The airways of individuals suffering from hypersecretion exhibit gland hypertrophy as well as a serous-mucous cell shift within the glands. These reflect disease-related abnormalities in gland growth and differentiation. The goal of the proposed studies is to gain information about the mechanisms controlling these processes. So that mechanisms relevant to normal development and disease can be compared, experimental studies will be performed in two model systems (1) the trachea of the newborn rat, in which glands develop between postnatal days 7 and 28 and (2) the trachea of the mycoplasma-infected adult rat, in which new glands develop between postinfection days 7 and 42. Studies listed under Specific Aim 1 will determine by confocal microscopy the size and distribution of tracheal submucosal glands at defined postnatal and post- infection time points in vivo as well as in an in vitro gland growth explant system. Studies listed under Specific Aim 2 will identify the cell type from which gland buds arise in each model, using double immunofluorescence to define cells that are both preparing to divide and express phenotype-specific markers. They will also determine by PCR, immunofluorescence and in situ hybridization whether elevations in the local concentration of specific growth factors and/or their receptors precede mitosis in these cells. Evidence for causal relationships between specific growth factors and gland growth will be sought using neutralizing antibodies in the in vitro gland growth explant system. Studies listed under Specific Aim 3 will identify extracellular matrix- degrading enzymes whose local tissue concentrations are elevated at the time of gland growth in each model and seek evidence for causal relationships between specific enzymes and gland growth using neutralizing antibodies in the in vitro gland growth explant system. Mechanisms controlling enzymes relevant to gland growth will be identified. Studies listed under Specific Aim 4 will examine serous differentiation mechanisms by identifying DNA-protein interactions controlling serous cell-specific lysozyme transcription. Rat lysozyme cDNAs and genomic clones will be isolated, the transcription start site mapped, and regulatory DNA sequences identified using routine methods. DNA regulatory sequences specifically involved in the initiation of lysozyme transcription during serous cell differentiation will be identified using a novel method of in vivo footprinting. Transcription factors interacting with these sequences will be isolated. Knowledge gained from these studies will hopefully suggest pharmacological interventions to inhibit excessive gland growth and differentiation abnormalities associated with hypersecretory airways.
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