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A life in allergy research: from bench to bedside
thesisposted on 2023-09-01, 14:35 authored by John Deighton
Background: Allergic diseases are some of the most common and significant causes of illness in developed countries. Allergen specific immunotherapy (SIT) has been developed as a novel treatment approach for allergic diseases based on administration of increasing doses of the causative allergen. One of the essential requirements for SIT, particularly in the areas of venom and peanut allergy, is the standardisation and characterisation of the allergen source, which can then be used not only in the development of accurate and sensitive assays to measure patient immunoglobulin and cytokine responses in-vivo but in clinical treatment as well. Methods: Wasp venom was collected from nest sites using electrical stimulation. The strength of the major enzymes present, hyaluronidase (Hyal) and phospholipase A1 (PLA1), were tested using lytic assays that I developed. In addition, several different biological immunoassays were also used to test each venom batch collected, one of which of was the assay I devised to measure the radioallergosorbent test (RAST) inhibition. Final confirmation of the strength of the venom pools was established by comparing the biological efficacy of the collected venom solutions in comparison to that currently in use in the clinic by skin testing on healthy volunteers. Enzymelinked- immunosorbent assays (ELISA) could then be developed measuring the in-vitro cytokine responses to bee and wasp venom in culture. The preparation of an immunoreactive crude peanut extract (CPE) containing all the major allergens was essential before immunoglobulin responses to peanut antigens in patients could be measured using a fully quantified ELISA. CPE could also be used in other in-vitro experiments. In particular, the elucidation of patient response to treatment both in the laboratory and in the clinical research facility. I achieved preparation of CPE by the extraction of the allergens from crushed peanuts followed by dialysis and lastly lyophilisation; gel electrophoresis and an ELISA, which measured immunoreactivity, confirmed this. Results: The development of these assays was vital in characterising the amount of allergen in the initial sample and the amount of antibody (or cytokine) produced by patients with bee and wasp allergies. Using these methods, our research team was able to demonstrate that the cytokine response to bee and wasp immunotherapy when successful resulted in a change in T-cell phenotype from a Th-2 response (high levels of the cytokine IL-4) to a Th-1 response (high levels of interferon-gamma). Peanut oral immunotherapy (POIT) is more complex, but preparation of the crude peanut extract was an essential starting point for most of what followed in both the in-vitro and in-vivo areas. Conclusions: The assays I developed in the laboratory were instrumental in allowing sensitive accurate and reproducible measurement of patient responses, which would not have been possible without full initial allergen standardisation and characterisation. This has ultimately led to better treatment for patients with venom allergy and was fundamental in the establishment of the first centre in the UK offering an effective treatment for peanut allergy.
InstitutionAnglia Ruskin University
- Accepted version