Methods: Flexible bronchoscopy and airway brushing was used to obtain large airway (LAE, 4-5 order) and small airway (SAE, 10-12 th th th order) airway epithelium from healthy nonsmokers (n= 22) and healthy smokers (n= 36). MSMB mRNA levels were assessed by th microarray (Affymetrix HG-U133 Plus 2.0), TaqMan and massive parallel RNA sequencing, with immunohistochemical assessment of Immunohistochemical analysis of cytopreps was performed on a random subset of individuals (n= 4 nonsmokers, n= 4 healthy cytopreps. smokers) to assess protein expression of MSMB by staining SAE and LAE with rabbit polyclonal anti-MSMB antibody (Novus Biologicals, Littleton, CO) and evaluating secretory, ciliated, and basal cells on each slide for MSMB expression. Microarray analysis demonstrated MSMB expression was increased in healthy smokers compared to healthy nonsmokers by

Results:

1.8-fold in the SAE (p< 0.01) and by 1.4-fold in the LAE (p< 0.02). TaqMan RT-PCR confirmed these changes with 2.5-fold increase in smoker SAE (p< 0.02) and 1.8-fold increase in the LAE (p< 0.01). The two isoforms of MSMB (PSP 94 and 57) were equally up-regulated in smokers compared to nonsmokers. These observations were confirmed by RNA sequencing (4.8-fold increase in PSP 94, p< 0.03, 4.3-fold increase in PSP 57, p> 0.09) and TaqMan RT-PCR (2.7-fold increase in PSP 94, p< 0.03, 2.4-fold increase in PSP 57, p> 0.08). Immunohistochemical analysis demonstrated that MSMB protein was expressed in a similar percentage of cells in the two groups (85% in smokers, 81% in nonsmokers, p> 0.8), but the intensity of staining was strikingly increased in the goblet/secretory cell airway epithelial cells of healthy There was no significant relationship between MSMB expression and gender (p> 0.8), age (p> 0.2), or extent of smoking in pack-yr smokers.(p> 0.5). Three months after smokers (n= 26) quit smoking, SAE MSMB expression decreased 1.9-fold (p< 0.01), but remained elevated 1.4-fold (p< 0.03) in comparison with MSMB mRNA levels of nonsmokers. Conclusions: The up-regulation of expression of MSMB by smoking in the small and large airway epithelium of smokers is likely secondary to airway goblet/secretory cell hyperplasia. These observations suggest that MSMB may be a useful biomarker of smoking-mediated goblet/secretory cell hyperplasia.