Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

Mold allergy: is it real and what do we do about it?

INTRODUCTION: fungi produce substances that contain pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps) which bind to pattern recognition receptors, stimulating innate immune responses in humans. they also produce allergens that induce production of specific ige. Areas covered: In this review we cover both innate and adaptive immune responses to fungi. Some fungal products can activate both innate and adaptive responses and in doing so, cause an intense and complex health effects. Methods of testing for fungal allergy and evidence for clinical treatment including environmental control are also discussed. In addition, we describe controversial issues including the role of Stachybotrys and mycotoxins in adverse health effects. Expert commentary: Concerns about long-term exposure to fungi have led some patients, attorneys and fungus advocates to promote fears about a condition that has been termed toxic mold syndrome. This syndrome is associated with vague symptoms and is believed to be due to exposure to mycotoxins, though this connection has not been proven. Ultimately, more precise methods are needed to measure both fungal exposure and the resulting health effects. Once that such methods become available, much of the speculation will be replaced by knowledge.

TLR9-dependent IL-23/IL-17 is required for the generation of Stachybotrys chartarum-induced hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease that develops after repeated exposure to inhaled particulate Ag. Stachybotrys chartarum is a dimorphic fungus that has been implicated in a number of respiratory illnesses, including HP. In this study, we have developed a murine model of S. chartarum-induced HP that reproduces pathology observed in human HP, and we have hypothesized that TLR9-mediated IL-23 and IL-17 responses are required for the generation of granulomatous inflammation induced by inhaled S. chartarum. Mice that undergo i.p. sensitization and intratracheal challenge with 10(6) S. chartarum spores developed granulomatous inflammation with multinucleate giant cells, accompanied by increased accumulation of T cells. S. chartarum sensitization and challenge resulted in robust pulmonary expression of IL-17 and IL-23. S. chartarum-mediated granulomatous inflammation required intact IL-23 or IL-17 responses and required TLR9, because TLR9(-/-) mice displayed reduced IL-17 and IL-23 expression in whole lung associated with decreased accumulation of IL-17 expressing CD4(+) and γδ T cells. Compared with S. chartarum-sensitized dendritic cells (DC) isolated from WT mice, DCs isolated from TLR9(-/-) mice had a reduced ability to produce IL-23 in responses to S. chartarum. Moreover, shRNA knockdown of IL-23 in DCs abolished IL-17 production from splenocytes in response to Ag challenge. Finally, the intratracheal reconstitution of IL-23 in TLR9(-/-) mice recapitulated the immunopathology observed in WT mice. In conclusion, our studies suggest that TLR9 is critical for the development of Th17-mediated granulomatous inflammation in the lung in response to S. chartarum.

Sta c 3 epitopes and their application as biomarkers to detect specific IgE.

Sta c 3 was determined to be one of the major allergens from Stachybotrys chartarum sensu lato. This is among the most common fungi on wet building materials. Various cognizant authorities have found that mold and dampness are associated with exacerbation of asthma and increased upper respiratory disease. The primary amino-acid sequence of this allergen was reported, however the IgE-binding epitopes were unknown. Using SPOT-synthesis techniques (SPOTs), one main linear epitope located between V91 and G105 was discovered by Western blot with atopic human sera. This was confirmed by inhibition ELISA assays with both synthesized epitopes and natural Sta c 3. Alanine scanning also revealed R100 and K101 are the critical amino acids for IgE binding. Using the binding-enhanced and alanine substituted epitopes as potential biomarkers, a reverse ELISA method was developed to detect the Sta c 3 sIgE in atopic human sera. This method employed streptavidin-biotinylated mutated epitopes as the capture matrix, probing the possibility of using small peptides for allergen/species specific IgE assays.

Stachybotrys chartarum-induced hypersensitivity pneumonitis is TLR9 dependent.

Hypersensitivity pneumonitis (HP), an inflammatory lung disease, develops after repeated exposure to inhaled particulate antigen and is characterized by a vigorous T helper type 1-mediated immune response, resulting in the release of IL-12 and interferon (IFN)-γ. These T helper type 1 cytokines may participate in the pathogenesis of HP. Stachybotrys chartarum (SC) is a dimorphic fungus implicated in a number of respiratory illnesses, including HP. Here, we have developed a murine model of SC-induced HP that reproduces pathology observed in human HP and hypothesized that toll receptor-like 9 (TLR9)-mediated dendritic cell responses are required for the generation of granulomatous inflammation induced by inhaled SC. Mice sensitized and challenged with 10(6) SC spores develop granulomatous inflammation with multinucleate giant cells, accompanied by increased accumulation of neutrophils and CD4(+) and CD8(+) T cells. SC sensitization and challenge resulted in robust pulmonary expression of tumor necrosis factor-α, IL-12, and IFN-γ. SC-mediated granulomatous inflammation required IFN-γ and was TLR9 dependent, because TLR9(-/-) mice displayed reduced peribronchial inflammation, decreased accumulation and/or activation of polymorphonuclear (PMN) and CD4(+) and CD8(+) T cells, and reduced lung expression of type 1 cytokines and chemokines. T-cell production of IFN-γ was IL-12 dependent. Our studies suggest that TLR9 is critical for dendritic cell-mediated development of a type 1 granulomatous inflammation in the lung in response to SC.

[Interstitial lung disease due to domestic moulds]. / Pneumopathie interstitielle diffuse secondaire aux moisissures domestiques.

Identifying the role of fungi present in the domestic environment in the development of interstitial pneumonia can be a difficult clinical problem. We report a case of interstitial lung disease case occurring in a 53-year-old patient. He presented with profound hypoxemia (PaO(2) 54mmHg). Chest CT showed diffuse ground glass opacities. Initial blood tests for allergy and autoimmune disease were negative. Faced with a worsening of his clinical status after returning home he was hospitalized several times. At fibreoptic bronchoscopy, multiple white deposits were observed. Bronchoalveolar lavage with differential cell count was performed, revealing a 23% lymphocytosis. Serology for specific household molds showed moderate reaction to various molds found in homes, especially Stachybotrys chartarum. Pulmonary function tests revealed a moderate restrictive pattern with impaired diffusion of carbon monoxide and a bronchiolocentric interstitial pneumonia was found at lung biopsy. After a permanent move to a new residence, clinical parameters, radiological, biological and functional normalized. The final diagnosis was interstitial lung disease related to mycotoxins of S. Chartarum. The diagnosis of hypersensitivity pneumonitis to domestic mold or interstitial lung disease secondary to mycotoxins should be considered in patients presenting with interstitial pneumonia and requires specific investigations to ensure that an environmental cause with an allergic or toxic role is not missed.

Production and characterization of IgM monoclonal antibodies against hyphal antigens of Stachybotrys species.

Stachybotrys is a hydrophilic fungal genus that is well known for its ability to colonize water-damaged building materials in indoor environments. Personal exposure to Stachybotrys chartarum allergens, mycotoxins, cytolytic peptides, and other immunostimulatory macromolecules has been proposed to exacerbate respiratory morbidity. To date, advances in Stachybotrys detection have focused on the identification of unique biomarkers that can be detected in human serum; however, the availability of immunodiagnostic reagents to Stachybotrys species have been limited. In this study, we report the initial characterization of monoclonal antibodies (MAbs) against a semi-purified cytolytic S. chlorohalonata preparation (cScp) derived from hyphae. BALB/c mice were immunized with cScp and hybridomas were screened against the cScp using an antigen-mediated indirect ELISA. Eight immunoglobulin M MAbs were produced and four were specifically identified in the capture ELISA to react with the cScp. Cross-reactivity of the MAbs was tested against crude hyphal extracts derived from 15 Stachybotrys isolates representing nine Stachybotrys species as well as 39 other environmentally abundant fungi using a capture ELISA. MAb reactivity to spore and hyphal antigens was also tested by a capture ELISA and by fluorescent halogen immunoassay (fHIA). ELISA analysis demonstrated that all MAbs strongly reacted with extracts of S. chartarum but not with extracts of 39 other fungi. However, four MAbs showed cross-reactivity to the phylogenetically related genus Memnoniella. fHIA analysis confirmed that greatest MAb reactivity was ultrastructurally localized in hyphae and phialides. The results of this study further demonstrate the feasibility of specific MAb-based immunoassays for the detection of S. chartarum.

Characterization of human antigenic proteins SchS21 and SchS34 from Stachybotrys chartarum.

BACKGROUND: SchS21 and SchS34 are proteins from Stachybotrys chartarum sensu latto that are antigenic in goats, mice and humans. Monoclonal antibodies to these proteins react with spores of S. chartarum and S. chlorohalonata but do not cross-react with a diverse taxonomic and ecological array of other fungi. METHODS: Based on partial sequences of the 21- and 34-kDa proteins, obtained from tandem mass spectra and Edman degradation, degenerate primers were designed for touchdown PCR and the resulting amplicons were sequenced. Subsequently, inverse-PCR was used to obtain genomic DNA sequences encoding SchS21 and SchS34. RT-PCR products were sequenced to predict the mature protein sequences of SchS21 and SchS34. Based on the speculation that SchS21 protein was a DNase, the enzymatic properties were investigated. RESULTS: Sequences of 435 and 666 bp in length were obtained from SchS21 and SchS34 cDNAs. The SchS21 open reading frame encodes a mature protein of 144 amino acids, while that of SchS34 is 221 amino acids in length. SchS21 is a secretory, alkaline, Mg-dependent exodeoxyribonuclease, while SchS34 is a secretory protein of unknown function. His-tagged forms of the mature SchS21 and SchS34 proteins were separately overexpressed in Escherichia coli and purified using Ni-NTA columns (0.5 mg/l yield). CONCLUSIONS: Based on Western blots, the expressed proteins were similar in molecular weight and bound to the respective monoclonal antibodies to SchS21 and SchS34 proteins from S. chartarum. Interactions with human sera IgE confirmed the expressed forms of SchS21 and SchS34 as naturally occurring allergens.

The relative allergenicity of Stachybotrys chartarum compared to house dust mite extracts in a mouse model.

A report by the Institute of Medicine suggested that more research is needed to better understand mold effects on allergic disease, particularly asthma development. The authors compared the ability of the fungus Stachybotrys chartarum (SCE) and house dust mite (HDM) extracts to induce allergic responses in BALB/c mice. The extracts were administered by intratracheal aspiration (IA) at several doses (0, 2.5, 5, 10, 20, 40, and 80 microg) 4 times over a 4-week period. Three days after the last IA exposure, serum and bronchoalveolar lavage fluid (BALF) were collected. The relative allergenicity of the extracts was evaluated based on the lowest dose that induced a significant response compared to control (0 microg) and the linear regression slope analysis across the dose range. SCE induced a more robust response than HDM for BALF some inflammatory cells (macrophage and neutrophils), whereas HDM induced more robust BALF lymphocyte and eosinophil responses. Although SCE induced a more robust serum total immunoglobulin E (IgE) response than did HDM, the induction of a similar response in a functional, antigen-specific IgE assay required approximately twice as much SCE as HDM. Even though SCE demonstrates the ability to induce allergic responses in the mouse model, considering the importance and relevance of eosinophil, lymphocyte, and antigen-specific IgE in allergic airway disease, it is concluded that HDM is more potent than SCE in the induction of allergic responses. These data suggest a threshold dose for SCE allergy induction. Furthermore, in damp water-damaged environments, exposure to S. chartarum might easily exceed the sensitization threshold for a susceptible population.

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.

Trichothecene mycotoxins activate inflammatory response in human macrophages.

Damp building-related illnesses have caused concern for years in many countries. Although the problem is extensive, the knowledge of the immunological reactions behind damp building-related illnesses is still quite limited. Trichothecene mycotoxins form one major group of toxins, which possibly contribute to the illnesses. Stachybotrys chartarum is a well-known, but also controversial damp building mold and many strains of this mold are capable of producing trichothecenes. In this report, we have examined the effect of S. chartarum and trichothecene mycotoxins on the proinflammatory cytokine response in human macrophages. As a result, satratoxin-positive S. chartarum activated inflammasome-associated caspase-1, which is needed for proteolytic processing of IL-1beta and IL-18. Furthermore, purified trichothecene mycotoxins, roridin A, verrucarin A, and T-2 toxin activated caspase-1, and these mycotoxins also strongly enhanced LPS-dependent secretion of IL-1beta and IL-18. The satratoxin-positive strain of S. chartarum and the trichothecenes also triggered the activation of caspase-3, which is an effector caspase of apoptosis. Satratoxin-negative S. chartarum was not able to activate either caspase-1 or caspase-3. In conclusion, our results indicate that human macrophages sense trichothecene mycotoxins as a danger signal, which activates caspase-1, and further enables the secretion of IL-1beta and IL-18 from the LPS-primed cells.

Low tumor necrosis factor alpha levels and neutrophil counts in nasal lavage after mold exposure.

BACKGROUND: The results of allergy tests against molds usually remain negative in patients with upper respiratory tract and conjunctival symptoms after microbial exposure in a water-damaged building. Most mold-exposed persons report nasal irritation. Immune mechanisms of the nasal symptoms have not been fully elucidated. OBJECTIVE: To investigate local inflammatory responses after mold exposure in the upper respiratory tract and the feasibility of nasal lavage in diagnosing work-related exposure. METHODS: Altogether, 26 mold-exposed and 20 nonexposed workers from the same hospital were selected for the present study. The work premises of the exposed workers had detectable moisture and microbial problems. All exposed workers and their nonexposed controls underwent clinical examination, laboratory tests to detect allergy to molds, and nasal lavage. Inflammatory cells and proinflammatory cytokines were measured in the nasal lavage fluid. Nasal lavages were performed again 6 months after a thorough renovation of the building. RESULTS: In the nasal lavage, the neutrophil count and the level of tumor necrosis factor alpha in the exposed employees were lower, whereas the macrophage and epithelial cell counts were higher than in the control group. After the renovation, no difference was found in inflammatory response between the study group and the control group. The mean concentration of serum IgG to Stachybotrys chartarum was higher in the exposed workers. CONCLUSIONS: These results suggest that exposure to toxin-producing microbial growth in a water-damaged building caused immunosuppression in nasal mucosa, leading to a decrease in neutrophil counts and tumor necrosis factor alpha levels. Nasal lavage is a suitable method for examining inflammatory responses in work-related mold exposure.

Immune response among patients exposed to molds.

Macrocyclic trichothecenes, mycotoxins produced by Stachybotrys chartarum, have been implicated in adverse reactions in individuals exposed to mold-contaminated environments. Cellular and humoral immune responses and the presence of trichothecenes were evaluated in patients with mold-related health complaints. Patients underwent history, physical examination, skin prick/puncture tests with mold extracts, immunological evaluations and their sera were analyzed for trichothecenes. T-cell proliferation, macrocyclic trichothecenes, and mold specific IgG and IgA levels were not significantly different than controls; however 70% of the patients had positive skin tests to molds. Thus, IgE mediated or other non-immune mechanisms could be the cause of their symptoms.

Characterization of monoclonal antibodies to an antigenic protein from Stachybotrys chartarum and its measurement in house dust.

Using sera from atopic patients we have isolated an extracellular protein, which is antigenic in humans, from Stachybotrys chartarum sesu lato. Here we report the production of monoclonal antibodies to the protein and the development of a sensitive and specific assay to the target protein as well as analyses in house dust samples spiked with spores. The detection limit for the target antigen in house dust was approximately 0.2 ng/g dry weight house dust. This detection limit is comparable to those for house dust mite allergen and the allergen of the fungus Aspergillus fumigatus but lower than that for the fungus Alternaria alternata.

Immunohistochemical and immunocytochemical detection of SchS34 antigen in Stachybotrys chartarum spores and spore impacted mouse lungs.

The purpose of this study was to evaluate the distribution of a 34 kD antigen isolated from S. chartarum sensu lato in spores and in the mouse lung 48 h after intra-tracheal instillation of spores by immuno-histochemistry. This antigen was localized in spore walls, primarily in the outer and inner wall layers and on the external wall surfaces with modest labelling observed in cytoplasm. Immuno-histochemistry revealed that in spore impacted mouse lung, antigen was again observed in spore walls, along the outside surface of the outer wall and in the intercellular space surrounding spores. In lung granulomas the labelled antigen formed a diffusate, some 2-3x the size of the long axis of spores, with highest concentrations nearest to spores. Collectively, these observations indicated that this protein not only displayed a high degree of specificity with respect to its location in spores and wall fragments, but also that it slowly diffuses into surrounding lungs.

The development of species-specific immunodiagnostics for Stachybotrys chartarum: the role of cross-reactivity.

Mold contamination and exposure to fungi in indoor environments has been associated with various adverse health effects but little is known about the significance of individual fungal species in the initiation or exacerbation of such effects. Using Stachybotrys chartarum as a model fungus we sought to demonstrate that monoclonal antibodies (mAbs) can provide species-specific diagnostic reagents and also be used to investigate immunological cross-reactivity patterns among fungi. Mice were immunized with S. chartarum spore walls and monoclonal antibodies were screened against 60 fungal species and 24 different isolates of S. chartarum using an indirect ELISA. One species-specific mAb (IgG(1)) reacted only with spore preparations but not mycelium of S. chartarum or propagules of any other fungus. Five cross-reactive mAbs (IgM) documented extensive cross-reactivity among nine related Stachybotrys species and several non-related genera including several species of Cladosporium, Memnoniella, Myrothecium and Trichoderma. We also found that the ELISA reactivity for cross-reactive antigens and different isolates of S. chartarum differed considerably for normalized total amounts of mycelial antigen. We demonstrate that mAbs and immunoassays have the potential to detect S. chartarum species-specifically. The observed reactivity patterns with cross-reactive mAbs suggest that several fungi may share common antigens and that the majority of antigens are expressed by spores and mycelia. The observed cross-reactivity patterns need to be considered for accurate interpretations of environmental and serological analyses.

Intranasal exposure to Stachybotrys chartarum enhances airway inflammation in allergic mice.

RATIONALE: Exposure to building dampness, often associated with growth of microbes such as Stachybotrys chartarum, has been linked to respiratory symptoms. We have shown previously in a murine model that exposure to S. chartarum can induce lung inflammation characterized by infiltration of neutrophils and lymphocytes; this process is regulated by proinflammatory cytokines and leucocyte-attracting chemokines. OBJECTIVES: Because an atopic predisposition may influence the response to microbes, we examined the effects of S. chartarum on allergic mice in an experimental model. BALB/c mice were sensitized to ovalbumin by intraperitoneal injections and exposed for 3 wk to spores of S. chartarum. MEASUREMENTS AND MAIN RESULTS: Numbers of eosinophils and neutrophils were drastically increased in bronchoalveolar fluid from these mice as compared with the ovalbumin-sensitized/challenged mice or those exposed to S. chartarum without ovalbumin sensitization. Histologic sections showed severe granulomatous inflammatory cell infiltrates in all compartments of the lung, including peribronchial, perivascular, and alveolar spaces. The mRNA levels of proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha and the chemokine CCL3/MIP-1alpha were also markedly increased in the lungs. Despite the enhancement of the pulmonary inflammatory reaction, exposure to S. chartarum spores significantly down-regulated airway hyperresponsiveness and showed a tendency to decrease levels of Th2 cytokines in the lung. CONCLUSION: Exposure to S. chartarum modulates the inflammatory reaction and airway hyperresponsiveness, depending on the allergic status of the exposed mice.