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Environment
Original article
Exposure to wood smoke particles produces inflammation in healthy volunteers
  1. Andrew J Ghio1,
  2. Joleen M Soukup1,
  3. Martin Case1,
  4. Lisa A Dailey1,
  5. Judy Richards1,
  6. Jon Berntsen2,
  7. Robert B Devlin1,
  8. Susan Stone1,
  9. Ana Rappold1
  1. 1The National Health and Environmental Effects Research Laboratory, US EPA, Research Triangle Park, North Carolina, USA
  2. 2TRC Environmental, Chapel Hill, North Carolina, USA
  1. Correspondence to Andrew Ghio, Environmental Public Health Division, HSF, US EPA, Campus Box 7315, 104 Mason Farm Road, Chapel Hill, NC 27711, USA; ghio.andy{at}epa.gov

Abstract

Objectives Human exposure to wood smoke particles (WSP) impacts on human health through changes in indoor air quality, exposures from wild fires, burning of biomass and air pollution. This investigation tested the postulate that healthy volunteers exposed to WSP would demonstrate evidence of both pulmonary and systemic inflammation.

Methods Ten volunteers were exposed to filtered air and, 3 weeks or more later, WSP. Each exposure included alternating 15 min of exercise and 15 min of rest for a total duration of 2 h. Wood smoke was generated by heating an oak log on an electric element and then delivered to the exposure chamber. Endpoints measured in the volunteers included symptoms, pulmonary function tests, measures of heart rate variability and repolarisation, blood indices and analysis of cells and fluid obtained during bronchoalveolar lavage.

Results Mean particle mass for the 10 exposures to air and WSP was measured using the mass of particles collected on filters and found to be below the detectable limit and 485±84 μg/m3, respectively (mean±SD). There was no change in either symptom prevalence or pulmonary function with exposure to WSP. At 20 h after wood smoke exposure, blood tests demonstrated an increased percentage of neutrophils, and bronchial and bronchoalveolar lavage revealed a neutrophilic influx.

Conclusions We conclude that exposure of healthy volunteers to WSP may be associated with evidence of both systemic and pulmonary inflammation.

  • Particulate matter
  • air pollution
  • inflammation
  • toxicology
  • particulates

https://doi.org/10.1136/oem.2011.065276

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    What this paper adds

    • Exposure to wood smoke particles (WSP) impacts on human health.

    • As few studies have directly evaluated human health endpoints following controlled WSP exposure, this investigation tested the postulate that healthy volunteers exposed to WSP would demonstrate evidence of both pulmonary and systemic inflammation.

    • Blood tests and bronchoscopy with lavage demonstrated increased neutrophils in both blood and lavage after wood smoke exposure.

    • These results indicate that human exposure to WSP may be associated with evidence of both systemic and pulmonary inflammation.

    Introduction

    The burning of wood impacts on domestic, occupational and environmental exposures. Domestic wood burning is the major source of particle exposure in many developed countries and can elevate indoor exposures to particulate matter (PM) to hundreds of micrograms per cubic metre.1 2 Firefighters and those living close to a forest fire are exposed to wood smoke particles (WSP) which can approach concentrations measured in milligrams per cubic metre.3 The burning of biomass (defined as wood, charcoal, agricultural residue or animal dung) is the main source of energy for domestic heating and cooking for more than two billion people worldwide and can expose individuals to smoke particle levels which intermittently exceed 10 mg/m3.4 Additionally, wood smoke can be a significant contributor to PM in ambient air5; in the USA, between 8% and 85% of fine PM is attributed to residential wood burning.2 6 7

    Wood smoke exposure acutely affects respiratory symptoms and exacerbates chronic obstructive pulmonary disease and bronchial asthma, leading to increased respiratory-related hospital admissions.3 8 9 Smoke from wildfires can also cause elevated levels of inflammatory biomarkers in the peripheral blood.10 11 Similarly, the burning of biomass, including wood, has been associated with an acute loss of lung function and a clinical and pathological presentation consistent with chronic obstructive pulmonary disease among non-smoking individuals.12–15 Finally, the burning of biomass has been included in the WHO's list of humanity's 10 greatest health concerns as a result of an association with acute lower respiratory infections, the chief cause of death in many populations around the world.4 16 17

    Few studies have directly evaluated human health endpoints following controlled WSP exposure.7 18–20 We tested the hypothesis that healthy volunteers exposed to WSP would demonstrate evidence of both pulmonary and systemic inflammation.

    Methods

    Study population

    Ten healthy, non-smoking volunteers were solicited by advertisements and included in this study. Subjects were between 18 and 40 years of age, lifetime non-smokers with no history of respiratory diseases and not taking any medication during participation in the study. Subjects were informed of the procedures and potential risks and each signed a statement of informed consent. The protocol and consent form were approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects.

    Study design

    Prior to the exposure (pre), symptoms were queried using a computerised questionnaire, peripheral blood was collected, and lung function and heart rate variability (HRV) were measured. Subjects then entered a chamber (approximately 1.83 m×1.83 m×2.44 m in size), sat on a recumbent bicycle ergometer, and were exposed to filtered air for 2 h. The exposure included alternating exercise for 15 min on the cycle ergometer with 15 min rest for a total duration of 2 h. The exercise was adjusted such that subjects breathed at a ventilatory rate of 25 l/m2 body surface area per minute. Heart rate, ECG and SpO2 were monitored continuously during the exposure. Immediately following the exposure (post), assessment of symptoms, blood tests, pulmonary function and HRV were repeated. The following morning (follow-up), there was a final round of symptom assessment, blood tests, pulmonary function testing and determination of HRV. Finally, the subject had bronchoscopy with lavage (approximately 20 h after exposure). After a minimum of 3 weeks, the entire protocol was repeated but with WSP exposure.

    Exposures

    The wood smoke was generated by heating red oak wood on an electric heating element (Brinkmann, Dallas, Texas, USA) in a Quadrafile 3100 woodstove (Colville, Washington, USA). The smoke was extracted from the chimney and injected into the chamber air stream. The smoke concentration was controlled using a tapered element oscillating microbalance (TEOM; Thermo Fisher Scientific, Franklin, Massachusetts, USA) to measure the chamber concentration. A Model DR-4000 DataRAM (Thermo Fisher Scientific) was also used to measure the concentration. The particle number concentration was measured with a Model 3022A CPC (TSI, Shoreview, Minnesota, USA) and the number size distribution was measured with a Model 3080L SMPS (TSI). The particle concentration was verified by weighing filters obtained using a versatile air pollution sampler (VAPS; URG, Chapel Hill, North Carolina, USA). Electrically heating the wood provided a controlled simulation of adding wood to an existing fire. The chamber temperature and humidity were controlled to approximately 22°C and 40%, respectively. Teflon filters from three exposures to air and WSP were washed in 1N HCl and metals determined using inductively coupled plasma optical emission spectroscopy (ICPOES; Model Optima 4300D, Perkin Elmer, Norwalk, Connecticut, USA).

    Symptom questionnaire

    Before, immediately after (within 30 min) and approximately 20 h after the exposure, volunteers completed a symptoms questionnaire enquiring about fatigue, headache, eye irritation, nasal irritation, rhinitis, inspiratory pain, shortness of breath, cough, wheezing and chest tightness.

    Blood samples

    Before, immediately after and approximately 20 h after the exposure, blood was drawn by standard venipuncture technique. Haemoglobin, haematocrit, white blood cell counts, differentials, indices of coagulation and inflammation, cytokines and carboxyhaemoglobin (COHb) were measured. Cytokine levels in the serum were measured using ELISA kits (R&D Systems, Minneapolis, Minnesota, USA). COHb in an anti-coagulated sample of venous blood was determined using the IL 682 Co-oximeter (Instrumentation Laboratory, Bedford, Massachusetts, USA).

    Lung function

    Spirometry and diffusing capacity were measured before, immediately after and approximately 20 h after the exposure. The subject inhaled completely and then exhaled rapidly and completely via a tube into a spirometer (Vmax, Viasys Healthcare System, Yorba Linda, California, USA). From this manoeuvre, forced vital capacity, forced expiratory volume in 1 s (FEV1) and peak flow (PEF) were derived. Diffusing capacity was also quantified and reported as the mean of two best values (Vmax). American Thoracic Society recommendations were followed in performing spirometry and measuring diffusing capacity.21 22

    Ambulatory electrocardiography, HRV and QT measurements

    Continuous ambulatory ECGs (Holter ECGs) were collected for approximately 24 h (Mortara Instrument, Milwaukee, Wisconsin, USA). Data were also collected during three 30 min periods (immediately prior to exposure and approximately 1 h and 20 h after the completion of each exposure) while the subjects rested quietly in a darkened room. Indices of HRV in both the time and frequency domains were calculated from edited records (ECG Superscribe). The time domain parameters were calculated over a 24 h period starting at the beginning of the first resting phase. Frequency domain and repolarisation parameters were calculated from the Holter ECGs obtained during each of the three 30 min resting periods.

    Bronchoscopy with lavage

    Using a standard protocol, volunteers underwent bronchoscopy with lavage 20 h after exposure to filtered air and WSP. The fibre-optic bronchoscope was wedged into a segmental bronchus of the right middle lobe. Six aliquots of sterile saline were instilled and immediately aspirated. The first was 20 ml and this fraction was labelled the bronchial lavage (BL) sample. The remaining five aliquots were 50 ml each and the return from this bronchoalveolar lavage (BAL) was designated the alveolar sample and is considered to reflect the environment of the distal respiratory tract. Cell differentials were performed on cytocentrifuged slides stained with a modified Wright stain (Leukostat Solution, Fisher Scientific). At least 200 cells per slide were counted. There was no difference in recovery of BL and BAL fluid between individuals exposed to filtered air and to WSP and all fluid recoveries were within 10% of one another. Consequently, soluble components were normalised per millilitre fluid. Cytokine levels were measured using ELISA kits (R&D Systems). Lavage protein and albumin concentrations were quantified using the Pierce Coomassie Plus Protein Assay Reagent (Pierce, Rockford, Illinois, USA) and an immunoprecipitin assay (Diasorin, Stillwater, Minnesota, USA), respectively.

    In vitro exposures to WSP

    WSP used for in vitro investigation was obtained by extraction from the stainless steel chimney above the woodstove. This material was sonicated in water (Sonic Dismembrator, Fisher Scientific, Pittsburgh, Pennsylvania, USA) to disaggregate the particles. Ionisable metal in the particle was quantified as that concentration displaced into 1N HCl (1.0 mg/1.0 ml) after 1 h agitation. Metal concentrations were quantified in triplicate using ICPOES. Acellular oxidant generation by WSP was measured employing thiobarbituric acid (TBA)-reactive products of deoxyribose.23

    BEAS-2B cells were grown in keratinocyte growth medium (KGM). Cellular oxidant generation was determined using DCF (dichlorodihydrofluorescein) fluorescence. BEAS-2B cells were loaded for 30 min with 10 μM DCFH-DA (dichlorodihydrofluorescein diacetate) and exposed to either PBS or 100 μg WSP/ml PBS. Fluorescence was measured on a spectrofluorimeter with excitation and emission set at 485 and 535 nm, respectively. Oxidant generation was expressed as the ratio of fluorescence relative to cells exposed to PBS after loading with DCFH-DS.

    BEAS-2B cells were then incubated with either KGM or 100 μg/ml WSP in KGM for 24 h. Cytotoxicity was measured using lactate dehydrogenase (LDH) release (ThermoFisher Diagnostic, Middletown, Virginia, USA). IL-8 concentrations in cell media were measured using an ELISA kit (R&D).

    Statistics

    Changes in biological parameters induced by the exposure to wood smoke were assessed using paired t tests. Lung function, blood and HRV frequency domain endpoints measured immediately following and approximately 20 h after the exposures were normalised by their pre-exposure levels (post/pre, follow-up/pre). The normalisation is used to control for within subject day to day variation. HRV frequency domain endpoints were additionally transformed by natural logarithm to satisfy the basic normality assumption. The magnitude of change between two exposures is summarised as per cent change relative to the pre-exposure levels in lung function and blood endpoints, and relative to air exposure in HRV frequency domain endpoints (RR). Normalisation was not used on the endpoints measured only at follow-up (eg, BAL and BL measures) or over a 24 h interval (HRV time domain) and these endpoints were compared directly between two exposures. Statistical analysis was performed using R statistical software (v 2.11.1). In vitro data were evaluated using one-way analysis of variance; the post hoc test employed was Duncan's multiple range test.

    Results

    Exposures to WSP included increased concentrations of particle, total hydrocarbon and carbon monoxide (see online supplementary table S1). Number median diameter approximated 0.1 μm and did not vary substantially between the subject exposures (see online supplementary table S1). Particles collected on filters during human wood smoke exposures were low in metals (see online supplementary table S2).

    Volunteers showed no changes in symptom prevalence with exposures to either filtered air or WSP. There were complaints of fatigue, headache and eye irritation, but these were noted by volunteers as frequently before as after both the filtered air and particle exposures. There was no nasal irritation, rhinitis, inspiratory pain, shortness of breath, cough, wheezing or chest tightness. Pulmonary function testing was normal and there were no significant changes following exposures to either filtered air or WSP (see supplementary online table S3).

    White blood cell count (figure 1A) and absolute numbers of lymphocytes and monocytes did not change significantly with exposure to WSP. The absolute neutrophil count in the blood increased by 23% (approaching significance with p=0.12, 95% CI −5% to 41%) after WSP exposure, while at follow-up the absolute count had increased significantly by 17% (p<0.01, 95% CI 11% to 44%). Similarly, expressed as per cent of total cells in the blood after the exposure to WSP, the percentage of neutrophils increased by 20% (p=0.09, 95% CI −3% to 40%; figure 1B) and by 11% at follow-up (p=0.04, 95% CI 1% to 23%; figure 2B). The percentage of lymphocytes and monocytes both decreased, but the changes did not reach significance (p=0.14 (95% CI −22% to 4%) at post, and p=0.06 (95% CI −33% to 1%) at follow-up for lymphocytes and p=0.09 (95% CI −0.34% to 0.03%) and p=0.19 (95% CI −34% to 8%) for monocytes figure 1C,D).

    Figure 1
    Figure 1

    Total white blood cell counts and per cent neutrophils, lymphocytes and monocytes in blood. Comparison of normalised values revealed significant increases in blood neutrophils 20 h after WSP relative to pre-exposure values (B). There were no changes in total white blood cell count (A), while decrements in lymphocytes (C) and monocytes (D) approached significance. *Significant difference between air and WSP exposures. WSP, wood smoke particles.

    Figure 2
    Figure 2

    Blood carboxyhaemoglobin (COHb) and lactic dehydrogenase (LDH) values. Exercise appeared to increase the concentration of COHb. However, comparison of normalised values revealed no significant changes in COHb between the air and WSP exposures (A). LDH was observed to increase both immediately after and approximately 20 h after WSP exposure (B). *Significant difference between air and WSP exposures. WSP, wood smoke particles.

    Baseline blood cytokine levels (IL-1β, IL-6, IL-8 and TNF-α) were very low as expected; nevertheless, IL-1β demonstrated a significant increase immediately following wood smoke exposure (p<0.0, 95% CI 23% to 59% at post; see online supplementary table S4). The concentration of COHb was decreased by 23% at follow-up but did not reach significance (p=0.11, 95% CI −54% to 7%). Blood levels of markers of thrombosis including von Willebrand's factor, d-dimer, plasminogen, plasminogen activator-1 and tissue plasminogen activator were not changed following exposure to WSP (see online supplementary table S5). However, blood LDH concentrations were elevated after particle exposure (p=0.11, 95 CI −3% to 19% and p=0.02, 95% CI 1% to 7%) immediately after the exposure and at follow-up, respectively) (figure 2B).

    Exposure to WSP resulted in minimal changes in autonomic nervous system indices as measured by Holter monitoring. Analysis of HRV in the time domain showed no significant changes in SDNN, pNN50 or RMSSD either immediately or 20 h after exposure. Similarly, no significant changes in repolarisation variables (QTc, P wave complexity, T wave complexity, QRS complexity and QTc dynamics) were seen at either time point following exposure. However, there were marginally significant increases in frequency domain end points (figure 3). Immediately following particle exposure, there was an 11.2% increase in the normalised high frequency (p=0.07) and a 19.4% increase in the high to low frequency ratio (p=0.10) relative to that after air exposure. There was also a 16.8% decrease in maximal heart rate immediately following particle exposure (p=0.016).

    Figure 3
    Figure 3

    Change in indices of heart rate variability. There was a significant decrement in maximal heart rate immediately following (post) but not 20 h after (follow-up) WSP exposure relative to air.

    In the BL and BAL fluid, neutrophils increased after WSP exposure relative to air (p<0.01 (95% CI 11.0 to 18.9) and p<0.01 (95% CI 6.1 to 12.7), respectively). The absolute increase in percentage neutrophils was small in both the BL and the BAL fluid (figure 4A,B). Following exposure to WSP, there was no change in concentrations of either cytokines or α-1 antitrypsin in the BAL fluid (see online supplementary table S6). There was no evidence in the lavage of an injury following exposures to WSP as measured by protein concentrations (47±20 following air exposure and 48±33 μg/ml following wood smoke exposure for total protein and 28±14 and 27±17 μg/ml for albumin, respectively).

    Figure 4
    Figure 4

    Bronchial and bronchoalveolar lavage neutrophils. The percentages of neutrophils in bronchial (A) and bronchoalveolar (B) lavage were increased after WSP relative to air exposure. *Significant increase using t test of independent means. WSP, wood smoke particles.

    Calcium was the metal found in the greatest concentration in the WSP displaced from the stainless steel chimney (see online supplementary table S2). Iron, magnesium, aluminium, zinc, chromium, nickel and copper were also quantifiable. These particles generated oxidants in an acellular assay for TBA reactive products of deoxyribose (figure 5A). This production of oxidants was significantly inhibited by the inclusion of either the metal chelator deferoxamine or the hydroxyl scavenger dimethylthiourea (both at a final concentration of 1.0 mM). In a similar manner, BEAS-2B cells produced oxidants measured as DCF fluorescence following exposure to 50 and 100 μg/ml WSP (figure 5B). This elevation was significant at both 1 and 2 h only for the exposure at 100 μg/ml. Finally, cytotoxicity and IL-8 were measured in BEAS-2B cells exposed to 0, 50 and 100 μg/ml WSP for 24 h. There was no change in LDH release following 24 h exposure to WSP. Relative to unexposed cells, the concentrations of IL-8 were increased following incubation with 50 and 100 μg/ml WSP (33±15, 81±28 and 104±31 pg/ml, respectively).

    Figure 5
    Figure 5

    In vitro oxidant generation by WSP. Acellular oxidant generation was measured employing thiobarbituric acid (TBA)-reactive products of deoxyribose at 532 nm. WSP (100 μg/ml) significantly increased oxidant generation, while the metal chelator deferoxamine and the hydroxyl scavenger DMTU (both at a final concentration of 1.0 mM) inhibited the production of hydroxyl radical (A). Similarly, there was significant production of oxidants by BEAS-2B cells, measured as DCF fluorescence, after exposure to 100 μg/ml WSP (B). No significant changes were noted between 1 and 2 h incubations. Differences between the individual values were compared using one-way analysis of variance. *Significant increase relative to incubation with saline and no inhibitor (A) and no WSP (B). WSP, wood smoke particles.

    Discussion

    Human exposures were almost exclusively to WSP. CO was the only gas that was present in significantly elevated levels in wood smoke exposures relative to clean air exposures, but the levels were not high enough to significantly affect blood COHb. WSP diameter size was approximately 0.1 μm, which is comparable to that in other studies of PM from wood combustion.24 There was little to no metal in the WSP collected during human exposure. The exposure used in this investigation (500 μg/m3) was high relative to the more common measurements of indoor levels of particles emitted from wood stoves. However, indoor exposures to WSP vary with the type and quality of wood, characteristics of the house, cooking and heating methods, activity patterns and season, resulting in a wide range of particle levels throughout the day. Use of a wood stove can elevate indoor PM to concentrations over 500 μg/m3.25 In homes using wood as the main energy source, PM levels can also exceed 500 μg/m3 and can even approach 1000 μg/m3.26

    Subjects demonstrated neither symptoms nor changes in pulmonary function tests with exposures to WSP; this lack of effect on symptoms and physiological endpoints is comparable to the results of a prior investigation into WSP at a concentration of 240–280 μg/m3.19 20 However, there were changes in blood indices of inflammation following WSP exposures. While the white blood cell count did not vary with exposure to WSP, the percentage and absolute number of neutrophils increased. Inflammatory changes in blood endpoints have been observed in other controlled human exposures to WSP. For example, volunteers exposed to 240–280 μg/m3 WSP for 4 h had increased concentrations of amyloid A protein.18–20 This is comparable to exposures to ambient particles which have demonstrated systemic inflammation with elevations in white blood cell counts and increases in C reactive protein.27 Despite some evidence of systemic inflammation following WSP exposure, there were no changes in blood cytokine concentrations. Similarly, there was no evidence of a pro-coagulative state after PM exposures. This contrasts with altered blood coagulation factors found in other studies of wood smoke exposure.18–20

    Minimal changes in cardiac endpoints were observed after exposure to WSP. Many studies have reported decreased high frequency (HF) indices of HRV following exposure to PM, but most of these studies examined elderly cohorts. Increased indices of HRV have been observed among younger people exposed to PM.28 29 HF is closely linked to vagal activity, while low frequency (LF) indices are thought to include both baroreceptor (parasympathetic) and vasomotor (sympathetic) influences. It is suggested that particle exposure causes a shift from parasympathetic to sympathetic tone. We found a decrease in maximal heart rate immediately after exposure, which we have not observed in any of our other PM studies, and are uncertain how to interpret this finding.

    BL and BAL revealed a neutrophilic influx into the lower respiratory tract following exposure to WSP. The magnitude of the neutrophilic influx was comparable to that observed following exposure to an approximately equivalent mass of concentrated ambient particles.30 While this is the first study to demonstrate elevations in neutrophils following WSP exposure, prior exposures to this particle similarly showed changes in endpoints reflecting pulmonary inflammation, including increased concentrations of NO in exhaled breath and malondialdehyde in condensate.18–20 In contrast, one study which exposed humans to WSP and employed bronchoscopy with lavage showed no evidence of inflammation in the lung as regards cell numbers, exhaled NO or mediator concentrations.7 Differences between our investigation and this negative study include dose (500 and 224 μg/m3, respectively) and duration of exposure (2 h and 3 h, respectively). The higher dose of WSP employed in our study may have affected the observed lung inflammation.

    Our exposure of healthy subjects to WSP resulted in no elevation of mediators in the lavage fluid. It is not clear what directed the transport of inflammatory cells into the lower respiratory tract. One possible explanation is differential binding of cytokines to the WSP, which has been previously reported.31 Alternatively, the lack of elevation of cytokines following exposures to WSP may be explained by the approximately 100-fold diluting effect of human BAL.

    The WSP obtained from the stainless steel chimney showed much higher metal concentrations than that collected on filters during human exposures. Ageing of the particles with complexation of available metals is likely to explain these differences. In vitro studies have previously demonstrated that WSP produce oxidative stress and pro-inflammatory events.1 32 33 Our in vitro investigation confirmed that WSP can generate oxidants in both acellular and cellular systems. Some of this oxidative stress appeared to be associated with metal in the PM. Incubation of BEAS-2B cells with WSP caused release of an inflammatory mediator (ie, IL-8). These data suggest that this specific emission source particle may follow the general model of biological activity for all PM with oxidative stress triggering phosphorylation-dependent cell signalling, activation and translocation of nuclear transcription factors (which control the activity of genes involved in inflammation) and increased expression of pro-inflammatory mediators.

    We conclude that human exposure to WSP is associated with both systemic and pulmonary inflammation. This is comparable to exposures to ambient PM and other emission source air pollution particles including diesel exhaust particles which can induce systemic and pulmonary inflammation.34–36 Diesel, wood smoke and biomass source-derived PM have compositional similarities with each other, but there can be differences in levels of particular organic and metal components (eg, concentrations of alkali salts are usually greater in WSP).1 37 While there are data to suggest that WSP is less toxic,31 our results suggest that this specific emission source particle follows the general model of biological activity for all PM. Differences between specific particles likely reflect disparities in oxidant generation (eg, surface functional groups, size/surface area and organics and metal concentrations) rather than dissimilarities in the molecular, biochemical and cellular pathways for expression of biological effect.

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    Supplementary materials

    Footnotes

    • Disclaimer: This report has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

    • Competing interests None.

    • Ethics approval This study was conducted with the approval of the University of North Carolina at Chapel Hill.

    • Provenance and peer review Not commissioned; externally peer reviewed.