Inhibition of degranulation and interleukin-6 production in mast cells derived from mice deficient in protein kinase Cbeta.
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The antigen-mediated activation of mast cells by means of IgE antibodies bound to the cell surface leads to direct interactions between FcepsilonRI receptor cytoplasmic domains and various intracellular proteins. These interactions initiate diverse signal-transduction pathways, and the activation of these pathways results in the immediate release of proinflammatory agents. A delayed response also occurs and includes the release of various cytokines. It is clear that the activation of kinases is a requirement for the exocytosis observed in mast cells. In addition to the tyrosine phosphorylation of the affected system by soluble tyrosine kinases, activity of protein kinase C (PKC) results in serine or threonine phosphorylation of multiple protein substrates. In this study, we found that mast cells derived from PKCbeta-deficient mice produce less interleukin 6 in response to IgE-Ag. The inhibition of exocytosis in the PKCbeta-deficient mast cells occurred whether the stimuli were due to the aggregation of the mast cell surface FcepsilonRI or to the calcium ionophore, ionomycin. However, no significant changes were observed in the proliferative response of the mast cells to interleukin 3 (IL-3) or in their apoptotic rate after IL-3 depletion. (Blood. 2000;95:1752-1757) (+info)
A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation.
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Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma 2-CH gamma 3-15aa linker-CH epsilon 2-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa D dimer that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases. (+info)
New farnesane-type sesquiterpenes, hedychiols A and B 8,9-diacetate, and inhibitors of degranulation in RBL-2H3 cells from the rhizome of Hedychium coronarium.
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Two new farnesane-type sesquiterpenes, hedychiols A and B 8,9-diacetate, were isolated from the methanolic extract of the fresh rhizome of Hedychium coronarium KOEN. cultivated in Japan. Their stereostructures were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of isolated constituents on the release of beta-hexosaminidase from RBL-2H3 cells were examined, and hedychilactone A and coronarin D were found to show the inhibitory activity. (+info)
Flow cytometry versus histamine release analysis of in vitro basophil degranulation in allergy to Hymenoptera venom.
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BACKGROUND: Flow cytometry (FCM) has been proposed for specific allergy in vitro testing. We investigated its biological significance for allergy to Hymenoptera venoms and compared it with the routinely performed basophil histamine release test (HRT). METHODS: Blood samples from 26 allergic and 8 nonallergic donors were incubated with venom at serial concentrations. Basophils were analyzed with anti-CD45-PE-Cyanin 5, Anti-IgE-FITC, and Anti-CD63-Phycoerythrine. HRT was measured by radioimmunoassay. RESULTS: FCM was as convenient as HRT for measuring basophil reactivity in at least 87% of allergic and 75% of nonallergic subjects. CD63 outer expression was specifically induced in 91% of releaser subjects (86% on HRT) and in 1 of 10 tests in nonallergic donors, or one of six tests (16% on HRT) in allergic patients tested with an irrelevant allergen. Both methods were concordant in 85.7% of the tests. The three discordant patients had low-grade reactions and borderline biological responses on FCM (n = 2) or HRT (n = 1). CONCLUSIONS: The dynamic, physiologic significance of CD63, the dose-response curve, and dependency on ethylene-diaminetetra acetic acid suggested that both tests reflect the same mechanism. (+info)
Flow-assisted quantification of in vitro activated basophils in the diagnosis of wasp venom allergy and follow-up of wasp venom immunotherapy.
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BACKGROUND: Correct identification of the culprit venom is a prerequisite for specific venom immunotherapy (VIT). Despite the efficacy of VIT, issues as how to monitor treatment and when to discontinue maintenance therapy remain to be established. METHODS: To evaluate diagnostic performances of the basophil activation test (BAT) in wasp venom allergy, 80 patients with a definite history of wasp venom anaphylaxis (systemic reactors) and 14 wasp-stung asymptomatic controls (stung controls) were enrolled. Venom-induced basophil activation was analyzed flow cytometrically by double-labeling with anti-IgE and anti-CD63. Results were compared to wasp IgE levels and results of a venom skin test (VST). To establish whether the BAT constitutes a candidate marker to monitor VIT, the BAT was repeated in 22 patients on the 5th day of a build-up course and after 6 months of maintenance VIT. Whether the BAT could contribute in the decision of discontinuing VIT was assessed in a cross-sectional analysis in 30 patients receiving treatment for 3 years. RESULTS: Comparison between systemic reactors and stung controls revealed a sensitivity of 86.4% and specificity of 100% for venom IgE, and sensitivity of 81.8% for VST, respectively. In contrast to stung controls, patients demonstrated dose-dependent venom-induced basophil activation. The BAT attained a sensitivity of 83.8% and specificity of 100%. At the end of the build-up course, no effect of VIT on the BAT was demonstrable. When the BAT was repeated after 6 months of treatment, submaximal stimulation of the cells demonstrated a significant decreased CD63 expression (P < 0.04). Patients having VIT for 3 years also demonstrated significantly lower venom-induced CD63 expression (P < 0.001). After 3 years, 60% of the patients had a negative BAT for submaximal stimulation of the cells whereas only 17.9% of the patients had negativation of wasp IgE. CONCLUSIONS: The BAT is a reliable instrument for the diagnosis of wasp venom anaphylaxis and might constitute an instrument to monitor wasp VIT. (+info)
Diclofenac induces basophil degranulation without increasing CD63 expression in sensitive patients.
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Diclofenac (Dc) induces an IgE-independent basophil (Ba) degranulation in susceptible individuals. CD63 Ba expression is utilized as an in vitro test for diagnosis of drug hypersensitivity. We tested the ability of Dc to induce CD63 Ba expression by flow cytometry (BAT) and Ba degranulation using light microscopy (HBDT) in patients sensitive to Dc. We studied 14 patients with diclofenac hypersensitivity, also two patients sensitive to Dermatophagoides pteronyssinus (Dp), and 12 normal controls. HBDT was performed by mononuclear cells toluidine blue staining. BAT determined CD63 expression in antiCD63/anti-IgE/anti-CD45-labelled whole blood. In each case, the percentage of activated Ba post-stimulation with 1 and 10 microg/ml Dc was determined. Positive controls included N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptide-induced activation. IgE-mediated Ba activation was induced with a Dp allergenic extract. With Dc 1 microg/ml, mean HBDT in Dc-susceptible individuals was 33.62 +/- 18.35% and 8.49 +/- 4.79% in controls (P = 0.0001). Mean BAT was 2.04 +/- 1.68% and 1.93 +/- 1.40% in controls (P = 0.8). Ba preincubation with Dc did not affect fMLP-induced CD63 expression, neither in Dc-sensitive individuals (P = 0.8) (n = 4) nor in subjects without Dc hypersensitivity (P = 0.25) (n = 4). Ba from the two patients sensitive both to Dc and Dp responded to Dp but not to Dc by BAT: Dc, 1.99 +/- 0.78%; Dp: 60.87 +/- 9.28%; but showed degranulation by HBDT: Dc, 30.53 +/- 1.02%, Dp: 48.78 +/- 22.17%. Dc induces Ba degranulation in sensitive patients in a way that does not induce CD63 expression and is different from IgE-mediated and fMLP-mediated degranulation. Our results suggest that CD63 expression is not a reliable diagnostic method for diclofenac allergy. (+info)
Hymenoptera venom allergy: taking the sting out of difficult cases.
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BACKGROUND: Correct identification of the culprit venom is a prerequisite for specific venom immunotherapy. OBJECTIVE: To assess whether the basophil activation test (BAT) constitutes an additional diagnostic instrument in patients with equivocal or negative specific immunoglobulin (Ig) E or venom skin test (VST) results. METHODS: One hundred eighteen patients with a compelling history of IgE-mediated hymenoptera venom allergy were enrolled. Venom-specific IgE was quantified by ImmunoCAP and VST was performed in all patients. Basophil activation was analyzed by flow cytometry after labeling with anti-IgE and anti-CD63. RESULTS: In 64 out of 118 patients, diagnosis was considered as definite and the entomologic description was confirmed by unequivocal and concordant positive specific IgE and VST results. In 53 of those 64 patients, BAT confirmed diagnosis, whereas the remaining 11 patients were nonresponsive in the BAT analysis. Forty-seven patients (40%) had a tentative diagnosis of venom allergy, as they had divergent specific IgE or VST results. In 31 of those patients, BAT was positive only for the suspected venom and helped to establish diagnosis of wasp and honeybee venom allergy in 28 and 3 patients, respectively. BAT was diagnostic in 7 patients with complete negative results for specific IgE and VST, despite clear entomologic identification. CONCLUSIONS: In about half the patients with diagnosis of venom allergy, unequivocal specific IgE and VST results are obtained and additional tests are not needed. In the remainder, diagnosis is less straightforward due to discrepant or negative specific IgE orVST results. In these patients, BAT constitutes a helpful additional instrument to identify the culprit venom and start venom immunotherapy accordingly. (+info)
Chronic idiopathic urticaria: comparison of clinical features with positive autologous serum skin test.
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BACKGROUND: Chronic idiopathic urticaria (CIU), in its extremely severe form, can pose a therapeutic challenge to the treating physician. It has been noted that in one third of such patients, autoantibodies against the IgE receptor are seen and such patients have more severe and unremitting urticaria. AIM: To compare clinical features of autoimmune urticaria with those of other CIU patients. METHODS: We conducted a prospective study in an attempt to correlate the clinical features with autoantibodies, indirectly detected via the autologous serum skin test (ASST), which is the simplest and the best in vivo clinical test for detection of basophil histamine-releasing activity. DISCUSSION: Out of 100 patients with chronic idiopathic urticaria, 34 showed a positive reaction to the autologous serum skin test and it was found that the frequency and severity of attacks was higher in these patients. CONCLUSION: ASST may be used as a simple and cost-effective test for the classification of chronic urticaria, which has proven to be a therapeutic challenge to the treating physician. (+info)