Oliviero Sacco

Epithelial cells and fibroblasts: structural repair and remodelling in the airways

Extensive lesions and changes in the architecture of the airway walls are commonly described in patients with respiratory infections, asthma, chronic bronchitis and interstitial lung diseases. Current knowledge identifies in airway epithelial cells and in fibroblasts the two cell types mainly involved in tissue repair after injury. During inflammatory respiratory disorders, extensive injury of airway epithelium may occur, with shedding of a large sheet of damaged cells in the bronchial and alveolar lumen but also with activation of the surviving epithelial cells and of the underlying fibroblasts. Indeed, besides acting as a physical and functional barrier to external agents, the epithelial surface of the bronchi has the capability to modulate the repair processes through the secretion of extracellular matrix proteins and the interaction with interstitial fibroblasts. Besides releasing pro-inflammatory cytokines and chemokines, the surviving epithelial cells and the underlying fibroblasts secrete factors contributing to airway repair, including the formation of the provisional extracellular matrix. This is indeed the substrate to which the epithelial cells at the edge of the lesion can attach to migrate in order to reconstitute the surface layer. In these processes airway epithelial cells receive the support of bronchial wall fibroblasts which actively release cytokines stimulating epithelial cell functions.

Bronchoalveolar lavage and esophageal pH monitoring data in children with "difficult to treat" respiratory symptoms.

Gastroesophageal reflux (GER) may be associated with chronic or recurrent asthma‐like symptoms secondary to bronchoconstrictor reflexes and/or inhalation of gastric content. The presence of lipid‐laden alveolar macrophages has been proposed as an index to establish the degree of gastric aspiration. We evaluated 20 children with “difficult to treat” respiratory symptoms and a clinical history suggestive of GER. All children underwent 24‐hr esophageal pH monitoring (pHm) and fiberoptic bronchoscopy with bronchoalveolar lavage (BAL). The amount of lipid per single macrophage was determined by a semiquantitative method, using fluorescence microscopy to detect Nile‐Red‐stained BAL cells and calculating a lipid‐laden macrophage index (LLMI).

Mineral oil lipoid pneumonia in a child with anoxic encephalopathy: treatment by whole lung lavage.

We describe a case of exogenous lipoid pneumonia in a child with anoxic encephalopathy who was taking mineral oil for constipation. Computed tomography produced images suggesting this condition, and the diagnosis was confirmed by demonstrating the presence of lipid‐laden alveolar macrophages in the bronchoalveolar lavage fluid. Despite discontinuing the offending agent, the pulmonary infiltrates did not improve; however, successful resolution was obtained by whole lung ravage. Pediatr Pulmonol. 1997; 23:235–239.

Downregulation of the expression of intercellular adhesion molecule (ICAM)-1 on bronchial epithelial cells by fenoterol, a β2-adrenoceptor agonist

Inflammatory airway disorders, such as asthma and chronic bronchitis, are characterized by overexpression of adhesion molecules on airway epithelial and endothelial cells. This phenomenon is associated with increased adherence and activation of polymorphonuclear leukocytes (PMNs). With the knowledge that beta2-adrenoceptor agonists demonstrate some anti-inflammatory activity in vitro, the present study was designed to evaluate whether fenoterol could interfere with adhesion molecule expression on airway epithelium. Human bronchial epithelial cells (HBECs), obtained by protease digestion from surgically resected bronchi, were stimulated with human recombinant interferon-gamma (rh IFN-gamma) in the presence of (a) fenoterol (10(-12)-10(-5) M); (b) dexamethasone (10(-12)-10(-5) M); and (c) fenoterol and dexamethasone. Because desensitization after high-dose exposure to agonists has been described for many membrane-associated receptors, in additional sets of experiments HBECs were preexposed to fenoterol and, as control, to dexamethasone for 8 hr, then washed and stimulated with rh IFN-gamma in the presence of fresh drugs. The cells were harvested after 24-hr culture and stained by specific monoclonal antibodies. The intensity of intercellular adhesion molecule-1 (ICAM-1) expression was then measured by flow cytometry analysis and expressed as mean fluorescence channel (mfc). The significant increase in ICAM-1 expression on HBECs induced by rh IFN-gamma was inhibited, in a dose-dependent manner, by the two drugs, but fenoterol was more efficient than dexamethasone at all of the concentrations tested (p < 0.05, all comparisons). In addition, the inhibitory activity of fenoterol was not enhanced by the simultaneous presence of dexamethasone in rh IFN-gamma-stimulated HBEC cultures (p > 0.05, all comparisons). Finally, preexposure to fenoterol or to dexamethasone did not induce any modification of the inhibitory effect of the two drugs on ICAM-1 expression (p > 0.05, all comparisons). These results suggest that clinical efficacy of fenoterol in patients with obstructive lung disease may include downregulation of adhesion molecule expression on airway epithelial cells.

An improved method to obtain highly differentiated monolayers of human bronchial epithelial cells.

SummaryElectrophysiological studies of human bronchial epithelial cells in vitro are limited by the scarcity of biological material available for primary culture. To overcome this problem, we set up a protocol in which the cell number is first enlarged in LHC9/RPMI 1640 serum-free medium for up to six passages, each passage giving a four- to eightfold amplification. The cells are then plated at high density on permeable supports. Cell differentiation, monitored by measuring transepithelial potential difference (PD) and electrical resistance (R), is induced with a medium containing serum and a cocktail of different supplements and hormones. Maximal values of PD and R, obtained after 4–7 d of culture on permeable supports, are around −50 mV and 3000–4000 ω/cm2, respectively. Ussing chamber experiments show that basal short-circuit current (ISC) is partially inhibited by the epithelial Na+ channel blocker amiloride. Stimulation with a cAMP-elevating agent induces a ISC increase that is inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Our culture protocol provides a large number of differentiated bronchial epithelial cell monolayers starting from a low amount of material. This characteristic is useful for in vitro studies of ion transport in airway epithelium.

An electrogenic amino acid transporter in the apical membrane of cultured human bronchial epithelial cells

We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolaterall-arginine (10-1,000 μM) increased the short-circuit current. Maximal effect and EC50 were ∼3.5 μA/cm2 and 80 μM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor ( N G-nitro-l-arginine methyl ester) did not decrease the arginine-dependent current. Apicall-lysine,l-alanine, andl-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50 values ranging from 26 to 971 μM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolateral L-arginine (10-1, 000 microM) increased the short-circuit current. Maximal effect and EC50 were approximately 3.5 microA/cm2 and 80 microM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester) did not decrease the arginine-dependent current. Apical L-lysine, L-alanine, and L-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50 values ranging from 26 to 971 microM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.

Total and allergen‐specific IgE levels in serum reflect blood eosinophilia and fractional exhaled nitric oxide concentrations but not pulmonary functions in allergic asthmatic children sensitized to house dust mites

Although elevated levels of serum immunoglobulin E (IgE) are considered the hallmark of atopic diseases, their clinical value in evaluating subjects with allergic disorders is under debate. To evaluate possible relationships between serum IgE levels and a variety of clinical parameters, 83 mild asthmatic children [10.98‐year‐old (2.95)], sensitized to house dust mites (HDM) Dermatophagoides pteronyssinus (Dp) or D. farinae (Df), were enrolled. As compared with normal control reference values detected in our laboratory, children with allergic asthma had higher blood eosinophil counts (expressed both as percentage and as absolute number) and higher fractional exhaled nitric oxide (FeNO) levels but similar values in pulmonary function parameters. In the allergic asthmatic population, serum levels of total, Dp‐specific or Df‐specific IgE correlated positively with eosinophil counts (Rho ≥ 0.30, p < 0.01, each correlation) and FeNO levels (Rho ≥ 0.33, p < 0.01, each correlation) but not with pulmonary function parameters (p > 0.1, each correlation). Finally, significant correlations, although moderate, were found in the allergic asthmatic population between eosinophil counts and FeNO levels (Rho ≥ 0.42, p < 0.001, each correlation). Thus, in atopic children sensitized to HDM with mild intermittent asthma, IgE levels in blood appear to reflect systemic (blood eosinophils) and organ‐specific (FeNO) markers of allergic inflammation but not pulmonary volumes or the degree of airflow limitation.

Pulmonary sarcoidosis: excess of helper T lymphocytes and T cell subset imbalance at sites of disease activity.

Different lymphocyte subpopulations have been evaluated in bronchoalveolar fluid and blood obtained from six patients with active and six with inactive pulmonary sarcoidosis and from six normal subjects by means of two recently described monoclonal antibodies, 5/9 and MLR4. The percentages of OKT4 positive (helper) and OKT8 positive (suppressor) T cells were also determined. Patients with active sarcoidosis had significantly higher proportions of 5/9 positive T cells in the bronchoalveolar fluid than patients with inactive disease (p less than 0.01) or normal subjects (p less than 0.001). In contrast, the proportions of 5/9 positive blood T cells were similar in the three groups studied. Patients with active sarcoidosis had also a greater proportion proportion of MLR4 positive T lymphocytes in bronchoalveolar fluid than patients with inactive disease or normal subjects (p less than 0.01 for each comparison), but similar proportions of MLR4 positive blood T cells were found in each group. The ratio of 5/9 positive to MLR4 positive T cells was higher in the bronchoalveolar fluid (but not in the blood) in patients with either active or inactive sarcoidosis than in normal subjects. These observations suggest that the MLR4 negative fraction rather than the MLR4 positive fraction of the 5/9 positive T cells is preferentially expanded in the lungs of patients with pulmonary sarcoidosis and may indicate a secondary role for the MLR4 positive T cells in producing lung injury in this disorder. Comparisons of the OKT4 positive and 5/9 positive T cells showed that in patients with active disease most of the lung T lymphocytes expressed both the OKT4 and the 5/9 surface antigens, so the 5/9 monoclonal antibody may be considered a good marker of activity in this disorder. Pulmonary sarcoidosis may be characterised by the preferential expansion of helper T cell subsets at sites of disease activity.

Weakly acidic gastroesophageal refluxes are frequently triggers in young children with chronic cough.

To evaluate whether the proportion of acid and weakly acidic refluxes preceding cough bursts could be different in infants, preschool‐ and school‐aged children with chronic, unexplained cough.

A 14-yr-old male with dyspnoea, productive cough and chest pain

A 14-yr-old White male was referred to the Urology Unit of the G. Gaslini Institute because of a post-traumatic urethral stenosis (arising from a bicycle accident) that, during the previous 18 months, had required repeated endoscopic urethral dilation manoeuvres at the patients local hospital. Apart from the urethral stenosis, the patient had been in excellent health until 2 months before admission, when slowly progressive exertional dyspnoea, associated with nonproductive cough and right-sided posterior chest pain, developed. On admission, the patient appeared in good clinical condition. Decreased percussion and auscultatory sounds were noted over the middle and lower portions of the right hemithorax. A summary of the results of the blood tests performed on admission is shown in table 1⇓. View this table: Table 1 Results of blood tests performed on admission Chest radiography (fig. 1⇓) and high-resolution computed tomography (HRCT) (fig. 2⇓) were performed. Fig. 1.— Chest radiograph. Fig. 2.— High-resolution computed tomography of the chest at two different window settings: a) “parenchymal”; and b) “soft tissue”. Thoracocentesis was performed and 500 mL haemorrhagic pleural fluid aspirated. Pleural fluid analysis did not show any cytological changes suggesting malignancies, amylase levels were within the normal range and microbiological evaluation results were negative for bacteria, fungi, mycoplasmata, mycobacteria and viruses. Plasma d‐dimer levels were slightly elevated but ultrasonography of the deep venous system did not show signs of thrombosis in the legs, penis or pelvis. Echocardiographic evaluation did not demonstrate any right ventricular dysfunction. Ultrasonographic examinations showed that there were no abnormalities of the abdominal organs and no peritoneal effusion and ruled out the presence of testicular or thyroid tumours. Fibreoptic bronchoscopy was then performed and did not reveal any airway abnormalities. Bronchoalveolar lavage analysis was nondiagnostic (no siderocytes suggestive of pulmonary haemorrhage, acid-fast bacilli, bacteria, viruses, fungi or malignant cells were identified in the epithelial lining fluid or lavage fluid …