Analytical and pharmacological validation of the quantification of phenol red in a mouse model: An optimized method to evaluate expectorant drugs.

INTRODUCTION: The evaluation of expectorant activity has been extensively studied in murine models, involving the secretion of phenol red in the trachea or bronchus to estimate the secretory capacity of lower airway mucosa. However, differences in the experimental protocols of several studies evidenced the need of to standardize the quantification of phenol red in the bronchoalveolar fluid (BALF). METHODS: The analytical methodology for the quantification of phenol red in the BALF was optimized by investigation of pH influence, quantity of the alkali agent added and appropriate wavelength for quantification of phenol red by UV-VIS spectroscopy. Different phenol red suspensions (0.05, 0.5, 1.25, 2.5 and 5%) were prepared and administered intraperitoneally in mice at doses 5, 25, 50, 250 or 500 mg/kg. RESULTS: It was shown that phenol red should be used at dose 500 mg/kg and intraperitoneal administration should be performed from a suspension at 1.25% (w/v). Furthermore, the alkalinizing agent of choice would be NaOH (0.1 M). The pharmacological validation of the analytical method showed that ambroxol (30, 60 or 120 mg/kg), guaifenesin (100 mg/kg), NH4Cl (2000 mg/kg) or salbutamol (4 mg/kg) can be used as positive controls. DISCUSSION: The phenol red quantification in the BALF is a rapid and low cost assay for the discovery of new expectorant drugs. Thus, it was proposed a standardization of the analytical and pharmacological methods to ensure the reliability of BALF processing and reproducibility of phenol red quantification for data analysis.

Differential elemental distribution of retained particles along the respiratory tract.

CONTEXT: Prolonged exposure to ambient particles is associated with premature mortality due to cardio-respiratory diseases and lung cancer. The size and composition of these particles determine their toxicity, which is aggravated by their long-term retention in the lungs. OBJECTIVE: To compare the elemental profile of particles retained along the bronchial tree and lymph nodes by combining laser capture microdissection (LCM) and elemental composition analysis through energy dispersive x-ray (EDX) and scanning electron microscopy (SEM). MATERIAL AND METHODS: Twenty-four right lung middle lobes from autopsied cases were obtained from two cities with different pollution backgrounds. Lung samples were collected from three distinct sites within the lung at the time of autopsy: peribronchial tissue, peripheral parenchyma and hilar lymph nodes. Areas of potentially increased particle deposition were microdissected using LCM and analyzed for elemental composition through EDX "allied" with SEM. RESULTS: Elemental analyses of the particles retained along the bronchial tree showed two groups of distribution: peribronchiolar or lymph node deposition. The elemental profile of peribronchial areas were significantly different between the two cities and were better discriminators of past air pollution exposure. CONCLUSION: Our data suggest that particle uptake varies along the bronchial tree and human lung tissue retains particles indicative of regional air pollution background.

Inflammatory cell mapping of the respiratory tract in fatal asthma.

BACKGROUND: The site and distribution of inflammation in the airways of asthmatic patients has been largely investigated. Inflammatory cells are distributed in both large and small airways in asthma. It has been demonstrated that distal lung inflammation in asthma may significantly contribute to the pathophysiology of the disease. The upper airways have also been implicated in the overall asthmatic inflammation. Although it is now accepted that lung inflammation is not restricted to the intrapulmonary airways in asthma, little is known about cell distribution in the other lung compartments and their relation to the intrapulmonary airways. OBJECTIVE: We aimed to map the inflammatory process in fatal asthma (FA), from the upper airways to the lung parenchyma. METHODS: Eosinophil, neutrophil, mast cell and lymphocyte content were determined in nasal mucosa, the trachea, intrapulmonary airways and parenchyma (peribronchiolar and distal) of 20 patients with FA and 10 controls. RESULTS: Eosinophil content was higher in all studied areas in FA compared with controls (P<0.02). Mast cell content was higher in the outer area of larger airways, small membranous bronchioles and in peribronchiolar parenchyma of FA compared with controls (P<0.04). CD3+, CD4+and CD20+cells showed increased content in FA intrapulmonary airways compared with controls (P<0.05). There was a positive correlation between CD4+cell content in nasal mucosa and larger airways in asthmatics. Increased neutrophil content was observed only in peribronchiolar parenchyma of FA (P=0.028). CONCLUSION: Eosinophils present a widespread distribution within the respiratory tract in FA, from the nasal mucosa to the distal lung. The outer wall of small membranous bronchioles is the main site of inflammatory changes in FA. There is a localized distribution of alveolar inflammation at the peribronchiolar region for mast cells and neutrophils. Our findings provide further evidence of the importance of the lung periphery in the pathophysiology of FA.

Response of bronchiolar Clara cells induced by a domestic insecticide. Analysis of CC10 kDa protein content.

Clara cells are the most reactive to xenobiotics among the mammalian respiratory tract cells. In this report, the response of Clara cells to acute or repetitive exposure to a commercial insecticide was studied, correlating the changes in the cell ultrastructure with the intracellular content of CC10 kDa protein as quantified by immunocytochemical morphometry. After a single exposure to insecticide, Clara cells reveal great expansion of their volume which is accompanied by a remarkable proliferation of smooth endoplasmic reticulum, swelling of the mitochondria, and changes in the nucleus. Morphometric analysis of CC10 bronchiolar content showed significant increases in both the number of Clara cells and the immunostained areas in individual cells. By western blot, CC10 immunoreactive bands strongly increased in lungs after insecticide treatment, but they were only slightly higher than the control when the vehicle of the insecticide was tested. By repetitive exposure to the insecticide, the rat bronchiolar epithelium undergoes extensive alterations, particularly on Clara cells, the number of which is considerably reduced. The remaining Clara cells shrink in size and the typical dome-like cytoplasm is lost. Secretory granule release is no longer seen and the changes of their shape and secretory content reflect a marked degradation and condensation process. Repetitive exposures to the insecticide produced a severe blockage of the proteinopoietic activity, particularly on the synthesis of CC10. Results reported here reveal that the acute inhalation of a commercial insecticide produces hypertrophy of Clara cells, a significant augmentation of CC10 synthesis, and probably differentiation de novo of Clara cells, and morphological changes compatible with a detoxification process. By contrast, exposure for 5 days provoked a general inhibitory effect on Clara cell activity with the loss of cell capability to synthesize and secrete CC10 kDa protein.

Decreased levels of nitrosothiols in the lower airways of patients with cystic fibrosis and normal pulmonary function.

Airway S-nitrosothiols (SNOs) are naturally occurring bronchodilators. SNOs, nitrate, and nitrite were measured in bronchoalveolar lavage fluid of 23 patients with cystic fibrosis (CF) and mild pulmonary disease (aged 6-16 years) and 13 healthy children (aged 8-15 years). Concentrations of SNOs were decreased in the lower airways of patients with CF and mild pulmonary disease (median, range: 0, 0-320 nmol/L vs 80, 0-970 nmol/L) despite normal levels of the inert nitric oxide metabolites nitrate and nitrite (mean +/- SEM: 3.7 +/- 0.5 micromol/L vs 4.8 +/- 0.9 micromol/L). S-nitrosolation- mediated bioreactivities may be impaired by depletion of the CF airway SNO reservoir.

Localization of immunoreactive tissue kallikrein in the seromucous glands of the human and guinea-pig respiratory tree.

An immunocytochemical study focused on the cellular localization of tissue kallikrein along the human and guinea-pig respiratory tracts is reported. A strong immunoreactivity for tissue kallikrein was observed in the seromucous glands of the nasal mucosa, trachea, and bronchi. In these glands, the immunostaining was restricted to the serous component of the acinus whereas mucous cells showed no staining. Since no immunoreactivity to kininogen was observed in any of the tissue constituents of the human and guinea-pig respiratory tree, transudation of the substrate from plasma was considered to be the preferred mode of delivery of the kininogen into the bronchopulmonary interstitium and lumen. Our results provide morphological evidence for the well documented presence of tissue kallikrein in bronchial lavage fluids and support the hypothesis that kinins may be one of the more important mediators involved during acute episodes of asthma and rhinitis.