Abstract

Between 12,000 to 17,000 people in the UK work with laboratory animals and are at risk of developing laboratory animal allergy (LAA), the major risk factor is exposure to laboratory animals (LA's). Though much research has been carried out, there is a lot we do not understand about LAA. In addition, in recent years there has been a shift away from the use of rats in open cages to mice in individually ventilated cages (IVCs). Studies suggest that this change has altered LA exposure patterns. As a result, past research has become outdated. SPIRAL (Safe Practice In Reducing Allergy in Laboratories) is a large cross-sectional study which investigated the risk of mouse work in modern research units. Between 2014 and 2017, 750 LA workers from seven UK research institutes were surveyed. Participants were tested for sensitisation to mice using a specific-IgE (S-IgE) assay and skin prick test (SPT). Participants also completed an online questionnaire and measurements of mouse allergen were collected from research facilities. Data from SPIRAL was used in this work. The thesis aim was to further the understanding of LAA immunology with an emphasis on diagnostic testing and the role of IgG4 in LAA. Identifying mouse-sensitised workers and reducing their exposure to mice is important for the prevention of disease progression. However, there is no gold-standard diagnostic test for sensitisation to mice, nor are there any recommendations on which mouse allergens/extracts should be used. In this work, common diagnostic tests for mouse sensitisation were compared: LA workers were tested for sensitisation to a commercial mouse epithelium SPT and for S-IgE to three ImmunoCAP mouse extracts; epithelium (e71); urine (e72); and a "combined" extract containing urine, epithelium and serum proteins (e88). 10.3% (n=62) of workers were sensitised to mice according to at least one diagnostic test (S-IgE assay and SPT). Of these, 32.3% (n=20) had a positive result for both tests (SPT and S-IgE to any ImmunoCAP extract), 64.5% (n=40) were positive for S-IgE to at least one of the ImmunoCAP extracts (e71, e72, e88) but had a negative SPT result, and two participants only had a positive SPT. Of the 60 workers positive for S-IgE to ImmunoCAP mouse extracts, the following were positive for S-IgE to the epithelium (e71, n=54), "combined" (e88, n=53) and urine (e72, n=42) extracts. The data indicated firstly that there was discordance between the two diagnostic tests, with the ImmunoCAP assay better than the SPT at detecting cases of sensitisation. Secondly, that results can vary depending on which ImmunoCAP extract is used. In support of this, western blots showed that (sensitised) SPIRAL participants had highly variable IgE-binding patterns to mouse allergens. In addition, three participants who had a negative SPT result but were positive for S-IgE to mouse epithelium (e71) had visible IgE-binding to mouse allergens: this suggested that additional cases of sensitisation detected by the ImmunoCAP assay were not false positives. Rodent specific IgG4 (S-IgG4) antibodies may have a role in tolerance to mouse allergy but study findings are inconsistent. SPIRAL participants were tested for mouse S-IgG4 and 28.2% (n=181) had detectable levels. Mouse S-IgG4 positive participants worked for significantly more years (8.62 years) with mice compared to IgG4 negative participants (5.38 years) (p<0.001), and technicians had significantly higher levels of S-IgG4 (7.00 AU/ml) compared to participants in other job roles (2.2 AU/ml) (p<0.001). Levels of mouse S-IgG4 were also higher in participants who were mouse-sensitised (p<0.001) and in those who reported one or more work-related symptoms (p<0.001). However, participants who were not sensitised to mice (p<0.001) and did not report work-related symptoms (p<0.001) had higher ratios of S-IgG4/IgE. The IgE-FAB assay is an in vitro model IgE-facilitated binding of allergen to CD23+ B cells. Inhibition of IgE-allergen binding to B cells was compared between two groups of SPIRAL participants: workers with the highest levels of mouse S-IgG4 (n=44) and workers who were negative for S-IgG4 (n=47). Workers with the highest levels of S-IgG4 inhibited IgE-allergen binding significantly more (72.6%) than S-IgG4 negative workers (14.1%) (p<0.001). Findings suggest that high exposures to mouse allergen may lead to the production of mouse S-IgG4 which through inhibition of allergic immune mechanisms may induce allergic tolerance. There is currently a need for a rapid, easy to use point-of-care (POC) test for mouse sensitisation. In this work, a lateral flow dipstick assay (LFDA) (precursor POC test) was developed and tested. The LFDA contained a test and control line. Preliminary data showed that the control line of the assay worked well, results also suggest that the test line of the assay was able to detect mouse specific IgE in serum. However, further work needs to be done to confirm these results and develop the assay further. The SPIRAL population was an accurate representation of LA workers across different research institutes, working practices and mouse exposures, which is the main strength of this work. The main limitation is that due to the limited volume of LA worker serum, assay repeats and experiment optimizations could not be repeated in some instances. Ideal future work would involve a longitudinal multicentred follow-up study of newly exposed LA workers; with periodic testing for mouse S-IgG4 and IgE; objective allergen exposure measurements; and real-time measures of physiological changes (i.e. lung function).

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