Bronchoalveolar Lavage Cell Counts Research Articles

Role of Claudin-5 in the Attenuation of Murine Acute Lung Injury by Simvastatin

The statins are now recognized to have pleiotropic properties, including augmentation of endothelial barrier function. To explore the mechanisms involved, we investigated the effect of simvastatin on endothelial cell (EC) tight junctions. Western blotting of human pulmonary artery ECs treated with simvastatin (5 μM) confirmed a significant time-dependent increase (16-48 h) in claudin-5 protein expression compared with controls, without detectable alterations in zonula occludens-1 or occludin. These effects were associated with membrane translocation of VE-cadherin, whereas translocation of vascular endothelial cadherin (VE-cadherin; silencing RNA) inhibited simvastatin-induced claudin-5 up-regulation. Moreover, simvastatin treatment of ECs induced increased phosphorylation of both FoxO1 and β-catenin, transcriptional regulators of claudin-5 expression mediated by VE-cadherin. Subsequently, we found no effect of claudin-5 silencing on EC barrier protection by simvastatin in response to thrombin stimulation, as measured by either transendothelial electrical resistance or by EC monolayer flux of FITC-dextran (2,000 kD). However, silencing of claudin-5 did significantly attenuate simvastatin-mediated EC barrier protection in response to thrombin, as measured by monolayer flux of sodium fluorescein (376 Da). Finally, employing a murine model of LPS-induced acute lung injury, there was no effect of claudin-5 silencing in vivo (intratracheal injection) on bronchoalveolar lavage fluid protein or cell counts, but LPS-induced lung tissue extravasation of the small molecular weight markers, sodium fluorescein and Hochst stain (562 Da), were significantly increased in claudin-5-silenced animals compared with simvastatin-treated control animals. These findings implicate a distinct mechanism underlying size-selective endothelial barrier-protective properties of statins, and may ultimately lead to new novel therapeutic targets for patients with acute lung injury.

Teaching Neuro Images : Awakening ptosis (unilateral hypnopompic eyelid palsy)

The increasing prevalence of allergic airway diseases in industrialized countries has been associated with high hygienic standards and reduced microbial exposure. Probiotic lactic acid bacteria, such as certain <i>Lactobacillus</i> strains, were shown to have potentially beneficial immunomodulatory properties. Therefore, we determined the effect of intranasal administration of <i>L. rhamnosus</i> GG (LGG) and GR-1 (LGR-1) in a mouse model of birch pollen-induced allergic asthma. The symptoms of allergic asthma were induced with 7 intranasal applications with 1- or 2-day interval with birch pollen extract. The asthma induction protocol was preceded by 8 intranasal administrations of LGG or LGR-1 at a concentration of 5 x 10⁸ CFU/dose over the course of two weeks on four consecutive days each week. We assessed serum antibody levels, bronchoalveolar lavage (BAL) cell counts, lung histology, lung cytokine levels, airway hyperreactivity. LGG administration resulted in a significant decrease in allergic airway inflammation (BAL eosinophils, lung IL-13 and IL-5 levels) and a significantly reduced number of respiratory exacerbations. Contrary to LGG, no decisive results were obtained for LGR-1. Longitudinal <i>in vivo</i> lung micro-computed tomography (micro-CT) is currently ongoing to determine inflammatory exacerbation. Complementary to micro-CT, fluorescence imaging (FLI) of a fluorescent LGG strain will be performed to understand the probiotic-host interactions at the whole-body and whole-organ level. In conclusion, bacterial strain-specific beneficial effects of intranasally administered LGG were observed in a mouse model of birch pollen-induced allergic asthma.

Effect of TiO&lt;sub&gt;2&lt;/sub&gt; Nanofibres on Selected Bronchoalveolar Parameters in Acute and Subacute Phase - Experimental Study

Titanium dioxide nanofibres (TiO2) were intratracheally instilled in dose of 4 mg/0.2 mL saline solution per animal (Wistar rats). After 48 hours and 14 days the animals were exsanguinated (under i.p. thiopental narcosis), bronchoalveolar lavage (BAL) was perfomed and cells from BAL fluid were isolated. Following inflammatory, cytotoxic and oxidative stress BAL parameters were examined: differential cell count (% of alveolar macrophages (AM), polymorphonuclears and lymphocytes); the viability and phagocytic activity of AM; the proportion of immature cells; the proportion of multinucleated cells; count of AM/mL lavage; count of BAL cells/mL lavage; the level of ascorbic acid and activity of superoxide dismutase, both in tissue homogenate and in bronchoalveolar lavage fluid. The majority of examined BAL parameters in the acute and subacute phase in our study suggest serious inflammatory and cytotoxic processes in lung after exposure to TiO2.

Development of a model of Ascaris Suum antigen-induced pulmonary inflammation in nonhuman primates

Airway inflammation is one of the hallmarks of asthma and can contribute to airway hyperresponsiveness. Using cynomolgus monkeys (Macaca fascicularis) that are naturally sensitized to Ascaris suum antigen in the wild, we developed a reproducible model of acute airway inflammation following segmental A.suum antigen challenge. A pediatric bronchoscope was used to perform bronchoalveolar lavage (BAL) for the collection of BAL cells and fluid (BALF). The bronchoscope was also used for the administration of a segmental lung A.suum challenge. Baseline BAL was performed on one lung prior to segmental antigen challenge on the opposite lung. BAL on the challenged lung was performed 24 hours post-challenge to assess pulmonary inflammation. As compared to baseline lavage, a single airway challenge with A.suum antigen, at a dose of 0.50 or 0.75 mg/challenge, resulted in a reproducible pulmonary inflammation. The inflammation was characterized by an increase in total BAL cell counts and increased eosinophils. From concentrated BALF samples, taken 24 hours post A.suum antigen challenge, there was also a trend towards increased IL-5 and eotaxin as measured by ELISA. In some studies, animals were treated with two doses of the steroid, dexamethasone (DEX), 1 mg/kg by IM injection, prior to antigen challenge. In these studies, administration of DEX prior to antigen challenge prevented airway inflammation. These data show that segmental challenge with A. suum antigen produced an acute pulmonary eosinophilic inflammation that was prevented by pretreatment with the steroid, dexamethasone. This model should be useful for testing the efficacy of selected drug candidates, as compared to proven anti-inflammatory therapy, in blocking the pulmonary inflammatory response to A. suum in a large animal model.

Human parainfluenza type 3 virus impairs the efficacy of glucocorticoids to limit allergy-induced pulmonary inflammation in guinea-pigs

Viral exacerbations of allergen-induced pulmonary inflammation in pre-clinical models reportedly reduce the efficacy of glucocorticoids to limit pulmonary inflammation and airways hyper-responsiveness to inhaled spasmogens. However, exacerbations of airway obstruction induced by allergen challenge have not yet been studied. hPIV-3 (human parainfluenza type3 virus) inoculation of guinea-pigs increased inflammatory cell counts in BAL (bronchoalveolar lavage) fluid and caused hyper-responsiveness to inhaled histamine. Both responses were abolished by treatment with either dexamethasone (20mg/kg of body weight, subcutaneous, once a day) or fluticasone propionate (a 0.5mg/ml solution aerosolized and inhaled over 15min, twice a day). In ovalbumin-sensitized guinea-pigs, allergen (ovalbumin) challenge caused two phases of airway obstruction [measured as changes in sGaw (specific airways conductance) using whole body plethysmography]: an immediate phase lasting between 4 and 6h and a late phase at about 7h. The late phase, airway hyper-responsiveness to histamine and inflammatory cell counts in BAL were all significantly reduced by either glucocorticoid. Inoculation of guinea-pigs sensitized to ovalbumin with hPIV-3 transformed the allergen-induced airway obstruction from two transient phases into a single sustained response lasting up to 12h. This exacerbated airway obstruction and airway hyper-responsiveness to histamine were unaffected by treatment with either glucocorticoid whereas inflammatory cell counts in BAL were only partially inhibited. Virus- or allergen-induced pulmonary inflammation, individually, are glucocorticoid-sensitive, but in combination generate a phenotype where glucocorticoid efficacy is impaired. This suggests that during respiratory virus infection, glucocorticoids might be less effective in limiting pulmonary inflammation associated with asthma.

IL-13 receptor α2 contributes to development of experimental allergic asthma

IL-13 receptor α2 (IL-13Rα2) binds IL-13 with high affinity and modulates IL-13 responses. There are soluble and membrane forms of IL-13Rα2 generated by alternative splicing in mice, but human subjects express only the membrane form of IL-13Rα2 (memIL-13Rα2). We determined the role of memIL-13Rα2 in the development of allergic inflammation in mouse models of asthma. IL-13Rα2-deficient and memIL-13Rα2 lung epithelium-specific transgenic mice were challenged with house dust mite (HDM). Airway hyperresponsiveness (AHR) and inflammation were assessed based on the airway pressure-time index, bronchoalveolar lavage (BAL) cell counts, and lung histology. Mucus production was determined by means of periodic acid-Schiff staining of lung sections, Western blot analysis of chloride channel calcium activated 3 (CLCA3) expression in lung homogenates, and ELISA of Muc5ac in BAL fluid. The expression of cytokines and chemokines was determined by using RT-quantitative PCR. In IL-13Rα2-deficient mice AHR and airway inflammation were attenuated compared with levels seen in wild-type mice after HDM challenge. Lung epithelial overexpression of memIL-13Rα2 in the IL-13Rα2-deficient mice reconstituted AHR and inflammation to levels similar to those observed in HDM-challenged wild-type mice. Mucus production was attenuated in lungs from HDM-treated IL-13Rα2-deficient mice, whereas lung epithelial overexpression of memIL-13Rα2 increased mucus production. Lung epithelial overexpression of memIL-13Rα2 had no effect on levels of the soluble form of IL-13Rα2 in serum or BAL fluid and did not affect IL-13-dependent signal transducer and activator of transcription 6 activation in the lungs. These data collectively support a distinct role for memIL-13Rα2 in the lung and suggest that memIL-13Rα2 might contribute to allergic inflammation.

Early treatment of chlorine-induced airway hyperresponsiveness and inflammation with corticosteroids

Chlorine (Cl2) is an industrial gas that is highly toxic and irritating when inhaled causing tissue damage and an acute inflammatory response in the airways followed by a long-term airway dysfunction. The aim of this study was to evaluate whether early anti-inflammatory treatment can protect against the delayed symptoms in Cl2-exposed mice.BALB/c mice were exposed by nose-only inhalation using 200ppm Cl2 during 15min. Assessment of airway hyperresponsiveness (AHR), inflammatory cell counts in bronchoalveolar lavage, occurrence of lung edema and lung fibrosis were analyzed 24h or 14days post-exposure. A single dose of the corticosteroid dexamethasone (10 or 100mg/kg) was administered intraperitoneally 1, 3, 6, or 12h following Cl2 exposure.High-dose of dexamethasone reduced the acute inflammation if administered within 6h after exposure but treated animals still displayed a significant lung injury. The effect of dexamethasone administered within 1h was dose-dependent; high-dose significantly reduced acute airway inflammation (100mg/kg) but not treatment with the relatively low-dose (10mg/kg). Both doses reduced AHR 14days later, while lung fibrosis measured as collagen deposition was not significantly reduced.The results point out that the acute inflammation in the lungs due to Cl2 exposure only partly is associated with the long-term AHR. We hypothesize that additional pathogenic mechanisms apart from the inflammatory reactions contribute to the development of long-term airway dysfunction. By using this mouse model, we have validated early administration of corticosteroids in terms of efficacy to prevent acute lung injury and delayed symptoms induced by Cl2 exposure.

Beneficial effects of arginase inhibition and inhaled l-arginine administration on airway histology in a murine model of chronic asthma

BackgroundIncreased arginase activity in the airways induces reduced bioavailability of l-arginine and cause deficiency of bronchodilatating and anti-inflammatory nitric oxide (NO). Therefore, arginine and arginase inhibitors may have therapeutic potential in the treatment of asthma. Using a murine model of asthma, we aimed to investigate the effects of inhaled l-arginine and arginase inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA) and co-treatment on airway histology of asthmatic lung tissue. MethodsForty-two BALB/c mice were divided into six groups: I (control), II (placebo), III, IV, V and VI. All mice except for control group were sensitised by an intraperitoneal injection of ovalbumin with alum adjuvant and then challenged with an aerosol of ovalbumin on three days of the week for eight weeks beginning from the 21st day of the study. Lung histology and bronchoalveolar lavage cell (BAL) counts were evaluated after treatment with inhaled l-arginine, nor-NOHA, l-arginine-nor-NOHA combination, budesonide and placebo. Interleukin(IL)-4 and IL-5 levels are determined in lung homogenates with ELISA. Resultsl-Arginine group was similar to budesonide group in lowering all histological parameters. Results of groups treated with nor-NOHA were also similar to budesonide group except for epithelial thickness. The number of eosinophils in BAL decreased significantly in groups receiving study drugs. Decrease was only noted in IL-4 levels in group receiving nor-NOHA. ConclusionWe demonstrated that inhaled l-arginine administration alleviated all histological parameters similar to budesonide and treatment with arginase inhibitor improved not all but some of the pathological changes in chronic asthma. Combination therapy had no additive effect on either treatment.

A novel two-hit rodent model of postoperative acute lung injury: priming the immune system leads to an exaggerated injury after pneumonectomy†

Postoperative acute lung injury (PALI) is a rare, poorly understood, usually fatal condition, accounting for the majority of deaths following lung resection. Its low frequency and unpredictable development make the identification of the mechanisms of injury from clinical studies alone almost impossible. Multiple validated 'two-hit models' exist for ALI secondary to other causes. We describe a novel rodent 'two-hit' model of PALI: a low-grade immune stimulus, such as sepsis, greatly aggravates the injury in the remaining lung observed following pneumonectomy. Under general anaesthesia, rats received either low-dose intratracheal lipopolysaccharide (IT-LPS) challenge (10 μg for 1 h) followed by left posterolateral thoracotomy, one-lung ventilation (OLV), pneumonectomy and 3 h of ventilation; 500 μl IT 0.9% saline followed by the same surgery or IT-LPS followed by sham surgery and ventilation. All other conditions were constant. Lung injury is heralded by neutrophil accumulation, which was determined by right lung bronchoalveolar lavage cell count. Data are presented as mean ± standard error of the mean. The T-test was used to compare normally distributed groups with correction for multiple comparisons. A dose-response curve identified the clinically relevant 'low dose' of LPS to be used in further studies. Ventilatory parameters were standardized to reflect clinical practice (volume-control, tidal volume of 6 ml/kg, positive end-expiratory pressure of 2 cmH2O, maximum airway pressure of <15 cmH2O). There was a degree of adaptation to obtain a consistent and robust model with retest validity. OLV and pneumonectomy alone produced a small lung injury (65.1 ± 5), as did 10 µg intratracheal LPS alone (50.7 ± 6.9). However, when OLV, pneumonectomy and 10 µg LPS were combined, an exaggerated injury occurred (161.4 ± 10.3), P = 0.007. Early results show that a two-hit model of PALI is viable and that sepsis aggravates the response to pneumonectomy. The model is now being further characterized. Once established, this model will offer the chance to better understand PALI and to develop and test novel therapies and risk reduction strategies for the condition.

Characterization of AQX‐1125, a small‐molecule SHIP1 activator

The efficacy of AQX-1125, a small-molecule SH2-containing inositol-5'-phosphatase 1 (SHIP1) activator and clinical development candidate, is investigated in rodent models of inflammation. AQX-1125 was administered orally in a mouse model of passive cutaneous anaphylaxis (PCA) and a number of rodent models of respiratory inflammation including: cigarette smoke, LPS and ovalbumin (OVA)-mediated airway inflammation. SHIP1 dependency of the AQX-1125 mechanism of action was investigated by comparing the efficacy in wild-type and SHIP1-deficient mice subjected to an intrapulmonary LPS challenge. AQX-1125 exerted anti-inflammatory effects in all of the models studied. AQX-1125 decreased the PCA response at all doses tested. Using bronchoalveolar lavage (BAL) cell counts as an end point, oral or aerosolized AQX-1125 dose dependently decreased the LPS-mediated pulmonary neutrophilic infiltration at 3-30 mg kg⁻¹ and 0.15-15 μg kg⁻¹ respectively. AQX-1125 suppressed the OVA-mediated airway inflammation at 0.1-10 mg kg⁻¹. In the smoke-induced airway inflammation model, AQX-1125 was tested at 30 mg kg⁻¹ and significantly reduced the neutrophil infiltration of the BAL fluid. AQX-1125 (10 mg kg⁻¹) decreased LPS-induced pulmonary neutrophilia in wild-type mice but not in SHIP1-deficient mice. The SHIP1 activator, AQX-1125, suppresses leukocyte accumulation and inflammatory mediator release in rodent models of pulmonary inflammation and allergy. As shown in the mouse model of LPS-induced lung inflammation, the efficacy of the compound is dependent on the presence of SHIP1. Pharmacological SHIP1 activation may have clinical potential for the treatment of pulmonary inflammatory diseases.

Effects of Diet-Induced Mild Obesity on Airway Hyperreactivity and Lung Inflammation in Mice

PurposeObesity has been suggested to be linked to asthma. However, it is not yet known whether obesity directly leads to airway hyperreactivity (AHR) or obesity-induced airway inflammation associated with asthma. We investigated obesity-related changes in adipokines, AHR, and lung inflammation in a murine model of asthma and obesity.Materials and MethodsWe developed mouse models of chronic asthma via ovalbumin (OVA)-challenge and of obesity by feeding a high-fat diet, and then performed the methacholine bronchial provocation test, and real-time PCR for leptin, leptin receptor, adiponectin, adiponectin receptor (adipor1 and 2), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) β, and tumor necrosis factor (TNF) α in lung tissue. We also measured cell counts in bronchoalveolar lavage fluid.ResultsBoth obese and lean mice chronically exposed to OVA developed eosinophilic lung inflammation and AHR to methacholine. However, obese mice without OVA challenge did not develop AHR or eosinophilic inflammation in lung tissue. In obese mice, lung mRNA expressions of leptin, leptin receptor, VEGF, TGF, and TNF were enhanced, and adipor1 and 2 expressions were decreased compared to mice in the control group. On the other hand, there were no differences between obese mice with or without OVA challenge.ConclusionDiet-induced mild obesity may not augment AHR or eosinophilic lung inflammation in asthma.

Hyperoxia Exacerbates Postnatal Inflammation-Induced Lung Injury in Neonatal BRP-39 Null Mutant Mice Promoting the M1 Macrophage Phenotype

Rationale. Hyperoxia exposure to developing lungs—critical in the pathogenesis of bronchopulmonary dysplasia—may augment lung inflammation by inhibiting anti-inflammatory mediators in alveolar macrophages. Objective. We sought to determine the O2-induced effects on the polarization of macrophages and the role of anti-inflammatory BRP-39 in macrophage phenotype and neonatal lung injury. Methods. We used RAW264.7, peritoneal, and bone marrow derived macrophages for polarization (M1/M2) studies. For in vivo studies, wild-type (WT) and BRP-39−/− mice received continuous exposure to 21% O2 (control mice) or 100% O2 from postnatal (PN) 1 to PN7 days, along with intranasal lipopolysaccharide (LPS) administered on alternate days (PN2, -4, and -6). Lung histology, bronchoalveolar lavage (BAL) cell counts, BAL protein, and cytokines measurements were performed. Measurements and Main Results. Hyperoxia differentially contributed to macrophage polarization by enhancing LPS induced M1 and inhibiting interleukin-4 induced M2 phenotype. BRP-39 absence led to further enhancement of the hyperoxia and LPS induced M1 phenotype. In addition, BRP-39−/− mice were significantly more sensitive to LPS plus hyperoxia induced lung injury and mortality compared to WT mice. Conclusions. These findings collectively indicate that BRP-39 is involved in repressing the M1 proinflammatory phenotype in hyperoxia, thereby deactivating inflammatory responses in macrophages and preventing neonatal lung injury.

Atrial natriuretic peptide attenuates agonist-induced pulmonary edema in mice with targeted disruption of the gene for natriuretic peptide receptor-A

Atrial natriuretic peptide (ANP) inhibits agonist-induced pulmonary edema formation, but the signaling pathway responsible is not well defined. To investigate the role of the particulate guanylate cyclase-linked receptor, natriuretic peptide receptor-A (NPR-A), we measured acute lung injury responses in intact mice and pulmonary microvascular endothelial cells (PMVEC) with normal and disrupted expression of NPR-A. NPR-A wild-type (NPR-A+/+), heterozygous (NPR-A+/-), and knockout (NPR-A-/-) mice were anesthetized and treated with thrombin receptor agonist peptide (TRAP) or lipopolysaccharide (LPS). Lung injury was assessed by lung wet-to-dry (W/D) weight and by protein and cell concentration of bronchoalveolar lavage (BAL) fluid. No difference in pulmonary edema formation was seen between NPR-A genotypes under baseline conditions. TRAP and LPS increased lung W/D weight and BAL fluid cell counts more in NPR-A-/- mice than in NPR-A+/- or NPR-A+/+ mice, but no genotype-related differences were seen in TRAP-induced increases in bloodless lung W/D weight or LPS-induced increases in BAL protein concentration. Pretreatment with ANP infusion completely blocked TRAP-induced increases in lung W/D weight and blunted LPS-induced increases in BAL cell counts and protein concentration in both NPR-A-/- and NPR-A+/+ mice. Thrombin decreased transmembrane electrical resistance in monolayers of PMVECs in vitro, and this effect was attenuated by ANP in PMVECs isolated from both genotypes. Administration of the NPR-C-specific ligand, cANF, also blocked TRAP-induced increases in lung W/D weight and LPS-induced increases in BAL cell count and protein concentration in NPR-A+/+ and NPR-A-/- mice. We conclude that ANP is capable of attenuating agonist-induced lung edema in the absence of NPR-A. The protective effect of ANP on agonist-induced lung injury and pulmonary barrier function may be mediated by NPR-C.

Inhalation of chlorine causes long-standing lung inflammation and airway hyperresponsiveness in a murine model of chemical-induced lung injury

Chlorine is highly irritating when inhaled, and is a common toxic industrial gas causing tissue damage in the airways followed by an acute inflammatory response. In this study, we investigated mechanisms by which chlorine exposure may cause reactive airways dysfunction syndrome (RADS) and we examined the dose-dependency of the development of symptoms. Mice were exposed to 50 or 200ppm Cl2 during a single 15min exposure in a nose-only container. The experiment terminated 2, 6, 12, 24, 48, 72h and 7, 14, 28 and 90 days post exposure. Inflammatory cell counts in bronchoalveolar lavage (BAL), secretion of inflammatory mediators in BAL, occurrence of lung edema and histopathological changes in lung tissue was analyzed at each time-point. Airway hyperresponsiveness (AHR) was studied after 24 and 48h and 7, 14, 28 and 90 days. The results showed a marked acute response at 6h (50ppm) and 12h (200ppm) post exposure as indicated by induced lung edema, increased airway reactivity in both central and peripheral airways, and an airway inflammation dominated by macrophages and neutrophils. The inflammatory response declined rapidly in airways, being normalized after 48h, but inflammatory cells were sustained in lung tissue for at least seven days. In addition, a sustained AHR was observed for at least 28 days. In summary, this mouse model of chlorine exposure shows delayed symptoms of hyperreactive airways similar to human RADS. We conclude that the model can be used for studies aimed at improved understanding of adverse long-term responses following inhalation of chlorine.

Autotaxin Emerges as a Therapeutic Target for Idiopathic Pulmonary Fibrosis

Autotaxin Emerges as a Therapeutic Target for Idiopathic Pulmonary Fibrosis

PCR based bronchoscopic detection of common respiratory pathogens in chronic cough: a case control study

BackgroundViral respiratory tract infection is the most frequent cause of acute cough and is reported at onset in about one third of patients with chronic cough. Persistent infection is therefore one possible explanation for the cough reflex hypersensitivity and pulmonary inflammation reported in chronic cough patients.MethodsBronchoscopic endobronchial biopsies and bronchoalveolar lavage cell counts were obtained from ten healthy volunteers and twenty treatment resistant chronic cough patients (10 selected for lavage lymphocytosis). A screen for known respiratory pathogens was performed on biopsy tissue. Chronic cough patients also underwent cough reflex sensitivity testing using citric acid.ResultsThere was no significant difference in incidence of infection between healthy volunteers and chronic cough patients (p = 0.115) or non-lymphocytic and lymphocytic groups (p = 0.404). BAL cell percentages were not significantly different between healthy volunteers and chronic cough patients without lymphocytosis. Lymphocytic patients however had a significantly raised percentage of lymphocytes (p < 0.01), neutrophils (p < 0.05), eosinophils (p < 0.05) and decreased macrophages (p < 0.001) verses healthy volunteers. There was no significant difference in the cough reflex sensitivity between non-lymphocytic and lymphocytic patients (p = 0.536).ConclusionsThis study indicates latent infection in the lung is unlikely to play an important role in chronic cough, but a role for undetected or undetectable pathogens in either the lung or a distal site could not be ruled out.Trials registrationCurrent Controlled Trials ISRCTN62337037 & ISRCTN40147207

Doxycycline impairs neutrophil migration to the airspaces of the lung in mice exposed to intratracheal lipopolysaccharide

BackgroundTetracyclines are broad-spectrum antibiotics that are also used to induce gene expression using the reverse tetracycline transactivator / tetracycline operator system (rtTA/tetO system). The system assumes that tetracyclines have no effects on mammals. However, a number of studies suggest that tetracyclines may have powerful anti-inflammatory effects. We report that the tetracycline, doxycycline, inhibits neutrophil (PMN) influx into the lungs of mice treated with bacterial endotoxin (LPS).MethodsMice were challenged with intratracheal LPS in the presence or absence of doxycyline. bronchoalveolar lavage cell counts and differential, total bronchoalveolar lavage protein, lung homogenate caspase-3 and tissue imaging were used to assess lung injury. In addition, PMN chemotaxis was measured in vitro and syndecan-1 was measured in bronchoalveolar lavage fluid.ResultsThe administration of doxycycline resulted in a significant decrease in the number of bronchoalveolar lavage PMNs in LPS-treated mice. Doxycycline had no effect on other markers of lung injury such as total bronchoalveolar lavage protein and whole lung caspase-3 activity. However, doxycycline resulted in a decrease in shed syndecan-1 in bronchoalveolar lavage fluid.ConclusionWe conclude that doxycycline has an important anti-inflammatory effect that can potentially confound the experiments in which the rtTA/tetO system is being used to study the immune response.

Iloprost improves endothelial barrier function in lipopolysaccharide-induced lung injury

The protective effects of prostacyclin and its stable analogue iloprost are mediated by elevation of intracellular cyclic AMP (cAMP) leading to enhancement of the peripheral actin cytoskeleton and cell-cell adhesive structures. This study tested the hypothesis that iloprost may exhibit protective effects against lung injury and endothelial barrier dysfunction induced by bacterial wall lipopolysaccharide (LPS). Endothelial barrier dysfunction was assessed by measurements of transendothelial permeability, morphologically and by analysis of LPS-activated inflammatory signalling. In vivo, C57BL/6J mice were challenged with LPS with or without iloprost or 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP) treatment. Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count and Evans blue extravasation. Iloprost and Br-cAMP attenuated the disruption of the endothelial monolayer, and suppressed the activation of p38 mitogen-activated protein kinase (MAPK), the nuclear factor (NF)-κB pathway, Rho signalling, intercellular adhesion molecular (ICAM)-1 expression and neutrophil migration after LPS challenge. In vivo, iloprost was effective against LPS-induced protein and neutrophil accumulation in bronchoalveolar lavage fluid, and reduced myeloperoxidase activation, ICAM-1 expression and Evans blue extravasation in the lungs. Inhibition of Rac activity abolished the barrier-protective and anti-inflammatory effects of iloprost and Br-cAMP. Iloprost-induced elevation of intracellular cAMP triggers Rac signalling, which attenuates LPS-induced NF-κB and p38 MAPK inflammatory pathways and the Rho-dependent mechanism of endothelial permeability.

Lymphocytic Bronchiolitis After Lung Transplantation Is Associated With Daily Changes in Air Pollution

Acute rejection represents a major problem after organ transplantation, being a recognized risk for chronic rejection and mortality. Recently, it became clear that lymphocytic bronchiolitis (LB, B-grade acute rejection) is more important than previously thought, as it predisposes to chronic rejection. We aimed to verify whether daily fluctuations of air pollution, measured as particulate matter (PM) are related to histologically proven A-grade rejection and/or LB and bronchoalveolar lavage (BAL) fluid cellularity after lung transplantation. We fitted a mixed model to examine the association between daily variations in PM(10) and A-grade rejection/LB on 1276 bronchoscopic biopsies (397 patients, 416 transplantations) taken between 2001 and 2011. A difference of 10 μg/m(3) in PM(10) 3 days before diagnosis of LB was associated with an OR of 1.15 (95% CI 1.04-1.27; p = 0.0044) but not with A-grade rejection (OR = 1.05; 95% CI 0.95-1.15; p = 0.32). Variations in PM(10) at lag day 3 correlated with neutrophils (p = 0.013), lymphocytes (p = 0.0031) and total cell count (p = 0.024) in BAL. Importantly, we only found an effect of PM10 on LB in patients not taking azithromycin. LB predisposed to chronic rejection (p < 0.0001). The risk for LB after lung transplantation increased with temporal changes in particulate air pollution, and this was associated with BAL neutrophilia and lymphocytosis. Azithromycin was protective against this PM effect.

Cytokine profile of bronchoalveolar lavage fluid in patients with combined pulmonary fibrosis and emphysema

Combined pulmonary fibrosis and emphysema (CPFE) is characterized by upper lobe emphysema together with lower lobe fibrosis. The aim of this study was to examine whether cytokine levels in the alveolar space are associated with emphysematous changes superimposed on pulmonary fibrosis. Consecutive patients (n = 102), diagnosed with pulmonary fibrosis were retrospectively evaluated. Cytokine levels and differential cell counts in bronchoalveolar lavage (BAL) fluid, pulmonary function, computed tomography (CT) scores and levels of serum markers were compared between patients with or without emphysema. Among the 102 patients (14 females, mean age 68 years), 38 (37%) had evidence of upper lobe emphysema on computed tomography (CT). Levels of epithelial neutrophil activating peptide 78 (ENA-78/CXCL5) and interleukin (IL)-8/CXCL8 in BAL fluid were significantly higher in patients with emphysema. Vital capacity (VC, % predicted) was greater, and ratio of forced expiratory volume in 1 s/forced vital capacity and diffusing capacity of carbon monoxide (DL(CO))/alveolar volume (V(A)) were lower in patients with emphysema. CXCL8 and CXCL5 levels were associated with percentage or absolute numbers of neutrophils in BAL fluid. In addition, CXCL8 levels were inversely correlated with VC and DL(CO)/V(A), and positively correlated with composite physiological index (CPI) and the extent of areas of low attenuation on CT. Increased CXC chemokine levels in the airspaces may be associated with emphysematous lung changes in patients with pulmonary fibrosis.