• BGFA, University Bochum, Germany
• Environmental Health Sciences Research Center, University of Iowa, USA
• Department of Microbiology, Utrecht University, Netherlands
• Department Infection and Immunity, Utrecht University, Netherlands

To optimize exposure assessment of microbiological agents in organic dust of indoor environments and characterize the antigenic, allergenic and/or proinflammatory potential of microbial associated molecular patterns (MAMPS) focusing on fungi-related agents.

• Sampling: Active and passive airborne dust sampling and reservoir dust sampling.
• Analysis: LAL-Assay, Beta-glucan Inhibition/Sandwich EIAs, EPS sandwich EIA
• Characterizing the antigenic, allergenic and/or proinflammatory potential: Serology, Whole Blood Assays
• Further: fungi culture, chromatographic methods

Exposure assessment (methodology):

We have developed and validated the applicability of an electrostatic dustfall collector( EDC). The EDC was tested during two 14-day periods in 7 non-farm and 9 farm homes, and in farm stables. In parallel, active airborne dust sampling was performed with Harvard impactors, and floor dust collected by vacuuming, using nylon sampling socks. We showed that endotoxin measured with the EDC is a more valid measure of average airborne endotoxin exposure than floor dust endotoxin levels, while reproducibility over time is similar. It thus showed to be a useful method, which allows to be used for self-assessment approaches. However, the dust yield is low and dust analysis requires high performance analytical procedures with low detection limits

Exposure assessment (agents):

Glucans. For the development of new glucan assays we collaborated with the Institute for Occupational Medicine (BGFA) in Bochum, Germany (dr. Ingrid Sander and prof. Monika Raulf-Heimsoth) and the Environmental Health Sciences Research Center (prof. Peter Thorne), Iowa University, Iowa City, USA, who both provided us with monoclonal and polyclonal anti-glucan antibodies. Three new highly sensitive sandwich EIAs were developed for measurement of airborne glucans. Although specificities differed, results in pilot series in the home environment showed strong correlations.

Heat Shock Proteins (HSP). The HSP research was performed in collaboration with the department Infection and Immunity of the Veterinary faculty of Utrecht University (prof. Willem van Eden and colleagues)

HSPs show immunoreactivity and therefore may play a role as protective particles like suggested in the "hygiene hypothesis". We aimed to develop a sensitive EIA for measurement of HSP in house dust. Two mouse monoclonal antibodies were provided by prof. Willem van Eden, LK1 specific for bacterial and mammalian HSP 60 and LK2 specific for bacterial HSP 60 (GroEL), as well as a recombinant human-HSP. We succeeded to setup two possible sandwich EIAs with the recombinant human-HSP as standard. However, we could not detect any HSP in house dust or stable dust samples, which may mean that either the antibodies available to us are not suitable to measure HSP in dust samples or that there is no or only very low levels of HSP in house dust and stable dust. The work will not be continued.

Fungal Extracellular Polysaccharides (EPS). The EPS EIA used in previous studies was restarted by screening different available anti-EPS rabbit sera for strong EPS antibody activity. We isolated the IgG fraction of the interesting sera by protein g purification and tested each fraction from the different sera for similar reactivity as compared to the EIA used in the published studies.

Whole blood stimulation. Whole blood stimulation/cytokine production experiments have and will be performed to assess the pro-inflammatory potency of various glucans, to study if cytokine reactivity and primary structure or source of the glucans are associated.

GABRIEL dust samples. In total 227 EDC and 229 mattress dust samples were extracted and analyzed for endotoxin and glucan levels. All mattress dust samples were additionally analyzed for EPS. Possible differences in exposure levels between groups of famers and non-farmers children, and associations between EDC and mattress dust levels will be determined.

Fungal cultures and fractionation. Much about the health effects of fungi is not yet understood. Fungal exposure might result in a variety of symptoms like infection due to viable fungal spores, an allergenic response resulting in specific antibodies to fungal allergens/components and non-specific inflammation induced by fungal components. Thus far, research in fungal allergy has focused on protein structures, while carbohydrates can cause similar symptoms.

Our objective is to define the immunogenicity and proinflammatory potential of fungal products more thoroughly and by that getting to better understand the diverse health effects caused by fungi. We will grow different strains of fungi under well-defined culture conditions and fractionate the cultures into components isolated from supernatant, after disruption of the cells and components from the cell wall. This research will be performed in collaboration with the department of microbiology of the Utrecht University (prof. Han Wösten and colleagues).

These fractions will be further analyzed and defined concerning composition (proteins, carbohydrates, lipids etc) and immunoreactivity (IgG-, IgE- serology) and proinflammatory potentials (cytokine stimulation in whole blood assay and/or PBMCs).

Finally, we would like to study, whether any of the existing methods (glucan EIAs, EPS EIA, Ergosterol or other measurement) give a valid proxy of the identified fungal components with either immunogenic or proinflammatory activities.

HITEA, EFRAIM