* Mary Ann Swetland Center for Environmental Health, and Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106, and
Saint Mary's University, Halifax, Nova Scotia, Canada B3H3C3
1 To whom correspondence should be addressed at Case Western Reserve University, 10620 Cedar Avenue, Cleveland, Ohio 441063029. E-mail: ixy{at}po.cwru.edu.
Received September 7, 2004; accepted December 1, 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Stachybotrys chartarum; trichothecenes; inflammatory response.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Macrocyclic trichothecenes produced by S. chartarum, which are among the most potent protein synthesis inhibitors (Ueno, 1977), have become the primary focus of several investigations. However, in addition to trichothecenes, this fungus is capable of producing other secondary metabolites such as spirodrimanes and atranones (Jarvis, 2002
) and a number of protein factors such as hemolysin (Vesper et al., 1999
, 2001
) and proteinases (Kordula et al., 2002
; Yike et al., 2002
), which may also contribute to disease onset.
Alcohol extraction of S. chartarum spores leads to a sharp decrease in pulmonary inflammation in rats when compared to the effects of intact spores. We have previously reported that such extraction practically eliminates trichothecene toxicity of the spores (Yike et al., 2001). These results and the reports of Rao et al. (2000a
,b
) suggested that trichothecenes were the primary factors responsible for induction of inflammatory response in the lungs of animals exposed to S. chartarum. However, alcohol extraction not only removes trichothecenes and other small molecules from the spores, but also denatures fungal proteins that may also contribute to pulmonary injury. Contrary to the earlier reports of Nikulin et al. (1996
, 1997
), we have observed strong inflammatory responses in infant rats when using strains of S. chartarum that have low trichothecene content (Yike and Dearborn, 2004
). Similar observations on mice were described by Flemming et al. (2004)
and Leino et al. (2003)
. Other fungi that do not produce trichothecenes are also known to elicit inflammatory effects (Cooley et al., 1999
), indicating the presence of other proinflammatory substances. The involvement of agents such as ß-D-glucan in the inflammatory process is well documented (Rylander, 1999
).
Proteolytic enzymes secreted by fungi have only recently been investigated as possible inducers of inflammation. It has been suggested that, in addition to their antigenic properties, fungal proteases act via protease-activated receptors and G proteins to activate production and release of proinflammatory cytokines (Kauffman et al., 2000). A serine protease from S. chartarum has been purified and characterized as having broad substrate specificity that would enable it to hydrolyze many of the physiologically significant proteins in the lung such as collagen, protease inhibitors, and neuropeptides (Kordula et al., 2002
). We have shown variable proteolytic activity and the presence of serine proteases in the extracts from the spores of different isolates of S. chartarum (Yike et al., 2002
).
Stachylysin, a hemolytic protein isolated from S. chartarum, may also contribute to tissue destruction and hemorrhage in the lung (Gregory et al., 2003; Vesper et al., 2001
; Vesper and Vesper, 2002
). The profile of toxin and hemolysin production by different strains of S. chartarum isolated from houses in Cleveland, OH, revealed that the strains originating from houses of patients suffering from pulmonary hemorrhage were likely to be highly hemolytic but did not always contain macrocyclic trichothecenes (Jarvis et al., 1998
; Vesper et al., 1999
).
In this study we examine the hypothesis that rapidly released trichothecene mycotoxins are primarily responsible for the acute changes in animal growth and proinflammatory cells and cytokines, while slowly released fungal proteins may be involved in the sustained lower level of inflammation. Comparing pathophysiological effects of spore preparations in which toxicity and ability to release biologically active proteins have been differentially altered allowed the assessment of the relative contribution of different classes of fungal components to the inflammation and lung injury in animals exposed to S. chartarum.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo release of satratoxin G and stachylysin.
The release of satratoxin G into the lungs was studied in a separate group of animals exposed to 4 x 105 sp/g BW (high trichothecene-producing isolate JS58-17, three animals per time point) and lavaged at 0, 15, 45, 60 min and 2, 3, 4, 5, 6, 16, and 24 h. A higher dose of fungal spores was chosen to facilitate toxin detection. Another group of rat pups exposed to 1 x 105 sp/g BW of highly hemolytic isolate JS 5806 (to facilitate stachylysin detection) and lavaged on day 1, 2, 3, 4, 7, 21, and 28 (three animals per time point) was used to study the release of stachylysin.
Fungal spores.
Spores of Stachybotrys chartarum strain JS58-17, originally isolated from a home in Cleveland, Ohio, were collected from dry wall cultures as described before (Vesper et al., 1999), and their toxicity measured using the luciferase translation inhibition assay (Yike et al., 1999
). Toxicity was expressed as Satratoxin G equivalents calculated by comparing the toxic effects of fungal spores with those of pure toxin. The viability of the spore preparations collected from dry wall was evaluated by plating them on potato dextrose agar and counting colonies after 48-h incubation at 30°C. Fungal spores collected from dry wall cultures were suspended in PBS with 0.1% Tween 20, quickly counted, and immediately injected directly into the trachea of 7-day-old rats at a concentration of 1.0 x 105 sp/g BW in a final volume of 20 µl. Extracted spores were prepared by suspending 2 x 106 spores in 10 ml of 95% ethanol. The suspensions were kept for 16 h at room temperature, whereupon they were placed in an ultrasonic bath for 30 min, still at room temperature. Centrifuged spores were resuspended in 10 ml of fresh 95% ethanol and sonicated again. Spore pellets were then washed three times with 20 ml of PBS and resuspended in a small volume of PBS with 0.1% Tween 20, counted, and instilled as described below. Autoclaved spores were collected from dry wall cultures treated at 120°C for 15 min.
The release of stachylysin was studied using the spores of highly hemolytic isolate JS58-06 grown on dry wall.
Animals.
Pregnant Sprague-Dawley female rats at 1819 days of gestation were obtained from Charles River, (Wilmington, Massachusetts). The rats were housed in microisolators in the animal facility and fed the standard diet of Teklad 8664 (Harlan, Madison, Wisconsin) and water ad libitum. Each litter contained between 1012 pups. The animal research protocol was received for compliance with the standards of humane treatment of animals and approved by the Case Western Reserve University's institutional animal care committee.
Instillation of fungal spores.
Seven-day-old newborn rats (mean weight 16.99 ± 0.23 g, SEM, n = 288) were anesthetized with methoxyflurane (Shering-Plough Animal Health Corp., Union, NJ). A transverse skin incision was made, and the trachea was exposed by blunt dissection, whereupon 1.0 x 105 sp/g BW suspended in 20 µl of PBS were injected directly into the trachea using a 24 G catheter needle attached to a sterile Hamilton syringe. The incision was closed and treated with New Skin liquid bandage (Medtech Laboratories Inc., Jackson, WY) to facilitate healing and decrease maternal cannibalism. Control animals received 20 µl of PBS/Tween-20 solution.
Histology.
Isolated lungs were inflation fixed at 20 cm H2O in 3% paraformaldehyde-PBS solution for 48 h, dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Lungs embedded in paraffin were sectioned until all left lung lobes were represented in the sectioning plane. Ten serial sections, each 3 µm thick were then cut. Sections 1 and 10 were then mounted onto a microscope slide and stained with Harris Haematoxylin and aqueous eosin (H&E).
Morphometry.
Morphological changes in the lungs of infant rats were evaluated by morphometric analysis. This methodology has been used before to study the lungs of mice exposed to S. chartarum (Rand et al., 2003) and provided us with a quantitative measure of changes in alveolar air space due to granuloma formation in lungs inflamed as a result of exposure to fungal spores. Alveolar space was defined as the percentage of air space in the lung parenchyma.
H&E-stained lung sections were used for quantitative analysis of alveolar space areas. Briefly, six animals were analyzed for each time point, and 10 regions of the lung parenchyma from each animal were randomly scanned using a 20x objective. Large airways and lung vessels were excluded. Randomly selected areas included granulomas with no alveolar space as well as sections of the lungs with very high percentage of air space.
An image field 4.18 x 104 m2 was defined over a region of parenchyma entirely filling the field of view. With PC image analysis software from PCI Genomics, pixel intensity values were established that defined the alveolar space with elimination of all the cells and cellular debris within the spaces. The alveolar space area was then determined within each region of interest field (ROI) as percentage values.
Bronchoalveolar lavage (BAL).
The rats were weighed, and the total lung capacity (TLC, 60 µl/g body weight) (Sahebjami, 1992
) was estimated for each animal. Anesthetized animals were exsanguinated via the right ventricle, and the lungs were perfused with PBS. The trachea was cannulated using a 24 G catheter attached to a 1-ml tuberculin syringe. The lungs were lavaged three times with a sterile PBS volume equal to 75% of TLC. Protease inhibitors, PMSF at final concentration of 0.1mM and ethylenediamine tetracetic acid at final concentration of 5 mM, were added to the BAL fluid, and 200 µl aliquots of the BAL fluid were transferred into separate microfuge tubes for hemoglobin measurements. The remaining fluid was centrifuged for 10 min at 100 x g at 4°C. The supernatants were filtered through 0.22-µm syringe filters and stored at 80°C prior to cytokine and urea assays. The pellets were suspended in 1 ml of ice-cold PBS solution, and the cell count was determined using a hemocytometer. Aliquots of the cell suspension were transferred to slides in a cytospin centrifuge. Slides were stained with the Wright-Giemsa stain, and differential cell count was performed to determine the cell composition of the BAL fluid. The dilution factor for BAL fluid and the volume of epithelial lining fluid (ELF) was calculated for each animal based on the urea concentration in serum and BAL fluid (Rennard et al., 1986
).
Determination of satratoxin G.
Satratoxin G toxicity of spore extracts was determined using the luciferase translation inhibition test (Yike et al., 1999). Satratoxin G content in BAL fluid was determined by an ELISA assay according to Chung et al. (2003)
, using reagents kindly provided by Dr. J. Pestka, University of Michigan.
Proteolytic and hemolytic activity of spore extracts.
Fungal spores were suspended in a solution containing 2 mM Tris /HCl pH 7.4, 100 mM KCl, 1.5% polyvinylpyrolidone and disrupted in a mini-bead beater with acid-washed glass beads. Cell breakage was evaluated microscopically. The suspensions were centrifuged for 20 min at 17,000 x g, and supernatants were collected. Enzymatic activity was measured using 25 µg protein of spore extracts and an EnzCheck® kit from Molecular Probes (Eugene, OR) with fluorescein-labeled gelatin as substrate. The activity was expressed as arbitrary fluorescence units per 106 spores. Hemolytic activity of spore extracts was assessed qualitatively by placing drops of extracts on sheep blood agar and incubating at 37°C for up to 72 h.
Stachylysin assay.
Stachylysin in the BAL fluid was determined by ELISA using an affinity purified antibody directed against this protein as described previously (Van Emon et al., 2003).
Electrophoretic analysis of bronchoalveolar lavage fluid (BALf) protein.
BAL fluid and serum samples (15 µg/lane) were subjected to SDSPAGE using 420% gradient gels from Bio-Rad (Hercules, CA) under reducing conditions. The gels were stained with Coommassie Blue R-250 (Bio-Rad, Hercules, CA).
Cytokine assay.
The cytokines were assayed by ELISA using kits from R&D Systems (Minneapolis, MN) containing affinity purified antibodies to rat cytokines. The limits of detection for both IL-1ß and TNF- were 5.0 pg/ml.
Protein assay.
Protein was measured according to the method of Bradford using the reagents from Bio-Rad (Hercules, CA).
Statistical analysis.
All data were checked for normality and analyzed by one- (factor: treatment) or two-way (factors: treatment and time) ANOVA using Tukey's test for all pairwaise multiple comparisons and Dunnettt's test for comparisons against the control group. While the weight and total BAL protein data followed normal distribution, other data required a logarithmic transformation. For morphometry data that were not normally distributed even after logarithmic transformation, nonparametric analysis of variance was performed. All statistical tests were performed using Sigmastat version 2.03 (Jandel Scientific, San Rafael, CA), and the results were expressed as mean ± SEM and considered statistically significant at p < 0.05 probability level.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The growth of surviving animals exposed to intact spores was significantly reduced at 24, 36, 48, and 72 h after exposure (p < 0.05; Fig. 1). At 48 h these animals reached their original weight and continued to grow, although they remained smaller than controls. The growth of animals treated with autoclaved spores was significantly different from controls only at 24, 36, and 72 h. Statistically significant differences (p < 0.05) between the animals treated with intact and autoclaved spores were noted at 36, 48, and 60 h. There was no discernable impact of ethanol-extracted spores on the growth of infant animals.
|
|
|
|
As shown in Figure 4C, the numbers of neutrophils were highly elevated at 16 h following exposure to intact spores of S. chartarum reaching the highest level at 24 h (p = 0.002 compared to PBS-treated control animals). This was followed by a fall in the number of neutrophils at 36 h and another increase at 48 h. After 60 h the number of neutrophils recovered in the BAL fluid started to decrease. Autoclaved spores elicited a much smaller neutrophil response observed between 16 and 60 h after exposure (p = 0.002 compared to PBS-treated animals). This response was even weaker but still statistically significant in the animals exposed to ethanol-extracted spores (p 0.002 compared to control animals). The levels of neutrophils in PBS-treated control group were low (4 x 104 cells/ml of epithelial lining fluid) and did not increase with time.
Increases in the levels of lymphocytes were small and highly variable in all treated animals until after 48 h, when they appeared to raise sharply in the animals treated with intact spores (Fig. 4D).
Proinflammatory cytokines (TNF- and IL-1ß) were detected in the BAL fluid of animals treated with different preparations of the spores of S. chartarum in contrast to PBS-treated group, where no cytokines could be detected. The highest increases were seen with intact spores, followed by autoclaved and ethanol-extracted spores. The concentrations of IL-1ß reached the highest level at 24 h post-exposure (Fig. 5A) in all of the experimental groups (p
0.015 compared to control animals). In rat pups treated with ethanol-extracted spores, the increases in the level of IL-1ß were relatively small, and the 24-h maximum concentration was not significantly different from those of 16 and 48 h. TNF-
reached its highest concentration at 16 h post-exposure (Fig. 5B, p = 0.002) in animals treated with intact spores and then decreased rapidly. Exposure to autoclaved spores led to a smaller induction of this cytokine with a peak at 24 h (p = 0.024). Extracted spores elicited a much weaker response, with the highest concentration detected at 48 h post-exposure (not statistically significant compared to control group).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intact, viable spores of S. chartarum had the strongest effect on survival and growth of the animals and on all of the inflammatory indices including erythrocytes, leukocytes, proinflammatory cytokines, and alveolar space. Those effects were progressively reduced in the animals exposed to autoclaved and ethanol-extracted spores. The time course of the responses observed in this study confirms reports of Rao et al. (2000a,b
) and our earlier findings (Yike et al., 2001
) indicating that the first 2448 h after exposure corresponds to the period of acute toxicity, which is then followed by partial recovery and sustained inflammation with developing granulomas. All exposure-related deaths occurred within 48 h. While ethanol-extracted spores had no effect on survival and growth of exposed rat pups, viable spores significantly affected the weight gain and resulted in 14% mortality. Autoclaved spores did not cause deaths but also significantly reduced the growth rate during the first 24 h. This reduction was followed by a recovery that was much faster compared to the animals exposed to intact spores. Thus, the effects of heat-stable mycotoxins without proteins were more short-lived. It has been postulated (Flemming et al., 2004
) that depressed weight change in mice may be related to proinflammatory cytokines induced by S. chartarum that may lead to appetite suppression (Kuby, 1997
). Both the levels and the time course of cytokine release in the BAL fluid agree with such interpretation.
The decreases of alveolar space in the animals exposed to different spore preparations paralleled other inflammatory parameters in that differences between the control animals and those exposed to the intact, autoclaved, and ethanol-extracted spores were highly significant, although there was no clear distinction between the effects of autoclaved and extracted spores. This may be related to technical difficulties in attaining sufficiently uniform levels of lung inflation. We have observed that the lungs of infant rats with large areas of inflammation tend to remain underinflated due to airway obstruction, which may undermine the accuracy of morphometric analysis, especially when small changes are considered (i.e., in case of extracted vs. autoclaved spores).
The differences between the effects of autoclaved and intact spores indicate that, in addition to trichothecenes, other components of the spores of S. chartarum are also involved in the lung inflammation and injury. While the autoclaved spores retain at least the macrocyclic trichothecene secondary metabolites compared to the intact spores, their proteinaceous components are clearly inactivated. Comparing the preparations of autoclaved spores to intact, untreated spores allows for differentiation of the effects of trichothecenes (and other heat-stable mycotoxins) from that of proteins and/or other components that are inactivated during autoclaving. Such comparison indicates that those components of the spores are as important if not crucial in the pathophysiology of spore inhalation. The residual effects observed with ethanol-extracted spores are likely to result from the response to ß-glucan. However, assuming that 13, ß-D-glucan is not significantly affected by the autoclaving and extraction procedures used, this spore component appears to be a minor contributor to the pathophysiology as suggested by Korpi et al. (2003)
, who observed only minor irritation when exposing mice to that cell wall polymer.
Flemming et al. (2004) observed that high trichothecene producing S. chartarum isolate JS58-17 evoked relatively fast responses in mice, generally within 24 h followed by a decline, while the responses stimulated by the nontoxic isolate JS58-06 increased throughout a 96-h period. These differences suggest that trichothecenes may be the main factor responsible for the early responses that peaked at 24 h. Later effects may depend more on the release of other fungal factors (i.e., proteinases and hemolysin). Such interpretation is in agreement with earlier studies of Creasia et al. (1987)
and more recent findings of Rand et al. (2002)
, who were unable to observe inflammatory lesions in the lungs of animals exposed to pure trichothecenes T-2 toxin and isosatratoxin-F. It has been suggested (Pang et al., 1987
) that T-2 toxin is rapidly absorbed from the lung, metabolized, and excreted. Demonstration that satratoxin G could only be detected in the BAL fluid of infant rats immediately following exposure to the spores further supports this view. Unpublished observations by Jarvis (personal communication) also suggest that S. chartarum, similar to other fungi (Demain, 1981
), releases a major portion of the trichothecene toxin from the surface of the spores. Immunochemical localization of satratoxin within the spores of S. chartarum found it to be primarily along the outer plasmalemma surface and in the inner wall layer (Rand et al., 2004
) consistent with rapid release from the spores.
In contrast to the rapid release and absorption of satratoxin, stachylysin, a hemolytic protein from S. chartarum (Vesper et al., 2001), was slowly released from the spores into the lung, reaching its maximal concentration in the BAL fluid 4 days after exposure, and could still be detected at 28 days. We have previously reported similar results (Gregory et al., 2003
) showing that more stachylysin can be labeled by immunohistochemistry at 72 h than at 24 h post-exposure.
Proinflammatory cytokines, neutrophils, and BAL fluid protein appear to be the most sensitive indicators of inflammation, because they either cannot be detected in PBS control animals (cytokines) or are detected at very low concentrations (neutrophils, protein) and increase rapidly after exposure to the spores of S. chartarum. The second peak of neutrophils in the BALf from animals treated with intact spores may be related to another influx of these cells in response to continued proteolytic injury from host proteinases in addition to fungal proteinases. The neutrophil decline between these peaks may reflect apoptotic death and clearing by macrophages. Trichothecenes including satratoxins have been shown to cause apoptosis of leukocytes (Yang et al., 2000). Proinflammatory cytokines including TNF-
have been implicated in the lethality of other toxins, i.e., Shiga toxins (Sasaki et al., 2002
).
Induction of proinflammatory cytokines measured in the BAL fluid of mice exposed to S. chartarum (Flemming et al., 2004) exhibited somewhat different time profiles, with the maximum response for TNF-
seen at 3 h. Although the exact reason for these differences remains unclear, it may stem from the apparent increased susceptibility of mice seen in different dose-related responses. This is not surprising in light of the reports of different susceptibility to S. chartarum observed within different strains of mice (Rosenblum et al., 2002
). Higher susceptibility of mice compared to rats to other toxic inhalants has also been reported (Csanady et al., 2003
).
It has been our working hypothesis that early effects such as growth impairment and significant increases in neutrophils, erythrocytes, and proinflammatory cytokines are mediated primarily by trichothecene mycotoxins, while the sustained lower level of inflammation could be attributed to the more slowly released fungal proteins. The rapid increase in total protein appears to support such interpretation. However, significant differences in inflammatory indices between intact and autoclaved spores, seen shortly after exposure, point to the involvement of fungal proteins also in the early events even before those proteins reach their peak concentrations and, perhaps, their synergistic action with trichothecenes. There seems to be no clear difference in timing between the cytotoxic effects of trichothecenes and the proinflammatory action of other fungal-derived agents as was suggested by others (Korpi et al., 2002; Nielsen et al., 2002
; Murtoniemi et al., 2001
).
While the increased responses seen with intact, viable spores compared to autoclaved spores tend to underscore the involvement of fungal proteins, at least some of those responses might be related to an increase in fungal mass (i.e., higher concentrations of mycotoxins, including trichothecenes, proteins, and other metabolites) that might take place right before and during germination of the spores. Although no germination or formation of fungal hyphae was noted under the experimental conditions of this study, we have previously observed germination and limited persistence of Stachybotrys chartarum in 4-day-old rat pups (Yike et al., 2003). Even if there is no germination in the lungs of 7-day-old pups, intense synthesis and release of metabolites by highly viable spores is possible and may, at least partially, account for what could be otherwise interpreted as synergistic effects of mycotoxins and fungal proteins. We have observed large increases in the concentration of total protein secreted from the spores during incubation in vitro, while the concentration of satratoxin G remained constant (unpublished). Thus, it appears that the spores may not synthesize satratoxin G within the first 72 h but apparently can produce large quantities of proteins.
The possibility of involvement of fungal proteins in the pathophysiology of spore inhalation sheds new light on the clinical significance of those isolates of S. chartarum that do not produce macrocyclic trichothecenes but have higher hemolytic and proteolytic activity than high trichothecene producers (Yike and Dearborn, 2004). In addition, the synergistic action of mycotoxins, proteins, and other fungal agents may explain the adverse pulmonary effects of the relatively low spore concentrations found in indoor air.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cooley, J. D., Wong, W. C., Jumper, C. A., and Straus, D. C. (1999). Cellular and humoral responses in an animal model inhaling Penicillium chrysogenum. In Bioaerosols, fungi and mycotoxins: Health effects, assessment, prevention and control. (E. Johanning, Ed.), pp. 403410. Eastern New York Occupational and Environmental Health Center, Albany, New York.
Creasia, D. A., Thurman, J. D., Jones, L. J., III, Nealley, M. L., York, C. G., Wannemacher, R. W., and Bunner, D. L. (1987). Acute inhalation toxicity to T-2 mycotoxin in mice. Fundam. Appl. Toxicol. 8, 230235.[CrossRef][ISI][Medline]
Csanady, G. A., Kessler, W., Hoffmann, H. D., and Filser, J. G. (2003). A toxicokinetic model for styrene and its metabolite styrene-7,8-oxide in mouse, rat and human with special emphasis on the lung. Toxicol. Lett. 138, 75102.[CrossRef][ISI][Medline]
Dearborn, D. G., Dahms, B. B., Allan, T. M., Sorenson, W. G., Montana, E., and Etzel, R. A. (2003). Clinical profile of 30 infants with acute pulmonary hemorrhage in Cleveland. Pediatrics 110, 627637.[CrossRef][ISI]
Demain, A. I. (1981). Why do microorganisms produce antimicrobials? In Fifty years of Antimicrobials: Past Perspectives and Future Trends (P. A. Hunter, G. K. Darby, and N. J. Russel, Eds.), pp. 205239. Cambridge University Press, New York.
Etzel, R. A., Montana, E., Sorenson, W. G., Kullman, J., Allan, T. M., and Dearborn, D. G. (1998). Acute pulmonary hemorrhage in infants associated with exposure to Stachybotrys atra and other fungi. Arch. Pediatr. Adolesc. Med. 152, 757762.
Flemming, J., Hudson, B., and Rand, T. G. (2004). Comparison of inflammatory and cytotoxic lung responses in mice after intratracheal exposure to spores of two different Stachybotrys chartarum strains. Toxicol. Sci. 78, 267276.
Gregory, L., Rand, G., Dearborn D., Yike, I., and Vesper, S. (2003). Immunocytochemical localization of a hemolysin-like protein in Stachybotrys chartarum spores and spore impacted mouse lung tissues. Mycopathologia 156, 109117.[CrossRef][ISI][Medline]
Jarvis, B. B., Sorenson, W. G., Hintikka, E.-L., Nikulin, M., Zhou, Y., Yang, 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.
Jarvis, B. B., (2002). Chemistry and toxicology of molds isolated from water damaged buildings. In Mycotoxins and Food Safety (J. W. DeVries, M. W. Trucksess, and L. S. Jackson, Eds.), pp. 4352. Kluver Academic/Plenum Publishers, New York, NY.
Johanning, E., Biagini, R., Hull, D., Morey, P., Jarvis, B., and Landsbergis, P. (1996). Health and immunology study following exposure to toxigenic fungi (Stachybotrys chartarum) in a water-damaged office environment. Int. Arch. Occup. Environ. Health 68, 207218.[CrossRef][ISI][Medline]
Kauffman, H. F., Tomee, J. F. C., van de Riet, M. A., Timmerman, A. J. B., and Borger, P. (2000). Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production. J. Allergy Clin. Immunol. 105, 11851193.[CrossRef][ISI][Medline]
Kordula, T., Banbula, A., Macomson, J., and Travis, J. (2002). Isolation and properties of Stachyrase A, a chymotrypsin -like serine protease from Stachybotrys chartarum. Infect. Immun. 70, 419421.
Korpi, A., Kasanen, J.-P., Kosma, V.-M., Rylander, R., and Pasanen, A.-L. (2003). Slight respiratory irritation but not inflammation in mice exposed to (13)-beta-D glucan aerosols. Mediators Inflamm. 12, 139146.[CrossRef][ISI][Medline]
Korpi, A., Kasanen, J.-P., Raunio, P., Kosma, V.-M., Virtanen, T., and Pasanen, A.-L. (2002). Effects of areosols from nontoxic Stachybotrys chartarum on murine airways. Inhal. Toxicol. 14, 521540.[CrossRef][ISI][Medline]
Kuby, J. (1997). Leukocytes migration and inflammation. In Immunology, 3rd ed., pp. 357378. W. H. Freeman and Company, New York.
Leino, M., Makelam M., Reijula, K., Haahtela, T., Mussalo-Rauhamaa, H., Tuomi, T., Hintikka, E.-L., and Alenius, H. (2003). Intranasal exposure to a damp building mould, Stachybotrys chartarum, induces lung inflammation in mice by satratoxin-independent mechanisms. Clin. Exp. Allergy 33, 16031610.[CrossRef][ISI][Medline]
Moffatt, J. D., Jeffrey, K. L., and Cocks, T. M. (2002). Protease activated receptor-2 activating peptide SLIGRL inhibits bacterial lipopolysaccharide-induced recruitment of polymorphonuclear leucocytes into airways of mice. Am. J. Respir. Cell Mol. Biol. 26, 680684.
Murtoniemi, T., Nevalainen, A., Suutari, M., Toivola, M., Komulainen, H., and Hirvonen, M. R. (2001). Induction of cytotoxicity and production of inflammatory mediators in raw 264.7 macrophages by spores grown on six different plasterboards. Inhal. Toxicol. 13, 233247.[CrossRef][ISI][Medline]
Nielsen, K. F., Huttunen, K., Hyvaerinen, A., Andersen, B., Jarvis, B. B., and Hirvonen, M.-R. (2002). Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages. Mycopathologia 154, 201205.[CrossRef][ISI][Medline]
Nikulin, M., Reijulam K., Jarvis, B.B., and Hintikka, E.-L. (1996). Experimental lung mycotoxicosis in mice induced by Stachybotrys atra. Int. J. Exp. Path. 77, 213218.[ISI][Medline]
Nikulin, M., Reijula, K., Jarvis, B. B., Veijalainenm P., and Hintikka, E.-L. (1997). Effects of intranasal exposure to spores of Stachybotrys atra in mice. Fund. Appl. Toxicol. 35, 182188.[CrossRef][ISI][Medline]
Pang, V. F., Lambert, R. L., Felsburg, P. J., Beasley, V. R., Buck, W. B., and Haschek, W. M. (1987). Experimental T-2 toxicosis in swine following inhalation exposure: Effects on pulmonary and systemic immunity and morphologic changes. Toxicol. Pathol. 15, 308319.[ISI][Medline]
Rand, T. G., Gregory, L., Dearborn, D., and J. Pestka. (2004). Localization of satratoxin-G in Stachybotrys chartarum spores and spore-impacted mouse lung tissues using immunocytochemistry. Toxicol. Pathol. 32, 2634.[ISI]
Rand, T. G., Mahoney, M., White, K., and Oulton, M. (2002). Microanatomical changes associated with alveolar type II cells in juvenile mice exposed to Stachybotrys chartarum and isolated toxin. Toxicol. Sci. 65, 239245.
Rand, T. G., White, K., Logan, A., and Gregory, L. (2003). Histological, immunohistochemical and morphometric changes in lung tissue in juvenile mice experimentally exposed to Stachybotrys chartarum spores. Mycopathologia 156, 119131.[CrossRef][ISI][Medline]
Rao, C. Y., Brain, J. D., and Burge, H. A. (2000a). Reduction of Pulmonary Toxicity of Stachybotrys chartarum spores by methanol extraction of mycotoxins. Appl. Environ. Microbiol. 66, 28172821.
Rao, C. Y., Burge, H. A., and Brain, J. D, (2000b). The time course of responses to intratracheally instilled toxic Stachybotrys chartarum spores in rats. Mycopathologia 149, 2734.[CrossRef][ISI][Medline]
Rennard, S. I, Basset, G., Lecossier, D., O'Donnell, K. M., Pinkston, P., Martin, P. G., and Crystal, R. G. (1986). Estimation of volume of epithelial lining fluid recovered by lavage using urea as a marker of dilution. J. Appl. Physiol. 60, 532538.
Rosenblum, J. H., Molina, R. M., Donaghey, T. C., and Brain, J. D, (2002). Murine pulmonary responses to Stachybotrys chartarum have genetic determinants. Am. J. Respir. Crit. Care Med. 165, A 537.
Rylander, R. (1999). Indoor air-related effects and airborne (13)-ß-D-glucan. Environ. Health Perspect. 107 (Suppl. 3), 501503.[ISI][Medline]
Sahebjami, H. (1992). Aging of the normal lung. In Comparative Biology of the Normal Lung (R. A. Parent, Ed.), pp.351366. CRC Press, Bocca Raton, Ann Arbor, Tokyo.
Sasaki, S., Omoem, K., Tagawam, Y., Iwakuram, Y., Sekikawa, K., Shinagawa, K., and Nakane, A. (2002). Roles of gamma interferon and tumor necrosis factor-alpha in Shiga toxin lethality. Microb. Pathog. 33, 4347.[CrossRef][ISI][Medline]
Ueno, Y. (1977). Mode of action of trichothecenes. Ann. Nutr. Aliment. 31, 885900.[ISI][Medline]
Van Emon, J. M., Reed, A. W., Yike, I., and Vesper, S. J. (2003). ELISA measurement of StachylysinTM in serum to quantify human exposures to the indoor mold Stachybotrys chartarum. J. Occup. Environ. Med. 45, 582591.[ISI][Medline]
Vesper, S. J., Dearborn, D. G., Yike, I., Sorenson, W. G., and Haugland, R. A. (1999). Hemolysis, toxicity and RAPD analysis of Stachybotrys chartarum strains from the Cleveland pulmonary hemorrhage outbreak and non-Cleveland strains. Appl. Environ. Microbiol. 65, 31753181.
Vesper, S. J., Magnuson, S., Dearborn, D., Yike, I., and Haugland, R. A. (2001). Initial characterization of the hemolysin from Stachybotrys chartarum. Infect. Immun. 69, 912916.
Vesper, S. J., and Vesper, M. (2002). Stachylysin may be a cause of hemorrhaging in humans exposed to Stachybotrys chartarum. Infect. Immun. 70, 20652069.
Yang, G.-H., Jarvis, B. B., Chung, Y.-J., and Pestka, J. J. (2000). Apoptosis induction by satratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPK, and SAPK/JNK activation. Toxicol. Appl. Pharmacol. 164, 149160.[CrossRef][ISI][Medline]
Yike, I., Allan, T., Sorenson, W., and Dearborn, D. (1999). Highly sensitive protein translation assay for trichothecene toxicity in airborne particulates: Comparison with cytotoxicity assays. Appl. Environ. Microbiol. 65, 8894.
Yike, I., and Dearborn, D. (2004). Pulmonary effects of Stachybotrys chartarum in animal studies. Adv. Appl. Microbiol. 55, 241273.[ISI][Medline]
Yike, I., Miller, M. J., Tomashefski, J., Walenga, R., and Dearborn, D. (2001). Infant rat model of Stachybotrys chartarum induced mycotoxicosis. Mycopathologia 154, 139152.[ISI]
Yike, I., Rand, T., and Dearborn, D. (2002). Proteases from the spores of S. chartarum. Am. J. Respir. Crit. Care Med. 165, A 537.
Yike, I., Vesper, S., Tomashefski, J., and Dearborn, D. (2003). Germination, viability and clearance of Stachybotrys chartarum in the lungs of infant rats. Mycopathologia 156, 6775.[CrossRef][ISI][Medline]