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

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

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 201-248.

Previous reports indicate that anti-tumor activity arising from systemically injected P. acnes is macrophage-mediated, whereas anti-tumor activity arising from locally injected P. acnes is T cell-mediated. It is possible these P. acnes-induced cytotoxic T cells arise via the Interleukin cascade. Therefore, this study investigated the involvement of Interleukin 2 (IL 2) and Interleukin 3 (IL 3), known components of the Interleukin cascade, in local P. acnes-mediated anti-tumor action. A 500 ug dose of heat-killed stationary phase P. acnes given simultaneously with 10⁴ tumor cells was found to inhibit tumor formation completely, therefore this amount was used as a standard dose throughout the study. Unvaccinated counterparts developed palpable tumors two weeks after tumor cell administration. Lower doses of vaccine protected animals from tumor growth to a lesser degree. A vaccine prepared from logarithmic phase P. acnes exerted a moderate anti-tumor effect in some cases. IL 2 and IL 3 levels were measured in vitro in normal BALB/c mice (N), tumor-bearing mice (TBH), normal vaccinated mice (N+V), and mice receiving both tumor cell and vaccine injection (T+V). IL 2 and IL 3 production was maintained in both N and N+V host splenocyte cultures throughout the study. In a similar fashion, levels of IL 2 and IL 3 in T+V host splenocyte cultures were comparable to those of N+V hosts. However, TBH splenocyte production of IL 2 and IL 3 began to decline when tumors became palpable, at Day 14 after tumor cell inoculation. By Day 28, TBH IL 2 and IL 3 levels were <15% of normal control levels. Causes for this suppression of IL 2 and IL 3 production in TBH were examined. From reports of others it appeared that suppression may be mediated through prostaglandin(s). Addition of the prostaglandin inhibitor indomethacin to splenocyte cultures greatly enhanced IL 2 production by N, N+V and T+V splenocytes, but failed to restore IL 2 production in TBH splenocyte cultures to normal levels. Thus, it appeared prostaglandins were not directly responsible for the majority of suppression seen in TBH. In the non-tumor-burdened host, prostaglandin appeared to play a homeostatic role regarding IL 2 production. Indomethacin-treatment had little effect on IL 3 production. Nylon wool fractionation of N, TBH, N+V and T+V splenocytes suggested a cell removed by nylon wool treatment was largely responsible for the suppression of IL 2 and IL 3 production in TBH. No obvious presence of functional suppressor cells was noted in N, N+V or T+V splenocytes. From these results, it appeared that P. acnes administration maintains and/or restores IL 2 and IL 3 production, thus favoring the production of CTL. In addition, the suppression of IL 2 and IL 3 production seen in TBH may be due to a nylon wool adherent suppressor cell. A model describing the effect of P. acnes administration on local anti-tumor activity was presented. / Master of Science

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

As vacinas pertussis celulares apresentam certa reatogenicidade e as acelulares, menos reatogênicas, têm seu uso restrito, devido a seu alto custo. O Instituto Butantan desenvolveu uma vacina pertussis celular (Plow), com baixo teor de lipopolissacarídeo e outra acelular (Pa), por metodologia simples e econômica. Essas preparações, combinadas aos toxóides diftérico e tetânico (DTPlow e DTPa, respectivamente), foram comparadas à DTP tradicional, com diferentes adjuvantes: vitamina A, surfactante pulmonar, BCG, monofosforil lipídeo A (MPL) e Al(OH)3. A resposta humoral em camundongos foi semelhante para as diferentes formulações e independente do adjuvante utilizado. As vacinas induziram níveis equilibrados de IgG1/IgG2a anti-pertussis e a DTPlow mostrou-se menos reatogênica, induzindo níveis significativamente menores de IL-6 sérica. A adição de MPL sugeriu tendência de proteção contra a colonização nasotraqueal no grupo imunizado com DTPa e levou à indução de IFN-g nos grupos imunizados com DTP e DTPa, sugerindo possível atividade imunomodulatória para Th1. / The whole cell pertussis vaccines present some reactogenicity and the acellular, less reactogenic, have prohibitive use due to its high cost. Instituto Butantan developed a less reactogenic whole cell pertussis vaccine (Plow), with low lipopolysaccharide content and an acellular vaccine (Pa), by simple and economic methodology. These preparations, combined to diphtheria and tetanus toxoids (DTPlow and DTPa, respectively), were compared with the traditional DTP, with different adjuvants: vitamin A, pulmonary surfactant, BCG, monophosphoryl lipid A (MPL) and Al(OH)3. The humoral immune response induced in mice by the different vaccine formulations, was similar and independent of the adjuvant used. The vaccines induced balanced levels of IgG1/IgG2a anti-pertussis and DTPlow showed to be less rectogenic, inducing lower levels of serum IL-6. The use of MPL suggested to induce higher protection against nasotracheal colonization in DTPa group and induced IFN-g in the DTP and DTPa groups, suggesting a possible immunemodulatory activity for Th1.

Paracoccidioidomicose (PCM) é uma doença sistêmica granulomatosa causada pelo fungo termo-dimórfico Paracoccidioides brasiliensis. A gp43 é o principal antígeno diagnóstico que contém o peptídeo de 15 aminoácidos designado como P10, que desenvolve a resposta imune do tipo Th1 dependente de IFN-<font face=\"Symbol\">g. No presente trabalho, comparamos a efetividade de diferentes adjuvantes em camundongos BALB/c infectados intratraquealmente com o isolado virulento Pb18. Alúmen, Adjuvante Completo e Incompleto de Freund, FliC flagelina e o lipídeo catiônico, foram testados em associação ou não com P10. Após 52 dias de infecção, o lipídeo catiônico mostrou os melhores resultados, com o menor número de UFCs, redução de fibrose e baixo número de células fúngicas no tecido pulmonar, além do incremento significativo de IFN-<font face=\"Symbol\">g e TNF-<font face=\"Symbol\">a e a redução dos níveis de IL-4 e IL-10. Estes resultados sugerem que a interação do peptídeo P10 com o lipídeo catiônico pode gerar uma melhor resposta imune mediada por células do tipo Th1, evitando a rápida disseminação da PCM experimental. / Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The gp43 is the major diagnostic antigen that contains the peptide P10. This peptide has 15 amino acids and develops a Th1- dependent IFN-<font face=\"Symbol\">g immune response. In this work, we compare the effectiveness of different adjuvants in BALB/c mice infected intratracheally with virulent isolate Pb18. Alum, Complete Adjuvant and Incomplete Freund\'s adjuvant, fliC flagellin from Salmonella enterica and cationic lipid were tested in combination or not with P10. After 52 days of infection, the cationic lipid showed the best results with the least number of colony forming units, reduction of fibrosis, low number of fungal cells in lung tissue and the significant increase of IFN-<font face=\"Symbol\">g and TNF-<font face=\"Symbol\">a with reduced levels of IL-4 and IL-10. These results suggest that, the interaction of peptide P10 with cationic lipids can lead to a better immune response mediated by Th1 type cells by avoiding the rapid spread of experimental PCM.