Kalli, Marina
(2021)
Use of humanised reporter cell lines for high-throughput detection of allergic sensitization.
PhD thesis, University of Nottingham.
Abstract
The prevalence of allergic diseases is growing rapidly in Europe. Today, it is estimated that more than 150 million individuals living in EU suffer from chronic allergic diseases, half of whom are underdiagnosed or poorly managed due to lack of awareness and shortage of medical specialists. Moreover, many patients do not report their symptoms or are not correctly diagnosed, while approximately 45% of the patients have never received an allergy diagnosis.
In vivo tests used for allergy diagnosis such as skin tests(e.g. SPT) and oral food challenge (OFC) may be uncomfortable for the patients, can pose a risk for anaphylaxis, and their results may be affected by the use of medications targeting the histamine receptor. On the other hand, in vitro tests are able to overcome most of these limitations. However, various in vitro methods used to detect allergen specific IgE (sIgE) in patients’ serum, such as the ImmunoCAP ISAC, do not always provide clinically relevant results. sIgE may not always be capable to crosslink the high-affinity receptor (FcεRI) in the presence of cognate antigen, an important mechanism that triggers mast cell and basophil degranulation and release of mediators responsible for most of the well-known allergy symptoms. In this project, we tested a new high-throughput in vitro method for allergy diagnosis using rat basophilic leukaemia (RBL) cell lines. After stable transfection of RBL cells expressing human FcεRIα with either FcεRIɣH or a nuclear factor of activated T cells (NFAT)-responsive intracellular fluorescence gene (DsRed), two reporter systems were generated: RBL NFAT-DsRed FCER1G and RBL SX-38 NFAT-DsRed-Express2, respectively. Transfection of RBL NFAT-DsRed with FcεRIɣH increased FcεRIαH chain expression levels, thus enhancing cell sensitivity compared to its parental cell line (NFAT-DsRed), albeit still not to a level comparable with RS-ATL8 expressing the highest level of FcεRIαH receptors/cell. Attempts to generate a more sensitive reporter system (the fluorescent equivalent of the RS-ATL8 luciferase reporter) were unsuccessful, as RBL SX-38 NFAT-DsRedExpress2 showed low levels of red fluorescence protein (RFP) expression after stimulation. Alternatively, using the RBL 703/21- NFAT-DsRed cells, generated previously in our lab, which express satisfactory levels of RFP after stimulation, we demonstrated a proof-of-concept high throughput reporter system with a potential to detect clinically relevant allergic sensitization. After assessing cell adhesion, printed-protein stability and cell activation onto three different polymer-treated surfaces, we showed that N-hydroxysuccinimide (NHS)-ester coated slides treated with fibronectin are a feasible proposition for further development of an allergy diagnosis system combining humanised RBL cells with printed allergens.
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