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.

Physiotherapeutic evaluation of patients with post COVID-19 condition: current use of measuring instruments by physiotherapists working in Austria and South Tyrol.

BACKGROUND: The implementation of standardized assessments in physiotherapeutic practice strongly supports diagnostic and treatment plans. Previous studies reported insufficient usage of standardized assessments due to lack of time, lack of knowledge, lack of resources and other barriers. Physiotherapy in outpatient settings became essential for the rehabilitation of patients with post COVID-19 condition but it remains unknown to what extent assessments are implemented into the evaluation of these patients. In this study, we explored the current use and barriers regarding the implementation of physiotherapeutic assessments to evaluate patients with post COVID-19 condition. METHODS: A cross-sectional online survey was carried out among 180 physiotherapists working in outpatient settings in Austria and South Tyrol. RESULTS: The majority of physiotherapists (88%) indicated that standardized assessments are useful, though less than a fifth of participants actually implement assessments in practice. Among implementation barriers, "insufficient experience" (41.8%) and "lack of knowledge" (36.6%) were mentioned most often. Concerning specific post COVID-19 assessments, the evaluation of "physical and respiratory function", "quality of life" and "activities of daily living" were stated to be of particular relevance. CONCLUSIONS: Our study revealed a low implementation rate and identified the main barriers regarding the non-usage of standardized assessments for post COVID-19 patients. TRIAL REGISTRATION: The Private University for Health Sciences and Health Technology (UMIT TIROL), and the Research Committee for Scientific Ethical Questions granted approval for the survey (RCSEQ, Hall in Tirol, Austria, Number 2834).

Surfactant protein D inhibits growth, alters cell surface polysaccharide exposure and immune activation potential of Aspergillus fumigatus.

Humoral immunity plays a defensive role against invading microbes. However, it has been largely overlooked with respect to Aspergillus fumigatus, an airborne fungal pathogen. Previously, we have demonstrated that surfactant protein D (SP-D), a major humoral component in human lung-alveoli, recognizes A. fumigatus conidial surface exposed melanin pigment. Through binding to melanin, SP-D opsonizes conidia, facilitates conidial phagocytosis, and induces the expression of protective pro-inflammatory cytokines in the phagocytic cells. In addition to melanin, SP-D also interacts with galactomannan (GM) and galactosaminogalactan (GAG), the cell wall polysaccharides exposed on germinating conidial surfaces. Therefore, we aimed at unravelling the biological significance of SP-D during the germination process. Here, we demonstrate that SP-D exerts direct fungistatic activity by restricting A. fumigatus hyphal growth. Conidial germination in the presence of SP-D significantly increased the exposure of cell wall polysaccharides chitin, α-1,3-glucan and GAG, and decreased ß-1,3-glucan exposure on hyphae, but that of GM was unaltered. Hyphae grown in presence of SP-D showed positive immunolabelling for SP-D. Additionally, SP-D treated hyphae induced lower levels of pro-inflammatory cytokine, but increased IL-10 (anti-inflammatory cytokine) and IL-8 (a chemokine) secretion by human peripheral blood mononuclear cells (PBMCs), compared to control hyphae. Moreover, germ tube surface modifications due to SP-D treatment resulted in an increased hyphal susceptibility to voriconazole, an antifungal drug. It appears that SP-D exerts its anti-A. fumigatus functions via a range of mechanisms including hyphal growth-restriction, hyphal surface modification, masking of hyphal surface polysaccharides and thus altering hyphal immunostimulatory properties.

Post-COVID-19 Rehabilitation: Perception and Experience of Austrian Physiotherapists and Physiotherapy Students.

The rehabilitation needs of COVID-19 survivors are increasingly recognized, with a focus on combating respiratory and neuromuscular dysfunctions. The aim here was to explore the perception of Austrian physiotherapists and physiotherapy students on post-COVID-19 rehabilitation care and to identify barriers for the application of sufficient rehabilitation. We analysed current knowledge and practical skills in respiratory physiotherapy, performing a cross-sectional national survey among physiotherapists working in outpatient settings and physiotherapy students in their last academic year of bachelor-level education in Austria. Out of 255 survey participants, one-third already had inquiries to treat post-COVID-19 patients, and the majority of respondents expected a further increased inflow of patients with rehabilitation needs (64.2%). Only 11.2% of respondents reported feeling sufficiently informed about post-COVID-19 rehabilitation. A total of 68.2% of students and up to 48.1% of physiotherapists favoured a COVID-19-specific adaptation already in the basic academic education, and 74.1% of survey participants indicated interest in attending specific training. Concerning respiratory physiotherapy, our data showed discrepancies between the estimation of the importance of specific examination and treatment techniques and the level of current experience. There is a clear lack of experience in implementing effective device-based respiratory therapy. Our data indicate an urgent need to develop new education and training programs with a focus on the interdisciplinary rehabilitation of patients with post-COVID-19 syndrome.

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.

Migrating Lung Monocytes Internalize and Inhibit Growth of Aspergillus fumigatus Conidia.

Monocytes are important players to combat the ubiquitously present fungus Aspergillus fumigatus. Recruitment of monocytes to sites of fungal A. fumigatus infection has been shown in vivo. Upon exposure to A. fumigatus in vitro, purified murine and human blood monocytes secrete inflammatory cytokines and fungicidal mediators. Mononuclear tissue phagocytes are phenotypically and functionally different from those circulating in the blood and their role in antifungal defenses is much less understood. In this study, we identified a population of migrating CD43+ monocytes in cells isolated from rat distal lungs. These cells are phenotypically different from alveolar macrophages and show distinct locomotory behavior on the surface of primary alveolar cells resembling previously described endothelial patrolling monocytes. Upon challenge, the CD43+ monocytes internalized A. fumigatus conidia resulting in inhibition of their germination and hyphal growth. Thus, migrating lung monocytes might play an important role in local defense against pulmonary pathogens.

Electrolyte Intake and Major Food Sources of Sodium, Potassium, Calcium and Magnesium among a Population in Western Austria.

Dietary intake of sodium, potassium, calcium and magnesium has a strong impact on personal health. In order to understand possible correlations with regional occurrence of diseases and to develop new dietary strategies, it is necessary to evaluate food choices in defined geographic areas. The aim of this study was to analyze daily consumption and major food sources of electrolytes with an emphasis on dietary patterns. In this representative, cross-sectional study, daily foods of 463 adults were assessed with two nonconsecutive 24-h recalls. Our results show high sodium and low potassium consumption in all age groups in both men and women. Furthermore, more than half of investigated persons had low calcium, and 40% indicated low dietary magnesium intake. Only 1% of our study population reached the recommended values for all electrolytes, while 13% consumed adequate levels of three electrolytes. Moreover, 14% did not reach reference levels for any of the four minerals. A further comparison of dietary patterns and food preferences showed significant differences in major food groups including nonalcoholic drinks, fruits, vegetables, legumes, milk products, vegetable oil, bread and sweets. Our results are important for further evaluations of nutrition intake and the development of new dietary strategies.

Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin ß-4-positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.-Quarta, S., Mitric, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.