Effects of solvent extracted organic matter from outdoor air pollution on human type II pneumocytes: Molecular and proteomic analysis.

Outdoor air pollution is responsible for the exacerbation of respiratory diseases in humans. Particulate matter with an aerodynamic diameter ≤2.5 µm (PM2.5) is one of the main components of outdoor air pollution, and solvent extracted organic matter (SEOM) is adsorbed to the main PM2.5 core. Some of the biological effects of black carbon and polycyclic aromatic hydrocarbons, which are components of PM2.5, are known, but the response of respiratory cell lineages to SEOM exposure has not been described until now. The aim of this study was to obtain SEOM from PM2.5 and analyze the molecular and proteomic effects on human type II pneumocytes. PM2.5 was collected from Mexico City in the wildfire season and the SEOM was characterized to be exposed on human type II pneumocytes. The effects were compared with benzo [a] pyrene (B[a]P) and hydrogen peroxide (H2O2). The results showed that SEOM induced a decrease in surfactant and deregulation in the molecular protein and lipid pattern analyzed by reflection-Fourier transform infrared (ATR-FTIR) spectroscopy on human type II pneumocytes after 24 h. The molecular alterations induced by SEOM were not shared by those induced by B[a]P nor H2O2, which highlights specific SEOM effects. In addition, proteomic patterns by quantitative MS analysis revealed a downregulation of 171 proteins and upregulation of 134 proteins analyzed in the STRING database. The deregulation was associated with positive regulation of apoptotic clearance, removal of superoxide radicals, and positive regulation of heterotypic cell-cell adhesion processes, while ATP metabolism, nucleotide process, and cellular metabolism were also affected. Through this study, we conclude that SEOM extracted from PM2.5 exerts alterations in molecular patterns of protein and lipids, surfactant expression, and deregulation of metabolic pathways of type II pneumocytes after 24 h of exposure in absence of cytotoxicity, which warns about apparent SEOM silent effects.

Renin-angiotensin system blockade on angiotensin-converting enzyme 2 and TMPRSS2 in human type II pneumocytes.

AIMS: Angiotensin-converting enzyme (ACE) 2 is the receptor for severe acute respiratory syndrome coronavirus 2 which causes coronavirus disease 2019 (COVID-19). Viral cellular entry requires ACE2 and transmembrane protease serine 2 (TMPRSS2). ACE inhibitors (ACEIs) or angiotensin (Ang) receptor blockers (ARBs) influence ACE2 in animals, though evidence in human lungs is lacking. We investigated ACE2 and TMPRSS2 in type II pneumocytes, the key cells that maintain lung homeostasis, in lung parenchymal of ACEI/ARB-treated subjects compared to untreated control subjects. MAIN METHODS: Ang II and Ang-(1-7) levels and ACE2 and TMPRSS2 protein expression were measured by radioimmunoassay and immunohistochemistry, respectively. KEY FINDINGS: We found that the ratio Ang-(1-7)/Ang II, a surrogate marker of ACE2 activity, as well as the amount of ACE2-expressing type II pneumocytes were not different between ACEI/ARB-treated and untreated subjects. ACE2 protein content correlated positively with smoking habit and age. The percentage of TMPRSS2-expressing type II pneumocytes was higher in males than females and in subjects under 60 years of age but it was not different between ACEI/ARB-treated and untreated subjects. However, there was a positive association of TMPRSS2 protein content with age and smoking in ACEI/ARB-treated subjects, with high TMPRSS2 protein levels most evident in ACEI/ARB-treated older adults and smokers. SIGNIFICANCE: ACEI/ARB treatment influences human lung TMPRSS2 but not ACE2 protein content and this effect is dependent on age and smoking habit. This finding may help explain the increased susceptibility to COVID-19 seen in smokers and older patients with treated cardiovascular-related pathologies.

Differential response of immobile (pneumocytes) and mobile (monocytes) barriers against 2 types of metal oxide nanoparticles.

BACKGROUND: Inhaled nanoparticles (NPs) challenges mobile and immobile barriers in the respiratory tract, which can be represented by type II pneumocytes (immobile) and monocytes (mobile) but what is more important for biological effects, the cell linage, or the type of nanoparticle? Here, we addressed these questions and we demonstrated that the type of NPs exerts a higher influence on biological effects, but cell linages also respond differently against similar type of NPs. DESIGN: Type II pneumocytes and monocytes were exposed to tin dioxide (SnO2) NPs and titanium dioxide (TiO2) NPs (1, 10 and 50 µg/cm2) for 24 h and cell viability, ultrastructure, cell granularity, molecular spectra of lipids, proteins and nucleic acids and cytoskeleton architecture were evaluated. RESULTS: SnO2 NPs and TiO2 NPs are metal oxides with similar physicochemical properties. However, in the absence of cytotoxicity, SnO2 NPs uptake was low in monocytes and higher in type II pneumocytes, while TiO2 NPs were highly internalized by both types of cells. Monocytes exposed to both types of NPs displayed higher number of alterations in the molecular patterns of proteins and nuclei acids analyzed by Fourier-transform infrared spectroscopy (FTIR) than type II pneumocytes. In addition, cells exposed to TiO2 NPs showed more displacements in FTIR spectra of biomolecules than cells exposed to SnO2 NPs. Regarding cell architecture, microtubules were stable in type II pneumocytes exposed to both types of NPs but actin filaments displayed a higher number of alterations in type II pneumocytes and monocytes exposed to SnO2 NPs and TiO2 NPs. NPs exposure induced the formation of large vacuoles only in monocytes, which were not seen in type II pneumocytes. CONCLUSIONS: Most of the cellular effects are influenced by the NPs exposure rather than by the cell type. However, mobile, and immobile barriers in the respiratory tract displayed differential response against SnO2 NPs and TiO2 NPs in absence of cytotoxicity, in which monocytes were more susceptible than type II pneumocytes to NPs exposure.

The plate body: 3D ultrastructure of a facultative organelle of alveolar epithelial type II cells involved in SP-A trafficking.

Plate bodies are facultative organelles occasionally described in the adult lungs of various species, including sheep and goat. They consist of multiple layers of plate-like cisterns with an electron dense middle bar. The present study was performed to elucidate the three-dimensional (3D) characteristics of this organelle and its presumed function in surfactant protein A (SP-A) biology. Archived material of four adult goat lungs and PFA-fixed lung samples of two adult sheep lungs were used for the morphological and immunocytochemical parts of this study, respectively. 3D imaging was performed by electron tomography and focused ion beam scanning electron microscopy (FIB-SEM). Immuno gold labeling was used to analyze whether plate bodies are positive for SP-A. Transmission electron microscopy revealed the presence of plate bodies in three of four goat lungs and in both sheep lungs. Electron tomography and FIB-SEM characterized the plate bodies as layers of two up to over ten layers of membranous cisterns with the characteristic electron dense middle bar. The membranes of the plates were in connection with the rough endoplasmic reticulum and showed vesicular inclusions in the middle of the plates and a vesicular network at the sides of the organelle. Immuno gold labeling revealed the presence of SP-A in the vesicular network of plate bodies but not in the characteristic plates themselves. In conclusion, the present study clearly proves the connection of plate bodies with the rough endoplasmic reticulum and the presence of a vesicular network as part of the organelle involved in SP-A trafficking.

A new mixed-ligand coordination polymer: protective activity on influenza a virus-induced COPD via regulating tlr3 gene expression on alveolar epithelial cells.

A new Cu(II) coordination polymer (CP) of [Cu5(µ3-OH)2(bcpt)4(bib)2] (1, bib = 1,4-bis(1-imidazoly)benzene and H2bcpt = 3,5-bis(3'-carboxyphenyl)-1,2,4-triazole) was synthesized by reaction of Cu(NO3)2·3H2O reacting with 3,5-bis(3'-carboxyphenyl)-1,2,4-triazole in the existence of 1,4-bis(1-imidazoly)benzene as the second ligand. The treatment activity of the compound on influenza A virus induced chronic obstructive pulmonary disease (COPD) was evaluated. First, the biological function of the lung was assessed by measuring the partial pressure for the carbon dioxide (PaCO2) and oxygen (PaO2) via the analysis of blood gas. Next, the inflammatory cytokines released by alveolar epithelial cells were determined via the ELISA test kit. In addition to this, the real-time RT-PCR was carried out to determine the inflammatory response relative expression in the alveolar epithelial cells. Finally, the relative expression of the TLR3 on the alveolar epithelial cells was revealed by western blot. Possible binding patterns were acquired from the post scoring software and molecular docking, which exhibited two possible functional side chain binding sites of TLR3 to compounds binding, possibly offering distinct regulatory mechanisms.

Role of alveolar collapse in idiopathic pleuroparenchymal fibroelastosis.

BACKGROUND: Zonal aggregates of elastic fibres (zonal elastosis) and intraalveolar collagenosis with septal elastosis are histologic components of subpleural fibroelastosis of idiopathic pleuroparenchymal fibroelastosis (IPPFE). Zonal elastosis is considered to result from alveolar collapse, but this mechanism has not been fully justified. METHODS: We immunohistochemically attempted to identify epithelial cells in zonal elastosis of 10 patients with IPPFE. The thickness of the zonal elastosis in relation to the total thickness of the fibroelastosis was examined to estimate the influence of zonal elastosis on the occurrence and development of IPPFE. RESULTS: In 9 of the 10 patients, multi-cytokeratin-positive cells were found lining the inner surface of slit-like spaces embedded in the zonal elastosis. Zonal elastosis was predominant when fibroelastosis was < 1 mm thick but less predominant when it was ≥1 mm. CONCLUSION: The zonal elastosis was proven to result from alveolar collapse, which might be an initial lesion in IPPFE. (Sarcoidosis Vasc Diffuse Lung Dis 2020; 37 (2): 212-217).

Surfactant-induced spreading of nanoparticles is inhibited on mucus mimetic surfaces that model native lung conditions.

We investigated the ability of surfactant-induced spreading to promote nanoparticle distribution on model mucus hydrogels. The hydrogels were formulated with viscoelastic properties and surface tensions that match those of native lung mucus. Nanoparticle-containing droplets with or without surfactant were deposited on the mucus surface and spreading patterns were monitored by time-course fluorescence imaging. Overall, surfactant-induced spreading of nanoparticles required an appropriate balance between Marangoni forces and viscoelastic subphase resistance. Spreading was enhanced on bare gels by increasing the concentration of surfactant in the droplets or reducing the viscoelastic properties of the subphase. However, with a pre-existing film of pulmonary surfactant on the mucus surface, spreading was dramatically inhibited as the surface tension gradient between the droplets and the surrounding subphase decreased. A complete lack of spreading was observed at surface tensions that matched those in the tracheobronchial region of the lungs, even with full-concentration Infasurf. These studies demonstrate that the magnitude of spreading on lung mucus-like surfaces is limited by native mucosal properties.

Proviral MicroRNAs Detected in Extracellular Vesicles From Bronchoalveolar Lavage Fluid of Patients With Influenza Virus-Induced Acute Respiratory Distress Syndrome.

Influenza A virus (IAV) causes severe respiratory infections and alveolar epithelial damage resulting in acute respiratory distress syndrome (ARDS). Extracellular vesicles (EVs) have been shown to mediate cellular crosstalk in inflammation by transfer of microRNAs (miRNAs). In this study, we found significant changes in the miRNA composition of EVs in the bronchoalveolar lavage fluid from patients with IAV-induced ARDS. Among the 9 significantly deregulated microRNAs, miR-17-5p was upregulated in patients' BALF and in EVs of IAV-infected lung epithelial cells (A549). In these cells, transfer of miR-17-5p strongly downregulated expression of the antiviral factor Mx1 and significantly enhanced IAV replication.

Intracellular localization of polymyxins in human alveolar epithelial cells.

Background: Current inhaled polymyxin therapy is empirical and often large doses are administered, which can lead to pulmonary adverse effects. There is a dearth of information on the mechanisms of polymyxin-induced lung toxicity and their intracellular localization in lung epithelial cells. Objectives: To investigate the intracellular localization of polymyxins in human lung epithelial A549 cells. Methods: A549 cells were treated with polymyxin B and intracellular organelles (early and late endosomes, endoplasmic reticulum, mitochondria, lysosomes and autophagosomes), ubiquitin protein and polymyxin B were visualized using immunostaining and confocal microscopy. Fluorescence intensities of the organelles and polymyxin B were quantified and correlated for co-localization using ImageJ and Imaris platforms. Results: Polymyxin B co-localized with early endosomes, lysosomes and ubiquitin at 24 h. Significantly increased lysosomal activity and the autophagic protein LC3A were observed after 0.5 and 1.0 mM polymyxin B treatment at 24 h. Polymyxin B also significantly co-localized with mitochondria (Pearson's R = 0.45) and led to the alteration of mitochondrial morphology from filamentous to fragmented form (n = 3, P < 0.001). These results are in line with the polymyxin-induced activation of the mitochondrial apoptotic pathway observed in A549 cells. Conclusions: Accumulation of polymyxins on mitochondria probably caused mitochondrial toxicity, resulting in increased oxidative stress and cell death. The formation of autophagosomes and lysosomes was likely a cellular response to the polymyxin-induced stress and played a defensive role by disassembling dysfunctional organelles and proteins. Our study provides new mechanistic information on polymyxin-induced lung toxicity, which is vital for optimizing inhaled polymyxins in the clinic.

Expression of cytochrome P450 mRNAs in Type II alveolar cells from subjects with chronic obstructive pulmonary disease.

Inhaled drugs are critical for the treatment of inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). To develop better therapeutics for pulmonary disease it is of potential importance to understand molecular mechanisms of local biotransformation in the lung. Alveolar epithelial type II (ATII) cells have a key role in homeostasis in the lung, but little is known about expression patterns of genes encoding cytochrome P450 (CYP) enzymes in ATII cells. In addition, alteration of CYP gene expression has not been fully defined in COPD. We previously established a method to purify ATII cells from the adult human lung using fluorescence-activated cell sorting. By employing this technique we determined gene expression patterns of 14 CYP enzymes in ATII cells from nonsmokers (n = 4) and smokers (n = 4), both having normal pulmonary function. Although most CYP genes are highly expressed in primary hepatocytes, we found that CYP1B1 mRNA expression was 7.2-fold higher in ATII compared to hepatocytes (P = .0275). Additionally we noted a 3.0-fold upregulation of CYP2C19 and 50% reduction in CYP2J2 mRNA expressions in ATII cells isolated from patients with COPD (n = 3) compared to smokers without COPD (n = 4). These data, for the first time, detail a comprehensive set of genes encoding CYP enzymes in human ATII cells and highlights differentially expressed CYP mRNAs of patients with COPD. Such understanding may have important implications for the development of novel inhaled drugs.

RAGE-induced changes in the proteome of alveolar epithelial cells.

The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor and member of the immunoglobulin superfamily. RAGE is constitutively expressed in the distal lung where it co-localizes with the alveolar epithelium; RAGE expression is otherwise minimal or absent, except with disease. This suggests RAGE plays a role in lung physiology and pathology. We used proteomics to identify and characterize the effects of RAGE on rat alveolar epithelial (R3/1) cells. LC-MS/MS identified 177 differentially expressed proteins and the PANTHER Classification System further segregated proteins. Proteins involved in gene transcription (RNA and mRNA splicing, mRNA processing) and transport (protein, intracellular protein) were overrepresented; genes involved in a response to stimulus were underrepresented. Immune system processes and response to stimuli were downregulated with RAGE knockdown. Western blot confirmed RAGE-dependent changes in protein expression for NFκB and NLRP3 that was functionally supported by a reduction in IL-1ß and phosphorylated p65. We also assessed RAGE's effect on redox regulation and report that RAGE knockdown attenuated oxidant production, decreased protein oxidation, and increased reduced thiol pools. Collectively the data suggest that RAGE is a critical regulator of epithelial cell response and has implications for our understanding of lung disease, specifically acute lung injury. SIGNIFICANCE STATEMENT: In the present study, we undertook the first proteomic evaluation of RAGE-dependent processes in alveolar epithelial cells. The alveolar epithelium is a primary target during acute lung injury, and our data support a role for RAGE in gene transcription, protein transport, and response to stimuli. More over our data suggest that RAGE is a critical driver of redox regulation in the alveolar epithelium. The conclusions of the present work assist to unravel the molecular events that underlie the function of RAGE in alveolar epithelial cells and have implications for our understanding of RAGE signaling during lung injury. Our study was the first proteomic comparison showing the effects of RAGE activation from alveolar epithelial cells that constitutively express RAGE and these results can affect a wide field of lung biology, pulmonary therapeutics, and proteomics.

Polarized light microscopy reveals physiological and drug-induced changes in surfactant membrane assembly in alveolar type II pneumocytes.

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general.

[Xinfeng Capsule improves lung function by regulating Notch/Jagged-HES axis of type II alveolar epithelial cells in adjuvant arthritis rats].

Objective To observe the effect of Xinfeng Capsule (XFC) on Notch/Jagged-HES of type II alveolar epithelial cells (AECII). Methods Rats were divided for four groups: normal control (NC) group, model control (MC) group, leflunomide (LEF) group, XFC group, with 10 rats in each group. Complete Freund's adjuvant (CFA) was injected in the right foot plantar skin of each rat except for the NC group. After adjuvant arthritis was successfully induced, LEF group was given LEF (0.5 mg/100 g), and XFC group was treated with XFC (0.034 g/100 g), once a day from the 13th day to the 42th day. The NC and MC groups were given normal saline instead. Swelling degree (SD), arthritis index (AI) and pulmonary function were observed. AECII was observed by transmission electron microscopy (TEM). The expressions of transforming growth factor ß1 (TGF-ß1), Notch1, Notch3, Jagged1 and HES1 proteins in AECII were detected by Western blotting. Results The pulmonary function parameters such as forced expiratory volume in 1 second (FEV1), maximum expiratory flow rate at 50% FVC (FEF50), instantaneous flow at 75% of expired volume (FEF75), peak expiratory flow (PEF) in the MC group were significantly lower than those in the NC group, and the expressions of TGF-ß1, Notch1, Notch3, Jagged1 and HES1 in AECII increased. The ultrastructure of AECII was damaged. Compared with the MC group, FEV1, FEF50, FEF75 and PEF increased, and TGF-ß1, Notch1, Notch3, Jagged1 and HES1 decreased in the XFC group. Compared with LEF group, the lung function was better in XFC group. Conclusion XFC can inhibit pulmonary fibrosis and improve pulmonary function by down-regulating TGF-ß1, Notch1, Notch3, Jagged1 and HES1 in rats with adjuvant arthritis.

Comparison of Omadacycline and Tigecycline Pharmacokinetics in the Plasma, Epithelial Lining Fluid, and Alveolar Cells of Healthy Adult Subjects.

The steady-state concentrations of omadacycline and tigecycline in the plasma, epithelial lining fluid (ELF), and alveolar cells (AC) of 58 healthy adult subjects were obtained. Subjects were administered either omadacycline at 100 mg intravenously (i.v.) every 12 h for two doses followed by 100 mg i.v. every 24 h for three doses or tigecycline at an initial dose of 100 mg i.v. followed by 50 mg i.v. every 12 h for six doses. A bronchoscopy and bronchoalveolar lavage were performed once in each subject following the start of the fifth dose of omadacycline at 0.5, 1, 2, 4, 8, 12, or 24 h and after the start of the seventh dose of tigecycline at 2, 4, 6, or 12 h. The value of the area under the concentration-time curve (AUC) from time zero to 24 h postdosing (AUC0-24) (based on mean concentrations) in ELF and the ratio of the ELF to total plasma omadacycline concentration based on AUC0-24 values were 17.23 mg · h/liter and 1.47, respectively. The AUC0-24 value in AC was 302.46 mg · h/liter, and the ratio of the AC to total plasma omadacycline concentration was 25.8. In comparison, the values of the AUC from time zero to 12 h postdosing (AUC0-12) based on the mean concentrations of tigecycline in ELF and AC were 3.16 and 38.50 mg · h/liter, respectively. The ratio of the ELF and AC to total plasma concentrations of tigecycline based on AUC0-12 values were 1.71 and 20.8, respectively. The pharmacokinetic advantages of higher and sustained concentrations of omadacycline compared to those of tigecycline in plasma, ELF, and AC suggest that omadacycline is a promising antibacterial agent for the treatment of lower respiratory tract bacterial infections caused by susceptible pathogens.

Mapping differential cellular protein response of mouse alveolar epithelial cells to multi-walled carbon nanotubes as a function of atomic layer deposition coating.

Carbon nanotubes (CNTs), a prototypical engineered nanomaterial, have been increasingly manufactured for a variety of novel applications over the past two decades. However, since CNTs possess fiber-like shape and cause pulmonary fibrosis in rodents, there is concern that mass production of CNTs will lead to occupational exposure and associated pulmonary diseases. The aim of this study was to use contemporary proteomics to investigate the mechanisms of cellular response in E10 mouse alveolar epithelial cells in vitro after exposure to multi-walled CNTs (MWCNTs) that were functionalized by atomic layer deposition (ALD). ALD is a method used to generate highly uniform and conformal nanoscale thin-film coatings of metals to enhance novel conductive properties of CNTs. We hypothesized that specific types of metal oxide coatings applied to the surface of MWCNTs by ALD would determine distinct proteomic profiles in mouse alveolar epithelial cells in vitro that could be used to predict oxidative stress and pulmonary inflammation. Uncoated (U)-MWCNTs were functionalized by ALD with zinc oxide (ZnO) to yield Z-MWCNTs or aluminum oxide (Al2O3) to yield A-MWCNTs. Significant differential protein expression was found in the following critical pathways: mTOR/eIF4/p70S6K signaling and Nrf-2 mediated oxidative stress response increased following exposure to Z-MWCNTs, interleukin-1 signaling increased following U-MWCNT exposure, and inhibition of angiogenesis by thrombospondin-1, oxidative phosphorylation, and mitochondrial dysfunction increased following A-MWCNT exposure. This study demonstrates that specific types of metal oxide thin film coatings applied by ALD produce distinct cellular and biochemical responses related to lung inflammation and fibrosis compared to uncoated MWCNT exposure in vitro.

A novel surfactant protein C L55F mutation associated with interstitial lung disease alters subcellular localization of proSP-C in A549 cells.

BACKGROUND: Heterozygous mutations of SFTPC, the gene-encoding surfactant protein C (SP-C), result in interstitial lung disease (ILD). However, characterization of mutations located in the mature domain of precursor SP-C (proSP-C) is limited. This study examined the molecular pathogenesis of such a mutation of ILD. METHODS: We employed sequencing of SFTPC and established A549 cells stably expressing several proSP-C mutants. Histopathology and transmission electron microscopy (TEM) of lung tissue from a pediatric patient with ILD were assessed. Effects of mutant proSP-C were evaluated by western blotting, immunofluorescence, and TEM. RESULTS: Sequencing of SFTPC revealed a novel heterozygous mutation, c.163C>T (L55F). In lung tissue, abnormal localization of proSP-C was observed by immunohistochemistry, and small and dense lamellar bodies (LBs) in type II alveolar epithelial cells (AECs) were detected by TEM. TEM of A549 cells stably expressing proSP-C(L55F) displayed abnormal cytoplasmic organelles. ProSP-C(L55F) exhibited a band pattern similar to that of proSP-C(WT) for processed intermediates. Immunofluorescence studies demonstrated that proSP-C(L55F) partially colocalized in CD63-positive cytoplasmic vesicles of A549 cells, which was in contrast to proSP-C(WT). CONCLUSION: We detected a novel c.163C>T mutation located in the mature domain of SFTPC associated with ILD that altered the subcellular localization of proSP-C in A549 cells.

Long-term ultrastructural indices of lead intoxication in pulmonary tissue of the rat.

In the present research long-term pulmonary toxicity of lead was investigated in rats treated by intraperitoneal administration of lead acetate for three consecutive days (25 mg/kg per day). Five weeks after treatment average lead content in the whole blood was 0.41 µg/dL ± 0.05, in the lung homogenates it measured 3.35 µg/g ± 0.54, as compared to the control values of 0.13 ± 0.07 µg/dL and 1.03 µg/g ± 0.59, respectively. X-ray microanalysis of lung specimens displayed lead localized mainly within type II pneumocytes and macrophages. At the ultrastructural level the effects of lead toxicity were found in lung capillaries, interstitium, epithelial cells, and alveolar lining. Alveolar septa showed intense fibrosis, consisting of collagen, elastin, and fibroblasts. Thinned alveolar septa had emphysematous tissue with some revealing signs of angiogenesis. Type II pneumocytes contained lamellar bodies with features of laminar destruction. Fragments of the surfactant layer were often detached from the alveolar epithelium. These findings indicate that 5 weeks after exposure, lead provokes reconstruction of the alveolar septa including fibrosis and emphysematous changes in the lung tissue.

Pulmonary haptoglobin (pHp) is part of the surfactant system in the human lung.

Since the existence of pHp was demonstrated, it has been shown that this molecule and its receptor CD163 are regulated by different stimuli. Furthermore, a comparably fast secretion of pHp was described as well as the immuno-stimulatory effects. The intention of this study was to elucidate the role of pHp in the human lungs further. Here we show, by means of confocal microscopy and immune-electron-microscopy, a clear co-localization of pHp with surfactant protein-B in lamellar bodies of alveolar epithelial cells type II. These results are underlined by immunohistochemical stainings in differently fixed human lung tissues, which show pHp in vesicular and released form. The images of the released form resemble the intended position of surfactant in the human alveolus. pHp is secreted by Alveolar epithelial cells type II as previously shown. Moreover, pHp is co-localized with Surfactant protein-B. We conclude that the presented data shows that pHp is a native part of the surfactant system in the human lung. VIRTUAL SLIDES: http://www.diagnosticpathology.diagnomx.eu/vs/2563584738239912.

Increased 25-hydroxycholesterol concentrations in the lungs of patients with chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: 25-Hydroxycholesterol (25-HC) is produced from cholesterol by the enzyme cholesterol 25-hydroxylase and is associated with atherosclerosis of vessels. Recently, 25-HC was reported to cause inflammation in various types of tissues. The aim of this study was to assess the production of 25-HC in the airways and to elucidate the role of 25-HC in neutrophil infiltration in the airways of patients with chronic obstructive pulmonary disease (COPD). METHODS: Eleven control never-smokers, six control ex-smokers without COPD and 13 COPD patients participated in the lung tissue study. The expression of cholesterol 25-hydroxylase in the lung was investigated. Twelve control subjects and 17 patients with COPD also participated in the sputum study. The concentrations of 25-HC in sputum were quantified by liquid chromatography/mass spectrometry/mass spectrometry analysis. To elucidate the role of 25-HC in neutrophilic inflammation of the airways, the correlation between 25-HC levels and neutrophil counts in sputum was investigated. RESULTS: The expression of cholesterol 25-hydroxylase was significantly enhanced in lung tissue from COPD patients compared with that from control subjects. Cholesterol 25-hydroxylase was localized in alveolar macrophages and pneumocytes of COPD patients. The concentration of 25-HC in sputum was significantly increased in COPD patients and was inversely correlated with percent of predicted forced vital capacity, forced expiratory volume in 1 s and diffusing capacity of carbon monoxide. The concentrations of 25-HC in sputum were significantly correlated with sputum interleukin-8 levels and neutrophil counts. CONCLUSIONS: 25-HC production was enhanced in the airways of COPD patients and may play a role in neutrophilic inflammation.

Disruption of ß-arrestins blocks glucocorticoid receptor and severely retards lung and liver development in mice.

In this study, the role of ß-arrestin 1 and ß-arrestin 2 in fetal lung and liver development was examined using Arrb1(-/-)Arrb2(-/-) mouse embryos. ß-Arrestin 1/2 dual-null mice died shortly after birth and morphological examination revealed an obvious pulmonary hypoplasia and severe hepatic impairment. Western blot analysis demonstrated that GR protein levels in Arrb1(-/-)Arrb2(-/-) lung and liver tissues were significantly decreased compared to wild type embryos. Expression of GR proteins was confirmed in the nuclei of type II pneumocytes of 18.5 day embryos (E18.5) by immunofluorescence. The production of hepatic glucose and mRNA level of gluconeogenic enzymes were dramatically reduced in E18.5 Arrb1(-/-)Arrb2(-/-) liver. These results suggest that GR is an important downstream effector of the ß-arrestin signaling pathway involved in regulation of lung and liver development. However, no obvious changes in GR expression following in vitro modulation of ß-arrestin 1/2 indicated the existence of an indirect regulatory relationship between GR and the ß-arrestin signaling pathway.