The neutrophils (50 aliquots) were placed in the upper wells, at a concentration of 3X106 cells/ml, in Hank’s solution with 0.1 percent BSA. Chambers were incubated for 30 min at 37°С in a humidified atmosphere of 95 percent air: 5 percent CO2; the filters were removed, fixed in absolute methanol, and stained with eosin-azur (Diff-Quik, Harleco; Gibbstown, NJ). The cells that migrated through the filter to the other side were counted. The NCA was measured as the mean number of cells per 10 high power fields (1,000X). The results were expressed as the percentage of the chemotactic response to FMLP 10-7 M. In each experiment, a negative control was assessed, using Hank’s solution for the BAL fluid and the fraction fluid further.
Gel-filtration chromatography was performed on a 75 column (1.6-cm diameter, 60-cm long, Superdex, Pharmacia). One-milliliter aliquots of a ten-fold concentrated BAL fluid (freeze-dried 10-ml aliquots solubilized in 1 ml of distilled water) were applied to a column (Superdex) previously equilibrated with phosphate buffered saline (PBS, pH 7.2; Gibco). Chromatography was performed at 4°C, with a flow rate of 1.0 ml/min; 5 ml of each fraction was collected. The column was calibrated with molecular weight markers (blue dextran, cytochrome c, insulin, and phenol red). For fractionations, 1.5 mg of protein was used.
All data were expressed as the means ± standard error of the mean. Correlation coefficients for relationships between pairs of variables were determined by linear regression analysis. For experiments with two groups, Student’s t test was used to determine statistical significance. Probability values of 0.05 or less were considered significant. Neutrophil chemotactic activity was measured in the pre- and post-EM treatment BAL fluid of the 13 DPB patients. As shown in Table 1, their mean duration of disease was 9.0 ±1.9 years; the onset was insidious. Sputum cultures at the time of admission yielded Hemophilus influenzae in four patients (cases 6, 8, 10, and 11), Pseudomonas aeruginosa in three (cases 4, 9, and 13), Staphylococcus aureus in one (case 1), and normal flora in 5 (cases 2,3,5,7, and 12).
Table 1—Clinical Characteristics of Ten Patients With Diffuse Panbronchiolitis
|Case No.||Age||Sex||ChronicSinusitis||Duration of EM tx, mo||Duration of Disease, yr||Sputum Culture||Diagnosis|
|1||21||M||+||6||2||S aureus||H influenzae||OLB*|
|2||62||F||+||12||10||Normal flora||Normal flora||OLB|
|3||21||M||+||6||5||Normal flora||H influenzae||OLB|
|4||52||M||+||12||10||P aeruginosa||Normal flora||OLB|
|5||63||M||+||8||20||Normal flora||P aeruginosa||Clinical|
|6||60||M||+||11||8||H influenzae||Normal flora||Clinical|
|7||49||F||+||6||26||Normal flora||Normal flora||OLB|
|8||25||M||+||12||10||H influenzae||Normal flora||OLB|
|9||19||F||+||12||5||P aeruginosa||Normal flora||Clinical|
|10||34||M||+||6||7||H influenzae||P aeruginosa||OLB|
|11||49||M||+||12||5||H influenzae||Normal flora||Clinical|
|12||14||M||+||6||4||Normal flora||Normal flora||OLB|
|13||41||M||+||12||5||P aeruginosa||Normal flora||OLB|
|mean+SE||39.2 ±4.9||9.3 ±0.8||9.0± 1.9|