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Cultures of primary human nasal epithelial cells (hNECs) differentiated at the air–liquid interface (ALI) represent a sophisticated and widely used model of the human upper respiratory epithelium. Despite the availability of various cell culture insert types and the well-established understanding that different culture media influence the cell culture characteristics, the possible impact of the insert brand remains rather underexplored. We cultured hNECs from nineteen healthy adult donors on three distinct brands of commercially available inserts—Corning® Transwell®, CELLTREAT®, and ThinCert®—and compared the ciliary activity and cellular composition of the cultures using high-speed video microscopy and flow cytometry, respectively. Additionally, we employed an alternative method of hNEC culture setup—the inverted condition—wherein the hNECs were seeded on the basal side of the insert with the idea to avoid mucus accumulation. Our results show that ciliary activity and cell type composition did not differ between insert types for both culture conditions. However, we found a higher ciliary beat frequency and a lower active (ciliated) area in the inverted setup compared to the conventional setup across all three insert brands. These findings indicate that all three mentioned insert types yield comparable cell cultures.

Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV) as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH) reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2, day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2). BSH supplementation further increased LAIV-induced granzyme B production (day2) in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses. ClinicalTrials.gov NCT01269723.

Many studies investigating the impact of individual risk factors on cord blood immune cell counts may be biased given that cord blood composition is influenced by a multitude of factors. The aim of this study was to comprehensively investigate the relative impact of environmental, hereditary and perinatal factors on cord blood cells. In 295 neonates from the prospective Basel-Bern Infant Lung Development Cohort, we performed complete blood counts and fluorescence-activated cell sorting scans of umbilical cord blood. The associations between risk factors and cord blood cells were assessed using multivariable linear regressions. The multivariable regression analysis showed that an increase per 10μg/m3 of the average nitrogen dioxide 14 days before birth was associated with a decrease in leukocyte (6.7%, 95% CI:-12.1,-1.0) and monocyte counts (11.6%, 95% CI:-19.6,-2.8). Maternal smoking during pregnancy was associated with significantly lower cord blood cell counts in multiple cell populations. Moreover, we observed sex differences regarding eosinophilic granulocytes and plasmacytoid dendritic cells. Finally, significantly increased numbers of cord blood cells were observed in infants exposed to perinatal stress. Cesarean section seems to play a significant role in Th1/Th2 balance. Our results suggest that all three: environmental, hereditary and perinatal factors play a significant role in the composition of cord blood cells at birth, and it is important to adjust for all of these factors in cord blood studies. In particular, perinatal circumstances seem to influence immune balance, which could have far reaching consequences in the development of immune mediated diseases.

Nasal epithelial cells (NECs) are among the first cells to be exposed to air pollutants and respiratory viruses. Although it is known that air pollution exposure and rhinovirus infections increase the risk for asthma development independently, it is unclear how these risk factors interact on a cellular level. Therefore, we aimed to investigate how exposure to diesel particulate matter (DPM) modifies the response of primary NECs to rhinovirus (RV) infection in vitro. Exposure of re‐differentiated, primary NECs (49 healthy children [0–7 years], 12 adults) to DPM modified the mRNA expression of viral cell‐surface receptors, pattern recognition receptors, and pro‐inflammatory response (also protein levels). After exposure to DPM, we additionally infected the NECs with RV‐1b and RV‐16. Viral loads (assessed by titration assays) were significantly higher in DPM‐exposed compared with non‐exposed NECs. Exposure to DPM prior to RV infection resulted in a significant upregulation of pro‐inflammatory cytokines (mRNA and protein level) and β‐defensins mRNA, and significant downregulation of pattern recognition receptors mRNA and CXCL10 (mRNA and protein levels). There was no difference between all outcomes of NECs from children and adults. We can conclude that exposure to DPM prior to RV infection increases viral loads by downregulation of viral defense receptors and upregulation of pro‐inflammatory cytokines. Our findings indicate a strong interaction between air pollution and the antiviral response to RV infection in NECs. We provide mechanistic evidence that exposure to air pollution increases susceptibility to RV infection. Exposure to diesel exhaust particles change the levels of immune factors and increase the susceptibility of respiratory epithelial cells to rhinovirus infections.

Smokers have increased susceptibility and altered innate host defense responses to influenza virus infection. Broccoli sprouts are a source of the Nrf2 activating agentsulforaphane, and short term ingestion of broccoli sprout homogenates (BSH) has been shown to reduce nasal inflammatory responses to oxidant pollutants. Assess the effects of BSH on nasal cytokines, virus replication, and Nrf2-dependent enzyme expression in smokers and nonsmokers. We conducted a randomized, double-blind, placebo-controlled trial comparing the effects of BSH on serially sampled nasal lavage fluid (NLF) cytokines, viral sequence quantity, and Nrf2-dependent enzyme expression in NLF cells and biopsied epithelium. Healthy young adult smokers and nonsmokers ingested BSH or placebo (alfalfa sprout homogenate) for 4 days, designated Days -1, 0, 1, 2. On Day 0 they received standard vaccine dose of live attenuated influenza virus (LAIV) intranasally. Nasal lavage fluids and nasal biopsies were collected serially to assess response to LAIV. In area under curve analyses, post-LAIV IL-6 responses (P = 0.03) and influenza sequences (P = 0.01) were significantly reduced in NLF from BSH-treated smokers, while quinoneoxidoreductasein NLF cells was significantly increased. In nonsmokers, a similar trend for reduction in virus quantity with BSH did not reach statistical significance. In smokers, short term ingestion of broccoli sprout homogenates appears to significantly reduce some virus-induced markers of inflammation, as well as reducing virus quantity. Nutritional antioxidant interventions have promise as a safe, low-cost strategy for reducing influenza risk among smokers and other at risk populations. ClinicalTrials.gov NCT01269723.

Background Preterm infants are susceptible to oxidative stress and prone to respiratory diseases. Autophagy is an important defense mechanism against oxidative-stress-induced cell damage and involved in lung development and respiratory morbidity. We hypothesized that autophagy marker levels differ between preterm and term infants. Methods In the prospective Basel-Bern Infant Lung Development (BILD) birth cohort we compared cord blood levels of macroautophagy (Beclin-1, LC3B), selective autophagy (p62) and regulation of autophagy (SIRT1) in 64 preterm and 453 term infants. Results Beclin-1 and LC3B did not differ between preterm and term infants. However, p62 was higher (0.37, 95% confidence interval (CI) 0.05;0.69 in log2-transformed level, p  = 0.025, p adj  = 0.050) and SIRT1 lower in preterm infants (−0.55, 95% CI −0.78;−0.31 in log2-transformed level, p adj  < 0.001). Furthermore, p62 decreased ( p adj -value for smoothing function was 0.018) and SIRT1 increased (0.10, 95% CI 0.07;0.13 in log2-transformed level, p adj  < 0.001) with increasing gestational age. Conclusion Our findings suggest differential levels of key autophagy markers between preterm and term infants. This adds to the knowledge of the sparsely studied field of autophagy mechanisms in preterm infants and might be linked to impaired oxidative stress response, preterm birth, impaired lung development and higher susceptibility to respiratory morbidity in preterm infants. Impact To the best of our knowledge, this is the first study to investigate autophagy marker levels between human preterm and term infants in a large population-based sample in cord blood plasma This study demonstrates differential levels of key autophagy markers in preterm compared to term infants and an association with gestational age This may be linked to impaired oxidative stress response or developmental aspects and provide bases for future studies investigating the association with respiratory morbidity

Natural killer (NK) cells play a crucial role in linking innate and adaptive immune responses, especially during viral infections and tumor surveillance. They have two major effector functions: the killing of stressed/abnormal cells and the release of cytokines. Their activity is regulated via inhibitory and activating surface receptors. At the same time that the production and use of engineered nanoparticles is steadily increasing, the risk for exposure to silver nanoparticles (AgNPs) from consumer products or biomedical applications is growing. Given this, we assessed the effects of 20-nm big AgNPs on NK cells, which represent an important part of the immune system. Our study involved overnight exposure of human blood NK cells to different concentrations of AgNPs, and silver (Ag) ion controls, and analyzing them for viability, surface receptor expression, intracellular markers, cytokine release, and killing potential. Exposure to AgNPs, but not to Ag ion controls, reduced the viability and the cytotoxic potential after polyriboinosinic-polyribocytidylic acid stimulation of NK cells and increased the expression of the inhibitory receptor CD159a. Exposure to AgNPs and Ag ion controls reduced the expression of the activating receptors CD335 and of CD16 and increased the expression of the activating receptor CD314. Overall, exposure to AgNPs changes NK cells’ function and phenotype and may present a risk for modulating human immune responses, which should be further investigated.

Primary ciliary dyskinesia (PCD) is a rare genetic disease characterized by dyskinetic cilia. Respiratory symptoms usually start at birth. The lack of diagnostic gold standard tests is challenging, as PCD diagnostics requires different methods with high expertise. We founded PCD-UNIBE as the first comprehensive PCD diagnostic center in Switzerland. Our diagnostic approach includes nasal brushing and cell culture with analysis of ciliary motility via high-speed-videomicroscopy (HSVM) and immunofluorescence labeling (IF) of structural proteins. Selected patients undergo electron microscopy (TEM) of ciliary ultrastructure and genetics. We report here on the first 100 patients assessed by PCD-UNIBE. All patients received HSVM fresh, IF, and cell culture (success rate of 90%). We repeated the HSVM with cell cultures and conducted TEM in 30 patients and genetics in 31 patients. Results from cell cultures were much clearer compared to fresh samples. For 80 patients, we found no evidence of PCD, 17 were diagnosed with PCD, two remained inconclusive, and one case is ongoing. HSVM was diagnostic in 12, IF in 14, TEM in five and genetics in 11 cases. None of the methods was able to diagnose all 17 PCD cases, highlighting that a comprehensive approach is essential for an accurate diagnosis of PCD.

Exposure to diesel exhaust (DE) is known to exacerbate allergic inflammation, including virus-induced eosinophil activation in laboratory animals. We have previously shown that in human volunteers with allergic rhinitis a short-term exposure to DE prior to infection with the live attenuated influenza virus (LAIV) increases markers of allergic inflammation in the nasal mucosa. Specifically, levels of eosinophilic cationic protein (ECP) were significantly enhanced in individuals exposed to DE prior to inoculation with LAIV and this effect was maintained for at least seven days. However, this previous study was limited in its scope of nasal immune endpoints and did not explore potential mechanisms mediating the prolonged exacerbation of allergic inflammation caused by exposure to DE prior to inoculation with LAIV. In this follow-up study, the methods were modified to expand experimental endpoints and explore the potential role of NK cells. The data presented here suggest DE prolongs viral-induced eosinophil activation, which was accompanied by decreased markers of NK cell recruitment and activation. Separate in vitro studies showed that exposure to DE particles decreases the ability of NK cells to kill eosinophils. Taken together, these follow-up studies suggest that DE-induced exacerbation of allergic inflammation in the context of viral infections may be mediated by decreased activity of NK cells and their ability to clear eosinophils.