Hofer Wong

Dose-dependent cigarette smoking-related inflammatory responses in healthy adults

The aim of this study was to determine the dose-response relationship between cigarette smoke exposure and pulmonary cell and cytokine concentrations in bronchoalveolar lavage (BAL). BAL cells and BAL supernatant concentrations of tumour necrosis factor-alpha (TNF alpha), interleukin (IL)-1 beta, IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 from 14 healthy smokers and 16 healthy nonsmokers were quantified. Statistically greater concentrations of neutrophils, macrophages, IL-1 beta, IL-6, IL-8 and MCP-1 were observed among smokers compared with nonsmokers (p < or = 0.0007 in all cases). Cigarette smoking, categorized ordinally as: less than one pack, one pack, or greater than one pack per day, was predictive of BAL macrophages (p < 0.0001), neutrophils (p = 0.015), IL-1 beta (p < 0.001) and IL-8 (p = 0.02). We conclude that concentrations of macrophages, neutrophils, IL-1 beta and IL-8 are elevated in the pulmonary microenvironment of smokers in a cigarette dose-dependent manner. Based on the present findings, we would caution against simple analyses that treat current smokers as a homogeneous group and which do not account for smoking intensity.

Analysis of induced sputum to examine the effects of prednisone on airway inflammation in asthmatic subjects

To determine whether induced sputum samples might provide a useful means for evaluating the effects of therapy on airway mucosal inflammation, we examined induced sputum samples obtained before and after 6 days of treatment with prednisone (0.5 mg/kg/day) or placebo in a randomized, double-blind study of 24 asthmatic subjects. Induced sputum was analyzed for total and differential cell counts and for concentrations of eosinophil cationic protein, albumin, and mucin-like glycoprotein. We found that the mean (+/- SEM) percentage of eosinophils in sputum samples from the prednisone-treated group fell from 14.1% +/- 5.0% at baseline to 1.8% +/- 0.8% after treatment, a decrease significantly greater than in the placebo-treated group (from 10.3% +/- 4.9% to 11.1% +/- 4.0%; p = 0.002). The absolute number of eosinophils also decreased significantly more in the prednisone-treated group than in the placebo-treated group (p = 0.04). In addition, eosinophil cationic protein levels in induced sputum fell more in the prednisone-treated group than in the placebo-treated group (from 324 +/- 131 ng/ml to 144 +/- 84 ng/ml vs 173 +/- 50 ng/ml to 188 +/- 47 ng/ml; p = 0.002). Furthermore, prednisone treatment was associated with a significant increase in peak expiratory flow, an effect that was significantly correlated with the decrease in eosinophil percentage in induced sputum (rs = 0.64, p = 0.04). Prednisone treatment was not associated with any significant change in the concentrations of albumin or mucin-like glycoprotein. We conclude that analysis of induced sputum is a useful noninvasive method for studying the effects of asthma therapy on airway eosinophilic inflammation.

Analysis of cellular and biochemical constituents of induced sputum after allergen challenge: A method for studying allergic airway inflammation

To determine whether analysis of the constituents of induced sputum permits detection of changes provoked by aerosolized antigen challenge, we performed sputum induction (20-minute inhalation of aerosolized 3% saline solution) before and after aerosolized allergen challenge in eight subjects with asthma. Total cell counts and cell differentials of nonsquamous cells in induced sputum samples were determined after the samples were homogenized in dithiothreitol. Centrifugation of the entire homogenized sputum sample yielded supernatant that could be analyzed for biochemical constituents. We found that the median percentage of eosinophils and neutrophils in induced sputum samples was significantly higher 4 hours after allergen challenge neutrophils in induced sputum samples was significantly higher 4 hours after allergen challenge than at baseline (12% vs 0.5%, p < 0.05; 30.5% vs 7.5%, p < 0.05) and remained high 24 hours after challenge. Median levels of eosinophil cationic protein and histamine in induced sputum supernatants were significantly higher 4 hours after challenge than at baseline (151.3 vs 39.8 ng/ml, p < 0.05; 19.4 vs 8.8 micrograms, p < 0.05) and remained significantly higher 24 hours after challenge. Tryptase was detectable in sputum from seven of the subjects, and in these subjects, we found a trend toward an increase in median tryptase levels 4 hours after allergen challenge (4.4 vs 2.2 U/L, p = 0.09). We conclude that analysis of induced sputum after aerosolized allergen challenge reveals changes in inflammatory cells and markers similar to those reported in bronchoalveolar lavage fluid and that sputum induction is a useful noninvasive method for studying allergic airway inflammation in asthma.

In vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects

BACKGROUND Human rhinoviruses (HRVs) characteristically cause upper respiratory tract infection, but they also infect the lower airways, causing acute bronchitis and exacerbating asthma. OBJECTIVE Our purpose was to study ex vivo the differences in the response to HRV infection of nasal and bronchial epithelial cultures from the same healthy and asthmatic individuals using conditions favoring development of fully differentiated, pseudostratified mucociliary epithelium. METHODS Cells from the inferior turbinates and bronchial tree of 5 healthy and 6 asthmatic individuals were cultured at an air-liquid interface. Cultures were infected with HRV-16, and after 48 hours, the degree of infection was measured. RESULTS Baseline median transepithelial resistance was lower in human bronchial epithelial (HBE) cell cultures than in human nasal epithelial (HNE) cell cultures (195 Omega.cm2 [95% CI, 164-252] vs 366 Omega.cm2 [95% CI, 234-408], respectively; P < .01). Virus replicated more easily in HBE cells than in HNE cells based on virus shedding in apical wash (log tissue culture infective dose of 50%/0.1 mL = 2.0 [95% CI, 1.0-2.5] vs 0.5 [95% CI, 0.5-1.5], P < .01) and on a 20- to 30-fold greater viral load and number of infected cells in HBE cell cultures than in HNE cell cultures. The increases in expression of RANTES and double-stranded RNA-dependent protein kinase were greater in HBE cell cultures than in HNE cell cultures, as were the concentrations of IL-8, IL-1alpha, RANTES, and IP-10 in basolateral medium. However, no significant differences between asthmatic and healthy subjects (including IFN-beta1 expression) were found. CONCLUSIONS Differentiated nasal epithelial cells might have mechanisms of increased resistance to rhinovirus infection compared with bronchial epithelial cells. We could not confirm previous reports of increased susceptibility to HRV infection in epithelial cells from asthmatic subjects.

Comparison of two methods of collecting induced sputum in asthmatic subjects

The method that we have previously reported for sputum induction involves collecting the entire expectorate produced over a 20 min inhalation of 3% saline aerosol. This method presents the potential disadvantage of a considerable and variable salivary contribution to the induced sputum sample. In this study, we examined whether separate collection of saliva and sputum represents a better method for collecting induced sputum during sputum induction. In 11 stable asthmatics, we compared the volume, total and differential cell counts, and eosinophil cationic protein (ECP) levels in four induced sputum samples, two performed using our previous method (Method A) and two using another method (Method B) in which subjects spit saliva into one container before coughing sputum into another. We found that the volume of sputum obtained with Method B was lower than that obtained with Method A (6.16 +/- 0.61 vs 20.1 +/- 2.7 mL; p = 0.003), as was the percentage of squamous cells (34 +/- 4 vs 47 +/- 6; p = 0.023). In addition, the ECP levels in samples collected by Method B were higher (261 +/- 42 vs 145 +/- 26 ng.mL-1; p = 0.01). The differential counts of nonsquamous cells were similar except for the percentage of neutrophils, which was lower in Method B (37 +/- 4 vs 50 +/- 5%; p = 0.019). The repeatability of measurements of eosinophil percentages and of ECP levels was similar for the two methods. We conclude that separate collection of saliva and sputum yields induced sputum samples with reduced amounts of saliva and is, therefore, a better method for collecting induced sputum.

Fractional analysis of sequential induced sputum samples during sputum induction: Evidence that different lung compartments are sampled at different time points

BACKGROUND The effect of the duration of sputum induction on markers of inflammation in induced sputum is unknown, and the optimal duration of sputum induction for research purposes in airway disease is uncertain. OBJECTIVE We sought to determine whether the duration of sputum induction influences the cellular or biochemical characteristics of induced sputum. METHODS Induced sputum was collected sequentially at 4-minute intervals during a 20-minute sputum induction in 12 subjects with mild and moderate asthma. Each 4-minute sample was collected and analyzed separately for total and differential cell counts and for levels of eosinophil cationic protein, fibrinogen, mucin-like glycoprotein, and surfactant protein SP-A. RESULTS The percentages of eosinophils and neutrophils were significantly higher at the beginning of the 20-minute sputum induction than at the end, whereas the percentage of macrophages was significantly lower at the beginning than at the end. In addition, the levels of eosinophil cationic protein and mucin-like glycoprotein were significantly higher at the beginning of the 20-minute induction than at the end, whereas the level of surfactant protein SP-A was significantly lower. CONCLUSIONS The duration of sputum induction significantly affects the cellular and biochemical composition of induced sputum in a manner suggesting that large airways are sampled at the beginning of sputum induction, whereas peripheral airways and alveoli are sampled at later time periods. Our data demonstrate the importance of standardizing the duration of sputum induction in clinical research studies, and on the basis of these data, we have chosen 12 minutes as the optimal duration for sputum induction in asthmatic subjects.

An Experimental Human Model of Metal Fume Fever

OBJECTIVE To examine the pathogenesis of metal fume fever in humans by studying functional, cellular, and biochemical responses after exposure to zinc welding fume. DESIGN Clinical experimental study. PARTICIPANTS We studied 14 welders recruited through public advertisements. INTERVENTIONS Participants welded galvanized steel. MEASUREMENTS We measured lung volumes, airflow, diffusing capacity for carbon monoxide, and airway reactivity at baseline as well as either 6 or 20 hours after welding. We carried out bronchoalveolar lavage either 8 hours (early follow-up, 5 participants) or 22 hours (late follow-up, 9 participants) after welding, assaying the fluid for total and differential cell counts and bronchoalveolar lavage supernatant concentrations of interleukin-1 and tumor necrosis factor (TNF). MAIN RESULTS Changes in pulmonary function and airway reactivity were minimal. Cumulative zinc exposure and polymorphonuclear leukocyte count in bronchoalveolar lavage fluid at late (r = 0.87; P less than 0.01) and early (r = 0.93; P less than 0.05) follow-up were positively correlated. Among the late follow-up group, the mean proportion of polymorphonuclear leukocytes was 37% (range, 19% to 63%), a statistically greater proportion than the 9% (range, 2% to 21%) seen among the early follow-up group (P less than 0.05). We did not detect TNF or more than a trace amount of interleukin-1 in the bronchoalveolar lavage supernatant. CONCLUSIONS Zinc oxide welding fume was associated with a marked dose-dependent increase in the number of polymorphonuclear leukocytes recovered in bronchoalveolar lavage fluid 22 hours after exposure but was not associated with a clinically significant change in pulmonary function or airway reactivity. Although we did not identify increases in either interleukin-1 or TNF levels in bronchoalveolar lavage fluid, cytokines or a cytokine-like mechanism may mediate the syndrome of metal fume fever.

Structural changes to airway smooth muscle in cystic fibrosis

Background: Chronic airway obstruction is characteristic of cystic fibrosis (CF) but there are few studies of airway smooth muscle remodelling in CF. Methods: Airway smooth muscle content and mean airway smooth muscle cell size were measured by applying design-based stereology to bronchoscopic biopsy specimens obtained from seven subjects with CF and 15 healthy controls. Results: The smooth muscle content increased by 63% in subjects with CF (mean (SD) 0.173 (0.08) v 0.106 (0.042) mm3 smooth muscle/mm3 submucosa, mean difference −0.067; 95% CI −0.12 to −0.013, p = 0.017) but there was no increase in mean cell size (2705 (351) v 2654 (757) μm3, mean difference −51; 95% CI −687 to 585, p = 0.87). Conclusions: These findings indicate hyperplasia of airway smooth muscle cells without hypertrophy and suggest that accumulation of airway smooth muscle cells may contribute to airway narrowing and bronchial hyperresponsiveness in CF.

Effect of a single dose of the selectin inhibitor TBC1269 on early and late asthmatic responses

Background Selectins participate in the initial phase of leucocyte migration from circulation to inflamed tissues and may play a role in inflammatory cellular influx into airways in asthma. In the sheep asthma model, TBC1269, a pan‐selectin antagonist, reduced late allergen response by 74%.

Exposure to medium and high ambient levels of ozone causes adverse systemic inflammatory and cardiac autonomic effects

Epidemiological evidence suggests that exposure to ozone increases cardiovascular morbidity. However, the specific biological mechanisms mediating ozone-associated cardiovascular effects are unknown. To determine whether short-term exposure to ambient levels of ozone causes changes in biomarkers of cardiovascular disease including heart rate variability (HRV), systemic inflammation, and coagulability, 26 subjects were exposed to 0, 100, and 200 ppb ozone in random order for 4 h with intermittent exercise. HRV was measured and blood samples were obtained immediately before (0 h), immediately after (4 h), and 20 h after (24 h) each exposure. Bronchoscopy with bronchoalveolar lavage (BAL) was performed 20 h after exposure. Regression modeling was used to examine dose-response trends between the endpoints and ozone exposure. Inhalation of ozone induced dose-dependent adverse changes in the frequency domains of HRV across exposures consistent with increased sympathetic tone [increase of (parameter estimate ± SE) 0.4 ± 0.2 and 0.3 ± 0.1 in low- to high-frequency domain HRV ratio per 100 ppb increase in ozone at 4 h and 24 h, respectively (P = 0.02 and P = 0.01)] and a dose-dependent increase in serum C-reactive protein (CRP) across exposures at 24 h [increase of 0.61 ± 0.24 mg/l in CRP per 100 ppb increase in ozone (P = 0.01)]. Changes in HRV and CRP did not correlate with ozone-induced local lung inflammatory responses (BAL granulocytes, IL-6, or IL-8), but changes in HRV and CRP were associated with each other after adjustment for age and ozone level. Inhalation of ozone causes adverse systemic inflammatory and cardiac autonomic effects that may contribute to the cardiovascular mortality associated with short-term exposure.