Experimental Lung Research最新文献

Signal disruptions in small animals during the realization of the Forced Oscillation Technique are a well-known cause of data loss as it leads to non-reliable estimations of the respiratory impedance. In this work, we assessed the effects of removing the disrupted epoch when a 3-seconds input signal composed of one and a half 2-seconds full cycle is used.

We tested our hypothesis in 25 SAMR1 mice under different levels of bronchoconstriction due to methacholine administration by iv bolus injections in different doses (15 animals) and by iv continuous infusion in different infusion rates (10 animals). Signal disruptions were computationally simulated as sharp drops in the pressure signal within a short timescale, and signal processing was performed using own developed algorithms.

We found that the model goodness of fit worsens when averaging techniques to estimate the input respiratory impedance are not used. However, no statistically significant differences were observed in the comparison between Constant Phase Model parameters of the full 3-s signal and the 2-s non disrupted epoch in all doses or infusion rates for both methacholine delivery strategies.

The proposed technique presents reliable outcomes that can reduce animal use in Forced Oscillation Technique realization.

Background: Excessive macrophage-mediated inflammation participates in the development of Staphylococcus aureus (S. aureus)-induced pneumonia. Checkpoint kinase 2 (Chek2) was screened out as macrophage-related infantile pneumonia gene after the differentially expressed analysis of RNAseq data derived from pam3CSK4 stimulated bone marrow-derived macrophages (BMDMs).

Methods: RAW264.7 macrophage cells were transfected with Chek2-specific gRNA, which were further overexpressed with wide-type Chek2 or Chek2 kinase activity mutant (Chek2 KD, D368N). At the same time, the relative protein and mRNA expression of inflammatory cytokines were determined. C57BL/6J WT mice were intranasally infected with S. aureus to induce S. aureus-induced pneumonia, which was treated with BML-277, an inhibitor of Chek2. The symptoms of pneumonia mice and inflammatory cytokines associated with the nuclear factor kappa B (NF-κB) signaling pathways were further examined.

Results: In vivo, BML-277 significantly promoted pneumonia symptoms, including mortality, lung infiltration of immune cells, and the abundance of lung pro-inflammatory cytokines. Mechanically, BML-277 did not affect BMDMs survival but up-regulated the mRNA expression of tumor necrosis factor (Tnf), nitric oxide synthase 2 (Nos2), interleukin (Il)23a, and the secretion of Tnf-α and Il-23a. At the same time, genetic complementation experiment testified that Chek2 KD did not inhibit NF-κB and relevant inflammatory cytokines expression.

Conclusion: Chek2 functions through the kinase mechanism to down-regulate the NF-κB pathway in macrophages to alleviate S. aureus-induced pneumonia in mice.

Purpose/Aim: In the context of asthma, airway bronchial remodeling and angiogenesis in the bronchial mucosa are well established. Cyclopeptidic-vascular endothelial growth inhibitor (cyclo-VEGI) is an inhibitor of the vascular endothelial growth factor (VEGF) receptor that increases the proliferation of endothelial cells and the formation of new vessels. However, changes in the bronchial arteries of patients with asthma have not been clearly elucidated. We investigated whether structural changes occurred in bronchial arteries, as well as the effects of cyclo-VEGI in a mouse model of chronic asthma (in vivo) and human fibroblasts (in vitro). Materials and Methods: A validated mouse model of allergic airway inflammation with ovalbumin (OVA) as the causative allergen was used for the study. Mice were treated with cyclo-VEGI or fluticasone during OVA challenge. In vitro experiments were conducted to determine whether fibroblasts proliferated following elastin exposure and the effects of cyclo-VEGI on them. Results: OVA sensitization and challenge led to greater perivascular smooth muscle area, more elastic fibers, and elevated expression of vascular cell adhesion molecule (VCAM)-1 antigen. These phenomena indicated changes to bronchial arteries. Cyclo-VEGI and fluticasone treatment both inhibited airway hyper-responsiveness and inflammation. Cyclo-VEGI-treated mice exhibited decreased perivascular smooth muscle area, elastin fibers, and VCAM-1 expression. Fluticasone-treated mice exhibited reductions in perivascular smooth muscle but not in perivascular elastin or VCAM-1 expression. In vitro, fibroblast proliferation was enhanced by elastin treatment, which was inhibited by cyclo-VEGI treatment. Eotaxin expression was elevated in elastin-treated fibroblasts and decreased with cyclo-VEGI treatment. Conclusions: Vascular remodeling occurred in our mouse model of chronic asthma. Cyclo-VEGI could reduce airway inflammation and hyper-responsiveness by inhibiting VCAM-1 expression and elastin deposition around the bronchial arteries.

Tobacco consumption is related to an increased risk to develop tuberculosis. Antimicrobial peptides are essential molecules in the response to Mycobacterium tuberculosis (Mtb) because of their direct antimicrobial activity. The aim of this study was to demonstrate that nicotine enters into Mtb infected epithelial cells and associates with the mycobacteria inducing genes related to antimicrobial peptides resistance. Epithelial cells were infected with virulent Mtb, afterwards cells were stimulated with nicotine. The internalization of nicotine was followed using electron and confocal microscopy. The lysX expression was evaluated isolating mycobacterial RNA and submitted to RT-PCR analysis. Our results indicated that nicotine promotes Mtb growth in a dose-dependent manner in infected cells. We also reported that nicotine induces lysX expression. In conclusion, nicotine associates to intracellular mycobacteria promoting intracellular survival.

Purpose: Subjects with obstructive sleep apnea (OSA) exhibit systemic and upper airway oxidative stress and inflammation, which cause mitochondrial dysfunction. The intend of this study is to estimate mitochondrial function (mitochondrial DNA/nuclear DNA [Mt/N] ratio) and protein levels of peroxisome proliferator-coactivated receptor gamma co-activator 1-alpha (PGC1-α) in the exhaled breath condensate (EBC) and plasma before and after continuous positive airway pressure (CPAP) treatment. Materials and methods: Twenty healthy individuals (control) and 40 subjects with severe or moderate OSA were recruited to undergo CPAP treatment and evaluation in a sleep study. The Mt/N ratio in the EBC and blood were assayed by quantitative real-time polymerase chain reaction. Enzyme-linked immunosorbent assay was used to measure the protein concentration of PGC1-α in the EBC and plasma. All experiments were performed after 3 months of CPAP treatment in subjects with OSA. Results: We observed no noteworthy differences between the control and treatment groups. Moreover, there were no differences in the Mt/N ratio in the blood and plasma levels of PGC1-α in subjects with OSA before and after treatment. However, the Mt/N ratio and protein levels of PGC1-α in the EBC of OSA subjects were higher than those in the control group and returned to normal levels after CPAP treatment. Conclusions: We successfully treated subjects with OSA by CPAP, which restored the Mt/N ratio and levels of PGC1-α in the EBC.

Transforming growth factor beta 1 (TGF-β1) induces pulmonary fibrosis by enhancing epithelial apoptosis and affects the enzymatic activity of transglutaminase 2 (TG2). The aim of this study was to determine the role of TG2 in TGF-β1-induced lung remodeling and alveolar macrophage modulation. We characterized the in vivo effects of TGF-β1 and TG2 on lung inflammation, fibrosis, and macrophage activity using transgenic C57BL/6 mice with wild and null TG2 loci. The effect of TG2 inhibition on in vitro TGF-β1-stimulated alveolar macrophages was assessed through mRNA analysis. TG2 was remarkably upregulated in the lungs of TGF-β1 transgenic (TGF-β1 Tg) mice, especially in alveolar macrophages and epithelial cells. In the absence of TG2, TGF-β1-induced inflammation was suppressed, decreasing the number of macrophages in the bronchoalveolar lavage fluid. In addition, the alveolar destruction and peribronchial fibrosis induced by TGF-β1 overexpression were significantly reduced, which correlated with decreases in the expression of fibroblast growth factor and matrix metallopeptidase 12, respectively. However, TG2 deficiency did not compromise the phagocytic activity of alveolar macrophages in TGF-β1 Tg mice. At the same time, TG2 contributed to the regulation of TGF-β1-induced macrophage activation. Inhibition of TG2 did not affect the TGF-β1-induced expression of CD86, an M1 marker, in macrophages, but it did reverse the TGF-β1-induced expression of CD206. This result suggests that TG2 mediates TGF-β1-induced M2-like polarization but does not contribute to TGF-β1-induced M1 polarization. In conclusion, TG2 regulates macrophage modulation and plays an important role in TGF-β1-induced lung inflammation, destruction, and fibrosis.

Although several studies demonstrate the anti-inflammatory effect of oxytocin in different pathophysiological processes, there are limited data describing the impact of oxytocin on acute respiratory distress syndrome (ARDS). We aimed to elucidate the protective effect of oxytocin in ARDS with histopathological evaluation and radiological imaging in addition to biochemical markers.

Fecal intraperitoneal injection procedure (FIP) was performed on 24 of 32 rats included in the study for creating a sepsis model. Rats were randomly assigned into four groups: control group (no procedure was applied, n = 8), untreated septic group [was operated (FIP) and received no treatment, n = 8], placebo group (FIP, treated with 10 ml/kg of saline at once, n = 8), and treated group (FIP, treated with 0.1 mg/kg of oxytocin at once, n = 8). Chest CT was performed for all rats 20 hours after the procedure and density of the lungs were measured manually by using HU. All animals were sacrificed for histopathological examination of lung damage and blood samples were collected for biochemical analysis.

Plasma malondialdehyde (MDA), lactic acid (LA), C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL 1-β) levels were significantly increased in the placebo (FIP + saline) and the untreated (FIP) groups, and plasma levels of all biomarkers were reversed by oxytocin. Further, the density of the lung parenchyma (Hounsfield unit) on CT images and the histopathological lung damage score values were closer to the control group in the oxytocin-treated group compared to the placebo group.

Our findings suggested that oxytocin could exert anti-inflammatory, antioxidant and protective effects in FIP-induced ARDS.

Current gene therapy delivery protocols for small animal lungs typically utilize indirect dose delivery via the nasal airways, or bolus delivery directly into the trachea. Both methods can result in variable transduction throughout the lung, as well as between animals, and cannot be applied in a targeted manner. To minimize variability and improve lung coverage we previously developed and validated a method to visualize and dose gene vectors into pre-selected lobes of rat lungs using a mini-bronchoscope. Lentiviral (LV) vectors are known to be fragile and can be inactivated easily by temperature or the application of shear stresses. There are several ways that the bronchoscope could be configured to deliver the LV vector, and these could result in different amounts of functional LV vector being delivered to the lung. This study evaluated several methods of LV vector delivery through the bronchoscope, and how flow rates and LV vector stabilizing diluents impact LV vector delivery. NIH-3T3 cells were exposed to LV vector containing the green fluorescent protein (GFP) reporter gene using various bronchoscopic delivery techniques and the number of GFP-positive cells produced by each was quantified by flow cytometry. The results showed that directly drawing the LV vector into the bronchoscope tip resulted in 80-90% recovery of viable vector, and was also the simplest method of delivery. The fluid delivery rate and the use of stabilizing serum in the vector diluent had no effect on the viability of the LV vector delivered. These findings can be used to optimize LV vector dose delivery into individual lung lobes of small animal models.

Aim of the Study: Many allergens have protease activities. Although the immunomodulatory effects of these antigens are well known, the effects attributed to their protease activities are not thoroughly investigated. We set out to determine the effects of house dust mite (HDM) allergens with varying protease activities on bronchial epithelial cell functions. Materials and methods: BEAS-2B cells were maintained in ALI-culture and stimulated with Der p1 (cysteine protease), Der p6 (serine protease), and Der p2 (non-protease) with and without specific protease inhibitors or heat denaturation. Cell viability and epithelial permeability were measured with MTT and paracellular flux assay, respectively. The effect of heat denaturation on allergen structure was examined using in silico models. Matrix metalloproteinases (MMPs) were investigated at the transcription (qPCR), protein (ELISA), and functional (zymography) levels. Results: Epithelial permeability increased only after Der p6 but not after Der p1 or Der p2 stimulation. Der p2 increased both MMP-2 and MMP-9 expression, while Der p1 increased only MMP-9 expression. The heat-denatured form of Der p1 unexpectedly increased MMP-9 gene expression, which, through the use of in silico models, was attributed to its ability to change receptor connections by the formation of new electrostatic and hydrogen bonds. IL-8 and GM-CSF production were increased after Der p1 and Der p2 but decreased after Der p6 stimulation. IL-6 decreased after Der p1 but increased following stimulation with Der p6 and heat-denatured Der p2. Conclusion: Allergens in house dust mites are capable of inducing various changes in the epithelial cell functions by virtue of their protease activities.

Purpose: IL-33 is known to induce corticosteroid-resistant eosinophilic inflammation and airway remodeling by activating type 2 innate lymphoid cells (ILC2s). Although the RAS signal pathway plays an important role in IL-33-induced ILC2s activation and airway remodeling, it is not known if RAS inhibitors are effective against refractory asthma. We examined the effects of the RAS inhibitor XRP44X in refractory asthma. Methods: RAS activity were examined by BAL fluid and T-cells isolated from spleen cells in Dermatophagoides pteronyssinus (Dp)-sensitized/challenged acute allergic airway inflammation model. A chronic allergic airway inflammation mouse model was generated by challenged with Dp. XRP44X and/or fluticasone were administrated nasally to different experimental groups. The effects of nasal simultaneous administration of XRP44X or fluticasone were assessed in mice administrated with IL-33 or Dp. Results: RAS activity in CD4⁺ T cells stimulated by Dp were suppressed by XRP44X. Although fluticasone and XRP44X only improved allergic airway inflammation in mice, XRP44X in combination with fluticasone produced further improvement in not only eosinophilic inflammation but also bronchial subepithelial thickness. XRP44X suppressed IL-5 and IL-13 production from ILC2s, although this effect was not suppressed by fluticasone. IL-33-induced airway inflammation resistant to fluticasone was ameliorated by XRP44X via regulating the accumulation of lung ILC2s. Conclusion: The RAS signal pathway plays a crucial role in allergen-induced airway remodeling associated with ILC2s. XRP44X may have therapeutic potential for refractory asthma.