Airway Eosinophilic Inflammation and Bronchial Hyperresponsiveness after Allergen Inhalation Challenge in Asthma

Allergen exposure in atopic asthmatic patients is associated with recruitment and activation of eosinophils in the airways. Once activated, eosinophils release toxic products, including the eosinophil cationic protein (ECP), able to damage bronchial structures and to increase bronchial hyperresponsiveness. With this background, the present study was designed to evaluate whether ECP levels in bronchoalveolar lavage (BAL) fluid could reflect, better than BAL eosinophil counts, the cellular activation that follows allergen exposure in atopic asthmatics. Twenty-two atopic patients attended the laboratory on two separate days. On the 1st day, they underwent methacholine (MCh) inhalation challenge to detect the degree of nonspecific bronchial hyperresponsiveness. On the 2nd day, they underwent fiberoptic bronchoscopy and BAL, at baseline or 4–6 h after allergen inhalation challenge. In this latter patient group, MCh challenge was repeated 3–5 h after allergen challenge, 1 h before fiberoptic bronchoscopy. The analysis of the mean baseline FEV₁ values and the degree of bronchial reactivity to MCh (MCh Pd₂₀) on the 1st study day did not demonstrate differences between the two patient groups (p > 0.1, each comparison). In addition, in the allergen-challenged group, MCh Pd₂₀ was decreased significantly after allergen challenge (151.4 μg/ml and 67.6 μg/ml, respectively, before and after challenge; p < 0.05). Evaluation of the different BAL cell types demonstrated that the proportions of eosinophils and epithelial cells were increased significantly in the allergen-challenged group compared with the group evaluated at baseline (p < 0.01 and p < 0.05, respectively). Moreover, ECP levels, corrected by the correspondent albumin levels (ECP/Alb), were higher in the allergen-challenged group compared with the group evaluated at baseline (p < 0.05). In addition, although a positive correlation was demonstrated between BAL eosinophil percentages and ECP/Alb values (r= 0.72, p < 0.05) in the group evaluated at baseline, no links were found between these parameters in the allergen-challenged group (p > 0.1). However, in this latter group, a weak positive correlation was demonstrated between eosinophil percentages and ΔMch, i.e., the increased nonspecific bronchial reactivity, which is observed after allergen challenge (r= 0.55; p < 0.05). Thus, in stable asthmatic patients an ongoing activation of eosinophils parallels their migration, but this eosinophilic inflammation is not strictly related to bronchial reactivity to Mch. By contrast, after allergen inhalation challenge, eosinophil recruitment and activation seem to follow different temporal kinetics, and eosinophilic inflammation may be partially associated with the degree of airway hyperresponsiveness. Accepted for publication: 15 September 1997     

β2-Agonist-Elevated Stress Response in Human Bronchial Epithelial Cells in Vivo and in Vitro

Recent studies report high baseline levels of stress (heat shock) proteins in bronchial epithelial cells from asthmatic individuals. The promoter of the gene encoding the 72-kDa heat shock protein has an element responsive to cAMP, which may be affected by β-agonists. This study examined stress protein levels in subjects enrolled in a segmental lung allergen challenge study to determine whether β-agonist medication could contribute to a stress response. Subjects were divided on the basis of no premedication (n= 17), metered dose inhalations of albuterol (n= 24), or placebo inhalation (n= 3) prior to bronchoscopy. Levels of the inducible stress protein Hsp72 and constitutive Hsp73 were quantitated in bronchial epithelial cells from brush biopsy of allergic nonasthmatic, allergic asthmatic, and normal individuals. Mean levels were increased significantly (p < 0.003 and p < 0.004, respectively) in those subjects who received albuterol premedication. No significant differences were found between clinical groups of individuals or for placebo inhalation vs nonpremedication. Albuterol in vitro increased the levels of Hsp72 and Hsp73 in epithelial cells from either nonpremedicated or placebo-treated donors; the Hsp72 levels correlated linearly with increased albuterol concentration (r= 0.81, p < 0.01). Therefore, β-agonists elevate or prolong an elevated stress response in epithelial cells, possibly through cAMP-mediated effects. Accepted for publication: 20 December 1996     

Methacholine Dose-Response Slopes from Maximal Bronchial Challenge Tests in Asthmatic Children: Methodological Aspects

To determine whether the slope of a maximal bronchial challenge test (in which FEV₁ falls by over 50%) could be extrapolated from a standard bronchial challenge test (in which FEV₁ falls up to 20%), 14 asthmatic children performed a single maximal bronchial challenge test with methacholine (dose range: 0.097–30.08 μmol) by the dosimeter method. Maximal dose-response curves were included according to the following criteria: (1) at least one more dose beyond a ΔFEV₁≥ 20%; and (2) a MFEV₁≥ 50%. PD₂₀ FEV₁ was calculated, and the slopes of the early part of the dose-response curve (standard dose-response slopes) and of the entire curve (maximal dose-response slopes) were calculated by two methods: the two-point slope (DRR) and the least squares method (LSS) in % ΔFEV₁×μmol⁻¹. Maximal dose-response slopes were compared with the corresponding standard dose-response slopes by a paired Student's t test after logarithmic transformation of the data; the goodness of fit of the LSS was also determined. Maximal dose-response slopes were significantly different (p < 0.0001) from those calculated on the early part of the curve: DRR_20% (91.2 ± 2.7 ΔFEV₁% ·μmol⁻¹) was 2.88 times higher than DRR_50% (31.6 ± 3.4 ΔFEV₁% ·μmol⁻¹), and the LSS_20% (89.1 ± 2.8% ΔFEV₁·μmol⁻¹) was 3.10 times higher than LSS_50% (28.8 ± 1.5% ΔFEV₁·μmol⁻¹). The goodness of fit of LSS_50% was significant in all cases, whereas LSS_20% failed to be significant in one. These results suggest that maximal dose-response slopes cannot be predicted from the data of standard bronchial challenge tests. Accepted for publication: 12 December 1996     

Budesonide Down-Regulates Eosinophil Locomotion But Has No Effects on ECP Release or on H2O2 Production

Treatment of allergic asthma with inhaled corticosteroids results in local down-regulation of proinflammatory cytokine synthesis and in marked decrease in tissue eosinophilia. Blood concentrations of inhaled corticosteroids, although significantly lower than those measured in the lung, may still have antiinflammatory effects on circulating eosinophils, reducing their ability to migrate. The aim of our study was to evaluate in vitro the activity of budesonide on blood eosinophils by measuring their chemotactic response, eosinophil cationic protein (ECP) release, and hydrogen peroxide (H₂O₂) production in the presence of different drug concentrations similar to those obtained at airway level (10⁻⁸ and 10⁻⁷ M) and at blood level (10⁻¹⁰ and 10⁻⁹ M). Partially purified blood eosinophils, isolated from 23 asthmatic subjects, were used to evaluate the activity of budesonide on: (1) chemotaxis toward the activated fifth component of complement (C5a, 0.1 μg/ml) or recombinant human (rh) interleukin (IL)-5 (200 pg/ml), (2) ECP release by cells stimulated with tetradecanoylphorbol acetate (TPA) and (3) H₂O₂ production by TPA-activated cells. The chemotactic response to C5a was down-regulated significantly by budesonide only by the highest concentrations tested (10⁻⁸ and 10⁻⁷ M); differently, budesonide was effective in inhibiting eosinophil migration toward rhIL-5, at all concentrations tested (p < 0.01, each comparison). By contrast, no drug-induced modifications were observed in ECP release or in H₂O₂ production (p > 0.05, each comparison). We conclude that concentrations of budesonide similar to those obtained in vivo are effective in inhibiting eosinophil locomotion but not in down-regulating the release of reactive oxygen species and granule-associated proteins. Accepted for publication: 11 February 1999     

Oxidative Inactivation of Surfactants

Reactive oxygen species (ROS) may play an important role in the chronic pulmonary morbidity of preterm infants. We therefore studied the magnitude and mechanisms of oxidative inactivation of a natural lung surfactant (NLS) and of two surfactants used for treatment of respiratory distress syndrome, beractant and KL₄ surfactant (KL₄). Incubation with Fenton reagents, 2-4 mM peroxynitrite (ONOO⁻) or 0.5 mM hypochlorous acid (OCl⁻), resulted in an increased minimum surface tension (MST) of all surfactants; the order of effect on MST was beractant > KL₄ > NLS. After incubation with Fenton reagents, NLS contained a higher concentration of conjugated dienes (p < 0.01) but lower concentration of malondialdehyde (p < 0.001) than beractant. Protein carbonyl concentrations after treatment with Fenton reagents were higher in NLS and KL₄ than in beractant (p < 0.05). Surface area cycling for 24 h with 2 mM ONOO⁻ or 0.5 mM OCl⁻ caused both beractant and KL₄ to increase the proportion of light subtypes from 8–10% to 26–29%; with Fenton reagents, there was disappearance of the light subtype and formation of ultraheavy subtype 74–91% with poor MST. Natural and therapeutic surfactants differ markedly in their sensitivity to ROS, which may be important for surfactants in therapeutic use because oxidative inactivation may limit their effect. Oxidation of natural surfactant may result in reduced function and contribute to chronic lung disease. Accepted for publication: 8 January 1999     

Antioxidant Function of Ambroxol in Mononuclear and Polymorphonuclear Cells in Vitro

This study quantifies the antioxidant function of ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine) in vitro. Polymorphonuclear cells (PMN) and mononuclear cells were isolated from the blood of healthy volunteers (n= 46) to determine reactive oxygen species (ROS) by luminol-enhanced chemiluminescence. Ambroxol or the controls N-acetylcysteine (NAC), nacystelyn (NAL), glutathione (GSH), superoxide dismutase (SOD), catalase, and the combination of SOD/catalase were incubated for 1 or 2 h with zymosan-activated cells in vitro using concentrations ranging from 10⁻⁶ to 10⁻³ mol/liter. Reduction of ROS-mediated luminescence was similar within the cell types. Ambroxol (10⁻⁴ mol/liter) reduced ROS about 75% (1-h incubation) and 98% (2-h incubation), respectively (p < 0.001). SOD and SOD/catalase, but not the H₂O₂-catalyzing substances (NAC, NAL, GSH, and catalase), reduced cellular ROS. This indicates that inflammatory cells predominantly generate O⁻ ₂, which can be scavenged by ambroxol. The antioxidant function of ambroxol with increasing incubation time suggests additional cellular antiinflammatory properties of this substance. Our results indicate that good antioxidant function of ambroxol is related mainly to direct scavenger function of reactive oxygen metabolites such as O⁻ ₂. However, an antioxidative effect of ambroxol may also be associated with the reduction of prooxidative metabolism in inflammatory cells. Concluding from this observation, and because of the well known high affinity of ambroxol for lung tissue, ambroxol may be an alternative in antioxidant augmentation therapy, particularly in pulmonary diseases characterized by an overburden of toxic oxygen metabolites. Accepted for publication: 5 December 1996     

Isoniazid Levels in the Bronchoalveolar Lavage Fluid of Patients with Pulmonary Tuberculosis

Isoniazid (INH) is one of the most important first line drugs in the treatment of tuberculosis. We utilized high performance liquid chromatography with a hydrazone extraction technique to measure INH in bronchoalveolar lavage (BAL) fluid specimens from six patients with active pulmonary tuberculosis. We found BAL fluid INH levels to be similar to 2-h peak serum levels. The concentration of INH in BAL fluid from lobes with infiltrate was similar to the concentration of INH in BAL fluid from lobes without infiltrate (0.062 μg/ml and 0.073 μg/ml, respectively). After adjusting for protein concentration in the BAL fluid, INH levels in lobes with infiltrate were threefold lower than in lobes without infiltrate. The correlation between the concentration of INH in serum and BAL fluid approached significance after correcting for protein (lobes with infiltrate, r ²= 0.60 (p= 0.07); lobes without infiltrate, r ²= 0.50 (p= 0.12)). INH penetrates into bronchoalveolar fluid, and concentrations of INH in the BAL fluid suggest that assessment of the INH serum concentration is adequate to evaluate bioavailability of the drug in patients with pulmonary tuberculosis. Accepted for publication: 26 June 1997     

The Peroxisome Proliferator Activated Receptor Gamma (PPARγ) Ligand Rosiglitazone Modulates Bronchoalveolar Lavage Levels of Leptin,Adiponectin, and Inflammatory Cytokines in Lean and Obese Mice

Obese mice that lack leptin receptor (db ⁻ /db ⁻) have been shown to have innate bronchial hyperresponsiveness (BHR). It has been proposed that the obesity-mediated BHR may involve a combination of increased leptin and reduced systemic adiponectin levels. The aim of this study was to determine if obesity modifies the airway concentration of leptin and adiponectin and whether treatment with a synthetic peroxisome proliferator-activated receptor gamma (PPARγ) ligand can reduce airway leptin and increase airway adiponectin. In this study, obese, leptin receptor-deficient (db ⁻ /db ⁻), or lean (db ⁺ /db ⁻) mice were treated with rosiglitazone (3 mg/kg/day) or vehicle by gavage daily for 1 week. Bronchioalveolar lavage (BAL) was subsequently performed to determine levels of leptin, adiponectin, and inflammatory cytokines. Treatment with rosiglitazone increased BAL adiponectin levels in lean (p = 0.04) and to a lesser extent in obese mice (p = 0.07). Rosiglitazone treatment lowered leptin levels in lean mice, but increased leptin levels in BAL fluid of obese mice (p < 0.01). The BAL levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) were lower in the lean rosiglitazone-treated group compared with the obese vehicle-treated group and lower in the obese rosiglitazone-treated group compared with the obese vehicle-treated group. These results demonstrate that obesity is associated with alterations in adipokine and cytokine levels in the airways that can be modulated by treatment with roziglitazone.     

Serum Eosinophil-Derived Neurotoxin (EDN) in Diagnosis and Evaluation of Severity and Bronchial Hyperresponsiveness in Childhood Asthma

This study sought to evaluate the use of serum eosinophil-derived neurotoxin (EDN), which has been proposed as a marker of airway inflammation in asthma in the diagnosis and evaluation of the severity and bronchial hyperresponsiveness in childhood asthma. We studied 72 children with atopic asthma, 36 children with nonatopic asthma, and 43 healthy controls. Skin prick tests, pulmonary function tests, and methacholine challenge tests were performed, in addition to total eosinophil count, serum ECP, and EDN being measured in all subjects. EDN levels were significantly higher in the atopic asthma group than those in the nonatopic asthma group or control group (p < 0.001), as were ECP levels (p < 0.001). EDN levels differed more significantly among groups divided by asthma severity (p < 0.001) than did ECP levels for these groups (p < 0.05). For the groups divided according to bronchial hyperresponsiveness, both EDN and ECP levels were significantly different (p < 0.005 and p < 0.01, respectively). Significant correlations were found between EDN and PC₂₀ (γ = −0.281; p < 0.001), between ECP and PC₂₀ (γ = −0.274; p < 0.005), and between EDN and ECP (γ = 0.443; p < 0.001). In conclusion, serum EDN, as another marker of eosinophilic inflammation together with ECP, may aid in the diagnosis of asthma, especially atopic asthma, and in the evaluation of the severity and bronchial hyperresponsiveness in childhood asthma.     

Thrombin in the Airways of Asthmatic Patients

The mechanism of airway remodeling in asthmatic patients is poorly understood. Thrombin is a multifunctional protease that, in addition to its critical role in thrombotic processes, has also been described as inducing cellular and molecular events relevant to tissue remodeling. The present investigation was undertaken to evaluate the activity of thrombin in the sputum of asthmatic patients and its potential role in airway remodeling. The study population comprised 8 healthy subjects and 14 stable patients with bronchial asthma. The concentrations of thrombin, thrombin-antithrombin complex (TAT), and tissue factor were measured in the sputum of all subjects. The concentrations of thrombin (p= 0.007), TAT (p= 0.01), and tissue factor (p= 0.02) in sputum were significantly higher in asthmatic patients than in healthy controls. The proliferative effects that sputum from asthmatic patients (p= 0.01) and thrombin (p= 0.03) have on cultured human smooth muscle cells was inhibited significantly in the presence of recombinant hirudin, a specific thrombin inhibitor. Significant statistical correlation was observed between the degree of bronchial responsiveness and the sputum concentrations of thrombin (r=−0.8; p= 0.02) and TAT (r=−0.9; p= 0.01). The results of this study showed that increased thrombin generation occurs in the airway of patients with asthma and that it may play a role in the pathogenesis of airway remodeling. Further studies should be carried out to assess whether these findings are also observed in other airway diseases. Accepted for publication: 6 April 1999     

Eosinophilic alveolitis in immunologic interstitial lung disorders

To analyze the role of eosinophils in alveolitis due to immunological interestitial lung disorders, 568 bronchoalveolar lavage (BAL) from 537 patients affected by 13 types of interstitial lung disease involving immunologic mechanisms were considered. An arbitrary cut-off of 4% of eosinophils in BAL was assumed. In five (idiopathic pulmonary fibrosis (IPF), allergic bronchopulmonary aspergillosis (ABPA), amiodarone-induced pneumonitis (AIP), chronic eosinophilic pneumonia (CEP), Churg-Strauss syndrome (CSS)) out of the thirteen groups we took into consideration, the level of eosinophils was >4%. In CEP and CSS in particular, the arbitrary cut-off of 4% was greatly exceeded (28.9%±27.4,p<0.01 and 33.6%±14.5,p<0.01, respectively). In the same two groups the increase of eosinophils in BAL was isolated with a direct correlation to the number of eosinophils in blood. By contrast, the increase of eosinophils in BAL of IPF, AIP and ABPA was of lesser extent (4.7%±5.7p<0.01, 5.0%±3.0p<0.01 and 6.1%±10.4p<0.01, respectively) and was accompanied by an increase of neutrophils in IPF, of lymphocytes in AIP and both in ABPA. These patterns are generally defined as “mixed alveolitis.” On the basis of these data we conclude that the term “eosinophilic alveolitis” should be reserved for CEP and CSS.     

Involvement of Tachykinins in Endotoxin-Induced Airway Hyperresponsiveness

Inhaled endotoxin, lipopolysaccharide (LPS), has been shown to result in bronchial hyperresponsiveness (BHR) to endogenous bronchoconstrictive mediators such as histamine. To determine the role of sensory neuropeptides released from bronchopulmonary C-fibers in LPS-induced BHR, 24 guinea pigs were allocated randomly to the following four groups. Animals in Groups I and IV were challenged with intratracheal instillation of 100 μl of saline vehicle, and those in Groups II and III with 1 mg of LPS (Escherichia coli, 0111:B4) in 100 μl of saline. Groups III and IV also received a high dose capsaicin (HDC) treatment to deplete tachykinins from C-fibers 1–2 weeks prior to the experiment. Animals were anesthetized and paralyzed, and total lung resistance (R_L) and compliance (C_dyn) were measured continuously during the experiment. Dose responses of R_L and C_dyn to histamine (0–8 μg/kg, intravenously) and capsaicin (0–1.6 μg/kg, intravenously), a specific C-fiber stimulant, were obtained prior to and at 1, 2, and 3 h following LPS/saline vehicle challenge. At 2 h after LPS, ΔR_L caused by histamine (8 μg/kg) was significantly higher in Group II (1.145%) than that in Group I (280%; p < 0.05); similarly, ΔR_L caused by capsaicin (1.6 μg/kg) was also increased after LPS (Group I, 107%; Group II, 267%; p < 0.05). Although HDC treatment completely abolished the bronchomotor response to capsaicin in both Groups III and IV, it enhanced the LPS-induced BHR to histamine (8 μg/kg; Group III, 1.834%; p < 0.05). In conclusion, these results suggest that the role of tachykinins in LPS-induced BHR may be dependent upon the type and the route of administration of the bronchoactive substance studied. Accepted for publication: 13 December 1996     

Initial Pleural Pressure Measurement in Spontaneous Pneumothorax

To assess whether pleural pressure (PP) measurement is helpful in evaluating the evolution of spontaneous pneumothorax (SP). To measure the inspiratory and expiratory PP at tidal volume in the SP in 85 cases. Ninety-one percent were cured with medical treatment, whereas the remaining 9% required surgery. In the cases medically resolved, the inspiratory pleural pressure (IPP) was −9 ± 5 mbar, and the expiratory pleural pressure (EPP) was −3 ± 6 mbar, whereas in the surgical cases, IPP was −7 ± 2 mbar (p= not significant), and EPP was 1 ± 3 mbar (p < 0.01). In the SP cases, which resolved in less than 7 days of drainage, IPP was −10 ± 5 mbar, and EPP was −3 ± 5 mbar, whereas IPP was −8 ± 3 mbar (p < 0.01), and EPP was 0 ± 4 mbar (p < 0.001) in those cases in which SP resolution required more than 7 days. The sensitivity to predict the need for surgery with an EPP ≥ 2 mbar was 100%, with a specificity of 49%. The cutoff of the curve was ≥ 1 mbar, with a sensitivity of 65% and specificity of 70%. The values of PP at the end of an expiration to tidal volume were negative in the SP cases, which were resolved by medical treatment, whereas they were atmospheric in those cases that required surgery. The static inspiratory and expiratory PP in the SP cases requiring thoracic drainage during <7 days showed more negative PP than those that resolved in a period longer than 7 days. Accepted for publication: 11 August 2000     

Furosemide-Induced Bronchodilation in the Rat Bronchus: Evidence of a Role for Prostaglandins

Pretreatment with inhaled fuorsemide has been shown to protect against bronchoconstrictive stimuli that indirectly activate airway smooth muscle. However, it is controversial as to whether furosemide acts directly on airway smooth muscle. To investigate this we studied the effect of furosemide on both methacholine (MCh)- and serotonin (5-HT)-induced bronchoconstriction in explanted rat airways. Lungs from 21 Sprague-Dawley rats (269 ± 15 g) were excised, inflated with agarose solution at 37°C (1% w/v, 48 ml/kg), embedded in 4% agarose, and refrigerated to gel the agarose. Lung slices (0.5–1.0 mm thick) were cultured overnight at 37°C. Explants were placed on a dissecting video microscope, and airway area was measured with an image analysis system. MCh or 5-HT was administered directly to explanted airways (final concentrations 3.8 × 10⁻⁶ M and 3.8 × 10⁻⁵ M, respectively). Five min later furosemide (3.7 × 10⁻⁵ M or 3.7 × 10⁻⁴ M) was added and airway area monitored 5, 10, 15, 30, and 60 min later. Results were expressed as a percentage of the maximal response. Significant bronchodilation was seen after 30 min in airways preconstricted with MCh and after 15 min in those preconstricted with 5-HT following 3.7 × 10⁻⁴ M furosemide (p < 0.05). 3.7 × 10⁻⁵ M furosemide caused bronchodilation only at 60 min in airways constricted with 5-HT. The effect was blocked by a 30-min incubation of explants with 10⁻⁶ M indomethacin. The furosemide-induced bronchodilation effect was not observed in airways strongly constricted with 3.8 × 10⁻⁵ M MCh. These findings indicate that in the rat at least, furosemide induces a weak bronchodilator effect present only at high doses, which seems to be dependent on the production of prostaglandins. This effect may be relevant to the observed therapeutic action of furosemide in asthmatics. Accepted for publication: 27 September 1996     

Bronchial Hyperresponsiveness in Farmers: Relation to Respiratory Symptoms, Lung Function, and Atopy

There is only limited information on the factors associated with nonspecific bronchial hyperresponsiveness (BHR) in farmers. Our purpose was to examine the relationship between BHR and respiratory symptoms, atopy, and abnormalities of lung function in a sample of French farmers. Farmers scheduled for a preventive medicine check-up in northeastern France were examined. Occupational exposure, respiratory symptoms, and work-related symptoms were assessed by questionnaire, sensitization to 34 common and agricultural allergens by skin prick tests, and BHR by the single-dose (1,200 μg) acetylcholine (ACh) challenge test. Data were obtained from 741 farmers (95% of those invited). Seventy-seven subjects (10.3%) had BHR defined as a fall in forced expiratory volume in 1 s (FEV₁) ≥ 10% after the inhalation of ACh or, for those with a poor lung function, an increase in FEV₁ > 10% and > 200 ml after the inhalation of 200 μg of salbutamol. The proportion of asthmalike symptoms, especially wheeze during work, positive skin tests to acarian (storage mites) and cereal dust allergens, and low levels of lung function was significantly greater among reactors than among nonreactors. Stepwise logistic regression analysis showed a significant and independent association between BHR and wheezing during work (OR = 4.99; 95% CI = 2.29–10.89; p= 0.0001) and baseline FEV₁ (OR = 1.49; 95% CI = 1.05–2.20; p= 0.026). In conclusion, hyperreactive farmers had significantly more asthmalike symptoms, positive skin tests, and abnormal lung function than normoreactive farmers. Work-related wheeze and low baseline FEV₁ were significantly and independently associated with BHR. Accepted for publication: 26 January 1999     

Inhibitory role of eosinophils on cell surface plasmin generation by bronchial epithelial cells: inhibitory effects of transforming growth factor beta

Eosinophilic bronchitis is an essential component of bronchial asthma, and eosinophils play an important role. We studied the effect of eosinophils on cell surface plasmin generation by bronchial epithelial cells, because plasmin is thought to be involved in bronchial tissue repair/remodeling by means of fibrinolysis and the activation of proteases such as matrix metalloproteases. Plasmin was generated from exogenous plasminogen on the cell surface of cultured bronchial epithelial cells, NCI-H292. Transforming growth factor β (TGF-β) treatment resulted in reduced cell surface plasmin generation and a large increase in plasminogen activator inhibitor-type 1 (PAI-1) antigen production in NCI-H292 cells, whereas no conspicuous effects were observed with IL-1β and TNFα treatment (regulators in pulmonary epithelial cells). On the other hand, this cell surface plasmin generation was reduced by co-incubation with Eol-1, an eosinophil cell line. The addition of TGF-β antisense and anti-TGF-β antibodies attenuated this adverse effect of Eol-1 cell co-incubation. These data suggest that eosinophils play an inhibitory role on cell surface plasmin generation by bronchial epithelial cells by means of the up-regulation of PAI-1 expression induced by TGF-β. Therefore, the accumulation of eosinophils in bronchial walls is thought to be involved in bronchial tissue repair/remodeling in asthma through this protease network. Accepted for publication: 10 January 2001     

Renal kallikrein in diabetic patients with hypertension accompanied by nephropathy

Summary We measured the 24-h excretion of urinary kallikrein in 27 patients with Type 2 (non-insulin-dependent) diabetes and in 10 normal control subjects. Mean (± SD) kallikrein excretion in diabetic patients with nephropathy (6.2±2.4 naphthyl units (NU)/day,n=13) was significantly lower than in control subjects (12.8±3.4NU/day,p<0.01) and in diabetic patients without nephropathy (9.4±3.4NU/day,n=14,p<0.05). Kallikrein excretion in hypertensive diabetic patients with nephropathy (5.1±1.6 NU/day,n=8) was significantly lower (p<0.05) than in normotensive patients with nephropathy (8.3±2.1 NU/day,n=5). There were no significant differences in kallikrein excretion rate (24-h excretion of urinary kallikrein/24-h creatinine clearance) among control subjects (9.9±4.3 NU/ml), diabetic patients with (9.0±3.2 NU/ml) and without (9.3±3.5 NU/ml) nephropathy. However, kallikrein excretion rate in hypertensive diabetic patients with nephropathy (7.7±3.3 NU/ml) was significantly lower (p<0.05) than in normotensive diabetic patients with nephropathy (11.8 ±2.0 NU/ml,n=10). Respective basal and post-stimulated (with intravenous furosemide 40 mg plus 60 min ambulation) plasma aldosterone concentrations measured in control subjects and in hypertensive diabetic patients with nephropathy were similar and increased to the same extent in the 2 groups (5.5±3.2 versus 5.3±3.2 and 9.3±2.6 versus 10.5±3.4 ng/ml), although the respective plasma renin activity tended to be lower in diabetic patients than in control subjects (0.7±0.6 versus 1.3±0.9 and 1.8±1.8 versus 3.0±2.6 ng⁻¹ · ml⁻¹ · h⁻¹). The results indicate that urinary kallikrein excretion is decreased in hypertensive diabetic patients with nephropathy, and that the decrease might not be attributable to an altered renin-aldosterone system.     

Acetyl-L-carnitine improves cognitive functions in severe hepatic encephalopathy: a randomized and controlled clinical trial

The aim of this study was to investigate the effects of ALC treatment on cognitive functions in patients with severe hepatic encephalopathy. This was a randomized, double-blind, placebo-controlled study. 61 patients with severe hepatic encephalopathy were recruited to the study. The 2 groups received either 2 g ALC twice a day (n = 30) or placebo (n = 30) for 90 days. Clinical and laboratory assessment, psychometric tests and automated electroencephalogram (EEG) analysis were performed for all patients. At the end of the study period, between the 2 groups we observed a significant difference in Everyday Memory Questionnaire −23.9 vs 4.4 (p < 0.001), Logical Memory (Paragraph recall) test 22.3 vs 0.7 (p < 0.001), Trail Making Test A −7.5 vs −2.6 (p < 0.001), Trail Making Test B −10.5 vs −3.1 (p < 0.001), Controlled Oral Word Association Test 4.2 vs 0.5 (p < 0.001), Hooper test 2.6 vs 0.1 (p < 0.05), Judgement of line orientation 2.8 vs 0.3 (p < 0.001), Digit Cancellation time −24.5 vs −2.4 (p < 0.001), NH₄⁺ 30.5 vs 13.5 (p < 0.001), prothrombin time 2 vs 2.4 (p < 0.05), alanine transaminase −10.7 vs −13.6 (p < 0.001). 88% of patients treated with ALC vs 72% of patients treated with placebo showed a significant improvement in EEG. The improvement of cognitive deficits, the reduction of ammonia, and the modification of EEG in patients treated with ALC suggest that ALC could represent a new tool in the treatment of severe hepatic encephalopathy.     

Response of upper and lower airway inflammation to bronchial challenge with house dust mite in Chinese asthmatics: a pilot study

BackgroundAllergen nasal challenge can induce increase of eosinophils in sputum, but report about eosinophilic inflammation in upper airway after allergen bronchial challenge in Chinese asthmatics was rare. The article aims to evaluate response of upper and lower airways to house dust mite (HDM) allergen bronchial challenge.MethodsHDM allergen bronchial challenge was carried out in asthmatic patients with allergic rhinitis (AR). Bronchial methacholine challenge and blood test were performed before and at 24 hours after allergen challenge. Nasal lavage and induced sputum for differential cells count and fractional exhaled nitric oxide (FeNO) measurement were performed before, 7 and 24 hours after allergen challenge.ResultsEighteen asthmatic patients with AR underwent HDM allergen bronchial challenge with no serious adverse events reported. Fifteen patients showed dual asthmatic response (DAR), while 2 patients showed early (EAR) and 1 late asthmatic response (LAR) only. At 24 hours after allergen bronchial challenge testing, average PC₂₀FEV₁ to methacholine significantly decreased (1.58 to 0.81 mg/mL, P=0.03), while both FeNO and the percentage of eosinophils in blood and sputum were significantly increased [52.0 (54.0) to 69.0 (56.0) ppb, P=0.01; 4.82% to 6.91%, P<0.001; 20.70% to 27.86%, P=0.03, respectively], but with no significant differences found in the percentage of eosinophils in nasal lavage (39.36% to 38.58%, P=0.89). However, at 7 hours after allergen challenge, the eosinophils in sputum were significant increased to 40.45% (P<0.001), but there was an increase (39.36% to 48.07%) with no statistical difference (P=0.167) found in nasal lavage.ConclusionsHDM allergen bronchial challenge induced different response of airway inflammation in upper and lower airways.     

Assessment of Ingestive and Oral Praxis Skills: Children with Cerebral Palsy vs. Controls

Eating impairments (dysphagia) in children with cerebral palsy (CP) have been well documented. However, individual components of ingestion, such as the feeding skills of eating and drinking and their relationship to oral-motor planning skills (praxis), remain largely undetermined. The purpose of the present study was to examine functional feeding and oral praxis skills in a group of children with CP and mild eating impairment and to compare their skills with a group of age-matched controls. As well, interobserver reliabilities and concurrent validity of these tests were examined to determine their reliability and to what extent they may be measuring similar constructs. Twenty-seven children with CP and 21 age-matched controls, aged 4.0–16 years, participated in this study. Two oral praxis tests (OFMF and OPT) and two standard ingestive skills tests (GVA and FFAm) were administered to each child. Children with CP scored consistently and significantly lower on the OFMF than controls (p < 0.001); similar results were achieved on the OPT (p < 0.001). Children with CP had difficulty with items where there was a high demand for repetition and smooth sequencing. On the ingestive skills tests children with CP took significantly longer for chewing a hard solid food texture than controls (p < 0.001), and functional feeding skills were also significantly poorer than in controls. Interrater reliability coefficients for the OPT and OFMF were excellent (all ICCs > 0.90). There was high concurrent validity between the OPT and OFMF (r= 0.90, p < 0.0001). The correlations for the FFAm and GVA tests were somewhat weaker (r=−0.54, p < 0.0001). Correlations were negative; as functional feeding scores increased (improved), chewing time decreased (improved). Excellent reliable and valid assessment instruments are available to the practicing clinician. They cover a wide range of oral-motor performance and, so, must be used judiciously and for the purpose that they have been developed.