* Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, New Mexico 87108 and
California Institute of Technology, Pasadena, California 91125
Received May 16, 2001; accepted September 24, 2001
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ABSTRACT |
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Key Words: wood smoke; particles; chemical composition; rat; pulmonary function; histopathology; cytokines; lymphocytes.
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INTRODUCTION |
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Epidemiologic studies have linked exposure to wood smoke with increased prevalence of respiratory illness in children and adults. Increased respiratory symptoms, decreased pulmonary function, and increased prevalence of chronic bronchitis have been associated with wood smoke exposure in Columbia (Dennis et al., 1996), Papua, New Guinea (Anderson, 1979
), and Nepal (Pandey, 1984
). Case-control studies showed that the risk of hospitalization with acute lower respiratory illnesses among Navajo children in Arizona living in households that cook and heat with wood was five times that for children living with families who cook with gas or electricity (Robin et al., 1996
). Other studies of children in Michigan (***Honicky et al., 1985
), Idaho (Butterfield et al., 1989
), Montana (Johnson et al., 1990
), Colorado (Larson and Koenig, 1994
), and Washington (Koenig et al., 1993
) show associations between fine particulate matter, with wood smoke being a contributing source, and decreased pulmonary function, cough, wheeze, and upper and lower respiratory infections. These studies with children lack the confounders of cigarette smoking or occupational exposures and suggest a causal relationship between elevated wood smoke levels and adverse health outcomes.
Wood smoke can be an important contributor to particle and gaseous material in ambient air and can account for as much as 80% of the airborne particle concentrations during the winter heating season in some locations (Larson and Koenig, 1994). Indoor exposures to wood smoke can occur not only from infiltration of outdoor air, but also from non-airtight or improperly operated wood stoves and other wood-burning appliances. Some studies report wood smoke concentrations as total suspended particulate matter from 2.7 to 25 mg/m3, much higher concentrations of particulate matter than those to which U.S. populations are currently exposed (up to 1 mg/m3 on a 24-h basis) (Larson and Koenig, 1994
).
Although wood smoke is an important environmental air contaminant in several locations in the United States, the toxicological database on subchronic or chronic exposures to wood smoke is very small (Larson and Koenig, 1994). Currently, extrapolation of results from animal studies to human populations living in areas with elevated wood smoke concentration is difficult, because the existing studies used exposure concentrations higher than those relevant to human populations in the U.S. (Kewal Lal et al., 1993
); (Liang et al., 1988
) or short, acute exposures (Fick et al., 1984
). Furthermore, the studies using subchronic exposures did not evaluate pulmonary function or symptoms of respiratory illness, key end points in epidemiological studies. The present study was designed to provide information on the potential respiratory health responses to subchronic wood smoke exposures in a Native American community in New Mexico. The incidence of childhood asthma in this community is about twice the national average (Clark et al., 1995
), and exposure to particulate matter from combustion sources may exacerbate asthma (Koenig et al., 1993
; Schwartz et al., 1993
). Some homes in this community burn wood that is found in the surrounding areas for heating and cooking and use a type of primitive conventional wood stove without a catalyst. The intention of this study was to mimic conditions observed in this community by using the same type of wood under similar burning conditions and wood smoke particle concentrations.
The effects of wood smoke exposure on pulmonary function and the respiratory tract epithelia in Brown Norway rats are described. Brown Norway rats were chosen for this study because this animal model mimics features of human allergic asthma in several aspects. This strain exhibits high IgE levels, early- and late-phase bronchoconstrictions, and a helper T-cell type 2 response to allergic sensitization (McMenamin et al., 1995; Ohtsuka et al., 1997
). These studies, therefore, will form the basis for further investigations on whether wood smoke exposure exacerbates asthma in rats sensitized or challenged to allergens.
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MATERIALS AND METHODS |
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After 2 weeks of quarantine, rats were randomly assigned by weight to three exposure groups (26 rats per group). At least 12 rats from each exposure group were removed for sacrifice after 30 or 90 days of exposure. Two additional rats were maintained in each group as backup in case of unexpected morbidity. All rats were given unique identification numbers by means of tail tattoo. Rats were maintained in the exposure chambers for 7 days for adaptation prior to their first wood smoke exposure. All animals were weighed not more than 1 week before exposures and at weekly intervals throughout the study. Clinical observation data were collected during each weighing session. Animals were checked once in the morning and once in the late afternoon at least 6 h apart 7 days a week for any clinical signs of abnormality, morbidity, or mortality. Food was removed from chambers before start of exposure, and then replaced when the day's exposure was complete.
Wood smoke exposure system.
Wood smoke was diluted with air to either 1 or 10 mg particles/m3 in multitiered, whole-body exposure chambers (H-1000, Lab Products, Inc., Aberdeen, MD) (Fig. 1). A third chamber was used for exposure to air as control. The wood stove (Vogelzang Boxwood Stove, Model BX-42E), similar to the type generally used in the homes of the Native American population of interest, was purchased new from a local source. The wood stove was a simple box stove without a catalyst. It was set up in a separate room from the exposure chambers; thus, the rats were not subjected to the heat generated by the fire. The stove flue was connected to an exhaust plenum maintained at approximately 5 inches water-negative to ambient pressure. A fraction of the wood smoke was diverted from the flue, cooled, and diluted to the desired concentrations. Ambient air for smoke dilution and control exposure was drawn through HEPA filters, tempered, and humidified. The exposure chambers were maintained at a temperature of 24 ± 1°C and a relative humidity of between 40 and 70%. The chambers were operated at 12 ± 2 chamber air changes/ h (200 ± 30 l/min). A computer-based system provided 24-h monitoring and recording of airflow rate, temperature, humidity, and pressure. The uniform distribution of wood smoke in the chambers was verified during this study. Chamber exhaust air was routed through HEPA filters, then discharged into the atmosphere at a position remote to the facility air intakes.
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Exposure characterization.
The particle concentration was determined using filter samples taken from a midchamber point. Filter holders were loaded with 25-mm diameter glass fiber filters, and flow was maintained at a constant volume of 2 l/min with a critical orifice. Filter samples were taken every 30 min throughout the 3-h exposure period from each chamber on each exposure day. Filters were weighed before and after the sampling period, and the weight differences were used to determine mass concentration.
The particle size distribution was determined using a micro-orifice uniform deposit impactor (MOUDI, MSP Corp., Minneapolis, MN) operated at a flow rate of 30 l/min. Probit analysis of the mass collected on the 10 stages and backup was used to determine the mass-median aerodynamic diameter (MMAD) and geometric standard deviation (SD) of the size distribution. A point-to-plane electrostatic precipitator was used to collect samples for electron micrographic analysis as described (Cheng et al., 1981). Electron micrographs were prepared using a Hitachi H7000 scanning/TEM (Hitachi Ltd., Tokyo, Japan).
Organic plus elemental carbon and speciated particle-bound organic compounds (Table 1) were analyzed at the Environmental Engineering Science Department, California Institute of Technology, Pasadena, CA (Cal-Tech). These samples were collected on seven separate occasions directly from the exposure chambers onto prebaked quartz fiber filters (Pall-Gellman; 47-mm Tissuequartz, QAO). Collected material was stored in baked glass containers at 80°C prior to shipment to Cal-Tech. A 1-cm2 portion of each filter was analyzed for total organic and elemental carbon by the thermal analysis method described by Birch and Cary (1996). Results of the carbon analysis did not include the mass of oxygen, hydrogen, or other noncarbon atoms present in organic material, which can contribute substantially to the mass of material that would be weighed on a filter. To facilitate comparison of weighed mass to organic material, a factor of 1.4 was used to correct for the nonmeasured components of organic material, as previously described for wood smoke studies (Kleeman et al., 1999
).
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Gaseous nitrogen oxide concentrations were measured by extracting gas samples into TedlarTM bags. Bag samples were then analyzed for NO and NOx using a chemiluminescence analyzer (Model 200A, Advanced Pollution Instruments, San Diego, CA). CO was measured by extracting gas samples through filters directly into an electrochemical measuring transducer (Multiwarn II, Draeger Safety, Inc., Pittsburgh, PA). Gas from each chamber was analyzed for 5 days each during weeks 2, 6, and 9.
Respiratory function tests.
The respiratory function of 810 rats per treatment group was measured by whole-body plethysmography using techniques described previously in detail (Mauderly et al., 1990). Rats were anesthetized with halothane, intubated orally with endotracheal and esophageal catheters, and measured in the prone position in a combination pressure and flow plethysmograph. During spontaneous breathing, respiratory pattern, dynamic lung compliance, and total pulmonary resistance were measured. Measurements during single-breath maneuvers induced by positive and negative airway pressures and during hyperventilation-induced apnea included quasistatic vital capacity and pressure-volume curves, functional residual capacity, single-breath N2 washout, forced vital capacity and flow-volume curve, and single-breath, CO-diffusing capacity. Total lung capacity and residual volume were calculated from measured volumes.
Histopathologic examination.
After 30 or 90 days of exposure, six rats from each group were killed for histopathologic examinations by intraperitoneal injection of 0.2 ml Euthasol (Delmarva Laboratories, Inc., Midlothian, VA) and exsanguination via the renal artery. The larynx and a part of the trachea were fixed in zinc formalin. The left lung was fixed in an expanded state by intratracheal instillation of zinc formalin at 25 cm of constant pressure for 6 h followed by immersion in the same fixative for 35 days. The fixed lungs were cut into five or six slices (each 5 mm thick) and embedded in paraffin. These slices were numbered from cephalad (slice 1) to caudad (slice 5 or 6).
To analyze the nasal epithelia, the head of each rat was removed from the carcass, as well as the lower jaw, skin, and musculature. Nasal passages were flushed retrograde through the nasopharyngeal orifice with zinc formalin, then immersed in a large volume of this fixative for at least 2 weeks. The noses were decalcified with 13% formic acid by immersion for 4 days. After decalcification, the nasal airways were transversely sectioned at four specific anatomic locations as described (Harkema and Hotchkiss, 1994). The following gross dental and palatine landmarks were used: immediately posterior to the upper incisor tooth (tissue block 1); at the incisive papilla (tissue block 2); at the second palatal ridge (tissue block 3); and in the middle of the front upper molar tooth (tissue block 4).
All tissues were fixed in zinc formalin for no less than 72 h. After fixation, tissue samples were embedded in paraffin, cut into 5-µm sections, and stained with hematoxylin and eosin or Alcian Blue (pH2.5)/Periodic Acid-Schiff (AB/PAS) as described previously (Tesfaigzi et al., 2000).
Histopathologic changes in two sections of the trachea, one section from the larynx, four sections from the nose, and two sections from the left lung were examined by a pathologist in a random and blinded fashion. Microscopic observations were graded for severity. Tissue sections from the trachea and lung were stained with AB/PAS and analyzed for changes in mucous cell numbers by counting the AB/PAS-positive cells per millimeter basal using the NIH image analysis software Scion Image (written by Wayne Rasband, U.S. National Institutes of Health) on a Power Macintosh G4. Epithelia lining the intrapulmonary axial airways at generations 3 and 4, which are represented in lung slices 3 and 4, were quantified. Furthermore, epithelia from two regions of the trachea were subjected to morphometric analyses.
Analysis of bronchoalveolar lavage fluid.
After 30 or 90 days of exposures, a set of six rats from each group was used for analysis of bronchoalveolar lavage (BALF). Rats were killed, thoracic contents exposed, and the lungs perfused through the pulmonary artery with saline. The larynx, trachea, and lungs were isolated. The entire lung was removed and lavaged through the trachea three times with 5 ml ice-cold phosphate-buffered saline without calcium and magnesium ions.
After removing the cells by centrifugation, levels of protein, lactate dehydrogenase (LDH), and ß-glucuronidase in the BALF were determined, because these values are useful indicators of cell damage or death. The levels of several cytokines (IL-1ß, IL-6, and TNF-) in the BALF were determined by ELISA as described previously (Conn et al., 1995
). These cytokines, which have generally been found to be present after an inflammatory process, were expected to be present after subchronic exposure to wood smoke. Cells recovered by BALF from the right lung lobes were enumerated using a hemocytometer. Cytological preparations were stained with Wright Giemsa, and differential counts were performed to determine the type of inflammation present in the BALF.
Exposures.
Animals were exposed to air as control and 1 or 10 mg/m3 wood smoke particle concentrations for 3 h/day, 5 days/week, for up to 90 days. The exposure concentrations were selected based on a pilot experiment, in which five rats each were exposed for 5 days to air as control, or to 2 or 20 mg particles of wood smoke/m3. These concentrations were based on previous reports showing minor health effects in Wistar rats exposed to 14.99 mg/m3 wood smoke concentrations for 19 months (Liang et al., 1988). The mean wood smoke concentrations for this pilot study were 2.1 (SD 0.948) and 20.5 (SD 3.407) mg particles/m3. Analysis of the BALF showed no changes in ß-glucuronidase activity. However, total protein and LDH levels were significantly elevated in the group exposed to 20 mg/m3 compared to the air controls. No differences were noted in the numbers of macrophages, lymphocytes, eosinophils, and neutrophils. The number of macrophages with < 10 and > 10 carbonaceous particles, presumably smoke particles, increased from 0 to 24% in the group exposed to 2 mg/m3. About 60% of macrophages contained > 10 particles in rats exposed to 20 mg/m3. Based on our findings in these 5-day exposures, we anticipated that exposures for 30 and 90 days at 1 and 10 mg/m3 wood smoke particle concentrations would produce measurable effects. Furthermore, concentrations of 1 mg/m3 would more closely resemble those measured in the Native Americans homes this study was intended to mimic.
Lymphocyte proliferation assay.
Lung-associated lymph nodes (LALNs) and spleens from six rats in each exposure group that were used to obtain lungs for lavage were removed at 30 or 90 days for lymphocyte proliferation assays. Immediately after removal of tissues, splenic and LALN cells were prepared, and their proliferation in response to Con A, a T-cellspecific mitogen, was analyzed as described (Singh et al., 2000).
Statistical analysis.
Data are expressed as mean ± standard error of the mean (SEM). Differences among groups were examined by analysis of variance (ANOVA) and t-tests using SAS software (Cary, NC) and by application of Tukey's or Bonferonni correction for multiple comparisons as appropriate. Differences were considered significant at p < 0.05.
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RESULTS |
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Clinical observations and body weight.
No clinical sign was noted on exposed rats, and there was no significant effect of exposure on body weight when exposed rats were compared with controls (Fig. 4).
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Dynamic lung compliance was higher in the exposed groups, and the difference was significant for the high-exposure group. In contrast, quasistatic compliance, a more specific measure of lung elastic recoil, was slightly (insignificantly) lower in the exposed groups. There was no significant difference in lung volumes between control and exposed groups, although vital capacity (both quasistatic and forced) was slightly smaller, and residual volume slightly larger, in the high-exposure group.
The 16% (nonsignificant) increase in the slope of phase III (alveolar plateau) of the single-breath N2 washout in both exposed groups suggested that gas distribution in the lung may have been slightly less uniform. Dynamic lung compliance was higher in exposed groups, but quasistatic lung compliance in the lung volume range of spontaneous breathing was slightly (nonsignificantly) decreased, rather than increased.
Histopathology.
At necropsy, the lungs of rats exposed to wood smoke were brownish to grayish in color, whereas the lungs of the air controls were pink. There was a minimal to mild chronic inflammation in the epiglottis of the larynx of several rats exposed to 1 or 10 mg/m3 after the 30- or 90-day exposure. After 90 days, only two rats in the high-exposure group and one in the low-exposure group had a minimal to mild squamous metaplasia in the body of the larynx posterior to the ventral laryngeal pouch. Scant inflammation was associated with the metaplasia. No squamous lesions were obvious in the nose or trachea of any rat in these experiments. Morphometric analysis of the upper tracheal epithelium showed a 2-fold increase in the number of PAS-positive cells in rats exposed to 10 mg/m3 for 30 days compared to the other groups (p < 0.05). However, after 90 days of exposure, the numbers decreased to levels observed in air-control rats. No changes in mucous cell metaplasia were noted in epithelia lining the larynx and trachea at the bifurcation after 30 or 90 days of exposure.
Increases in the number and pigmentation of alveolar macrophages (Figs. 5A and 5B) were related to increased wood smoke exposure. Alveolar macrophage numbers were increased in the high-exposure group after the 30-day sacrifice and were generally more severe after 90 days of exposure (data not shown). After 30 and 90 days of exposure, the severity of pigmentation was increased with exposure concentration. After 90 days, both exposure groups (Fig. 6B
), but not the air controls (Fig. 6A
), exhibited a slight thickening of the alveolar septae with hypertrophic alveolar epithelial cells and a few mononuclear cells in some alveoli that contained clumps of particle-laden macrophages. Some lungs from all groups had inflammatory lesions, which have been described by Charles River Laboratories as idiopathic granulomatous pneumonia of Brown Norway rats. These lesions were noted, but considered as background lesions.
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BALF Cell differentials.
After 30 days of exposure, the numbers of lymphocytes and neutrophils were slightly but not significantly increased in the BALF of rats exposed to 10 mg/m3 compared with the air control and 1 mg/m3 groups (data not shown). No difference in lymphocyte and neutrophil numbers was observed between groups after the 90-day exposure. However, the number of macrophages in the BALF was decreased significantly by about half (p < 0.05) in the 10 mg/m3 group compared to the 1 mg/m3 group (Fig. 7).
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Lymphocyte proliferation assay.
T lymphocytes isolated from both the LALNs and the spleen were not affected in their proliferative response to Con A by any wood smoke exposure level or length of exposure.
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DISCUSSION |
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The carbonaceous component of wood smoke particles from the exposure chambers constituted about 75 % of the weighed mass, which is consistent with previous reports on the chemistry of wood smoke (Hildemann et al., 1991; McDonald et al. 2000
; Rogge et al., 1998
). More than 20% of the particulate mass was unaccounted for by chemical analysis in the present study. The difference between the measured chemical species and the weighed mass can be attributed at least in part to known wood smoke constituents (e.g., sulfates, potassium, chloride), which were not analyzed in our study. Although particle-bound water was not measured, it can contribute to the difference between the sum of chemical species and weighed mass (Van Loy et al., 2000
). In addition, the weight of the organic material was estimated by applying a correction factor to the organic-carbon content measured by the thermal analysis. The thermal analysis measures only carbon in its elemental form, and without knowledge of the exact contribution of the noncarbon elements to the mass of organic material, the correction factor applied is only an estimate. Because organic carbon accounts for the majority of the particulate material, a small error in this estimate from the true value could significantly affect the total amount of organic carbon reported.
The measured chemical species in this study accounted for about 15% of the organic carbon present in wood smoke. The organic analysis employed here is comparable to contemporary comprehensive characterizations of both ambient air (Schauer and Cass, 2000) and air pollution sources such as diesel exhaust (Schauer et al., 1999
) and wood combustion (Schauer et al., 2001
). In general, characterizations of ambient and source-based organic material in the literature range from accounting for a fraction of a percent to as high as 50% of the total particle-bound organic material, because of limitation in current technology.
Most of the quantified chemical species in the wood smoke particles were composed of cellulose/hemicellulose- (sugar derivatives, furans) and lignin- (guaiacols, substituted benzenes/phenols) derived products. These findings reflect the structure and chemical composition of wood, which comprises approximately 6575 % cellulose fibers bound together by lignin, an aromatic polymer making up 2030 % of the overall mass (Rowell, 1984; Larson and Koenig, 1993
). Many other compounds identified, such as the resin acids and diterpenoids, are also unique to wood smoke.
Health Effects
Results of respiratory function tests indicated a small, exposure-related reduction in function consisting of reduced gas exchange at the alveolar membrane, and nonsignificant reductions in lung size and the uniformity of gas distribution within the lung. Overall, the impact was small and would be considered of little clinical importance. Only the reduction in gas exchange was consistent across multiple test parameters, and the difference from controls was less than 20%. This degree of reduction would be at the borderline of clinical significance in human subjects for whom test values are compared to normal ranges and thus, although statistically significant, is a modest effect. The slightly (insignificantly) smaller lungs of the high-exposure group, indicated by total lung capacity and vital capacity, and their slightly (insignificantly) larger residual volume may have been related to exposure. Because the lungs of rats are growing during the age range of this study (Mauderly and Gillett, 1992), the impact on lung size more likely reflects slower lung growth than an actual reduction in lung size. This conclusion is supported by the concurrence of slightly higher lung volume and body weight in the low-exposure group and slightly lower values in the high-exposure group than in controls, although neither the lung-volume nor body-weight differences were significant. The larger residual volume was consistent with earlier airway closure during exhalation, which in turn was consistent with the smaller quasistatic and forced vital capacity. It could be speculated that had the exposure continued, these differences might have progressed, consistent with changes observed in smokers.
The reason for the different effects on dynamic (increased) and quasistatic (slightly decreased) compliance cannot be determined from the measurements made in this study. Such an outcome might be possible if the increased resistance to airflow caused the exposed rats to breathe at a different set-point on the pressure-volume curve than the control rats. This explanation is unlikely, however, because functional residual capacity (volume at end expiration) was nearly identical in the control and high-exposure groups and represented very similar portions of total lung capacity (26% and 28%, respectively).
Although alveolar macrophage hyperplasia and pigmentation were increased with exposure concentration, decreased numbers of macrophages were found in the BALF in the high-exposure group. Significantly reduced numbers of macrophages were also found in the BALF of Fischer-344/Crl rats exposed for 12, 18, and 24 months to whole diesel exhaust diluted to a soot concentration of 3.5 mg/m3 compared with air controls (Mauderly et al., 1989), although exposures to diesel exhaust particles at 1.5 mg/m3 showed no difference compared to controls (Strom, 1984
). These findings suggest that macrophages containing carbon particles remain in the lung tissue and may be harder to retrieve by bronchoalveolar lavage, possibly as a result of the observed clumping of particle-laden macrophages in alveolar spaces of exposed rats.
The mild mucous cell metaplasia observed in the upper trachea of rats exposed for 30 days was resolved after 90 days of exposure. However, the increase of the AB-positive mucous cells was caused by exposure to 1 mg/m3 for 90 days. This change in staining pattern denotes a change in the content from neutral (PAS-positive) to acidic (AB-positive) glycoproteins (Samet and Cheng, 1994). Many glycoprotein-containing epithelial cells in rat airways are serous cells, which produce primarily neutral glycoproteins (Forrest and Lee, 1991
), and they undergo a change to contain acidic glycoproteins in response to inhaled irritants (Jeffrey and Reid, 1977
). It is possible that these changes occurred early in the high-exposure group and returned to control levels at 30 and 90 days of exposure.
After subchronic exposures to wood smoke, the cytokines TNF-, IL-1ß, and IL-6 were undetectable. Inflammatory cells, such as neutrophils, lymphocytes, or eosinophils, were not present in significantly increased numbers in the BALF. The absence of these indicators of early inflammation after subchronic exposures is not surprising; a reduction in inflammation with continued exposure has been reported for exposures to endotoxin (Shimada et al., 2000
). However, the concentration levels of wood smoke may have been too low to elicit inflammation in the lung. These results and the lack of change in the proliferative response of T lymphocytes from the LALNs of exposed and nonexposed rats in response to Con A suggest that wood smoke alone may not affect the immune system.
In summary, our observations suggest that exposure to wood smoke caused minor, but significant effects on pulmonary function and on epithelia lining the nasal and tracheobronchial regions of Brown Norway rats. Future studies will use rats sensitized to allergens to determine whether wood smoke may affect a compromised immune system as fine particulate matter does in asthmatic children (Koenig et al., 1993).
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ACKNOWLEDGMENTS |
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NOTES |
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Research conducted on this study was sponsored by the National Environmental Respiratory Center (NERC) with support from multiple government and industry sponsors., including the U. S. Environmental Protection Agency. This paper is not intended to represent the views or policies of any NERC sponsor.
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