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- Materials and methods
Background: Sensitization to rats and mice can develop in laboratory animal workers exposed to only one species. Reasons for this dual sensitization are unclear but may reflect a genetic predisposition to developing allergy (atopy) or alternatively cross-reactivity between rat and mouse urinary allergens. We examined cross-reactivity between rat and mouse urine and the effect atopy has on dual sensitization in laboratory animal workers.
Methods: In a cross-sectional study the frequency of sensitization to rat and/or mouse was analysed in 498 employees exposed to both rat and mouse at work and 220 to rat only. RAST inhibitions, western blots and blot inhibitions were carried out on a subset of five individuals to assess cross-reactivity.
Results: Fourteen per cent of workers were sensitized to rats and 9% to mouse. Over half (62%) of rat sensitized individuals were also mouse sensitized and the majority (91%) of mouse sensitized individuals were also rat sensitized. IgE cross-reactivity was demonstrated between rat and mouse urine using RAST inhibitions. Rates of atopy did not differ between rat only sensitized individuals compared with those sensitized to both species. Sensitization to cats and rabbits was more common amongst those with dual sensitization.
Conclusions: Dual sensitization to rat and mouse reflects IgE cross-reactivity rather than atopy. Individuals with dual sensitization are more likely to be sensitized to other animal allergens. These findings will have implications for individuals working with only one rodent species who develop sensitization and symptoms to be aware of the potential for allergy to other species.
Individuals working with laboratory animals are at risk of developing occupational allergy and asthma. Rats and mice frequently used in animal research are the most common causes of laboratory animal allergy (LAA). Sensitization to both rat and mouse can develop in employees exposed to only one species, although the reason for this dual sensitization is unclear. It may reflect a predisposition to developing allergy and this is supported by the fact that atopy is a major risk factor for the development of LAA (1–6). Alternatively because rat and mouse allergens are closely related immunological cross-reactivity may exist between allergens from these two species.
The major rat and mouse allergens, respectively, Rat n 1 and Mus m 1, belong to the lipocalin family of proteins and share 66% homology in their amino acid sequence (7). Since Rat n 1 and Mus m 1 share a high degree of sequence homology and an overall 3D structure they may also share cross-reactive IgE binding determinants. However, several very early studies failed to find evidence of cross-reactivity between rat and mouse allergens (8–10) although one study did observe a partial identity between rat, mouse and guinea pig urine in a single rat allergic patient’s serum (11).
For the individual working with either rat or mouse and becoming sensitized to the urinary allergens, it is important to elucidate the degree of cross-reactivity between the allergens from both species, as this may have an important impact on their employment. Moreover identifying cross-reactive structures may also allow for the development of efficient modes of immunotherapy in which specific cross-reactive epitopes can be used to treat multiple allergies.
In a large cross-sectional study of laboratory animal workers, we investigated atopy and the frequency of sensitization to rat and/or mouse allergens in association with specific exposures in order to understand whether sensitization to both species reflected atopy or immunological cross-reactivity. Using RAST, RAST inhibition and western blotting techniques, we investigated the degree of cross-reactivity between rat and mouse urine in a subset of individuals from this cross-sectional study.
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- Materials and methods
Allergy to both rat and mouse is common amongst individuals working with laboratory animals. We have demonstrated within a workforce of laboratory animal workers that more than half (63%) of rat sensitized individuals also have evidence of specific IgE to mouse urine and the majority of mouse sensitized individuals are also sensitized to rats (90%). We hypothesized that this dual sensitivity could be explained by either a predisposition to allergy, for example, atopy or cross-reactivity between the allergens from these two species.
Rates of atopy did not differ significantly between individuals sensitized to rats only compared with those sensitized to both species, indicating that a predisposition to allergy was not an important factor in whether an individual was likely to have dual sensitization. Furthermore, we observed similar rates of sensitization to mouse in both rat only exposed workers compared with those exposed to both species suggesting cross-reactivity as the most likely explanation for these findings.
We investigated cross-reactivity between rat and mouse urinary proteins using detailed inhibition experiments and observed that rat urine inhibited the binding of specific IgE to mouse urine (53–90%) more strongly than mouse urine was capable of inhibiting specific IgE binding to rat urine (35–79% inhibition). Self-inhibition with the homologous antigen was high. Similarly, immunoblotting results also demonstrated cross-reactivity between the separated proteins of rat and mouse urine to which the individual had specific IgE. Thus, we have demonstrated cross-reactivity between rat and mouse urine using both RAST inhibition and blot inhibition techniques. Furthermore, these results suggest that there are common epitopes within the urinary allergens of rat and mouse, but proteins from rat urine in particular contain a greater number of unique epitopes.
The B cell epitopes of Rat n 1 have not as yet been determined. Bayard et al. attempted to map the IgE binding regions of Rat n 1 using overlapping peptides but found great individual variation between sera from rat allergic individuals in their binding to these peptides (18) although some were recognized more frequently than others. Of particular interest in our study was the fact that all sensitized individuals exhibited unique inhibition profiles. This may have been the result of differing patterns of IgE binding to the various allergens within the rat and mouse urine by each individual. Taken together, the findings of Bayard et al. and results from our study demonstrate that Rat n 1 and Mus m 1 are likely to contain multiple B cell epitopes some of which are unique and others which are shared between the two allergens.
Mapping of the identical residues on the surfaces of the 3D structure of Rat n 1 and Mus m 1 revealed a substantial area in which several of the identical amino acids in the primary sequence (regions 2, 6, 7 and 8) join together to form a large accessible site for potential IgE binding. The majority of these residues are located at the C terminus of the molecule, with the exception of region 2 which is located at the N-terminus. This is consistent with reports of B cell epitopes being more likely to be located in flexible and protruding regions such as the terminal parts of the protein (19). Interestingly, regions 2 and 7 (Fig. 4) correlated with the peptides that were recognized by IgE antibodies in sera from rat allergic subjects in the study by Bayard et al. (18) and region 7 is located in the conserved region of the molecules, indicating that this area may be a common cross-reactive epitope amongst all lipocalin allergens. Furthermore, the regions within the cow lipocalin allergens, Bos d 2, and Bos d 5 of most importance for IgE binding were located in the carboxy terminal portion of the molecules (20–22). Thus it is likely that both Rat n 1 and Mus m 1 contain a conformational epitope made up of identical linear sequences from the terminal ends of their primary structure to which IgE antibodies are directed, resulting in cross-reactivity between the two proteins.
A recent study demonstrated IgE cross-reactivity between Mus m 1 from mouse and Equ c 1 from horse (23). Mus m 1 and Equ c 1 have only a 43% sequence homology; however they contain an area of similar amino acid sequence which is likely to be the cross-reactive epitope between both molecules. Rat allergen was not included in this study but it is possible given the cross-reactivity between rat and mouse urinary allergens that we have observed that Rat n 1 contains an epitope that is also cross reactive with Equ c 1.
We were interested to find in our study that individuals who were sensitised to both rat and mouse had a significantly higher IgE titre to rat urine compared with those only sensitized to rats. Given that we have demonstrated cross-reactivity between rat and mouse urinary allergens, it is likely that IgE directed to epitopes on mouse allergens that cross react with epitopes on rat allergens are responsible for the IgE titre to rat urine being raised in those with dual sensitization. Furthermore, positive skin prick tests to other animals such as cat and rabbit were more commonly seen amongst those sensitized to both rodent species rather than rats only. These findings indicate that dual sensitization to rat and mouse is associated with immune responses to a broader spectrum of animal allergens.
This study was carried out on a large, carefully defined cross-sectional population and is the first to date to carry out detailed immunological techniques to assess cross-reactivity between rat and mouse urinary allergens. One limitation was that we were unable to determine whether rat only exposed individuals, despite never handling mice in the past or at the time of the study, were also being exposed to mouse allergen within the animal houses where they worked with rats. Nevertheless our study has confirmed that cross-reactivity does indeed exist between rat and mouse urinary allergens and the degree of cross-reactivity is likely to be dependent on the various epitopes being recognized on the major allergens from these two species. Furthermore, we have shown that allergy to mouse in the absence of allergy to rats is uncommon in individuals working with laboratory animals and allergy to other animal species is less common amongst rat monosensitized individuals compared with those sensitized to both rat and mouse. These findings have important implications for those working with laboratory animals. In particular it is necessary for individuals working with only one rodent species who develop sensitization and symptoms to be aware of the potential for allergy to other species. Strategies developed for immunotherapy would need to be aware of the cross-reactive epitopes between Rat n 1 and Mus m 1.