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SF Hamm-Alvarez, Y Wang, C Mazurek, N Kasahara; Transduction of Primary Rabbit Lacrimal Acini With Replication-Incompetent Adenovirus Serotype 5 Inhibits Exocytosis and Transcytosis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3139.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To explore the effects of replication incompetent adenovirus serotype 5 (Ad) on lacrimal acinar secretory functions. Methods: Primary rabbit lacrimal acini cultured for two days were exposed to Ad containing either green fluorescent protein or ß-galactosidase reporter genes at a MOI of 5 for 24 hrs prior to measurement of basal and carbachol (CCH)-stimulated (100 µM) release of protein, ß-hexosaminidase, and secretory component (SC) derived from the polymeric immunoglobulin A receptor. Ad-induced changes in membrane and cytoskeletal organization were assessed by confocal microscopy using appropriate primary and secondary fluorophore-conjugated antibodies or fluorescent probes. Results: Transduction of lacrimal acini with Ad containing either reporter gene significantly (p <0.05) increased basal protein and ß-hexosaminidase release by 2-fold while concomitantly and significantly inhibiting the CCH-stimulated release of these same markers by 2-fold. While basal release of SC was not altered by Ad, the CCH-stimulated release of SC was significantly reduced by 2-fold. Ad transduction did not decrease cell viability or protein synthesis, as determined by trypan blue exclusion and pulse-labeling, respectively. No major changes in cytoskeletal filaments, Golgi membranes or immature VAMP2-enriched secretory vesicles could be detected by confocal microscopy. However, Ad transduction did elicit a major depletion of apical mature secretory vesicles enriched in rab3D. Conclusions: Replication-incompetent Ad markedly alters lacrimal acinar secretory responses, decreasing CCH-stimulated exocytosis and transcytosis while elevating basal exocytosis. The loss of apical rab3D-enriched secretory vesicles suggests that Ad may alter secretory vesicle maturation, thereby uncoupling stimulated and basal exocytosis. Ocular gene therapy strategies using Ad-derived materials may therefore alter the quantity and composition of tear fluid.
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