FUNGAL ALLERGENS AND ANTIGENS - THEIR CHARACTERIZATION AND BIOLOGICAL EFFECTS IN MICE AFTER INHALATION EXPOSURE

Project leader: Anna-Liisa Pasanen, University of Kuopio, Department of Environmental Sciences, P.O.Box 1627,
FIN-70211 Kuopio, Finland, tel. +358-17-163 580, e-mail: Annal.Pasanen@uku.fi
 
 

PUBLICATIONS

TIIVISTELMÄ SUOMEKSI

Researchers:
Anne Korpi, University of Kuopio, Department of Environmental Sciences, tel. 358-17-163 594, e-mail: Anne.Korpi@uku.fi (Part 2)
Marja Kärkkäinen, University of Kuopio, Department of Environmental Sciences, tel. 358-17-162 707, e-mail: Marja.Karkkainen@uku.fi (Part 1)
Päivi Raunio, University of Kuopio, Department of Environmental Sciences, tel. 358-17-163 593, e-mail: Paivi.Raunio@uku.fi (Part 1)
Jukka Pekka Kasanen, University of Kuopio, Department of Environmental Sciences, tel. 358-17-163 220, e-mail: Kasanen@uku.fi (Part 2)
Jaakko Rautiainen, University of Kuopio, Department of Clinical Microbiology, tel. 358-17-162 712, e-mail: Jaakko.Rautiainen@uku.fi (Part 1)
Soili Saarelainen, University of Kuopio, Department of Clinical Microbiology, tel. 358-17-162 707, e-mail: Soili.Saarelainen@uku.fi (Part 2)
Tuomas Virtanen, University of Kuopio, Department of Clinical Microbiology, tel. 358-17-162 715, e-mail: Tuomas.Virtanen@uku.fi

Financing SYTTY organization: The Academy of Finland (Part 1 and 2), Tekes (Part 1), Medix Biochemica Ltd (Part 1)
Funding from SYTTY / Total funding of project (€): 376438 / 396620
Person-months of work funded by SYTTY / Total person-months of work: 130 / 153,5

KEY WORDS: biological responses, immunoassays, inhalation exposure, Stachybotrys chartarum, specific antigens
 

EXTENDED ABSTRACT

PART 1. Characterisation of allergic components of Stachybotrys chartarum

1 Introduction

The final goal of this project is to develop immunochemical test kits to be used for the detection of Stachybotrys chartarum fungus from environmental samples on the site of contamination and to evaluate occupants' exposure to the fungus based on specific antibody measurements. The most important presupposition for this aim was to identify, purify and to characterise the molecular structure of an antigenic component that is specific to the fungus. Extensive studies were first conducted to clarify the cross-reactivity between S. chartarum and other fungal species that are likely to exist with S. chartarum in the environment as well as the effect of growth media on the composition of antigenic components of S. chartarum. In addition, immunogenic nature of the candidate components were verified. After the recognition of a promising component, the next step was to isolate and purify the component and to collect a sufficiently large amount of the purified component for the sequence analyses. At this moment, the sequencing is under process and the isolation of the respective gene will be completed by the end of March 2002.

2 Methods

The protein and antigenic compositions of S. chartarum extracts prepared from five fungal strains were characterized by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting methods. The fungus was cultured on malt extract and cellulose broth to investigate the impact of a medium on the antigen composition. The cross-reactivity between S. chartarum and 10 other fungal species was identified by the inhibition-immunoblotting method as described by Raunio et al. 2001 and 2002.

Two cDNA libraries were constructed from S. chartarum cultures. Different aged mycelia were separated from the extracts and the total RNA was isolated from which messanger RNA was purified using the Qiagen Oligotex poly A+ mRNA purification method. The cDNA libraries were screened with the S. chartarum specific rabbit polyclonal immune sera and the sera of patients with a verified exposure to S. chartarum.
Acetate precipitation combined with the anion exchange method and MonoQ -column (Tris-buffer pH 7.6, 90 mL NaCl gradient from 0 to 1 M) was the best combination for separation of specific components from the S. chartarum extract. Partly purified specific proteins were used for the immunoblotting, sugar, and sequencing analysis.

Glycoproteins of S. chartarum extract were identified with series of lectins that selectively recognise the terminal sugars. Both the crude extract and purified fractions were processed with the periodate treatment to remove sugar moieties. The immunoblotting analysis was done to both untreated and periodate treated strips to find out, whether the sugars removal had an effect on antibody binding (Raunio et al. 2001).

The partly purified, the most promising component of S. chartarum was transferred onto polyvinylidene difluoride membrane and the proteins were stained with Coomassie brilliant blue. Applied Biosystems 477A Pulsed Liqued Phase protein sequencer was used for determination of the N-terminal amino acid sequence of the protein. The in-gel protease digestion method and Edman degradation analyses were also used for amino acid sequencing of peptides of the same S. chartarum component. Sample preparation and amino acid sequencing were performed by Dr. Nisse Kalkkinen, Institute of Biotechnology, Viikki, University of Helsinki. Furthermore, the amino acid structure of selected immunogenic components in S. chartarum extract was examined by mass spectrometry by Dr. Seppo Auriola, Department of Pharmaceutical Chemistry, University of Kuopio.

Partial amino acid sequences of the selected component were used to design degenerated primers for amplification of the respective DNA with PCR and S. chartarum cDNA was used as a template (translated directly from mRNA). PCR-products were sequenced by automated DNA sequencing service at A.I.V institute, University of Kuopio.  Selected DNA fragments will be used as probes for screening the S. chartarum cDNA- and genomic libraries.

3 Results and Discussion

The S. chartarum extract cultured in cellulose broth contained a higher number of protein and antigenic components than the extract prepared from malt extract broth. Four antigenic components of S. chartarum cultured in cellulose broth were most dominant. In the cross-reactivity analysis, two of them were most specific for S. chartarum. Various S. chartarum strains showed similar protein and antigenic profiles and had equal inhibitory potencies.  The most promising candidate for a specific component (50 kDa) was selected to the further studies. This component is easily extracted from S. chartarum cultures, S. chartarum spores are enriched with this component, and the component dominates in S. chartarum cultures in cellulose rich media, e.g. gypsum board.

The cDNA libraries for S. chartarum extracts contained 30000 and 300000 phaques. The screened with the S. chartarum specific immune sera and the patients' sera produced no positive plaques. Specific DNA-probes and heterologous hybridisation methods will be used for screening cDNA and genomic libraries during February-March 2002.

Many antigenic components of S. chartarum were glycoproteins. Mannose was the major sugar moiety. The 50 kDa component contained only a faint galactose-beta(1-4)-N-acetylglucosamine moiety. Four components of  S. chartarum lost partly their antibody binding activity, while the 50 kDa component was not affected by the periodate treatment and the antibody binding activity remained unchanged.

Using 10% acryamide gel instead of previously used 15% gel, two bands were detected instead of one (50 kDa) in the SDS-PAGE and immunoblotting analyses. Both bands, about 47 and 50 kDa, showed immunoreactivity, but the 50 kDa component was more easily purified was therefore selected for the amino acid sequencing. Amino acid structure of nine peptides of the 50 kDa component was identified. The mass spectrometry analyses indicated that trypsin digested components of 47 and 50 kDa contain similar peptide profiles. Thus, both the components are similar or different in structure, due to the fact that the expression of one fungal gene may lead  to several slightly modified translation products, or several genes may be expressed simultaneously.  DNA sequencing of  the amplified PCR products showed more detailed information about the DNA structure and the gene regulation system of the component.

4 Conclusions

The 50 kDa component was proved to be most characteristic of S. chartarum, was recognised and isolated and its molecular structure is now partly identified. The project will be continued by the end of March 2002 when hopefully the whole DNA structure of the gene is characterised and the project may proceed to the production and testing of recombinant proteins and monoclonal antibodies that will provide a good basis for the further development of specific tests for S. chartarum.
 

PART 2. Exposure of mice by inhalation to fungi, irritating effect in the respiratory tract and immune responses

1 Introduction

Occupants of buildings in moisture or mould damages often complain of eye and upper respiratory tract irritation. These symptoms have been postulated to be caused partly by microbially produced volatile organic compounds (MVOCs). Also the detected ß -1,3-D-glucan levels indoors have been reported to be related to irritation. The risk of chemicals for provoking upper airway irritation in humans can be estimated with a recommended indoor air level (RIL) concept. At first, a concentration of a chemical evoking a 50 % decrease in the respiratory rate of mice (i.e., RD50) is established indicating the sensory irritation potency of a chemical. By using a validated safety factor, the RD50 value can be used to set a safe indoor air level for the compound. Although the majority of symptoms caused by indoor fungal exposure are those reflecting non-specific airway inflammation, a classic IgE-mediated sensitisation may develop against fungi. In experimental studies, increased total IgE and/or fungus-specific IgG1 levels have been reported after fungal exposure.

The objective of this study was to detect possible upper respiratory tract irritative effects in mice resulting from inhalation of fungal antigens, MVOCs, or a fungal cell-wall component (ß-glucan). Also, immunological effects due to exposure to fungal antigen extract were investigated, and RD50 values and respective RIL values were determined for three individual MVOCs and a mixture of five MVOCs.

2 Methods

The immunological effects caused by non-toxic Stachybotrys chartarum (Sc) antigens were evaluated by immunizing mice with Sc grown on malt extract broth, and after repeated inhalation exposures to Sc grown on cellulose broth. Sc aerosol exposure schemes were either twice a week for three weeks or altogether 5 exposures within 10 days, and exposure periods lasted from 15 to 30 min. During the exposures, respiratory patterns of the animals were monitored. Certain antibody levels in the sera of mice as well as certain cytokines produced by stimulated spleen cells were determined by ELISA. In addition, the ability of the Sc extract to cause cell proliferation with different Sc concentrations was examined.

The respiratory parameters of mice were constantly monitored upon single and/or repeated exposures to MVOCs, or to aerosols of Aspergillus versicolor, or ß-glucan in order to reveal sensory and pulmonary irritation and airflow limitation in the conducting airways. MVOC exposures lasted for 30 min, and repeated exposures were performed on four consecutive days. RD50 values were established for three single MVOCs and a mixture of 5 MVOCs. The risk of MVOCs for provoking irritation symptoms in humans was estimated with a RIL concept. Inflammatory signs in the tissue samples from the lungs were determined from the mice exposed to MVOCs. A. versicolor and ß-glucan aerosol exposures lasted from 15 to 20 min, and the repeated ß-glucan exposures were performed at 7 exposures within 22 days. After the exposure to b-glucan, inflammatory signs in the tissue samples from the respiratory tract were determined.

3 Results and Discussion

Immunisation of mice with Sc resulted in increased antibody levels of serum total IgE, and Sc-specific IgG1, IgG2a and IgA. The schedule of the repeated Sc aerosol exposures was found to affect the total IgE and Sc-specific IgG2a antibody production, whereas the levels of Sc-specific IgG1 and IgA remained unchanged regardless of aerosol exposure. Sc caused a strong and significant antigen concentration-dependent cellular stimulation (proliferation) and IL-4 production in splenocytes of Sc-immunised mice. These responses, and additionally the production of IL-10, were further enhanced in the Sc-immunised mice after inhalation exposure to Sc. Sc aerosols induced sensory irritation response in both the Sc-immunised and non-immunised mice already during the first exposure. No signs of inflammation were present in the nasal cavities of the animals, whereas a slight influx of inflammatory cells was seen in the alveoli 3 or 10 days after the last Sc exposure. These results indicate that Sc caused other than mycotoxin-related biological effects by stimulating the murine immune system and by causing a distinct sensory irritation response in the murine airways.

MVOCs provoked sensory irritation, and no signs of inflammation in the lung tissues were observed. The RILs for these compounds suggest that MVOCs could cause upper respiratory tract irritation in humans at the levels of mg/m3 for individual compounds, but such concentrations only rarely occur in moldy buildings.

Also A. versicolor aerosols provoked sensory irritation. Inflammatory markers were not analysed from these mice. The ß-glucan aerosol exposure induced a very mild sensory irritation response that was not concentration-dependent. Occasionally, also slight pulmonary irritation response was observed. Tissue samples from these mice have not yet been analyzed. Thus, sensory irritation response observed with fungal extracts is not unambiguously caused by ß-glucan present in the extract.

4 Conclusions

MVOCs, alone, cannot be considered to be responsible for symptoms of upper airway irritation in microbially contaminated indoor environments. Sensory irritation can be provoked by fungal extract aerosols, but one of the fungal cell-wall components, ß-glucan, does not fully account for the response. However, other individual cell wall components (e.g., ergosterol) and fungal glycoproteins should be investigated before detailed conclusions on the practical importance of this finding can be drawn. Additionally, only after the causative agents (to sensory irritation response) have been characterised, recommendations and standardisation of reliable methods for the fungal exposure assessment can be presented.
 
 

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