This suggests that in previous protocols allergen sensitisation was still ongoing during challenge and an increased period between the two was required for the generation of a full response. This modification restores the gap between sensitisation and challenge to the duration used Epigenetic Reader Domain inhibitor in this laboratory’s original sensitisation protocol (Lewis et al.,

1996) which had decreased with previous modifications (Smith & Broadley, 2007). Notwithstanding the reduced time between final sensitisation dose and challenge when increasing to 3 sensitisations, there was still a loss of allergic responses with protocol 1 compared to previous studies. The addition of a 3rd sensitisation injection on day 7 resulted in a further shortening of the sensitisation period to 8 days. 8 days between the final allergen sensitisation and challenge may not be enough time to produce a full immunological response, except when the sensitisation conditions are increased to a certain extent, as seen in guinea-pigs sensitised with an increased adjuvant

concentration. The late asthmatic response is associated with an influx of a range of inflammatory cells including eosinophils and T lymphocytes (Nabe et al., 2005). Eosinophilia is correlated with the magnitude of the LAR, both being significantly this website increased in humans and animal models following allergen challenge (Dente et al., 2008, Evans et al., 2012 and Gauvreau et al., 1999). Additionally, corticosteroids which reduce eosinophil and lymphocyte numbers also decrease the LAR (Kawayama et al., 2008 and Leigh et al., 2002). The present study demonstrated that increases in both eosinophils and lymphocytes coincided with the development of

a LAR, supporting a link between these parameters. Although we examined cellular influx at Thiamine-diphosphate kinase 24 h after Ova challenge and not at the peak of the LAR, our previous studies with earlier versions of this model have shown significant increases in neutrophils, macrophages and eosinophils at the time of the LAR (Danahay et al., 1999 and Toward and Broadley, 2004). However, not all results from this study support this relationship; eosinophils were also increased in protocols 1–4, which did not demonstrate a LAR. Studies in humans have also demonstrated similar results. Blocking OX40, a co-stimulator receptor important in generating allergic responses significantly attenuated eosinophilia with no effect on the LAR (Gauvreau et al., 2014). Additionally, older studies have demonstrated that anti-IL-5 therapy reduced eosinophilia but not AHR and the LAR in humans (Leckie et al., 2000). Overall, the role of eosinophils in the LAR remains uncertain. The investigation of factors such as the activation status of eosinophils may be more revealing than cell number.