Bochum, Universiẗat, Diss., 2000.

Soil-transmitted helminths are a major cause of morbidity for humans and their livestock. A combination of better sanitation, anthelminthic drugs and vaccines are predicted to reduce the morbidity of these parasites in humans. The drugs currently used to treat these infections, albendazole and mebendazole, are fairly ineffective against Trichuris trichiura (human whipworm), and there are reports of drug resistance arising within parasite populations in Vietnam and Zanzibar. There are also no commercially available vaccines against human STH species, and very few against their veterinary counterparts. The murine whipworm, T. muris, has been used for over 50 years as a model for T. trichiura. These parasites share homology at the genomic and transcriptomic levels, and the immune responses associated with both acute and chronic infection have been well studied using the T. muris mouse model. T. muris excretory/secretory products have been studied in the context of vaccination for over four decades, however relatively little progress has been made towards identifying the molecular components that stimulate protective immunity following vaccination or during acute infection. Here, a stringent selection protocol was developed using chromatography and mass spectrometry methods combined with a measurement of T cell cytokine production. The work presented in this thesis provides a novel framework for identifying potential immunogenic candidates within adult T.muris excretory/secretory products. Exosome-like vesicles isolated from adult T. muris ES were also explored as a source of host protective material. Vaccination with exosome-like vesicles protected male C57BL/6 mice from a subsequent low dose infection, which would ordinarily progress to chronicity, and a number of potential immunogenic candidates were identified. Over the course of this thesis, several important observations were made relating to characteristics of the immune response induced by vaccination with ES. Firstly, proteinaceous material is likely to be responsible for the host protective properties of ES. Secondly, vaccination with ES products stimulates long-lasting immunity. Thirdly, vaccination with ES collected from both larval and adult stages stimulates protective immunity. The number of potential immunogenic candidates has also been narrowed down from over four hundred to just eleven. Given the homology between T. muris and T. trichiura at both the genomic and transcriptomic levels, this work has the potential to advance vaccine design for T. trichiura and other Trichuris parasites.

Thesis--Catholic Univ. of America.

Düsseldorf, Universiẗat, Diss., 2004.

Circadian rhythms have been implicated in severity and outcome of infection and disease. Commonly, LPS and bacterial infection have been used to identify the mechanisms behind the difference in immune responses depending on the time of day of the challenge. In this thesis, the colon dwelling nematode parasite Trichuris muris, which elicits a Th2 immune response in resistant mice, was used to identify if circadian rhythms influence infection outcome 3 weeks post infection. C57BL/6 mice infected with 200 eggs of T. muris at ZT0 (7am, lights on) expelled the parasite more efficiently than mice infected at ZT12 (7pm, lights off), which expelled with a delay of several days compared to ZT0 infected mice. Analysis of cell infiltration into the colon during the first days of infection suggested that there was no visible difference in the local immune response. There also were no differences in macrophage and dendritic cell numbers in colon tissue of naïve mice at ZT0 or ZT12. Further experiments examined immunomodulation of the immune response to T. muris by pushing the immune response towards a Th1, by low dose infection, or a Th2 response, by vaccination with excretory/secretory antigen. In both cases any circadian influence was overwritten. Mice infected at ZT0 or ZT12 with only 40 eggs of T. muris were equally susceptible to infection and mice infected at ZT3 10 days after vaccination at ZT0 or ZT12 were equally resistant to infection. Mice food restricted to mid-light phase and infected at ZT0 were not significantly delayed in their worm expulsion or polarised more towards a Th1 immune response compared to ZT0 infected mice fed during the dark phase. Therefore it is unlikely that feeding behaviour during the first days of infection is able to polarise towards a Th1 response and lead to delayed worm expulsion. Transgenic mice were used to dissect the mechanism underlying the delay in worm expulsion in ZT12 infected mice. mPer2::luc mice were used to confirm rhythmic Per2 expression in colon tissue and dendritic cells. Infection of mPer2::luc mice at ZT0 or ZT12 with T. muris showed similar worm expulsion, antibody and cytokine production when infected at ZT0 or ZT12. Bmal1floxLysMcre mice, which lack rhythmic clock gene expression in macrophages and granulocytes, produced a stronger Th2 antibody response in a primary infection at ZT3 than wild-type littermate controls. Newly generated mPer2::lucBmal1floxCD11ccre mice showed the no difference in worm burden and parasite specific antibody production between ZT0 and ZT12 infected mice. Only IL-10 and IL-6 levels were significantly lower in ZT12 infected mPer2::LucBmal1floxCD11ccre mice compared to ZT12 infected wild-type littermates. Confirmation of removal of exon 8 of the Bmal1 gene was not achieved; therefore it is not clear if circadian rhythm in dendritic cells has any impact on the immune response to T. muris or if the mPer2::LucBmal1floxCD11ccre mice and littermate controls both contain circadian rhythm in dendritic cells and therefore cannot be used to identify the role of the dendritic cell clock in the time of day differences in infection outcome. This thesis shows that time of day of the infection impacts on the outcome of infection with Trichuris muris.

Biological and immunological aspects of the host-parasite relationship were examined in mice which are susceptible (A/J) and resistant (B10.A) to Giardia muris. B10.A exhibited a shorter latent period, lower cyst output during the acute phase of the infection and shorter period of cyst release compared to A/J. Characteristics of the infection transmitted from mouse-to-mouse and those induced by oral inoculation with cysts or trophozoites were similar. The infection was longer in male A/J and B10.A mice compared to females. Susceptibility and resistance during both the acute and elimination phases of the infection were under non-H-2-linked multigenic control. A/J and B10.A differed in non-specific serum IgG and IgA, but not in the specific IgG and IgA to G. muris. Specific antibodies participated in complement-mediated killing of trophozoites. Spleen, mesenteric lymph node and peritoneal cells from A/J and B10.A mice had a similar ability to kill trophozoites. The capacity of B10.A to mount inflammatory responses was greater than that of A/J. A/J were more immunosuppressed than B10.A during the infection, particularly at mucosal sites. Macrophage-like suppressor cells were shown to be the mediators of this suppression.

No description available.

Infection with human gastrointestinal (GI) parasites, such as Trichuris trichiura, affects more than billion people worldwide, causing significant morbidity and health problems especially in poverty-stricken developing countries. Despite extensive research, the mechanisms of induction and regulation of effector immune responses against these parasites are incompletely understood, which hinders the development of anti-parasite therapies. Infection with GI parasite is usually chronic suggesting that parasites are capable of modulating immune responses of their host to prevent expulsion. However, mechanisms by which parasites control host immunity to allow infection are still ill-defined. The aim of this PhD study was to characterise the role of different immunoregulatory mechanisms in immunity to GI parasite infection, with a focus on dendritic cells (DCs), regulatory T cells (Tregs) and the regulatory cytokine transforming growth factor β (TGF-β).Here we showed for the first time that loss of TGF-β-activating integrin alphavβ8 specifically on DCs resulted in protection from chronic infection with Trichuris muris, a mouse model of T. trichiura infection in man. Accelerated expulsion was immune-mediated and although increased levels of protective Th2 cytokines were observed very early during infection, elevated levels of non-protective Th1 cytokines were also detected. Partial depletion of CD4+ or FcεRI+ cells had no effect on the observed phenotype. Since deletion of alphavβ8 on DCs results in decreased numbers of Tregs in the gut, we tested whether depletion of Tregs using a mouse model that allows conditional ablation of Foxp3+ Tregs (DEREG mice) would alter infection development. Although transient Treg depletion at the beginning of infection had no major effect on expulsion kinetics, we observed a tendency for enhanced Th2 responses in DEREG mice. Moreover, even though DC-mediated TGF-β activation via alphavβ8 integrin was essential for T. muris infection development, transient depletion of DCs had no effect on the induction of Th2 responses or parasite expulsion. These data indicate a novel role for the TGF-β-activating integrin alphavβ8 and DCs in regulating effector immune responses during T. muris infection and may contribute to the development of new anti-parasite therapies.

In mice infected with 10$ sp4$ Giardia muris cysts, a peak lymphocyte proliferation in the spleen and Peyer's patches in response to Giardia extract occurred during the elimination and latent phases, respectively. This shows that the Peyer's patch cells are more responsive than the spleen to Giardia infection. Th2-type cytokines produced by Peyer's patch cells may play a protective role during the latent and acute phases. Th1-type cytokines may contribute to this production during the elimination phase. Cytokine production in response to Giardia extract in vitro was observed in mice immunized with this extract, but not in control mice. Therefore, Giardia antigen can induce cytokine production in vitro in a specific manner.

Trichuris trichiura is a gastrointestinal dwelling nematode that infects almost 500 million people worldwide. T. muris occurs naturally in mice and is very closely related the human whipworm, making it a suitable model to dissect the immune response against the parasite. Studies using the Trichuris muris system have identified CD4+ T cells as dictators of the outcome of infection. In wild type mice, infection with a high dose of T. muris eggs leads to resistance and worm expulsion, which are dependent on a Th2 response and the secretion of type 2 cytokines especially interleukin (IL) 13. Chronicity is dependent on a Th1 response and occurs when mice are infected with a low dose of T. muris eggs. It is well established that metabolic changes are essential to promoting T cell activation and effector function. Moreover, during chronic infection the host immune system is continuously exposed to parasite antigen, which represents a metabolic challenge. This thesis has investigated the importance of T cell metabolism during response against T. muris. Data presented here show that low and high dose T. muris infections promote upregulation of the glycolytic pathway in CD4+ T cells. During later stages of chronic infection, CD4+ T cells displayed supressed glycolysis and mitochondrial respiration, and may be due to metabolic modulation imposed by the parasite. Leucine uptake via the amino acid transporter Slc7a5 was previously shown to be required for mTORC1 activation and for T cell effector function. Data presented here show that in early stages following a high dose T. muris infection, mice that lack Slc7a5 in T cells have delayed worm expulsion, impaired production of antibodies, and lower levels of IL-13. Their CD4+ T cells present reduced glycolytic rates when compared to cells from cohoused infected wild type mice. However, at later stages of infection, antibody, IL-13 and glycolytic levels were restored together with worm expulsion. CD4+ T cells from the early stage of infection showed reduced phosphorylation of mTOR, which suggested that impairment of function was mTOR dependent. Indeed, mice lacking mTOR in T cells fail to expel a high dose of parasites. They showed abrogation of IL-13 production, impairment in antibody class switching and their CD4+ T cells failed to upregulate glycolysis. Thus, this thesis shows that mTOR is essential for the proper functioning of T cells during T. muris infection and efficient amino acid transport plays a significant role. Taken together, these data show that metabolic orchestration of T cell function influences the capacity to effectively control helminth infection and that even subtle changes in T cell metabolic control can have a major effect on response phenotype.