, 2005 and Makwana Ferroptosis inhibitor et al., 2012). The latter, released from eosinophils, can damage the epithelium and expose underlying sensory nerves, increasing sensitivity to bronchoconstrictor stimuli like histamine (Homma et al., 2005). In the present study, lavage eosinophil numbers increased at 24 h, concomitant with the development of AHR. However, this relationship is not clear cut since the original Ova protocol used in this study (protocol 1) resulted in significant eosinophilia but with no AHR. Similarly, in other models and

humans, eosinophilia and AHR have been observed to be dissociated (Birrell et al., 2003 and Leckie et al., 2000). Cell counts can differ between lavage fluid and lung sections which could explain this result (Maestrelli et al., 1995). However, it was observed in this study that eosinophil numbers were moderately related between assessment methods, although tissue assessment seemed less likely to discern small changes. This suggests that the number of eosinophils may not

be important in AHR. It does not discount that some other factor such as eosinophil activation status could AG-14699 be more critical. The AHR observed in the present study can be assumed to be non-specific as previous studies with earlier version of our model have shown increases in sensitivity to a wide range of spasmogens (Spruntulis & Broadley, 1999). Allergen sensitisation begins with the uptake of antigen by antigen presenting cells (APCs) which process and present it to lymphocytes, which in turn undergo either apoptosis or activation (Hammad et al., 2010). Activation leads to the development of an allergic immune response. The extent of this response is dependent on the

sensitisation conditions. Increased immune stimulation during sensitisation results in increased lymphocyte priming and consequently stronger responses when the allergen is re-encountered. In the present study, cumulative modifications to the sensitisation conditions including increased number of injections, Ova concentration and Al(OH)3 concentration caused a progressive increase in total and eosinophil counts. Al(OH)3 enhances sensitisation to antigens via a variety of mechanisms including enhanced antigen uptake, T-cell proliferation, uric acid formation, inflammasome formation and promotion of Th2 ADP ribosylation factor type responses (Eisenbarth et al., 2002, Kool et al., 2008, Morefield et al., 2005 and Sokolovska et al., 2007). In accordance with this, increased Al(OH)3 concentration significantly increased lymphocyte influx and induced the development of a LAR, suggestive of enhanced sensitisation. Al(OH)3 produces these effects in a concentration-dependent manner, with an excess of free adjuvant required for increased immune stimulation (Majgaard Jensen & Koch, 1988). Allergen sensitisation takes several weeks to develop, involving the production of IgE and activation of lymphocytes.