Repeated Allergen Exposure in A/J Mice Causes Steroid-Insensitive Asthma via a Defect in Glucocorticoid Receptor Bioavailability.

The importance of developing new animal models to assess the pathogenesis of glucocorticoid (GC)-insensitive asthma has been stressed. Because of the asthma-prone background of A/J mice, we hypothesized that asthma changes in these animals would be or become resistant to GCs under repeated exposures to an allergen. A/J mice were challenged with OVA for 2 or 4 consecutive d, starting on day 19 postsensitization. Oral dexamethasone or inhaled budesonide were given 1 h before challenge, and analyses were done 24 h after the last challenge. Airway hyperreactivity, leukocyte infiltration, tissue remodeling, and cytokine levels as well as phosphorylated GC receptor (p-GCR), p-GATA-3, p-p38, MAPK phosphatase-1 (MKP-1), and GC-induced leucine zipper (GILZ) levels were assessed. A/J mice subjected to two daily consecutive challenges reacted with airway hyperreactivity, subepithelial fibrosis, and marked accumulation of eosinophils in both bronchoalveolar lavage fluid and peribronchial space, all of which were clearly sensitive to dexamethasone and budesonide. Conversely, under four provocations, most of these changes were steroid resistant. A significant reduction in p-GCR/GCR ratio following 4- but not 2-d treatment was observed, as compared with untreated positive control. Accordingly, steroid efficacy to transactivate MKP-1 and GILZ and to downregulate p-p38, p-GATA-3 as well as proinflammatory cytokine levels was also seen after two but not four provocations. In conclusion, we report that repeated allergen exposure causes GC-insensitive asthma in A/J mice in a mechanism associated with decrease in GCR availability and subsequent loss of steroid capacity to modulate pivotal regulatory proteins, such as GATA-3, p-p38, MKP-1, and GILZ.

Nebulized lidocaine prevents airway inflammation, peribronchial fibrosis, and mucus production in a murine model of asthma.

BACKGROUND: Evidence suggests that nebulized lidocaine is beneficial in asthma therapy, but to what extent and the mechanisms underlying this effect remain poorly understood. The aim of this study was to assess the impact of lidocaine treatment using a murine model of allergic asthma characterized by expression of pivotal features of the disease: inflammation, mucus production, and lung remodeling. METHODS: A/J mice sensitized with ovalbumin were treated with inhaled lidocaine or vehicle immediately after ovalbumin intranasal challenges. Lung function, total and differential leukocytes in bronchoalveolar lavage fluid, peribronchial eosinophil density, interleukin (IL)-4, IL-5 and eotaxin-1 levels, epithelial mucus, collagen, extracellular-matrix deposition, matrix metalloproteinase-9 activity, and GATA-3 expression were evaluated. Between five and eight animals per group were used. RESULTS: Inhaled lidocaine inhibited ovalbumin-induced airway hyperreactivity to methacholine, and accumulation of lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid 24 h after the last allergen provocation. Lidocaine administration also prevented other pathophysiological changes triggered by ovalbumin in lung tissue, including peribronchial eosinophil and neutrophil infiltration, subepithelial fibrosis, increased content of collagen and mucus, matrix metalloproteinase-9 activity, and increased levels of IL-4, IL-5, IL-13, and eotaxin-1. Furthermore, inhaled lidocaine inhibited lung tissue GATA-3 expression in ovalbumin-challenged mice. We also demonstrated that lidocaine inhibited the expression of GATA-3 in ovalbumin-stimulated T cells in vitro. CONCLUSIONS: Inhaled lidocaine prevents eosinophilic inflammation, overproduction of mucus, and peribronchial fibrosis in a murine model of asthma, and impaired airway hyperreactivity, possibly by inhibiting allergen-evoked GATA-3 expression and the subsequent up-regulation of proinflammatory cytokines and chemokines.

Two-dimensional difference gel electrophoresis (DiGE) analysis of plasmas from dengue fever patients.

Dengue fever is the world's most important arthropod-born viral disease affecting humans. To contribute to a better understanding of its pathogenesis, this study aims to identify proteins differentially expressed in plasmas from severe dengue fever patients relative to healthy donors. The use of 2-D Fluorescence Difference Gel Electrophoresis to analyze plasmas depleted of six high-abundance proteins (albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin) allowed for the detection of 73 differentially expressed protein spots (n = 13, p < 0.01), of which 37 could be identified by mass spectrometry. These 37 spots comprised a total of 14 proteins, as follows: 7 had increased expression in plasmas from dengue fever patients (C1 inhibitor, alpha1-antichymotrypsin, vitamin D-binding protein, fibrinogen gamma-chain, alpha1-acid glycoprotein, apolipoprotein J and complement component C3c), while 7 others had decreased expression in the same samples (alpha-2 macroglobulin, prothrombin, histidine-rich glycoprotein, apolipoproteins A-IV and A-I, transthyretin and complement component C3b). The possible involvement of these proteins in the inflammatory process triggered by dengue virus infection and in the repair mechanisms of vascular damage occurring in this pathology is discussed in this study.

Nutritional tryptophan restriction impairs plasticity of retinotectal axons during the critical period.

The use-dependent specification of neural circuits occurs during post-natal development with a conspicuous influence of environmental factors, such as malnutrition that interferes with the major steps of brain maturation. Serotonin (5-HT), derived exclusively from the essential aminoacid tryptophan, is involved in mechanisms of development and use-dependent plasticity of the central nervous system. We studied the effects of the nutritional restriction of tryptophan in the plasticity of uncrossed retinotectal axons following a retinal lesion to the contralateral retina during the critical period in pigmented rats. Litters were fed through their mothers with a low tryptophan content diet, based on corn and gelatin, a complemented diet with standard tryptophan requirements for rodents or standard laboratory diet. The results suggest a marked reduction in the plasticity of intact axons into denervated territories in the tryptophan restricted group in comparison to control groups. Tryptophan complementation between PND10-21 completely restored retinotectal plasticity. However, the re-introduction of tryptophan after the end of the critical period (between PND28-P41) did not restore the sprouting ability of uncrossed axons suggesting a time-dependent effect to the reversion of plasticity deficits. Tryptophan-restricted animals showed a reduced activity of matrix metalloproteinase-9 and altered expressions of phosphorylated forms of ERK1/2 and AKT. Our results demonstrate the influence of this essential aminoacid as a modulator of neural plasticity during the critical period through the reduction of serotonin content which alters plasticity-related signaling pathways and matrix degradation.

Matrix metalloproteinase-9 is involved in the development and plasticity of retinotectal projections in rats.

OBJECTIVE: During postnatal development, retinotectal projections undergo a process of misplaced axon elimination, leading to a topographical matching between the retinal surface and the superior colliculus. Matrix metalloproteinases (MMPs) have been implicated in the development and plasticity of the nervous system. We studied the expression and role of MMPs during normal development of retinotectal projections and after monocular enucleation-induced plasticity. MATERIAL AND METHODS: Lister hooded rats at different postnatal ages received subpial ethylene vinyl acetate 40W implants to deliver an MMP inhibitor or vehicle to the superior colliculus. Animals received intraocular injections of horseradish peroxidase for anterograde tracing of ipsilateral projections. For immunoblotting and zymography, colliculi were removed without fixation. RESULTS: We observed the highest MMP activity in the first postnatal week, with decreasing activity thereafter. Monocular enucleation at postnatal day 10 yielded a rapid increase in MMP activity, 24 h following denervation of the contralateral colliculus. Importantly, inhibition of MMP activity in vivo induced a marked delay of axonal clustering along the medial aspect of colliculus. CONCLUSIONS: Our data indicate that MMPs are crucial in retinotectal development concurring to the fine tuning of topographical order and synaptic specificity of these connections.

PO41, a snake venom metalloproteinase inhibitor isolated from Philander opossum serum.

PO41 was isolated from Philander opossum serum by DEAE-Sephacel, Phenyl Superose and Superdex 200 chromatographies and showed a molecular mass of 41,330 Da by MALDI-TOF MS. Molecular masses of 81.5 and 84.5 kDa were obtained by size exclusion chromatography and dynamic laser light scattering, respectively, suggesting that PO41 is dimeric. Its isoelectric point was estimated to be lower than 3.5. PO41 presented similar amino terminal sequence to those of DM40 and DM43, two antihaemorrhagins previously isolated from Didelphis marsupialis serum and was recognized by polyclonal antibodies raised against D. marsupialis antibothropic fraction. To study the inhibitory properties of this protein, the metalloproteinases bothrolysin and jararhagin were isolated from Bothrops jararaca venom by chromatographies on Superdex 200 and Phenyl Superose. Jararhagin was further submitted to a Mono Q column. The proteolytic and haemorrhagic effects of these haemorrhagins were neutralized by PO41. Both snake venom metalloproteinases formed stable complexes with PO41. The stoichiometry of the complex PO41-jararhagin was one inhibitor subunit to one molecule of the enzyme. These results show that PO41 has physicochemical, structural, immunoreactive and biological properties similar to other metalloproteinase inhibitors belonging to the supergene family of immunoglobulins.