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Previous studies of the ontogeny of the mucosal immune system have shown a significant increase in salivary Immunoglobulin A levels occurring at about five years of age. This study has monitored a group of 3 and 4 year old children during one year of attendance at Pre-School to examine whether such an increase could be linked to increased antigenic exposure associated with moving into a school like environment. Saliva samples were collected at regular intervals and analysed for immunoglobulin and total protein levels. Daily health records were maintained for each child, and a detailed social and medical history was collected for each child at the beginning of the study. The elevated mucosal immune response observed in previous studies involving children in day care centres and attending school was not seen in this study. No significant difference was observed between children who had previously attended Pre-School or child care centres and those who were attending for the first time. However, a marked seasonal increase in mean salivary IgA during the winter months was observed and this increase correlated with an increase in respiratory infections. Hence, in studies of developmental aspects of mucosal immune response it is essential that modifiers such as season and infection be recorded.

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.

Mucosal immune system comprises not only the major compartment of the immune system but also important interface with the outer environment. It is responsible in maintaining an intricate balance with the danger and non-danger stimuli of the outer world by employing specific anatomical features and unique functional mechanisms. Mucosal immune system has been long understudied, perhaps due to the limited accessibility, and its biological importance is thus still underevaluated. However, it has become evident that it is important to study mucosal immune system not only in local mucosal affections but also when uncovering pathogenic mechanisms and novel prevention strategies of organ specific autoimmune diseases such as type 1 diabetes. Thus, the first, more clinically oriented part of this thesis is focused on mucosal immune system of the upper respiratory tract in disease conditions - in nasal polyposis (NP). Because there is a substantial accumulation of eosinophils and neutrophils in the most frequent type of NP, we investigated and described increased expression of chemokine receptors CCR1 and CCR3 in NP versus nasal mucosa. Both innate immune mechanisms as well as homeostasis of epithelial cells may participate in NP. We have documented increased numbers of iNOS-positive and insulin-like growth...

Mucosal immune system comprises not only the major compartment of the immune system but also important interface with the outer environment. It is responsible in maintaining an intricate balance with the danger and non-danger stimuli of the outer world by employing specific anatomical features and unique functional mechanisms. Mucosal immune system has been long understudied, perhaps due to the limited accessibility, and its biological importance is thus still underevaluated. However, it has become evident that it is important to study mucosal immune system not only in local mucosal affections but also when uncovering pathogenic mechanisms and novel prevention strategies of organ specific autoimmune diseases such as type 1 diabetes. Thus, the first, more clinically oriented part of this thesis is focused on mucosal immune system of the upper respiratory tract in disease conditions - in nasal polyposis (NP). Because there is a substantial accumulation of eosinophils and neutrophils in the most frequent type of NP, we investigated and described increased expression of chemokine receptors CCR1 and CCR3 in NP versus nasal mucosa. Both innate immune mechanisms as well as homeostasis of epithelial cells may participate in NP. We have documented increased numbers of iNOS-positive and insulin-like growth...

The mucosal system, which forms a barrier between internal organ systems and the external environment, is frequently exposed to pathogenic microorganisms. Immunoglobulin A (IgA) antibody secreting cells (ASCs) localize in the lamina propria, and produce IgA antibodies which help protect mucosal tissues. The concept of a common mucosal immune system in which IgA ASCs have the ability to populate any mucosal site has been proposed (1, 2). However, recent research has suggested that IgA ASCs primed in different mucosal sites might possess different sets of chemokine receptors, and therefore migrate specifically to particular mucosal locations (3). In this study, the specific compartmentalization of IgA ASCs in two mouse salivary glands: sublingual gland (SLG), and submandibular gland (SMG) was studied. It was observed that SLG had 12 times more IgA ASCs per gram of gland than that of SMG (p<0.01). This suggested that IgA ASCs migrated to the two salivary glands with different efficiencies. Since the migration of lymphocytes is mediated by interactions between tissue specific chemokines and chemokine receptors, I hypothesized that the specific compartmentalization of IgA ASCs in the SLG and SMG was mediated by the differential expression of IgA ASC attracting chemokines. Quantitative PCR was used and showed that SLG expressed high levels of CCL28 and its receptor CCR10, which correlated to the distribution of IgA ASCs in the two salivary glands. In agreement with QPCR data, reduced levels of IgA ASCs were found in the SLG of CCR10 deficient mice when compared to wild type (WT) mice. Adoptive transfer of CCR10 deficient mice with WT spleen cells reconstituted the WT phenotype. It was therefore concluded that the interaction between CCL28 and CCR10 play an important role in mediating the migration of IgA ASCs into SLG. These results suggested that the accumulation of IgA ASC to distinct salivary glands is a highly selective process. These data also suggested that homing within mucosal sites is not common but rather a highly regulated process with specific subsets of cells homing to different tissues within the mucosal immune system.

Gastrointestinal microflora has been shown to have a bi-directional relationship with the host immune system. A variety of fermentable carbohydrate polymers largely pass through the small intestine, providing fermentable substrates for gut microflora. Dietary fibre supplementation may provide a strategy for manipulating the intestinal bacterial profile, changing the interaction with the mucosal immune system, thereby modulating the host immune system. We used a BBc rat animal model to evaluate the effects of oat bran and wheat bran dietary fibre on the immune system. Previous collaborative efforts have shown that these dietary fibres can change the intestinal microflora, with wheat bran fibre showing a greater ability to influence colonic microbial community diversity. We have shown that dietary wheat bran fibre led to reduced IL-4 levels in the liver and T lymphocyte numbers in the Mesenteric Lymph Node and may be involved in reduced IgA levels in the cecal contents. In addition, IgA in the cecal contents was decreased while MLN B cell numbers increased in response to dietary wheat bran fibre. It was observed that neither wheat bran or oat bran treatments exerted any pro-inflammatory effects, with oat bran actually improving antioxidant status. These results suggest that both oat and wheat bran fibre treatments induce changes in the intestinal microflora, and that the microflora changes due to wheat fibre are associated with immunomodulatory effects on the host. This type of dietary fibre supplementation could ultimately provide a potential strategy for promoting health through microflora-associated effects on the immune system.