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Epidemiologic evidence suggests a link between morbidity and mortality and levels of particulate matter in the atmosphere. We studied the inflammatory response to inhalation of diesel exhaust particulates (DEP) in normal volunteers. DEP were collected from the exhaust of a stationary diesel engine and were resuspended in an exposure chamber. Ten nonsmoking healthy volunteers were exposed for 2 h at rest to a controlled concentration of DEP (monitored at 200 µg/m3 particulate matter of less than 10 µm aerodynamic diameter [PM10]) or air in a double-blind, randomized, crossover study. Exposures were followed by serial spirometry and measurement of pulse, blood pressure, exhaled carbon monoxide (CO), and methacholine reactivity, as well as sputum induction and venesection for up to 4 h after exposure, and a repeat of all these procedures at 24 h after exposure. There were no changes in cardiovascular parameters or lung function following exposure to DEP. Levels of exhaled CO were increased ater exposure to DEP, and were maximal at 1 h (air: 2.9 ± 0.2 ppm [mean ± SEM]; DEP: 4.4 ± 0.3 ppm; p < 0.001). There was an increase in sputum neutrophils and myeloperoxidase (MPO) at 4 h after DEP exposure as compared with 4 h after air exposure (neutrophils: 41 ± 4% versus 32 ± 4%; MPO: 151 ng/ml versus 115 ng/ml, p < 0.01), but no change in concentrations of inflammatory markers in peripheral blood. Exposure to DEPs at high ambient concentrations leads to an airway inflammatory response in normal volunteers.
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INTRODUCTION |
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INTRODUCTION
METHODS
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Road traffic pollution is a significant public health problem, with particulate pollution from diesel exhaust of increasing concern. Diesel combustion products include gases, semivolatile organic substances, and particles of respirable size (particulate matter of less than 10 µm aerodynamic diameter; PM10). Increased PM10 concentrations are associated with acute episodes of respiratory ill health and increased mortality from both cardiovascular and respiratory causes (1). For example, asthma symptoms increase by 20% when daily PM10 concentrations increase by 50 µg/m3, and increases of 100 µg/m3 and 200 µg/m3 cause 20% increases in hospital admissions and mortality, respectively (2).
Workers exposed to diesel exhaust report respiratory symptoms (3) accompanied by reversible decreases in lung function (6). The lung function changes appear to be caused by the particulate fraction of diesel exhaust, since filtering out the particulates reduces these changes (7, 8). Controlled exposure to diesel exhaust provokes symptoms (9), increases in airway resistance (10), and inflammatory changes within the lung and in peripheral blood (11). The studies in which the latter findings were made used whole diesel exhaust, which does not allow evaluation of the individual contributions of gases or particulates.
Diesel exhaust particles (DEP) are thought to consist of a
carbon core surrounded by trace metals, such as nickel, and
salts to which are adsorbed organic hydrocarbons (12). A
number of these components have inflammatory effects in the
lungs of laboratory animals. For example, intratracheal instillation of ultrafine carbon particles in rats leads to neutrophil
influx into the lungs, and to increases in bronchoalveolar lavage fluid (BALF) concentrations of tumor necrosis factor-
(TNF)-
(13). Intratracheal instillation of nickel in rats causes
severe and sustained inflammation, with generation of free
radicals (14). Inhalation of hydrocarbons also leads to lung inflammation. For example, increases in the activity of a number
of enzyme markers of acute inflammation were found in rabbit lung homogenates after exposure to n-hexane (15). The
foregoing observations indicate that diesel particles themselves can induce airway inflammation. To date, there has
been no evaluation of the effects of diesel particulates alone in
human volunteers. Consequently, our aim was to investigate
the effect of diesel particulates on clinical measures and airway inflammation, using inflammatory markers in induced
sputum, and using exhaled carbon monoxide (CO) as an index
of oxidative stress.
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Subjects
Ten healthy, nonsmoking volunteers (three males and seven females), aged 28 ± 3 yr (mean ± SEM) and with normal lung function (baseline FEV1: 99.8 ± 3.3% predicted) and normal bronchial reactivity (provocative concentration of methacholine causing a 20% decline in FEV1 [PC20] > 64 mg/ml), were recruited. None was atopic on skin-prick testing to common aeroallergens (cat, grass pollen, house dust mite, and Aspergillus fumigatus). The subjects reported no history of respiratory or allergic disease, and were taking no medications. None had suffered an upper respiratory tract infection for at least 8 wk before the study. The study was approved by the ethics committee of the Royal Brompton Hospital and the National Heart & Lung Institute, and written informed consent was obtained from all volunteers.
Study Design
The study was conducted in a double-blind manner, with randomized exposures to DEP or clean air. At screening, subjects underwent spirometry, measurement of exhaled CO, and sputum induction. Before each exposure, baseline spirometry was conducted, baseline measurements of pulse rate, blood pressure, and exhaled CO were made, and blood was taken. Subjects were then exposed at rest to DEP (200 µg/m3 PM10) or to air for 2 h in a challenge chamber. After exposure, spirometry was repeated and pulse, blood pressure, and exhaled CO were measured immediately, and a further sample of blood was taken. Clinical measurements were then repeated half-hourly for 4 h. At 4 h, methacholine challenge was performed and an induced sputum sample was collected. Subjects returned at 24 h after exposure and all measurements other than sputum induction were repeated. After a 4-wk washout period, subjects returned for the alternative (air or DEP) exposure.
Particulate Exposures
DEP were collected with cyclone collectors installed on the exhaust vent of a stationary diesel engine (16). In order to maximize the weight of the particulate mass collected at the end of each engine run, the engine was run at 3,000 rpm with a 300- to 500-kPa brake mean effective pressure (b.m.e.p.) or load, and a 20% exhaust-gas recirculation fraction: conditions chosen to represent the operating conditions of a light-duty vehicle. This included the use of a heat exchanger, four long cyclones, and a standard exhaust gas recirculation route (EGR) (i.e., the EGR flow was tapped at a standard position upstream of the heat exchanger). The collected powder was passed through a 20-µm sieve and was then stored in tightly stoppered glass vials until used. Particles were resuspended through use of a commercial powder disperser (Palas RBG1000). Subjects were exposed for 2 h at rest in a chamber (1.4 × 1.7 × 2.3 m) into which resuspended DEP was injected from a single point. Two fans provided sufficient air movement for dispersion of DEP through the chamber. A stable equilibrium concentration (DEP input matched by losses from settling and exchange of air through the chamber) was reached rapidly after the powder disperser was activated, and could be maintained throughout the challenge period. By altering the rate of activity of the powder disperser, a range of particle concentrations suitable for human challenge studies could be achieved. Particulate concentration within the chamber, close to the head of the volunteer, was monitored with a dust analyzer (Version 5.30E; Grimm Labortechnik GmbH, Germany). This provided real-time PM10 concentrations (expressed as µg/m3), allowing the concentration within the chamber to be maintained at the desired level. Before exposures, the analyzer was calibrated by gravimetric analysis of all particles collected onto a filter during a 2-h test exposure to account for the density of the DEP and any mass contributed by particles smaller than 0.75 µm. The physical structure of DEP was investigated through scanning electron microscopy (SEM), and the particle size distribution of resuspended DEP was examined on a single occasion with a Las-X spectrometer (Particle Measuring Systems, Boulder, CO), which optically measures particles with diameters as small as 0.09 µm.
Assay of DEP for Endotoxin
DEP were analyzed for endotoxin content with the Limulus amebocyte lysate assay (17). Assuming a respiratory volume of 1.5 to 2 m3/h, a 2-h exposure to DEP at a concentration of 200 µg/m3 leads to a total inhaled dose of 600 to 800 µg. Consequently, DEP were prepared at a concentration of 1 mg/ml in endotoxin-free water, and were vigorously vortexed. A 1 ml sample was centrifuged at 18,500 × g (Mikro 24-48R centrifuge; Hettich, Tuttlingen, Germany). The supernatant and a whole sample of DEP solution were analyzed in duplicate for endotoxin, using a commercially available kit (E-Toxate; Sigma Chemicals, Poole, UK) according to the manufacturer's instructions. The lower detection limit of the assay was 6 pg/ml (0.06 endotoxin units/ml).
Lung Function Measurements
FEV1 and FVC were measured with a dry wedge spirometer (Vitalograph, Buckingham, UK). FEV1 values are expressed as percentages of the predicted value. Baseline values were measured after 15 min rest, and the highest of three readings was used. Single readings only were taken at other times. The level of bronchial reactivity was assessed through methacholine challenge performed according to a standardized technique (18). The dose causing a 20% decrease in FEV1 (PC20) was determined by linear interpolation of the concentration-versus-FEV1 response curve.
Carbon Monoxide Levels
Levels of CO were measured with a hand-held CO analyzer (Micro Smokerlyzer CO monitor; Bedfont Scientific Ltd., Upchurch, UK) as previously described (19). Subjects exhaled orally without a noseclip, from TLC to RV, at a flow rate of 5 to 6 L/min, and the plateau CO value at end expiration was recorded. Ambient air CO was measured before each reading, and was subtracted from the highest of three readings. The mean ambient CO concentration for all 220 measurements (i.e., 22 measurements for each of 10 subjects) in the study was 0.5 ppm (SEM = 0.1 ppm), with a mean measured concentration of 3.8 ppm (SEM = 1.0 ppm) before subtraction of the ambient value. The mean ratio of ambient to measured CO was 0.14 (SEM = 0.01), indicating an acceptable signal-to-noise ratio. The reproducibility of exhaled CO measured on two separate days in 30 normal subjects showed an intraclass correlation coefficient of 0.93.
Sputum Induction and Processing
Sputum was induced by inhalation for 15 min of 3.5% saline via an ultrasonic nebulizer (Model 2000; DeVilbiss Co., Heston, UK), as previously described (20). Subjects discarded saliva into a bowl, and washed
their mouths before each expectoration. Secretions collected during
the first 5 min were discarded in order to minimize contamination
with squamous epithelial cells. Subjects were encouraged to cough
deeply at 5-min intervals and at any other time they felt the need to
do so. Secretions expectorated during the final 10 min were kept at
4° C for not more than 2 h before processing. The whole sputum sample was diluted with Hanks' balanced salt solution (HBSS) containing
dithiothreitol (DTT) (Sigma Chemicals), and the solution was vortexed at room temperature. When the solution was homogeneous, the
volume was recorded and the sample was diluted further with HBSS
to a final concentration of 0.05% DTT, and was centrifuged at 300 × g
for 10 min. The supernatant was separated and frozen at 
70° C until further analysis. The cell pellet was resuspended in HBSS. Total cell
counts were made on a hemocytometer slide, using Kimura stain, and
slides were prepared with a Cytospin (Shandon, Runcorn, UK) and
stained with May-Grunwald-Giemsa stain. Differential cell counts
were made by a blinded observer. Three hundred nonsquamous cells
were counted on two slides for each sample. Differential cell counts
are expressed as percentages of nonsquamous cells.
Sputum Supernatant Assays
Tumor necrosis factor (TNF)- and interleukin (IL)-8 concentrations
in sputum supernatants were measured with respective amplified sandwich enzyme-linked immunosorbant assays (ELISAs) as previously described (21) (IL-8 ELISA was from Genzyme Diagnostics,
West Malling, UK). The lower limits of detection of the assays were 8 pg/ml for TNF- and 16 pg/ml for IL-8, respectively. Sputum supernatant levels of myeloperoxidase (MPO) were measured with a commercially available kit (R&D Systems, Abingdon, UK), which employs a sandwich ELISA with a lower limit of detection of 1.5 ng/ml.
Standards for all assays were made up in 0.05% DTT.
Plasma Cytokine Assays
Ten milliliters of venous blood were collected into sodium citrate-containing tubes and centrifuged at 3,000 rpm for 10 min. Plasma was
then removed and immediately frozen at 70° C. P-selectin and IL-6
were measured with commercially available kits (R&D Systems) that
employ a quantitative sandwich immunoassay technique. Samples were
diluted 1:20 (vol/vol) with sample diluent before P-selectin assay.
TNF- was assayed with the same ELISA method as used for sputum
samples, with standards made up in phosphate-buffered saline/10% fetal calf serum.
Statistical Analysis
All comparisons were made between exposures. Where repeated measurements were made (spirometry and exhaled CO), the value representing the greatest change from baseline was used as a summary value in analysis. Spirometric and exhaled CO data were normally distributed and compared through the use of paired t tests. Sputum cell counts and supernatant analyte concentrations were not normally distributed, and were compared through Wilcoxon's signed-ranks test. Repeatability of exhaled CO values was calculated as the intraclass correlation coefficient (Ri) (22) according to the formula: Ri = intersubject variance/(intra- + intersubject variance).
A value of p < 0.05 was considered significant throughout.
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RESULTS |
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Particle Characterization
SEM of resuspended DEP samples showed particle aggregates with a "grape-bunch" appearance. The Las-X spectrometer indicated that approximately 85% of the mass of resuspended DEP was within or below the 1.1-µm size class. The volume mean diameter of the DEP was 0.72 µm. The endotoxin content of both the whole DEP solution and the supernatant was below the detection limit of the assay used (i.e., < 6 pg/ml).
Adverse Events
All subjects completed the trial, and none experienced symptoms during exposure. One subject complained of a sore throat following air exposure, which remitted by the next day.
Cardiovascular Parameters
Pulse rate dropped by ~ 16 beats/min immediately after both air and DEP exposures (Figure 1). However, there were no differences in pulse rate or blood pressure from one exposure to another at any time.
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Lung Function Measurements
There were no differences between mean baseline FEV1 measurements on the first and second exposure days (air: 99.9 ± 3.6% predicted; DEP: 99.2 ± 3.6% predicted), and no changes in FEV1 or FVC after either exposure. There were no changes in bronchial reactivity to methacholine at 4 h or 24 h after either exposure.
Exhaled Carbon Monoxide
There was no difference in mean baseline exhaled CO on the first and second study days. There was a 50% increase in mean exhaled CO levels after DEP as compared with air exposure, with a maximal increase at 1 h after exposure (1 h postexposure values: air = 2.9 ± 0.2 ppm, PM10 = 4.4 ± 0.3 ppm; p < 0.0005; Figure 2).
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Plasma Assays
There was no difference in preexposure values of P-selectin,
IL-6, or TNF- concentrations in plasma on the two exposure
days (Table 1). There was no change in the levels of any of
these factors after either air or DEP exposure (Table 1).
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Sputum Cell Counts and Supernatant Assays
There was a significant, 9% increase in the differential neutrophil count at 4 h after DEP as compared with air exposure (p < 0.01; Figure 3A). This was accompanied by a 7% decrease in
the differential macrophage count (p < 0.05; Figure 3B). There
were no differences in the differential counts of lymphocytes,
eosinophils, or epithelial cells after the air or DEP exposures
(Table 2). There was a significant increase in sputum supernatant MPO concentrations after DEP as compared with air exposure (p < 0.01; Figure 4). There was no significant difference
between the air and DEP exposures in the sputum supernatant
concentrations of IL-8 (air: 2.1 ng/ml, range: 0.4 to 5.2 ng/ml;
DEP: 1.7 ng/ml, range: 0.8 to 29.0; p = 0.2) or TNF- (air: 22.4 pg/ml, range 0.0 to 46.1 pg/ml; DEP: 35.3 pg/ml, range: 0.0 to 80.0 pg/ml; p = 0.1), although in two samples the level of
TNF- was below the limit of detection of the assay.
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We found that inhalation of DEP caused an airway inflammatory response in normal subjects, with increases in neutrophils and MPO in induced sputum, and increased exhaled CO levels. As far we are aware, this is the first published study of the isolated effects of controlled exposure to DEP in humans.
The concentration of 200 µg/m3 PM10 was chosen to reflect high ambient or occupational exposure. Mean PM10 concentrations in cities in the United Kingdom are 20 to 30 µg/m3, with maximum hourly averages of 100 to 300 µg/m3 (23). Traffic is estimated to contribute 83% of PM10 emissions in London (24), with the majority of this derived from diesel vehicles. Monitoring studies show personal exposures of up to 123 µg/ m3 PM10 for traffic wardens over an 8-h shift (25), and of 139 µg/m3 respirable suspended particles for cyclists (26). Truck drivers and car-ferry workers can be exposed to 100 to 1,000 µg/m3 of dust (6, 27).
No chemical analysis was done of the diesel particulates collected in the present study, and we have made no attempt to determine the effect of varying engine load on the chemical composition of particles. However, samples of resuspended DEP showed particle aggregates similar in appearance to the grape-bunch aggregates observed in environmental samples (28), and the < 1.1 µm particle size distribution of 85% of the resuspended DEP in the present study compares well with a recent estimate of the size distribution of ambient diesel exhaust, to which particles smaller than 1 µm contribute 85% of mass (24). Thus, results of the Las-X spectrometry and the SEM inestigations in our study indicate that the resuspension process produces DEP with a similar size distribution and aggregation pattern to that of ambient diesel exhaust (24, 28). We also showed that controlled levels of DEP can be generated and maintained for the duration of human challenge studies. We acknowledge that some loss of volatile organic compounds may have resulted from our storage of collected particles before resuspension of DEP for the challenge studies. However, the results of the current work indicate that pulmonary inflammatory responses still occur with DEP despite a possible reduction in its full irritant capacity. Further investigations are required to determine the exact chemical composition of DEP and the effect that storage may have on their chemical composition.
To date, few exposure studies have been performed on human subjects. Inhalation of DEP has previously been shown to affect lung function in normal subjects, leading to increases in both airway resistance and specific airway resistance (10). However, changes have not been seen in the less sensitive measures of FEV1 and FVC (6, 9). This is consistent with finding in the present study of no effect on FEV1 or FVC in normal subjects after exposure to 200 µg/m3 PM10. No reports exist of whether inhalation of DEP affects breathing pattern, and this was not measured in the present study. It would be of interest to know whether or not DEP exposure causes changes in tidal volume and respiratory rate, since this may magnify or attenuate the irritant effects of inhaled DEP. Further studies of this are warranted.
Unlike those in previous studies, our subjects did not complain of any adverse effects or symptoms from the exposure to DEP itself. Previous exposure studies have shown generally poor tolerance of exposures (9, 10), with subjects complaining of eye and nasal irritation, unpleasant smell, headache, dizziness, nausea, tiredness, and cough. These symptoms were generally short-lived and disappeared by 30 min after exposure. The most likely explanation for this discrepancy is that the symptoms seen in previous studies were caused not by diesel particulates but by other components of diesel exhaust. Diesel exhaust contains many chemicals, and some, when in the gas and particulate phase, such as NO2 and hydrocarbons, including aldehydes, are known irritants. Coughing, nasal irritation, and laryngeal symptoms have been described after exposure to 1 ppm NO2 (29). Occupational exposure to formaldehyde at concentrations of 0.16 to 0.54 mg/m3 causes eye irritation (30), and the unpleasant smell of diesel exhaust may arise from oxygenated and aromatic compounds within the exhaust mixture (31).
The mechanism by which exposure to diesel exhaust causes adverse respiratory effects is poorly understood. Diesel particles may induce oxidant stress in the airways. A study of intratracheal instillation of PM10 in rats (32) demonstrated increases in neutrophils in BALF, accompanied by decreases in reduced glutathione concentrations. The oxidative effect of PM10 was confirmed in vitro by depletion of supercoiled plasmid DNA, an effect reversed by mannitol, a specific hydroxyl radical scavenger (32). Indirect evidence of an oxidant effect of diesel particulates has also been demonstrated in humans. One-hour exposure to diesel exhaust caused a 12-fold increase in nasal lavage concentrations of ascorbic acid (33), which may act as an antioxidant defense.
We indirectly examined the role of oxidative stress in the response to inhaled diesel particulates by measuring exhaled CO levels in the breath of our subjects. Exhaled CO was increased after DEP exposure, indicating an increase in oxidative stress. CO is formed during the degradation of heme to bilirubin, a reaction catalyzed by heme oxygenase (HO) (34). Induction of HO-1 by oxidant stress contributes to antioxidant defense within the lung (35, 36), and bilirubin itself is an antioxidant (37). Therefore, HO-1 induction has been suggested to protect against oxidant-mediated cell injury (38). There is controversy in the literature about the value of exhaled CO as a marker of airway inflammation. Exhaled CO levels are increased in untreated asthmatic individuals, and are reduced with corticosteroid treatment (19, 39). However, a recent abstract reported no difference in exhaled CO levels of atopic subjects and steroid-naive asthmatic individuals as compared with normal controls, despite clear differences in exhaled NO levels (40). Therefore, further work is required to clarify the role of oxidative stress in the response to DEP.
We also found an increase in differential neutrophil counts in induced sputum after DEP exposure. This agrees with previous studies showing increased neutrophils after exposure to whole diesel exhaust in normal subjects (11, 41). Although the increase in neutrophils was small, it was consistent, with an increase in eight of the 10 subjects exposed. This suggests a real inflammatory effect of DEP exposure, although the clinical relevance to this of the observed changes in neutrophil counts is not clear. We have previously shown that inhalation of endotoxin leads to a neutrophilic inflammatory response in induced sputum in normal volunteers (21). This is unlikely to account for the present results, since there was no measurable endotoxin in the DEP samples.
Neutrophil influx may occur in response to release of inflammatory mediators. In vitro, DEP cause release of IL-8, granulocyte-macrophage colony-stimulating factor, and IL-6 from human bronchial epithelial cells (42). However, we found no change in sputum supernatant concentrations of IL-8, and a previous study found no increase in BALF IL-8 concentrations after exposure to whole diesel exhaust (11). This may represent differences between in vitro and in vivo exposures, or may be due to the time point studied. The in vitro work involved long incubations with DEP, with the earliest changes not detected until 7 to 8 h after exposure (43). However, there was a significant increase in sputum supernatant MPO concentrations after DEP exposure. MPO is a protein released from neutrophils, and is a marker of neutrophil activation, which indicates that there is an influx of activated neutrophils in response to DEP exposure. Induced sputum samples are presumed to originate in the lower airways (45). However, inflammatory cells originating in the nose may contaminate induced sputum samples (postnasally in the pharynx). This is relevant to the present study, since the nasal passage is the initial site of contact with inhaled DEP during quiet breathing.
Exposure to whole diesel exhaust has previously been shown
to lead to a systemic response in normal subjects, with significant increases in peripheral blood neutrophils and platelets
(11). In order to further assess the systemic response to inhaled
diesel particulates, we investigated the effects of diesel particulate exposure on serum levels of IL-6, TNF-, and P-selectin.
IL-6 is released from human epithelial cells in response to in
vitro exposure to diesel particulates (44), and IL-6 messenger
RNA (mRNA) is upregulated after nasal challenge with diesel
particulates in human volunteers (46). P-selectin is involved in
the initial stages of neutrophil recruitment into the lung (47).
However, we found no change in plasma concentrations of either of these parameters following exposure to DEP.
In conclusion, the present study confirms that exposure to ambient concentrations of DEP provokes an inflammatory response in the airways of normal subjects that is characterized by an influx of activated neutrophils accompanied by an increase in exhaled CO levels, indicative of oxidant stress. Furthermore, the study gives direct evidence of an important role for the particulate fraction of diesel exhaust in provoking this inflammatory response in human subjects.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. J. A. Nightingale, Department of Thoracic Medicine, National Heart & Lung Institute, Dovehouse Street, London SW3 6LY, UK.
(Received in original form August 20, 1999 and in revised form December 8, 1999).
Acknowledgments: The authors thank Ms. C. Kelly and Mrs. S. Meah for technical help with clinical measurements.
Supported by a pilot grant from the Medical Research Council.
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References |
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REFERENCES
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