Smoke-induced inhalation injury: effects of retinoic acid and antisense oligodeoxynucleotide on stability and differentiated state of the mucociliary epithelium.

Rabbit tracheal explants, exposed to burning pine wood smoke, were cultured in a chemically defined medium with and without retinoic acid (+/- RA). Exposures of 15-20 minute led to RA-independent degeneration of the mucociliary epithelial sheath. In 10 minute exposures tissue integrity was retained, but epithelial morphology changed from normal pseudostratified columnar to the flattened appearance typical of the squamous phenotype. Despite the dramatic shift in morphology, explants exhibited normal RA-dependent mucin gene expression characteristic of the mucociliary phenotype. Furthermore, electron micrographs showed continued presence of both secretory granules and cilia. RA(+) cultures also showed a normal pattern of adherent epithelial cells. In RA(-) cultures, however, there were prominent intercellular spaces indicating an RA dependence for maintaining adhesive contacts following smoke exposure. An 18-mer mucin antisense oligomer that suppressed mucin gene expression also unexpectedly blocked the smoke induced metaplasia in RA(+) cultures, but the sense oligomer had no effect.

Differentiation of respiratory epithelium: the effects of retinoic acid and carcinogens on the expression of mucociliary vs. squamous phenotype.

Changes in ultrastructural characteristics and mucin gene expression were examined in rat tracheal explants cultured in a synthetic medium +/- retinoic acid (RA), benzo[a]pyrene (B[a]P) and N-methyl-N-nitrosourea (NMNU). In the RA(+) cultures, no changes in either ultrastructural features or mucin gene expression were detected after 48 h incubation. After 96 h incubation, however, the ultrastructural features associated with the squamous phenotype were characteristics of cultures containing the two carcinogens and the mucin gene expression was slightly reduced. Thus, in the presence of retinoic acid, the carcinogen induced changes in cytology to the squamous phenotypes were not matched by a marked loss of mucin gene expression. Explants cultured for 48 h without RA and +/- carcinogens showed none of the cytological changes associated with onset of the squamous phenotype. While mucin mRNA was still detected, it was clearly reduced compared to 48 h cultures in RA(+) medium. However, 48 h later, all explants exhibited pronounced squamous metaplasia and the mucin message decreased to trace levels. Thus, the results of these experiments with B[a]P and NMNU in RA(+) and RA(-) media indicates that at least the early carcinogen induced changes may be distinct from those associated with the retinoid pathway controlling expression of the mucin component of the mucociliary epithelium.

Retinoic acid-regulated cellular differentiation and mucin gene expression in isolated rabbit tracheal-epithelial cells in culture.

Rabbit tracheal epithelial cells were cultured in a serum-free and hormone-supplemented medium with and without retinoic acid. The cells showed time-dependent mucin gene expression when cultured in the medium with retinoic acid. In the absence of retinoic acid, however, mucin mRNA was barely detectable in the cells. When retinoic acid was added back to the medium, the mucin message was prominent again. Actinomycin D and cycloheximide did not inhibit mucin gene expression. The mucin message was slightly elevated by cAMP agonists. A mucin antisense oligomer inhibited the retinoic acid-induced mucin mRNA expression and secretion, thus offering an alternate approach in the management of mucus hypersecretion in upper airway respiratory diseases such as chronic bronchitis, asthma, and cystic fibrosis.

Retinoic acid modulation of mucin mRNA in rat tracheal explants: response to actinomycin D, cycloheximide, signal transduction effectors and antisense oligodeoxynucleotide.

Factors affecting the retinoic acid modulated expression of mucin mRNA in rat tracheal cultures were studied. Actinomycin D had no effect on mucin mRNA in cultures grown with retinoic acid (RA+). The usual precipitous drop in mucin mRNA in cultures lacking retinoic acid (RA-) was prevented by actinomycin D. Cycloheximide also had no effect on mucin mRNA in RA+ cultures, but, like actinomycin D, it prevented the precipitous drop in mucin mRNA in RA- cultures. cAMP agonists had some marginal effects on the mucin mRNA, but none as dramatic as those noted by actinomycin D and cycloheximide in the RA- cultures. An antisense oligomer (18 bases) to rat mucin cDNA inhibited the mucin mRNA expression in RA+ cultures.

Effect of retinoic acid on mucin gene expression in rat airways in vitro.

Ultrastructural examination of rat tracheal explants at various times of culture in a serum-free and hormone-supplemented medium containing retinoic acid showed that the cytological characteristics of the epithelium were well preserved for at least 192 h. Hybridization analyses for mucin core protein mRNA in the explants were performed with a 30-base oligonucleotide probe, the design of which was based on the tandem repeat sequence of the rat intestine mucin core protein. The probe reacted with total RNA prepared from trachea, intestine and colon, but not with total RNA obtained from liver or alveolar region of the lung. Type-I keratin expression was observed in the explant grown at different periods of time in a medium with and without retinoic acid. The hybridization probe gave a prominent reaction with RNA preparations obtained from tracheal explants incubated for as long as 192 h in a medium containing retinoic acid. In the absence of retinoic acid, however, the mucin message was evident at the 24 h time point but thereafter decreased to barely detectable levels. When retinoic acid was added at 96 h to the latter cultures, the mucin mRNA was prominent again after additional incubation for 24 and 48 h. Northern-blot analyses of tracheal RNA showed a diffuse band at approx. 7.5 kb. Addition of a variety of chemical and pharmacological agents to explants cultured in the presence of retinoic acid had no dramatic induction or inhibitory effects on the mucin mRNA. Only the steroid prednisolone had a reproducible inhibitory effect.

In vitro effects of drugs on production of mucins in rabbit tracheal epithelial cells expressing mucin gene: a model system for studying upper airway respiratory diseases.

Rabbit tracheal epithelial cells, cultured on collagen-coated dishes in serum-free and hormone-supplemented medium, were found to incorporate [3H]glucosamine into high-molecular-weight components that were secreted in the medium. The chemical analysis of the secreted products resulted in a profile that resembled that of mucous glycoproteins (mucins). When examined by dot blot analysis, the total RNA isolated from these cells hybridized to an antisense 30-mer oligonucleotide corresponding to a rat intestine mucin peptide sequence, indicating that mucin gene was expressed in these cell lines. Lung and liver tissues of rabbit did not express this gene. Transmission electron microscopy exhibited secretory granules in these cells. The incorporation of [3H]glucosamine into mucins was inhibited by three aryl-N-acetyl-galactosaminides and a chemical carcinogen, N-nitroso-N-ethyl urea, whereas 5-azacytidine enhanced the proliferation of cells as well as the radiolabeling of mucins. Parasympathetic agent (pilocarpine), cholinergic antagonist (atropine), and beta-adrenergic agonist (isoproterenol) alone have little effect on the secretion of mucins. The cholinergic agonist, methacholine, was found to increase the production of mucins and addition of atropine to the medium before methacholine blocked this stimulation. Histamine was found to stimulate mucin production in these cells.

Ethanol-induced urticaria: a case report.

Urticaria, after ingesting ethanol, is rare. A 36-year-old Caucasian male developed multiple, generalized, pruritic urticarial lesions 5 to 15 minutes after drinking alcoholic beverages of any type. A blinded challenge with 5 mL of chemically pure 95% ethanol in concentrated grape juice caused urticaria and an elevation of plasma histamine. Pure grape juice alone was unreactive. Prick skin tests with Brewers' yeast, ethanol, acetaldehyde, and acetic acid were negative. Hydroxyzine (25 mg, p.o., q.i.d.), given for three days prior to challenge, inhibited skin response and histamine release. Biopsy of the urticarial lesions caused by ethanol ingestion showed mast cell degranulation. In this subject, ethanol appeared to directly affect mast cell mediator release by non-IgE mechanisms.

Factors that affect the cell cycle of Mycobacterium avium.

Mycobacterium avium, a pathogen of both animals and humans, is an acid-fast bacterium that is very drug-resistant and pleomorphic in colony and cellular morphology. By selective filtration, cells 1 micrometer long could be obtained. When placed in fresh medium, these small cells elongated to form filaments that aggregated during about a 40-hr incubation period. The filamentous cells divided rapidly for an additional 40 hr, with a doubling time of approximately 6 hr. Fission ceased, and the resulting culture consisted of coccobacilli. The cell cycle would not proceed if the cells were starved for either fatty acid or ammonium ion. During elongation (the growth phase), protein, DNA, and triglycerides were synthesized exponentially. During the fission stage, the triglycerides were utilized and redistributed among other cellular constituents. It is proposed that the cell cycle offers a unique system by which to test drugs that may inhibit growth of, or be bactericidal for, M. avium.