MiR-424 overexpression protects alveolar epithelial cells from LPS-induced apoptosis and inflammation by targeting FGF2 via the NF-κB pathway.

Acute respiratory distress syndrome (ARDS) is a multifactorial, inflammatory lung injury disease with high morbidity and mortality. However, the underlying pathogenic mechanism remains unknown. In this study, lipopolysaccharide (LPS)-stimulated alveolar epithelial cells were used to mimic the inflammatory pathogenesis of ARDS in vitro. We here investigated the role of miR-424 in LPS-stimulated alveolar epithelial cells and found it to be substantially downregulated. Overexpression of miR-424 inhibited apoptosis and inflammation in LPS-stimulated alveolar epithelial cells, and the miR-424 inhibitor exhibited the opposite effect. A bioinformatic analysis revealed a potential binding site of miR-424 in the 3'-UTR of fibroblast growth factor 2 (FGF2). A luciferase reporter assay suggested that miR-424 targeted FGF2 in alveolar epithelial cells. The level of FGF2 protein was inhibited by miR-424 mimic, whereas was significantly upregulated after miR-424 suppression in LPS-stimulated alveolar epithelial cells. MiR-424 also exhibited the protective role in LPS-induced apoptosis and inflammation by directly targeting FGF2 via the NF-κB pathway. In conclusion, our results demonstrate that miR-424 had a protective role in LPS-induced apoptosis and inflammation of alveolar epithelial cells by targeting FGF2 via regulating NF-κB pathway. This might contribute novel evidence to help identify a therapeutic target for treating ARDS.

Staphylococcus aureus impairs sinonasal epithelial repair: Effects in patients with chronic rhinosinusitis with nasal polyps and control subjects.

BACKGROUND: The effect of Staphylococcus aureus on nasal epithelial repair has never been assessed in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). OBJECTIVE: This study aimed to determine whether (1) nasal epithelial cell cultures from patients with CRSwNP and control subjects repair differently; (2) S aureus exoproducts compromise nasal epithelial repair; (3) S aureus alters lamellipodial dynamics; and (4) deleterious effects could be counteracted by the Rho-associated coiled-coil kinase inhibitor Y-27632. METHODS: Primary nasal epithelial cells (pNECs) collected during surgeries were cultured and injured under 3 conditions: (1) basal conditions, (2) exposed to S aureus exoproducts, and (3) exposed to S aureus exoproducts and Y-27632. Epithelial repair, lamellipodial dynamics, and cytoskeletal organization were assessed. RESULTS: Under basal conditions, pNEC cultures from patients with CRSwNP presented significantly lower repair rates and reduced lamellipodial protrusion length and velocity than those from control subjects. S aureus exoproducts significantly decreased repair rates and protrusion dynamics in both control subjects and patients with CRSwNP; however, the effect of S aureus on cell protrusions was more sustained over time in patients with CRSwNP. Under basal conditions, immunofluorescence assays showed significantly reduced percentages of cells with lamellipodia at the wound edge in patients with CRSwNP compared with control subjects. S aureus altered cell polarity and decreased the percentage of cells with lamellipodia in both groups. Finally, Y-27632 prevented the deleterious effects of S aureus exoproducts on CRSwNP repair rates, as well as on lamellipodial dynamics and formation. CONCLUSIONS: S aureus exoproducts significantly alter epithelial repair and lamellipodial dynamics on pNECs, and this impairment was more pronounced in patients with CRSwNP. Importantly, Y-27632 restored epithelial repair and lamellipodial dynamics in the presence of S aureus exoproducts.

Solitary chemosensory cells are a primary epithelial source of IL-25 in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: IL-25 can function as an early signal for the respiratory type 2 response characteristic of allergic asthma and chronic rhinosinusitis with nasal polyps (CRSwNP). In the mouse gut, tuft cells are the epithelial source of IL-25. However, the source of human airway epithelial IL-25 has remained elusive. OBJECTIVE: In this study we sought to determine whether the solitary chemosensory cell (SCC) is the predominant source of IL-25 in the sinonasal epithelium. METHOD: Flow cytometry and immunofluorescence for SCCs and IL-25 were used to interrogate polyp and turbinate tissue from patients with CRSwNP. Mucus was collected during acute inflammatory exacerbations from patients with CRSwNP or chronic rhinosinusitis without nasal polyps and IL-25 levels determined by using ELISA. Lastly, sinonasal epithelial cultures derived from polyp and turbinate tissue were stimulated with IL-13 and analyzed for SCC proliferation and IL-25 production. RESULTS: This study demonstrates that a discrete cell type, likely an SCC, characterized by expression of the taste-associated G protein gustducin and the intestinal tuft cell marker doublecortin-like kinase 1, is the predominant source of IL-25 in the human upper airway. Additionally, we show that patients with CRSwNP have increased numbers of SCCs in nasal polyp tissue and that in vitro IL-13 exposure both increased proliferation and induced apical secretion of IL-25 into the mucosal layer. CONCLUSIONS: Inflammatory sinus polyps but not adjacent turbinate tissue show expansion of the SCC population, which is the source of epithelial IL-25.

Repeated Domperidone treatment modulates pulmonary cytokines in LPS-induced acute lung injury in mice.

The dopaminergic antagonist drug Domperidone has immunomodulatory effects. We investigated the effects of repeated Domperidone treatment in a model of Lypopolyssacharide (LPS)-induced acute lung inflammation. Adult C57BL/6J mice were treated with either Vehicle or Domperidone for 5days, and challenged intranasally with LPS in the following day. The behavior of mice was analyzed in the open field and elevated plus-maze test before and 24h after LPS challenge. The bronchoalveolar lavage fluid, blood and lung tissue were collected 24h and 48h after LPS challenge. Domperidone treatment increased LPS-induced tumor necrosis factor (TNF) and interleukin (IL)-6 production in the bronchoalveolar lavage fluid, without altering tissue damage and the number of immune cells in the lungs and circulation. Locomotor and anxiety-like behavior were unchanged after Domperidone and/or LPS treatment. Cytokine data indicate that Domperidone promotes a change in activity of other cell types, likely alveolar epithelial cells, without affecting immune cell migration in the present model. Due to the role of these cytokines in progression of inflammation, Domperidone treatment may exacerbate a subsequent inflammatory injury.

An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.

KEY POINTS: Extracellular ATP, in association with [Ca2+ ]i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. ABSTRACT: Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca2+ ]i -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca2+ ]i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml-1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1-100 µm) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1 mm), associated with inflammatory conditions, lowered basal CBF by reducing [Ca2+ ]i and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.

Detection of a novel stem cell probably involved in normal turnover of the lung airway epithelium.

Regeneration of the lung airway epithelium after injury has been extensively studied. In contrast, analysis of its turnover in healthy adulthood has received little attention. In the classical view, this epithelium is maintained in the steady-state by the infrequent proliferation of basal or Clara cells. The intermediate filament protein nestin was initially identified as a marker for neural stem cells, but its expression has also been detected in other stem cells. Lungs from CD1 mice at the age of 2, 6, 12, 18 or 24 months were fixed in neutral-buffered formalin and paraffin-embedded. Nestin expression was examined by an immunohistochemical peroxidase-based method. Nestin-positive cells were detected in perivascular areas and in connective tissue that were in close proximity of the airway epithelium. Also, nestin-positive cells were found among the cells lining the airway epithelium. These findings suggest that nestin-positive stem cells circulate in the bloodstream, transmigrate through blood vessels and localize in the lung airway epithelium to participate in its turnover. We previously reported the existence of similar cells able to differentiate into lung chondrocytes. Thus, the stem cell reported here might be a bone marrow-derived mesenchymal stem cell (BMDMSC) able to generate several types of lung tissues. In conclusion, our findings indicate that there exist a BMDMSC in healthy adulthood that participates in the turnover of the lung airway epithelium. These findings may improve our knowledge about the lung stem cell biology and also provide novel approaches to therapy for devastating pulmonary diseases.

A lumped mucosal wave model of the vocal folds revisited: recent extensions and oscillation hysteresis.

This paper examines an updated version of a lumped mucosal wave model of the vocal fold oscillation during phonation. Threshold values of the subglottal pressure and the mean (DC) glottal airflow for the oscillation onset are determined. Depending on the nonlinear characteristics of the model, an oscillation hysteresis phenomenon may occur, with different values for the oscillation onset and offset threshold. The threshold values depend on the oscillation frequency, but the occurrence of the hysteresis is independent of it. The results are tested against pressure data collected from a mechanical replica of the vocal folds, and oral airflow data collected from speakers producing intervocalic /h/. In the human speech data, observed differences between voice onset and offset may be attributed to variations in voice pitch, with a very small or inexistent hysteresis phenomenon.

Stereological estimation of the surface area and oxygen diffusing capacity of the respiratory stomach of the air-breathing armored catfish Pterygoplichthys anisitsi (Teleostei: Loricariidae).

The stomach of Pterygoplichthys anisitsi has a thin, translucent wall and a simple squamous epithelium with an underlying dense capillary network. In the cardiac and pyloric regions, most cells have short microvilli distributed throughout the cell surface and their edges are characterized by short, densely packed microvilli. The mucosal layer of the stomach has two types of pavement epithelial cells that are similar to those in the aerial respiratory organs. Type 1 pavement epithelial cells, resembling the Type I pneumocyte in mammal lungs, are flat, with a large nucleus, and extend a thin sheet of cytoplasm on the underlying capillary. Type 2 cells, resembling the Type II pneumocyte, possess numerous mitochondria, a well-developed Golgi complex, rough endoplasmic reticulum, and numerous lamellar bodies in different stages of maturation. The gastric glands, distributed throughout the mucosal layer, also have several cells with many lamellar bodies. The total volume (air + tissue), tissue, and air capacity of the stomach when inflated, increase along with body mass. The surface-to-tissue-volume ratio of stomach varies from 108 cm(-1) in the smallest fish (0.084 kg) to 59 cm(-1) in the largest fish (0.60 kg). The total stomach surface area shows a low correlation to body mass. Nevertheless, the body-mass-specific surface area varied from 281.40 cm(2) kg(-1) in the smallest fish to 68.08 cm(2) kg(-1) in the largest fish, indicating a negative correlation to body mass (b = -0.76). The arithmetic mean barrier thickness between air and blood was 1.52 +/- 0.07 microm, whereas the harmonic mean thickness (tau(h)) of the diffusion barrier ranged from 0.40 to 0.74 microm. The anatomical diffusion factor (ADF = cm(2) microm(-1) kg(-1)) and the morphological O(2) diffusion capacity (D(morphol)O(2) = cm(3) min(-1) mmHg(-1) kg(-1)) are higher in the smallest specimen and lower in the largest one. In conclusion, the structure and morphometric data of P. anisitsi stomach indicate that this organ is adapted for oxygen uptake from air.

Gross morphology and surface ultrastructure of the gills of Odontesthes argentinensis (Actinopterygii, Atherinopsidae) from a Southwestern Atlantic coastal lagoon.

Odontesthes argentinensis was collected from Mar Chiquita Coastal Lagoon, the Southernmost coastal Atlantic Lagoon of Argentina. The morphology of the gills was analyzed by scanning electron microscopy. The morphology of the superficial structures of the gill filaments and pharyngeal region of the gill arch was discussed and related to their functional aspects. The gills arches are structurally similar to those of other teleosts and bring out the osmoregulatory capacity of this species. The epithelium that covers the surface of the filaments and the pharyngeal region of the gill arch is formed by polygonal pavement cells with conspicuous microridges. These folds in the membrane are not denoted in the epithelium of the respiratory lamellae. Apical crypts of chloride cells are present on the afferent and interlamellar filament surfaces, but are absent elsewhere on the gill arch. The highest density of mucous cells is observed into the gill filament and the pharyngeal region which indicates the existence of a protective strategy of the respiratory lamellae and the pharynx. The epithelium of the gill arches and the rakers is studded with spines. There are taste buds along the whole pharyngeal region that may be associated with their participation in tasting at this zone.

Larynx and cervical trachea in humidification and heating of inhaled gases.

To evaluate the participation of the larynx and cervical trachea in conditioning inspired gases, we randomly allocated 16 mixed-breed dogs to two groups: group TT (tracheal tube; n = 8) and group LMA (laryngeal mask airway; n = 8). The dogs were anesthetized with pentobarbital sodium and mechanically ventilated for 3 hours. The parameters studied were temperature and absolute humidities of ambient, inhaled, and tracheal air. There was a small increase in tracheal air temperature compared to inhaled air temperature, but no significant difference between groups. The absolute humidity of tracheal air was greater in group LMA than in group TT (23 mg H2O * L(-1) and 14 mg H2O * L(-1), respectively; p < .0001). The difference in absolute humidity between the tracheal air and the inhaled air was higher in group LMA at all times (p < .0001). We conclude that the larynx and cervical trachea of the dog participate in humidification and heating of inhaled air by means of air contact with mucosa in this airway segment.

The indirect effect of Tityus discrepans on rabbit pulmonary vasculature.

Serum (IS) was obtained 0.5, 2, 4 or 6 h after inoculating s.c. six rabbits (approximately 2 kg) in each time period with 1 mg/kg of Tityus discrepans (Td) venom; the control was serum obtained from four rabbits 4 h after injecting them 1 ml s.c. of 0.9% NaCl. IS produced a transient (<25 min) rise in pulmonary artery pressure of isolated and perfused rabbit lungs, other lung parameters were not altered. We found that both scorpion venom and IS produced a approximately 50% transient increase of transendothelial electric resistance in cultured tissue human umbilical cord vein. Neither venom nor IS changed the transepithelial electrical resistance of tissue cultured human airway epithelia. The experiments suggest that humoral factors contained in the inoculated serum modify vascular endothelium in a much more effective manner than the venom by itself. These experiments also make it unlikely that vascular endothelium is the source of the humoral factors contained in inflammatory serum.