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Peri-natal egg and milk allergen exposure in relation to tolerance or allergic sensitisation to food in infancy
Project Code: T07044
Allergy and Inflammation Sciences, University of Southampton.
Aims and objectives
In order to address the hypothesis that there is a bell shaped curve of susceptibility to allergic disease in relation to maternal exposure to food allergens during pregnancy we established a high risk pregnancy cohort. This involved recruiting pregnant women with uncomplicated pregnancies from the community between 8 and 16 weeks after conception. In order to fulfil the recruitment criteria either the pregnant lady or her partner was required to have a clinical history of allergic disease and at least one positive allergy skin prick test to a range of common inhalant and ingestant allergens. The study has focused on the intake of egg (as in T07005 to attempt to replicate the findings in the non-intervention limb), and cows’ milk. These are the two commonest food allergens to affect young children. We added milk additionally because we anticipated a much wider range of exposures during pregnancy.
In order to monitor the intake of these two foods during pregnancy a food frequency questionnaire was designed to be administered over two one-month periods between 20 and 24 weeks’ and 34 and 38 weeks’ gestation. Additionally if the mother chose to breastfeed a third food frequency questionnaire was completed between 4 and 8 weeks after delivery.
The food frequency questionnaire was validated in 50 women, 25 of whom were pregnant, by comparing the questionnaire as administered in the study population with a 7 day food diary which is sometimes considered to be the gold standard for evaluation of dietary intakes.
Blood samples were taken from the mother at 24 and 38 weeks’ gestation. Cord blood was collected at the delivery of the babies. Subsequently breast milk samples were collected 8 weeks after delivery where breastfeeding was proceeding. It was intended that the babies would be followed as part of Phase II of the study at 6, 12 and 18 months of age.
The assays done on the blood samples have required development and optimisation in the laboratory. They involve the measurement of the circulating levels of a component of egg known as ov-albumin and a component of cows’ milk known as beta lacto globulin. In addition the mother’s antibodies to these two food proteins have also been measured. This has included the allergy antibody (IgE) as well as a non-allergy antibody known as IgG. Levels of these have been related to the levels of the actual food proteins themselves as well as to the amount of the egg and milk eaten by the mothers.
Similarly, levels of the egg and milk proteins were to be measured in the breast milk specimens. Subsequently all of this information was to be analysed in relation to the development of egg and milk allergy and eczema in the babies at 6, 12 and 18 months of age. The clinical assessments on the babies involve history, examination and blood sampling to measure the allergy antibody levels in the blood.
An additional spin-off from this study is that cells from the cord blood sample have been separated and stored in liquid nitrogen in order that we can do some detailed studies of the nature of the cells to establish whether there is any possibility of not only predicting subsequent allergic disease but also the nature of that disease. This involves examining molecules on the surface of the cells that help the cells to move towards specific tissues such as the skin, the gut or the lungs. We hypothesized that these so-called tissue homing molecules if present will indicate the nature of the allergic diseases that the child will develop. Thus if skin homing molecules are expressed on the surface of allergy sensitised cells then we would predict this would be associated with later development of eczema.
Outcome / Key Results
The original proposal was to recruit a cohort of 400 in order to have 320 who completed the study. However, recruitment proved to be more difficult than anticipated. This was offset to some extent by being able to re-calculate the likelihood of the study achieving meaningful results based on more recent published data and a preliminary analysis of the first 50 subjects entering the study. However, the extended recruitment period also meant that the follow-up period of the cohort would have had to be reduced from 2 years to 18 months and the study would only be statistically powered to look at the effects of dietary intake of egg or milk during pregnancy on eczema and sensitization in the infant, rather than clinical food allergy. By the close of the study we had recruited 251 subjects and the drop out rate from the study was running at 15%.
Validation of the food frequency questionnaires
These have shown good reproducibility and an excellent correlation between the food frequency questionnaire and the food diary. We have concluded from the validation study that dietary intake data collected by food frequency questionnaire gives appropriate measurements of egg and protein intake over a month period which can be used to assess the way in which variations affect outcomes.
The milk protein intakes have ranged between 28g and 270g per week which is equivalent to 1.5 pints of milk per week up to 1.5 pints per day. For egg protein the range is 1g to 20g per week which is equivalent to one egg in eight weeks up to three eggs per week.
The levels of proteins from cows’ milk and egg measured at 24 and 38 weeks’ gestation correlated well with one another suggesting both that mothers stuck to a consistent diet and that the degree of absorption of allergens through the bowel into the circulation were consistent through pregnancy. However, there was no correlation between the levels of these proteins in the blood and the amount of milk and egg consumed by the mothers in the previous month. This however is not surprising as the amounts in the blood are representative only of what has been eaten in the previous two to six hours.
Milk and egg antibodies
The levels of antibodies to milk or egg measured at 24 and 38 weeks’ gestation correlated with one another showing that there is little change in these levels through pregnancy. Furthermore this appeared to be independent of dietary intake in that there was no correlation between the intake of egg or milk and the levels of the respective antibodies in the blood. Ostensibly this might appear disappointing but it in fact allows us to discriminate whether any effects subsequently on the baby are due directly to exposure to the food protein or indirectly are a consequence of the transfer of antibodies to those foods from the mother to the baby. It suggests that the degree of antibody response is influenced by factors other than those related purely to amount of dietary exposure.
By completion of Phase 1 of the study, the number of babies who had reached the six month assessment point was small and therefore any results need to be interpreted with great caution and could not be published unless the full cohort has been assessed. However, thus far there is a suggestion that our hypothesis in relation to high dose antenatal exposure to food proteins reduces the risk of subsequent allergy is partially true. In relation to the development of eczema there was a lower maternal intake of egg during pregnancy compared with those babies who subsequently have not developed eczema. This difference is significant. However there is no significant difference in relation to the presence or absence of eczema and milk intake in pregnancy.
What it means
This is Phase 1 of a study which was intended to continue until all the subjects recruited have reached at least 18 months of age. Thus at the moment our conclusions are limited. The lack of correlation between maternal egg or milk intake and the maternal antibody levels to ova albumin and beta lacto globulin offers an opportunity to disentangle the influences of exposure to the food and the effects of transfer of antibody from mother to fetus in relation to allergy outcomes. There is already a suggestion that a high pregnancy exposure is associated with low risk of eczema in relation to egg but currently not milk. Whether this means that there is something special about egg in terms of its impact on allergy outcomes remains to be established. A particular focus for further study should be on the potential that endotoxin contamination of egg actually reduces the risk of allergic sensitization.
It was hoped that the outcome of this research once Phase 2 was completed would aid advice to be given to families about strategies to reduce food allergy and eczema in children. However, phase 2 was not funded and follow-up of the subjects could not be sustained.
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