Expression of acquired immunity to gastrointestinal parasites usually breaks down during the periparturient period, which is characterised by an increased worm burden and nematode egg excretion. It is believed that this breakdown of immunity may have a nutritional basis, and that by reducing nutrient scarcity the lactating animal will be able to reduce her worm burden. Therefore, the aim of this thesis was to carry out four experiments to investigate the potential use of metabolisable protein as an alternative way to control gastrointestinal parasitism in periparturient animals. A lactating rat model was used to address this issue as lactating rats exhibit a breakdown of immunity to the gastrointestinal nematode Nippostrongylus brasiliensis. The first experiment (chapter two) aimed to verify that a reduction in worm burden is indeed related to changes in nutrient supply and not associated with changes in the gut environment. This was achieved by manipulating nutrient (litter) demand whilst nutrient supply was maintained constant. The results showed that the periparturient breakdown of immunity to N. brasiliensis (measured by a reduced worm burden) was sensitive to changes in nutrient demand and that these effects were independent of changes in the gut environment. The second experiment (Chapter Three) tested the effect of increased protein supply or reduced protein demand on the resistance to parasites in lactating rats whilst energy intake was kept constant. Under these conditions effects of protein supply could not be confounded with effects of any nutrient or energy intake. The results supported the view that under a restricted feeding regime, breakdown of immunity to N. brasiliensis (measured by a reduced number of eggs in the colon content) was sensitive to changes in protein scarcity. Following on from this, the next experiment (Chapter Four) assessed the effects of a gradual increase in protein supply on resistance and immune responses to N. brasiliensis in lactating rats. It was shown that as protein contents of the diets progressively increased, the number of worms and eggs present in the colon decreased. Although we found that differences in protein supply affected parasite burden, we found no affects of protein supply on local immune responses. This may have been due to the single sampling point used. Therefore, the objective of the last experiment (Chapter Five) was to assess temporal effects of increased protein supply on resistance and immune responses to N. brasiliensis. In agreement with previous experiments, the results showed that an increase in protein at times of protein scarcity improved resistance to N. brasiliensis, illustrated by a lower number of nematode eggs in the colon. The results also showed that local immune responses such as immunoglobulin levels (IgA, IgE & IgG2a), RMCP II levels and goblet cell counts were affected by differences in protein supply at various time points post secondary infection. The potential application of using a lactating rat as a suitable model to fully understand the underlying immunological basis of relaxation in immunity during the periparturient period and its sensitivity to nutrient scarcity is considered in the General Discussion (Chapter Six).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1965. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1964. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 59-62.

Approximately two billion people are infected with helminths worldwide. In order to develop a vaccine against these pathogens, more needs to be known about the immune response to helminths. Eosinophils are important for resistance to some helminth species and their recruitment to infected tissues, attachment to parasites and degranulation may all be critical processes for immunity. Complement may contribute to these processes via generation of chemotactic factors (C3a and C5a) or opsonisation of the parasite with C3b/iC3b. The importance of complement during helminth infection is unclear, though complement does promote leukocyte-mediated killing of several helminth species in vitro. The aim of the present study was to investigate the role of complement in immunity of mice to Nippostrongylus brasiliensis, with a focus on whether complement facilitates eosinophildependent resistance to this parasite. A new fluorescence-based method for quantifying in vitro complement deposition and leukocyte adherence on N. brasiliensis was developed. C3 from human serum was deposited on infective-stage L3 via the classical or lectin complement pathways. In contrast, the alternative complement pathway mediated binding of mouse C3 and eosinophil-rich mouse peritoneal leukocytes to L3. Interestingly, the ability of complement and leukocytes to bind to the parasite changed as it matured. Larvae recovered from the skin 30 min post-injection (p.i.) were coated with C3, however those harvested 150 min p.i. exhibited reduced C3 binding capacity. Binding of C3 and eosinophils to larvae recovered from the lungs 24-48 h p.i. (L4) was also diminished compared to that seen on L3. Adult intestinal worms bound C3 and leukocytes only when treated ex vivo with serum and cells. Mice lacking in classical (C1q-deficient), alternative (factor B-deficient) or all complement pathways (C3-deficient) were then employed to determine if complement was important for resistance of mice to N. brasiliensis. IL-5 Tg mice deficient in individual complement genes were generated to assess whether complement contributed to eosinophildependent resistance to the parasite. Factor B deficient mice exhibited impaired C3 deposition on larvae, eosinophil recruitment, eosinophil degranulation and larval aggregation in the skin 30 min p.i. Eosinophil recruitment was similarly abolished by treatment of mice with the C5aR inhibitor PMX53. However at 150 min p.i., larval aggregation, eosinophil and neutrophil recruitment, leukocyte adherence and eosinophil degranulation were largely complement-independent. Ablation of factor B or C3 caused minor but significant increases in lung-larval burden during primary, but not in secondary, infections. Critically, a lack of C3 or factor B in IL-5 Tg mice failed to greatly impair the strong innate anti-parasite resistance typical of these animals, suggesting that eosinophils can provide immunity to N. brasiliensis infection in the absence of complement. This was unexpected, given the evidence from this and previous studies which suggested that in vitro, complement is important for promoting eosinophil-dependent killing of N. brasiliensis and other helminth species. The mechanism(s) by which eosinophils kill N. brasiliensis remain unknown, but may involve the coordination of the complement system with complement-independent factors that act in the early stages of infection. Critically, the influence of complement is limited, because soon after entry into the host, the parasite develops the ability to resist complement activation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311182 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

ADAM10 is a zinc-dependent metalloprotease. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. In this study, we examined the role of ADAM10 in the immune system. Here, we show that knocking out ADAM10 on the mature B2 cell causes a defect in the development of secondary lymphoid architecture that becomes more severe post-immunization. We also show that overexpression of ADAM10 leads to a defect in hematopoiesis, which eliminates B2 lymphocyte development. This defect additionally induces accumulation of myeloid derived suppressor cells, MDSCs. ADAM10Tg MDSCs function synonymous to tumor MDSCs. Of the two subpopulations of MDSCs, granulocytic MDSCs increase parasitic clearance in a model of N. brasiliensis. Monocytic MDSCs are more immunosuppressive in regards to tumor. Both subpopulations are dependent on the presence of mast cells for activity. It is thought that this relationship is mediated through histamine and IL-13. During N. brasiliensis infection, ADAM10Tg mice, lacking B2 B cells but having intact B1 B cells, makes increased IgE over WT mice. This production of IgE is thought to be produced by the B1 cells. Of the two types of B1 cells, B1a cells make the majority of the IgE. This IgE production is enhanced by MDSC accumulation and can be induced by MDSC adoptive transfer in a parasite-free mouse. Lastly, ADAM10 is the key sheddase for CD23 on B2 cells. When IgE is bound to its antigen to form an immune complex, IC, it binds CD23 and is internalized. After CD23 bound to IgE ICs is internalized, it is sorted into bexosomes. These bexosomes are transferred to dendritic cells which are responsible for presenting to T cells and inducing an increased antigen-specific immune response. Overall, ADAM10 is important for many aspects of the immune response.

Animals acquire immunity against gastrointestinal (GI) nematode infection depending on their age and continual exposure to larvae, however, expression of this acquired immunity is often penalized during pregnancy and lactating period. This is described as periparturient relaxation in immunity (PPRI), and suggested to have nutritional basis. Although dietary protein has positive effect on immunity against GI nematode infection in mammalian host, we have not fully achieved to characterise the detailed interaction between PPRI and dietary protein. Therefore, this PhD aimed to further investigate this interaction in a well-established Nippostrongylus brasiliensis re-infected lactating rat model. Feeding high protein diet (HP) as opposed to low protein diet (LP) during pregnancy was necessary in maintaining sufficient maternal performances and systemic immune response (Chapter 2 and 3). Accumulation of host’s body protein reserve during pregnancy was significantly higher in HP fed animals compared to LP fed counterparts, which led to improvement in both maternal performances and immunity during the early stage of lactation. However, as lactation period progressed and re-infection of N. brasiliensis took place, importance of current dietary status, rather than the accumulated protein reserve, became evident for maternal performances and immunity. Indeed, animals fed HP during lactation showed significantly heavier pup weight compared to LP fed animals; HP animals showed higher serum immunoglobulin levels and reduced worm burden compared to LP. N. brasiliensis, however, goes through systemic migration, entering host’s skin and migrating to lung parenchyma through blood vessels before reaching the intestine. In Chapter 4, a detailed lung pathology study following N. brasiliensis infection was performed to generate data on the effects of the nematode in the lung of the rat host as such data were scarce. Rat host showed similar lung pathology to that of mice; up-regulation of genes related to type 2 immunity and development of emphysema-like pathology were observed following N. brasiliensis re-infection. In Chapter 5, the effect of dietary protein supplementation on lung and intestinal histology and gene expression analysis was investigated. It was shown that HP fed animals showed higher expression of genes related to type 2 immunity compared to LP in the lung. This effect of protein supplementation in the lung may have contributed to fewer worm burdens in HP fed rats compared to LP in the intestine. Dietary protein supplementation significantly affected the expression of genes related to goblet cells; it resulted in up-regulation of the expression of Retnlb and down-regulation of Agr2 and Tff3 in HP fed animals compared to LP. It is evident that dietary protein is modulating intestinal immunity, and this may be targeted towards specific pathways. In addition, the effect of dietary protein supplementation on immune cell populations of secondary lymphoid organs was analysed. Marked increase in the percentage of macrophage in the spleen and T cell in the mesenteric lymph node was observed following protein supplementation, highlighting the importance of dietary protein on systemic immunity during the parasite infection. These results demonstrate that dietary protein supplementation is effective for improving both maternal performances and immune responses, not only at the intestinal phase but also at the migrating phase, when animal is undergoing PPRI during N. brasiliensis infection. Such information is expected to define strategic utilisation of nutrient supply and to result in development of sustainable parasite control strategies in mammals.

Recent advances in the field of host immunity against parasitic nematodes have revealed the importance of macrophages in trapping tissue migratory larvae. Protective immune mechanisms against the rodent hookworm Nippostrongylus brasiliensis (Nb) are mediated, at least in part, by IL-4-activated macrophages that bind and trap larvae in the lung. However, it is still not clear how host macrophages recognize the parasite. An in vitro co-culture system of bone marrow-derived macrophages and Nb infective larvae was utilized to screen for the possible ligand-receptor pair involved in macrophage attack of larvae. Competitive binding assays revealed an important role for β-glucan recognition in the process. We further identified a role for CD11b and the non-classical pattern recognition receptor ephrin-A2 (EphA2), but not the highly expressed β-glucan dectin-1 receptor, in this process of recognition. This work raises the possibility that parasitic nematodes synthesize β-glucans and it identifies CD11b and ephrin-A2 as important pattern recognition receptors involved in the host recognition of these evolutionary old pathogens. To our knowledge, this is the first time that EphA2 has been implicated in immune responses to a helminth.