Neus Prats

Resident Cell Chemokine Expression Serves as the Major Mechanism for Leukocyte Recruitment During Local Inflammation

The mechanistic relationships between initiating stimulus, cellular source and sequence of chemokine expression, and leukocyte recruitment during inflammation are not clear. To study these relationships in an acute inflammatory process, we challenged a murine air pouch with carrageenan. A time-dependent increase in TNF-α, monocyte chemottractant protein-1 (MCP-1), macrophage-inflammatory protein-1α (MIP-1α), RANTES, KC, and MIP-2 was found in the exudates preceding cell recruitment, but displaying different kinetic profiles. Air pouches generated for 2, 6, or 9 days before initiating inflammation demonstrated a proportional increase in the number of cells lining the cavities. Two hours after carrageenan stimulation, the synthesis of TNF-α and all chemokines but RANTES increased in proportion to the lining cellularity, although no differences in infiltrating leukocytes were found, suggesting that the early source of these mediators is resident cells. To assess the contribution of neutrophils to chemokine synthesis at later time points, we used neutropenic animals. Neutrophil depletion caused a decrease in TNF-α (51%), KC (37%), MIP-1α (30%), and RANTES (57%) levels and a 2-fold increase in monocytes 4 h after challenge. No effect on MIP-2 and MCP-1 levels was observed. The selective blockade of CXCR2 or CCR1 inhibited neutrophil recruitment by 74% and 54%, respectively, without a significant inhibition of monocytes. A differential effect on TNF-α and MCP-1 levels was observed after these treatments, indicating that the two receptors did not subserve a mere redundant chemotactic role. Overall, our results suggest that chemokines synthesized by resident cells play an important role in the evolution of the inflammatory response.

The sphingosine-1-phosphate receptor-1 antagonist, W146, causes early and short-lasting peripheral blood lymphopenia in mice

Agonists of the sphingosine-1-phosphate (S1P) receptors, like fingolimod (FTY720), are a novel class of immunomodulators. Administration of these compounds prevents the egress of lymphocytes from primary and secondary lymphoid organs causing peripheral blood lymphopenia. Although it is well established that lymphopenia is mediated by S1P receptor type 1 (S1P1), the exact mechanism is still controversial. The most favored hypothesis states that S1P1 agonists cause internalization and loss of the cell surface receptor on lymphocytes, preventing them to respond to S1P. Hence, S1P1 agonists would behave in vivo as functional antagonists of the receptor. For this hypothesis to be valid, a true S1P1 antagonist should also induce lymphopenia. However, it has been reported that S1P1 antagonists fail to show this effect, arguing against the concept. Our study demonstrates that a S1P1 antagonist, W146, induces a significant but transient blood lymphopenia in mice and a parallel increase in CD4+ and CD8+ lymphocytes in lymph nodes. Treatment with W146 also causes the accumulation of mature T cells in the medulla of the thymus and moreover, it induces lung edema. We show that both the S1P1 antagonist and a S1P1 agonist cause lymphopenia in vivo in spite of their different effects on receptor expression in vitro. Although the antagonist purely blocks the receptor and the agonist causes its disappearance from the cell surface, the response to the endogenous ligand is prevented in both cases. Our results support the hypothesis that lymphopenia evoked by S1P1 agonists is due to functional antagonism of S1P1 in lymphocytes.

Effects of Aclidinium Bromide in a Cigarette Smoke–Exposed Guinea Pig Model of Chronic Obstructive Pulmonary Disease

Long-acting muscarinic antagonists are widely used to treat chronic obstructive pulmonary disease (COPD). In addition to bronchodilation, muscarinic antagonism may affect pulmonary histopathological changes. The effects of long-acting muscarinic antagonists have not been thoroughly evaluated in experimental models of COPD induced by chronic exposure to cigarette smoke (CS). We investigated the effects of aclidinium bromide on pulmonary function, airway remodeling, and lung inflammation in a CS-exposed model of COPD. A total of 36 guinea pigs were exposed to CS and 22 were sham exposed for 24 weeks. Animals were nebulized daily with vehicle, 10 μg/ml, or 30 μg/ml aclidinium, resulting in six experimental groups. Pulmonary function was assessed weekly by whole-body plethysmography, determining the enhanced pause (Penh) at baseline, after treatment, and after CS/sham exposure. Lung changes were evaluated by morphometry and immunohistochemistry. CS exposure increased Penh in all conditions. CS-exposed animals treated with aclidinium showed lower baseline Penh than untreated animals (P = 0.02). CS induced thickening of all bronchial wall layers, airspace enlargement, and inflammatory cell infiltrate in airways and septa. Treatment with aclidinium abrogated the CS-induced smooth muscle enlargement in small airways (P = 0.001), and tended to reduce airspace enlargement (P = 0.054). Aclidinium also attenuated CS-induced neutrophilia in alveolar septa (P = 0.04). We conclude that, in guinea pigs chronically exposed to CS, aclidinium has an antiremodeling effect on small airways, which is associated with improved respiratory function, and attenuates neutrophilic infiltration in alveolar septa. These results indicate that, in COPD, aclidinium may exert beneficial effects on lung structure in addition to its bronchodilator action.

Combined anti CXC receptors 1 and 2 therapy is a promising anti-inflammatory treatment for respiratory diseases by reducing neutrophil migration and activation

Neutrophil infiltration and activation in the lung are important pathophysiological features in COPD, severe asthma and bronchiectasis mostly mediated by CXCL8 and CXCL1 via CXCR1 and CXCR2. No thorough study to date has been performed to compare the anti-inflammatory effect profile of dual CXCR1/2 vs. selective CXCR2 antagonists in relevant human neutrophil assays and pulmonary inflammation models. Dual CXCR1/2 (SCH527123, diaminocyclobutandione-1) and selective CXCR2 (SB265610, thiopyrimidine-1) antagonist activity and receptor residence time were determined by [(35)S]GTPγS binding in human (h)- and guinea pig (gp)-CXCR1 and CXCR2 overexpressing membranes. h-neutrophil chemotaxis, degranulation and ROS production were established using CXCL8 or CXCL1 to evaluate dual CXCR1/2- or selective CXCR2-dependent activities. LPS-induced lung inflammation in gp was selected to assess in vivo potency. Dual CXCR1/2 antagonists blocked both CXCL8 and CXCL1-induced h-neutrophil functions and [(35)S]GTPγS binding. In contrary, selective CXCR2 antagonists displayed significantly reduced potency in CXCL8 -mediated h-neutrophil responses despite being active in CXCR2 assays. Upon LPS challenge in gp, administration of SCH527123 inhibited the increase of neutrophils in BALF, modestly reduced blood neutrophils and induced minor neutrophil accumulation in bone marrow. Differentiation of CXCR1/2 vs. CXCR2 antagonists could not be extended to in vivo due to differences in CXCR1 receptor homology between h and gp. Dual CXCR1/2 therapy may represent a promising anti-inflammatory treatment for respiratory diseases reducing more effectively neutrophil migration and activation in the lung than a CXCR2 selective treatment. However, the in vivo confirmation of this claim is still missing due to species differences in CXCR1.

Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B Adenosine Receptor Antagonist.

The structure-activity relationships for a series of pyrazine-based A2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2 cytokine production, and OVA-specific IgE levels.

Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome

Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10–15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

A novel inhaled Syk inhibitor blocks mast cell degranulation and early asthmatic response

Spleen tyrosine kinase (Syk) is essential for signal transduction of immunoreceptors. Inhibition of Syk abrogates mast cell degranulation and B cell responses. We hypothesized that Syk inhibition in the lung by inhaled route could block airway mast cells degranulation and the early asthmatic response without the need of systemic exposure. We discovered LAS189386, a novel Syk inhibitor with suitable properties for inhaled administration. The aim of this study was to characterize the in vitro and in vivo profile of LAS189386. The compound was profiled in Syk enzymatic assay, against a panel of selected kinases and in Syk-dependent cellular assays in mast cells and B cells. Pharmacokinetics and in vivo efficacy was assessed by intratracheal route. Airway resistance and mast cell degranulation after OVA challenge was evaluated in an ovalbumin-sensitized Brown Norway rat model. LAS189386 potently inhibits Syk enzymatic activity (IC50 7.2 nM), Syk phosphorylation (IC50 41 nM), LAD2 cells degranulation (IC50 56 nM), and B cell activation (IC50 22 nM). LAS189386 inhibits early asthmatic response and airway mast cell degranulation without affecting systemic mast cells. The present results support the hypothesis that topical inhibition of Syk in the lung, without systemic exposure, is sufficient to inhibit EAR in rats. Syk inhibition by inhaled route constitutes a promising therapeutic option for asthma.

A longitudinal assessment of miR-122 and GLDH as biomarkers of drug-induced liver injury in the rat

Abstract Context: There is an ongoing search for specific and translational biomarkers of drug-induced liver injury (DILI). MicroRNA-122 (miR-122) has previously shown potential as a sensitive, specific, and translational biomarker of DILI in both rodent, and human studies. Objective: To build on previous work within the field, we examined biomarker kinetics in a rat model of acetaminophen (APAP)-induced liver injury to confirm the sensitivity, and specificity of miR-122 and glutamate dehydrogenase (GLDH). Materials and methods: qRT-PCR and a standard enzymatic assay were used for biomarker analysis. Results: Both miR-122 and GLDH were demonstrated to be more readily-detectable biomarkers of APAP-DILI than alanine aminotransferase (ALT). Peak levels for all biomarkers were detected at 2 days after APAP. At day 3, miR-122 had returned to baseline; however, other biomarkers remained elevated between 3 and 4 days. We were also able to demonstrate that, although miR-122 is present in greater quantities in exosome-free form, both exosome-bound and non-vesicle bound miR-122 are released in a similar profile throughout the course of DILI. Discussion and conclusions: Together, this study demonstrates that both GLDH and miR-122 could be used during preclinical drug-development as complementary biomarkers to ALT to increase the chance of early detection of hepatotoxicity.

Double-stranded RNA evokes exacerbation in a mouse model of corticosteroid refractory asthma.

RNA viruses are a major cause of respiratory infections and are known to exacerbate asthma and other respiratory diseases. Our aim was to test the ability of poly(I:C) (polyinosinic:polycytidylic acid), a viral surrogate, to elicit exacerbation in a model of severe asthma driven by HDM (house dust mite) in FCA (Freunds complete adjuvant). Poly(I:C) was administered intranasally around the HDM challenge in FCA-HDM-sensitized animals. Changes in AHR (airway hyperresponsiveness), BALF (bronchoalveolar lavage fluid) inflammatory infiltrate, HDM-specific immunoglobulins and cytokine/chemokine release were evaluated at different points after the challenge. The effect of oral dexamethasone was also assessed. Exacerbation was achieved when poly(I:C) was administered 24 h before the HDM challenge and was characterized by enhanced AHR and an increase in the numbers of neutrophils, macrophages and lymphocytes in the BALF. Th1, Th2 and Th17 cytokines were also elevated at different time points after the challenge. Peribronchial and alveolar inflammation in lung tissue were also augmented. AHR and inflammatory infiltration showed reduced sensitivity to dexamethasone treatment. We have set up a model that mimics key aspects of viral exacerbation in a corticosteroid-refractory asthmatic phenotype which could be used to evaluate new therapies for this condition.

Characterization of a model of tracheal plasma extravasation in passively sensitized rats using anti-allergic and anti-inflammatory drugs by oral and intratracheal route

The aim of the following study was to characterize a passive systemic anaphylaxis rat model of dinitrophenyl (DNP)-induced plasma extravasation in the trachea to determine if the model is appropriate for the evaluation of new drugs targeting airway mast cells by oral and intratracheal (i.t.) route. To this purpose we have used fluticasone and a range of anti-allergic drugs including compounds either active on mast cell activation, such as cromoglycate and the Syk inhibitor R406, or active on mast cell mediators, such as cetirizine and montelukast. To further characterize the model, the effect of fluticasone, cromoglycate and R406 on rat tracheal mast cell degranulation was also assessed histologically. DNP-induced tracheal plasma extravasation was inhibited by cromoglycate (i.v. and i.t.) and R406 (p.o.), but not by fluticasone (i.t.), cetirizine or montelukast (p.o.). Cromoglycate and R406 also showed inhibition of tracheal mast cell degranulation, whereas fluticasone was inactive. These results suggest that the DNP-induced tracheal plasma extravasation model constitutes a useful animal model for the evaluation, by oral and i.t. route, of new anti-allergic drugs intended to target airway mast cells.