Inflammation-associated gene transcription and expression in mouse lungs induced by low molecular weight compounds from fungi from the built environment.

Few metabolites from fungi found indoors have been tested for inflammatory mediators endpoints in primary cultures of alveolar macrophages or in vivo. In this study, mice were intratracheally instilled with a single dose comprising 4x10(-5)moletoxin/kg lung wt dose of either atranone C, brevianamide, cladosporin, mycophenolic acid, neoechinulin A & B, sterigmatocystin or TMC-120A. These toxins are from fungi common on damp building materials. The dose used was comparable to the estimated doses of possible human exposure. Hematoxylin and eosin (H&E) histology and Alcian Blue/Periodic Acid Schiff (AB/PAS) histochemistry were used to evaluate lungs for time course (4h and 12h post-exposure (PE)) inflammatory and toxic changes. Reverse-transcription (RT)-PCR based arrays were also employed to evaluate time course inflammation-associated gene transcription in lung tissues of the different toxins. Immunohistochemistry (IHC) was used to probe MIP-2 and Tnf-alpha protein expression in treatment lungs to determine whether responses correspond with gene transcription data. Both histology and histochemistry revealed that toxin exposed lungs at 12h PE showed evidence of inflammation. H&E revealed that bronchioli were lined with irregularly thickened and sometimes sloughing epithelium and bronchiolar spaces supported infiltration of leukocytes, cellular and mucus-like debris while alveolar spaces supported swollen macrophages and modest amorphous debris accumulations. All toxin-instilled lungs exhibited copious mucus production and alveolar macrophages with red stained cytoplasm on bronchiolar surfaces, especially at 12h PE. Array analysis of 83 inflammation-associated genes extracted from lung tissue demonstrated a number of patterns, compared to controls. 82 genes assayed at 4h PE and 75 genes at 12h PE were significantly altered (p< or =0.05; >or =1.5-fold or < or =-1.5-fold change) in the different treatment animal groups. Expression of transcriptionally regulated genes was confirmed using immunohistochemistry that demonstrated MIP-2 and Tnf-alpha staining in respiratory bronchiolar epithelia, alveolar macrophages and alveolar type II cells. The transcriptional regulation in these genes in the treatment groups suggests that they may serve central roles in the immunomodulation of toxin-induced pro-inflammatory lung responses. Hierarchical cluster analysis revealed significant patterns of gene transcription linking the response of the toxins at equimolar doses in three groups: (1) brevianamide, mycophenolic acid and neoechinulin B, (2) neoechinulin A and sterigmatocystin, and (3) cladosporin, atranone C and TMC-120. The results further confirm the inflammatory nature of metabolites/toxins from such fungi can contribute to the development of non-allergenic respiratory health effects.

The effect of elevated dietary cholesterol on pulmonary surfactant function in adolescent mice.

It has been established that phospholipids and cholesterol interact in films of pulmonary surfactant (PS). Generally it is thought that phospholipids increase film stability whereas cholesterol increases film fluidity. To study this further, we modified dietary cholesterol in mice which received either standard rodent lacking cholesterol (sd), or high cholesterol (2%) diet (hc) for 1 month. Phospholipid stability was investigated by a capillary surfactometer (CS), which measures airflow resistance and patency. PS was collected by bronchiolar lavage and centrifuged to obtain the surface-active film (SAF). Results showed that the hc-SAF had significantly more cholesterol than sd-SAF. CS analyses at 37 degrees C showed no significance differences in airflow resistance between hc-SAF and sd-SAF. However, at 37 degrees C, sd-SAF showed greater ability to maintain patency compared to hc-SAF, whereas at 42 degrees C hc-SAF showed patency ability similar to sd-SAF. The results suggested that increased cholesterol in hc-SAF induced less stability in the SAF possibly due to cholesterol's fluidizing effect on phospholipids at physiological temperatures.

DNA fragmentation in developing lung fibroblasts exposed to Stachybotrys chartarum (atra) toxins.

Stachybotrys chartarum (atra) is a toxic mold that grows on water-damaged cellulose-based materials. Research has revealed also that inhalation of S. chartarum spores caused marked changes in respiratory epithelium, especially to developing lungs. We analyzed the epigenetic potential of S. chartarum spore toxins on developing rat lung fibroblasts using single cell gel electrophoresis (comet assay). Isolated fetal lung fibroblasts were exposed to S. chartarum spore toxins for 15 min, 3, 14, or 24 hr and control cells were exposed to saline under the same conditions. Cells were embedded in agarose, electrophoresed under alkaline conditions and silver stained. DNA damage was assessed in terms of fragmentation as measured by comet tail length (DNA migration) and intensity (% DNA contained within head and tail). Upon visual inspection, control fibroblasts showed no DNA fragmentation whereas S. chartarum-treated cells had definable comets of various degrees depending upon the time-course. Analyses of the comets revealed that exposure to S. chartarum spore toxins for at least 15 min to 14 hr, induced increased DNA fragmentation in a time-dependent manner. The fact that exposure to toxins for 24 hr showed less damage suggested that developing lung fibroblasts may have the capability of repairing DNA fragmentation.

Comparison of immunomodulator mRNA and protein expression in the lungs of Stachybotrys chartarum spore-exposed mice.

Stachybotrys chartarum is an important toxigenic fungus that has been associated with respiratory disease onset in animals and humans. It can be separated into macrocyclic trichothecene-producing and nonproducing chemotypes based on secondary metabolite production. However, effects of spores of the two chemotypes on lung inflammatory responses are poorly understood. In this study, real-time reverse-transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) were used to investigate time-course (1, 3, 6, 24, and 48 h post-instillation [PI]) relationships in mice intratracheally exposed to 300 spores/g body weight of a macrocyclic trichothecene-producing (JS 58-17) and a nonproducing (JS 58-06) S. chartarum isolate and of Cladosporium cladosporioides. There were marked differences in the magnitude and temporal patterns of mouse lung immune responses to intratracheal exposure to spores of these species at this spore dose. Both macrophage inflammatory protein 2 (MIP-2) and surfactant protein-D (SP-D) mRNA expression were significantly upregulated in lungs of JS 58-17-treated animals compared to that of all other treatment animals at 6 and 24 h PI. Heightened mRNA expression of these immunomodulators combined with comparatively depressed MIP-2 and tumor necrosis factor (TNF)-a protein expression suggests that the action of macrocyclic trichothecenes sequestered in 58-17 spores is involved. Interestingly, TNF-a protein expression in all spore treatment animal groups was also significantly increased over that in saline controls. Similarities in expression among all spore treatment animals suggest that chemicals other than toxic secondary metabolites, and possibly spore-sequestered 1,3-beta-D-glucan, may contribute to lung pathogenesis.

Comparison of inflammatory and cytotoxic lung responses in mice after intratracheal exposure to spores of two different Stachybotrys chartarum strains.

Stachybotrys chartarum is an important toxigenic fungus that has been associated with respiratory disease onset in animals and humans. While it can be separated into macrocyclic trichothecene- and atranone-producing chemotypes based on secondary metabolite production, effects of spores of the two chemotypes on lungs are poorly understood. In this study we used bronchoalveolar lavage fluid (BALF) to investigate dose-response (30, 300, 3000 spores/g body weight [BW]) and time-course (3, 6, 24, 48, 96 h post instillation [PI]) relationships in mice to exposure of macrocyclic trichothecene- (JS 58-17) and atranone-producing (JS 58-06) S. chartarum strains, as well as Cladosporium cladosporioides spores. BALF total protein, albumin, pro-inflammatory cytokine (IL-1beta, IL-6, and tumor necrosis factor-alpha [TNF-alpha]), and lactate dehydrogenase (LDH) concentrations showed significant (p < 0.05) fungal species (S. chartarum vs. C. cladosporioides) and strain (58-17 vs. 58-06), spore dose and time dependent changes. The no adverse effect level (NOAEL) due to exposure to spores of JS 58-17 and JS 58-06 was < 30 spores/g BW; for C. cladosporioides it was < 300 spores/g BW. At moderate and high S. chartarum doses, BALF composition reflects differences in strain toxicity while at the lowest dose, BALF composition of either S. chartarum strain were similar. This suggests that at low spore doses, it is spore sequestered factors common to both strains not strain dependent toxins that are contributing to lung disease onset.

Immunocytochemical localization of stachylysin in Stachybotrys chartarum spores and spore-impacted mouse and rat lung tissue.

Stachylysin is a proteinaceous hemolytic agent that is produced by Stachybotrys chartarum. Stachylysin was found, using immunohistochemical and immunocytochemical methods, to be localized in S. chartarum spores/mycelia primarily in the inner wall suggesting that it is constitutively produced. Spores instilled in mouse or rat lung tissues resulted in granuloma formation, which showed the highest stachylysin concentration in the inner wall of the spore and near the spore, with less at distance indicating that it had diffused out from the spore. The in vitro high stachylysin producing strain (58-06) was also highest in vivo, based on immunohistochemistical staining. More stachylysin was observed in the mouse lung tissue at 72 h than at 24 h indicating that production/release is a relatively slow process. The localization of stachylysin in macrophage phagolysosomes suggests that these cells may be involved with hemolysin inactivation. This would be consistent with what is known about asp-hemolysin produced by Aspergillus fumigatus.

Histological, immunohistochemical and morphometric changes in lung tissue in juvenile mice experimentally exposed to Stachybotrys chartarum spores.

Stachybotrys chartarum is an important toxigenic fungus often associated with chronically wet cellulose-based building materials. The purpose of this study was to evaluate some histological, immunohistochemical and morphometric changes in mouse lung tissues exposed intratracheally to either 50 microl of 1.4 x 10(6) S. chartarum spores (< or = 35 ng toxin/kg BW), isosatratoxin-F (35 ng/kg BW), 50 microl of 1.4 x 10(6) Cladosporium cladosporioides spores, or 50 microl saline. Exposure of lung tissues to S. chartarum or C. cladosporioides spores resulted in granuloma formation at the sites of spore impaction. Some of the lung tissues impacted by S. chartarum spores also showed erythrocyte accumulation in the alveolar air space, dilated capillaries engorged with erythrocytes, and hemosiderin accumulation at spore impaction sites, which were features not noted in the C. cladosporioides-spore treated animals. Immunohistochemistry revealed reduced collagen IV distribution in lung granulomas in S. chartarum-treated animals especially at 48 and 72 hr post-exposure compared to that in lungs of mice with C. cladosporioides-spore induced granulomas. Quantitative analysis of pooled S. chartarum and C. cladosporioides spore impacted lungs revealed significant depression (P < 0.05) of alveolar air space from 71.4 +/- 6.1% in untreated animals to 56.04 +/- 6.1% in the S. chartarum- and 60.24 +/- 5.5% in the C. cladosporioides-spore treated animals. It also revealed that alveolus air space in S. chartarum treated animals declined significantly from 63.74 +/- 3.1% at 12 hr post-exposure to 42.94 +/- 7.9% at 72 hr post-exposure and was increased to 54.84 +/- 5.2% at 96 hr post-exposure. Alveolus air space in C. cladosporioides treated animals also decreased significantly from 64.84 +/- 7.1% at 12 hr exposure to 54.94 +/- 5.4% at 48 hr post-exposure and was increased to 64.64 +/- 10.1% at 96 hr post-exposure. It also revealed significant (P < 0.05) alveolar accumulation of erythrocytes from 1.24 +/- 1.4% in the untreated animals to 3.44 +/- 1.5% in the pooled S. chartarum spore treated animals. Erythrocyte abundance in S. chartarum treated animals increased significantly (P < 0.001) from 2.14 +/- 1.7% at 12 hr post-exposure to 5.54 +/- 1.5% at 72 hr and 4.94 +/- 1.4% at 96 hr post-exposure. These results further reveal that exposure to S. chartarum spores elicit tissue responses in vivo significantly different from those associated with exposure to pure trichothecene toxin and to spores of a non-toxigenic fungus.

Microanatomical changes in alveolar type II cells in juvenile mice intratracheally exposed to Stachybotrys chartarum spores and toxin.

Stachybotrys chartarum is an important environmental fungus. We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores and to the trichothecene, isosatratoxin-F, both in vitro and in vivo, in a juvenile mouse model. This sensitivity is manifest as significant changes in the composition and normal metabolic processing of pulmonary surfactant. This study evaluated the effects of a single intratracheal exposure of S. chartarum spores and toxin on ultrastructure and dimensions of alveolar type II cells from juvenile mice. This was to determine whether there are concurrent morphological and dimensional changes in the alveolar type II cell that reflect the metabolic alterations in pulmonary surfactant that we observed in the treated mice. Marked ultrastructural changes were associated with alveolar type II cells in both S. chartarum and isosatratoxin-F treated animals compared to untreated, saline, and Cladosporium cladosporioides spore treated animals. These ultrastructural changes included condensed mitochondria with separated cristae, scattered chromatin and poorly defined nucleolus, cytoplasmic rarefaction, and distended lamellar bodies with irregularly arranged lamellae. Point count stereological analysis revealed a significant increase (p < 0.05) in lamellar body volume density in S. chartarum and isosatratoxin-treated animals after 48 h exposure. Mitochondria volume density was significantly lower in the isosatratoxin-F (48 h exposure) and S. chartarum treated (24 and 48 h exposure) animals compared to those in the other treatment groups. These results reveal that exposure to S. chartarum spores and toxin elicit cellular responses in vivo differently from those associated with exposure to spores of a nontoxigenic mold species. They also indicate that accumulation of newly secreted pulmonary surfactant in the alveolar space of S. chartarum and isosatratoxin-F treated animals might be a consequence of cellular trauma resulting in lamellar body volume density changes leading to increased release of pulmonary surfactant into the alveolar space.

Effects of Stachybotrys chartarum on surfactant convertase activity in juvenile mice.

We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores, both in vitro and in an in vivo juvenile mouse model. In mice, this sensitivity is manifest in part as a significant increase in the newly secreted, biologically active, heavy aggregate form of alveolar surfactant (H) and the accumulation of the lighter, "metabolically used", biologically inactive alveolar surfactant forms (L(vivo)) in the interalveolar space. Conversion of the heavy, surface-active alveolar surfactant to the light metabolically used, nonsurface active forms is believed to involve the activity of an enzyme, namely convertase, which is thought to be derived from lamellar bodies (LB) in alveolar type II cells. The purpose of this study was to evaluate the effects of S. chartarum spores on mouse H and LB convertase activity by measuring their rates of conversion to L(vivo) using the in vitro surface area cycling technique. It was determined whether there were concurrent changes in the protein and phospholipid concentrations of the raw bronchoalveolar lavage fluid (RL) and LB fractions that could be correlated with changes in convertase activity. Conversions of H to L(vivo) in untreated control mice and saline-, isosatratoxin F-, and Cladosporium cladosporioides-exposed mice were not significantly different (p > 0.05). However, conversion from H to L(vivo) in the mice exposed to S. chartarum spores was significantly higher than all other treatment groups (p < 0.001). LB to L(vivo) conversions in untreated and saline-exposed mice were not significantly different, although they were significantly higher than the H to L(vivo) conversions in these two animal treatment groups (p < 0.005), which supports the position that LB is a source of convertase activity in animals. LB to L(vivo) conversion from C. cladosporioides-, isosatrotoxin F-, and S. chartarum-exposed mice were all significantly depressed (p < 0.003) compared to the LB to L(vivo) conversion values obtained from untreated and saline-exposed mice. Protein concentrations in RL, H, L(vivo), and LB from mice exposed to S. chartarum spores were significantly elevated compared to those from the other treatment groups (p < 0.001). Protein concentration in H isolated from C. cladosporioides-exposed mice was also significantly elevated above untreated and saline control animal levels. Phospholipid concentrations in H isolated from S. chartarum-exposed mice were significantly elevated compared to those from other treatment groups, while LB phospholipid concentrations were significantly increased compared to saline and untreated control animal groups. These results show that S. chartarum spores significantly alter convertase activity in both the H and LB surfactant fractions in juvenile mice and that these changes can be related to changes in protein and phospholipid concentrations in alveolar lavage fractions. As surfactant promotes lung stability by reducing the surface tension of the air-alveolar interface, these results further support our position that inhalation exposure to S. chartarum spores in exposed individuals may lead to altered surfactant metabolism, and possibly to lung dysfunction through diminished alveolar surfactant surface tension attributes, and lung stability.

Ichthyophonus irregularis sp. nov. from the yellowtail flounder Limanda ferruginea from the Nova Scotia shelf.

A previously described unusual form of the protistan parasite Ichthyophonus, differing in morphological and developmental features from I. hoferi sensu Plehn & Mulsow, was recovered from yellowtail flounder Limanda ferruginea Storer from the Brown's Bank area of the Nova Scotia shelf. The nuclear gene encoding the rRNA of the small ribosomal subunit was amplified from this unusual form of Ichthyophonus using the polymerase chain reaction, sequenced and aligned with other eukaryote small subunit (ssu)-rDNAs. Inferred phylogenetic trees clearly show that its ssu-rDNA is distinct from those of 2 isolates of I. hoferi sensu Plehn & Mulsow from different hosts and geographical locations (herring in the North Sea, and yellowtail flounder from the Nova Scotia shelf). We consider the unusual form to be a separate species, I. irregularis. The occurrence of a second, distinct type of Ichthyophonus within a single host species raises the possibility that ichthyophoniasis could be produced by different (although related) pathogens, and in some cases, by concurrent infections of the two.

Effects of Stachybotrys chartarum (atra) conidia and isolated toxin on lung surfactant production and homeostasis.

This study evaluated the effects of Stachybotrys chartarum conidia and a trichothecene, isosatratoxin-F, on choline incorporation into DSPC by fetal rabbit alveolar type II cells and on alveolar surfactant subtypes in mice. Exposure of fetal rabbit type II cells to S. chartarum conidia at concentrations of 10(3) to 10(6) conidia ml(-1) significantly depressed [3H] choline incorporation after 24 h of exposure. Exposure of the rabbit cells to 10(5) to 10(6) conidia ml(-1) also resulted in significantly depressed [3H] choline uptake after 48 h. Additionally, fetal rabbit alveolar type II cells exposed to isosatratoxin-F in concentrations ranging from 10(-9) to 10(-4) M showed a significant reduction in [3H] choline incorporation into DSPC. Alveolar surfactant phospholipid concentrations in the different metabolic subfractions of lung lavage fluid of mice intratracheally exposed to either 50 microl of 10(7) ml(-1) S. chartarum conidia or 50 microl 10(-7) M isosatratoxin-F showed some significant changes at 12, 24, 48, and 72 h post-exposure, compared to the surfactant subfractions of control mice which were either untreated, exposed to saline or to 50 microl of 10(-7) ml(-1) Cladosporium cladosporioides conidia. In both the S. chartarum- and the isosatratoxin-F-treated mice, exposure significantly increased P10, P100, and S100 phospholipid concentrations, while the P60 phospholipid concentrations were depressed. In contrast, C. cladosporioides-treated mice showed only one significant change in subfraction phospholipid concentration: P60 was depressed at 48 h post-exposure. These results reveal that alveolar type II cells are sensitive to exposure to S. chartarum conidia and to isosatratoxin F. Sensitivity is manifest by alterations in the normal metabolic processing of alveolar surfactant. In exposed mice, this effect appears to involve a significant increase in newly secreted surfactant and an accumulation of the used surfactant forms.

A novel clade of protistan parasites near the animal-fungal divergence.

Sequences of nuclear-encoded small-subunit rRNA genes have been determined for representatives of the enigmatic genera Dermocystidium, Ichthyophonus, and Psorospermium, protistan parasites of fish and crustaceans. The small-subunit rRNA genes from these parasites and from the "rosette agent" (also a parasite of fish) together form a novel, statistically supported clade. Phylogenetic analyses demonstrate this clade to diverge near the animal-fungal dichotomy, although more precise resolution is problematic. In the most parsimonious and maximally likely phylogenetic frameworks inferred from the most stably aligned sequence regions, the clade constitutes the most basal branch of the metazoa; but within a limited range of model parameters, and in some analyses that incorporate less well-aligned sequence regions, an alternative topology in which it diverges immediately before the animal-fungal dichotomy was recovered. Mitochondrial cristae of Dermocystidium spp. are flat, whereas those of Ichthyophonus hoferi appear tubulovesiculate. These results extend our understanding of the types of organisms from which metazoa and fungi may have evolved.

Effects of Ichthyophonus hoferi on condition indices and blood chemistry of experimentally infected rainbow trout (Oncorhynchus mykiss).

Body condition, hepatosomatic index and blood chemistry of Oncorhynchus mykiss experimentally infected with a tissue dwelling fish pathogenic fungus, Ichthyophonus hoferi, were monitored over a 6 wk period. This was to determine whether the infection constituted a stress manifest by changes in the hypothalamic-pituitary interrenal axis, and especially plasma cortisol levels. Infection caused anaemia and leucopenia but did not change the condition, hepatosomatic indices, or plasma chloride, cholesterol, cortisol, creatinine, glucose, osmolarity, potassium, total protein, sodium and T4. It is suggested that increased cortisol levels may not be a normal component of the stress response of fish to disease caused by invasive infectious agents.