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The Effect of Omalizumab on Basophil and Mast Cell Responses using an Intranasal Cat Allergen Challenge
S196 Abstracts
751
The Effect of Omalizumab on Basophil and Mast Cell Responses using an Intranasal Cat Allergen Challenge J. A. Eckman, P. M. Sterba, D. Kelly, V. Alexander, B. S. Bochner, D. W. MacGlashan, Jr, S. S. Saini; Johns Hopkins Asthma and Allergy Center, Baltimore, MD. RATIONALE: Omalizumab treatment suppresses basophil FceRI expression faster than skin mast cells. We utilized omalizumab to elucidate the relative contributions of basophil versus mast cell activation in a nasal allergen challenge (NAC) model. METHODS: Eleven cat-allergic subjects were enrolled in a 3.5-month, double-blinded, randomized (3.5:1), placebo-controlled trial of omalizumab using standard allergic asthma dosing. Cat allergen was used for all study procedures. At baseline, subjects underwent NAC-1 with lavage for PGD2 measurement, skin prick test titration (SPTT), and blood sampling for basophil histamine release (BHR) and basophil IgE/IgE-receptor measurements. Basophil studies were repeated at day 3 and then weekly until cat allergen-induced BHR was <20% of baseline or until day 45. Baseline visit procedures were repeated after the BHR reduction (NAC2) and at the treatment period’s completion (NAC-3). RESULTS: In subjects with a >80% reduction in their basophil IgE receptors by day 10 (n 5 8), BHR to their optimal dose (0.1 or 1 BAU/mL) of cat allergen was significantly lower by NAC-2 compared to NAC-1 (71% decrease; p 5 0.03). Compared to NAC-1, these subjects demonstrated significant decreases in total sneeze counts (59% decrease, p 5 0.03) and combined nasal symptom scores (53% decrease, p 5 0.02) by NAC-2. In contrast, no significant (p > 0.05) shifts in SPTT or nasal lavage PGD2 were seen until NAC-3. Subjects without a significant change in their basophil IgE receptors (n 5 3) had no significant shifts in any outcome. CONCLUSIONS: Reduction in nasal symptom scores occurred when the basophil, but not mast cell, response was reduced. NAC responses appear partially basophil dependent.
752
MONDAY
Survey of Aspirin Administration in Systemic Mastocytosis J. H. Butterfield; Mayo Clinic, Rochester, MN. RATIONALE: Previous recommendations for aspirin administration in systemic mastocytosis (SM) have encompassed doses between 3.9 and 5.2 g/day, to achieve plasma salicylate levels between 20 to 30 mg/dl. These high levels of salicylate may not be tolerated by patients and moreover may not be necessary for control of prostaglandin (PG) D2 production. METHODS: Twenty SM patients who had been given aspirin were identified and their medical records were reviewed retrospectively for baseline tryptase levels, percentage of bone marrow involvement by SM and changes in the excretion of the PGD2 metabolite 11b-PGF2a and of N-methylhistamine in response to aspirin. RESULTS: In this series, 1 patient reported a prior adverse reaction to aspirin and required aspirin desensitization in hospital after developing a delayed reaction as an outpatient. A variety of initial aspirin doses and schedules were effectively used to administer and up-dose aspirin. In 20 of 20 patients with documented SM and elevated baseline urinary excretion of 11b-PGF2a aspirin therapy caused a reduction to normal in the excretion of this metabolite. There was no significant change in the excretion of Nmethylhistamine. Doses of aspirin required ranged from 81 mg twice daily to 500 mg twice daily. Improved clinical symptoms were reported in 14 of 20 patients given aspirin. CONCLUSIONS: The majority of SM patients does not experience untoward reactions to aspirin and can safely be given aspirin if their prior history is negative for aspirin or NSAID intolerance. Control of elevated PGD2 levels in SM can be achieved with lower doses of aspirin than previously reported as necessary in this disorder.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
753
RGS13 Controls G-Protein-Coupled Receptor-Evoked Responses of Human Mast Cells J. A. DiVietro, G. Bansal, H. S. Kuehn, A. M. Gilfillan, K. M. Druey; NIH, Bethesda, MD. RATIONALE: Since RGS13 is a regulator of GPCR signaling and is highly expressed in human mast cells we characterized how RGS13 affects GPCR-mediated functions of human mast cells. METHODS: RGS13 was depleted by specific siRNA or shRNA in two human mast cell lines, HMC-1 and LAD2. RGS13 was also overexpressed in HCM-1 cells. Ca21 mobilization, Akt phosphorylation, chemotaxis, cytokine secretion, and degranulation were then measured in response to different agonists. RESULTS: RGS13 depletion increased Ca21 mobilization in response to GPCR ligands adenosine, C5a, S1P, and CXCL12. Akt phosphorylation, chemotaxis, and IL-8 secretion induced by CXCL12 was also greater in shRGS13 HMC-1 cells compared to control, while overexpressing RGS13 decreased CXCL12 mediated Ca21 mobilization, Akt phosphorylation, and chemotaxis. Transient RGS13 knockdown in LAD2 cells led to increased degranulation to S1P but not to antigen/IgE or C3a. CONCLUSIONS: These results suggest that RGS13 restricts certain GPCR-mediated biological responses of human mast cells.
754
Stimulation of Human Mast Cells by Heterotypic Adhesion to T Cells Leads to Activation of Ras via. RasGRP1 A. Mor, I. Shefler, Y. A. Mekori; Meir Medical Center, Kfar Saba, Israel. RATIONALE: We have recently shown that T cells can activate human mast cells by heterotypic adhesion. This pattern of activation involves the MAPK system and results in the release of different cytokines. Since Ras proteins are the key upstream elements in the MAPK pathway, we aimed in this work to study the spatiotemporal pattern of Ras activation in human mast cells stimulated by activated T cells. METHODS: To study the localization of Ras we over-expressed the fluorescent probe for activated Ras, YFP-RBD (the Ras binding domain of Raf-1 fused with yellow fluorescent protein) in LAD2 human mast cells and imaged the cells after stimulation with activated Jurkat T cells. We also studied the localization of the guanine nucleotide exchange factors RasGRP1 and RasGRP4 under the same conditions. RESULTS: In resting cells the YFP-RBD probe was distributed homogeneously throughout the cytosol. Upon stimulation with activated T cells, the probe re-localized to the plasma membrane. RasGRP1, whose presence was essential for this process, followed the same pattern of translocation. CONCLUSIONS: Stimulation of human mast cells by heterotypic adhesion to T cells leads to activation of Ras via. RasGRP1.
751
The Effect of Omalizumab on Basophil and Mast Cell Responses using an Intranasal Cat Allergen Challenge J. A. Eckman, P. M. Sterba, D. Kelly, V. Alexander, B. S. Bochner, D. W. MacGlashan, Jr, S. S. Saini; Johns Hopkins Asthma and Allergy Center, Baltimore, MD. RATIONALE: Omalizumab treatment suppresses basophil FceRI expression faster than skin mast cells. We utilized omalizumab to elucidate the relative contributions of basophil versus mast cell activation in a nasal allergen challenge (NAC) model. METHODS: Eleven cat-allergic subjects were enrolled in a 3.5-month, double-blinded, randomized (3.5:1), placebo-controlled trial of omalizumab using standard allergic asthma dosing. Cat allergen was used for all study procedures. At baseline, subjects underwent NAC-1 with lavage for PGD2 measurement, skin prick test titration (SPTT), and blood sampling for basophil histamine release (BHR) and basophil IgE/IgE-receptor measurements. Basophil studies were repeated at day 3 and then weekly until cat allergen-induced BHR was <20% of baseline or until day 45. Baseline visit procedures were repeated after the BHR reduction (NAC2) and at the treatment period’s completion (NAC-3). RESULTS: In subjects with a >80% reduction in their basophil IgE receptors by day 10 (n 5 8), BHR to their optimal dose (0.1 or 1 BAU/mL) of cat allergen was significantly lower by NAC-2 compared to NAC-1 (71% decrease; p 5 0.03). Compared to NAC-1, these subjects demonstrated significant decreases in total sneeze counts (59% decrease, p 5 0.03) and combined nasal symptom scores (53% decrease, p 5 0.02) by NAC-2. In contrast, no significant (p > 0.05) shifts in SPTT or nasal lavage PGD2 were seen until NAC-3. Subjects without a significant change in their basophil IgE receptors (n 5 3) had no significant shifts in any outcome. CONCLUSIONS: Reduction in nasal symptom scores occurred when the basophil, but not mast cell, response was reduced. NAC responses appear partially basophil dependent.
752
MONDAY
Survey of Aspirin Administration in Systemic Mastocytosis J. H. Butterfield; Mayo Clinic, Rochester, MN. RATIONALE: Previous recommendations for aspirin administration in systemic mastocytosis (SM) have encompassed doses between 3.9 and 5.2 g/day, to achieve plasma salicylate levels between 20 to 30 mg/dl. These high levels of salicylate may not be tolerated by patients and moreover may not be necessary for control of prostaglandin (PG) D2 production. METHODS: Twenty SM patients who had been given aspirin were identified and their medical records were reviewed retrospectively for baseline tryptase levels, percentage of bone marrow involvement by SM and changes in the excretion of the PGD2 metabolite 11b-PGF2a and of N-methylhistamine in response to aspirin. RESULTS: In this series, 1 patient reported a prior adverse reaction to aspirin and required aspirin desensitization in hospital after developing a delayed reaction as an outpatient. A variety of initial aspirin doses and schedules were effectively used to administer and up-dose aspirin. In 20 of 20 patients with documented SM and elevated baseline urinary excretion of 11b-PGF2a aspirin therapy caused a reduction to normal in the excretion of this metabolite. There was no significant change in the excretion of Nmethylhistamine. Doses of aspirin required ranged from 81 mg twice daily to 500 mg twice daily. Improved clinical symptoms were reported in 14 of 20 patients given aspirin. CONCLUSIONS: The majority of SM patients does not experience untoward reactions to aspirin and can safely be given aspirin if their prior history is negative for aspirin or NSAID intolerance. Control of elevated PGD2 levels in SM can be achieved with lower doses of aspirin than previously reported as necessary in this disorder.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
753
RGS13 Controls G-Protein-Coupled Receptor-Evoked Responses of Human Mast Cells J. A. DiVietro, G. Bansal, H. S. Kuehn, A. M. Gilfillan, K. M. Druey; NIH, Bethesda, MD. RATIONALE: Since RGS13 is a regulator of GPCR signaling and is highly expressed in human mast cells we characterized how RGS13 affects GPCR-mediated functions of human mast cells. METHODS: RGS13 was depleted by specific siRNA or shRNA in two human mast cell lines, HMC-1 and LAD2. RGS13 was also overexpressed in HCM-1 cells. Ca21 mobilization, Akt phosphorylation, chemotaxis, cytokine secretion, and degranulation were then measured in response to different agonists. RESULTS: RGS13 depletion increased Ca21 mobilization in response to GPCR ligands adenosine, C5a, S1P, and CXCL12. Akt phosphorylation, chemotaxis, and IL-8 secretion induced by CXCL12 was also greater in shRGS13 HMC-1 cells compared to control, while overexpressing RGS13 decreased CXCL12 mediated Ca21 mobilization, Akt phosphorylation, and chemotaxis. Transient RGS13 knockdown in LAD2 cells led to increased degranulation to S1P but not to antigen/IgE or C3a. CONCLUSIONS: These results suggest that RGS13 restricts certain GPCR-mediated biological responses of human mast cells.
754
Stimulation of Human Mast Cells by Heterotypic Adhesion to T Cells Leads to Activation of Ras via. RasGRP1 A. Mor, I. Shefler, Y. A. Mekori; Meir Medical Center, Kfar Saba, Israel. RATIONALE: We have recently shown that T cells can activate human mast cells by heterotypic adhesion. This pattern of activation involves the MAPK system and results in the release of different cytokines. Since Ras proteins are the key upstream elements in the MAPK pathway, we aimed in this work to study the spatiotemporal pattern of Ras activation in human mast cells stimulated by activated T cells. METHODS: To study the localization of Ras we over-expressed the fluorescent probe for activated Ras, YFP-RBD (the Ras binding domain of Raf-1 fused with yellow fluorescent protein) in LAD2 human mast cells and imaged the cells after stimulation with activated Jurkat T cells. We also studied the localization of the guanine nucleotide exchange factors RasGRP1 and RasGRP4 under the same conditions. RESULTS: In resting cells the YFP-RBD probe was distributed homogeneously throughout the cytosol. Upon stimulation with activated T cells, the probe re-localized to the plasma membrane. RasGRP1, whose presence was essential for this process, followed the same pattern of translocation. CONCLUSIONS: Stimulation of human mast cells by heterotypic adhesion to T cells leads to activation of Ras via. RasGRP1.