Antigen-induced mast cell expansion and bronchoconstriction in a mouse model of asthma.

Lung mastocytosis and antigen-induced bronchoconstriction are common features in allergic asthmatics. It is therefore important that animal models of asthma show similar features of mast cell inflammation and reactivity to inhaled allergen. We hypothesized that house dust mite (HDM) would induce mastocytosis in the lung and that inhalation of HDM would trigger bronchoconstriction. Mice were sensitized with intranasal HDM extract, and the acute response to nebulized HDM or the mast cell degranulating compound 48/80 was measured with respiratory input impedance. Using the constant-phase model we calculated Newtonian resistance (Rn) reflecting the conducting airways, tissue dampening (G), and lung elastance (H). Bronchoalveolar lavage fluid was analyzed for mouse mast cell protease-1 (mMCP-1). Lung tissue was analyzed for cytokines, histamine, and α-smooth muscle actin (α-SMA), and histological slides were stained for mast cells. HDM significantly increased Rn but H and G remained unchanged. HDM significantly expanded mast cells compared with control mice; at the same time mMCP-1, α-SMA, Th2 cytokines, and histamine were significantly increased. Compound 48/80 inhalation caused bronchoconstriction and mMCP-1 elevation similarly to HDM inhalation. Bronchoconstriction was eliminated in mast cell-deficient mice. We found that antigen-induced acute bronchoconstriction has a distinct phenotype in mice. HDM sensitization caused lung mastocytosis, and we conclude that inhalation of HDM caused degranulation of mast cells leading to an acute bronchoconstriction without affecting the lung periphery and that mast cell-derived mediators are responsible for the development of the HDM-induced bronchoconstriction in this model.

Inhibitory effects of (-)-epigallocatechin-3-gallate and pterostilbene on pancreatic cancer growth in vitro.

BACKGROUND: It has been previously shown that the naturally occurring antioxidant (-)-epigallocatechin-3-gallate (EGCG), found in green tea, and pterostilbene, a stilbenoid derived from blueberries, inhibit pancreatic cancer in vitro when used individually. We hypothesized that the combination of EGCG and pterostilbene would reveal additive effects in vitro. METHODS: Using the pancreatic cancer cell lines MIA PaCa-2 and PANC-1, efficacy and synergism were evaluated for cell proliferation and viability (3-(4,5-dimethyltiazol-2-y1)-2,5-diphenltetrazolium bromide assays, cell cycle analysis) and mitochondrial apoptosis (mitochondrial depolarization, cytochrome C release, caspase-3/7 activity, cell death detection using enzyme-linked immunosorbent assay). RESULTS: Cell proliferation assays revealed significant additive antiproliferative effects with pterostilbene and EGCG in both cell lines at the later, 72-h, point (P < 0.05). MIA underwent S-phase arrest with the combination (10-12% increase); however, cell cycle arrest was not observed in PANC. The combination induced mitochondrial depolarization and upregulated cytochrome C (P < 0.05) in MIA, but these effects were not observed in PANC. EGCG increased caspase-3/7 in MIA; however, the combination did not significantly increase the activity in either cell line (P < 0.05). Apoptosis was only observed in PANC (P < 0.05). The reduction in proliferation in MIA in the 3-(4,5-dimethyltiazol-2-y1)-2,5-diphenltetrazolium bromide assays with the combination indicated that cell death occurs, possibly through another mechanism. CONCLUSIONS: Our results are encouraging regarding the future use of EGCG and pterostilbene to improve traditional pancreatic cancer therapies. In conclusion, EGCG and pterostilbene have additive, antiproliferative effects in vitro and alter the apoptotic mechanisms in both cell lines by modulation at different points in the mechanism.

Anti-inflammatory effects of levalbuterol-induced 11ß-hydroxysteroid dehydrogenase type 1 activity in airway epithelial cells.

Airway epithelial NF-κB activation is observed in asthmatic subjects and is a cause of airway inflammation in mouse models of allergic asthma. Combination therapy with inhaled short-acting ß2-agonists and corticosteroids significantly improves lung function and reduces inflammation in asthmatic subjects. Corticosteroids operate through a number of mechanisms to potently inhibit NF-κB activity. Since ß2-agonists can induce expression of 11ß-HSD1, which converts inactive 11-keto corticosteroids into active 11-hydroxy corticosteroids, thereby potentiating the effects of endogenous glucocorticoids, we examined whether this mechanism is involved in the inhibition of NF-κB activation induced by the ß-agonist albuterol in airway epithelial cells. Treatment of transformed murine Club cells (MTCC) with (R)-albuterol (levalbuterol), but not with (S)- or a mixture of (R + S)- (racemic) albuterol, augmented mRNA expression of 11ß-HSD1. MTCC were stably transfected with luciferase (luc) reporter constructs under transcriptional regulation by NF-κB (NF-κB/luc) or glucocorticoid response element (GRE/luc) consensus motifs. Stimulation of NF-κB/luc MTCC with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNFα) induced luc activity, which was inhibited by pretreatment with (R)-, but not (S)- or racemic albuterol. Furthermore, pretreatment of GRE/luc MTCC with (R)-, but not with (S)- or racemic albuterol, augmented 11-keto corticosteroid (cortisone) induced luc activity, which was diminished by the 11ß-HSD inhibitor glycyrrhetinic acid (18ß-GA), indicating that there was a conversion of inactive 11-keto to active 11-hydroxy corticosteroids. LPS- and TNFα-induced NF-κB/luc activity was diminished in MTCC cells treated with a combination of cortisone and (R)-albuterol, an effect that was inhibited by 18ß-GA. Finally, pretreatment of MTCC cells with the combination of cortisone and (R)-albuterol diminished LPS- and TNFα-induced pro-inflammatory cytokine production to an extent similar to that of dexamethasone. These results demonstrate that levalbuterol augments expression of 11ß-HSD1 in airway epithelial cells, reducing LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production through the conversion of inactive 11-keto corticosteroids into the active 11-hydroxy form in this cell type.