exposed cultured DRG neurons in vitro to concentrations of eosinophils and their products based on practical constraints of the eosinophil isolation procedure, on mediator concentrations previously shown to exhibit activity in a variety of other systems, and on published values of their concentrations in sputum or lavage fluid from human asthmatic subjects. It is thought that eosinophils kill parasites by releasing their granule contents directly at the parasite surface, resulting in large local concentrations of eosinophil-derived substances (3). By extension, the concentration of eosinophil-derived mediators at the neuronal surfaces in our coincubation experiments could conceivably be either much greater or much less than that achieved at airway nerve terminals in vivo. Indeed, the observation by Costello et al. (9) that antigen challenge of sensitized guinea pigs causes eosinophil infiltration within intrapulmonary nerves supports the latter possibility.

Fourth, it is conceivable that the disparate species of origin of the neonatal DRG neurons (rat) and the eosinophils (human) used in this study influenced our results. However, we could not use rat eosinophils because we could not collect the large numbers required for study, and we could not, of course, obtain human DRG for culture. It is also possible that cultured DRG cells obtained from adult, rather than neonatal, rats might have behaved differently. We chose to study cultured neonatal rat DRG cells because the large majority exhibit phenotypic features of C fiber neurons (19), and we have previously demonstrated that their secretion of sensory neuropeptides in response to diverse stimuli in vitro at least qualitatively parallels corresponding responses in vivo (19, 54).

In conclusion, we demonstrate that activated human eosinophils directly stimulate rat DRG neurons in primary cell culture to release SP and that MBP alone exerts a similar effect. Based on this result, we speculate that the cell-cell interactions demonstrated here may also operate in intact airway tissues during eosinophilic inflammation and so may contribute to the pathobiology of human asthma.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants AI-34566, HL-56399, HL-46368, HL-48257, HL-08519, and HL-02376 and by grants from the American Lung Association and the Sprague Foundation.

    FOOTNOTES

Address for reprint requests: J. Solway, University of Chicago, MC 6026, 5841 S. Maryland Ave., Chicago, IL 60637.

Received 11 February 1997; accepted in final form 21 August 1997.

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Abstract
Introduction
Methods
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Discussion
References

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