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Second generation antihistamines are recognised as being highly effective treatments for allergy-based disease and are among the most frequently prescribed and safest drugs in the world. However, consideration of the therapeutic index or the benefit/risk ratio of the H1 receptor antagonists is of paramount importance when prescribing this class of compounds as they are used to treat non-life threatening conditions. There are many second generation antihistamines available and at first examination these appear to be comparable in terms of safety and efficacy. However, the newer antihistamines in fact represent a heterogeneous group of compounds, having markedly differing chemical structures, adverse effects, half-life, tissue distribution and metabolism, spectrum of antihistaminic properties, and varying degrees of anti-inflammatory effects. With regard to the latter, there is growing awareness that some of these compounds might represent useful adjunct medications in asthma therapy. In terms of safety issues, the current second generation grouping includes compounds with proven cardiotoxic effects and others with the potential for adverse drug interactions. Moreover, some of the second generation H1 antagonists have given cause for concern regarding their potential to cause a degree of somnolence in some individuals. It can be argued, therefore, that the present second generation grouping is too large and indistinct since this was based primarily on the concept of separating the first generation sedating compounds from nonsedating H1 antagonists. Although it is too early to talk about a third generation grouping of antihistamines, future membership of such a classification could be based on a low volume of distribution coupled with a lack of sedating effects, drug interactions and cardiotoxicity.

In the lung the expression of xenobiotic-metabolizing enzymes such as cytochromes P450 (CYP) and glutathione S-transferases (GST) may be affected by inhaled pollutants. Toluene diisocyanate (TDI) is a highly volatile chemical compound known to induce a wide array of diseases in workers exposed to vapors or sprays, including respiratory allergy and asthma. We investigated the effect of inhaled TDI on expression of CYP 1A1, 2B1, 2E1, and 3A1 and of alpha-, mu-, and pi-GST in rat lung. Animals were exposed to targeted concentrations of 0.01, 0.1, or 1 ppm TDI vapors or to cleaned filtered air for 8 h. Expression of CYP and GST was analyzed 18 24 h after the end of exposure using western blotting, northern blotting, and immunohistochemistry. Constitutive levels of CYP 2B1 and 3A1 proteins were found in lung tissue from control rats, whereas CYP 1A1 and 2E1 proteins were not detectable. Animal exposure to TDI vapors neither modified CYP 3A1 protein expression, nor led to any detectable expression of CYP 1A1 or 2E1. In contrast, exposure to 1 ppm TDI induced a 40% reduction in CYP 2B1 protein levels. This decrease was associated with a 33% decrease in CYP 2B1 mRNA levels. Additionally, CYP 2B1 immunolabeling localized to ciliated epithelial cells, Clara cells, and type II alveolar cells in the lung tissue of control rats was markedly decreased in animals exposed to 1 ppm TDI. Constitutive levels of alpha-, mu-, and pi-GST proteins were found in lung tissue from control rats. Exposure to TDI had no effect on lung expression of either of the GST. In conclusion, this study clearly shows a selective decrease in CYP 2B1 expression by TDI vapors in rat lung. The contribution of CYP 2B1 to metabolize further xenobiotics is therefore altered.

We have compared the inhibitory effects of ebastine (10 mg), ebastine (20 mg) and cetirizine (10 mg) on histamine-induced wheal and flare skin reactions 24 h following a 6-day-long treatment. This was a double-blind, randomised, crossover, placebo-controlled study involving 24 healthy volunteers (18-65 years) with negative skin prick tests and the absence of specific IgEs to common allergens. Subjects were randomised to receive each of the following treatments once daily for 6 days: ebastine (10 mg), ebastine (20 mg), cetirizine (10 mg) or placebo with a washout period of 5 days. Twenty-four hours after the last dose of each treatment, histamine skin prick tests were performed (0, 0.5, 1, 2.5, 5, 10, 20, 50, 100 and 200 mg/mL), and wheal and flare responses were measured. All active treatments produced significant inhibition of the wheal responses compared to placebo (P < 0.001). Wheal response inhibition was significantly better with 20 mg of ebastine compared with 10 mg of ebastine and 10 mg of cetirizine. In a comparison to histamine concentrations required to produce a wheal surface area of 10 mm2, 20 mg of ebastine was also significantly better than ebastine 10 mg and cetirizine (P < 0.001), and 10 mg ebastine was significantly better than cetirizine (P < 0.05). Highly significant (P < 0.001) effects on the flare response were observed with each active treatment compared to placebo, with no difference between groups. The frequency of adverse events, primarily somnolence, was similar among the four treatment groups. Our results clearly indicate that ebastine, at either recommended dosage of 10 and 20 mg, and cetirizine produced significant inhibition of the histamine-induced wheal and flare reaction compared to placebo for up to 24 h. A superior efficacy of 20 mg of ebastine is observed compared with 10 mg of ebastine and 10 mg of cetirizine on the skin wheal response 24 h after the last dose of a 6-day-long treatment. This study clearly proves ebastine to be an effective, truly once-daily antihistamine.

The aim of this study was to investigate whether repeated exposure to subclinical doses of cat allergens, not inducing asthma symptoms, could affect eosinophil cationic protein (ECP) levels in bronchoalveolar lavage (BAL) or in peripheral blood, without the appearance of clinical symptoms. Twelve patients with mild asthma, all sensitized to cats and not exposed to cat allergen at home, underwent a series of inhalations of cat allergen or placebo for 8 days over 2 weeks. A methacholine challenge was performed before and after the allergen and saline exposures, and BAL and blood were sampled for ECP measurements and eosinophil counts. No patients experienced asthma symptoms. However, PD20 methacholine (geometric mean) decreased significantly from 263 μg before to 126 μg after inhalation of allergen. Inhalation of saline did not induce any significant change in PD20. The change in log PD20 before and after cat allergen exposure was statistically different from the change in log PD20 before and after saline. Median ECP levels in BAL and serum increased significantly after allergen exposure, from 0.8 to 3.1 μg/l (p

A mast cell infiltration of the bronchial smooth muscle layer has been reported in patients sensitized to common allergens. Stem cell factor (SCF) is a chemotactic and survival factor for mast cells. SCF is expressed as a soluble (sSCF) and a membrane-bound (mSCF) form, after alternative splicing of the exon encoding the proteolytic cleavage site. SCF expression by human bronchial smooth muscle cells in culture was evaluated, comparing it to that of human lung fibroblasts in culture. sSCF released in the culture supernatant was assessed by an enzyme-linked immunosorbent assay. Total SCF messenger ribonucleic acid (mRNA) was measured by competitive polymerase chain reaction (PCR) after reverse transcription. Expression of the two forms of SCF mRNA was assessed by PCR, with primers spanning the alternatively spliced exon. Smooth muscle cells produced sSCF (21.9+/-2.6 pg x mL(-1)), although at lower levels than fibroblasts (35.9+/-3.5 pg x mL(-1)); the expression of total SCF mRNA was also at lower levels than in fibroblasts (8.6+/-0.2 and 19.0+/-2.0 amol x fmol glyceraldehyde 3-phosphate dehydrogenase complementary deoxyribonucleic acid(-1), respectively). However, smooth muscle cells expressed proportionally more (1.7-fold) mSCF mRNA than did fibroblasts. In conclusion, this study shows that bronchial smooth muscle cells express stem cell factor, with a relatively high expression of membrane-bound stem cell factor. This might be related to the presence of mast cells within the bronchial smooth muscle layer, i.e. at the site of bronchoconstriction, with possible implications in the pathophysiology of asthma.

Bronchial innervation is interrupted at lung transplantation. Nerve fibers with cell bodies above the section, such as sensory C fibers, should degenerate. Using histofluorescence, we evaluated calcitonin gene-related peptide (CGRP) immunoreactivity in syngeneic Lewis rats 1 and 5 mo after unilateral lung transplantation and in controls. CGRP-immunoreactive (IR) neuroendocrine cells were located within the epithelium of large and small bronchi. At 1 mo after transplantation, their number had significantly increased in large bronchi and had normalized 5 mo after transplantation. The density of CGRP-IR fibers in control lungs gradually decreased from large (0. 35 +/- 0.02 micron/micron basal lamina) to small (0.23 +/- 0.02) and peripheral bronchi (0.12 +/- 0.01). At 1 mo after lung transplantation, few CGRP-IR fibers were observed in large bronchi (0.17 +/- 0.02), fewer in small bronchi (0.04 +/- 0.01) (P0.01), and none in peripheral bronchi. At 5 mo after lung transplantation, transplanted lungs still had fewer CGRP-IR fibers in large (0.22 +/- 0.02) and small (0.11 +/- 0.02) bronchi (P0.02) than did controls, but there were, nonetheless, more in the small bronchi than at 1 mo after transplantation (P0.01). Additionally, few CGRP fibers were present in the peripheral bronchi (0.03 +/- 0.01) (P0.01). These results clearly demonstrate the occurrence of denervation followed by partial reinnervation with CGRP-IR fibers after transplantation in rat lung.

Cathepsin S (Cat S), expressed predominately on antigen presenting cells, has been proposed as a therapeutic target for asthma. We used genetic and pharmacological tools to investigate the role of Cat S in murine models of allergic asthma. Mice null for Cat S were protected from OVA-induced pulmonary inflammation, but exhibited no protection from OVA-induced airway hyper-reactivity. To determine the role of Cat S during the challenge phase, we identified a potent and selective Cat S inhibitor, Compound A (Cpd A, IC50 mCat S = 0.6 nM, ≥470 fold selective vs mCat B, K, L), which inhibited antigen presentation in a mouse cell-based assay (IC50 = 44 nM). The prodrug of Cpd A, Cpd B, gave excellent plasma levels of Cpd A when dosed in mice by gavage, or in food. In vivo competition in mice with an irreversible pan-selective cysteine cathepsin probe showed that Cpd B gave selective inhibition of lung and spleen Cat S at 1 mpk, but lost selectivity at 50 mpk. Cpd B was dosed in mice (10, 100 mpk in food) over 4 days of the challenge period in the murine ovalbumin model. Both doses had no effect on bronchoalveolar lavage infiltrating cells, despite showing high levels of Cat S inhibition. Thus, Cat S inhibition in a therapeutic mode does not attenuate airway inflammation in antigen sensitised mice suggesting that anti-Cat S therapy would not be an effective asthma treatment.

The influence of adenosine and selective A1 and A2 agonists and antagonists was investigated on the cholinergic and the excitatory non-cholinergic (e-NC) contractions induced by electrical field stimulation in the guinea-pig bronchi. Adenosine (10 nM-1 mM) induced a concentration-dependent inhibition of the e-NC contraction (EC50 = 90 +/- 14 microM), whereas the cholinergic peak was only slightly affected. Preincubation of the tissue with the adenosine uptake blocker dipyridamole (10 microM) significantly shifted the concentration-inhibition curve to adenosine to the left (EC50 = 10 +/- 1 microM), suggesting an interaction with extracellular adenosine receptors of A1 and/or A2 subtype. To characterize the receptor type involved in this effect, selective adenosine derivatives were studied. The agonist to both A1 and A2 adenosine receptors, 5'-N-ethylcarboxamidoadenosine (NECA) was more potent than the selective A1 agonist, (-)-R-6-phenylisopropyladenosine (R-PIA), in inhibiting the e-NC contraction (EC50 = 0.10 +/- 0.04 and 0.60 +/- 0.12 microM, respectively, with a maximal inhibition of 70 and 45%, respectively). The concentration-response curve to NECA was shifted to the right by the A2 receptor selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (10 microM) (EC50 = 1.4 +/- 0.5 microM) as well as by the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 microM) (EC50 = 0.7 +/- 0.3 microM). The inhibitory effect induced by the association of both antagonists, DPCPX and DMPX, was considerably potentiated (EC50 > 22 +/- 2.5 microM). The effect of R-PIA was also shifted to the right by DPCPX (EC50 = 8.2 +/- 1.6 microM) but was not modified by DMPX. The contractile response to exogenous substance P was unaffected by NECA pretreatment (0.3 microM). Altogether, these results suggest that adenosine-induced inhibition of e-NC contraction of guinea-pig bronchi is mediated through activation of both A1 and A2 adenosine receptors linked to inhibition of the release of neuropeptides from C-fibre nerve endings.

Evidence suggests that cytokines may modulate smooth muscle cell function in a variety of inflammatory diseases. In the present study, we characterized the specific receptor subtypes that mediate tumor necrosis factor alpha (TNF alpha) effects on myocyte proliferation and on agonist-induced calcium transients in cultured human tracheal smooth muscle cells (TSMC). Pretreatment of human TSMC with TNF alpha potentiated cytosolic calcium [(Ca2+)i] transients evoked by carbachol. In a similar manner, selective TNF alpha-p55 receptor agonists such as htr-9, an activating monoclonal antibody, or a recombinant TNF-p55 (rTNF-p55), which specifically activates the TNF alpha-p55 receptor but not the TNF alpha-p75 receptor, also augmented [Ca2+]i transients evoked by carbachol. In parallel experiments, TNF alpha, rTNF alpha-p55, and htr-9 induced human TSMC proliferation as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Interestingly, activation of the TNF alpha-p75 receptor with a selective agonist, recombinant TNF alpha-p75 (rTNF alpha-p75), or inhibition of the TNF alpha-p75 receptor with utr-1, an inhibitory anti-TNF alpha-p75 receptor antibody, had no effect on TNF alpha-augmented calcium transients or on myocyte growth. To further confirm the receptor specificity of these findings, immunocytochemical studies were performed using receptor-specific antibodies. These studies demonstrated marked cell-surface expression of the TNF alpha-p55 receptor compared with expression of the TNF alpha-p75 receptor on human TSMC. Taken together, our results suggest that TNF alpha modulates agonist-induced calcium transients and induces human TSMC proliferation by specific activation of the TNF alpha-p55 receptor. Further studies addressing the cellular and molecular mechanisms regulating cytokine modulation of airway smooth muscle function may provide new insight into mechanisms that induce airway hyperresponsiveness in asthma.

We have investigated the nasal response to substance P after pollen exposure in seasonal allergic rhinitic patients. Seven patients with strictly seasonal allergic rhinitis were studied during the pollen season, 24 h after nasal challenge with pollen. They received increasing doses of nebulized substance P (0 to 80 nmol) in each nostril. Responses were assessed by measurement of nasal airway resistance by posterior rhinomanometry and quantification of albumin, histamine, and inflammatory cells in the nasal lavage fluid. Nasal airway resistance increased in a dose-dependent manner after substance P challenge. Protein and albumin in nasal lavage fluids increased after administration of substance P: from 2.6 +/- 0.3 to 6.8 +/- 1.1 mg for protein (P < 0.01) and from 0.2 +/- 0.1 to 3.1 +/- 0.6 mg for albumin (P < 0.02). Expressed as a percentage of total protein, albumin increased from 10.5 +/- 3.6% to 39.9 +/- 3.5% (P < 0.02), suggesting occurrence of plasma leakage. No histamine release was observed after challenge with substance P. Total cell counts significantly increased from 11.4 +/- 2.4 to 41.8 +/- 17.3 x 10(3) cells/ml after substance P (P < 0.05). Eosinophils were already numerous before substance P challenge (2.1 +/- 0.7 x 10(3) cells/ml), and the number of eosinophils markedly increased in all patients after substance P (for the whole group, 25.8 +/- 13.3 cells/ml, P < 0.05). In contrast, the number of neutrophils only slightly increased in five patients, and changes did not reach significance for the group as a whole. Our results show that substance P induces nasal obstruction and albumin extrusion in allergic rhinitic patients after repeated pollen exposure. These vascular phenomena are associated with recruitment of eosinophils. Since substance P is known to be released after nasal allergen challenge, our data suggest a role for substance P in the chronic eosinophilic inflammation of the nasal mucosa observed in symptomatic allergic rhinitis.

The effects of nasal administration of increasing doses of exogenous substance P have been studied in patients with allergic rhinitis treated with placebo or with the H1 antagonist cetirizine (10 mg twice daily for 3 days). Responses to substance P were assessed by posterior rhinomanometry (measuring nasal airway resistance) and by measure of histamine, protein and albumin production and cell recovery in nasal lavage fluids before and after challenge. Substance P induced a dose-dependent increase in nasal airway resistance which was similar after treatment with either cetirizine or placebo (maximal increase in nasal airway resistance was 4.2-fold greater than the baseline with the placebo and 4.7-fold greater than the baseline with cetirizine). No histamine release was observed. Similar increases in protein and albumin production were observed after stimulation with substance P along with the placebo (protein: from 0.35 +/- 0.11 to 3.31 +/- 0.62 mg and albumin: from 0.09 +/- 0.04 to 2.08 +/- 0.39 mg) and when combined with cetirizine treatment (proteins: from 0.42 +/- 0.09 to 3.62 +/- 0.77 and albumin: from 0.17 +/- 0.04 to 2.19 +/- 0.51 mg). After stimulation with substance P, percentages of neutrophils recovered in nasal fluids increased from 26.2 +/- 11.5 to 54.5 +/- 9.5 with the placebo and from 35.5 +/- 11.0 to 53.6 +/- 9.5 with cetirizine. Eosinophils were inconsistently found after substance P stimulation during both treatments. In conclusion, nasal response to substance P is not modified by cetirizine which suggests that the effect of substance P is not secondary to histamine release in the nose in man.

Guinea-pigs pretreated with phosphoramidon or saline were treated with an aerosol of substance P (SP) or saline. 24 h later, the pulmonary inflation pressure (PIP) to substance P or to cumulative doses of acetylcholine or of histamine was recorded. The PIP response to SP itself was significantly enhanced in animals treated with phosphoramidon+SP as compared with phosphoramidon+saline (2.5-fold increase 1 min after the end of the inhalation, P0.001). The response to acetylcholine and to histamine was also significantly enhanced in phosphoramidon+substance P-treated as compared with phosphoramidon+saline-treated guinea-pigs (PC200 = 38.9 and 1.6 as compared with 77.6 and 3.9 micrograms/ml, P0.01 and P0.05 respectively). The production of superoxide anions by alveolar macrophages in response to f-MLP was also enhanced after treatment with phosphoramidon+SP as compared with phosphoramidon+saline (6.4 +/- 0.7 and 3.8 +/- 0.3 cpm, P0.001 respectively). In animals treated with saline+SP or saline+saline, the PIP responses and the production of superoxide anion were similar. Altogether these results suggest that SP contributes to the bronchial hyper-responsiveness in asthma and this probably through activation of alveolar macrophages.

We have characterized the muscarinic cholinergic receptor population by binding assay in left and right lung membranes from syngeneic Lewis rats one month after unilateral left lung transplantation and from controls. The density of muscarinic receptors measured by [3H](-)QNB binding was similar in left and right lung membranes one month after unilateral left lung transplantation (Bmax = 28.2 +/- 2.2 and 29.0 +/- 1.4 fmol/mg protein, respectively) and was similar between transplanted and control rats. Binding affinity also was similar on both sides (Kd = 28.5 +/- 9.5 and 32.3 +/- 2.7 pM, respectively). Binding parameters (Ki) of four selective muscarinic antagonists determined from competitive binding experiments of 0.5 nM [3H](-)QNB showed an order of potency: atropine4-DAMPAF-DX 116 BSpirenzepine both in transplanted and in control lungs. There was no significant difference in Ki values between left transplanted and right non transplanted lungs (1.8 +/- 0.1 and 1.9 +/- 0.1 nM; 4.3 +/- 0.5 and 3.5 +/- 0.5 nM; 80.9 +/- 13.8 and 78.9 +/- 18.8 nM; 480.4 +/- 40.9 and 481.7 +/- 78.2 nM, respectively for atropine, 4-DAMP, AF-DX 116 BS and pirenzepine). Values were also similar between transplanted and control lungs. These observations suggest a similar population of muscarinic receptors in the transplanted and the non transplanted lung in the Lewis rat. This result is in accordance with the similar physiological effect of exogenous acetylcholine that we previously observed in transplanted and non transplanted bronchi.

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.

We previously reported that NF-κB activation is essential for the overexpression of the major mast cell growth factor SCF in inflammation (Da Silva et coll., Faseb J 2003). We show here the activation of the nuclear kinase MSK1 as the key step of NF-kB activation, and the consequences in a murine asthma model. NF-κB p65 was phosphorylated at Ser276 upon IL-1β treatment as shown by Western blotting. Chromatin immunoprecipitation experiment showed binding of NF-κB, of MSK1 and of CBP to the SCF gene κB intronic enhancer. Further on, IL-1β upregulated SCF expression and this was abolished by S276C p65 mutation, MSK1 kinase-dead mutation, anti-MSK1 siRNA transfection, or by pharmacological inhibition by the H89 kinase inhibitor compound, indicating a role for MSK1 mediated S276 p65 phosphorylation in SCF expression. Additionally, we evaluated the effect of this kinase inhibitor, compound H89 (10 mg/kg), as compared to solvent in a murine asthma model created by sensitization and challenge of mice with ovalbumin + Al(OH)3. The eosinophilic infiltrate and the associated remodelling of the airways including mucus secretion were inhibited by 80% and 60%, respectively. In conclusion, we therefore propose MSK1 as an interesting therapeutic target to combat mast cell-associated inflammation in particular in asthma.

Introduction Our previous works showed that peroxisome proliferator-activated receptor-a (PPARα) downregulated airway eosi-nophilic inflammation induced by allergen in sensitized mice [1]. However, inflammation associated with severe asthma involves mixed eosinophil and neutrophil infiltrate and increased levels of IL-5, IL-8 and other inflammatory cell chemoattractants. Concomitant exposure to allergen and lipopolysaccharides (LPS) reproduces some features of this inflammatory response in sensitized mice [2]. We have therefore addressed the anti-inflammatory activity of PPARα in allergen-sensitized and challenged mice exposed to LPS. Methods Ovalbumin (OA)-sensitized wild-type (PPARa +/+ ) vs homozygous knockout (PPARa) mice, as well as OA-sensitized C57BL/6 mice treated with the PPARα agonist, fenofibrate (15 mg/day), were challenged by intranasal instillation of OA in the presence of 100 ng LPS. Airway inflammation was assessed by quantification of cell number and chemoattractants in bronchoal-veolar lavage fluid (BALF). Results Upon challenge with OA in the presence of LPS, PPARα -/- mice exhibited greater eosinophil, neutrophil and macrophage number (5.6-, 2.3- and 3.1- fold, respectively, p +/+ animais. PPARα-/- mice dis-played also increased levels of IL-5 (3.3-fold, p Conclusions All together, our data provide evidence that PPARα reduces the mixed inflammatory cell infiltrate and the che-moattractant production induced by co-exposure to allergen and LPS in sensitized mice. This suggests that PPARα activation may represent an effective therapeutic approach in severe asthma.

Introduction Peroxisome proliferator-activated receptor-a (PPARa) is a transcription factor activated by natural and synthetic ligands. Our previous work showed that PPARa deficiency resulted in a greater airway eosinophilic inflammation upon allergen challenge in sensitized mice, whereas PPARa activation by fenofibrate downregulated this inflammation [1]. This suggests that PPARα activators may be useful in the treatment of asthma. However, little is known so far about PPARa expression in the lung. The aim of our study was 1) to assess the presence of PPARa in the lung from naive and from allergen-sensitized and challenged mice and 2) to study regulation of PPARa lung expression by fenofibrate or dexa-methasone, known as PPARa inducers in the liver. Methods Naive or ovalbumin (OA)-sensitized and challenged C57BL/6 mice were treated with fenofibrate (7.5 mg/day), dexa-methasone (0.03 mg/day) or their vehicle (1% carboxymethylcellu-lose in water) administered per os for 10 days. PPARa lung expression was assessed by quantification of PPARa mRNA using polymerase chain reaction after reverse-transcription of total lung RNA. Airway inflammation was assessed by determining total and differential cell count in bronchoalveolar lavage fluid. Results PPARa mRNA was found at significant levels in lung tissue from naive mice. This expression was upregulated by 65% (p Conclusions Our data show that PPARa is expressed in mouse lung and regulated upon allergie inflammation or in response to fenofibrate or dexamethasone. Upregulation of PPARa expression by PPARa activators or glucocorticoids could account for their anti-inflammatory activity.

Introduction Le stem cell factor (SCF) agit sur la croissance, le chimiotactisme et l’activation des mastocytes. Il pourrait donc etre implique dans les maladies associees a une augmentation du nombre de mastocytes, comme l’asthme. Des travaux anterieurs ont montre que l’expression du SCF est augmentee par l’IL - 1β, sous controle du facteur de transcription NFκB et des MAP kinases p38 et ERK1/2. En l’absence de stimulus, NFκB est sequestre dans le cytoplasme par liaison au complexe IκB . L’IL-lβ provoque la phos-phorylation d’i κ B et sa degradation, l’entree de NFκB libre dans le noyau, sa liaison a l’ADN et l’augmentation de l’expression de genes cibles, comme le SCF. De nombreux sites de phosphorylation de la sous-unite p65 de NFκB ont ete decrits, qui pourraient controler son activite. Nous avons etudie l’interrelation entre les voies des MAP kinases p38 et ERK1/2 et la phosphorylation de NFκB, aboutissant au controle de l’expression du SCF. Materiel et methodes Le promoteur du SCF a ete sous-clone dans un vecteur codant pour une luciferase. Une seconde construction a ete realisee, dans laquelle l’element de reponse a NFκB a ete delete. L’activite luciferase a ete mesuree dans des fibroblastes pulmonaires humains transfectes par ces constructions, apres traitement par IL-ip. L’implication des MAP kinases p38 et ERK1/2 a ete etudiee par l’utilisation des inhibiteurs selectifs SB202190 et PD98059, respectivement. L’etat de phosphorylation d’i κ B et de la sous-unite p65 de NFκB a ete mesure par Western blotting. La liaison de p65 au promoteur du SCF a ete etudiee par immunopre-cipitation de la chromatine (ChIP). Resultats L’augmentation d’activite du promoteur du SCF par l’IL-β est abolie par une deletion de l’element de reponse a NFκB ainsi que par l’association des inhibiteurs SB202190 et PD98059. Ces inhibiteurs n’ont aucun effet sur la phosphorylation d’iKB et sa degradation, mais bloquent la phosphorylation de la serine 276 de p65. La liaison de p65 au promoteur du SCF est egalement inhibee par les composes SB202190 et PD98059. Conclusion NFκB est essentiel a la regulation de l’activite du promoteur du SCF par l’IL-1β. L’activation des voies p38 et ERK1/2 est necessaire a la phosphorylation de NFκB pour sa liaison au promoteur du SCF. Une meilleure connaissance des kinases et des phosphatases controlant cette phosphorylation offrirait de nouvelles cibles therapeutiques pour le controle des genes sous la dependance de NFκB, comme le SCF.

Introduction Un role du facteur de croissance des nerfs ou NGF a ete propose dans l’inflammation des voies aeriennes et l’hyperreac-tivite bronchique. Nous avons envisage dans cette etude un role du NGF dans le remodelage bronchique, en etudiant son effet prolife-ratif dans les cellules musculaires lisses bronchiques humaines en culture primaire. Methodes La proliferation induite par le NGF (0,1-10 pM) est etudiee en presence ou non d’inhibiteurs de kinases par dosage de 1) la metabolisation du sel de tetrazolium XTT dans les cellules viables, et 2) l’incorporation dans l’ADN de 5Bromo-2-deoxyuridine (BrdU), un analogue de la thymidine. L’etude de la phosphoryla-tion du recepteur TrkA du NGF et des MAPK (mitogen-activated protein kinase) est realisee par immunoprecipitation et/ou Western blotting. Resultats Le NGF induit une proliferation dose-dependante des cellules musculaires lisses bronchiques et induit la phosphorylation du recepteur TrkA, effets bloques par l’inhibiteur de tyrosine-kinase K252a. Les inhibiteurs de PI3K (phosphatidyl-inositol-3-kinase) et de la MAPK JNK (c-Jun N-terminal kinase) ne modifient pas l’effet proliferatif du NGF, montrant l’absence d’implication de ces kinases dans les voies de signalisation du recepteur TrkA dans la proliferation. En revanche, les inhibiteurs de la proteine kinase C (PKC) bloquent partiellement la proliferation induite par le NGF et abolissent selectivement la phosphorylation de la MAPK p38 sans modifier celle de la MAPK ERK1/2 (extracellular-regulated protein kinase 1/2). Un inhibiteur de la raf-kinase diminue egalement la proliferation induite par le NGF, et bloque selectivement la phosphorylation de ERK1/2 sans modifier celle de p38. Les inhibiteurs de ERK1/2 et p38 inhibent la proliferation des cellules musculaires lisses bronchiques induite par le NGF, confirmant l’implication de ces deux MAPK dans les voies de signalisation activees par le recepteur TrkA et impliquees dans l’effet proliferatif du NGF. Conclusion Nous avons montre dans cette etude l’activation du recepteur TrkA par le NGF dans les cellules musculaires lisses bronchiques humaines en culture, conduisant a lactivation de deux voies de signalisation bien distinctes, la proteine kinase C activant selectivement p38, et la voie ras/raf activant selectivement ERK1/2. Ces deux voies de signalisation sont necessaires et suffisantes a l’effet proliferatif du NGF.

BackgroundInflammation is a hallmark of acute lung injury and chronic airway diseases. In chronic airway diseases, it is associated with profound tissue remodeling. Peroxisome proliferator-activated receptor-α (PPARα) is a ligand-activated transcription factor, that belongs to the nuclear receptor family. Agonists for PPARα have been recently shown to reduce lipopolysaccharide (LPS)- and cytokine-induced secretion of matrix metalloproteinase-9 (MMP-9) in human monocytes and rat mesangial cells, suggesting that PPARα may play a beneficial role in inflammation and tissue remodeling.MethodsWe have investigated the role of PPARα in a mouse model of LPS-induced airway inflammation characterized by neutrophil and macrophage infiltration, by production of the chemoattractants, tumor necrosis factor-α (TNF-α), keratinocyte derived-chemokine (KC), macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1), and by increased MMP-2 and MMP-9 activity in bronchoalveolar lavage fluid (BALF). The role of PPARα in this model was studied using both PPARα-deficient mice and mice treated with the PPARα activator, fenofibrate.ResultsUpon intranasal exposure to LPS, PPARα-/-mice exhibited greater neutrophil and macrophage number in BALF, as well as increased levels of TNF-α, KC, MIP-2 and MCP-1, when compared to PPARα+/+mice. PPARα-/-mice also displayed enhanced MMP-9 activity. Conversely, fenofibrate (0.15 to 15 mg/day) dose-dependently reduced the increase in neutrophil and macrophage number induced by LPS in wild-type mice. In animals treated with 15 mg/day fenofibrate, this effect was associated with a reduction in TNF-α, KC, MIP-2 and MCP-1 levels, as well as in MMP-2 and MMP-9 activity. PPARα-/-mice treated with 15 mg/day fenofibrate failed to exhibit decreased airway inflammatory cell infiltrate, demonstrating that PPARα mediates the anti-inflammatory effect of fenofibrate.ConclusionUsing both genetic and pharmacological approaches, our data clearly show that PPARα downregulates cell infiltration, chemoattractant production and enhanced MMP activity triggered by LPS in mouse lung. This suggests that PPARα activation may have a beneficial effect in acute or chronic inflammatory airway disorders involving neutrophils and macrophages.

The effect of ouabain on the histamine secretion induced by compound 48/80 has been studied using rat peritoneal mast cells. Ouabain did not modify histamine release in the presence of millimolar concentrations of extracellular calcium. However, when mast cells were previously washed with a calcium-free buffer, ouabain strongly potentiated histamine release elicited by compound 48/80. The full potentiation of mast cell secretion by ouabain required 30 min preincubation before adding compound 48/80. It was inhibited by lanthanum and EGTA. Potassium deprivation mimicked the effect of ouabain. A 30 min preincubation time without potassium was also required. Potassium concentrations below 2.7 mM increased the effect of ouabain whereas higher potassium concentrations reversed this effect. The potentiation of compound 48/80-induced histamine release by ouabain or potassium deprivation was not immediately reversed by washing away ouabain or by adding potassium, respectively. The data confirm that sodium-potassium ATPase is involved, through a calcium-dependent process, in the regulation of histamine release from mast cells.

The inhibition of sodium-potassium ATPase by ouabain or potassium-deprivation led to a potentiation of histamine release induced by antigen or compound 48/80 from rat mast cells. The fullest potentiation required 30 min preincubation of cells with ouabain or without potassium before adding the triggering agent. The potentiation was observed provided that the calcium concentration was less than 0.5 mM. The effect of sodium-potassium ATPase blockade was reversed slowly by washing out ouabain or by adding potassium. Metabolic inhibition with deoxyglucose and dinitrophenol-blocked histamine release observed under all conditions described. Histamine release from rat mast cells was also elicited when the cells were incubated in sodium-deficient media where sodium was replaced by sucrose. Potassium-deprivation potentiated this secretion process previously shown to be dependent upon metabolic energy and sensitive to millimolar calcium concentrations.

Clenbuterol 10(-8) to 10(-6) M inhibited antigen-induced histamine release from passively sensitized human lung tissue. This inhibition was not antagonized by propranolol, whereas the inhibitions observed with isoprenaline and fenoterol were reduced by propranolol. Clenbuterol also inhibited compound 48/80-induced histamine release and immunological histamine secretion from actively sensitized peritoneal rat mast cells. High concentrations of clenbuterol were required (10(-5) to 10(-3) M) and propranolol did not antagonize these inhibitions of histamine release. Isoprenaline and salbutamol did not modify the secretion from rat mast cells. The potential anti-anaphylactic activity of clenbuterol, might be partly related to its calcium antagonist property.

Increased non-specific bronchial hyperresponsiveness (BHR) has been reported after positive reaction to isocyanates in patients with isocyanate-sensitive asthma. The increased responsiveness may, however, also precede the asthma attack. We therefore compared non-specific BHR to a cholinergic agent before and after exposure to toluenediisocyanate (TDI) that induced no asthma symptoms in 11 workers with isocyanate-related asthma. Patients were exposed for 3 consecutive days to progressively increasing doses of TDI (5, 10, and 20 ppb min −1 for 20 min) in an exposure chamber with continuous TDI monitoring. No immediate nor late asthmatic bronchial reaction was observed in any patient after any dose of TDI during or after challenge. A significant increase in non-specific BHR was noted 24 h after the last dose of TDI challenge, however. This increase was at least one doubling dose for seven of 11 patients. In conclusion, our study shows that, in patients with isocyanate-induced asthma, exposure to TDI induces a slight but significant increase in non-specific BHR in the absence of any immediate or late bronchial response to isocyanate. This result, which requires further confirmation, may justify a proposal to measure non-specific BHR, even after a negative specific inhalation test to TDI, as an additional diagnostic element for TDI-induced occupational asthma, to help lower the percentage of the undetected occupational asthma cases.

Trafficking of the TrkA receptor after stimulation by NGF is of emerging importance in structural cells in the context of airway inflammatory diseases. We have recently reported the expression of functional TrkA receptors in human airway smooth muscle cells (HASMC). We have here studied the TrkA trafficking mechanisms in these cells. TrkA disappearance from the cell membrane was induced within 5 min of NGF (3pM) stimulation. Co-immunoprecipitation of clathrin-TrkA was revealed, and TrkA internalisation inhibited either by clathrin inhibitors or by siRNA inducing downregulation of endogenous clathrin. TrkA internalised receptors were totally degraded in lysosomes, with no recycling phenomenon. Newly synthesized TrkA receptors were thereafter re-expressed at the cell membrane within 10 h. TrkA re-synthesis was inhibited by blockade of clathrin-dependent internalisation, but not of TrkA receptors lysosomal degradation. Finally, we observed that NGF multiple stimulations progressively increased TrkA expression in HASMC, which was associated with an increase in NGF/TrkA-dependent proliferation. In conclusion, we show here the occurrence of clathrin-dependent TrkA internalisation and lysosomal degradation in the airway smooth muscle, followed by upregulated re-synthesis of functional TrkA receptors and increased proliferative effect in the human airway smooth muscle. This may have pathophysiological consequences in airway inflammatory diseases.

L'effet de l'inhibition de la Na, K-ATPase a ete etudie sur la liberation d'histamine induite par l'antigene a partir de mastocytes peritoneaux de rats sensibilises. Il apparait qu'en presence de Ca 2+ , l'ouabaine jusqu'a 10 −7 M ne reussit pas a produire une modification significative de la liberation d'histamine. Cependant dans un milieu depourvu de Ca, la tres faible liberation obtenue en reponse a l'antigene a ete nettement accrue de 10 −5 M a 10 −3 M

1 Cyclic adenosine 3',5'-monophosphate (cyclic AMP)- and cyclic guanosine 3',5'-monophosphate (cyclic GMP)-phosphodiesterase activities from rat lung were selectively inhibited by ZK 62711 and M & B 22948, respectively. Theophylline and papaverine inhibited both activities. 2 Rat lung strips contracted by carbachol were relaxed by 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 26711, EC25 = 7 x 10(-8)M) and 2-O-propoxyphenyl-8-azapurin-6-one (M & B 22948, EC25 = 5 x 10(-7)M) indicating relaxant properties of both cyclic AMP and cyclic GMP. 3 The antigen-induced histamine release from human basophils was inhibited by ZK 62711 (IC25 = 8 x 10(-7)M), whereas M & B 22948 had no effect. On the contrary, the release from rat mast cells was inhibited by M & B 22948 (IC25 = 10(-6)M), while ZK 62711 had no effect. 4 These data show an inhibitory effect of cyclic AMP on histamine release to be involved with basophils, whereas cyclic GMP is predominantly involved with mast cells. Is is suggested that the antianaphylactic properties of cyclic nucleotide phosphodiesterase inhibitors are mainly linked to the increase of cyclic GMP.

A role of opioids as inhibitors of the non-adrenergic non-cholinergic (NANC) excitatory nerves has been studied in the guinea-pig bronchi using electrical field stimulation. Morphine and DAGO gave dose-dependent inhibition of NANC contraction, which was reversed by naloxone, whereas the cholinergic nerve and substance P responses were unaffected. [Met5]enkephalin and [Leu5]enkephalin weakly inhibited NANC contraction, whereas dynorphin-(1-13) had no effect. These results suggest that opiates inhibit NANC bronchoconstriction via mu-opioid receptors.

Background Full-root aortic valve replacement with stentless xenografts has potentially superior hemodynamic performance compared to stented valves. However, a number of cardiac surgeons are reluctant to transform a classical stented aortic valve replacement into a technically more demanding full-root stentless aortic valve replacement. Here we describe our technique of full-root stentless aortic xenograft implantation and compare the early clinical and midterm hemodynamic outcomes to those after aortic valve replacement with stented valves. Methods We retrospectively compared the pre-operative characteristics of 180 consecutive patients who underwent full-root replacement with stentless aortic xenografts with those of 80 patients undergoing aortic valve replacement with stented valves. In subgroups presenting with aortic stenosis, we further analyzed the intra-operative data, early postoperative outcomes and mid-term regression of left ventricular mass index. Results Patients in the stentless group were younger (62.6 ± 13 vs. 70.3 ± 11.8 years, p

Background Potential involvement of the CCR10/CCL28 axis was recently reported in murine models of allergic asthma. If confirmed, blockade of the CCR10 receptor would represent an alternative to current asthma therapies. We evaluated the effect of a novel Protein Epitope Mimetic CCR10 antagonist, POL7085, in a murine model of allergic eosinophilic airway inflammation. Methods Mice were sensitized and challenged to ovalbumin. POL7085, a CCR10 antagonist (7.5 and 15 mg/kg), dexamethasone (1 mg/kg) or vehicle were administered intranasally once daily 1h before each allergen challenge. On day 21, airway hyperresponsiveness, bronchoalveolar lavage inflammatory cells and Th2 cytokines, and lung tissue mucus and collagen were measured. Results Allergen challenge induced airway hyperresponsiveness in vehicle-treated animals as measured by whole body barometric plethysmography, and eosinophilia in bronchoalveolar lavage. POL7085 dose-dependently and significantly decreased airway hyperresponsiveness (34 ± 16 %) and eosinophil numbers in bronchoalveolar lavage (66 ± 6 %). In addition, the highest dose of POL7085 used significantly inhibited lung IL-4 (85 ± 4 %), IL-5 (87 ± 2 %) and IL-13 (190 ± 19 %) levels, and lung collagen (43 ± 11 %). Conclusions The Protein Epitope Mimetic CCR10 antagonist, POL7085, significantly and dose-dependently decreased allergen-induced airway hyperresponsiveness and airway inflammation after once daily local treatment. Our data give strong support for further investigations with CCR10 antagonists in asthmatic disease. Electronic supplementary material The online version of this article (doi:10.1186/s12931-015-0231-5) contains supplementary material, which is available to authorized users.

Antianaphylactic properties have been attributed to cyclic nucleotide phosphodiesterase inhibitors through increase of cyclic AMP levels, according to the concept that increases in cyclic AMP reduce release and increases in cyclic GMP enhance release. However, Coulson et al. [3] showed that the inhibition of histamine release from human lung is correlated to the inhibition of cyclic GMP hydrolysis. We studied the effect of specific inhibitors of cyclic AMP and cyclic GMP hydrolysis on the antigen-induced mediator release from rat mass cells and human basophils and on airways relaxation [4]. The results suggested that the modulation of mediator release was different from one cell type to the other, enhancement of cyclic AMP levels leading to the inhibition of release from basophils, while cyclic GMP appears to be predominantly involved in mast cells. The present paper shows that high concentrations of sodium nitrite, a stimulating agent of guanylate cyclase, inhibit histamine release from rat mast cells in the presence or absence of MB 22948, a selective cyclic GMP phosphodiesterase inhibitor.

The binding of the chemokine CXCL12 to its receptors CXCR4 and CXCR7 may be prevent by a CXCL12 neutraligand, chalcone 4, having an anti -inflammatory activity in a mouse model of allergic asthma. Our work consisted in proposing strategies for the development of active and bioavailable molecules, able to promote local action, and to study the in vivo mechanism of action of the CXCL12 neutraligands. We initially developed a short and reproducible mouse model of allergic asthma, suitable for a rapid assessment of the anti -inflammatory activity of new compounds, in order to ensure rational development of the proposed strategies. We developped three soluble prodrugs, adapted to local administration, inactive but rapidly cleaved in active chalcone 4. In vivo, the prodrugs have an anti-inflammatory activity at suitable doses to minimize side effects. To promote the benefit of local anti-inflammatory action of CXCL12 neutraligand, we synthesized an antedrug, carbonitrile-chalcone 4, active locally in vivo after local administration and rapidly degraded before its distribution in the body.Finally, the study of chalcone 4 allowed us to highlight a significant asthma activity. An activity related to the rapid elimination of the CXCL12 chemokine from the lung. The trapping of CXCL12 by the neutraligand reduced M1 macrophage activation and their release of pro inflammatory cytokines, as decreases the recruitment of CXCR4+ eosinophils and lymphocytes. In conclusion, our work provided mechanistic elements related to the roles of the chemokine CXCL12 in asthma, and present two interesting strategies adapted to local administration to limit adverse effects., La liaison de CXCL12 à ses récepteurs CXCR4 et CXCR7 peut-être bloquée par un neutraligand de CXCL12, la chalcone 4, qui présente une activité anti-inflammatoire dans un modèle d’asthme chez la souris. Notre travail a consisté à proposer des stratégies permettant le développement de molécules biodisponibles et actives localement, et d’étudier le mécanisme d'action in vivo des neutraligands de CXCL12. Nous avons développé un modèle court et reproductible d'asthme allergique chez la souris, adapté à une évaluation rapide de l’activité anti-inflammatoire des nouveaux composés et un développement raisonné des stratégies envisagées. Nous avons synthétisé trois prodrogues solubles, adaptées à une administration locale, inactives et rapidement clivées en chalcone 4 active. In vivo, les prodrogues sont anti-inflammatoires à des doses susceptibles de limiter les effets indésirables. Pour favoriser davantage l'action anti-inflammatoire locale du neutraligand de CXCL12, nous avons synthétisé une ante-drogue, la carbonitrile-chalcone 4, active in vivo par administration locale et rapidement dégradée en deux composés inactifs avant sa distribution dans l'organisme. Enfin, l'étude de la chalcone 4 a mis en évidence une activité antiasthmatique significative, liée à l'élimination rapide de la chimiokine CXCL12 du poumon. La capture de CXCL12 par le neutraligand réduit la différenciation des macrophages M1 et leur libération de cytokines pro-inflammatoires, ainsi que le recrutement des éosinophiles et des lymphocytes CXCR4+. En conclusion, nos travaux ont permis de conforter les rôles de la chimiokine CXCL12 dans l'asthme et présenter deux stratégies aptes à limiter les effets indésirables.