Investigation of sensitization mechanisms and recombinant allergen-based diagnosis for American cockroach allergy

• Main Authors: Szu-Wei Liu, 劉思偉
• Other Authors: Nancy M. Wang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/78943930853478440489

碩士 === 國立彰化師範大學 === 生物技術研究所 === 97 === Cockroaches are known to produce potent aeroallergens which elicit IgE-mediated allergy and strongly associated with asthma. Traditional methods of cockroach allergy diagnosis and treatment are based on the use of crude extracts containing wide variety of undesirable protein, which leaves rooms for improvability regarding the test sensitivity and specificity. In the Part I of this study, we would like to utilize recombinant proteins (rPer a 1.0104, 3.0203 and 7.0101) to replace cockroach crude extracts in clinical diagnosis and immunotherapy. A total of 118 sera from cockroach-allergic patients were collected and analyzed for their specific IgE against rPer a 1.0104, 3.0203 and 7..0101 IgE ELISA results showed that the prevalence of IgE antibodies to rPer a 1.0104, 3.0203 and 7.0101 were 66.1%, 67.8% and 57.6%, respectively. Current data demonstrated that Per a 1.0104, 3.0203 and 7.0101are important P. americana major allergens that should be considered for clinical use. The use of component-resolved diagnostics may be useful to evaluate the allergen cocktails for immunotherapy by monitoring patient’s IgE directed to relevant allergens. Beside, the mechanisms underlying epithelial cell activation by American cockroach allergens (Cra A) are unclear. Interleukin (IL) -8 plays a critical role in the persistence of the inflammatory process in allergy. In the Part II, we aimed to investigate the mechanism by which Cra A triggers IL-8 released from human airway epithelial cells. Firstly, we determined the effects of Cra A on IL-8 expression and secretion in A549 cells. Cra A stimulated the production of IL-8 at protein and mRNA levels with dose- and time-dependent manners. Secondly, the effect of Cra A on IL-8 release was blocked by serine-protease inhibitors, PMSF and aprotinin; while it could not be suppressed by cysteine- or aspartic protease inhibitors. It demonstrated that serine protease activities were found in the extract of American cockroach. Thirdly, A549 cells expressed all four protease-activated receptors (PARs) at mRNA levels as assessed by real-time PCR. When epithelial cells were stimulated with Cra A, PAR-2 and PAR-3 mRNAs reached up to 1.73 and 1.94-folds increase than nonstimulated cells. Finally, we investigated the role of mitogen-activated protein kinases (MAPK) from PAR-2 or PAR-3 activation in IL-8 synthesis. Western blotting showed that an increase in ERK1/2 and JNK phosphorylation after Cra A stimulation. Furthermore, when A549 cells were preincubated with U0126 (ERK 1/2 kinase inhibitor) or SP600125 (JNK kinase inhibitor), it resulted in a reduction 100% and 44% of IL-8 production, respectively. We conclude that Cra A induced IL-8 release in airway epithelial cells and this is dependent on activation of PAR-2 and-3, and coordinating with the ERK1/2 and JNK signaling pathways.