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Exposure to farm environment has been shown to both protect from allergic diseases and increase the risk of respiratory syndromes. Mechanisms have been previously investigated by using farm dust extracts or specific components of dust. The use of authentic farm dust would better reflect the natural exposure. The aim of our study was to highlight the importance of proper assessment of the cow stable dust characteristics before conducting further investigations. For this purpose, we characterized microbiome and size distribution of unprocessed cow stable dust and its toxicological properties, as they have been often overlooked in search of protective factors. Stable dust samples from four Finnish dairy farms were collected by utilizing two different collection methods. Toxicological potential was analysed by stimulating co-cultures of lung epithelial and macrophage-like cells with dust. Size and mass distributions of airborne particles in the stables and bacterial and fungal microbiota of the dust were analysed. Stimulation with dust did not affect viability, but heightened oxidative stress responses and cytokine secretion, and slightly reduced the metabolic activity. There were a few differences in responses between farms, however, the differences were mainly in the intensity and not in the direction of the response. Cellular responses induced by dusts collected by different sampling methods did not differ substantially. Unprocessed stable dust samples showed relatively low direct toxicity but were able to trigger immune responses in studied cell model. This suggest that these dust collection methods could be utilized when investigating e.g. asthma-protective mechanisms.

The effect of cryopreservation on antigen-presenting cells (APCs) is understudied. It is important to understand the effects of cryopreservation on these cells as they play a major role in immune responses, and they could be utilized in different clinical applications. In this study, we compared fresh and cryopreserved PBMCs in regards of their general immune responsiveness and, furthermore, the effect of cryopreservation on the circulating APCs among PBMCs. We stimulated fresh and cryopreserved PBMCs (N = 6) with LPS or Poly(I:C).Cytokine production of PBMCs and expression of functional markers CD80 and ILT4 on major types of APCs, dendritic cells (DCs) and monocytes, were analysed. We also analysed whether cryopreservation affects different subtypes of DCs (plasmacytoid and myeloid DCs) differently. Cryopreserved PBMCs produced less cytokines than fresh cells in response to stimulation, but the response profiles were comparable. Cryopreservation had also an effect on the relative proportions of APCs. Stimuli-induced responses were somewhat parallel but weaker than those observed in fresh cells. This study suggests that the use of cryopreserved cells is more suitable in studies that assess general responses to stimuli instead of measuring exact levels of reactions. Thus, the interpretation and comparison of the results of different studies should not be done without considering the differences in cryopreservation techniques and their effects on PBMCs and, more specifically, on APCs.