John Costello

Anti-inflammatory effects of low-dose oral theophylline in atopic asthma

Theophylline, in addition to its bronchodilator effect, may attenuate inflammation in asthma. We did a double-blind placebo-controlled study of the effect of oral theophylline on the inflammatory response of the bronchial mucosa to inhalation of allergen in 19 atopic asthmatic subjects. Bronchoscopy and bronchial biopsy were done 24 hours after allergen inhalation before and after six weeks of treatment with oral slow-release theophylline, 200 mg 12 hourly. The mean serum concentration was 36.6 mumol/L, which is below the currently-accepted therapeutic range. After treatment with theophylline there was a significant reduction in the number of EG2-positive activated eosinophils (5.9 before and 2.1 after treatment, Wilcoxon signed rank p < 0.05) and total eosinophils (16.7 before and 7.6 after treatment, p < 0.05) beneath the epithelial basement membrane. We conclude that low-dose oral theophylline attenuates airway inflammatory response to allergen inhalation in atopic asthma.

The effect of an oral leukotriene antagonist L-649,923 on histamine and leukotriene D4-induced bronchoconstriction in normal man☆

We have studied the effect of prior treatment with an oral leukotriene (LT) antagonist L-649,923, on histamine and LTD4-induced bronchoconstriction in 12 normal male subjects in a double-blind, placebo-controlled, randomized, crossover trial. L-649,923 had no effect on histamine-induced bronchoconstriction but antagonized LTD4-induced bronchoconstriction, causing a mean shift to the right of the dose-response curves of 3.8-fold. This study demonstrates that LT antagonism is possible and safe in man and that LTD4-inhalation challenge testing can be used to detect LT antagonism.

Participation of the cysteinyl leukotrienes in the acute bronchoconstrictor response to inhaled platelet activating factor in man.

To determine whether the effects of platelet activating factor on the airways may be due to the production of leukotrienes we studied the effects of pretreatment with the selective cysteinyl leukotriene antagonist SK&F 104353-Z2 on the airway and cellular responses to inhaled platelet activating factor. Eight healthy men were studied in a randomised, double blind placebo controlled cross-over study. A single dose of platelet activating factor that caused a fall of at least 35% in specific airways conductance (sGaw) was determined initially for each subject. Challenge with this dose of platelet activating factor was then carried out on two further occasions after pretreatment with a single nebulised dose of SK&F 104353-Z2 or placebo. The % reductions in specific airways conductance and of partial flow at 30% of vital capacity (PVmax30) were less after SK&F 104353-Z2 than after placebo (22 versus 34 for sGaw, 19 versus 31 for PVmax30). The mean (95% confidence limits (CL] differences in the maximum % fall from control values for SK&F 104353-Z2 and placebo were -12.6 (-23.8, -1.4) for sGaw and -12.5, (-20.8 -4.2) for PVmax30. The mean % fall in neutrophil count was similar after SK&F 104353-Z2 (46%) and after placebo (50%) (95% CL of difference 13.6, 6.6). Bronchial responsiveness to methacholine did not increase above baseline values in any subject when measured two weeks after challenge by platelet activating factor. This study suggests that leukotrienes play a part in the response to platelet activating factor in man.

Effect of intravenous corticosteroid on ex vivo leukotriene generation by blood leucocytes of normal and asthmatic patients

BACKGROUND The cysteinyl-leukotrienes (LTC4, LTD4, LTE4) are critical bronchoconstrictor and eosinophilotactic mediators in asthma while LTB4 is a potent neutrophil chemoattractant. Glucocorticosteroids are front line anti-inflammatory treatment for asthma but the evidence that they reduce leukotriene (LT) synthesis in vivo is poor. METHODS In a randomised, double blind, placebo controlled, crossover trial immunoassays were used to measure ex vivo synthesis of LTC4and LTB4 by calcium ionophore stimulated blood leucocytes and bronchoalveolar lavage (BAL) cells of eight normal subjects and eight patients with mild allergic asthma 4–6 hours after intravenous administration of a single 100 mg dose of methylprednisolone. RESULTS Ionophore stimulated synthesis of LTC4 (but not LTB4) in blood granulocytes tended to be higher in asthmatic subjects (mean 9.7 ng/106 cells) than in normal subjects (4.2 ng/106 cells; p = 0.08) and intravenous methylprednisolone reduced synthesis of LTC4 (but not LTB4) to normal levels (2.9 ng/106 cells; 95% CI for the reduction 1.0 to 12.5 ng/106 cells; p = 0.03). In blood mononuclear cells methylprednisolone reduced LTC4synthesis in asthmatic subjects from 1.26 to 0.79 ng/106cells (95% CI for the reduction 0.26 to 0.79, p = 0.014) and tended to reduce LTC4 synthesis in normal subjects from 1.51 to 0.86 ng/106 cells (p = 0.08). Methylprednisolone also significantly reduced synthesis of LTB4 in mononuclear cells from both subject groups (p = 0.014). It had no effect on LT synthesis in BAL cells from either group nor on LT levels in BAL fluid. CONCLUSIONS Intravenous methylprednisolone can reduce synthesis of leukotrienes in blood granulocytes and mononuclear cells within six hours of a single intravenous dose.

The biotransformation in vitro of cysteinyl leukotrienes in blood of normal and asthmatic subjects.

The metabolism of exogenous leukotriene C4 (LTC4), LTD4 and LTE4 (10(-8) M) was studied in vitro in blood of normal and asthmatic subjects for up to 2 hr by reverse-phase high performance liquid chromatography. In whole blood, incubation of LTC4 (T1/2 = 11.5 min) resulted in the formation of LTD4 and LTE4 whose biosynthesis was inhibited by serine borate (30 mM). Similar experiments performed with LTD4 (T1/2 = 5 min) produced a single metabolite (LTE4) which was inhibited by L-cysteine (10 mM). On the other hand, LTE4 represented a highly stable product in our in vitro system. The bioconversion of LTC4 or LTD4 was slower in plasma but this effect appeared more pronounced for the cysteinylglycinyl derivative. The bioconversion of LTD4 in whole blood or plasma was almost twice as rapid as LTC4. Experiments performed with asthmatic blood showed no significant difference in the survival of LTC4. These results suggest that blood may play a role in regulating the bioavailability of cysteinyl-containing LTs which could be of relevance to their excretion in man.

Platelet activating factor does not cause a reproducible increase in bronchial responsiveness in normal man

The reproducibility of acute effects of inhaled PAF on airway calibre, circulating neutrophil count and any subsequent increase in bronchial responsiveness has been studied in six normal subjects and compared to the effects of inhaled lyso‐PAF, the inactive precursor and metabolite of PAF. PAF caused acute bronchoconstriction and a transient fall in neutrophil count on two separate occasions in five out of six subjects (minimum percentage of baseline values (mean): first PAF challenge; sGaw 69%, V̇max30 72%; neutrophil count 70%; second PAF challenge; sGaw 61 %, V̇max30 74%, neutrophil count 63%). In one subject inhaled PAF caused bronchoconstriction and a transient fall in neutrophil count once, but a second challenge resulted in no detectable changes. There was no significant increase in bronchiai responsiveness to methacholine in any subject studied on five occasions over a 2‐week period following each PAF challenge. Challenge with lyso‐PAF did not cause acute effects or any subsequent changes in bronchial responsiveness. These findings demonstrate that any effects of inhaled PAF on bronchial responsiveness in normal man are small and probably not of clinical significance. It would also be inappropriate to use this human model to study the mechanisms of bronchial hyperresponsiveness or for the preliminary assessment of potential new anti‐asthma drugs.

Shape based region growing using derivatives of 3D medical images: application to semiautomated detection of pulmonary nodules

This paper presents a new method for shape based segmentation of 3D medical images. 3D geometric information is calculated for each voxel by computing the partial derivatives of the 3D image. The shape features of the iso-intensity surfaces are subsequently extracted. The extracted shape features are combined with 3D intensity-based region growing to give accurate separation of connected objects having different shapes but similar intensity values. We have applied this method to both synthetic and real 3D CT lung images. The experimental results demonstrate that the presented method, unlike the traditional intensity-based method, is able to segment connected objects accurately. A sphere can be differentiated from a connected cylindrical shape within the synthetic data. In the case of the real 3D CT lung images, all of the nodules can be detected and separated accurately from adjoining blood vessel or from the lung wall.

The detection of 5-lipoxygenase and cyclo-oxygenase products in sputum of patients with chronic bronchitis and bronchiectasis

Leukotrienes (LTs) and prostanoids (Ps) were detected in sputum of patients with chronic bronchitis and/or bronchiectasis (CB/B) using selective superfusion bioassay and radioimmunoassay (RIA) techniques. Analysis of sputum extracts showed a 4-fold increase in the level of LTB4 compared to the cysteinyl-containing LTs (LTC4/LTD4). The measurement of cyclo-oxygenase products (COPs) indicated relatively greater amounts of the vasodilator prostaglandin E2 (PGE2) and prostacyclin (PGI2) compared to the vasoconstrictor prostaglandin F2 alpha (PGF2 alpha) and thromboxane A2 (TxA2) agents (70:30% of total COPs respectively). The presence of eicosanoids (LTs and Ps) in sputum of patients with CB/B suggest that these biologically active substances may act as mediators of bronchoconstriction and inflammation in these diseases.

The generation and metabolism of leukotrienes in the ionophore-stimulated blood of normal and asthmatic subjects

The generation and metabolism of leukotrienes (LTs) B4, C4, D4, and E4 were studied in vitro in the A23187-stimulated whole blood of normal (N) and atopic asthmatic (AA) human subjects. Using a combination of reversed-phase high performance liquid chromatography and radioimmunoassay, we have demonstrated that the blood cells of atopic asthmatic patients have an enhanced ability to release LTB4 and LTC4 when compared to those of normal subjects. The release of LTB4 and LTC4 in response to ionophore is dose- and time-dependent. Half-maximal doses of ionophore caused the generation of high, sustained levels of LTB4, which are significantly higher in the AA blood than in N blood. Incubations of 3H-LTB4 in ionophore-stimulated N and AA blood revealed a slow metabolism to 20-OH-LTB4 and 20-COOH-LTB4. LTC4 is generated in smaller amounts than LTB4, with an early peak after 10 min which is significantly higher (p less than 0.01) in the AA blood compared to the N blood. Subsequent metabolism of LTC4 elicits significantly greater amounts of LTD4, and consistently higher levels of LTE4, in the AA blood. Parallel incubations of 3H-LTC4 in ionophore-stimulated N and AA blood demonstrated rapid metabolism of LTC4 by the glutathione detoxification pathway. The elevated production of LTB4 and LTC4 in AA blood was not accounted for by differences in leukocyte sub-type counts in the two groups, nor by differences in their rates of catabolism. The novel, selective 5-lipoxygenase inhibitor BW A4C [N-(3-phenoxycinnamyl) acetohydroxamic acid] caused dose-dependent inhibition of LTB4 and LTC4 generation and was equipotent in N and AA blood.

Reduction in leukotriene B4 generation by bronchoalveolar lavage cells in asthma.

BACKGROUND--Leukotrienes are inflammatory mediators implicated in the pathogenesis of asthma. The capacity of inflammatory cells within the airways to generate leukotrienes may be altered in asthma. This hypothesis was tested using bronchoalveolar lavage (BAL) to sample cells within the airways from atopic asthmatic and normal subjects, and by measuring their capacity to generate leukotriene B4 (LTB4) and leukotriene C4 (LTC4) in response to A23187, a potent stimulus of leukotriene generation. METHODS--Bronchoalveolar lavage was performed in 12 mild asymptomatic atopic asthmatic patients and 12 normal subjects. Mixed BAL cell aliquots (approximately 80% alveolar macrophages) were incubated with 0-20 microM A23187 for 10 minutes and with 4 microM A23187 for 0-30 minutes, and leukotrienes were measured by radioimmunoassay and high performance liquid chromatography. RESULTS--Mixed BAL cells from asthmatic subjects generated less LTB4 than cells from normal subjects in dose response and time course experiments (area under the curve 81.5 (0.0-228.5) ng.min.10(-6) cells in asthmatic subjects and 197.9 (13.9-935.6) ng.min.10(-6) cells in normal subjects. There were no differences in LTC4 generation between BAL cells from asthmatic and normal subjects. CONCLUSIONS--Generation of LTB4 by BAL cells from atopic asthmatic subjects in response to A23187 was reduced. As the alveolar macrophage is the major source of LTB4 in BAL cells, these results probably reflect reduced generation of LTB4 by alveolar macrophages from asthmatic patients. This may be a consequence of monocyte migration into the lung, or altered alveolar macrophage function in asthma, or both.