Regulation of Surfactant Protein Gene Expression by Aspergillus fumigatus in NCl-H441 Cells.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes serious lung diseases in immunocompromised patients. The lung surfactant produced by alveolar type II and Clara cells in the lungs is an important line of defense against A. fumigatus. The surfactant consists of phospholipids and surfactant proteins (SP-A, SP-B, SP-C and SP-D). The binding to SP-A and SP-D proteins leads to the agglutination and neutralization of lung pathogens as well as the modulation of immune responses. SP-B and SP-C proteins are essential for surfactant metabolism and can modulate the local immune response; however, the molecular mechanisms remain unclear. We investigated changes in the SP gene expression in human lung NCI-H441 cells infected with conidia or treated with culture filtrates obtained from A. fumigatus. To further identify fungal cell wall components that may affect the expression of SP genes, we examined the effect of different A. fumigatus mutant strains, including dihydroxynaphthalene (DHN)-melanin-deficient ΔpksP, galactomannan (GM)-deficient Δugm1 and galactosaminogalactan (GAG)-deficient Δgt4bc strains. Our results show that the tested strains alter the mRNA expression of SP, with the most prominent and consistent downregulation of the lung-specific SP-C. Our findings also suggest that secondary metabolites rather than the membrane composition of conidia/hyphae inhibit SP-C mRNA expression in NCI-H441 cells.

Response of salivary biomarkers to an empathy triggering film sequence-a pilot study.

Empathy is a multifaceted phenomenon that is difficult to measure. Self-report questionnaires are the most common and well-validated measures while currently no validated protein biomarkers associated with the empathic reaction have been established. Trigger films have been previously used in psychological research to evoke emotions. Thus, in the present randomized cross-over study we investigated the responses of nine salivary biomarkers that have been related to emotions and stress following an empathy triggering and a control film sequence. Additionally, questionnaires for empathy (Saarbrucken Personality Questionnaire (SPQ)) and current mental stress were applied and participants were asked to assess the film protagonists' emotions using the Positive and Negative Affect Schedule. Data from 46 participants were included in the analysis. α-Amylase, IgA, IL-1ß and estradiol showed a significantly different response between the empathy and control intervention. Moreover, normalized levels of these biomarkers significantly correlated with single scales of the SPQ (control film sequence: α-amylase and IgA with personal distress; estradiol with empathic concern; IL-1ß with fantasy; empathy triggering film sequence: IgA with empathic concern, fantasy and the total empathy score). These findings indicated that the observed changes in salivary biomarker levels were reflective of a physiological response to the empathy triggering film sequence. Future studies using different triggers and settings will show if the identified biomarkers can be considered as surrogate markers for empathic reactions in general.

Evaluation of Stable LifeAct-mRuby2- and LAMP1-NeonGreen Expressing A549 Cell Lines for Investigation of Aspergillus fumigatus Interaction with Pulmonary Cells.

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.

Stimulation of surfactant exocytosis in primary alveolar type II cells by A. fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen with small airborne spores (conidia) that may escape clearance by upper airways and directly impact the alveolar epithelium. Consequently, innate alveolar defense mechanisms are being activated, including professional phagocytosis by alveolar macrophages, recruitment of circulating neutrophils and probably enhanced secretion of pulmonary surfactant by the alveolar type II (AT II) cells. However, no data are available in support of the latter hypothesis. We therefore used a coculture model of GFP-Aspergillus conidia with primary rat AT II cells and studied fungal growth, cellular Ca2+ homeostasis, and pulmonary surfactant exocytosis by live cell video microscopy. We observed all stages of fungal development, including reversible attachment, binding and internalization of conidia as well as conidial swelling, formation of germ tubes and outgrowth of hyphae. In contrast to resting conidia, which did not provoke immediate cellular effects, metabolically active conidia, fungal cellular extracts (CE) and fungal culture filtrates (CF) prepared from swollen conidia caused a Ca2+-independent exocytosis. Ca2+ signals of greatly varying delays, durations and amplitudes were observed by applying CE or CF obtained from hyphae of A. fumigatus, suggesting compounds secreted by filamentous A. fumigatus that severely interfere with AT II cell Ca2+ homeostasis. The mechanisms underlying the stimulatory effects, with respect to exocytosis and Ca2+ signaling, are unclear and need to be identified.