Liberation of spores and hyphal fragments from fungal cultures (Aspergillus fumigatus (AF), Aspergillus versicolor (AV) and Penicillium chrysogenum (PC)) was studied using 2 aerosol generators, 3 culture media, 2 air velocities and 2 and 8 week old cultures. Sub-? particles were liberated from the cultures in addition to spores, spore aggregates and other larger particles. Most sub-micrometer particles originated from the mycelium (filamentous network of fungal cells). Fragments of spores were also detected in experiments with the Aspergillus species. Fragments with the same size and shape as spores were observed. It is likely that previous studies have overestimated the liberation of spores. Observation of fragmented spores indicate that previous findings may be partially based on experimental artefacts. Detection of fragments from mycelium incl. sub-? particles in environmental samples requires specific labeling because these particles lack characteristic morphological features. A method was developed based on immuno-gold labeling with IgY antibody against AV spores and detection by field emission scanning electron microscopy during a research stay in USA. Samples collected during renovation of moisture damage were analysed. This method is an important supposition for assessment mycelium fragments which may have allergic effects in the airways.
Three articles have been published. The candidate defended his thesis at NMBU 22.5.2015. Spores from AF and PC were produced in the aerosol generator but AV and Stachybotrys chartarum (SC) cultures liberated too few spores for the toxicological studies. Spores from AV and SC were therefore produced by washing off culture plates. Fragments were prepared by harvesting mycelium before spore formation, drying and crushing in a ball mill. The respirable fraction was collected by aspiration through a respirable cyclone.
Hyphal fragments and spores were measured in dwellings with visible fungal problems. Hyphal fragments were 53% of the fungal particles in indoor air and 21% in outdoor air. 1-3% were small fragments (manuscript).
Spores and hyphae fragments from AF, PC AV and SC were cultured, sampled and characterized by number and shape of particles, and mycotoxin, ?-glucan and protease content/activity. Pro-inflammatory properties of mold particles were examined in human THP-1 monocytes, THP-1 macrophages and bronchial epithelial cells (BEAS-2B). All preparations contained either spores or hyphae, except spores from PC and SC which also to some extent contained hyphae. Each mold species had mainly one type of protease activity. Mycotoxin levels were low. ?-glucans and proteases were detected mainly in hyphae preparations. THP-1 macrophages were the most sensitive model giving an increased secretion of cytokines at very low doses of AF hyphae (10 ng/cm2). There were large differences in potency between the species with regard to pro-inflammatory responses. However, the differences could not be easily explained by any of the characteristics above. AF and PC hyphae were as expected much more potent inducer of cytokines than spores, surprisingly this did not seem to be the case for AV and SC. A paper published in Indoor Air 2017.
A comparison between AF spores and hyphae inactivated by X-ray or heat versus untreated, showed that none of the inactivation methods destroyed the inflammatory responses. Studies on mechanism involved in the release of cytokine following exposure to AF and AV spores and hyphae in different macrophages (THP-1-, blood monocyte derived- (MDM), and lung macrophages) are completed. AF was by far most potent. Furthermore, the various macrophage type responded quantitative and qualitative very differently. The responses obtained in MDM seemed most relevant. In these macrophages, the response from AF spores and hyphae seemed to involve mainly toll-like receptor (TLR) 4 and to a lesser degree TLR2. The various cytokine responses appeared to go through the same receptor. The manuscript will be submitted in 2018.
The immune responses in lung epithelial cells to AF and PC are further characterized. Untreated AF spores formed more readily hyphae when cultured with epithelial cells than without cells. In contrast to what was observed in macrophages, TLR-2 seems to be involved in both AF and PC hyphae-induced cytokine responses. Manuscript will be submitted in 2018.
Co-exposure of mineral particles and organic components (AF-hyphae) resulted in a synergistic release of IL-1b. AF-hyphae induced synthesis of pro IL-1?, and partly released IL-1?. Mineral particle caused synergistic effects by cleaving the preform induced by AF hyphae, resulting in an additional release of IL-1b. This effect were characterized in THP-1 macrophages, and confirmed in the other macrophage and lung epithelial cells models. Manuscript is submitted.
The writing of the PhD-thesis, based on 4 publications, has started and will be submitted in February 2019.
Hyfefragmenter har forskjellige og til dels sterkere biologiske effekter enn sporer. Videre kan dagens måter å karakterisere inneklima mhp sopp ikke direkte overføres til biologiske responser. Flere biologiske metoder in vitro er etablert, videreutviklet og sammenlignet mhp inflammatoriske responser. Testbatteriet er nytt og klart mer relevant.Resultatene viser at dagens forståelse av sammenhengen mellom eksponering for sopp og sykdom er meget mangelfull.Målinger av sopp partikler i innemiljø med en ny metode for hyfefragmenter utviklet i prosjektet viste at en vesentlig del av sopp partiklene er fragmenter.Hyfefragmenter bør derfor kvantifiseres separat i fremtidig forskning på sammenhengen mellom eksponering for sopp partikler og helse.
Prosjektet har vist at det finnes flere gode supplerende metoder for karakterisering av ulike sopp partikler som bør utprøves og utvikles videre slik at deres fulle potensiale mhp informasjon for kartlegging av inneklimaplager kommer klarere frem.
Fungal spores have obtained widespread attention as possible causal agents for adverse indoor air associated health problems. It has been reported that fungi may adversely affect human health through allergic and non-allergic airway inflammation, by infec tion and by the toxicity of mycotoxins. However, it is still not clear if spores inhaled in indoor environments really have all the adverse health implications suggested, as the indoor levels of airborne spores are often found to be too low to explain ass ociations with health effects other than allergy.
Recent studies have drawn attention to fungal hyphae and smaller-sized fungal fragments as tentative causes of indoor health problems. Furthermore, experimental studies have suggested that the inflammator y properties of fungal spores are modulated by their viability showing allergic responses to viable spores and non-allergic responses to non-viable spores. However, since no method for measurement of fungal fragments exists, the airborne levels indoor and outdoor, and their toxic properties are largely unknown. Thus, possible health implications of fungal particles besides spores are still very unclear.
The goals for this project are therefore to i) develop measurement methods for viable and non-viable sp ores, hyphae and fragments, ii) assess their airborne levels in a small proof-of-principle study in common indoor environments and in moisture-damaged buildings, iii) characterize these fungal particles by chemical and biological methods, and iv) study th eir toxic properties in vitro and in vivo. For this purpose, viable and non-viable spores, hyphae and fragments will be prepared from pure cultures of fungal species associated with moisture-damage in buildings.
The results are expected to provide new me thods for exposure assessment of fungal particles to be used in future epidemiological studies, and new insights in the possible role that fungal growth in indoor environments may have on respiratory health.