Sun, Shanshan (2014) Evaluation of IgE and non-IgE mediated inflammatory pathways generated through exposure to prawn proteins. PhD thesis, James Cook University.

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Abstract

The purpose of this study was to evaluate the role of two major allergic pathways — IgE and non-IgE antibody mediated food allergy reaction (FAR) and lung inflammation disease using crustacean extracts. Although IgE antibody mediated food allergy reaction (IFAR) are defined as major mechanism in the past several decades, proteases from different sources acting similar to allergens as well as the long-term inflammatory stimulus through activation of protease activation receptors (PARs) are important. This opens new insight into non-IgE antibody mediated food allergy (NFAR) due to its dual effects. Therefore, it is necessary to investigate the interactions of these two pathways for a better understanding of the currently poor correlation between clinical symptoms and IgE specific diagnostics.

The current studies employed a humanized rat basophilic leukemia (RBL) cell line as well as a human lung epithelial cell line (A549) due to the presence of IgE receptors and PARs. Flow cytometric (FACS) and real time PCR (qPCR) were applied to analyze two types of receptors expression. Up-regulation of different cytokines was quantified by ELISA and qPCR. Allergens identification and signalling proteins detection were performed by western blotting. Protease identification was determined by zymography and mass spectrometry (MS) analysis. The purification of the identified trypsin, was performed by HPLC (C18 column). The trypsin DNA sequence was established by designed primers based on multiple alignments of published crustacean trypsin, obtained from GeneBank by homology search. The computer generated structure model of Black tiger prawn (Penaeus monodon) trypsin (PT) was calculated with the program Swiss-Model.

This novel PT was characterised as a highly autoproteolytic resistant enzyme. The RBL cell line was established as a functional model to examine IgE responses to crustacean allergens, however high human IgG antibody titers and intrinsic enzyme activity from sample extracts, limited the application of this system. Nevertheless, the presence of PARs was for the first time described, and in addition the enhanced PAR2 expression by IgE/anti-IgE. Furthermore, IL4/IL13 mRNA levels were dramatically upregulated through the IgE/anti-IgE complex or PT. Furthermore, PT stimulation of A549 cells, co-cultured with IL4/IL13, boosted surprisingly PAR2 and PAR4 expressions in addition to nine different cytokines. MAPK phosphorylation was detected in both stimulated cell lines.

This is the first study to demonstrate that IgE mediated activation can improve PARs expression. Furthermore, different cytokines upregulated through PARs activation (non-IgE mediated) may also regulate IgE production in sensitised allergic subjects. Therefore, PARs seems to be a crucial link in IgE mediated and non-IgE mediated food allergic reactions.

Item ID:	40729
Item Type:	Thesis (PhD)
Keywords:	allergy; anti-immunoglobulin E autoantibodies; cysteine proteinases; food allergy; IgE; immunoglobulin E; immunology; inflammation; inflammatory; non-IgE; prawn allergy; prawns; proteases; seafood allergy; serine proteinases; shellfish allergy; shrimp allergy; shrimps; trypsin
Date Deposited:	14 Oct 2015 02:17
FoR Codes:	11 MEDICAL AND HEALTH SCIENCES > 1107 Immunology > 110701 Allergy @ 100%
SEO Codes:	97 EXPANDING KNOWLEDGE > 970111 Expanding Knowledge in the Medical and Health Sciences @ 30% 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 70%
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