Furrie, Elizabeth; (1993) Intestinal modification of a protein antigen and its effect on oral tolerance induction. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The mechanisms regulating the induction of systemic immunological hyporesponsiveness to fed protein antigens (oral tolerance) have been studied in mice using ovalbumin (OVA) and bovine serum albumin (BSA). Intestinal processing of OVA and BSA was studied in vivo by transfer of serum collected from OVA and BSA fed mice at specific timepoints and transfer of that serum into naive recipient mice. The nature of the "gut processed" OVA was subsequently analysed in vitro using affinity chromatography, gel filtration chromatography, SDS-PAGE, immunoblotting techniques and enzyme linked immunosorbent assays for antigen detection in serum collected from OVA fed animals. An earlier observation that immunoreactive OVA can be detected in serum 5 minutes after a feed of OVA was confirmed. However, such serum was not tolerogenic in recipient mice and the tolerogenic effect did not appear until 30 minutes after feeding with full tolerance in recipients observed only with serum collected 60 minutes after a feed. In vitro pepsin and acid treatment of OVA and BSA was used to mimic the conditions in the murine stomach. Pepsin treatment of OVA did not render OVA tolerogenic in recipient mice whereas, pepsin treated BSA was able to induce full suppression of delayed type hypersensitivity (DTH) reactions in recipient mice. This suggests that different mechanisms may be responsible for generating tolerogenic material depending on the nature of the antigen used. In order to investigate the role of the antigen specific immune system in the production of a transferable "tolerogen", Scid (severe combined immunodeficient i.e. no mature B and T cells) mice were used as donors in the serum transfer model of oral tolerance. It was found that Scid mice were unable to render fed OVA tolerogenic in serum transfer to naive BALB/c recipient mice. However serum collected from BSA fed Scid mice did induce partial BSA tolerance in recipient mice. This data suggests that although a functioning immune system is essential for the production of an OVA tolerogen it is of less importance in the production of a BSA tolerogen. The failure of Scid mice to render OVA tolerogenic was not due to their lack of gut flora since germ free mice generated tolerogenic OVA which was transferable to naive BALB/c recipients. However, limited histological analysis of small intestine from Scid, germ free BALB/c and conventionally reared BALB/c mice demonstrated that there is a difference in the cellular composition and physiology of the Scid mouse gut. The transfer of spleen cells from mice 7 days after a feed of OVA or BSA induced antigen specific suppression of DTH in recipient mice but only in the presence of antigen administered immediately after the transfer of cells to recipients. Investigations of serum collected from BALB/c mice 5 minutes and 60 minutes after feeding OVA and collected from Scid mice 60 minutes after an OVA feed permitted the identification of candidate tolerogenic fractions of OVA having molecular weights of approximately 21kDa and 24kDa. These studies suggest that processing of fed protein antigens by the gut and associated lymphoid tissues to produce a transferable OVA or BSA tolerogen plays an important role in the ontogeny of oral tolerance induction in mice.

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