Proper immune function is indispensable, as failure to mount an immune response against a pathogen can lead to serious complications or even death. T cells act by enhancing the activation of phagocytic cells as well as the activation of B cells. Their widespread influence on an immune response makes optimal T cell activation vital. Maximal T cell proliferation and survival is accomplished by stimulation with antigen, a costimulatory signal, and an adjuvant. However, excessive T cell activation can lead to chronic B cell activation and the production of autoantibodies, a hallmark of autoimmune disease. In this thesis, optimal T cell stimulation was studied using an in vivo adoptive transfer model. Results showed that antigen stimulation of T cells along with ligation of the costimulatory molecule OX40 led to an accumulation of antigen-specific cells. OX40 ligation allowed the antigen specific cells to proceed through more cell cycles than cells stimulated with antigen alone. The addition of the adjuvant lipopolysaceharide (LPS) to this system allowed for increased cell survival. However, the continual presence of an adjuvant may also have injurious effects. This was highlighted with the appearance of "Toxic Oil Syndrome" (TOS) in which an adulterated rapeseed oil, an oil with known adjuvant activity, was sold for human consumption. People developed an autoimmune condition characterized by polyclonal B cell activation and autoantibody production. A genetic predisposition was implicated with TOS and was further investigated in this thesis. Although the A. SW mouse has the genetically susceptible genotype, these mice did not develop TOS following exposure to "toxic oil" indicating that other factors may be important in TOS susceptibility. Extending the techniques used in these studies and applying them to the canine immune system was the final topic investigated in this series of studies. Understanding immune pathways of the mammalian immune system is particularly important for comparative studies when dogs are used as models to investigate human immune system disorders. These studies combined will allow for a better understanding of the balance between an optimal immune response and an imbalance leading to hypersensitivity or immunosuppression, as well as interspecies relationships. / Graduation date: 2002

Mesoporous silica particles offer great potential benefits as vehicles for drug delivery and in other biomedical applications. They present a high loading capacity due their ordered and size-tuneable pores that allow molecules to be loaded and released. In addition, they offer the possibility to enhance oral bioavailability of drugs with limited aqueous solubility and to protect pH sensitive drugs from the acidic conditions in the stomach on their way to the intestine. The aim of this thesis was to evaluate the biocompatibility and effects of mesoporous silica particles on immunocompetent cells. Subsequently, two potential life sciences applications were investigated: as adjuvants and as weight reduction agents. Adjuvants are used in vaccines in order to enhance the immunological response towards attenuated and poorly immunogenic antigens. Their function can be mediated through dendritic cells which have a central role in the control of adaptive immunity including immunological memory. Our results show that different types of mesoporous silica particles were able to tune the development of T cells both in human cell cultures and in mice. In contrast to the approved adjuvant alum (aluminium salts) which is a specific inducer of Th2-type immune responses, the particles induced more Th1-like responses, which may be desired in vaccines against allergy and intracellular pathogens such as viruses. Particle exposure to macrophages did not affect their cell function which is crucial for tissue homeostasis, wound repair and in prevention of autoimmune responses. Likewise, the cytokine secretion was not affected, which suggest that macrophages would not modulate the immune response towards the particles. Furthermore, mesoporous silica particles were highly tolerated at daily oral administrations of up to 2000 mg/kg doses for some of the materials prepared. Large pore mesoporous silica particles were shown to act as weight and body fat reduction agents without other observable pathological signs when administered in the diet of obese mice. Together; those results are promising for the development of mesoporous silica as drug delivery systems and adjuvants for oral administration of drugs or vaccines. Additionally, large pore mesoporous silica materials are potential agents for the treatment of obesity.

Thesis (Ph. D.)--Mississippi State University. Department of Biochemistry and Molecular Biology. / Title from title screen. Includes bibliographical references.

Thesis (MD (Anaesthesiology))--University of Pretoria, 2003. / Includes bibliographical references.

published_or_final_version / abstract / toc / Molecular Biology / Doctoral / Doctor of Philosophy

African animal trypanosomiasis is a major threat to African agriculture causing a loss estimated to 4.5 billion US$ per annum. Trypanosoma congolense is the major causative agent in African animal trypanosomiasis and is transmitted by tsetse flies of the Glossina spp. Congopain, a major cathepsin L-like cysteine peptidase in T. congolense is associated with trypanotolerance in N‘Dama cattle and is a target for an anti-disease vaccine. It is suggested that trypanotolerant cattle control the disease by antibody mediated neutralisation of congopain, and that immunisation of cattle against congopain can mimic trypanotolerance resulting in minimised disease pathology. Susceptible cattle immunised with recombinant catalytic domain of congopain, C2, produced high levels of anti-congopain IgG specific antibodies against congopain, maintained weight and exhibited less severe anaemia. However, there was no effect on the establishment of T. congolense infection and acute anaemia development in trypanosusceptible cattle. It has been suggested that failure of congopain to give full protection of the host may be due to poor presentation to the immune system by conventional adjuvants used in previous studies. The aim of the present study was to improve the presentation of the catalytic domain of congopain (C2) to the immune system, by linking it to the proposed molecular adjuvant, BiP, an ER localised HSP70. A further aim was to localise the domain(s) of BiP where the adjuvant properties reside. BiP consists of an ATPase domain (ATPD), a peptide binding domain (PBD) and a C-terminal domain (C-term). Consequently, BiP69, BiP69 lacking the C-terminal domain (BiP60), BiP coding fragments (ATPD, PBD and C-term) and the C2 coding sequence were amplified by PCR from either genomic T. congolense DNA or plasmid DNA. The PCR products were each sub-cloned into a pTZ57RT vector, and C2 cloned into a pET-28a expression vector. The BiP coding fragments were inserted into the recombinant pET-28a-C2 vector, resulting in pET-28a-BiP69-C2, pET-28a-BiP60-C2, pET-28a-ATPD-C2, pET-28a-PBD-C2 and pET-28a-C-term-C2 coding chimeras. The fusion proteins were expressed in an E. coli system as insoluble inclusion bodies at the expected sizes of 96 kDa (BiP69-C2), 88 kDa (BiP60-C2), 47 kDa (PBD-C2), 34 kDa (C-term-C2) and 27 kDa (C2). However, the ATPD-C2 fusion protein was expressed at a larger and smaller size in different attempts. Protein expression was confirmed by western blots using anti-BiP antibodies and anti-congopain N-terminal peptide antibodies. Recombinantly expressed peptide binding domain (PBD)-C2, C-terminus-C2, BiP69-C2, BiP60-C2 chimeras and a BiP69 fusion protein were purified and refolded by a Ni-NTA based one-step on-column refolding method. Bacterial proteins co-purifying with BiP69-C2 and BiP60-C2 chimeras were removed by incubation with 5 mM ATP in the dissociation buffer, but poor yields resulted in using these chimeras as non-pure proteins. Immunisation of Balb/c mice with the BiP69-C2 fusion protein chimera induced a higher antibody response to C2 compared to immunisation with the BiP69/C2 mixture or with C2 in Adjuphos/Quil A. BiP69-C2 and PBD-C2 chimeras and BiP69/C2 mixture induced a robust antibody response to BiP69, but no correlation could be made with the contribution to control of parasitemia and disease induced pathology. Mice immunised with BiP69-C2 and PBD-C2 chimeras showed a better booster effect of T. congolense infection with higher anti-C2 antibody stimulation compared to control groups. Immunisation did not change the establishment of T. congolense infection and anaemia development in most immunised groups. However, mice immunised with the BiP69/C2 mixture and with the PBD-C2 chimera produced anti-C2 antibodies possible contributing to clearing parasites 10 days and 16 days earlier respectively, than mice immunised with BiP69-C2, C-term-C2 and BiP60-C2 chimeras and PBS, C2 and C2 in Adjuphos/Quil A control groups and showed no clinical symptoms of the disease. There was no significant difference in percentage mice survival between BiP-C2 chimera immunised mice and control groups immunised with C2 alone or with a mixture of Adjuphos/Quil A or immunised with PBS. In the present study, it was shown that BiP69 has adjuvant effects when linked to C2 and that its peptide binding domain acts as an adjuvant. It is possible that the removal of the C-terminal domain reduced the adjuvant potency of the peptide binding domain suggesting a prominent role in the adjuvant effect of the BiP molecule. Finding the exact role of the C-terminal domain in the adjuvant effect of BiP would be of utmost interest, and would involve comparing anti-C2 antibody response produced by immunisation with C2 linked to the peptide binding domain with or without the C-terminal domain. Future work includes repeating this study in trypanosusceptible cattle to confirm these findings. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser.