The ability to obtain airway epithelial cells from the lower respiratory tract in living human donors will facilitate study of the biologic properties of these cells. We report our experience harvesting tracheobronchial epithelial cells from living human donors by brushing the mucosal surface of the trachea and mainstem bronchi. Cells were obtained on 21 occasions from 18 healthy adult subjects under direct vision with a brush-tipped catheter during fiberoptic bronchoscopy. The average number of cells harvested per subject was 14 +/- 2 x 10(6), and cell viability determined by trypan blue exclusion averaged 36 +/- 4%. Of note, cell viability was significantly enhanced when lidocaine was confined to the nares. Lidocaine was also observed to diminish cell viability in vitro in a dose-dependent fashion. Morphologic and staining properties were used to classify harvested cells into the three major cell types present in the mucosa (i.e., ciliated, secretory, and basal cells). All three subtypes were obtained. The percentage of ciliated, secretory, and basal-like cells was 24 +/- 2%, 11 +/- 1%, 29 +/- 1%, respectively, while the remaining 36% were difficult to type. In one subject in whom brushing was performed on three occasions over a 7-wk period, the percentage of each of the three subtypes was similar across procedures. Harvested cells could be successfully placed in primary culture with a plating efficiency of 50 to 60% and could be subcultured for up to seven passages. Acutely dissociated cells could be used to study the beta-adrenergic receptor adenylyl cyclase system since they produced cAMP in response to isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

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Beta-adrenergic agonist-mediated activation of the beta receptor coupled-adenylyl cyclase (beta AR-AC) system expressed by human airway epithelial cells alters airway function. However, little is known about the magnitude of expression, subtype, and function of the beta receptor-adenylyl cyclase (beta AR-AC) system in human airway epithelial cells from healthy, nonsmoking subjects. Therefore, we characterized beta AR number and subtype and the cAMP response to isoproterenol (iso) in acutely dissociated human tracheocytes harvested from 22 healthy, nonsmoking adults during fibroptic bronchoscopy. Moreover, because the regulation of beta AR-AC system function in response to beta-agonists or inflammatory mediators released into the airway in asthma is poorly understood, we examined the cAMP response to iso after 30 min exposure of cells to iso or the protein kinase C activator, sn-1,2-dioctanoyl glycerol (diC8). The beta AR-AC system was highly expressed and functional in human airway epithelial cells. Group mean beta AR density (i.e., Bmax), equilibrium dissociation constant (Kd), and the percentage of beta 2AR subtypes assessed by radioligand binding were approximately 8,900 receptors/cell, 45 pM, and approximately 80%, respectively. Mean maximum cAMP production was approximately 42 pmol/10(5) cells, and the mean EC50 of the response to iso was 131 nM. However, Bmax and cAMP responses to iso varied considerably across subjects. For example, Bmax varied ninefold, and the EC50 of the cAMP response varied 39-fold interindividually. The EC50 was inversely related to beta AR density (r = -0.81, p < 0.05), suggesting that sensitivity of the cAMP response to iso was in part dependent on beta AR density. In all experiments, cAMP responses to iso stimulation were markedly desensitized in dose-dependent fashion by 30 min pretreatment with iso or diC8. For example, pretreatment with iso 10 microM or diC8 100 microM reduced maximum cAMP production to 22 and 63% of control values, respectively. These data indicate that: (1) the beta AR-AC system is highly expressed on acutely dissociated airway epithelial cells from normal adult, but beta AR expression and its functional coupling to adenylyl cyclase vary considerably interindividually; and (2) the beta AR-AC system of normal human airway epithelial cells is rapidly desensitized by exposure to beta-adrenergic agonists or activators of PKC.

IMPORTANCECompared with the operating room (OR), office-based intravitreal injection (IVI) is considered a more cost-effective and convenient approach, yet clinical outcomes of IVIs with anti-vascular endothelial growth factor (VEGF) agents in different settings (office-based vs OR) have not been systematically evaluated.OBJECTIVE To evaluate the safety outcomes of IVI with anti-VEGF agents in the OR vs office-based setting.DATA SOURCES PubMed, Embase, Cochrane Library, Web of Science, and ClinicalTrials.gov were searched from inception to July 2020.STUDY SELECTION Eligible studies reporting on patients who received IVIs with anti-VEGF drugs with a clearly stated injection setting of the office or OR.DATA EXTRACTION AND SYNTHESIS Two reviewers independently screened studies, extracted data, and assessed risk of bias. A meta-analysis was conducted to determine the rates of endophthalmitis (EO) and culture-positive EO. MAIN OUTCOMES AND MEASURESRates of EO and culture-positive EO following anti-VEGF IVIs in the OR and office-based setting.RESULTS Thirty-one studies with a total of 1 275 815 injections were included. Comparative analysis suggested no difference between rates of EO after IVIs performed in the office and OR settings (odds ratio, 3.06; 95% CI, 0.07-139.75; P = .57; I 2 = 80%) were identified, yet a higher rate of culture-positive EO was found in the office setting (odds ratio, 21.52; 95% CI,; P = .006; I 2 = 0%). The pooled rates of EO following anti-VEGF IVIs were 0.03% (95% CI, 0.03-0.04) and 0.02% (95% CI, 0.01-0.04) in office and OR settings, respectively, and the pooled rates of culture-positive EO were 0.01% (95% CI, 0.01-0.02) and 0.01% (95% CI, 0-0.02). The pooled rates of other ocular and systemic adverse events were low. CONCLUSIONS AND RELEVANCEThe rate of clinically suspected or culture-positive EO following anti-VEGF IVIs was low whether the procedure was performed in the office or OR setting. Bacterial spectrum could differ between the 2 settings. This meta-analysis could not determine if it is more appropriate to give treatment in the OR for safety reasons in low-income compared with higher-income regions in the world.

Stimulation of adenylyl cyclase mediates the effects of beta-adrenergic agonists and prostaglandin E2 (PGE2) on tracheobronchial epithelial cell function by increasing intracellular cyclic adenosine monophosphate (cAMP). In turn, increases in cAMP affect airway function by modulating ciliary beating, chloride and water transport, mucus secretion, and release of bronchoactive substances. This study examined the function and regulation of the beta-adrenergic receptor-adenylyl cyclase system (beta AR-AC) in tracheal epithelial cells isolated from the rabbit, a frequently used animal model of airway reactivity, inflammation, and electrolyte transport. beta AR number, assessed by ligand binding using the non-subtype-specific beta-antagonist [125I]iodopindolol, averaged approximately 10,700 beta AR/cell (400 fmol/mg membrane protein). Greater than 85% of the receptors were of the beta 2 subtype as determined by competitive antagonist displacement of iodopindolol by selective beta 1- (betaxolol) and beta 2- (ICI 118,551) antagonists. cAMP synthesis was stimulated with isoproterenol, PGE2, and forskolin in a time- and concentration-dependent fashion. Preincubation of epithelial cells for 30 min with either isoproterenol (10 microM) or the peptide inflammatory mediator, bradykinin (100 microM), markedly depressed subsequent isoproterenol-stimulated cAMP synthesis. Isoproterenol-induced beta AR-AC desensitization appeared to be homologous since cAMP responses to PGE2 and forskolin, a direct activator of adenylyl cyclase, were not reduced. The effect of bradykinin on isoproterenol-stimulated cAMP response was mimicked by preincubation with either dioctanoyl glyceride or phorbol myristate acetate, activators of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)