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Notes: We evaluated the effect of pre-treatment with 1 and 2 mg b.i.d. of ketotifen on the early response to nasal challenge in a double-blind cross-over trial. Weekly nasal challenges were performed in 10 allergic subjects after 1 hr and 1, 2, 3 and 4 weeks of ketotifen administration. The response to nasal challenge was monitored by counting the number of sneezes, the assessment of subjective symptoms, and by measuring the levels of histamine and TAME-esterase activity in recovered nasal lavages. The number of sneezes diminished significantly after a single dose of drug with both the 1 and 2 mg doses. Prolonged pre-treatment did not improve the results. The levels of histamine and TAME-esterase activity in recovered nasal lavages were not changed significantly by either pre-treatment at either dose. Although the number of subjects was small, our data suggest that ketotifen diminishes allergic symptomatology by its antihistaminic properties rather than by inhibiting histamine release from mast cells. As we did not look into the effects of ketotifen on other products generated by mast cells (prostanoids, leukotrienes and tryptase), we cannot fully rule out an effect on mast cells.

Notes: The mechanism(s) leading to the development of late phase allergic reactions is (are) unknown. Previous studies have indicated that a relationship between serum IgE and the late phase exists.To explore the relationships between allergen-specific immunoglobulins in bronchoalveolar lavage (BAL) fluids and the magnitude of airflow limitation during the late phase response to inhaled allergen.Ragweed-specific IgE, IgA, secretory IgA (sIgA) and IgG were measured in BAL fluid and in the serum 1–5 weeks before whole lung antigen challenge with ragweed extract, in 16 ragweed allergic asthmatics. In addition, BAL and serum eosinophil cationic protein (ECP) and BAL fibrinogen levels were determined and BAL cells counted and differentiated. The latter procedures were repeated in a second BAL performed 24 h after the end of the ragweed challenge. After the challenge, lung function was monitored hourly for 8 h, to record the magnitude of airflow limitation.Ragweed-specific immunoglobulins were detected in 25% to 37.5% of BAL samples. Compared to the subjects with undetectable BAL fluid ragweed-specific IgE levels at baseline, those with detectable antibodies had stronger late phase reactions as determined by the nadir of FEV1 between hours 4 and 8 after the ragweed inhalation challenge (P = 0.0007). Allergen-induced changes in BAL ECP and fibrinogen levels were also higher in those subjects with detectable ragweed-specific IgE in baseline fluids (P = 0.03 and P = 0.005, respectively). Significant relationships between BAL antigen-specific IgA, serum ragweed-specific IgE and IgA and the late phase reaction were also found.The results of this study point towards the possibility that allergen-specific IgE and IgA may be independently involved in the pathogenesis of the late phase reaction. This notion merits further exploration.

Notes: Background Human basophils undergo anaphylactic degranulation, characterized by extrusion of membrane-free granules, and piecemeal degranulation, characterized by progressive removal of granule contents in the absence of granule extrusion. F-Met peptide stimulates a degranulation continuum in human basophils that includes both forms of secretion. Charcot-Leyden crystal protein is stored in tbe granules of unstimulated human basopbils.Objective The objective of this study was to determine the subcellular localization of the Charcot-Leyden crystal protein in individual morphological basophil phenotypes that are stimulated by f-Met peptide and are associated with secretion.Methods A post-embedding immunogold analysis was used to detect changes in the subcellular sites of Charcot-Leyden crystal protein in human basophils stimulated with f-Met peptide. Human basophils from nonnal donors were purified by countercurrcnt centrifugal elutriation and Percoll density gradients, stimulated to degranulate with 1 μm f-Met peptide (or incubated in buffer controls), and recovered for histamine assay, electron microscopy and immunogold labelling. Specificity controls Included omission of the primary antibody and substitution of the primary antibody with non-immune normal rabbit IgG or with Charcot-Leyden crystal protein-Sepharose-absorbed primary antibody.Results The results showed new sites of labelling and different densities of labelling for Charcot-Leyden crystal protein in distinctive basophil phenotypes stimulated by f-Met peptide. New sites for Charcot-Leyden crystal protein included nucleus, cytoplasm, degranulation channel, degranulation channel membrane, plasma membrane, and a newly recognized granule population similar to primary granules in eosinophils. These new sites, as well as previously documented sites of Charcot-Leyden crystal protein (granules, intragranular Charcot-Leyden crystals, cytoplasmic vesicles) showed variahic labelling when analysed by phenotype. Other sites (besides intragranular Charcot-Leyden crystals) of formed Charcot-Leyden crystals included cytoplasm, degranulation channel, extracellular space and rarely, nucleus. Analysis of cytoplasmic vesicles, total granules and altered granules, and gold particles in subcellular compartments in seven identifiable phenotypes revealed that f-Met peptide stimulated human basophils to empty their granules by transporting Charcot-Leyden crystal protein in vesicles to the plasma membrane in the absence of granule extrusion in cells exbibiting piecemeal degranulation. In cells exhibiting anaphylactic degranulation. gold-labelled Charcot-Leyden crystals were extruded to the cells' exterior in concert with granule particles and concentric dense membranes contained within granules. Completely degranulated cells had a high density of plasma membrane gold label that was associated with numerous Correspondence: Ann M. Dvorak MD, Department of Pathology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215, USA. gold-laden endocytotic cytoplasmic vesicles. Basophils reconstituted their main granule population, within which Charcot-Leyden crystals resided, in part by endocytosis of previously released plasma membrane-bound Charcot-Leyden crystal protein. Completely recovered cells displayed decreased Charcot-Leyden crystal protein labelling of the plasma membrane and vesicle compartments, the presence of a highly labelled new granule subset that resembled Charcot-Leyden crystal protein-containing primary granules in eosinophils, and the highest density of granule and intragranular Charcot-Leyden crystal gold labelling of all phenotypes tbat developed after stimulation.Conclusion Seven individual f-Met peptide-activated human basophil phenotypes labelled by an ultrastructural immunogold method to detect subcellular sites of Charcot-Leyden crystal protein showed changing distributions of this protein which document the capability of human basopbils to undergo complex release and recovery reactions that may be pertinent to the functions of Charcot-Leyden crystal protein and the capabilities of these cells in vivo in a variety of diseases.

Notes: Background Desloratadine is a non-sedating, clinically effective, anti-allergic therapy that has been shown to exhibit anti-inflammatory properties that extend beyond its ability to antagonize histamine at H1-receptor sites. This latter effect has been shown in vitro to be both IgE-dependent and -independent.Objective In this study, we addressed the ability of desloratadine to inhibit the in vitro generation of interleukin (IL)-4 and IL-13 from human basophils while concurrently comparing its efficacy in preventing mediator release by these cells.Methods Basophil-enriched suspensions were treated with various concentrations of desloratadine for 15 min before stimulating with either anti-IgE antibody, calcium ionophore, IL-3 or phorbol ester. Histamine (fluorimetry), LTC4 (RIA) and IL-4 (ELISA) were all assayed using the same 4-h culture supernatants. IL-13 (ELISA) was measured in supernatants harvested after 20 h incubation. IL-4 mRNA expression (dilutional RT-PCR) was also examined.Results Desloratadine was found to be nearly six–seven times more potent in preventing the secretion of IL-4 and IL-13 induced by anti-IgE than it was at inhibiting the release of histamine and LTC4. These cytokines were equally inhibited by desloratadine following activation with ionomycin despite the lack of an effect on the histamine induced with ionomycin. Desloratadine had a lesser effect regarding inhibition of the IL-13 secreted in response to IL-3 and PMA. There was no evidence that desloratadine mediated its inhibitory effects by causing decreased cell viability. Finally, IL-4 mRNA accumulation was remarkably inhibited, by as much as 80%, following pretreatment with desloratadine.Conclusion While capable of inhibiting histamine and LTC4 release by human basophils, desloratadine is more effective at targeting the signals regulating IL-4 and IL-13 generation in these cells. This inhibitory effect on cytokine generation provides additional evidence that this antihistamine exerts anti-inflammatory properties.

Notes: Background Multiple mediators including prostaglandin D2 and leukotriene B4 have been shown to increase in nasal secretions during the early response to nasal challenge with antigen.Objective Our objective was to investigate the time course of prostanoid and leukotriene B4 release into nasal secretions on both the ipsilateral and contralateral side after a unilateral nasal allergen challenge.Methods We performed a controlled, randomized trial. Six volunteers were challenged unilaterally with antigen or diluent in a randomized order and discs were used to collect nasal secretions from both nostrils at 2 min intervals for 20 min after the challenge. Prostanoids and leukotriene B4 (LTB4) in recovered nasal secretions were measured by combined capillary gas chromatography-negative ion chemical ionization mass spectrometry (GC/MS).Results Nasal allergen challenge resulted in a significant and immediate increase in symptoms and sneezing. PGD2 was significantly elevated above diluent values (0.6 ± 0.6pg) 30s after removal of the allergen disc (P〈0.05), reached its peak (423.2 ± 182.4pg) at 2 min and then slowly decreased. PGD2 also increased on the contralateral side after unilateral allergen challenge, reaching peak values about six times lower than on the ipsilateral side (70.8 ± 2l.7pg at 6 min). Levels of 9a, Ub-PGF2 after antigen provocation became significantly higher than after diluent (0 ± 0pg) on the ipsilateral side at 2 min (17.2 ± 5.9 pg), and reached peak levels at 4 mm (25.1 ± 8.0 pg). LTB4 also increased significantly on the side of ehallenge. For the other prostanoids measured (PGF2, PGF2α TxB2, 6kPGF1α), no significant changes in either ipsilateral or contralateral secretions were observed after allergen challenge.Conclusions Our study described the kinetics of PGD2 and LTB4 release as well as the contralaterai release of PGD2.

Notes: Backgrottnd Basophils arc circulaling, secretory grunulocytes that are generally considered to be end-stage cells. In one species of guinea-pigs, basophilic leucocytes have been shown to recover from stimulated secretion in short-term cultures. Similar studies have not been done using human basophils.Objective The purpose of this study was to examine human hasophils in short-term recovery intervals following stimulation of secretion to determine whether visual evidence of recovery occurred.Methods We examined the ultrastrucural morphology of early recovery (l0 min-6h) of human basophils following secretion stimulated by formyl-mcthionyl-lcucyl-phenyl-alanine (FMLP). A combined technique for electron microscopy consisted of postfixation exposure to cationizcd ferritin and reduced osmium, providing maximum quality images and allowing identification of intracellular spaces/organelles that opened to the cell surface, often out of the plane of section.Results The ultrastructural evaluation revealed that control basophils (0 time–6h) did not undergo regulated secretion or develop the morphologies associated with recovery following secretion. FMLP-stimulated basophils underwent an overlapping continuum of piecemeal degranulation - anaphylactic degranutation (0 time- I min), producing vesicle- and granule-free, completely degranulated, viable, mature basophils with polylobed nuclei. The early recovery period (10 min-h) following FMLP stimulation was characterized by reconstitution of granules. Morphological mechanisms for granule reconstitution included a mixture of conservation, condensation, and synthetic events.Coticlttsion Human basophils, like guinea pig basophils, have the polential to recover from regulated secretion.