A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear.Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelialendothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber.We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4.Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection. @ERSpublications Influenza A virus damages tight junctions, and specifically claudin-4, of respiratory epithelial cells

BackgroundTo date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.MethodsThus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.ResultsRegarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.ConclusionWe present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

BackgroundThe impact of climate on biodiversity is indisputable. Climate changes over geological time must have significantly influenced the evolution of biodiversity, ultimately leading to its present pattern. Here we consider the paleoclimate data record, inferring that present-day hot and cold environments should contain, respectively, the largest and the smallest diversity of ancestral lineages of microbial eukaryotes.Methodology/Principal FindingsWe investigate this hypothesis by analyzing an original dataset of 18S rRNA gene sequences from Western Greenland in the Arctic, and data from the existing literature on 18S rRNA gene diversity in hydrothermal vent, temperate sediments, and anoxic water column communities. Unexpectedly, the community from the cold environment emerged as one of the richest observed to date in protistan species, and most diverse in ancestral lineages.Conclusions/SignificanceThis pattern is consistent with natural selection sweeps on aerobic non-psychrophilic microbial eukaryotes repeatedly caused by low temperatures and global anoxia of snowball Earth conditions. It implies that cold refuges persisted through the periods of greenhouse conditions, which agrees with some, although not all, current views on the extent of the past global cooling and warming events. We therefore identify cold environments as promising targets for microbial discovery.

Drug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial cell line (ISO-HAS-1) were used in an experimental set up resembling the alveolar-capillary barrier of the lung. In terms of IL-8 and sICAM Sicastar resulted in harmful effects at higher concentrations (60 μg/ml) in conventional monocultures but not in the coculture, whereas AmOrSil showed no significant effects. Immunofluorescence counterstaining of endosomal structures in NP-incubated cells showed no evidence for a clathrin- or caveolae-mediated uptake mechanism. However, NPs were enclosed in flotillin-1 and -2 marked vesicles in both cell types. Flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. In addition, we examined the transport of NPs across this in vitro model of the alveolar-capillary barrier forming a tight barrier with a transepithelial electrical resistance of 560±8 Ω cm(2). H441 in coculture with endothelial cells took up much less NPs compared to monocultures. Moreover, coculturing prevented the transport of NP from the epithelial compartment to the endothelial layer on the bottom of the filter insert. This supports the relevance of coculture models, which favour a differentiated and polarised epithelial layer as in vitro test systems for nanoparticle uptake.

Amorphous silica nanoparticles (aSNPs) gain increasing popularity for industrial and therapeutic claims. The lung with its surface area of 100–140 m2 displays an ideal target for therapeutic approaches, but it represents also a serious area of attack for harmful nanomaterials. The exact nature of the cytotoxic effects of NPs is still unknown. Furthermore, cellular pathways and the destiny of internalized NPs are still poorly understood. Therefore, we examined the cytotoxicity (MTS, LDH) and inflammatory responses (IL-8) for different-sized aSNPs (30, 70, 300 nm) on our lung epithelial cells line NCI H441 and endothelial cell line ISO-HAS-1. Additionally, colocalization studies have been conducted via immunofluorescence staining for flotillin-1- and flotillin-2-bearing endocytic vesicles. Subsequently, the relevance of flotillins concerning the viability of aSNP-exposed epithelial cells has been evaluated using flotillin-1/2 depleted cells (siRNA). This study reveals the relevance of the nanoparticle size regarding cytotoxicity (MTS, LDH) and inflammatory responses (IL-8), whereat the smaller the size of the nanoparticle is, the more harmful are the effects. All different aSNP sizes have been incorporated in flotillin-1- and flotillin-2-labelled vesicles in lung epithelial and endothelial cells, which display a marker for late endosomal or lysosomal structures and appear to exhibit a clathrin- or caveolae-independent mode of endocytosis. Flotillin-depleted H441 showed a clearly decreased uptake of aSNPs. Additionally, the viability of aSNP-exposed cells was reduced in these cells. These findings indicate a contribution of flotillins in as yet unknown (clathrin or caveolae-independent) endocytosis mechanisms and (or) endosomal storage.