Number Of Cells In Bronchoalveolar Lavage Research Articles

Lung injury and oxidative stress induced by inhaled chlorine in mice is associated with proinflammatory activation of macrophages and altered bioenergetics

Chlorine (Cl2) gas is a highly toxic and oxidizing irritant that causes life-threatening lung injuries. Herein, we investigated the impact of Cl2-induced injury and oxidative stress on lung macrophage phenotype and function. Spontaneously breathing male C57BL/6J mice were exposed to air or Cl2 (300 ppm, 25 min) in a whole-body exposure chamber. Bronchoalveolar lavage (BAL) fluid and cells, and lung tissue were collected 24 h later and analyzed for markers of injury, oxidative stress and macrophage activation. Exposure of mice to Cl2 resulted in increases in numbers of BAL cells and levels of IgM, total protein, and fibrinogen, indicating alveolar epithelial barrier dysfunction and inflammation. BAL levels of inflammatory proteins including surfactant protein (SP)-D, soluble receptor for glycation end product (sRAGE) and matrix metalloproteinase (MMP)-9 were also increased. Cl2 inhalation resulted in upregulation of phospho-histone H2A.X, a marker of double-strand DNA breaks in the bronchiolar epithelium and alveolar cells; oxidative stress proteins, heme oxygenase (HO)-1 and catalase were also upregulated. Flow cytometric analysis of BAL cells revealed increases in proinflammatory macrophages following Cl2 exposure, whereas numbers of resident and antiinflammatory macrophages were not altered. This was associated with increases in numbers of macrophages expressing cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), markers of proinflammatory activation, with no effect on mannose receptor (MR) or Ym-1 expression, markers of antiinflammatory activation. Metabolic analysis of lung cells showed increases in glycolytic activity following Cl2 exposure in line with proinflammatory macrophage activation. Mechanistic understanding of Cl2-induced injury will be useful in the identification of efficacious countermeasures for mitigating morbidity and mortality of this highly toxic gas.

Targeted nanotherapy with everolimus reduces inflammation and fibrosis in scleroderma-related interstitial lung disease developed by PSGL-1 deficient mice.

Interstitial lung disease (ILD) is the main cause of mortality in systemic sclerosis (SSc), and current therapies available are of low efficacy or high toxicity. Thus, the identification of innovative less toxic and high efficacy therapeutic approaches to ILD treatment is an urgent need. The interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin initiates leukocyte extravasation and deletion of the corresponding gene (Selplg) induces a SSc-like syndrome with high incidence of ILD in aged mice. Aged PSGL-1 KO (Selplg-/- ) mice were used to assess the therapeutic effects of nanotherapy with everolimus, included in liposomes decorated with high MW hyaluronic acid (LipHA+Ev) and administered intratracheally to specifically target CD44-expressing lung cells. PSGL-1 KO mice had increased numbers of CD45+ and CD45- cells, including alveolar and interstitial macrophages, eosinophils, granulocytes and NK cells, and myofibroblasts in bronchoalveolar lavage (BAL). CD45+ and CD45- cells expressing pro-inflammatory and pro-fibrotic cytokines were also increased. Lungs from PSGL-1 KO mice showed increased immune cell infiltration and apoptosis and exacerbated interstitial and peribronchial fibrosis. Targeted nanotherapy with LipHA+Ev decreased the myofibroblasts in BAL, cells producing proinflammatory and profibrotic cytokines, and the degree of lung inflammation at histology. LipHA+Ev treatment also decreased the severity of peribronchial and interstitial lung fibrosis, from moderate to mild levels. In PSGL-1 KO mice, targeted nanotherapy with LipHA+Ev was an effective treatment for SSc-ILD, reducing the number of inflammatory and fibrotic cells in BAL and reducing inflammation and fibrosis in lungs.

Characterization of a flexible AAV-DTR/DT mouse model of acute epithelial lung injury.

Animal models are important to mimic certain pathways or biological aspects of human pathologies including acute and chronic pulmonary diseases. We developed a novel and flexible mouse model of acute epithelial lung injury based on adeno-associated virus (AAV) variant 6.2-mediated expression of the human diphtheria toxin receptor (DTR). Following intratracheal administration of diphtheria toxin (DT), a cell-specific death of bronchial and alveolar epithelial cells can be observed. In contrast to other lung injury models, the here described mouse model provides the possibility of targeted injury using specific tropisms of AAV vectors or cell-type-specific promotors to drive the human DTR expression. Also, generation of cell-specific mouse lines is not required. Detailed characterization of the AAV-DTR/DT mouse model including titration of viral genome (vg) load and administered DT amount revealed increasing cell numbers in bronchoalveolar lavage (BAL; macrophages, neutrophils, and unspecified cells) and elevation of degenerated cells and infiltrated leukocytes in lung tissue, dependent of vg load and DT dose. Cytokine levels in BAL fluid showed different patterns with higher vg load, e.g., IFNγ, TNFα, and IP10 increasing and IL-5 and IL-6 decreasing, whereas lung function was not affected. In addition, laser-capture microdissection (LCM)-based proteomics of bronchial epithelium and alveolar tissue revealed upregulated immune and inflammatory responses in all regions and extracellular matrix deposition in infiltrated alveoli. Overall, our novel AAV-DTR/DT model allows investigation of repair mechanisms following epithelial injury and resembles specific mechanistic aspects of acute and chronic pulmonary diseases.

Dual Effect of Low-Molecular-Weight Bioregulators of Bacterial Origin in Experimental Model of Asthma.

Asthma is one of the most common noncommunicable diseases, affecting over 200 million people. A large number of drugs control asthma attacks, but there is no effective therapy. Identification of reasons for asthma and preventing this disease is a relevant task. The influence of bacterial components is necessary for the normal development of the immune system and the formation of an adequate immune response to antigens. In the absence of microorganisms or their insufficient exposure, the prerequisites are formed for excessive reactivity to harmless antigens. In the present study, we analyzed cellular and humoral factors in a standard mouse model of OVA-induced asthma modified by 5-fold intraperitoneal injection of bacterial cell wall fragments of glucosaminylmuramyl dipeptide (GMDP) 5 μg/animal or 1 μg lipopolysaccharide (LPS) per animal for 5 days before sensitization by ovalbumin (OVA). Preliminary administration of LPS or GMDP to animals significantly reduced goblet cells as well as the number of neutrophils, lymphocytes, and eosinophils in bronchoalveolar lavage, wherein GMDP corrected neutrophilia to a 2-fold degree, and LPS reduced the severity of eosinophilia by 1.9 times. With OVA administration of GMDP or LPS at the sensitization stage, an increase in the total number of bronchoalveolar lavage cells due to neutrophils, macrophages, lymphocytes, and eosinophils in relation to the group with asthma without GMDP or LPS was observed. The administration of GMDP or LPS to normal mice without asthma for 5 days had no statistically significant effect on the change in the number and population composition of cells in bronchoalveolar lavage in comparison with the control group receiving PBS. As a result of a study in a mouse model of asthma, a dual effect of LPS and GMDP was established: the introduction of LPS or GMDP before sensitization reduces neutrophilia and eosinophilia, while the introduction of LPS or GMDP together with an allergen significantly increases neutrophilia and eosinophilia. The study of the immunoglobulin status shows that in normal-asthma mice, GMDP and LPS slightly increase IgA in bronchoalveolar lavage; at the same time, in the asthma model, injections of GMDP or LPS before sensitization contribute to a significant decrease in IgA (2.6 times and 2.1 times, respectively) in BALF and IgE (2.2 times and 2.0 times, respectively) in blood serum. In an experimental model of asthma, the effect of GMDP and LPS was multidirectional: when they are repeatedly administered before sensitization, the bacterial components significantly reduce the severity of the allergic process, while in the case of a joint injection with an allergen, they increase the influx of macrophages, lymphocytes, and neutrophils into the lungs, which can aggravate the course of pathological process. Thus, the insufficient effect of antigens of a bacterial nature, in particular, with prolonged use of antibiotics can be compensated for by substances based on low-molecular-weight bioregulators of bacterial origin to establish the missing signals for innate immunity receptors, whose constant activation at a certain level is necessary to maintain homeostasis.

Intratracheal administration of solutions in mice; development and validation of an optimized method with improved efficacy, reproducibility and accuracy

Animal models are still vital in the field of respiratory disease research. To improve the accuracy and consistency of the dose of specific compounds administered specifically in the respiratory tract, it is important to optimize and to compare the technique to currently available techniques. In this study, an optimized intubation-mediated intratracheal administration (IMIT) technique is described and compared to oropharyngeal aspiration (OA).Adult female Balb/c mice were treated with Evans Blue using IMIT or OA and sacrificed after a short recovery to observe the distribution of solutions throughout the lungs. Additionally, mice were treated with increasing doses of lipopolysaccharide (LPS) or saline to compare efficacy of both techniques. Inflammatory cell numbers in bronchoalveolar lavage were quantified 24 h post-administration.Evans Blue staining revealed a more homogeneous distribution and less variability among animals treated using IMIT as compared to OA. Higher inflammatory cell numbers were observed in IMIT mice compared to OA mice after exposure to vehicle or the lowest LPS concentration.This study shows that the optimized IMIT is superior to OA with regards to efficacy, reproducibility and accuracy. This IMIT method can be deployed to refine 3R animal welfare aspects of the experimental design and improve the reproducibility of respiratory disease mouse models.

Hexarelin modulates lung mechanics, inflammation, and fibrosis in acute lung injury.

Introduction: Acute respiratory distress syndrome (ARDS) is an acute form of diffuse lung injury characterized by (i) an intense inflammatory response, (ii) increased pulmonary vascular permeability, and (iii) the loss of respiratory pulmonary tissue. In this article we explore the therapeutic potential of hexarelin, a synthetic hexapeptide growth hormone secretagogue (GHS), in an experimental model of ARDS. Hexarelin has anti-inflammatory properties and demonstrates cardiovascular-protective activities including the inhibition of cardiomyocyte apoptosis and cardiac fibrosis, both of which may involve the angiotensin-converting enzyme (ACE) system. Methods: In our experimental model, ARDS was induced by the instillation of 100 mM HCl into the right bronchus; these mice were treated with hexarelin (320 μg/kg, ip) before (Pre) or after (Post) HCl challenge, or with vehicle. Respiratory system compliance, blood gas analysis, and differential cell counts in a selective bronchoalveolar lavage (BAL) were determined 6 or 24 hours after HCl instillation. In an extended study, mice were observed for a subsequent 14 days in order to assess lung fibrosis. Results: Hexarelin induced a significant improvement in lung compliance and a reduction of the number of total immune cells in BAL 24 hours after HCl instillation, accompanied with a lower recruitment of neutrophils compared with the vehicle group. At day 14, hexarelin-treated mice presented with less pulmonary collagen deposition compared with vehicle-treated controls. Conclusions: Our data suggest that hexarelin can inhibit the early phase of the inflammatory response in a murine model of HCl-induced ARDS, thereby blunting lung remodeling processes and fibrotic development.

Lavage lipidomics signatures in children with cystic fibrosis and protracted bacterial bronchitis

BackgroundBalanced composition of a well-functioning pulmonary surfactant is crucial and essential for normal breathing. Here, we explored whether the composition of lipids recovered by broncho-alveolar lavage (BAL) in children with cystic fibrosis (CF) differ from children with protracted bacterial bronchitis (PBB) and controls. We wanted to differentiate, if alterations are primarily caused by the disease process or secondary due to an increased amount of cell-membrane lipids derived from inflammatory cells. MethodsComprehensive lipidomics profiles of BAL fluid from children diagnosed with CF, PBB and controls were generated by electrospray ionization tandem mass spectrometry analysis. BAL cell differential and numbers were examined. Results55 children (37 patients with CF, 8 children with PBB and 10 controls) were included in this study. Results showed comparable total quantities of lipids in all groups. Phospholipids were the major lipid fraction and similar in all groups, whereas the fractions of cholesteryl esters were less and of free cholesterol were increased in children with CF. Among the phospholipids, patients with CF had higher proportion of the non-surfactant membrane-lipids in the classes phosphatidylethanolamine based plasmalogens (PE P), phosphatidylethanolmine (PE) and phosphatidylserine (PS), but a lower proportion of phosphatidylcholine (PC) compared to healthy controls. No such changes were identified in the BAL fluid of children diagnosed with PBB. No differences were observed for the surfactant lipids dipalmitoyl-phosphatidylcholin (PC 32:0) and phosphatidylglycerol (PG). ConclusionsIn CF patients with neutrophilic airway inflammation the lipid composition for surfactant phospholipid components were unchanged, whereas alteration in lipid profile were characteristic for those found in membranes of inflammatory cells. We suspect that the changes in CF were caused by the prolonged inflammation in contrast to a relatively short standing process in PBB.

Bacterial secretion of histamine within the gut influences immune responses within the lung.

Histamine is an important immunomodulator influencing both the innate and adaptive immune system. Certain host cells express the histidine decarboxylase enzyme (HDC), which is responsible for catalysing the decarboxylation of histidine to histamine. We and others have shown that bacterial strains can also express HDC and secrete histamine; however, the influence of bacterial-derived histamine on the host immune responses distant to the gut is unclear. The Escherichia coli BL21 (Ecoli BL21) strain was genetically modified to express the Morganella morganii (Mmorganii)-derived HDC gene (Ecoli BL21_HTW). Ecoli BL21 and Ecoli BL21_HTW were gavaged to ovalbumin (OVA) sensitized and challenged mice to investigate the effect of bacterial-derived histamine on lung inflammatory responses. Oral administration of Ecoli BL21_HTW, which is able to secrete histamine, to wild-type mice reduced lung eosinophilia and suppressed exvivo OVA-stimulated cytokine secretion from lung cells in the OVA respiratory inflammation mouse model. In histamine receptor 2 (H2R)-deficient mice, administration of histamine-secreting bacteria also reduced inflammatory cell numbers in bronchoalveolar lavage (BAL). However, the suppressive effect of bacterial-derived histamine on BAL inflammation was lost in HDC-deficient mice. This loss of activity was associated with increased expression of histamine degrading enzymes and reduced histamine receptor expression. Histamine secretion from bacteria within the gut can have immunological consequences at distant mucosal sites, such as within the lung. These effects are influenced by host histamine receptor expression and the expression of histamine degrading enzymes.

Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases.

MicroRNA-7 Deficiency Ameliorates the Pathologies of Acute Lung Injury through Elevating KLF4.

Recent evidence showed that microRNA-7 (miR-7) played an important role in the pathologies of lung-related diseases. However, the potential role of miR-7 in acute lung injury (ALI) still remains poorly understood. Here, we assessed the effect of miR-7 deficiency on the pathology of ALI. We, first, found that the expression of miR-7 was upregulated in lung tissue in murine LPS-induced ALI model. Notably, we generated miR-7 knock down mice by using miRNA-Sponge technique and found that miR-7 deficiency could ameliorate the pathologies of lung as evidenced by accelerated body weight recovery, reduced level of bronchoalveolar lavage (BAL) proinflammatory cytokines and decreased number of BAL cells in ALI mice. Moreover, the proportion and number of various immune cells in BAL, including innate immune cell F4/80+ macrophages, γδT cells, NK1.1+ T cells, and CD11c+DCs, as well as adaptive immune cell CD4+ T cells and CD8+ T cells, also significantly changed, respectively. Mechanistic evidence showed that KLF4, a target molecule of miR-7, was upregulated in lung tissues in ALI model, accompanied by altered transduction of NF-κB, AKT, and ERK pathway. These data provided a previously unknown role of miR-7 in pathology of ALI, which could ultimately aid the understanding of development of ALI and the development of new therapeutic strategies against clinical inflammatory lung diseases.

Investigation of the anti‑asthmatic activity of Oridonin on a mouse model of asthma.

Oridonin is an extract obtained from a traditional Chinese medicinal herb, Xihuangcao. Previous studies have demonstrated that Oridonin exerts various pharmaceutical effects, such as anti-tumor and immunosuppressive effects, as well as modulating cytokine balance. The present study identified that Oridonin could regulate the Th1/Th2 cytokine balance in mice. However, as the anti-asthmatic effect of Oridonin is currently unknown a mouse model of asthma was used in the present study. BALB/c mice were sensitized using ovalbumin (OVA), then the sensitized mice were treated with Oridonin prior to OVA challenge. The in vivo study indicated that Oridonin decreased the OVA-induced airway hyper-responsiveness significantly (P<0.05). In addition, the results indicated that in Oridonin-treated mice, the eosinophil number and total inflammatory cell number in bronchoalveolar lavage (BAL) fluid decreased significantly in the Oridonin group when compared with the control group. Further study indicated that Oridonin significantly decreased the level of inflammatory cytokines, which were induced by OVA, in BAL fluid. Histological studies were performed to evaluate the effect of Oridonin on eosinophilia and mucus in the airway, the results indicated that Oridonin significantly inhibited the eosinophilia and mucus production in the lungs. Therefore the present study demonstrated that Oridonin regulates Th1/Th2 balance in mice and exhibited anti-asthmatic effects in a mouse model of asthma. These findings indicate that Oridonin may serve as a potential therapeutic compound for the treatment of asthma in future.

Efeito do estresse pré-natal na regulação da inflamação alérgica pulmonar no modelo murino de asma experimental

Devido ao seu rápido crescimento, o feto é particularmente vulnerável a insultos e modificações no millieu hormonal. Este fato sugere que situações adversas experimentadas pela mãe grávida podem alterar o desenvolvimento e a saúde da prole, explicado principalmente pela permeabilidade da barreira placentária a diversos hormônios e substâncias. O objetivo deste trabalho foi estudar o efeito do estresse pré-natal na regulação da inflamação alérgica pulmonar, empregando o modelo murino de asma experimental. Para este propósito foram utilizadas camundongas virgens da linhagem Swiss, com 50 dias de idade. Foi empregado o modelo de choque nas patas para promover o estresse pré-natal e o modelo do “metrô de Nova Efeito do estresse pré-natal na regulação da inflamação alérgica pulmonar no modelo murino de asma experimental 65 Iorque” para o estresse pós-natal. As fêmeas foram distribuídas em 4 grupos experimentais: CC: fêmeas não estressadas;CE: fêmeas estressadas pós-natalmente aos 60 dias de idade (PND60), EC: fêmeas nascidas de mães estressadas entre o dia 15 (GD15) e 18 de gestação (GD18); EE: fêmeas nascidas de mães estressadas entre o GD15 e GD18 e estressadas pós-natalmente aos PND60. A indução da inflamação alérgica pulmonar foi realizada através da sensibilização dos animais com solução de ovalbumina (OVA) 0,1 mg.Kg-1 sc para avaliação do leucograma, lavado broncoalveolar (BAL), celularidade hematopoiética medular e neuroquímica. Os experimentos foram realizados 24h após a última sessão de nebulização.O número de células do BAL foi significantemente maior nos animais do grupo EE, em relação àqueles dos grupos CC (P&lt;0.01), CE (P&lt;0.01) e EC (P&lt;0.001). Na contagem diferencial do BAL os linfócitos e macrófagos do grupo EE foram significantemente maiores que em os outros grupos avaliados (P&lt;0.05). Na contagem diferencial de células no sangue não foram observadas alterações (P&gt;0.05) para os linfócitos, neutrófilos, eosinófilos e monócitos; porém, observou-se diferenças significativas (P&lt;0.05) entre o número de bastonetes dos grupos, sendo maior nos animais do grupo CC em relação àquelesdo grupo EC. O número de células hematopoiéticas da medula óssea foi significantemente (P&lt;0.05) menor nos animais do grupo EE, em relação àqueles do grupo CC. No córtex pré-frontal, há diferenças significantes na relação Ácido Homovanílico/Dopamina (HVA/DA) (P&lt;0.05), sendo maior nos animais do grupo EC, em relação àqueles do grupo CE. Em conclusão,o estresse pré-natal levou a modulação de células do sistema imune (SI) dos neonatos, evidenciado após a exposição a estresse agudo pós-natal, amplificando a resposta alérgica pulmonar. Sugere-se que a maior susceptibilidade dos animais do grupo EE seja consequência de alterações induzidas pelo estresse pré-natal no eixo hipotálamo-pituitária-adrenal (HPA).

Elevated levels of FN1 and CCL2 in bronchoalveolar lavage fluid from sarcoidosis patients.

BackgroundSarcoidosis is a granulomatous systemic inflammatory disease in which more than 90 % of all patients develop pulmonary manifestations. Several gene associations have previously been described, but established and clinically useful biomarkers are still absent. This study aimed to find proteins in bronchoalveolar lavage (BAL) fluid that can be associated with the disease.MethodsWe developed and performed profiling of 94 selected proteins in BAL fluid and serum samples obtained from newly diagnosed and non-treated patients with sarcoidosis. Using multiplexed immunoassays, a total of 317 BAL and 217 serum samples were analyzed, including asthmatic patients and healthy individuals as controls.ResultsOur analyses revealed increased levels of eight proteins in sarcoidosis patients compared to controls. Out of these, fibronectin (FN1) and C-C motif chemokine 2 (CCL2) revealed the strongest associations. In addition, cadherin 5 (CDH5) was found to correlate positively with lymphocyte cell numbers in BAL fluid.ConclusionsApplying a high throughput proteomics screening technique, we found proteins of potential clinical relevance in the context of sarcoidosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0381-0) contains supplementary material, which is available to authorized users.

Angiotensin-(1-7) improves oxygenation, while reducing cellular infiltrate and fibrosis in experimental Acute Respiratory Distress Syndrome.

BackgroundThe renin-angiotensin system (RAS) plays a role in the pathogenesis of ARDS, Angiotensin II (Ang-II) contributing to the pathogenesis of inflammation and fibrogenesis. Angiotensin-(1-7) (Ang-(1-7)) may antagonize the effects of Ang-II. This study was aimed at evaluating the potential for Ang-(1-7) to reduce injury, inflammation and fibrosis in an experimental model of ARDS in the acute and late phases.MethodsMale Sprague Dawley rats underwent an instillation of 0.1 M hydrochloric acid (HCl, 2.5 ml/kg) into the right bronchus. In an acute ARDS study, acid-injured rats were subjected to high stretch mechanical ventilation (18 ml/kg) for 5 h and randomized to receive an intravenous infusion of either vehicle (saline), Ang-(1-7) at low dose(0.27 μg/kg/h) (ALD), or high dose (60 μg/kg/h) (AHD) starting simultaneously with injury or 2 h afterwards. Arterial blood gas analysis and bronchoalveolar lavage (BAL) were performed to assess the injury. For the late ARDS study, after HCl instillation rats were randomized to either vehicle or high dose Ang-(1-7) (300 μg/kg/day) infused by mini osmotic pumps for two weeks, and lung hydroxyproline content measured.ResultsIn the acute ARDS study, Ang-(1-7) led to a significant improvement in oxygenation (PaO2/FiO2 : vehicle 359 ± 86; ALD 436 ± 72; AHD 44 442 ± 56; ANOVA p = 0.007) and reduced white blood cells counts (vehicle 4,519 ± 2,234; ALD 2,496 ± 621; AHD 2,744 ± 119/mm3; ANOVA p = 0.004). Only treatment with high dose Ang-(1-7) reduced inflammatory cell numbers in BAL (vehicle 127 ± 34; AHD 96 ± 34/ μl; p = 0.033). Interestingly also delayed administration of Ang-(1-7) was effective in reducing injury. In later ARDS, Ang-(1-7) decreased hydroxyproline content (649 ± 202 and 1,117 ± 297 μg/lung; p < 0.05).ConclusionsAngiotensin-(1-7), decreased the severity of acute lung injury and inflammation induced by combined acid aspiration and high stretch ventilation. Furthermore, continuous infusion of Ang-(1-7) reduced lung fibrosis 2 weeks following acid aspiration injury. These results call for further research on Ang-(1-7) as possible therapy for ARDS.

Potential therapeutic effect of Allium cepa L. and quercetin in a murine model of Blomia tropicalis induced asthma.

BackgroundAsthma is an inflammatory condition characterized by airway hyperresponsiveness and chronic inflammation. The resolution of inflammation is an essential process to treat this condition. In this study we investigated the effect of Allium cepa L. extract (AcE) and quercetin (Qt) on cytokine and on smooth muscle contraction in vitro and its therapeutic potential in a murine model of asthma.MethodsAcE was obtained by maceration of Allium cepa L. and it was standardized in terms of quercetin concentration using high performance liquid chromatography (HPLC). In vitro, using AcE 10, 100 or 1000 μg/ml or Qt 3.5, 7.5, 15 μg/ml, we measured the concentration of cytokines in spleen cell culture supernatants, and the ability to relax tracheal smooth muscle from A/J mice. In vivo, Blomia tropicalis (BT)-sensitized A/J mice were treated with AcE 100, 1000 mg/kg or 30 mg/kg Qt. We measured cell influx in bronchoalveolar lavage (BAL), eosinophil peroxidase (EPO) in lungs, serum levels of Bt-specific IgE, cytokines levels in BAL, and lung histology.ResultsWe observed a reduction in the production of inflammatory cytokines, a relaxation of tracheal rings, and a reduction in total number of cells in BAL and EPO in lungs by treatment with AcE or Qt.ConclusionAcE and Qt have potential as antiasthmatic drugs, as they possess both immunomodulatory and bronchodilatory properties.

Pulmonary and Atherogenic Effects of Multi-Walled Carbon Nanotubes (MWCNT) in Apolipoprotein-E-Deficient Mice

Rapid growth in nanotechnology has raised concerns regarding adverse health effects due to human exposure to manufactured nanoparticles. Carbon nanotubes (CNT) are among the most extensively used nanoparticles. This study examined pulmonary and atherosclerotic effects of multiwalled CNT (MWCNT) in a mouse model of atherosclerosis. Female apolipoprotein E-deficient (apoE–/–) mice were exposed to 40 μg MWCNT, once each week for 16 consecutive weeks by pharyngeal aspiration. On d 1 after the last administration, tissues were extracted from half the group, while the remaining animals were sacrificed at d 7. Bronchoalveolar lavage (BAL) was performed to obtain BAL fluid. In addition, plasma, lung, and aortas were extracted to assess pulmonary inflammation and atherosclerotic lesion formation. Polymorphonuclear leukocytes and total BAL cell number increased significantly in MWCNT-exposed mice on d 1 and 7 postexposure. Cell-free BAL fluid obtained from MWCNT-exposed mice at d 1 and 7 postexposure contained significantly elevated levels of total protein, lactate dehydrogenase (LDH), surfactant protein-D, and mucin. Although MWCNT exposure increased pulmonary injury and inflammation, the aortic intimal surface covered by atherosclerotic lesions was not significantly different between control apoE–/– mice and apoE–/– MNCNT-treated animals. Total plasma cholesterol concentrations also were not markedly affected by MWCNT exposure. These results demonstrate that pulmonary exposure to MWCNT affects local airway inflammation but did not appear to augment progression of atherosclerosis in female apoE–/– mice.

Antiallergic Effects of Caffeic Acid in Blomia Tropicalis Murine Model of Experimental Asthma

Background: Atopic asthma is a chronic airway inflammatory disorder, which is characterized by reversible airway obstruction, airway hyperresponsiveness and production of Th2-dominated response with the production of cytokines IL-4, IL-5 and IL-13. The most common drugs used to treat allergic respiratory diseases, control their symptoms, but also induce long-term side effects. Therefore, in order to mitigate these effects, new treatments with alternative drugs must be pursued to improve asthma control. Among several compounds investigated by the scientific community, caffeic acid (CA), a phenolic acid has shown important biological properties, such as antioxidant and anti-inflammatory. In this study we investigated the antiallergic effects of caffeic acid on inflammatory cytokines, in vitro, and in a murine model of experimental asthma induced by the Blomia tropicalis extract (Bt). Methods: We measured the concentration of cytokines (IL-4, IL-5 and IL-13) on spleen cell culture supernatants in vitro, using 25, 50 or 100 ?M of CA. For in vivo experiments, AJ mice were sensitized (100 ?g/ animal s.c) and challenged (10?g/ animal i.n) with Bt. Sensitized A/J mice were treated or not with CA (10, 100 or 200mg/Kg) or with 3mg/kg of Dexametazone (Dex) and the following parameters were analyzed: number of total cells in bronchoalveolar lavage (BAL); eosinophil peroxidase activity (EPO) in BAL; serum level of specific IgE, IgG1 and IgG2a; and histopathological changes in the lungs. Results: The CA oral treatment caused a reduction in total number of cells in BAL, EPO in lungs, serum level of specific IgG1 and histopathological changes showed attenuate allergic inflammation in the lungs. Additionally, we observed a significant decrease of Th2-type cytokines (IL-4, IL-5 and IL-13) on spleen cell culture supernatants. Conclusion: These results suggest that oral treatment with caffeic acid could be a promising alternative treatment for bronchial asthma.

Retnla overexpression attenuates allergic inflammation of the airway.

Resistin-like molecule alpha (Retnla), also known as ‘Found in inflammatory zone 1’, is a secreted protein that has been found in bronchoalveolar lavage (BAL) fluid of ovalbumin (OVA)-induced asthmatic mice and plays a role as a regulator of T helper (Th)2-driven inflammation. However, the role of Retnla in the progress of Th2-driven airway inflammation is not yet clear. To better understand the function of Retnla in Th2-driven airway inflammation, we generated Retnla-overexpressing (Retnla-Tg) mice. Retnla-Tg mice showed increased expression of Retnla protein in BAL fluid and airway epithelial cells. Retnla overexpression itself did not induce any alteration in lung histology or lung function compared to non-Tg controls. However, OVA-sensitized/challenged Retnla-Tg mice had decreased numbers of cells in BAL and inflammatory cells accumulating in the lung. They also showed a reduction in mucus production in the airway epithelium, concomitant with a decreased Muc5ac level. These results were accompanied by reduced levels of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, with no effect on levels of OVA-specific immunoglobulin isotypes. Furthermore, phosphorylation of ERK was markedly reduced in the lungs of OVA-challenged Retnla-Tg mice. Taken together, these results indicates that Retnla protects against Th2-mediated inflammation in an experimental mouse model of asthma, suggesting that therapeutic approaches to enhance the production of Retnla or Retnla-like molecules could be valuable for preventing allergic lung inflammation.

Antisense Oligonucleotide Treatment Enhances the Recovery of Acute Lung Injury through IL-10–Secreting M2-like Macrophage-Induced Expansion of CD4+ Regulatory T Cells

MicroRNAs (miRNAs) have been shown as an important regulator in the pathologies of acute lung injury (ALI). However, the potential effect of miRNA-based therapeutic studies in ALI remains poorly understood. We assessed the effect of antisense oligonucleotides (ASOs) against miR-155 on the development of ALI using a murine ALI model. We found that miR-155 ASO treatment could enhance the recovery of ALI as evidenced by accelerated body weight back, reduced level of bronchoalveolar lavage (BAL) protein and proinflammatory cytokines, and reduced number of BAL cells. Adoptive cell transfer assay in RAG1(-/-) mice showed that CD4(+)CD25(+) regulatory T cells (Tregs) mediated the enhanced recovery of ALI. Mechanistic evidence showed that enhanced expansion of Tregs in vivo, dominantly induced by IL-10-secreting M2-like macrophages, was critical for their elevated proportion in miR-155 ASO-treated ALI mice. Finally, we report that C/EBPβ, a target molecule of miR-155, was upregulated and associated with IL-10 secretion and M2-like phenotype of macrophages. These data provided a previously unknown mechanism for miRNA-based therapy against ALI, which could ultimately aid the understanding of recovery of ALI and the development of new therapeutic strategies against clinical inflammatory lung disease.

Influence of hepatic P450‐mediated Amiodarone metabolism on Amiodarone‐induced pulmonary toxicity

Amiodarone (AMI), a widely used, effective anti‐arrhythmic drug, has severe side effects, including acute lung toxicity and pulmonary fibrosis. Our aim is to test the hypothesis that a decline in liver microsomal P450‐meditated AMI metabolism leads to increased accumulation of AMI in lung and consequently greater pulmonary toxicity. We studied three mouse strains, all on B6 background: wild type (WT), LCN (liver‐specific P450 reductase (CPR) null; has little P450 activity in hepatocytes), and Cpr‐low (has global suppression of CPR expression in all cells). Adult, male mice were treated with AMI (160 mg/kg, i.p.) or vehicle alone, once/day for 3 days. On day 4, tissue AMI levels were determined, and bronchoalveolar lavage (BAL) was analyzed for signs of lung toxicity. Hepatic AMI levels were 1.1‐and 2.7‐fold higher, whereas lung AMI levels were 1.3‐and 4.3‐fold higher, in Cpr‐low and LCN mice, respectively, than in WT mice. As a general marker of lung toxicity, total numbers of cells in BAL were increased by 1.7‐and 2.3‐fold, in Cpr‐low and LCN mice, respectively, compared to treated WT mice. Furthermore, a 2‐fold increase in the number of lymphocytes in BAL was seen in LCN, compared to WT, following AMI treatment. These results support a role of liver P450 in protecting the lung against AMI's acute toxicity. Studies are ongoing to determine if a decrease in hepatic AMI metabolism leads to greater rate of pulmonary fibrosis.