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Concept: Respiratory epithelium

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ABSTRACT The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

Concepts: Immune system, Cytokine, Virus, Interferon, Influenza, Severe acute respiratory syndrome, Respiratory epithelium, Coronavirus

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Motility helps many pathogens swim through the highly viscous intestinal mucus. Given the differing outcomes of Campylobacter concisus infection, the motility of eight C. concisus strains isolated from patients with Crohn’s disease (n=3), acute (n=3) and chronic (n=1) gastroenteritis and a healthy control (n=1) were compared. Following growth on solid or liquid media the eight strains formed two groups; however, the type of growth medium did not affect motility. In contrast, following growth in viscous liquid medium seven of the eight strains demonstrated significantly decreased motility. In media of increasing viscosities the motility of C. concisus UNSWCD had two marked increases at viscosities of 20.0 and 74.7 centipoises. Determination of the ability of UNSWCD to swim through a viscous medium, adhere to and invade intestinal epithelial cells showed that while adherence levels significantly decreased with increasing viscosity, invasion levels did not significantly change. In contrast, adherence to and invasion of UNSWCD to mucus-producing intestinal cells increased upon accumulation of mucus, as did bacterial aggregation. Given this aggregation, we determined the ability of the eight C. concisus strains to form biofilms, and showed that all strains formed biofilms. In conclusion, the finding that C. concisus strains could be differentiated into two groups based on their motility may suggest that strains with high motility have an increased ability to swim through the intestinal mucus and reach the epithelial layer.

Concepts: Bacteria, Epithelium, Stomach, Viscosity, Biofilm, Respiratory epithelium, Diarrhea, Intestinal epithelium

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Childhood exposure to environmental particulates increases the risk of development of asthma. The underlying mechanisms may include oxidant injury to airway epithelial cells (AEC). We investigated the ability of ambient environmental particulates to contribute to sensitization via the airways, and thus to the pathogenesis of childhood asthma. To do so, we devised a novel model in which weanling BALB/c mice were exposed to both ambient particulate pollutants and ovalbumin for sensitization via the respiratory tract, followed by chronic inhalational challenge with a low mass concentration of the antigen. We also examined whether these particulates caused oxidant injury and activation of AEC in vitro. Furthermore, we assessed the potential benefit of minimizing oxidative stress to AEC through the period of sensitization and challenge, by dietary intervention. We found that characteristic features of asthmatic inflammation developed only in animals which received particulates at the same time as respiratory sensitization, and were then chronically challenged with allergen. However, these animals did not develop airway hyper-responsiveness. Ambient particulates induced epithelial injury in vitro, with evidence of oxidative stress, and production of both pro-inflammatory cytokines and Th2-promoting cytokines such as IL-33. Treatment of AEC with an anti-oxidant in vitro inhibited the pro-inflammatory cytokine response to these particulates. Ambient particulates also induced pro-inflammatory cytokine expression following administration to weanling mice. However, early-life dietary supplementation with anti-oxidants did not prevent the development of an asthmatic inflammatory response in animals that were exposed to particulates, sensitized and challenged. We conclude that injury to airway epithelium by ambient environmental particulates in early life is capable of promoting the development of an asthmatic inflammatory response in sensitized and antigen-challenged mice. These findings are likely to be relevant to the induction of childhood asthma.

Concepts: Immune system, Inflammation, Cytokine, Asthma, Antioxidant, Interleukin, Endothelium, Respiratory epithelium

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Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established.

Concepts: Protein, Microbiology, Virus, Infection, Influenza, Respiratory epithelium, Mucus, Orthomyxoviridae

38

Irreversible destruction of bronchi and alveoli can lead to multiple incurable lung diseases. Identifying lung stem/progenitor cells with regenerative capacity and utilizing them to reconstruct functional tissue is one of the biggest hopes to reverse the damage and cure such diseases. Here we showed that a rare population of SOX9+ basal cells (BCs) located at airway epithelium rugae can regenerate adult human lung. Human SOX9+ BCs can be readily isolated by bronchoscopic brushing and indefinitely expanded in feeder-free condition. Expanded human SOX9+ BCs can give rise to alveolar and bronchiolar epithelium after being transplanted into injured mouse lung, with air-blood exchange system reconstructed and recipient’s lung function improved. Manipulation of lung microenvironment with Pirfenidone to suppress TGF-β signaling could further boost the transplantation efficiency. Moreover, we conducted the first autologous SOX9+ BCs transplantation clinical trial in two bronchiectasis patients. Lung tissue repair and pulmonary function enhancement was observed in patients 3-12 months after cell transplantation. Altogether our current work indicated that functional adult human lung structure can be reconstituted by orthotopic transplantation of tissue-specific stem/progenitor cells, which could be translated into a mature regenerative therapeutic strategy in near future.

Concepts: Pulmonology, Asthma, Lung, Cure, Epithelium, Regeneration, Respiratory epithelium, Basal cell carcinoma

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Cells lining the respiratory tract are equipped with mechanisms that dampen the effects of oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a mediator involved in regulating oxidative stress. Recent data indicate Nrf2 also controls expression of secretory leukocyte protease inhibitor (SLPI). Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, enhances Nrf2 activity. Therefore, we hypothesized that SFN supplementation induces SLPI secretion in the nasal mucosa in an Nrf2 dependent manner. Healthy nonsmoking adults ingested SFN-containing broccoli shake homogenate (BSH) for 3 consecutive days. Nasal lavage fluid (NLF) was collected before and after BSH ingestion and analyzed for SLPI protein levels. In follow up in vitro experiments, differentiated primary nasal epithelial cells were used to evaluate the relationship between SFN, Nrf2, and SLPI. Epithelial cells were transduced with Nrf2-specific shRNA to examine the regulatory role of Nrf2 on SLPI expression. Supplementation with BSH significantly increased SLPI levels in NLF. SFN supplementation in vitro significantly enhanced SLPI secretion and these effects were significantly decreased in cells transduced with Nrf2-specific shRNA. Our data support a relationship between nutritional supplementation, Nrf2 activation, and SLPI secretion. Therefore, ingestion of SFN-containing foods has therapeutic potential to augment SLPI expression in the nasal mucosa.

Concepts: Nutrition, Respiratory system, Respiratory epithelium, Mucus, Sulforaphane, Cruciferous vegetables, Brassica oleracea, Vegetables

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We report a hitherto not documented case of primary mucinous cystadenoma arising in the spermatic cord within the right inguinal canal of a78-year-old man. The tumor was painless, hard and mobile. A computed tomography scan on the pelvis revealed an oval shaped, low attenuation mass, measuring 5.0x2.5x2.1 cm, that was present adjacent to the vas deferens. Grossly, the excised mass was multicystic mucinous tumor, filled with thick mucoid materials. Microscopically, the cystic wall was irregularly thickened. The cystic epithelium commonly showed short papillae lined by a single layer of columnar to cuboidal mucinous epithelial cells without significant stratification or cytologic atypia. Goblet cells were also frequently present. Immunohistochemically, the neoplastic cells showed positive reaction to carcinoembryonic antigen, cytokeratin 20, CDX2, epithelial membrane antigen, and CD15. However, they were negative for PAX8 and Wilms' tumor 1 protein. Pathological diagnosis was a papillary mucinous cystadenoma of the spermatic cord. Although mucinous cystadenoma in this area is extremely rare, it is important that these lesions be recognized clinically and pathologically in order to avoid unnecessary radical surgery. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1720965948762004.

Concepts: Cancer, Oncology, Anatomical pathology, Cystic fibrosis, Neoplasm, Respiratory epithelium, Vas deferens, Spermatic cord

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To date, there is only a fragmentary understanding of the fundamental mechanisms of airway mucociliary transport. Application of the latest measurement techniques can aid in deciphering the complex interplay between ciliary beat and airway surface liquid (ASL) transport. In the present study, direct, quasi-simultaneous measurements of the cilia-induced fluid and bead transport were performed to gain a better insight into both transport mechanisms. In this study cilia-induced periciliary liquid (PCL) transport is measured by means of micro Particle Image Velocimetry (μPIV) with neutrally buoyant tracers. Particle Tracking Velocimetry (PTV) with heavier polystyrene-ferrite beads is performed to simulate particle transport. Contrary to recent literature, in which the presence of mucus was deemed necessary to maintain periciliary liquid (PCL) transport, effective particle and fluid transport was measured in our experiments in the absence of mucus. In response to muscarine or ATP stimulation, maximum fluid transport rates of 250μm/s at 15μm distance to the tracheal epithelia were measured while bead transport rates over the epithelia surfaces reached 200μm/s. We estimated that the mean bead transport is dominated by viscous drag compared to inertial fluid forces. Furthermore, mean bead transport velocities appear to be two orders of magnitude larger compared to bead sedimentation velocities. Therefore, beads are expected to closely follow the mean PCL flow in non-ciliated epithelium regions. Based on our results, we have shown that PCL transport can be directly driven by the cilia beat and that the PCL motion may be capable of driving bead transport by fluid drag.

Concepts: Fluid dynamics, Measurement, Viscosity, Arithmetic mean, Respiratory epithelium, Particle image velocimetry, Particle tracking velocimetry, Velocimetry

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Collections of cells must be patterned spatially during embryonic development to generate the intricate architectures of mature tissues. In several cases, including the formation of the branched airways of the lung, reciprocal signaling between an epithelium and its surrounding mesenchyme helps generate these spatial patterns. Several molecular signals are thought to interact via reaction-diffusion kinetics to create distinct biochemical patterns, which act as molecular precursors to actual, physical patterns of biological structure and function. Here, however, we show that purely physical mechanisms can drive spatial patterning within embryonic epithelia. Specifically, we find that a growth-induced physical instability defines the relative locations of branches within the developing murine airway epithelium in the absence of mesenchyme. The dominant wavelength of this instability determines the branching pattern and is controlled by epithelial growth rates. These data suggest that physical mechanisms can create the biological patterns that underlie tissue morphogenesis in the embryo.

Concepts: Lung, Epithelium, Tissues, Skin, Respiratory epithelium, Pattern, Endoderm

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Scaffold design is an important aspect of in vitro model development. In this study, nanoscaffold surface modification, namely UV-radiation and genipin cross-linking to immobilize collagen on the surface of electrospun Poly (methylmethacrylate) (PMMA) nanofibers sheet was investigated. Samples were divided into 4 groups; PMMA nanofibers (PMMA), collagen-coated PMMA nanofibers (PMMACOL), Genipin-crosslinked-collagen coated PMMA nanofibers (PMMAGEN), and UV-irradiated collagen-coated PMMA nanofibers (PMMAUV). 6 hours of UV radiation significantly reduced the hydrophobicity of PMMA nanofibers from (131.88°±1.33°) to (110.04°±0.27°) (p<0.05). The amount of collagen immobilized was significantly higher in PMMAGEN group (239.36±16.63 µg collagen/mg nanofibers) (p<0.05) compared to the other groups. RECs on all scaffold expressed epithelial cell specific markers (CK18 and CK14), mucin producing cell marker (MUC5Ac) and were actively proliferating, based on the positive expression of Ki67. Total number of attached cells was significantly highest in PMMAUV group on day 9 (6.44x10(4)±2.77x10(4) cells/cm(2)) and it has the highest proliferation rate from day 4 to 9 (0.005±0.003 h(-1)) compared to the other groups. Eventhough PMMAGEN group showed highest collagen adsorption, in terms of cells attachment and proliferation, PMMAUV group showed a better outcome compared to the other groups. Thus, PMMAUV scaffold is more suitable to be used in construction of in vitro respiratory epithelial model.

Concepts: DNA, Gene expression, Ultraviolet, Epithelium, Cornea, Respiratory epithelium, Connective tissue