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Background: Exhaled breath condensate (EBC) is simple to collect and as such a non-invasive method that has attracted substantial interest in the last few years. However, several methodological concerns have been raised and it has been difficult to reproduce results between different centres. Because of low concentrations of inflammatory markers, potential loss in the sampling system may have great influence. The aim of the present study was to se if evaporation and plastic coating could facilitate detection. Methodology: Through methodological improvements, we have now made it possible to measure EBC concentrations of eicosanoids and cytokines in our system. Due to absorbance of both fatty acid derivates and proteins to several plastics, the first step is coating of all surfaces with bovine serum albumin and Tween 20. Since several assays are sensitive to these factors, the methodology has to be standardised to avoid false results. Secondly, larger amounts of EBC have to be vacuum-dried, and thereafter resolved in the respective assay buffers. The EBCs have to be concentrated 5-10 times, depending on samples and assay sensitivity. Results: Due to these improvements we can measure, for example, cysteinyl-leukotrienes, leukotriene B"4, prostaglandin E and 8-isoprostane. High sensitivity assays have also made it possible to measure cytokines, for example, interleukin (IL)-1@b, IL-8 and IL-13. Summary: We are aware of different results from other labs. However, it seems essential to coat and to concentrate the samples in order to achieve reliable and measurable results.

We hereby report on a three year project initiative undertaken by our research team encompassing large-scale protein expression profiling and annotations of human primary lung fibroblast cells. An overview is given of proteomic studies of the fibroblast target cell involved in several diseases such as asthma, idiopatic pulmonary disease, and COPD. It has been the objective within our research team to map and identify the protein expressions occurring in both activated-, as well as resting cell states. The JGGL database www.2DDB.org has been built around these data, allowing advanced hypothesis building using the interactive query bioinformatic tools developed. Gene ontology has been applied to these annotations, classifying and correlating protein expressions to function. The localization as well as the biological processes involved for the annotations are being presented including an annotation-, and sequence-identification strategy, resulting in close to 2000 protein identities. Both gel based, high resolution 2D-gels, and liquid-phase separation (three-dimensional HPLC), as well as the combination of gel- and LC-based approaches (1D-gels and nano-capillary LC, reversed-phase) were utilized. Protein sequencing and structure identities were acquired by a combination of MALDI-, and electrospray−mass spectrometry techniques. Phenotypical and morphological characterizations were also made for this human disease target cell in both stimulated- and resting-cell states. The use of functional assays that demonstrate the key regulating role of growth factors and cytokine stimuli such as PDGF, TGF-β, and EGF and the effect of ECM molecules such as Biglycan, are also presented and discussed. Keywords: fibroblast • nucleus • proteome • lung • mass spectrometry • TGF-β