Structurally diverse polyketides and phenylspirodrimanes from the soft coral-associated fungus Stachybotrys chartarum SCSIO41201.

Four undescribed polyketide derivatives, named arthproliferins A-D (1-4), and one undescribed phenylspirodrimane derivative, named arthproliferin E (7), along with 11 known metabolites (5, 6, 8-16) were isolated from the soft coral-associated fungus Stachybotrys chartarum SCSIO41201. Their structures were determined through spectroscopic methods, X-ray crystallography, and ECD analysis. Compounds 1 and 3-15 were evaluated for their cytotoxic, and antibacterial activities. Among them, compounds 1 and 15 displayed moderate inhibitory activity against methicillin-resistant Staphylococcus aureus ATCC 29213 with an MIC value of 78 and 39 µg/mL, respectively. Furthermore, compound 15 displayed strong cytotoxic activities against the tested cell line with IC50 values less than 39 nM.

New Bisabosquals from Stachybotrys sp. PH30583 Elicited on Solid Media.

Stachybotrys sp. PH30583 cultured in liquid medium only led to one structure type of novel isochroman dimers. Using the one strain-many compounds strategy, the reinvestigation of the metabolites from Stachybotrys sp. PH30583 cultured in rice solid medium led to the isolation of four triprenyl phenols, including two new bisabosquals and two known phenylspirodrimanes. Nitrobisabosquals A and B (1 and 2) are the first case of pyrrolidone-bisabosquals reported in literature. Totally different compounds were isolated using rice solid medium, compared with those isolated using liquid medium, so that rice solid medium presents a key factor in the production of triprenyl phenols. Compound 1 exhibited cytotoxicity against tumor cells, A-549, HL-60, MCF-7 SMMC-7721, and SW480, as well as weak anticoagulant activity with activated partial thromboplastin time (APTT) of 32.1 ± 0.17 s (p < 0.05 vs. Con.) at a concentration of 5 mM. Triprenyl phenol metabolites could be used as chemotaxonomic markers for Stachybotrys.

Isoprene Side-chain of SMTP is Essential for Soluble Epoxide Hydrolase Inhibition and Cellular Localization.

SMTPs, a family of natural small molecules that effectively treat ischemic stroke, are subject to clinical development. SMTPs enhance plasminogen activation and inhibit soluble epoxide hydrolase (sEH), leading to promotion of endogenous thrombolysis and anti-inflammation. The SMTP molecule consists of atricyclic γ-lactam moiety, an isoprene side-chain, and an N-linked side-chain. Here, we investigate the yet-to-be-characterized function of the isoprene side- chain of SMTPs in sEH inhibition and cellular distribution. The results demonstrated that oxidative modification as well as truncation of the side-chain abolished epoxide hydrolase inhibition. The introduction of a terminal hydroxy group exceptionally unaffected epoxide hydrolase, but led to impaired cellular localization, resulting in diminution of cellular epoxide hydrolase inhibition. Thus, the isoprene side-chain of SMTP is an important pharmacophore for epoxide hydrolase inhibition and cellular localization.

Sesquiterpenoids and xanthones derivatives produced by sponge-derived fungus Stachybotry sp. HH1 ZSDS1F1-2.

A new (2) and four known (1, 8-10) sesquiterpenoids, two new (3 and 4) and eight known (5-7, 11-15) xanthone derivatives were isolated from the cultures of sponge-derived fungus Stachybotry sp. HH1 ZDDS1F1-2. The structure of the compounds 1-15 was determined mainly by analysis of the one-dimensional and two-dimensional NMR spectroscopic data and by analogy with the data of those reported. Compound 1 was confirmed by X-ray crystallography. All the compounds were tested for their cytotoxic, antiinflammatory and antiviral (EV71) effects. Compounds 5, 7 and 11 showed significant cytotoxicity against selected human tumor cell lines. Compounds 3, 4 and 11 also displayed significant inhibitory activity against cycloooxygenase (COX-2). Compounds 4, 5 and 11 showed activities against intestinal virus EV71.

Occurrence of Stachybotrys chartarum chemotype S in dried culinary herbs.

Stachybotrys (S.) chartarum is an omnipresent cellulolytic mould which produces secondary metabolites, such as the highly toxic macrocyclic trichothecenes. While it is known to occur in animal feed like hay and straw as well as in water-damaged indoor environments, there is little knowledge about the occurrence of S. chartarum and its secondary metabolites in food. The objective of the present study was to examine selected dried culinary herbs for the presence of S. chartarum chemotype S, to assess the potential risk of a contamination of foods with macrocyclic trichothecenes. In total, 50 Stachybotrys isolates from different types of culinary herbs (n=100) such as marjoram (Origanum majorana Linné (L.)), oregano (Origanum vulgare L.), thyme (Thymus vulgaris L.), and savory (Satureja hortensis L.) were examined by MTT-cell culture test (effect-based bioassay), ELISA, and by liquid chromatography tandem mass spectrometry (LC-MS/MS). Selected toxic and non-toxic isolates (n=15) were genetically characterized by PCR and sequencing. Five isolates (10%) were highly toxic in the MTT-cell culture test, and the production of macrocyclic trichothecenes was proven by ELISA and LC-MS/MS. These five isolates were genetically confirmed as S. chartarum chemotype S. To the best of our knowledge, this is the first report about a contamination of dried culinary herbs with toxigenic S. chartarum.

Fungal and microbial volatile organic compounds exposure assessment in a waste sorting plant.

In the management of solid waste, pollutants over a wide range are released with different routes of exposure for workers. The potential for synergism among the pollutants raises concerns about potential adverse health effects, and there are still many uncertainties involved in exposure assessment. In this study, conventional (culture-based) and molecular real-time polymerase chain reaction (RTPCR) methodologies were used to assess fungal air contamination in a waste-sorting plant which focused on the presence of three potential pathogenic/toxigenic fungal species: Aspergillus flavus, A. fumigatus, and Stachybotrys chartarum. In addition, microbial volatile organic compounds (MVOC) were measured by photoionization detection. For all analysis, samplings were performed at five different workstations inside the facilities and also outdoors as a reference. Penicillium sp. were the most common species found at all plant locations. Pathogenic/toxigenic species (A. fumigatus and S. chartarum) were detected at two different workstations by RTPCR but not by culture-based techniques. MVOC concentration indoors ranged between 0 and 8.9 ppm (average 5.3 ± 3.16 ppm). Our results illustrated the advantage of combining both conventional and molecular methodologies in fungal exposure assessment. Together with MVOC analyses in indoor air, data obtained allow for a more precise evaluation of potential health risks associated with bioaerosol exposure. Consequently, with this knowledge, strategies may be developed for effective protection of the workers.

Co-cultivated damp building related microbes Streptomyces californicus and Stachybotrys chartarum induce immunotoxic and genotoxic responses via oxidative stress.

Oxidative stress has been proposed to be one mechanism behind the adverse health outcomes associated with living in a damp indoor environment. In the present study, the capability of damp building-related microbes Streptomyces californicus and Stachybotrys chartarum to induce oxidative stress was evaluated in vitro. In addition, the role of oxidative stress in provoking the detected cytotoxic, genotoxic, and inflammatory responses was studied by inhibiting the production of reactive oxygen species (ROS) using N-acetyl-l-cysteine (NAC). RAW264.7 macrophages were exposed in a dose- and time-dependent manner to the spores of co-cultivated S. californicus and S. chartarum, to their separately cultivated spore-mixture, or to the spores of these microbes alone. The intracellular peroxide production and cytotoxicity were measured by flow cytometric analysis, nitric oxide production was analyzed by the Griess method, DNA damage was determined by the comet assay, and cytokine production was measured by an immunochemical ELISA (enzyme-linked immunosorbent assay). All the studied microbial exposures triggered oxidative stress and subsequent cellular damage in RAW264.7 macrophages. The ROS scavenger, NAC, prevented growth arrest, apoptosis, DNA damage, and cytokine production induced by the co-culture since it reduced the intracellular level of ROS within macrophages. In contrast, the DNA damage and cell cycle arrest induced by the spores of S. californicus alone could not be prevented by NAC. Bioaerosol-induced oxidative stress in macrophages may be an important mechanism behind the frequent respiratory symptoms and diseases suffered by residents of moisture damaged buildings. Furthermore, microbial interactions during co-cultivation stimulate the production of highly toxic compound(s) which may significantly increase oxidative damage.

Purification and comparative neurotoxicity of the trichothecenes satratoxin G and roridin L2 from Stachybotrys chartarum.

Satratoxin G (SG), a macrocyclic trichothecene produced by Stachybotrys chartarum, induces apoptosis in cultured neuronal cells as well as nasal olfactory sensory neurons (OSN) in the nose and brain of mice exposed intranasally to this toxin. The purposes of this study were to (1) develop a facile method for production and purification of both SG and its putative biosynthetic precursor, roridin L2 (RL2), from S. chartarum cultures and (2) compare their relative neurotoxicity in vitro and in vivo. Stachybotrys chartarum 29-58-17 was cultured in Fernbach flasks on rice (5 x 10(5) spores/250 g rice) for 4 to 6 wk. Following extraction with acetonitrile, the extract was dried, dissolved in dichloromethane, and subjected to Michel-Miller silica-gel chromatography using a stepwise acetonitrile-dichloromethane gradient with SG and RL2 eluting in the 30 and 40% acetonitrile fractions, respectively. Purification of the two compounds was completed by C18 semipreparative reverse-phase liquid chromatography using an acetonitrile-water gradient, and purity was confirmed by electrospray ionization/collision-induced dissociation (ESI-CID) tandem mass spectroscopy. Although viability significantly decreased in PC-12 neuronal cells treated with 10 to 25 ng/ml of SG, RL2 at concentrations up to 1000 ng/ml was not toxic. Flow cytometry and agarose DNA fragmentation assays revealed that SG at 10 to 25 ng/ml induced apoptotic death in the PC-12 cells, while RL2 at concentrations up to 1000 ng/ml was without effect. In a similar fashion, intranasal exposure of mice (female B6C3F1) to SG at 100 microg/kg body weight (bw) induced marked OSN apoptosis and atrophy of the olfactory epithelium, whereas RL2 at the equivalent dose did not exhibit toxicity. Taken together, an optimized protocol for production and isolation of trichothecenes from S. chartarum cultures is described and further demonstrates that while the macrocyclic SG was neurotoxic in vitro and in vivo, its biosynthetic precursor, RL2, was nontoxic.

DNA damage and p53 in RAW264.7 cells induced by the spores of co-cultivated Streptomyces californicus and Stachybotrys chartarum.

Our recent studies have revealed that the co-cultivation of environmental microbes, Streptomyces californicus and Stachybotrys chartarum, potentiates the immunotoxic properties of the spores. In the present study, the spore-induced genotoxic potential of these microbes was investigated. Dose related differences in genotoxic and cytotoxic effects and in p53 level in mouse RAW264.7 macrophages were studied after 24h exposure to the spores of separately cultivated Streptomyces californicus or Stachybotrys chartarum alone, a simple spore-mixture of these microbes as well as to the spores of co-cultivated microbes. The genotoxic effect of the exposures was determined by the Comet assay and p53 level was analyzed by immunoblotting. Cytotoxicity was assessed by using flow cytometric analysis and also by the MTT test. The results revealed that the spores of co-cultivated microbes evoked DNA damage, p53 accumulation and cytotoxicity at a lower dose than the other exposures, and at the highest dose there was a 2.5-fold increase in DNA damage compared to control. In addition, the spores of Streptomyces californicus alone induced a 1.5-fold increase in DNA damage compared to control, dose dependent p53 accumulation and also extensive cytotoxicity. In contrast, the mixture of separately cultivated spores or the spores of Stachybotrys chartarum alone did not induce DNA damage with any tested dose although they triggered significant cytotoxicity and a slightly increased p53 level. Our results suggest that the detected genotoxic responses are the result of DNA damage in RAW264.7 cells by some genotoxically active metabolite(s) and the production of this compound was stimulated in Streptomyces californicus when it was co-cultivated with Stachybotrys chartarum.

Global gene expression changes underlying Stachybotrys chartarum toxin-induced apoptosis in murine alveolar macrophages: evidence of multiple signal transduction pathways.

The overall mechanism(s) underlying macrophage apoptosis caused by the toxins of the indoor mold Stachybotrys chartarum (SC) are not yet understood. In this direction, we report a microarray-based global gene expression profiling on the murine alveolar macrophage cell line (MH-S) treated with SC toxins for short (2 h) and long (24 h) periods, coinciding with the pre-apoptotic (<3 h) and progressed apoptotic stages of the treated cells, respectively. Microarray results on differential expression were validated by real-time RT-PCR analysis using representative gene targets. The toxin-regulated genes corresponded to multiple cellular processes, including cell growth, proliferation and death, inflammatory/immune response, genotoxic stress and oxidative stress, and to the underlying multiple signal transduction pathways involving MAPK-, NF-kB-, TNF-, and p53-mediated signaling. Transcription factor NF-kB showed dynamic temporal changes, characterized by an initial activation and a subsequent inhibition. Up-regulation of a battery of DNA damage-responsive and DNA repair genes in the early stage of the treatment suggested a possible role of genotoxic stress in the initiation of apoptosis. Simultaneous expression changes in both pro-survival genes and pro-apoptotic genes indicated the role of a critical balance between the two processes in SC toxin-induced apoptosis. Taken together, the results imply that multiple signaling pathways underlie the SC toxin-induced apoptosis in alveolar macrophages.

Co-cultivation of Streptomyces californicus and Stachybotrys chartarum stimulates the production of cytostatic compound(s) with immunotoxic properties.

We have recently shown that the actinobacterium Streptomyces californicus and the fungus Stachybotrys chartarum originating from moisture damaged buildings possess both immunotoxic and immunostimulatory characteristics, which are synergistically potentiated by microbial interaction. In the search for the causative agent(s) behind the immunotoxicity, the cytostatic effects of the co-cultivated spores of S. californicus and S. chartarum were compared to those caused by widely used cytostatic agents produced by streptomycetes. The RAW264.7 macrophages were exposed to four doses of doxorubicin (DOX), actinomycin D (AMD), mitomycin C (MMC) or phleomycin (PHLEO) for 24 h. Kinetics of the spores of the co-cultivated and the separately cultivated microbes (1x10(6) spores/ml) was compared to DOX (0.15 muM). Apoptotic responses were analyzed by measuring DNA content and mitochondria membrane depolarization with flow cytometer, and by the fluorometric caspase-3 assay. The present data indicate that interactions during co-cultivation of S. californicus and S. chartarum stimulate the production of an unidentified cytostatic compound(s) capable of inducing mitochondria mediated apoptosis and cell cycle arrest at S-G(2)/M. The spores of co-cultivated microbes caused a 4-fold collapse of mitochondrial membrane potential and an almost 6-fold caspase-3 activation and DNA fragmentation when compared to control. Similar responses were induced by DNA cleaving compounds, especially DOX and AMD, at the relatively low concentrations, but not the spores of the same microbes when they were grown separately. These data suggest that when growing in the same habitat, interactions between S. californicus and S. chartarum stimulates the production of an unknown cytostatic compound(s) which evoke immunotoxic effects similar to those by chemotherapeutic drugs.

Acute inflammatory responses to Stachybotrys chartarum in the lungs of infant rats: time course and possible mechanisms.

Stachybotrys chartarum has been linked to building-related respiratory problems including pulmonary hemorrhage in infants. The macrocyclic trichothecenes produced by S. chartarum have been the primary focus of many investigations. However, in addition to trichothecenes this fungus is capable of producing other secondary metabolites and a number of protein factors. This study examines the effects of intact, autoclaved, and ethanol-extracted spores on the lungs of infant rats as an approach to differentiate between secondary metabolites and protein factors. Seven-day-old infant rats were exposed intratracheally to 1 x 10(5) spores/g body weight (toxic strain JS58-17) and sacrificed at various times up to 72 h. The inflammatory response was measured by morphometric analysis of the lungs and determination of inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid. Alveolar space was greatly reduced in animals exposed to fungal spores compared to phosphate buffered saline (PBS)-treated controls. The largest effects were observed in pups treated with intact spores where alveolar space 24 h after treatment was 42.1% compared to 56.8% for autoclaved spores, 51.1% for ethanol-extracted spores, and 60.6% for PBS-treated controls. The effects of different spore preparations on inflammatory cells, cytokine, and protein concentrations in the BAL fluid can be ranked as intact > autoclaved > extracted. Tumor necrosis factor alfa (TNF-alpha), interleukin 1-beta (IL-1beta), and neutrophils were the most sensitive indicators of inflammation. The difference between autoclaved (100% trichothecene toxicity, denatured/enzymatically inactive proteins) and intact (100% trichothecene activity, unaltered/released proteins) spores indicates the involvement of fungal proteins in the inflammatory response to S. chartarum and sheds new light on the clinical importance of "nontoxic" strains.

Localization of satratoxin-G in Stachybotrys chartarum spores and spore-impacted mouse lung using immunocytochemistry.

Satratoxin-G (SG) is the major macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum (atra) and has been implicated as a cause of a number of animal and human health problems including pulmonary hemorrhage in infants. However, there is little understanding where this toxin is localized in the spores and mycelial fragments of this species or in the lung impacted by SG-sequestered spores. The purpose of this study was to evaluate the distribution of SG in S. chartarum spores and mycelium in culture, and spore-impacted mouse lung in vivo, using immunocytochemistry. SG was localized predominately in S. chartarum spores with moderate labelling of the phialide-apex walls. Labelling was primarily along the outer plasmalemma surface and in the inner wall layer. Only modest labelling was observed in hyphae. Toxin localization at these sites supports the position that spores contain the highest satratoxin concentrations and that the toxin is constitutively produced. In impacted mouse lung, highest SG labelling was detected in lysosomes, along the inside of the nuclear membrane in nuclear heterochromatin and RER within alveolar macrophages. Alveolar type II cells also showed modest labelling of the nuclear heterochromatin and RER. There was no evidence that the toxin accumulated in the neutrophils, fibroblasts, or other cells associated with the granulomas surrounding spores or mycelial fragments. These observations indicate that SG displays a high degree of cellular specificity with respect to its uptake in mouse lung. They further indicate that the alveolar macrophages play an important role in the sequestration and immobilization of low concentrations of the toxin.

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.

Indoor mold, toxigenic fungi, and Stachybotrys chartarum: infectious disease perspective.

Damp buildings often have a moldy smell or obvious mold growth; some molds are human pathogens. This has caused concern regarding health effects of moldy indoor environments and has resulted in many studies of moisture- and mold-damaged buildings. Recently, there have been reports of severe illness as a result of indoor mold exposure, particularly due to Stachybotrys chartarum. While many authors describe a direct relationship between fungal contamination and illness, close examination of the literature reveals a confusing picture. Here, we review the evidence regarding indoor mold exposure and mycotoxicosis, with an emphasis on S. chartarum. We also examine possible end-organ effects, including pulmonary, immunologic, neurologic, and oncologic disorders. We discuss the Cleveland infant idiopathic pulmonary hemorrhage reports in detail, since they provided important impetus for concerns about Stachybotrys. Some valid concerns exist regarding the relationship between indoor mold exposure and human disease. Review of the literature reveals certain fungus-disease associations in humans, including ergotism (Claviceps species), alimentary toxic aleukia (Fusarium), and liver disease (Aspergillys). While many papers suggest a similar relationship between Stachybotrys and human disease, the studies nearly uniformly suffer from significant methodological flaws, making their findings inconclusive. As a result, we have not found well-substantiated supportive evidence of serious illness due to Stachybotrys exposure in the contemporary environment. To address issues of indoor mold-related illness, there is an urgent need for studies using objective markers of illness, relevant animal models, proper epidemiologic techniques, and examination of confounding factors.

Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages.

The metabolite profiles of 20 Stachybotrys spp. isolates from Finnish water-damaged buildings were compared with their biological activities. Effects of purified compounds on cytotoxicity and production of inflammatory mediators such as nitric oxide, IL-6 and TNFalpha in murine RAW264.7 macrophage cells were studied. The 11 isolates belonging to the satratoxin-producing chemotype were highly cytotoxic to the macrophages. The isolates inducing inflammatory mediators all belonged to the atranone-producing chemotype, but pure atranones B, and D did not elicit a response in the bioassay. Altogether, cytotoxicity of Stachybotrys sp. isolates appear to be related to satratoxin production whereas the specific component inducing inflammatory responses in atranone-producing isolates remains obscure.

Effects of satratoxins and other macrocyclic trichothecenes on IL-2 production and viability of EL-4 thymoma cells.

The macrocyclic trichothecenes are a group of potent protein synthesis inhibitors that have been encountered in indoor air and food as a result of infestation by the fungus Stachybotrys. To evaluate the capacity of these mycotoxins to alter immune functions, the effects of satratoxin G, H, F, roridin A, and verrucarin A on interleukin 2 (IL-2) production and viability were evaluated in a murine T-cell model. EL-4 thymoma cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin and concurrently exposed to various concentrations of the trichothecenes. Enzyme-linked immunosorbent assay (ELISA) of supernatants revealed that IL-2 concentrations at 24 and 72 h were significantly increased in cultures that were incubated in the presence of 0.5 to 1 ng/ml of satratoxin H, 1 to 5 ng/ml of isosatratoxin F, 0.1 to 0.5 ng/ml of roridin A, and 0.25 to 0.5 ng/ml of verrucarin A. However, IL-2 levels at these time points were significantly depressed when incubated in the presence of higher concentrations of satratoxin G (> or =2.5 ng/ml), satratoxin H and isosatratoxin F (> or =5 ng/ml), and roridin A and verrucarin A (> or =1 ng/ml). Cell viability, as measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, was depressed by each of the trichothecenes in a concentration-dependent manner. MTT responses were significantly decreased by as little as 0.5 ng/ml satratoxin G, roridin A, and verrucarin A and by 2.5 ng/ml of isosatratoxin F and satratoxin H. When these data were compared to those found in EL-4 cells for the 8-ketotrichothecene vomitoxin (deoxynivalenol), a common food contaminant, the macrocyclic trichothecenes were at least 100 times more potent. The results indicate that, at low concentrations, macrocyclic trichothecenes as a group could superinduce IL-2 production even while partially decreasing cell viability, whereas higher concentrations suppressed cytokine production and were markedly cytotoxic. The capacity of these compounds to dysregulate cytokine production in a biphasic fashion may play an etiologic role in outbreaks of human illnesses associated with indoor Stachybotrys contamination.

Production of reactive oxygen metabolites by opsonized fungi and bacteria isolated from indoor air, and their interactions with soluble stimuli, fMLP or PMA.

Changes in the levels of free intracellular calcium ([Ca2+]i) and the production of reactive oxygen metabolites (ROM) induced by opsonized indoor air fungi and bacteria in human polymorphonuclear leukocytes (PMNL) were measured. Moreover, modification of a chemotactic peptide (fMLP)-and a tumor promoter (PMA)-induced production of ROM by opsonized fungi and bacteria were studied. The cells were exposed to graded doses of opsonized Candida sp., Aspergillus sp., Cladosporium sp., Stachybotrys sp., Penicillium sp., Paecilomyces sp., or A4 or A91 Streptomyces sp. alone, or together with fMLP or PMA. All the organisms were isolated from air samples of mold-problem buildings. None of the fungi or bacteria induced changes in [Ca2+]i or the production of ROM without opsonization with human serum. Of all opsonized fungi and bacteria, only Candida sp. elevated [Ca2+]i. All fungi and bacteria, except Paecilomyces sp. and Stachybotrys sp., markedly increased the production of ROM in PMNL. Furthermore, A91 Streptomyces sp. and Aspergillus sp. amplified fMLP-induced production of ROM. Only Candida sp. increased PMA-induced phenomen that normally occurs in the lung, was required for biological activity of the fungi and bacteria. Amplification by opsonization of fungi- or bacteria-induced leukocyte activation revealed remarkable changes between these biologically active particles. The present results suggest that many indoor air fungi and bacteria may activate leukocytes to produce oxidative stress, perhaps associated with harmful effects in exposed individuals.