Liquid Chromatography Tandem Mass Spectrometry Based Label-Free Quantification Method for Assessment of Allergen-Induced Anaphylactoid Reactions.

Mast cells are essential in mediating inflammatory processes. When activated, mast cells can rapidly release characteristic granules and various mediators into the interstitium. Tryptase (TPS) and ß-hexosaminidase (HEXB) are typical protease mediators stored in granules and released upon activation. They have been recognized as important biomarkers of anaphylaxis, and the released level is associated with the severity of allergic reactions. In this study, a sensitive, accurate, and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneously quantifying the two biomarkers was developed and validated in LAD2 cell culture supernatant, and P14R was used as internal standard. Good linearity was observed in the range of 50-2500 ng/mL for TPS and 10-2000 ng/mL for HEXB both with R2 > 0.99. The matrix effect and recovery were both within acceptable limits. We quantified TPS and HEXB released from Laboratory of Allergic Disease 2 (LAD2) mast cells treated with several potential allergens, and the results demonstrate that the method can be used to investigate TPS and HEXB levels in LAD2 mast cell model during allergy research. We anticipate our approach to be a robust and sensitive assessment method for more biomarkers with similar kinetics characteristics and to be a major tool of allergic drug assessment or antiallergic drug development in research.

Detection of low-molecular-weight mast cell-activating factors in serum from patients with chronic spontaneous urticaria

Background: Functionally active autoantibodies to IgE and to the high-affinity IgE receptor (FcεRI) can be detected in serum in about 40% of patients with chronic spontaneous urticaria (CSU). Recent studies showed that serum from patients with CSU can induce activation of mast cells, irrespective of whether they carry high-affinity IgE receptors. Objective: To evaluate mast cell activation induced by factors in the serum of CSU patients with a molecular weight lower than that of autoantibodies. Methods: Eight CSU patients and 5 healthy controls were evaluated. Whole serum and serum fractionated at 100, 50, and 30 kDa were used to stimulate in vitro LAD2 mast cells. The enzymatic activity of β-hexosaminidase was evaluated in supernatants and cell pellets as a measure of mast cell degranulation. Results: Mean (SEM) release of mast cell β-hexosaminidase induced by whole serum from CSU patients was higher than that induced by serum from the healthy controls (14.4 [2.7%] vs 5.1 [2.4%]; P =.027). In addition, serum fractions below 100 kDa and below 50 kDa from CSU patients induced mast cell degranulation that was significantly higher than that induced by the corresponding fractions in sera from healthy controls (10.2% [1.4%] vs 3.8% [1.9%] [P=.024] and 10.1% [1.2%] vs 3.9% [1.7%] [P=.012], respectively). In 4 CSU patients, we evaluated serum fractions <30 kDa, which retained their capacity to activate mast cells (11.0% [0.7%]). Conclusions: This study shows that sera from CSU patients may contain low-molecular-weight mast cell-activating factors other than autoantibodies. These factors could be an additional mechanism contributing to the pathogenesis of CSU (AU)

Introducción: Los autoanticuerpos IgE funcionalmente activos y los receptores de alta afinidad para la IgE (FcεRI) pueden ser detectados n el suero de aproximadamente un 40% de los pacientes con urticaria crónica espontánea (UCE). Estudios recientes muestran que el uero de estos pacientes puede inducir activación de mastocitos con o sin receptores de alta afinidad para la IgE. Objetivo: El objetivo de este estudio fue evaluar la actividad de los factores séricos de los sueros de pacientes con UCE con un peso molecular inferior al de los autoanticuerpos. Métodos: Para ello se estudiaron 8 pacientes con UCE y 5 controles sanos. El suero completo de cada uno de ellos y el fraccionado a 100,50 y 30 kDA se utilizó para estimular in vitro mastocitos LAD2. La actividad enzimática de la β-hexosaminidasa se determinó en los sobrenadantes y en el botón celular con el fin de cuantificar la degranulación mastocitaria. Resultados: En cuanto a los resultados obtenidos se observó una liberación de β-hexosaminidasa mastocitaria inducida por los sueros completos de los pacientes con UCE (14,4±2,7%, media ± EE de la media) significativamente mayor que la inducida por sueros de controles sanos (5,1±2,4%; p=0,027). Así mismo, las fracciones séricas inferiores a 100 kDa e inferiores a 50 kDa de los pacientes con UCE indujeron degranulación mastocitaria significativamente superior a la inducida por las fracciones correspondientes de sueros controles (10,2±1,4% vs 3,8±1,9% [p=0,024] and 10,1±1,2% vs 3,9±1,7% [p=0,012], respectivamente). En 4 pacientes con UCE observamos que las fracciones inferiores a 30 kDa mantenían la capacidad de activar a los mastocitos (11,0±0,7%). Conclusiones: En conclusión, este estudio muestra que el suero de los pacientes con UCE puede contener factores de bajo peso molecular diferentes a los autoanticuerpos que son capaces de activar a los mastocitos. Este hallazgo podría contribuir a conocer los mecanismos de la patogénesis de la UCE (AU)