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Title: Towards the prediction and diagnosis of drug hypersensitivity reactions
Author: Watkinson, Joel
ISNI:       0000 0004 9352 0071
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Adverse drug reactions (ADRs) are becoming an increasing burden for both the healthcare system and the pharmaceutical industry. Indeed, up to 6.5% of all hospital admissions are attributed to ADRs. Drug hypersensitivity reactions (DHRs) are of particular interest to us - ADRs with an immune aetiology - especially T cell mediated delayed type reactions. They are idiosyncratic in nature, and as such are not predictable based on the pharmacology of the drug. Genetic studies have elucidated associations between specific HLA alleles and DHR, but in most cases a reaction is not observed in the patient. In vivo drug challenge tests have been used to diagnose and predict the likelihood of an DHR, but are not always appropriate due to their invasive nature and inaccurate predictive capabilities. What is desperately needed in order to advance the field and prevent DHR are accurate, repeatable, and reliable in vitro tests for both the diagnosis and prediction of these reactions. Whilst dendritic cell T cell co-culture systems enable us to determine the ability of a compound to prime and activate naïve T cells, there are some problems that must be addressed. Existing assays do not take into account the precursor frequency of naïve T cells for a specific drug. Thus, naïve T cell priming to certain drugs cannot be detected, as there not enough T cells to reach the precursor frequency required for activation. The assay is also too complicated, with cells being cultured and moved between plates multiple times for each assay. Additionally, only one donor at a time can be analysed. For this reason, within this thesis I have generated two new assays; the T cell multi-well assay (T-MWA), and the T cell multi-donor assay (T-MDA). Post-dendritic cell generation, the T-MDA and T-MWA take place in a single 96-well plate, miniaturising and streamlining the assay. The T-MDA allows for the comparison of up to 16 donors in a single assay, with positive responses being able to be examined in further detail by the T-MWA. In each assay nitroso sulfamethoxazole (SMX-NO) led to positive priming in all donors (either SI > 1.5 or p < 0.05 in MannWhitney statistical test), whilst the β-lactam antibiotic piperacillin led to positive priming in only one donor in each assay. Bandrowski's base (BB), an oxidation product of the contact allergen p-phenylenediamine led to positive priming in 4 out of 8 donors. The T-MWA allows for the analysis of 40 wells per drug treatment (opposed to 3), which accounts for the T cell precursor frequency associated to a drug reaction. The T-MWA was used to establish naïve T cell priming against SMX-NO, BB, piperacillin, and sulfamethoxazole (SMX). The greatest level of priming in terms of both statistical significance and the strength of response was observed to SMX-NO and BB as these regularly had good or strong responses (SI > 2) and dot plots of all wells had p < 0.05. Priming was still achieved to piperacillin and SMX, albeit with fewer responsive wells responding, with weaker levels of proliferation. This enables SMX-NO to be used as a positive control, and something by which in the future researchers can compare the immunogenicity of other drugs. Despite the success of the T-MDA and T-MWA, it was important to understand whether the assay recreates accurate immune regulation, and whether this would change dependent on the test drug and have a bearing on the observed results. T regulatory cells (Tregs) as well as programmed death-1 (PD-1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) all have immunosuppressive actions on the immune system and can determine whether or not T cell activation occurs. Tregs were generated during the course of priming in 3 out of 3 donors to both SMX-NO (Tregs < 1% of CD4+ goes to mean of 3% of CD4+ cells) and piperacillin (Tregs < 1% of CD4+ goes to mean of 2.5% of CD4+ cells); whilst PD-1 and CTLA-4 expression overall increased in all donors. Upon blockade of PD-1 and CTLA-4 in the T-MWA, the strength of the response increased, but negative priming was not reversed upon blockade of the co-inhibitory pathways due to increased background proliferation (P > 0.05). The lymphocyte transformation test (LTT) is the current gold standard of in vitro diagnostic assays, but its applicability domain is limited in vulnerable paediatric patient cohorts who often suffer DHR due to their extended exposure to multiple drugs. Out of 13 patient samples tested for drug specific PBMC proliferative responses: 8 of them resulted in negative results (p > 0.05), despite the patient presenting with suspected DHR from clinical observation. Four resulted in a single potential culprit drug being positive in LTT, whilst 1 flagged multiple potential culprit drugs. This makes determining the correct drug to withdraw extremely difficult in cases of multiple drug treatment. Additionally, the LTT takes 6 days to complete, which is too long for a diagnostic assay. miRNA are known to regulate a range of biological processes and regulate expression of up to 50% of genes. They are also known to play a regulatory role in the immune system and may determine whether or not activation occurs. miR 9, 18, 21, and 155 are known to be upregulated upon immune activation. We used a miR upregulation assay whereby upregulation of these known miR could be quantified and correlated with T cell activation in 3 days instead of 6. miR 9, 18, 21, and 155 were differentially upregulated in antigen specific T cell clones and patient PBMC all of which were positive by LTT or ELISpot, proving the concept that miR upregulation could be used in the future to determine T cell activation more rapidly than the LTT. These studies were successful in bringing about improvements to in vitro assays such as the naïve T cell priming assay and LTT. However, further studies into the immune regulatory pathways involved at the cellular and transcriptional level will be the key to improving and therefore moving towards truly dependable predictive and diagnostic in vitro assays for DHR.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral