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Rate of Negativization of the Basophil Activation Test (BASOTEST) and RAST in Patients with Immediate Allergic Reactions to Amoxicillin
Antigen-Induced Production of S1P Leads to Cell Migration Independently of S1P Receptor in RBL-2H3 Cells I. JUNG, O. H. CHOI; JHAAC, Johns Hopkins University, Baltimore, MD. RATIONALE: Cross-linking of Fc RI has been shown to cause cell migration by producing sphingosine-1-phosphate (S1P). The present study examined whether S1P produced by antigen stimulation would act intracellularly or extracellularly via S1P receptors in RBL-2H3 cells. METHODS: Chemotaxis was measured in a modified Boyden chamber using polycarbonate filters. Antigen (10 pg/ml) or S1P (10 nM) was added to the lower chamber and cells (5 104) were added to the upper chamber. Migrated cells were counted after 4 hr incubation. Chemotaxis was also measured in RBL-2H3 cells overexpressing sphingosine kinase1 (SK1), SK1 with an N-terminal extension (long-SK1) and kinase-inactive SK1. RESULTS: To determine the mechanism by which antigen causes cell migration, antigen- or S1P-induced cell migration was measured in the presence or absence of an SK inhibitor dimethylsphingosine (DMS) or a Gi protein inhibitor pertussis toxin (PTX). Antigen-induced cell migration was completely inhibited by DMS but not by PTX, while S1P-induced migration was completely inhibited by PTX but not by DMS. Furthermore, overexpression of SK1 or long-SK1 increased baseline migration, but only long-SK1 affected antigen-induced migration. As expected, antigen-induced cell migration was inhibited by overexpression of kinaseinactive long-SK1. CONCLUSIONS: These results suggested that antigen-induced migration was absolutely dependent on the production of S1P but independent of S1P receptor. Conversely, S1P-induced migration was dependent on S1P receptors but not the production of S1P. Funding: National Institutes of Health
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Analysis of Global Protein Content in Mast Cell Exosomes
K. Ekström, H. Valadi, N. Almqvist, A. Bossios, M. Sjöstrand, J. Lötvall; Lung Pharmacology Group. Dept. of Resp. Med and Allergol, Göteborg, SWEDEN. RATIONALE: Exosomes are small membrane vesicles of endocytic origin. They are secreted from a variety of cells and display markers and functional properties from the cell of origin. Mast cell derived exosomes may play an important role in regulating allergic inflammation. The aim of this study was to identify the protein content of these exosomes and compare it with the proteins in exosomes from other sources. METHODS: Exosomes released from the murine mast cell line MC/9 were isolated and detected using electron microscopy (EM). Exosomal proteins were extracted and collected in a SDS-PAGE gel. Total proteins were cut and purified from the gel. The proteins were trypsinated and analysed using the nanoflow LC-MS/MS followed by MASCOT program to identify proteins. RESULTS: Exosomes could be detected using EM. More than 100 proteins were identified using nanoflow LC-MS/MS. These include cytosolic proteins such as heat-shock proteins, annexines, cytoskeleton proteins and membrane-bound proteins such as CD63, CD54, CD43 and MHCI. CONCLUSIONS: The protein content of mast cell-derived exosomes, identified by nanoflow LC-MS/MS, were compared with other exosomal proteins. A number of universal exosomal proteins were identified in addition to a number of mast cell exosome specific proteins. Funding: Swedish Heart-Lung Foundation, Vårdal Foundation and the Swedish MRC. Carboxypeptidase as a Marker of Mast Cell Heterogeneity in Human Tissues M. G. Buckley, S. He, Y. He, S. Goda, J. Gelnar, A. F. Walls; Immunopharmacology Group, University of Southampton, Southampton, UNITED KINGDOM.
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RATIONALE: Mast cell subpopulations have frequently been categorized according to the expression of the serine proteases tryptase and chymase. Carboxypeptidase is established as a major constituent of mast cell secretory granules, but the distribution of this protease in human mast cell populations has been little examined. METHODS: A series of monoclonal antibodies were prepared against mast cell carboxypeptidase purified from human tissues, and employed in immunohistochemistry to examine the distribution of this protease in two conditions associated with mast cell infiltration of the affected tissues: bronchial asthma and osteoarthritis. RESULTS: The new antibodies reacted in ELISA and in immunoblotting with carboxypeptidase purified from human skin or from recombinant expression systems. There was selective binding to mast cells in immunohistochemistry with a range of tissues. Immunohistochemistry with double labeling or the application of camera lucida techniques with sequentially cut thin sections indicated that cells staining for carboxypeptidase were generally stained also for tryptase and for chymase. Cells staining for carboxypeptidase represented a small proportion of those staining for tryptase in the bronchial mucosa and there was little difference in numbers between asthmatic and non-asthmatic subjects. In non-diseased synovial tissues, some 20% of mast cells expressed both of these proteases, but in tissues from cases of osteoarthritis the proportion was much higher (80%; p<0.009). Relative numbers of carboxypeptidase and chymase-staining cells were similar in all tissues examined. CONCLUSIONS: Mast cell carboxypeptidase appears to be restricted to the chymase-containing subset of human tissues in health and disease. Funding: Sir Jules Thorn Charitable Trust, London Law Trust, Thrasher Research Fund Rate of Negativization of the Basophil Activation Test (BASOTEST) and RAST in Patients with Immediate Allergic Reactions to Amoxicillin T. D. Fernandez1, M. J. Torres2, C. Mayorga1, E. Martin2, J. L. RodriguezBada1, C. Antunez1, N. Blanca2, J. A. Cornejo-Garcia1, M. Blanca2; 1Research Laboratory, Carlos Haya Hospital, Malaga, SPAIN, 2Allergy Service, Carlos Haya Hospital, Malaga, SPAIN. RATIONALE: The in vitro methods most used for diagnosing immediate allergic reactions to penicillins are the basophil activation test (BAT) and immunoassays (CAP and RAST). No studies of the rate of negativization of these techniques are available. METHODS: 41 patients with immediate allergic reactions to amoxicillin, and with RAST and/or BAT positive, were followed up for four years at six month intervals. RESULTS: Survival analysis (Kaplan-Meier test) showed differences in the rate of negativization between RAST and BAT (p=0.0167), with BAT becoming negative sooner. The same comparison, but depending on the hapten used, only showed differences for amoxicillin (p=0.05). We also performed this analysis for both tests depending on the different clinical manifestations, and only found differences in the negativization rate in patients with urticaria (p=0.0035). In order to analyze the role of skin testing in the maintenance of the in vitro response, we compared those patients who were skin tested during the study with those from whom blood samples were obtained. Differences were found in the RAST (p=0.0676), with a faster negativization in those patients who did not have a skin test. CONCLUSIONS: The rate of negativization of both in vitro tests differs, and is faster for BAT. These differences are mostly detected when amoxicillin is the hapten used and in those patients with urticaria. Contact with penicillins, even just a small amount as used in skin tests, can influence the rate of negativization, although only in the RAST assay ~ola de Alergologia e Funding: FIS and Fundacion Sociedad Espan inmunologia Clinica
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Abstracts S69
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2
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Analysis of Global Protein Content in Mast Cell Exosomes
K. Ekström, H. Valadi, N. Almqvist, A. Bossios, M. Sjöstrand, J. Lötvall; Lung Pharmacology Group. Dept. of Resp. Med and Allergol, Göteborg, SWEDEN. RATIONALE: Exosomes are small membrane vesicles of endocytic origin. They are secreted from a variety of cells and display markers and functional properties from the cell of origin. Mast cell derived exosomes may play an important role in regulating allergic inflammation. The aim of this study was to identify the protein content of these exosomes and compare it with the proteins in exosomes from other sources. METHODS: Exosomes released from the murine mast cell line MC/9 were isolated and detected using electron microscopy (EM). Exosomal proteins were extracted and collected in a SDS-PAGE gel. Total proteins were cut and purified from the gel. The proteins were trypsinated and analysed using the nanoflow LC-MS/MS followed by MASCOT program to identify proteins. RESULTS: Exosomes could be detected using EM. More than 100 proteins were identified using nanoflow LC-MS/MS. These include cytosolic proteins such as heat-shock proteins, annexines, cytoskeleton proteins and membrane-bound proteins such as CD63, CD54, CD43 and MHCI. CONCLUSIONS: The protein content of mast cell-derived exosomes, identified by nanoflow LC-MS/MS, were compared with other exosomal proteins. A number of universal exosomal proteins were identified in addition to a number of mast cell exosome specific proteins. Funding: Swedish Heart-Lung Foundation, Vårdal Foundation and the Swedish MRC. Carboxypeptidase as a Marker of Mast Cell Heterogeneity in Human Tissues M. G. Buckley, S. He, Y. He, S. Goda, J. Gelnar, A. F. Walls; Immunopharmacology Group, University of Southampton, Southampton, UNITED KINGDOM.
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RATIONALE: Mast cell subpopulations have frequently been categorized according to the expression of the serine proteases tryptase and chymase. Carboxypeptidase is established as a major constituent of mast cell secretory granules, but the distribution of this protease in human mast cell populations has been little examined. METHODS: A series of monoclonal antibodies were prepared against mast cell carboxypeptidase purified from human tissues, and employed in immunohistochemistry to examine the distribution of this protease in two conditions associated with mast cell infiltration of the affected tissues: bronchial asthma and osteoarthritis. RESULTS: The new antibodies reacted in ELISA and in immunoblotting with carboxypeptidase purified from human skin or from recombinant expression systems. There was selective binding to mast cells in immunohistochemistry with a range of tissues. Immunohistochemistry with double labeling or the application of camera lucida techniques with sequentially cut thin sections indicated that cells staining for carboxypeptidase were generally stained also for tryptase and for chymase. Cells staining for carboxypeptidase represented a small proportion of those staining for tryptase in the bronchial mucosa and there was little difference in numbers between asthmatic and non-asthmatic subjects. In non-diseased synovial tissues, some 20% of mast cells expressed both of these proteases, but in tissues from cases of osteoarthritis the proportion was much higher (80%; p<0.009). Relative numbers of carboxypeptidase and chymase-staining cells were similar in all tissues examined. CONCLUSIONS: Mast cell carboxypeptidase appears to be restricted to the chymase-containing subset of human tissues in health and disease. Funding: Sir Jules Thorn Charitable Trust, London Law Trust, Thrasher Research Fund Rate of Negativization of the Basophil Activation Test (BASOTEST) and RAST in Patients with Immediate Allergic Reactions to Amoxicillin T. D. Fernandez1, M. J. Torres2, C. Mayorga1, E. Martin2, J. L. RodriguezBada1, C. Antunez1, N. Blanca2, J. A. Cornejo-Garcia1, M. Blanca2; 1Research Laboratory, Carlos Haya Hospital, Malaga, SPAIN, 2Allergy Service, Carlos Haya Hospital, Malaga, SPAIN. RATIONALE: The in vitro methods most used for diagnosing immediate allergic reactions to penicillins are the basophil activation test (BAT) and immunoassays (CAP and RAST). No studies of the rate of negativization of these techniques are available. METHODS: 41 patients with immediate allergic reactions to amoxicillin, and with RAST and/or BAT positive, were followed up for four years at six month intervals. RESULTS: Survival analysis (Kaplan-Meier test) showed differences in the rate of negativization between RAST and BAT (p=0.0167), with BAT becoming negative sooner. The same comparison, but depending on the hapten used, only showed differences for amoxicillin (p=0.05). We also performed this analysis for both tests depending on the different clinical manifestations, and only found differences in the negativization rate in patients with urticaria (p=0.0035). In order to analyze the role of skin testing in the maintenance of the in vitro response, we compared those patients who were skin tested during the study with those from whom blood samples were obtained. Differences were found in the RAST (p=0.0676), with a faster negativization in those patients who did not have a skin test. CONCLUSIONS: The rate of negativization of both in vitro tests differs, and is faster for BAT. These differences are mostly detected when amoxicillin is the hapten used and in those patients with urticaria. Contact with penicillins, even just a small amount as used in skin tests, can influence the rate of negativization, although only in the RAST assay ~ola de Alergologia e Funding: FIS and Fundacion Sociedad Espan inmunologia Clinica
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Abstracts S69
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2