Aggregation of the high affinity IgE receptor (Fc⑀RI) in a mast cell line resulted in activation of the p42 and the stress-activated p38 mitogen-activated protein (MAP) kinases. Selective inhibition of these respective kinases with PD 098059 and SB 203580 indicated that p42 MAP kinase, but not p38 MAP kinase, contributed to the production of the cytokine, tumor necrosis factor-␣, and the release of arachidonic acid in these cells. Neither kinase, however, was essential for Fc⑀RI-mediated degranulation or constitutive production of tumor growth factor-␤. Studies with SB 203580 and the p38 MAP kinase activator anisomycin also revealed that p38 MAP kinase negatively regulated activation of p42 MAP kinase and the responses mediated by this kinase.Stimulation of mast cells by aggregation of membrane IgE receptors (Fc⑀RI), leads to recruitment of the tyrosine kinase Syk and activation of Syk-dependent signaling cascades (1, 2). These cascades include activation of phospholipase C and sphingosine kinase for mobilization of calcium ions and PKC 1 (3, 4) and the activation of p42 MAP kinase cascade through Ras (2, 5). These cascades lead ultimately to secretion of intracellular granules, a response primarily driven by the increase in [Ca 2ϩ ] i and activation of PKC (6), and a cPLA 2 -mediated release of arachidonic acid. The activation of cPLA 2 is dependent on increase of [Ca 2ϩ ] i and phosphorylation by MAP kinase (2,7,8).Stimulated mast cells also produce a variety of cytokines that include interleukins 1, 3, 4, 5, and 6 as well as TNF␣ and granulocyte-macrophage colony-stimulating factor (9, 10). Typically, increased expression of cytokine mRNA and protein is detectable 30 min to several hours after the addition of stimulant (11). These cytokines, particularly TNF␣, are thought to mediate pathologic inflammatory reactions (10) and protective responses to bacterial infection (12). The production and release of TNF␣ are regulated through signals transduced by calcium and PKC, although there are indications that additional Fc⑀RI-mediated signals may operate for optimal production of TNF␣ in cultured RBL-2H3 mast cells. Compared with antigen, other stimulants are relatively weak inducers of TNF␣ production when doses of stimulants are matched for maximal stimulation of degranulation (13). Also, concentrations of Ro31-7549 that block PKC, secretion of granules, and release of TNF␣ only partially block production of TNF␣ (13).The present objective was to determine whether stimulation of MAP kinases induces additional signals for production of TNF␣. A linkage between these events has not been established in mast cells. Antigen-induced stimulation of p42 MAP kinase coincides with the activation of its upstream regulators, Ras, Raf, and MEK-1 (2, 5), and persists through the period when production of TNF␣ would be most apparent (14). As noted in this paper, however, RBL-2H3 cells also possess the mammalian homologue of the yeast HOG-1 protein kinase, p38 MAP kinase. We have utilized the MEK-1 inhibitor, PD 098059 (15, 16)...

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The distribution of responses in study populations provides a novel method of comparing the benefit of two treatments. This 6-week, randomised, placebocontrolled, double-blind study compared the effectiveness of oral montelukast with inhaled beclomethasone in chronic asthma by assessing the distribution and overlap of patient responses to therapy, as measured by a clinical outcome (asthma control days).A total of 730 adult patients with asthma, age 15-65 yrs, with a forced expiratory volume in one second (FEV1) at baseline of 50-85% of predicted and o15% improvement in FEV1 after inhaled b-agonist were enrolled. After a 2-week placebo run-in period, patients were randomly allocated to receive montelukast (10 mg once daily), inhaled beclomethasone (200 mg twice daily) or placebo. The primary end-point (per cent of asthma control days) was compared between treatments as the overlap in the response distributions.The overlap of the distribution of responses between the montelukast and beclomethasone groups was 89% for per cent asthma control days and 96% for change from baseline in FEV1. The mean (¡SD) per cent asthma control days in the montelukast and beclomethasone groups was significantly higher than that in the placebo group (placebo 40.0 ¡ 35.8, montelukast 50.7 ¡ 37.1, beclomethasone 57.9 ¡ 36.1). The mean differences between montelukast and placebo, beclomethasone and placebo, and montelukast and beclomethasone were significant. The mean per cent change (¡SD) from baseline in FEV1 was 12.1¡18.7 and 13.9¡20.8 in the montelukast and beclomethasone groups, respectively, and significantly greater than that in the placebo group (6.4¡20.1); there was no significant difference between the montelukast and beclomethasone groups in mean values or response distribution. There was also no difference among treatment groups in the frequency of adverse experiences.A comparison of the response distribution is an important approach to comparing therapies; montelukast and beclomethasone provided similar response distributions for the end-point of per cent asthma control days over a 6-week treatment period. Eur Respir J 2003; 21: 123-128

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In these trials, COPD subjects administered nebulized arformoterol demonstrated significant and sustained improvement in lung function over 12 weeks.

PurposeTo evaluate the relationship between the IOP-lowering effect of trabodenoson and the associated structural and functional changes in the trabecular meshwork (TM).MethodsSix independent cohorts of young and aged mice were exposed to three different topical once-a-day formulations of trabodenoson and eyes were compared to those treated with placebo drops. IOP was measured daily just before drug administration using rebound tonometry. Outflow facility was measured in enucleated eyes. Flow patterns and morphology of conventional outflow tissues were monitored using tracer beads and standard histology, respectively. In parallel, three-dimensional human TM tissue constructs (3D-HTM) were grown and used in experiments to test effect of trabodenoson on the expression of collagen IV, fibronectin, matrix metalloproteinase (MMP)-2 and MMP-14 plus MMP-2 activity.ResultsTopical administration of trabodenoson significantly lowered IOP on every day tested, up to 7 days. After 2 days of treatment, outflow facility increased by 26% in aged mice and 30% overall (young and aged mice), which was significantly different from vehicle (P < 0.05). Outflow facility was 15% higher than controls after 7 days of treatment (P = 0.07). While gross morphology was not affected by treatment, the intensity of tracer bead distribution increased by day 7 (P = 0.05). Parallel experiments in 3D-HTM showed that trabodenoson treatment significantly increased MMP-2 activity and MMP-14 abundance, while decreasing fibronectin and collagen IV expression.ConclusionsTrabodenoson alters ECM turnover by TM cells and increases conventional outflow facility, which accounts for its ability to lower IOP in young and aged mice.

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