Effect of a leukotriene B4 receptor antagonist, LY293111, on allergen induced responses in asthma.

BACKGROUND: Leukotriene (LT) B4 is a potent neutrophil chemoattractant and also stimulates eosinophils in vitro, but its role in asthmatic inflammation is unknown. METHODS: The effect of the novel LTB4 receptor antagonist, LY293111, was examined using allergen challenge as a model for asthmatic inflammation in 12 atopic asthmatic subjects in a double blind placebo controlled crossover trial. Subjects with an established early (EAR) and late asthmatic response (LAR) to allergen at screening received oral LY293111 in a dose of 112 mg three times daily for seven days or placebo before further allergen challenge. Each treatment was separated by a washout period of 28 days. Individuals underwent histamine challenge one hour before and three hours after allergen challenge. Bronchoalveolar lavage (BAL) fluid was obtained at bronchoscopy 24 hours after allergen challenge. RESULTS: There was no difference in baseline lung function, EAR, LAR, or in airway responsiveness to histamine before and after allergen between placebo and LY293111. By contrast, treatment with LY293111 significantly reduced the number of neutrophils in BAL fluid expressed as both absolute cell numbers and percentage cell differential counts: absolute cell counts, median (range) 0.04 (0.02-0.15) x 10(6) after LY293111, 0.09 (0.02-0.43) x 10(6) after placebo; percentage differential cell counts 0.35 (0.1-2.0) after LY293111, 0.80 (0.1-3.6) after placebo (p < 0.05). Eosinophils, macrophages, and lymphocytes in BAL fluid did not differ between treatments. There was a significant reduction in the concentration of myeloperoxidase (MPO) with both placebo (16 (6.6) ng/ml) and LY293111 (3.5 (1.8) ng/ml) and of LTB4 (placebo 4.6 (1.2) pg/ml, LY293111 2.2 (0.2) pg/ml). Concentrations of LTC4 and interleukin 8 were reduced, although not significantly, whereas concentrations of interleukin 6, GM-CSF, and TNF-alpha were unchanged by LY293111. CONCLUSIONS: These results demonstrate an influence of LTB4 on neutrophil influx and activation in the airway following allergen challenge. Despite this anti-inflammatory effect, there was no measured physiological benefit and this questions the functional role of the neutrophil in the pathophysiology of allergen induced asthma.

A kinetic assay for eosinophil peroxidase activity in eosinophils and eosinophil conditioned media.

The activity of eosinophil peroxidase (EPO) is commonly employed as a measure of eosinophil activation in biologic fluids. Determination of product formation by this enzyme by end-point measurement may be affected profoundly by substrate concentrations, reaction time and degradation of end-product and enzyme. To determine more accurately EPO concentrations in media conditioned by isolated, purified eosinophils, we have developed a kinetic, colorimetric assay to measure EPO concentration as a function of maximum velocity of reaction (Vmax). An automated method for determining Vmax in a 96-well microplate colorimetric assay was utilized over a wide range of substrate concentrations. Concentrations greater than or equal to 3 x 10(-8) g/ml could be determined reliably with this assay. Peroxidase activity was inhibited in a concentration-dependent manner by the addition of 3-amino-1,2,4-triazole (AMT). The EPO concentration in eosinophils determined by this kinetic method was approximately 1.1 x 10(-5) g/10(6) eosinophils. Eosinophil activation with 10(-6) M f-Met-Leu-Phe (fMLP) caused substantial EPO secretion (9.0 +/- 1.7% vs. 2.9 +/- 0.6% total EPO content for control, P = 0.05) and decrease in eosinophil EPO concentration (92.3 +/- 4.2% of control, P = 0.038). Secretion was enhanced by the addition of 5 micrograms/ml cytochalasin B to 10(-6) M fMLP (25.9 +/- 12.7% total EPO content, P = 0.043 vs. control); similar decreases were noted in eosinophil EPO concentration (71.7 +/- 16.1% of control, P = 0.043). These data demonstrate that determination of EPO secretion by measurement of Vmax is a reliable, accurate method for precise quantification of this enzyme in media containing purified eosinophils or eosinophil products in the absence of other forms of peroxidase activity.

Evidence of a novel association of unsaturated fatty acids with Gc (vitamin D-binding protein).

The strong sequence homology described recently between Gc (Vitamin D-binding protein) and albumin, and the ability of the latter, to bind 2-p-toluidinylnaphthylene sulfonate (TNS) promoted similar binding studies with Gc. TNS bound to native Gc as demonstrated by fluorescence, but chloroform:methanol extraction of Gc and gas chromatography revealed that large amounts of unsaturated fatty acids - 16:1, 18:1, 18:2 and 20:4 were also associated with Gc. In addition, TNS fluorescence was abolished by delipidation of Gc, and restored upon subsequent reconstitution with fatty acid. Finally, 70-80% of [3H]-arachidonic acid added to whole serum bound to Gc. These results demonstrated that TNS fluorescence of the native molecule reflects associated lipid, and suggest a novel role for Gc as a reservoir for a circulating pool of unsaturated fatty acids.

Group-specific component (vitamin D binding protein) prevents the interaction between G-actin and profilin.

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.

Isolation of a highly enriched sarcolemma membrane fraction from canine heart.

A highly enriched sarcolemma preparation was isolated by differential centrifugation of a canine ventricular homogenate followed by centrifugation of a membrane fraction layered over 22% (w/v) sucrose. Ouabain binding, ouabain-sensitive potassium phosphatase activity and 5'-nucleotidase activity were enriched 19--27 fold over the homogenate whereas Ca2+-ATPase and succinate dehydrogenase activities were 0.75 and 0.36, respectively, of that for the homogenate. The isolation procedure was relatively rapid and yielded about 2.0 mg protein/100 g of ventricular muscle. The highest salt concentration used in the procedure was 0.6 M KCl and no detergents were employed. Initial characterization studies suggested that the sarcolemma-enriched fraction consists predominantly if not totally of freely permeable membrane vesicles and that the sarcolemma does not manifest a Ca2+-ATPase activity, at least within the limits of the assay procedures employed. This preparation was concluded to be about 1.5- to 4-fold more highly enriched with sarcolemmal markers than preparations obtained by previously published procedures. Accordingly, the preparation provides an improved basis for the probe of calcium movements that occur across the sarcolemma in association with the excitation-contraction-relaxation sequence of the mammalian myocardial cell.

The effect of thermal injury on plasma carnitine in rats.

The plasma concentration of L-carnitine in scalded rats was determined to be greater (p less than or equal to 0.05) than that of control rats at 6 h following the administration of a 20% body surface, full-thickness burn produced by scalding in a 100 degrees C water bath for 15 sec.