Maija-Riitta Hirvonen, Ph.D., docent, National Public Health Institute, Division of Environmental Health, Kuopio, Finland, e-mail: maija-riitta.hirvonen@ktl.fi

Epidemiological evidence shows that building moisture and microbial growth are associated with respiratory symptoms related to inflammatory reactions, ie. irritation, infections and asthma. At present, it is not known which are the most important causative microbes able to induce these adverse effects, what are the specific cellular effects and, particularly, what are the mechanisms of them. These data are, however, needed for proper risk assessment of the moldy house problem and the measures taken to solve it. There is an urgent need on experimental work on cell cultures and laboratory animals with the microbes isolated from moldy buildings suspected to be harmful. Such data is at present to most extent missing but the present plan is aimed to provide it comprehensively.

We have recently observed that 1) streptomycetes induce production of inflammatory mediators i.e. nitric oxide (NO), cytokines and reactive oxygen species (ROS) and cause cell death in mice macrophages in vitro, 2) these responses are not dependent on the viability of the spores of streptomycetes, and preliminary: 1) growth conditions play an important role in the ability of these microbes to induce the production of inflammatory mediators and to cause cytotoxicity 2) streptomycetes produce NO and cause cytotoxicity also in human lung epithelial cell line, and 3) the strains of the streptomycetes active in vitro also elevate the same inflammatory mediators in bronchoalveolar lavage fluid (BAL) in rats after an intratracheal instillation to lungs. Altogether, these results suggest that certain moldy house microbes are able to induce inflammatory responses and/or to cause cell death in mammalian cells. This may play a central role in the cascade of events leading to the adverse health effects.At this phase, it is inevitable to study which other microbes characteristic to moldy houses cause similar effects and what are the effects of these microbes in lungs.

The overall aim of the study is to find out which microbes among the mixed population of the microbes present in the moldy houses are able to cause adverse respiratory health effects and what are the mechanisms of them. The focus is on inflammatory responses and cytotoxicity in human and mice cells and local toxicity in lungs and effects on respiration in animals. Effects of six typical microbes isolated from moldy buildings are studied: Streptomyces anulatus, Sreptomyces californicus, Aspergillus versicolor, Stachybotrys atra, Fusarium, and mycobacteria. This plan evaluates effects of these microbes in vitro in both human and mice cell cultures, deepens our previous work with streptomycetes to new mechanisms and expands studies to animals in vivo. The specific aims for the in vitro studies are 1) to study in detail the effects and the mechanisms of cell death and inflammatory responses in human and mice macrophages, induced by these microbes and their combinations, 2) to study cytotoxicity and the inflammatory responses induced by these microbes in human lung epithelial cells 3) to study the relation between growing conditions of the microbes and their ability to induce inflammatory responses and cytotoxicity. The specific aims for in vivo studies are 1) to study effects of streptomycetes and the microbes proving to be harmful in in vitro studies in lungs of mice after intranasal instillation (inflammation, local toxicity in lungs), 2) to identify the target cells of effects in the airways 3) to evaluate the effects of the microbes on respiration in guinea pigs after intratracheal instillation. This study identifies potentially harmful microbes present in moldy houses to cause respiratory effects, describes those effects in the lungs of laboratory animals and elucidates the cellular mechanisms of moldy house effects. These data will form a new fundamental basis for risk assessment of the health effects of those microbes and help to develop methods for biomonitoring of harmful microbial exposure. Identification of the most harmful microbes is also the basis for decisions to solve the moldy house problems. In vitro studies: to investigate the effects induced by occupational exposure to microbes present in moldy houses on nasal functions and production of inflammatory mediators in nasal lavage fluid (NAL) cells in healthy and symptomatic subjects.