J. Med. Microbio1.-Vol. 22 (1986), 265-268 0 1986 The Pathological Society of Great Britain and Ireland Loss of ciliary activity in organ cultures of rat trachea treated with lipo-oligosaccharide from Haemophilus influenzae A. P. JOHNSON and T. J. INZANA" Division of Sexually Transmitted Diseases, M R C Clinical Research Centre, Harrow, Middlesex, HA I 3UJ, and *Department of Veterinary Microbiology- Pathology, College of Veterinary Medicine, Washington State University, WA 991647040, USA Summary. Organ culture of rat trachea was used as an experimental model to examine the ability of lipo-oligosaccharide from Haemophilus influenzae to damage respiratory tract mucosal tissue. Lipo-oligosaccharide from two strains of H. injuenzae produced a significant decrease in the ciliary activity of tracheal rings observed over a 3-5 day period. No loss of ciliary activity was observed with the lipid-free moiety of the lipo- oligosaccharide. Introduction Haemophilus injuenzae is the commonest cause of bacterial meningitis in infants and young chil- dren and may also cause other serious systemic infections in this age group. It is also associated with exacerbations of chronic bronchitis in adults (Turk, 1982). Despite the importance of H. injluenzae as a human pathogen, its pathogenicity is incompletely understood. To increase our understanding of this, several experimental systems, including an animal model of meningitis (Moxon et al., 1974) and the use of organ cultures of respiratory mucosa inocu- lated with H. injluenzae (Denny, 1974; Johnson et al., 1983) have been developed. With the latter system it was shown that ciliary activity in cultures of tracheal rnucosa inoculated with H. injluenzae was lost because of sloughing of epithelial cells (Denny, 1974; Johnson et al., 1983). It was also observed that sterile supernates from infected organ cultures contained a soluble, heat-stable factor that caused loss of ciliary activity when transferred to fresh organ cultures (Denny, 1974). Although the heat-stable toxic factor was not identified, Denny speculated that it might be H. influenzae endotoxin. In the present study, the concept that H. injuenzae endotoxin might be capable of damaging tracheal mucosa has been investigated by determining the effect that purified lipo-oligosaccharide from H. influenzae has on the ciliary activity of organ cultures of rat trachea. Received 12 Mar. 1986; accepted 19 Mar. 1986 Materials and methods Lipo-oligosaccharide Lipo-oligosaccharide (LOS) was extracted from H. influenzae strains 1060 and S-2. Strain 1060 was isolated from a child with meningitis at the Texas Children's Hospital, Houston, TX. Strain S-2 is a capsule-deficient mutant of strain Eagan (Moxon et a)., 1984). The LOS was isolated from each strain by enzyme digestion and phenol-water extraction (Johnson and Perry, 1976; Inzana, 1983). Lipid-free oligosaccharide was isolated from strain Eagan as described by Inzana et al. (1985). The samples of LOS and oligosaccharide were prepared in the laboratory of one of the authors (TJI) and after lyophilisation, were sent to APJ for assessment of their biological activity in rat trachea organ cultures. Preparation of organ cultures of rat trahcea Young male and female specific-pathogen-free Spra- gue-Dawley or Lewis rats, bred at the Clinical Research Centre, were used. Tracheas were removed aseptically from animals by the method of Johnson et al. (1983) and placed in a petri-dish containing Eagle's minimal essential medium (MEM) buffered at pH 7.2 with 50 mM HEPES and supplemented with ampicillin 10 pglml. The MEM was prepared with sterile pyrogen-free water as described by Johnson et al. (1983). Each trachea was cut into a series of rings which were placed individually in tissue-culture tubes containing 1 ml of MEM and then incubated at 37°C on a roller drum (Cherry and Taylor-Robinson, 1970). Treatment of organ cultures In initial experiments, organ cultures were incubated 265