Idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis are associated with surfactant system dysfunction, alveolar collapse (derecruitment), and collapse induration (irreversible collapse). These events play undefined roles in the loss of lung function. The purpose of this study was to quantify how surfactant inactivation, alveolar collapse, and collapse induration lead to degradation of lung function. Design-based stereology and invasive pulmonary function tests were performed 1, 3, 7, and 14 days after intratracheal bleomycin–instillation in rats. The number and size of open alveoli was correlated to mechanical properties. Active surfactant subtypes declined by Day 1, associated with a progressive alveolar derecruitment and a decrease in compliance. Alveolar epithelial damage was more pronounced in closed alveoli compared with ventilated alveoli. Collapse induration occurred on Day 7 and Day 14 as indicated by collapsed alveoli overgrown by a hyperplastic alveolar epithelium. This pathophysiology was also observed for the first time in human IPF lung explants. Before the onset of collapse induration, distal airspaces were easily recruited, and lung elastance could be kept low after recruitment by positive end-expiratory pressure (PEEP). At later time points, the recruitable fraction of the lung was reduced by collapse induration, causing elastance to be elevated at high levels of PEEP. Surfactant inactivation leading to alveolar collapse and subsequent collapse induration might be the primary pathway for the loss of alveoli in this animal model. Loss of alveoli is highly correlated with the degradation of lung function. Our ultrastructural observations suggest that collapse induration is important in human IPF.

Objective: To analyze the potential role of the Notch signaling pathway in pancreatic cancer angiogenesis and invasion. Background: Angiogenesis, pain, and early neuroinvasion are clinical features of pancreatic cancer. Blood vessels and nerves develop together and use common routes through the organism. The Notch pathway (Notch-1/4, Jagged-1/2, Delta-1) appears crucial in this process. The current study analyzed the Notch pathway in pancreatic cancer and characterized its angiogenic and invasive effects. Methods: Five PaCa cell lines were cultured for the in vitro experiments. Real-time quantitative RT-PCR was done to quantify mRNA expression in 31 human PaCa specimens, and immunohistochemistry was used to localize protein expression within tumor specimens. Activation of the Notch signaling was done by transfection of PaCa cells with a constitutive active Notch-1 mutant (Notch-IC). Overexpression of Jagged and Delta was achieved by transfection of full-length cDNA. Spheroid assays were used to study angiogenesis and ELISAs to measure VEGF, bFGF, and angiogenin expression. Matrigel invasion assays were used to analyze tumor cell invasion. Results: Notch-3 and Notch-4 mRNA were significantly (P < 0.001) overexpressed in PaCa. Immunohistochemistry revealed protein accumulation of Notch-1 as well. All ligands were significantly up-regulated. A positive immunosignal of ligands was seen in nerves, blood vessels, and ductal tumor cells. Transfection of PaCa cells with the constitutive active Notch-IC mutant and with Jagged-1 revealed increased levels for VEGF. Concomitantly, recombinant Jagged-1 increased sprouting of endothelial cells in the spheroid assay. Conclusion: The Notch pathway most likely regulates neurovascular development in pancreatic cancer. Activation of this signaling pathway by constitutive Notch-1 mutants and by Jagged-1 causes an angiogenic and invasive tumor phenotype. Specific blockade of Notch signaling may therefore be beneficial for patients with pancreatic cancer.

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-β1 (TGF-β1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-β1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation Emilie Seydoux,1,2 Barbara Rothen-Rutishauser,1,3 Izabela M Nita,1 Sandor Balog,3 Amiq Gazdhar,1 Philip A Stumbles,4,5 Alke Petri-Fink,3,6 Fabian Blank,1,* Christophe von Garnier1,*1Department of Respiratory Medicine, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; 3Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland; 4School of Veterinary and Life Sciences, Molecular and Biomedical Sciences, Murdoch University, Perth, WA, Australia; 5Telethon Kids Institute, Perth, WA, Australia; 6Department of Chemistry, University of Fribourg, Fribourg, Switzerland*These authors contributed equally to the manuscript Introduction: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods: Bone marrowâ??derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4+ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results: The frequency of PS particleâ??positive CD11c+/CD11b+ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4+ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion: These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4+ T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles. Keywords: polystyrene particles, nanoparticles, immune modulation, mouse dendritic cells, CD4+ T-cells

Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic lung disease, resulting in respiratory insufficiency and reduced survival. Pulmonary fibrosis is a result of repeated alveolar epithelial microinjuries, followed by abnormal regeneration and repair processes in the lung. Recently, stem cells and their secretome have been investigated as a novel therapeutic approach in pulmonary fibrosis. We evaluated the potential of induced pluripotent stem cells (iPSC) conditioned media (iPSC-cm) to regenerate and repair the alveolar epithelium in vitro and improve bleomycin induced lung injury in vivo. Methods: IPSC-cm was collected from cultured iPSC derived from human foreskin fibroblasts and its biological effects on alveolar epithelial wound repair was studied in an alveolar wound healing assay in vitro. Furthermore, iPSC-cm was intratracheally instilled 7 days after bleomycin induced injury in the rat lungs and histologically and biochemically assessed 7 days after instillation. Results: iPSC-cm increased alveolar epithelial wound repair in vitro compared with medium control. Intratracheal instillation of iPSC-cm in bleomycin-injured lungs reduced the collagen content and improved lung fibrosis in the rat lung in vivo. Profibrotic TGFbeta1 and α-smooth muscle actin (α-sma) expression were markedly reduced in the iPSC-cm treated group compared with control. Antifibrotic hepatocyte growth factor (HGF) was detected in iPSC-cm in biologically relevant levels, and specific inhibition of HGF in iPSC-cm attenuated the antifibrotic effect of iPSC-cm, indicating a central role of HGF in iPSC-cm. Conclusion: iPSC-cm increased alveolar epithelial wound repair in vitro and attenuated bleomycin induced fibrosis in vivo, partially due to the presence of HGF and may represent a promising novel, cell free therapeutic option against lung injury and fibrosis.