* Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606;
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, 86 T. W. Alexander Drive, MD92, Research Triangle Park, North Carolina, 27711; and
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268
Received June 10, 2002; accepted July 29, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: allergy; asthma; Stachybotrys chartarum; respiratory exposure.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A variety of microscopic fungi, also known as molds, are known to cause a number of diseases in humans, ranging from frank colonization of tissues to immune responses in atopic individuals (Pieckova and Jesenska, 1999). The toxigenic mold Stachybotrys chartarum has been associated with a number of health effects, including acute pulmonary hemorrhage in infants (Elidemir et al., 1999
; Etzel et al., 1998
; Montana et al., 1997
; Novotny and Dixit, 2000
; Tripi et al., 2000
), and sick-building syndrome (Cooley et al., 1998
; Mahmoudi and Gershwin, 2000
). In addition, both anecdotal and published reports have associated exposure to this mold with asthma (Hodgson et al., 1998
). Previous research has focused on the effects of the potent trichothecene mycotoxins produced by S. chartarum (Croft et al., 1986
; Jarvis et al., 1995
, 1998
; Sorenson et al., 1987
), and the recently described hemolytic protein stachylysin (Vesper et al., 2001
). However, the potential exists for genetically predisposed individuals inhabiting buildings or homes contaminated with S. chartarum to be directly exposed to aerosolized S. chartarum (whole or fragmented conidia and/or mycelium) and to develop allergic responses, resulting in or exacerbating existing respiratory disease. The goal of the current study was to assess the allergenic potential of S. chartarum in a controlled animal study.
Animal models have been used to study inflammatory and immunological responses of the respiratory tract to allergens, and to investigate airway hyperresponsiveness and inflammation. Initially, in vivo measurements of lung conductance and compliance following intravenous infusions of bronchoconstrictive agents (Martin et al., 1988), and to aerosolized antigen (Renz et al., 1992
), were performed in anesthetized, tracheostomized, mechanically ventilated mice. However, the effect of the surgical procedure and anesthesia on respiratory physiological responses was not fully known, pulmonary responses to bronchoconstrictor agents administered by intravenous injection were not well defined, and the animals could only be subjected to one experimental measurement, being killed at its conclusion. To address these shortcomings, barometric whole-body plethysmography was developed, allowing measurement of airway responsiveness to aerosolized agents to be determined in unrestrained conscious mice (Travis et al., 1982
). This method allows repeated measurements to be performed on the same subject, allowing both the acute and late-phase respiratory physiological responses characteristic of allergic asthma to be studied in a single group of animals, and has been validated by direct comparison with the more invasive procedures previously mentioned (Hamelmann et al., 1997
).
Previous work in this laboratory with Metarhizium anisopliae, a fungal pesticide licensed for indoor control of cockroaches, has demonstrated that extracted fungal proteins administered to BALB/c mice are capable of increasing endpoints characteristic of allergic asthma, including eosinophilia and increased serum IgE levels (Ward et al., 1998), as well as airway hyperresponsiveness and inflammatory changes in lung pathology (Ward et al., 2000
). These endpoints are considered to be pathognomonic of human allergic asthma. In this study, we used this animal model to demonstrate a cause-effect relationship for exposure to S. chartarum and allergic asthma-type responses.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fungal Antigen Preparation
Five S. chartarum isolates (5807, 5816, 5817, 5818, and 6301, described in Vesper et al., 1999) originally obtained from residences in Cleveland, OH (Etzel et al., 1998
) were grown on a nylon filter resting on plates of sheeps blood agar or cellulose agar at 23°C for one month and combined in approximately equal weight amounts. This mass was extracted by grinding with a sterile mortar and pestle for approximately 12 min in a total of 15 volumes (by weight) of Hanks balanced salt solution (HBSS, Gibco BRL, Life Technologies Inc., Rockville, MD) + 0.05% Tween 80 (Fisher Scientific, Pittsburgh, PA). The resulting suspension was stirred overnight at 4°C, and then centrifuged at 12,500 x g for 1 h at 4°C. The supernatant was decanted, adjusted to pH 6.0 with HCl, and filter sterilized with a 1.2 µm syringe filter followed by a 0.2 µm media filter. This sterile filtrate was concentrated with a stirred-cell concentrator (Amicon, Inc., Beverly, MA) using YM3 membranes (molecular weight
3000 Da retained). The crude antigen preparation (S. chartarum extract 1 [SCE-1]) was assayed for total protein concentration, as described below, and stored at 80°C until use. Endotoxin levels of the individual extracts were measured using a Limulus amebocyte lysate test kit (BioWhittaker, Walkersville, MD); levels ranged from 13.757.2 pg/ml (for comparison, USP standard for water for injection, 25 pg/ml).
Experimental Design
Three groups of 24 mice each were exposed by involuntary aspiration (IA) 4 times over a four-week period, as previously described in (Ward et al., 1998), to 20, 10, or 5 µg SCE-1 in 50 µl total volume. Briefly, mice were anaesthetized by halothane inhalation, and the dose of SCE-1 was deposited in the oropharynx. Animals inhaled the SCE-1 when their noses were gently occluded with a gloved fingertip. A group of control mice (vehicle control) was IA exposed concurrently 4 times over a four-week period to HBSS alone. There were no significant differences in the responses to the various doses; therefore, the data presented in Results is from the 10-µg dose group compared to the HBSS controls. An additional group of mice were IA exposed to 3 doses of HBSS and a final single dose of 20 µg SCE-1 in 50-µl total volume to serve as a nonspecific inflammation control. The responses of these animals were compared to results obtained from naïve animals. Additionally, a group of mice was exposed to 4 doses of 10 µg bovine serum albumin (BSA) in 50-µl total volume to serve as a nonallergenic protein control for both the amount of protein administered and for the route of exposure. For the experiments measuring airway responsiveness, mice were exposed 4 times to 10 µg SCE-1, to a single 10 µg dose of SCE-1, or to 4 doses of 10 µg BSA, as described above. Antigen-specific immediate responses were measured after each dose, and responsiveness to methacholine was measured on Days 1 and 3 following the final dose, as described below.
Bronchoalveolar Lavage and Blood Collection
Blood and bronchoalveolar fluid (BALF) samples were collected as previously described (Ward et al., 1998). Briefly, mice were anaesthetized with sodium pentobarbital and blood samples were collected by cardiac puncture. The blood was allowed to clot for 12 h at room temperature prior to centrifugation, and the serum was stored at 80°C until analysis. The lungs were lavaged twice with 1 ml aliquots of HBSS, and the bronchoalveolar lavage fluid (BALF) aliquots were pooled and stored on ice. The BALF was centrifuged at 100 x g for 15 min at 4°C to pellet the cells, the supernatant was decanted, and the cells were resuspended in 500 µl of HBSS. Aliquots of BALF supernatant were assayed for total protein and lactate dehydrogenase (LDH) as described below, and the remainder was stored at 20°C for IgE and cytokine assays. Total BALF cell counts were performed using a hemacytometer and viability assessed by Trypan-blue dye exclusion. Cytology slides were made by centrifuging 100150 µl of resuspended cells onto glass slides at 200 rpm for 10 min using a Cytospin 2 centrifuge (Shandon Inc., Pittsburgh, PA). The BALF cells were stained with Wright-Giemsa (Fisher Scientific) on a Hema-Tek 2000 slide stainer (Miles, Inc., Elkhart, IN), and were differentially counted at 200 cells per slide (one slide per animal).
Total Protein and Lactate Dehydrogenase (LDH) Assays
BALF samples were assayed for total protein using Pierce Coomassie-Plus Protein Assay Reagent (Pierce, Rockford, IL). Concentrations were determined from a standard curve using BSA standards obtained from Sigma Chemical Co. (St. Louis, MO). Additionally, the BALF samples were assayed for LDH using a commercially prepared kit and controls (Sigma). The assay reagent proportions remained the same but volumes were reduced for use on a Cobas Fara II Centrifugal Spectrophotometer (Hoffman-LaRoche, Branchburg, NJ).
Total IgE, IgA, and IL-5 Enzyme-Linked Immunosorbent Assays (ELISAs)
Total IgE ELISA.
All reagents and incubation periods were at room temperature to enhance assay precision and accuracy, and all volumes added were 100 µl unless otherwise noted. Certified 96-well microtiter plates (Costar Corp., Cambridge, MA) were coated with rat anti-mouse IgE (PharMingen, San Diego, CA) at 3.0 µg/ml in phosphate buffered saline (PBS), pH 7.3, sealed, and incubated overnight at 4°C. The plates were washed 3 times between each incubation with PBS + 0.5% Tween 20 (Fisher Scientific). To block nonspecific binding, 200 µl of PBS + 1% BSA (Sigma) was added to plates. After 30 min, plates were washed as described above and either BALF samples (undiluted) or mouse serum (diluted 1:30 and 1:90 in blocking buffer), and purified mouse IgE standards (PharMingen, in 2-fold dilutions from 800 ng/ml to 0.78 ng/ml) were applied. Following a 1-h incubation and wash as described above, biotinylated rat anti-mouse IgE (PharMingen) at 2.5 µg/ml in blocking buffer was added to all wells. After a 1-h incubation and 6 washes, streptavidin-horseradish peroxidase conjugate (Zymed Laboratories Inc., San Francisco, CA) at 0.16 µg/µl was added and the plates incubated for 20 min. Following 6 washes, a 3,5-5,5 tetramethylbenzidine substrate solution (TMB One-Step Substrate Solution, Dako Corporation, Carpinteria, CA) was added and incubated for at least 20 min. Optical density was read on a SpectraMax 340PC Plate Reader (Molecular Devices Corp., Menlo Park, CA) at a wavelength of 650 nm. Softmax Pro® software (version 2.6.1, Molecular Devices Corp.) was used for data collection and conversion from optical density to protein concentrations.
Total IgA ELISA.
The IgA ELISA was performed using the same protocol as described for the IgE ELISA. Briefly, microtiter plates were coated with anti-mouse IgA (PharMingen) at 3.0 µg/µl in PBS (pH 7.3). BALF samples were diluted 1:100, and the IgA standard (purified mouse IgA, K isotype, PharMingen) was added in 2-fold dilutions, providing a range of from 500 ng/ml to 0.49 ng/ml. The biotinylated anti-mouse IgA (PharMingen) was added at 2.5 µg/µl. Streptavidin-horseradish peroxidase (Zymed Labs) and TMB substrate (Dako Corp.) were applied and optical density determined as described above. Softmax Pro® software was used for data collection and conversion from optical density to protein concentrations.
IL-5 ELISA.
The IL-5 ELISA was performed as previously described (Ward et al., 2000). Briefly, microtiter plates were coated with anti-mouse IL-5 (PharMingen) at 4.0 µg/µl in 0.1 M sodium phosphate buffer (pH 9.0), sealed, and incubated overnight at 4°C. The plates were blocked with diluent buffer (PBS + 1.0% BSA + 0.05% Tween 20) for 1 h at room temperature, the IL-5 standard (PharMingen) was added in 2-fold dilutions (20001.95 pg/ml range), and the BALF samples were applied undiluted. The plates were sealed and incubated again overnight at 4°C. The biotinylated detection antibody (biotin-anti-mouse rIL-5, PharMingen) was diluted in diluent buffer, conjugated streptavidin-horseradish peroxidase (Zymed Labs) and TMB substrate (Dako Corp.) were applied, and optical density was determined as described above. Softmax Pro® software was used for data collection and conversion from optical density to protein concentrations.
Histopathology
Following bronchoalveolar lavage, the lungs were inflated in situ with 10% buffered formalin acetate (Fisher Scientific, Fair Lawn, NJ), embedded in paraffin, and sectioned. One slide per mouse was prepared by sectioning the lung along the main stem bronchus and staining with hematoxylin-eosin solution prior to pathological analysis. The entire lung was examined at several magnifications and the relative degree of severity of inflammatory, degenerative, and proliferative changes was graded on a scale of 1 to 5 (1, minimal; 2, slight/mild; 3, moderate; 4, moderately severe; 5, severe/high). The pathology scores for lung sections were summarized as follows: incidence, the number of mice exhibiting certain pathology; severity, the numeric average of the pathology scores for a particular lesion per treatment group (protocol used by Experimental Pathology Laboratories, Inc., Research Triangle Park, NC). Diffuse alveolar macrophage accumulation consisted of activated macrophages present in alveolar air spaces in a large area of the lung parenchyma. Alveolitis consisted of focal thickening of alveolar septae, often with inflammatory cells in the affected alveolar spaces. Edema was diagnosed when there was a distinct distended area surrounding vessels that contained proteinaceous fluid.
Measurements of Airway Responsiveness
Antigen-specific immediate responses.
Antigen-specific airway responsiveness was measured immediately after each exposure to SCE-1 (Days 28, 14, 7, 0) in unrestrained mice, using whole body plethysmography (Buxco Electronics, Troy, NY). In each plethysmograph, a pressure signal is generated from the pressure difference between the main chamber, containing the unrestrained mouse, and a reference chamber that cancels atmospheric disturbances. Signals were analyzed using BioSystem XA software (SFT3812, version 2.0.2.48, Buxco Electronics) to derive whole body flow parameters including respiratory rate, tidal volume, inspiratory time (Ti), expiratory time (Te), peak inspiratory flow (PIF), peak expiratory flow (PEF), and relaxation time (RT). These parameters are used by the BioSystem XA software to calculate enhanced pause (PenH), a unitless parameter that strongly correlates with lung resistance (Hamelmann et al., 1997). PenH reflects changes in pulmonary resistance during bronchoconstriction according to the following equation: PenH = ((Te RT) ÷ RT) x (PEF ÷ PIF). Baseline PenH measurements for each animal were recorded for 10 min and averaged. Animals were then IA exposed to SCE-1, and placed back in the chambers within 5 min of dosing. PenH readings were then monitored and averaged over a 1-hour post-instillation period.
Airway responsiveness to methacholine aerosol.
Airway responsiveness to methacholine (MCh) aerosol was determined by measurement of time-integrated changes in PenH in response to MCh on Days 1 and 3 after the final exposure to SCE-1. After measurement of baseline PenH for 5 min, either saline or MCh in increasing concentrations (4, 8, 16, and 32 mg/ml) was nebulized and delivered through an inlet of the chamber for 1 min, and measurements of PenH were made for 4, 5, 6, 7 and 11 min, respectively, after each dose. After subtracting baseline values, time-integrated changes in PenH were calculated and expressed as areas under the curve (AUC) for each concentration of MCh (PenH units x s). Immediate responses and methacholine responsiveness were analyzed across treatment groups and within individual mice on different days of the protocol. The interaction between the treatment and day effects was also examined.
Statistics
The data were analyzed using analysis of variance. Pairwise comparisons were performed as subtests of the overall model. In cases of comparing a control group to other groups, Dunnetts test was used. Significance was attributed to an effect or difference if the probability was less than 0.05. Adjustments in significance levels of multiple comparisons were made, using a modified Bonferroni correction.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is also interesting to note the dramatic increase in total BALF IgA levels we observed in mice receiving multiple doses of SCE-1. Serum IgA levels are increased in active cases of farmers lung disease (Kaukonen et al., 1993), as well as in induced sputum from asthmatics (Nahm et al., 1998
). While both IgG and IgA are capable of triggering eosinophil degranulation, secretory IgA is the most potent isotype (Abu-Ghazaleh et al., 1989
), as well as the principal antibody found on mucosal surfaces. Animals that received one dose of SCE-1 showed a small but significant increase in IgA levels at day 7, as one would expect in the development of a humoral response, whereas BSA-treated mice did not exhibit an IgA response. We propose IgA could be another indicator of an allergic response, when accompanied by other accepted parameters, such as eosinophilia and increased IgE levels.
Airway narrowing following exposure to an inhaled allergen is the primary physiological response in allergic asthma, and airway hyperresponsiveness to a nonspecific stimulus such as methacholine is a diagnostic tool used in patients with suspected asthma. We used a barometric whole-body plethysmography system to measure both immediate airway bronchoconstriction and airway hyperresponsiveness in unanesthetized, unrestrained mice (Hamelmann et al., 1997). This method allowed repeated measurements to be performed, enabling us to determine both the acute immediate responses and longer-lasting airway hyperresponsiveness following allergen exposure in the same animals. Further, this system allowed us to administer the bronchoconstricting agent methacholine as an inhaled nebulized mist, rather than by injection. This avoids potential interactions of other organ systems that would have been exposed to higher concentrations of a nonspecific cholenergic agonist, and uses the same route of administration and concentrations as those used in clinical settings.
In the present study, mice previously sensitized to SCE-1 had both increased immediate bronchoconstriction upon SCE-1 challenge and increased airway hyperresponsiveness to the nonspecific methacholine challenge. We observed a small statistically significant increase in bronchoconstriction after the 2nd exposure. This indicates the first dose was sufficient to cause sensitization, and a single challenge was sufficient to evoke physiological responses correlating with the allergic and inflammatory biochemical and pathological alterations observed. The subsequent 3rd and 4th exposures appeared to drive the responses toward a peak with no evidence of a decline, indicative of tolerance, after the last dose. In contrast, animals exposed to a single dose of SCE-1 did not demonstrate immediate bronchoconstriction nor increased airway hyperresponsiveness to a subsequent nonspecific challenge, despite the presence of biochemical and pathological indices of pulmonary inflammation. This argues that the acute and methacholine-induced respiratory physiological responses we observed are allergen-induced, and not an artifact of a nonspecific inflammatory response. Further, the biochemical and pathological responses, particularly increases in IgE and the presence of eosinophils, were predictive of physiological changes in exposed mice.
Another concern of studies utilizing the respiratory route of sensitization and challenge was that any protein introduced into the lungs in this fashion could cause inflammation and/or allergic responses similar to the ones we observed. BSA was selected as a candidate for a negative control protein, based on the works reported by Dearman et al.(2000) and Hilton et al.(1997)
. Their work, while utilizing different routes of exposure and greater amounts of protein, suggested that BSA either would not provoke an allergic response, or would provoke a weak response. BSA did not cause increases in any of the inflammatory or allergic parameters we measured. Therefore, our findings indicate that BSA is both nonallergenic and noninflammatory when administered by aspiration at the dose level we used (10 µg), even when administered multiple times, and it can serve as a negative control for this experimental protocol in future studies.
There was a possibility that the amino acid sequences of BSA and mouse serum albumin were similar enough to be viewed as "self" by the immune system, and therefore, incapable of provoking an immune response. A BLAST comparison of BSA (accession no. AAA51411) and mouse serum albumin (accession no. CAD29888) revealed an amino acid identity of only 68% (416 of 608), indicating substantial sequence differences between these molecules. Also, both (Hilton et al.1997) and Dearman et al.(2000)
reported that intranasal exposure to BSA provoked a vigorous IgG response, indicating BSA was recognized as a foreign protein by the mouse immune system. Thus, BSA appears to be antigenic, but not allergenic, when administered without adjuvant to BALB/c mice.
We have shown responses in mice exposed to S. chartarum extract that are consistent with human allergic airway disease. Therefore, we conclude there are proteins in this aqueous extract capable of inducing allergy in a susceptible population. Future studies to isolate and characterize the specific allergens are planned, as are studies to determine if nonallergenic components of the extract function as an adjuvant. The BALB/c mouse model provides the means to address these issues and should also be useful in assessing the potential allergic responses and relative potencies of other fungal organisms found to contaminate the indoor environment.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
This research paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. 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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CDC (2001). Self-reported asthma prevalence among adultsUnited States, 2000. MMWR Morb. Mortal. Wkly. Rep 50, 682686.[Medline]
Cooley, J. D., Wong, W. C., Jumper, C. A., and Straus, D. C. (1998). Correlation between the prevalence of certain fungi and sick-building syndrome. Occup. Environ. Med. 55, 579584.[Abstract]
Croft, W. A., Jarvis, B. B., and Yatawara, C. S. (1986). Airborne outbreak of trichothecene toxicosis. Atmospher. Environ. 20, 549552.[ISI]
Dearman, R. J., Caddick, H., Basketter, D. A., and Kimber, I. (2000). Divergent antibody isotype responses induced in mice by systemic exposure to proteins: A comparison of ovalbumin with bovine serum albumin. Food Chem. Toxicol. 38, 351360.[ISI][Medline]
Elidemir, O., Colasurdo, G. N., Rossmann, S. N., and Fan, L. L. (1999). Isolation of Stachybotrys from the lung of a child with pulmonary hemosiderosis. Pediatrics 104, 964966.
Etzel, R. A., Montana, E., Sorenson, W. G., Kullman, G. J., Allan, T. M., Dearborn, D. G., Olson, D. R., Jarvis, B. B., and Miller, J. D. (1998). Acute pulmonary hemorrhage in infants associated with exposure to Stachybotrys atra and other fungi. Arch. Pediatr. Adolesc. Med. 152, 757762.
Hamelmann, E., Schwarze, J., Takeda, K., Oshiba, A., Larsen, G. L., Irvin, C. G., and Gelfand, E. W. (1997). Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am. J. Respir. Crit. Care Med. 156, 766775.
Hilton, J., Dearman, R. J., Sattar, N., Basketter, D. A., and Kimber, I. (1997). Characteristics of antibody responses induced in mice by protein allergens. Food Chem. Toxicol. 35, 12091218.[ISI][Medline]
Hodgson, M. J., Morey, P., Leung, W. Y., Morrow, L., Miller, D., Jarvis, B. B., Robbins, H., Halsey, J. F., and Storey, E. (1998). Building-associated pulmonary disease from exposure to Stachybotrys chartarum and Aspergillus versicolor. J. Occup. Environ. Med. 40, 241249.[ISI][Medline]
Holt, P. G., Macaubas, C., Stumbles, P. A., and Sly, P. D. (1999). The role of allergy in the development of asthma. Nature 402, B1217.[ISI][Medline]
Jarvis, B. B., Salemme, J., and Morais, A. (1995). Stachybotrys toxins 1. Nat. Toxins 3, 1016.[Medline]
Jarvis, B. B., Sorenson, W. G., Hintikka, E. L., Nikulin, M., Zhou, Y., Jiang, J., Wang, S., Hinkley, S., Etzel, R. A., and Dearborn, D. (1998). Study of toxin production by isolates of Stachybotrys chartarum and Memnoniella echinata isolated during a study of pulmonary hemosiderosis in infants. Appl. Environ. Microbiol. 64, 36203625.
Kaukonen, K., Savolainen, J., Viander, M., Kotimaa, M., and Terho, E. O. (1993). IgG and IgA subclass antibodies against Aspergillus umbrosus in farmers lung disease. Clin. Exp. Allergy 23, 851856.[ISI][Medline]
Kobayashi, T., Miura, T., Haba, T., Sato, M., Serizawa, I., Nagai, H., and Ishizaka, K. (2000). An essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model. J. Immunol. 164, 38553861.
Mahmoudi, M., and Gershwin, M. E. (2000). Sick building syndrome. III. Stachybotrys chartarum. J. Asthma 37, 191198.[ISI][Medline]
Martin, T. R., Gerard, N. P., Galli, S. J., and Drazen, J. M. (1988). Pulmonary responses to bronchoconstrictor agonists in the mouse. J. Appl. Physiol. 64, 23182323.
Montana, E., Etzel, R. A., Allan, T., Horgan, T. E., and Dearborn, D. G. (1997). Environmental risk factors associated with pediatric idiopathic pulmonary hemorrhage and hemosiderosis in a Cleveland community. Pediatrics 99, E5, 18.
Nahm, D. H., Kim, H. Y., and Park, H. S. (1998). Elevation of specific immunoglobulin A antibodies to both allergen and bacterial antigen in induced sputum from asthmatics. Eur. Respir. J. 12, 540545.
Novotny, W. E., and Dixit, A. (2000). Pulmonary hemorrhage in an infant following 2 weeks of fungal exposure. Arch. Pediatr. Adolesc. Med 154, 271275.
Pieckova, E., and Jesenska, Z. (1999). Microscopic fungi in dwellings and their health implications in humans. Ann. Agric. Environ. Med. 6, 111.[Medline]
Renz, H., Smith, H. R., Henson, J. E., Ray, B. S., Irvin, C. G., and Gelfand, E. W. (1992). Aerosolized antigen exposure without adjuvant causes increased IgE production and increased airway responsiveness in the mouse. J. Allergy Clin. Immunol. 89, 11271138.[ISI][Medline]
Sanderson, C. J. (1992). Interleukin-5, eosinophils, and disease. Blood 79, 31013109.[ISI][Medline]
Sorenson, W. G., Frazer, D. G., Jarvis, B. B., Simpson, J., and Robinson, V. A. (1987). Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra. Appl. Environ. Microbiol. 53, 13701375.[ISI][Medline]
Travis, E. L., Brightwell, D., Aiken, M., and Boyd, M. R. (1982). Whole body plethysmography as a noninvasive assay of toxic lung injury in mice: Studies with the pulmonary alkylating agent and cytotoxin, 4-ipomeanol. Toxicol. Appl. Pharmacol 66, 193200.[ISI][Medline]
Tripi, P. A., Modlin, S., Sorenson, W. G., and Dearborn, D. G. (2000). Acute pulmonary haemorrhage in an infant during induction of general anaesthesia. Paediatr. Anaesth. 10, 9294.[ISI][Medline]
Vesper, S. J., Dearborn, D. G., Yike, I., Sorenson, W. G., and Haugland, R. A. (1999). Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains. Appl. Environ. Microbiol. 65, 31753181.
Vesper, S. J., Magnuson, M. L., Dearborn, D. G., Yike, I., and Haugland, R. A. (2001). Initial characterization of the hemolysin stachylysin from Stachybotrys chartarum. Infect. Immun. 69, 912916.
Ward, M. D., Madison, S. L., Sailstad, D. M., Gavett, S. H., and Selgrade, M. K. (2000). Allergen-triggered airway hyperresponsiveness and lung pathology in mice sensitized with the biopesticide Metarhizium anisopliae. Toxicology 143, 141154.[ISI][Medline]
Ward, M. D., Sailstad, D. M., and Selgrade, M. K. (1998). Allergic responses to the biopesticide Metarhizium anisopliae in Balb/c mice. Toxicol. Sci. 45, 195203.[Abstract]
Weiss, K. B., and Sullivan, S. D. (2001). The health economics of asthma and rhinitis: I. Assessing the economic impact. J. Allergy Clin. Immunol. 107, 38.[ISI][Medline]