We used a double-blind, randomized, placebo-controlled, crossover design to evaluate the systemic, pulmonary, and mucosal inflammatory effects of inhaled nebulized endotoxin, corn dust extract, soybean dust extract, and pyrogen-free, calcium-free, and magnesium-free Hanks’ balanced salt solution (HBSS) (Cell-Gro; Media-Tech, Inc., Herndon, Va).
Subjects were required to be healthy never-smokers taking no prescription medicines, with no history of agricultural work. While no bronchoprovocative testing or skin testing was done, they were required to have no history consistent with asthma and no history suggestive of atopy (seasonal allergies or rhinitis). Subjects were recruited by a newspaper advertisement. Of 16 individuals volunteering for this investigation, 3 men and 4 women met the inclusion criteria and were selected as participants in the study. The mean age of the subjects was 26.9 years (range, 19 to 36 years). canadianneighborpharmacy.com
Subjects were exposed to each of four solutions (HBSS, endotoxin, corn, and soybean dust extracts) via inhalation challenge. Although the order in which the solutions were administered was randomly assigned, corn dust extract was the first or second inhaled solution for all subjects, and endotoxin was the third or fourth substance inhaled in six of the seven subjects (Table 1). Vital signs (temperature, BP, respiratory rate, and heart rate) and spirometry were measured before inhalation challenge, 30 min after, 1 h after, hourly until 8 h after challenge, and then again at 24 h. Peripheral blood leukocyte and differential cell counts were obtained prior to inhalation challenge and 6 h later. Nasal lavage was performed 24 h after inhalation challenge. Each inhalation challenge was separated by at least 10 days.
Preparation of Inhaled Solutions
Soybean dust was collected from an air filtration system in a soybean processing plant. Settled corn dust was collected from a grain (corn) elevator. While the sites at which the dusts were collected processed their respective grains exclusively, the degree to which these samples represent dust from similar grain facilities was not investigated. Extracts were produced by mixing 3 g of the dust with 30 ml HBSS followed by shaking for 60 min, centrifugation at 3,000 rpm, then filter sterilization of the supernatant using a 0.45 fim polyvinylidine difluoride (PVDF) (Acrocap; Gelman Sciences, Ann Arbor, Mich) filter. The endotoxin concentrations of both solutions were 7 mg/L as determined by the chromogenic Limulus amebocyte lysate assay (QCL-1000; Whittaker Bioproducts, Walkersville, Md). The pH of the corn dust extract was 5.3, and the pH of the soybean dust extract was 5.8. Lyophilized Escherichia coli endotoxin (serotype 0111:B4, Sigma Chemical Co, St. Louis, Mo) was diluted in HBSS to attain a final concentration of 7 mg/L. The pH of all solutions (including HBSS) was adjusted to 5.8. Sterility was confirmed by culture on trypticase soy agar at 30°C and 52°C, MacConkey s agar at 35°C, and malt extract agar at 25°C. All solutions were stored at — 70°C until they were used.
The solutions were administered via a nebulizer (DeVilbiss 646) and dosimeter (DeVilbiss, DeVilbiss Health Care Inc, Somerset, Fa), operated at 20 psi air pressure. The subjects controlled the timing of each nebulized dose and were instructed to inhale through the mouthpiece of the nebulizer and exhale through their nose. With this system and technique, we were able to maximize the amount of grain dust extract delivered to the mucosa of the airways. The mean extract dose delivered was 0.08 ml/kg. This resulted in delivery of between 4.5 and 8.1 ml to each subject, corresponding to between 30 and 60 jig of endotoxin. This dose corresponds to the expected range of endotoxin inhaled during a workshift in farming environments.
Pulmonary Function Testing
The pulmonary function tests consisted of serial spirometry using a spirometer (MedCraphics CPF-S, MedGraphics Corporation, St. Paul, Minn). These maneuvers were performed using standard protocols and the American Thoracic Society guidelines.
Nasal lavage was performed 24 h after inhalation challenge. The subject tilted his head back, held his breath, and a 5-ml aliquot of sterile saline solution was instilled in one nostril, held for 10 s, then forcibly expelled. This was performed three times in each nostril. The lavage fluid was filtered through two layers of gauze, and it was centrifuged twice with resuspension of the cell pellet in 1 ml of HBSS. Cell counts were determined (Coulter Counter, Coulter Electronics Inc, Hialeah, Fla), and differential cell counts were performed using cytospin preparation and staining (Diff-Quick, Baxter Scientific Products, Miami, Fla).
The crossover design of this study dictated the analysis. Paired comparisons of spirometric data, vital signs, and peripheral blood leukocyte and differentia] cell counts, and nasal lavage cell and differential cell counts were made between each solution type at each time point. Comparisons were also made to baseline data for peripheral blood leukocyte and differential cell counts for each grain type. The Wilcoxon Signed Rank Test was used for all analyses.
Table 1—Order of Administration of Extracts