Exhaled breath condensate cysteinyl leukotrienes and airway remodeling in childhood asthma: a pilot study

BACKGROUND: It has been suggested that cysteinyl leukotrienes (cysLTs) play an important role in airway remodeling. Previous reports have indicated that cysLTs augment human airway smooth muscle cell proliferation. Recently, cysLTs have been measured in exhaled breath condensate (EBC). The aim of this study was to evaluate the relationship between cysLTs in EBC and another marker of airway remodeling, reticular basement membrane (RBM) thickening, in endobronchial biopsies in children. METHODS: 29 children, aged 4–15 years, with moderate to severe persistent asthma, who underwent bronchoscopy as part of their clinical assessment, were included. Subjects underwent spirometry and EBC collection for cysLTs analysis, followed by bronchoscopy and endobronchial biopsy within 24 hours. RESULTS: EBC cysLTs were significantly lower in asthmatic children who were treated with montelukast than in those who were not (median (interquartile range) 36.62 (22.60–101.05) versus 249.1 (74.21–526.36) pg/ml, p = 0.004). There was a significant relationship between EBC cysLTs and RBM thickness in the subgroup of children who were not treated with montelukast (n = 13, r = 0.75, p = 0.003). CONCLUSION: EBC cysLTs appear to be associated with RBM thickening in asthma

Pro-asthmatic cytokines regulate unliganded and ligand-dependent glucocorticoid receptor signaling in airway smooth muscle

To elucidate the regulation of glucocorticoid receptor (GR) signaling under pro-asthmatic conditions, cultured human airway smooth muscle (HASM) cells were treated with proinflammatory cytokines or GR ligands alone and in combination, and then examined for induced changes in ligand-dependent and -independent GR activation and downstream signaling events. Ligand stimulation with either cortisone or dexamethsone (DEX) acutely elicited GR translocation to the nucleus and, comparably, ligand-independent stimulation either with the Th2 cytokine, IL-13, or the pleiotropic cytokine combination, IL-1β/TNFα, also acutely evoked GR translocation. The latter response was potentiated by combined exposure of cells to GR ligand and cytokine. Similarly, treatment with either DEX or IL-13 alone induced GR phosphorylation at its serine-211 residue (GRSer211), denoting its activated state, and combined treatment with DEX+IL-13 elicited heightened and sustained GRSer211phosphorylation. Interestingly, the above ligand-independent GR responses to IL-13 alone were not associated with downstream GR binding to its consensus DNA sequence or GR transactivation, whereas both DEX-induced GR:DNA binding and transcriptional activity were significantly heightened in the presence of IL-13, coupled to increased recruitment of the transcriptional co-factor, MED14. The stimulated GR signaling responses to DEX were prevented in IL-13-exposed cells wherein GRSer211 phosphorylation was suppressed either by transfection with specific serine phosphorylation-deficient mutant GRs or treatment with inhibitors of the MAPKs, ERK1/2 and JNK. Collectively, these novel data highlight a heretofore-unidentified homeostatic mechanism in HASM cells that involves pro-asthmatic cytokine-driven, MAPK-mediated, non-ligand-dependent GR activation that confers heightened glucocorticoid ligand-stimulated GR signaling. These findings raise the consideration that perturbations in this homeostatic cytokine-driven GR signaling mechanism may be responsible, at least in part, for the insensirtivity to glucocorticoid therapy that is commonly seen in individuals with severe asthma

PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells

Background: Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G(q)-and G(s)-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.Methods: IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.Results: The beta(2)-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.Conclusion: Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p

Cysteinyl leukotrienes: multi-functional mediators in allergic rhinitis

Cysteinyl leukotrienes (CysLTs) are a family of inflammatory lipid mediators synthesized from arachidonic acid by a variety of cells, including mast cells, eosinophils, basophils, and macrophages. This article reviews the data for the role of CysLTs as multi-functional mediators in allergic rhinitis (AR). We review the evidence that: (1) CysLTs are released from inflammatory cells that participate in AR, (2) receptors for CysLTs are located in nasal tissue, (3) CysLTs are increased in patients with AR and are released following allergen exposure, (4) administration of CysLTs reproduces the symptoms of AR, (5) CysLTs play roles in the maturation, as well as tissue recruitment, of inflammatory cells, and (6) a complex inter-regulation between CysLTs and a variety of other inflammatory mediators exists.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75432/1/j.1365-2222.2006.02498.x.pd

Effect of permanent bed planting combined with controlled traffic on soil chemical and biochemical properties in irrigated semi-arid Mediterranean conditions

Improving agricultural soil quality in semi-arid regions is necessary for reducing soil erosion and improving water use. Conservation agriculture (CA) can increase soil quality and biodiversity, and reduce operational costs without losing crop productivity both under irrigated or rainfed conditions. However, few studies on soil chemical and biochemical status in irrigated farms under CA in the Mediterranean region are available. Permanent beds with crop residue retention (PB) have been proposed as an alternative to conventionally tilled beds with residue incorporation into the soil (CB). These two soil management systems combined with controlled traffic were compared during two different seasons (2009 and 2010) in a loamy alluvial Typic Xerofluvent soil under a maize (Zea mais L.)-cotton (Gossypium hirsutum L.) crop rotation trial established in 2007 in Southern Spain. Total organic carbon (TOC), water soluble carbon (WSC), Kjeldahl nitrogen (Kjel-N), dehydrogenase (DHA) and β-glucosidase (β-Glu) activities and microbial biomass carbon (MBC) and nitrogen (MBN) were analysed in soil from beds and furrows after crop harvest. Results indicated that Kjel-N, TOC and enzymatic activities were significantly higher in soil from furrows in PB than in CB, but practically no differences were found in soil from the bed zone. Moreover, traffic did not affect chemical and biochemical parameters in spite of its compacting effect. Major differences were found between samplings due to different quantity and nature of the residues (maize vs. cotton). Principal component analysis confirmed that TOC, Kjel-N and β-Glu (and DHA to a less extend) are useful indicators of soil management impact on soil quality in this irrigated Mediterranean conditions; however, this is not the case of WSC, a common indicator in rainfed conditions. Results confirmed that conservation agriculture is the better option to increase soil biological and biochemical quality in irrigated farms under Mediterranean semi-arid conditions.This work was supported by the Spanish Ministry of Science and Innovation (project AGL2010-22050-C03) and FEDER funds. The authors thank Dr J.M. Murillo for his useful comments on the manuscript. M. Panettieri and I. Carmona thank CSIC for his “JAE-Predoc” fellowship and her “JAE-Tec” contract, respectively.Peer Reviewe