An in vitro model of the follicle-associated epithelia that overlie the Peyer's patches of the
small intestine was developed and validated to examine the mechanisms of mucosal antigen
sampling. This model displays many phenotypic and physiological characteristics of M cells
including apical expression of [alpha]5[beta]l integrin and enhanced energy dependent participate
transport. CD4+ T-cells were shown to be an important influence on the development of Mlike
cells.
The model was used to examine the M cell mediated uptake of several putative whole-cell
killed bacterial vaccines. Greater numbers of non-typeable Haemophilus influenzae NTHi
289, NTHi 2019, Escherichia coli 075 HMN and Streptococcus pneumoniae were
transported by model M cells compared to control Caco-2 enterocyte-like cells. Studies in
isolated murine intestine segments confirmed the selective uptake of NTHi 289 and
Escherichia coli demonstrating that intestinal mucosal sampling of these antigens is
performed by M cells. Pseudomonas aeruginosa was not absorbed as whole cell bacteria but
as soluble antigen, as indicated by the presence of bacterial DNA in the cytoplasm of
epithelial cells. These results suggest that bacteria such as NTHi and E. coli are sampled by
the mucosal immune system in a different manner to that of bacteria such as Pseudomonas.
A number of potential cell surface receptors were investigated to identify which molecules
are responsible for intestinal uptake whole-cell killed bacteria. Immunofluorescence studies
detected the presence of toll-like receptor-2, toll-like receptor-4, PAF-R and [alpha]5[beta]l integrin
on in vitro M-like cell cultures. Examinations of murine intestine confirmed the presence of
TLR-4 and PAF-R. TLR-4 was found in small quantities and on M cells. In contrast to the M
cell model, TLR-2 expression in the murine intestine was sparse.
Receptor inhibition experiments provided evidence for the involvement of TLR-4, PAF-R
and [alpha]5[beta]l integrin in M cell uptake of killed bacteria both in vitro and in vivo.
This thesis has contributed valuable information regarding the mechanisms of uptake of
whole-cell killed bacteria by the intestinal mucosal immune system. For the first time, M cell
sampling of whole-cell killed bacteria has been demonstrated. Furthermore, the receptors
involved in these processes have been identified. This information will be of great use in the
development and optimisation of new oral vaccines.
| Identifer | oai:union.ndltd.org:ADTP/218727 |
| Date | January 2004 |
| Creators | Tyrer, Peter Charles, n/a |
| Publisher | University of Canberra. Health Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | ), Copyright Peter Charles Tyrer |
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