Evaluation of RAST inhibition as a method for the standardization of house-dust extracts

RAST-inhibition was evaluated as a method for the in vitro standardization of house dust allergens, using four lyophilized house-dust preparations of different degree of purification (R1, R2, R3 and R4). When a serum pool of highly house-dust sensitive patients was used, a qualitatively similar potency sequence by RAST-inhibition between the different preparations could be established as with in vivo skin testing. When, however, individual sera were examined, striking differences were observed in both the qualitative and the quantitative potency relationships. Furthermore, large variations were noted in duplicate measurements in RAST-inhibition. From this study it became clear that in vitro RAST inhibition and in vivo skin tests can have only statistical meaning for the standardization of house-dust allergen. In individual patients, striking differences may occur in the response to different preparations, thereby making standardization by these techniques rather unsatisfactory. By applying these techniques, we gained the impression that patients were being characterized rather than allergens

Characterization of (-)-[3H]dihydroalprenolol binding to intact and broken cell preparations of human peripheral blood lymphocytes

In this study we compared characteristics of (-)-[3H]dihydroalprenolol ([3H]DHA) binding sites in crude membrane preparations of human peripheral blood lymphocytes with those of intact, viable cells. A valid determination of specific β-adrenergic receptor binding in both preparations was obtained by defining non-specific [3H]DHA binding with 10−6 M l- or dl-propranolol or 10−3 M l-isoproterenol. Higher concentrations of propranolol were used in prior reports on lymphocyte membranes. We showed that these concentrations may inhibit non-specific binding, causing non-saturability and inhomogeneity of the estimated ‘specific’ binding. In the intact cell preparations, inclusion of 10−4 M phentolamine was necessary to reduce the high degree of non-specific binding. By contrast, phentolamine (10−4 M) showed no effect on the [3H]DHA binding to membrane preparations. At 37°C the [3H]DHA binding to β-adrenergic receptor sites in both intact and broken cell preparations was rapid and reversible. The sites were stereoselective, as l-propranolol was about two orders of magnitude more potent to inhibit [3H]DHA binding than was the d-isomer. In both preparations, agonists competed for specific binding with a rank order of potency isoproterenol > epinephrine > norepinephrine, which indicated a β2-type of adrenergic receptor. The specific [3H]DHA binding was saturable and Scatchard analysis revealed comparable numbers of homogenous, non-cooperative binding sites (approximately 1250 receptors/cell in the membrane preparations and 1700 receptors/cell in the intact cells). In spite of these similarities the membrane sites showed a lower affinity for the antagonists [3H]DHA and propranolol than did the intact cell sites, whereas their affinity for the agonists was increased. These differences indicate that the membrane system might be less suited to provide physiologically significant information about the β-adrenergic receptor system

The excretion of leukotriene E4 into urine following inhalation of leukotriene D4 by human individuals

Healthy volunteers underwent bronchial challenge with increasing doses of nebulized leukotriene D4 (0.007 - 200 nmol) at 15 min intervals. Total amounts of 200 nmol (females) and 400 nmol (males) were inhaled, corresponding to approximately 100 nmol and 200 nmol deposited in the lung, respectively. Of the latter amounts 3 +/- 1% (mean +/- S.E.M., n = 5) was found to be excreted as leukotriene E4 into the urine within 12 h. No further excretion after this period was observed. Approximately 50% of the total urinary leukotriene E4 was excreted during the first 2 h. These results suggest that a possible formation of sulfidopeptide leukotrienes in the lung in vivo can be monitored by measuring leukotriene E4 excretion into the urin

Specific leukotriene formation by purified human eosinophils and neutrophils

Human granulocytes isolated from peripheral blood have been described to synthesize both LTB4 and LTC4 from arachidonic acid. We have observed that the amount of LTC4 produced by human granulocyte preparations is strongly dependent on the relative amount of eosinophils. To investigate a possibly significant difference in leukotriene synthesis of the eosinophilic and neutrophilic granulocytes, we developed a purification method to isolate both cell types from granulocytes obtained from the blood of healthy donors. Leukotrienes were generated by incubation of the purified cells with arachidonic acid, calcium ionophore A23187, calcium-chloride and reduced glutathione. Surprisingly, eosinophils were found to produce almost exclusively the spasmogenic LTC4. In contrast, neutrophils produce almost exclusively the chemotactic LTB4, its omega-hydroxylated metabolite 20-hydroxy-LTB4 and two non-enzymically formed LTB4 isomers