Bronchoalveolar Lavage Fluid Supernatant Research Articles

Application of dexmedetomidine for lung injury in elderly patients undergoing one-lung ventilation.

This study aimed to evaluate the effects of dexmedetomidine (DEX) on lung injury, the oxygenation index and perioperative pulmonary complications in elderly patients who underwent thoracotomy with one-lung ventilation (OLV). A total of 120 elderly patients with lung cancer were included in the present study. According to the random number table method, these patients were randomly divided into two groups: group D and group C. Patients in group D were intravenously pumped with 0.5 μg/kg/h of DEX before anesthesia. The infusion was completed within 15 min, and anesthesia was induced by venous injection. Patients in group C were pumped with equal volumes of normal saline. At T2 and T3, compared with group C, group D had a significant decrease in cardiac index, mean arterial pressure and central venous pressure (p < 0.05). At T2, T3 and T4, compared with group C, group D had a significant increase in pH and PaO2 (p < 0.05). At T2, T3 and T4, compared with group C, group D had a significant decrease in Qs/Qt (p < 0.05). At T6, compared with group C, group D had a significant decrease in the supernatant of bronchoalveolar lavage fluid of tumor necrosis factor-α and interleukin 6 (p < 0.05). At T5, compared with group C, group D had a significant decrease in Visual Analogue Scale score (p < 0.05), and a significant increase in Ramsay Sedation Scale score (p < 0.05), and the number of respiratory and cardiovascular events also decreased (p < 0.05). In elderly patients, dexmedetomidine can reduce Qs/Qt and increase PaO2 during OLV in surgery. It can reduce lung injury. Moreover, DEX reduced respiratory and cardiovascular complications in the perioperative period.

Geranylgeranyl diphosphate synthase 1 knockout ameliorates ventilator-induced lung injury via regulation of TLR2/4-AP-1 signaling

ObjectiveTo investigate the role of geranylgeranyl diphosphate synthase 1 (GGPPS1) in ventilator-induced lung injury along with the underlying mechanism. MethodsA murine VILI model was induced by high-tidal volume ventilation in both wild-type and GGPPS1 knockout mice. GGPPS1 expression was detected in the bronchoalveolar lavage fluid (BALF) supernatants of acute respiratory distress syndrome (ARDS) patients and healthy volunteers, as well as in lung tissues and BALF supernatants of the VILI mice using enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), western bolt and immunohistochemical (IHC). The wet/dry ratio, total BALF proteins, and lung injury score were analyzed. The percentage of neutrophils was detected by flow cytometry and IHC. Inflammatory cytokine levels were measured by ELISA and qRT-PCR. The related expression of Toll-like receptor (TLR)2/4 and its downstream proteins was evaluated by western blot. ResultsGGPPS1 in BALF supernatants was upregulated in ARDS patients and the VILI mice. Depletion of GGPPS1 significantly alleviated the severity of ventilator induced lung injury in mice. Total cell count, neutrophils and inflammatory cytokines (interleukin [IL]-6, IL-1β, IL-18 and tumor necrosis factor-α) levels in BALF were reduced after GGPPS1 depletion. Moreover, addition of exogenous GGPP in GGPPS-deficient mice significantly exacerbated the severity of ventilator induced lung injury as compared to the PBS treated controls. Mechanistically, the expression of TLR2/4, as well as downstream proteins including activator protein-1 (AP-1) was suppressed in lung tissues of GGPPS1-deficient mice. ConclusionGGPPS1 promoted the pathogenesis of VILI by modulating the TLR2/4–AP-1 signaling pathway, and GGPPS1 knockout significantly alleviated the lung injury and inflammation in the VILI mice.

A novel chalcone derivative exerts anti-inflammatory and anti-oxidant effects after acute lung injury.

We explored the effects of compound 33, a synthetic chalcone derivative with antioxidant activity, on lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compound 33, dexamethasone or vehicle was administered intragastrically to mice 6 h before intratracheal instillation of LPS. After 24 h, the effects of compound 33 on alveolar structural damage were evaluated by assessing lung morphology and the wet/dry weight ratio. Protein and proinflammatory cytokine levels and superoxide dismutase activity were also examined in the cell free supernatant of bronchoalveolar lavage fluid. Additionally, we investigated the anti-inflammatory and antioxidant activity of compound 33 in vitro and its effects on the MAPK/NF-κB and Nrf2/HO-1 pathways. Pretreatment with compound 33 prevented LPS-induced structural damage, tissue edema, protein exudation, and overproduction of proinflammatory mediators. The effects of compound 33 were similar to or greater in magnitude than those of the positive control, dexamethasone. Moreover, compound 33 exerted anti-inflammatory and antioxidant effects in vitro by inhibiting the MAPK/NF-κB pathway and activating the Nrf2/HO-1 pathway. Compound 33 may therefore be a promising candidate treatment for ALI.

Proteomic analysis of bronchoalveolar lavage fluid in rat exposed to TiO2 nanostructured aerosol by inhalation

The pulmonary toxicological properties of inhaled titanium dioxide were studied using bronchoalveolar lavage fluid (BALF) cytology and proteomics analyses. Fischer 344 rats were exposed to 10 mg/m3 of TiO2 nanostructured aerosol by nose-only inhalation for 6 h/day, 5 days/week for 4 weeks. Lung samples were collected up to 180 post-exposure days. As previously described, cytological analyses of BALF showed a strong inflammatory response up to 3 post-exposure days, which persisted however, at a lower intensity up to 180 days. In addition, using Multidimensional Protein Identification Technology (MudPIT), we identified a total of 107, 50 and 45 proteins (UniprotKB identifiers) differentially expressed in exposed rats immediately, 3 and 180 days after the end of exposure respectively. Increased levels of inflammatory proteins, members of proteasome, various histones, proteins involved in cytoskeleton organization, were noticed up to 3 days (short-term response). Some of these proteins were linked with Neutrophil Extracellular Trap formation (NETosis). Long-term response was also characterized by a persistent altered expression of proteins up to 180 days. Altogether, these results suggest that exposure to low toxicity low solubility nanomaterials such as TiO2 may induce long-term changes in the pulmonary protein expression pattern of which the physio-pathological consequences are unknown. SignificanceThis paper describes in rats, at the pulmonary level, the effects of inhaled nanostructured aerosol of TiO2 on the secreted proteins found in the broncho-alveolar space by comparing the proteomic profile in broncho-alveolar lavage fluid supernatants of control and exposed animals. This work brings new insights about the early events occurring following the end of exposure and suggests the formation of Neutrophil Extracellular Traps (NETosis) that could be interpret as a potential early mechanism of defense against TiO2 nanoparticles. This work also describes the long term effects (180 post-exposure days) of such an exposure and the change in secreted protein expression in the absence of significant histopathological modifications.

Abstract 4463: Tumor-derived cell-free DNA from bronchoalveolar lavage fluid (BALF): A potential liquid biopsy analysis in lung cancer patients

Abstract Background: Bronchoalveolar lavage (BAL) is the most common technique for sampling the components of the alveolar space. Here, we evaluated the potential use of BAL fluid (BALF) in liquid biopsy in lung cancer. Methods: This study enrolled 56 lung cancer patients. And 57 BALF (separated supernatant and precipitate) samples and 56 peripheral blood lymphocytes samples were collected. 57 formalin-fixed Paraffin-embedded (FFPE) tissues were also obtain. We utilized a 1021-gene NGS panel in matched tissue DNA, BALF supernatant cfDNA and BALF precipitate DNA to identify somatic mutations with white blood cell DNA as a germline control. Results: Mutations were identified in 52 (~91.22%) BALF precipitate samples and 55 (~96.49%) BALF supernatant samples, comparing to 53 (92.98%) in tumor samples. EGFR mutations were observed in 22 BALF precipitate samples and 24 supernatant samples. Among these, 23 were also identified in matched tumor samples. One BALF precipitate samples specific and two BALF supernatant samples specific EGFR mutations were confirmed by Cobas assay. In addition, gene fusions including ALK, ROS1 and RET were found in 6 tumors, 4 BALF precipitate samples and 5 supernatant samples, respectively. In total, there were 195 mutations identified in the tumor samples, of which 147 (~75.4%) mutations were detected in the matched BALF precipitate samples, 168 (~86.1%) mutations were detected in the matched BALF supernatant samples and 145 (~74.4%) mutations were shared in three types of samples. Tumor mutation burden (TMB) were calculated, and the consistency between tissue and BALF precipitate was 90% and the consistency between tissue and BALF supernatant was 94%. Conclusions: Liquid biopsy using BALF shown high potential to profiling genetic alterations of patients with lung cancer, which might be implemented and standardized into clinical use. Citation Format: Yuhua Gong, Xinyu Zhang, Chun Li, Weiran Wang, Maosong Ye, Yancheng Zhao, Xin Yi, Yaping Xu, Qin Hu, Yanfang Guan, Ling Yang, Xuefeng Xia, Min Zhou, Jian'an Huang, Hua Zhang, Tao Ren, Qian Shen, Kai Wang, Yingyong Hou, Xin Zhang. Tumor-derived cell-free DNA from bronchoalveolar lavage fluid (BALF): A potential liquid biopsy analysis in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4463.

Epigallocatechin gallate ameliorates airway inflammation by regulating Treg/Th17 imbalance in an asthmatic mouse model

Epigallocatechin gallate (EGCG) is a polyphenol that is found in green tea that has been shown to ameliorate airway inflammation in an ovalbumin-sensitized asthmatic mouse model. The purpose of this study was to investigate whether the immunomodulatory and anti-inflammatory effects of EGCG by regulating the regulatory T cell (Treg)/Th 17 cells balance in this model. Female BALB/c mice were sensitized and challenged with ovalbumin by intraperitoneal injection. EGCG was administered to asthmatic mice intraperitoneally 1 h before each OVA challenge. Airway hyperresponsiveness (AHR) was measured, and lung inflammatory infiltrations were assessed by hematoxylin and eosin (HE) staining. Serum OVA-specific IgE levels, Interleukin-10 (IL-10) levels and Interleukin-17A (IL-17A) levels in the bronchoalveolar lavage fluid (BALF), serum, and splenocyte culture supernatants were measured by ELISA. Flow cytometry was used to assess the effects of EGCG on the frequency of CD4+CD25+Foxp3+Treg cells in the splenocytes and real-time PCR method was used to measure the expression of Forkhead box P3 (Foxp3) mRNA and retinoid-related orphan receptor gammat (RORγt) mRNA in the lung tissue. The results showed that administration of EGCG significantly decreased AHR and OVA specific IgE in the serum, increased IL-10 levels in the BALF, serum, and splenocyte culture supernatant, and the frequency of CD4+CD25+Foxp3+Treg cells in the splenocytes in asthmatic mice. Administration of EGCG also ameliorated airway inflammation and eosinophil infiltrations in asthmatic mice. These results suggested that EGCG likely ameliorated OVA-induced airway inflammation by increasing the production of IL-10, the number of CD4+CD25+Foxp3+Treg cells and expression of Foxp3 mRNA in the lung tissue, and it could be an effective agent for treating asthma.

Dynamics of Ventilatory Pattern Variability (VPV), Cardio‐Respiratory Coupling (CRC) and Cytokines in an E. coli Sepsis Model

Cardio‐respiratory control is disrupted in sepsis. We hypothesized that increases in inflammatory cytokines that are expressed in the brainstem in a rat model of Escherichia coli (E. coli) peritonitis would correlate with decreases in VPV and the uncoupling of CRC. We implanted fibrin clots containing either 25×106 or 0 E. coli in the peritoneal cavity of chronically instrumented, adult Sprague‐Dawley Rats (n=19). We recorded cardiorespiratory patterns of awake, freely moving rats at 3, 6, 12, 24, and 48 hours after implantation. At each time point, blood, bronchoalveolar lavage fluid (BALF), peripheral and central nervous system (CNS) tissues were collected to measure cytokine concentrations. We quantified VPV and CRC using a set of complementary measures sensitive to both linear and nonlinear dynamics. We measured interleukin 1β (IL‐1b), interleukin 6 (IL‐6) and tumor necrosis factor α (TNF‐a) concentrations in serum, BALF and tissue supernatants by Luminex assay. Compared to the sham group, infected rats developed a systemic inflammatory response by 3h [increased serum IL‐6 (p&lt;0.05), IL‐1b (p=0.05)], which peaked at 6h and then decreased progressively. Also, brainstem inflammation was evident at 3h post inoculation, with increases in IL‐1β in the medulla (p&lt;0.01) and pons (p&lt;0.05). Brainstem inflammation was associated with increased predictability in VPV, indicated by increases in mutual information (p&lt;0.05) and nonlinear complexity index (NLCI). In our initial analysis of CVC (n=8 rats at 24h), the X2 values of the distribution of R‐wave timings relative to the onset of inspiration were highly significant but were not different between sham and septic groups. We conclude that decreases in VPV (i.e., increased pattern predictability) were present early in the course of systemic inflammation following E. coli infection in rats and these changes were associated with elevated serum and brainstem cytokines. In contrast, the persistence of CVC in the inoculated rats was unexpected; however, at 24h the NLCI was decreasing indicating that these rats may be recovering from the peritonitis.Support or Funding InformationVA Research Service (I01BX004197); NIH (U01 EB021960)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Correlation between galectin-3 level in bronchoalveolar lavage fluid and cellular immunity in children with refractory Mycoplasma pneumoniae pneumonia

To study the correlation of galectin-3 level in bronchoalveolar lavage fluid (BALF) with Mycoplasma pneumoniae (MP) load and cellular immunity of neutrophils and macrophages in the airway in children with refractory MP pneumonia (RMPP). A total of 64 children with RMPP who were hospitalized from January 2013 to January 2017 were enrolled. In addition to the conservative medical treatment, all the 64 children with RMPP were given bronchoalveolar lavage in the acute stage (5-7 days after admission) and 48 out of the 64 children were given bronchoalveolar lavage in the recovery stage (10-14 days after admission). Four milliliters of BALF of the affected lung lobe or segment were collected. ELISA was used to measure the level of galectin-3 in BALF supernatant. RT-PCR was used to measure MP load. Hematoxylin and eosin staining was used to measure the percentage of neutrophils and macrophages. Six children with bronchial foreign bodies were enrolled as the control group. The RMPP group had a significantly higher level of galectin-3 in BALF in both the acute and recovery stages than the control group (P<0.01), and the level of galectin-3 in the acute stage was significantly higher than in the recovery stage (P<0.01). The RMPP group had a significantly higher percentage of neutrophils in BALF in both the acute and recovery stages than the control group (P<0.01), and the percentage of neutrophils in the acute stage was significantly higher than in the recovery stage (P<0.01). The RMPP group had a significantly lower percentage of macrophages in BALF in both the acute and recovery stages than the control group (P<0.01), but there was no significant difference in the percentage of macrophages between the acute and recovery stages (P>0.05). The RMPP group had a significantly higher MP load in BALF in both the acute and recovery stages than the control group (P<0.01), and the MP load in the acute stage was significantly higher than in the recovery stage (P<0.01). In the children with RMPP, galectin-3 level in BALF in the acute stage was positively correlated with MP load and the percentage of neutrophils (rs=0.789 and 0.726 respectively; P<0.01). Galectin-3 is involved in the process of airway inflammation in children with RMPP, and the level of galectin-3 in BALF is positively correlated with MP load. RMPP is a cellular immune inflammatory lesion with the increase of neutrophils and the reduction in macrophages. Galectin-3 is closely associated with neutrophil chemotaxis and luminal infiltration in children with RMPP. MP load gradually decreases with the recovery from RMPP, but it is not completely eliminated by the immune system in the recovery stage. MP infection can increase the consumption of macrophages in children with RMPP.

Calpain 1 in bronchoalveolar lavage fluid is associated with poor prognosis in lepidic predominant pulmonary adenocarcinoma

Calpain 1 is a proinflammatory calcium-activated cysteine protease, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the externalization of calpain 1 and the release of soluble TLR2 during tumor progression of pulmonary lepidic predominant adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry and soluble TLR2 by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (P=0.003). TLR2 was expressed on PMN and tumor cells and decreased after calpain exposure. Soluble fragment of TLR2 in BALF supernatants was correlated to the extracellular calpain 1 concentration (r=0.624; P<0.001), and its high level was associated with tumor progression and a pro-inflammatory environment. Extracellular calpain 1 secreted by tumor cells, could participate in inflammatory microenvironment and tumor progression through TLR2 in LPA.

Siraitia grosvenorii residual extract attenuates ovalbumin-induced lung inflammation by down-regulating IL-4, IL-5, IL-13, IL-17, and MUC5AC expression in mice

BackgroundSiraitia grosvenorii fruits are used in traditional medicine to treat cough, sore throat, bronchitis, and asthma. PurposeThis study aimed to investigate the anti-inflammatory and anti-asthmatic effects of S. grosvenorii residual extract (SGRE) on ovalbumin (OVA)-induced asthma in mice. MethodsAsthma was induced in BALB/c mice by systemic sensitization to OVA, followed by intratracheal, intraperitoneal, and aerosol allergen challenges. SGRE was orally administered for four weeks. We investigated the effects of SGRE on airway hyper-responsiveness, OVA-specific IgE production, histological analysis of lung and trachea, immune cell phenotyping, Th1/Th2 cytokine production in bronchoalveolar lavage fluid (BAL) fluid and splenocytes, and gene expression in the lung. ResultsSGRE ameliorated OVA-driven airway hyper-responsiveness, serum IgE production, and histopathological changes in the lung and trachea. SGRE reduced the total number of cells in the lung and BAL, the total number of lymphocytes, neutrophils, monocytes, and eosinophils in the lung and BAL, the absolute number of CD4+/CD69+ T cells in the lung, and the absolute number of CD4+/CD8+ T cells and CD11b+/Gr-1+ granulocytes in the lung and BAL. SGRE also reduced Th2 cytokines (IL-4, IL-5, and IL-13) and increased the Th1 cytokine IFN-γ in the BAL fluid and supernatant of splenocyte cultures. SGRE decreased the OVA-induced increase of IL-13, TARC, MUC5AC, TNF-α, and IL-17 expression in the lung. ConclusionSGRE exerts anti-asthmatic effects via the inhibition of Th2 and Th17 cytokines and the increase of Th1 cytokines, suggesting that SGRE may be a potential therapeutic agent for allergic lung inflammation, such as asthma.

Novel Application of Aptamer Proteomic Analysis in Cystic Fibrosis Bronchoalveolar Lavage Fluid.

Biomarkers are needed in cystic fibrosis (CF) to understand disease progression, assess response to therapy, and enrich enrollment for clinical trials. Aptamer-based proteomics have proven useful in blood samples. The aim is to evaluate proteins in bronchoalveolar lavage fluid (BALF) in CF children compared to controls and identify endotypes during CF exacerbations. BALF is collected clinically from 50 patients with CF and nine disease controls, processed, and stored per protocol. BALF supernatants are analyzed for 1129 proteins by aptamer approach (SOMAscan proteomics platform). Proteins are compared across groups and used for pathway analysis. Endotypes are identified within the CF group. CF BALF has increased concentrations of neutrophil elastase, myeloperoxidase, and decreased concentration of protein folding and host defense proteins. Pathways that distinguished CF subjects included interferon gamma signaling, membrane trafficking, and phospholipid metabolism. In the CF group, unbiased analysis of proteins identified two distinct endotypes that differed based on BALF white blood cell and neutrophil counts and detection of CF pathogens. Proteomic analysis of the CF airway demonstrates a complex environment of proteins and pathways. This work provides evidence that aptamer-based proteomics can differentiate between groups and can determine endotypes within CF.

Increased levels of interleukin 27 in patients with early clinical stages of non-small cell lung cancer.

INTRODUCTION Interleukin 27 (IL‑27) is a cytokine secreted mostly by antigen‑presenting cells. It is important for the immune polarization of T helper‑1 (Th1) cells, and its role in interstitial lung diseases (ILDs) and lung cancer has been investigated. OBJECTIVES We assessed IL‑27 expression in the lower airways of patients with selected ILDs and early‑stage non-small cell lung cancer (NSCLC). PATIENTS AND METHODS IL‑27 concentrations were examined by an enzyme‑linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid supernatants collected from patients with pulmonary sarcoidosis (PS; n = 30), extrinsic allergic alveolitis (EAA; n = 14), idiopathic pulmonary fibrosis (IPF; n = 12), nonspecific interstitial pneumonia (NSIP; n = 14), and NSCLC stages I to IIa (n = 16) with peripheral localization, and in controls (n = 14). The major lymphocyte subsets in BAL fluid were phenotyped, and intracellular IL‑27 expression was evaluated by flow cytometry. RESULTS IL‑27 concentrations in BAL fluid supernatants were significantly increased in Th1‑mediated conditions such as EAA and PS, but not in IPF or NSIP. The highest IL‑27 levels (median [SEM], 16.9 [17.5] pg/ml) were reported for NCSLC, and the lowest-for controls (median [SEM], 0.4 [0.2] pg/ml). IL‑27 was undetectable in corticosteroid‑treated patients with PS. Both CD4+ and CD8+ lymphocytes were positive for IL‑27; they were a possible local source of IL‑27 because the cytokine levels were positivelysignificantly correlated with the total number of lymphocytes, including CD4+ cells. CONCLUSIONS Our results support the Th1‑linked activity of IL‑27in ILDs. Early‑stageNSCLC is characterizedby high IL‑27expression in the lower airways. IL‑27 is produced by a high percentage of CD4+ and CD8+ cells in BAL fluid, both in patients and controls.

Evaluation of oxidative stress in caprine bronchoalveolar lavage fluid of pneumonic and normal lungs

Research in the area of oxidative stress in pneumonic pathology still requires attention in small ruminants especially with the use of bronchoalveolar lavage (BAL) which may be a more sensitive indicator of respiratory diseases than blood. This investigation evaluates the role of oxidative stressin the pathogenesis of caprine pneumonia using BAL fluid (BALf) from healthy and pneumonic goats. A BALf from 192 goats (whose pneumonic histopathology had been characterized using standard techniques) was biochemically assayed for anti-oxidants and pro-oxidants. Malondialdehyde (MDA), hydrogen peroxide generation (H2O2), myeloperoxidase (MPO) and reduced glutathione (GSH) contents were measured to assess free radical activity in the BALf. Superoxide dismutase (SOD), Glutathione transferase (GST) and Glutathione peroxidase (GPx) activity were also determined colourimetrically. There were significant increases in the BALf supernatant of MDA, H2O2 and MPO with decreases in GSH level and SOD activity in the pneumonic goats (P<0.05). There was also significant correlation of BALf oxidative assay to the type and severity of pneumonia. The levels of MDA, H2O2, and MPO increased significantly (P<0.05) in bronchopneumonia and bronchointerstitial pneumonia than other pneumonic conditions and normal lungs. The management of caprine pneumonia should often incorporate antioxidant supplementation to correct the imbalance in pro-oxidant and anti-oxidant levels.

Lung microbiome and host immune tone in subjects with idiopathic pulmonary fibrosis treated with inhaled interferon-γ

Therapies targeting inflammation reveal inconsistent results in idiopathic pulmonary fibrosis (IPF). Aerosolised interferon (IFN)-γ has been proposed as a novel therapy. Changes in the host airway microbiome are associated with the inflammatory milieu and may be associated with disease progression. Here, we evaluate whether treatment with aerosolised IFN-γ in IPF impacts either the lower airway microbiome or the host immune phenotype.Patients with IPF who enrolled in an aerosolised IFN-γ trial underwent bronchoscopy at baseline and after 6 months. 16S rRNA sequencing of bronchoalveolar lavage fluid (BALF) was used to evaluate the lung microbiome. Biomarkers were measured by Luminex assay in plasma, BALF and BAL cell supernatant. The compPLS framework was used to evaluate associations between taxa and biomarkers.IFN-γ treatment did not change α or β diversity of the lung microbiome and few taxonomic changes occurred. While none of the biomarkers changed in plasma, there was an increase in IFN-γ and a decrease in Fit-3 ligand, IFN-α2 and interleukin-5 in BAL cell supernatant, and a decrease in tumour necrosis factor-β in BALF. Multiple correlations between microbial taxa common to the oral mucosa and host inflammatory biomarkers were found.These data suggest that the lung microbiome is independently associated with the host immune tone and may have a potential mechanistic role in IPF.

Insulin Modulates Cytokine Release, Collagen and Mucus Secretion in Lung Remodeling of Allergic Diabetic Mice.

The role of insulin in lung remodeling in a model of asthma in healthy and diabetic mice was evaluated. Diabetic male BALB/c mice (alloxan, 50 mg/kg, intravenous) and controls were sensitized by subcutaneous (s.c.) injection of ovalbumin (OA, 20 µg) in aluminum hydroxide (Al(OH)3, 2 mg) 10 days after the alloxan injection and received the same dose 12 days later. Six days after the last sensitization, animals were nebulized with OA solution for 7 days. The first set of diabetic and control mice received 2 and 1 IU, respectively, of s.c. neutral protamine Hagedorn (NPH) insulin and were analyzed 8 h later. The second set of diabetic and control mice received 2 and 1 IU, respectively, of insulin 12 h before the OA challenge and half doses of insulin 2 h before each the seven OA challenges. Twenty-four hours after the last challenge, the following analyses were performed: (a) quantification of the cells in the bronchoalveolar lavage fluid (BALF), the white cell count, and blood glucose; (b) morphological analysis of lung tissues by hematoxylin and eosin staining; (c) quantification of collagen deposition in lung tissues and mucus by morphometric analysis of histological sections stained with Masson's trichrome and periodic acid-Schiff (PAS), respectively; and (d) quantification of the cytokine concentrations (IL-4, IL-5, and IL-13) in the BALF supernatant. Compared to controls, diabetic mice had significantly reduced inflammatory cells (81%) in the BALF, no eosinophils in the BALF and peripheral blood and reduced collagen deposition and mucus in the lungs. BALF concentrations of IL-4 (48%) and IL-5 (31%) decreased and IL-13 was absent. A single dose of insulin restored peripheral blood eosinophils and BALF mononuclear cells but not BALF eosinophils, collagen deposition, and mucus levels. However, multiple doses of insulin restored both total cells and eosinophils in the BALF and peripheral blood, BALF cytokines, and collagen deposition and mucus secretion into the lungs. The results suggest that insulin modulates the production/release of cytokines, cell migration, deposition of collagen, and mucus secretion in lung remodeling of a mouse model of asthma.

JNK Inhibitor SP600125 Attenuates Paraquat-Induced Acute Lung Injury: an In Vivo and In Vitro Study.

Acute lung injury (ALI) is a major complication soon after paraquat poisoning and rapidly progresses with high mortality. However, the specific mechanism underlying paraquat-induced ALI is still unclear. In this study, the mechanism underlying the protective effects of SP600125 on paraquat-induced ALI was investigated according to oxidative stress, inflammation, and apoptosis. The rats were randomly assigned into the control group (CON), the paraquat poisoning group (PQ), and the PQ + SP600125 group (SP). A549 cells were divided into the Con group, Pq group, and Sp group. H&E staining and detection of lung wet/dry ratio were employed to evaluate lung injury. Annexin V-PI staining was done to evaluate A549 cell apoptosis. Dihydroethidium fluorescence was used to measure reactive oxygen species (ROS) in the lungs and A549 cells. ELISA was performed to detect TNF-α and IL-6 in the supernatant of bronchoalveolar lavage fluid (BALF) and A549 cells. RT-qPCR was done to measure the messenger RNA (mRNA) expression of TNF-α and IL-6 in the lungs and A549 cells. Western blotting assay was performed to detect the protein expression of phospho-JNK, total JNK, and cleaved caspase-3. Electrophoretic mobility shift assay was employed to detect the DNA binding activities of AP-1 and P-p65. JNK inhibitor SP600125 reduced JNK phosphorylation, downregulated cleaved caspase-3 protein level, decreased AP-1 transcriptional activity and ROS level, and reduced the transcription and expression of TNF-α and IL-6, which improved ALI and cell apoptosis after paraquat poisoning. Our results indicate that JNK/AP-1 mediates ALI as well as oxidative stress and inflammation deterioration secondary to paraquat poisoning.

The Natural Course of Atopic Dermatitis and the Association with Asthma.

In this paper, we aimed to explore the potential mechanism underlying atopic dermatitis (AD) and its association with asthma. The BALB/c mice were randomly assigned to three groups, including the vehicle control (VD) group, the AD group, and the treatment (TR) group. The AD mice model was successfully constructed in the AD and TR group. The dermatitis severity scores and skin lesions were significantly increased in AD mice after DNCB application. Airway responsiveness in the AD group was significantly higher than in the TR group. The number of inflammatory cells was increased in skin lesions and bronchoalveolar lavage fluid (BALF) of AD mice. The levels of IL-4, IL-5, IFN-γ, and OVA-IgE in BALF supernatants of mice in the AD group were higher than those in the VC group. All the changes in AD mice were decreased by tacrolimus. These results indicate that AD may be a significant risk factor for atopic asthma development.

Metalloproteinases and their inhibitors are influenced by inhalative glucocorticoid therapy in combination with environmental dust reduction in equine recurrent airway obstruction

BackgroundOverexpression of matrix-metalloproteinases (MMPs) has been shown to lead to tissue damage in equine recurrent airway obstruction (RAO), as a misbalance with their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), occurs. This favors irreversible pulmonary fibrosis formation. Increased levels of MMPs, TIMPs or altered ratios between them can be used as biomarkers of respiratory disease. We hypothesized that levels of MMPs, TIMPs and their ratios correlate with improvement in clinical findings and bronchoalveolar lavage fluid (BALF) cytology after 10 days of inhalative glucocorticoid therapy and environmental dust reduction (EDR) and may be used to monitor treatment success.Ten horses with a history of RAO participated in a prospective clinical study. Clinical and cytological scoring was performed before and after inhalative therapy using budesonide (1500 μg BID over 10 days) and EDR (bedding of wood shavings and wet hay as roughage). Gelatin zymography was performed for qualitative and semi-quantitative evaluation of MMP-2 and MMP-9 in BALF supernatant, while fluorimetry was used to evaluate MMP-8 activity. Additionally, specific equine ELISA assays were used for quantitative assessment of MMP-2, MMP-9, TIMP-1 and TIMP-2.ResultsA significant reduction in the total and several single parameters of the clinical score were found after 10 days of inhalative therapy and EDR. The concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 (ELISA) as well as their activities (MMP-2 and MMP-9 zymography and MMP-8 fluorimetry) were significantly decreased after therapy. Significant improvements in MMP-8/TIMP-1 and MMP-8/TIMP-2 ratios were also found, differences between other ratios before and after therapy were insignificant.ConclusionsMetalloproteinases and their inhibitors, in particular MMP-9 and TIMP-2, are valuable markers for clinical improvement in RAO.

Metabolomic biomarkers predictive of early structural lung disease in cystic fibrosis.

Neutrophilic airway inflammation plays a role in early structural lung disease in cystic fibrosis, but the mechanisms underlying this pathway are incompletely understood.Metabolites associated with neutrophilic inflammation were identified by discovery metabolomics on bronchoalveolar lavage fluid supernatant from 20 preschool children (2.9±1.3 years) with cystic fibrosis. Targeted mass-spectrometric detection of relevant metabolites was then applied to 34 children (3.5±1.5 years) enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) who underwent chest computed tomography and bronchoalveolar lavage from two separate lobes during 42 visits. Relationships between metabolites and localised structural lung disease were assessed using multivariate analyses.Discovery metabolomics identified 93 metabolites associated with neutrophilic inflammation, including pathways involved in metabolism of adenyl purines, amino acids and small peptides, cellular energy and lipids. In targeted mass spectrometry, products of adenosine metabolism, protein catabolism and oxidative stress were associated with structural lung disease and predicted future bronchiectasis, and activities of enzymes associated with adenosine metabolism were elevated in the samples with early disease.Metabolomics analyses revealed metabolites and pathways altered with neutrophilic inflammation and destructive lung disease. These pathways can serve as biomarkers and potential therapeutic targets for early cystic fibrosis lung disease.

Characteristics of Allergic Pulmonary Inflammation in CXCR3Knockout Mice Sensitized and Challenged with House Dust Mite Protein.

Chemokine C-X-C motif receptor 3 (CXCR3) is a chemokine receptor that is mainly expressed by activated T lymphocytes. T cells play important roles in allergic pulmonary inflammation, which is a hallmark of asthma and elicits the localized accumulation of activated T cells in the lung. In China, a marked increase in the incidence rate of chronic allergic pulmonary inflammation has made it a major public health threat. In the present study, we investigated the role of CXCR3 and its ligands in airway inflammation induced by house dust mite protein (HDMP) in a CXCR3 knockout (CXCR3KO) asthma mouse model. Pathological manifestations in the lung, cell counts and bronchoalveolar lavage fluid (BALF) classifications were studied using hematoxylin and eosin (H&E) staining. The levels of IL-4 and IFN-γ in the BALF and splenocyte supernatants were measured using ELISA. CD4+ and CD8+ T cells in the lung and spleen were analyzed by flow cytometry. RT-PCR was applied to measure the mRNA transcript levels of monokines induced by IFN-γ(CXCL9) and IFN-γ inducible protein 10(CXCL10). The total cell counts, eosinophil counts, and IL-4 levels in the BALF and cultured splenocyte supernatants were significantly increased, while the levels of IFN-γ were reduced in the HDMP groups(P<0.01). Changes in the total cell counts, eosinophil counts, and lymphocyte counts, as well as the total protein levels in the BALF, the levels of IL-4 in splenocyte supernatants, and the pathological manifestations in the lung, were all greater in CXCR3KO mice than in C57BL/6 wild-type mice. Furthermore, the expression levels of CXCL9 and CXCL10 mRNA transcripts in the lungs of CXCR3KO mice were lower than those in C57BL/6 wild-type mice (P<0.05). CXCR3 and its ligands (i.e., CXCL9 and CXCL10) may play anti-inflammatory roles in this animal model. Promoting the expression of CXCR3 and its ligands may represent a novel therapeutic approach for preventing and curing asthma.