Mycobacterium terrae isolated from indoor air of a moisture-damaged building induces sustained biphasic inflammatory response in mouse lungs

Abstract

.html USA) at 30°C for 5 weeks. Then the bacterial cells were washed, suspended in Hank’s bal- anced salt solution (HBSS) (Gibco, Paisley, UK), and sonicated in a water bath (Ultrasonic, Lahti, Finland) for 15 min. The microbe con- centration was counted with an epifluorescence microscope by using the acridine orange stain- ing method adjusted for mycobacteria (Hobbie et al. 1977; Katila and Mäntyjärvi 1982). Before dosing, the cell suspension was sonicated in a water-bath sonicator on ice for 40 min to ensure a single cell suspension. Animals. Specific pathogen-free male NIH/S mice (8–9 weeks old) were obtained from the breeding colony of the National Public Health Institute, Division of Environmental Health (Kuopio, Finland). They were transferred from the barrier unit to a conventional animal room and housed singly in metal cages on aspen wood chips (FinnTapvei, Kaavi, Finland) 1 week before the experiment. Animals received water and food (R36 Maintenance Diet for rats and mice, Lactamin, Stockholm, Sweden) ad libi- tum. The mice were on a 12-hr light/dark cycle (lights on 700 hr–1900 hr) at 21°C and 27–38% relative humidity. The experimental protocols were approved by the Research Animal Committee of the University of Kuopio and the Provincial State Office. Experimental design. The dose depen- dence of the effects induced by the microbe was investigated by exposing mice (10 ani- mals/group) to a single dose of M. terrae (1 × 107, 5 × 107, or 1 × 108 microbes/animal) or HBSS (carrier control; Gibco) for 24 hr by intratracheal instillation (50 µL/animal) under 6% sevoflurane (Sevorane; Abbot, Abbott Park, IL, USA) anesthesia. The time course of the effects was studied at 6 and 24 hr and at 3, 7, 14, 21, and 28 days after the exposure (10 animals/group) to a single dose of 1 × 108 cells of M. terrae or HBSS. Exposure to microbes. The bacteria were instilled into mouse lungs as described previ- ously (Jussila et al. 2001). In brief, an anes- thetized mouse was laid in a 66° upward posture and the incisors were placed on a wire. The dos- ing was performed under visual control by using a cold-light source (KL 1500 electronic; Schott, Mainz, Germany) placed against the throat. The nostrils were blocked, the tongue was extended, and the dose was delivered onto the vocal folds with a Finn pipette tip. Sample collection. At the indicated expo- sure times, the mice were anesthetized with pentobarbital (100 mg/kg) and exsanguinated by cardiac puncture. Blood was collected and serum separated (Capiject T-MG; Terumo, Elkton, MD, USA) for cytokine analyses. Lungs (filled with 10% phosphate-buffered formalin), liver, spleen, and lymph nodes (mediastinal, axillary, brachial, and both superficial and deep cervical nodes) of three or four mice of each group were preserved in 10% phosphate-buffered formalin for histopathologic evaluation. The tracheas of the remaining animals (six or seven per group) were cannulated with polyethylene tubing. At first, lungs were la

Symptom Clusters