The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: Habib Zaghouani. "May 2008" Includes bibliographical references.

<p>The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines.<p>\ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Orientador: Iracilda Zeppone Carlos / Resumo: A esporotricose é uma micose de distribuição universal causada por fungos termodimórficos do complexo de espécies Sporothrix schenckii (S. schenckii). No Brasil, a esporotricose é considerada endêmica, sendo normalmente adquirida pela inoculação acidental do seu agente causal através da pele ou através da transmissão zoonótica por gatos infectados. As formas clínicas podem variar entre cutânea, linfocutânea e sistêmica, esta última sendo mais comumente observada em pacientes imunodeprimidos. A ineficácia do tratamento antifúngico contra esta micose, especialmente em pacientes imunocomprometidos, tem levado à busca de terapias mais eficazes e seguras. Com base em vários estudos que mostram a eficiente utilização de células dendríticas como ferramenta para o desenvolvimento de vacinas contra diferentes fungos, o objetivo deste trabalho foi avaliar a capacidade protetora de células dendríticas derivadas da medula óssea (BMDCs) ativadas com as proteínas da superfície celular de S. schenckii (PSCs) em camundongos infectados com S. schenckii strictu sensu. As BMDCs foram estimuladas com PSCs e analisadas quanto à expressão superficial de moléculas co-estimulatórias, bem como à secreção de citocinas pró-inflamatórias. Posteriormente, camundongos sádios foram vacinados com uma ou duas doses de BMDCs para avaliar a sua imunogenicidade e, por último, foi avaliado o efeito das BMDCs em camundongos infectados por S. schenckii. Nossos resultados mostram que as PSCs foram capazes de ativar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sporotrichosis is a universally distributed mycosis caused by thermodimorphic fungi of the Sporothrix schenckii (S. schenckii) species complex. In Brazil, sporotrichosis is considered endemic and is usually acquired by accidental inoculation of its causative agent through the skin or through zoonotic transmission by infected cats. Clinical forms may vary between cutaneous, lymphocutaneous and systemic, the latter being more commonly observed in immunosuppressed patients. The ineffectiveness of antifungal treatment against this mycosis, especially in immunocompromised patients, has led to the search for more effective and safe therapies. Based on several studies showing the efficient use of dendritic cells as a tool for the development of different fungal vaccines, the aim of this work was to evaluate the protective capacity of bone marrow derived dendritic cells (BMDCs) activated with cell surface proteins of S. schenckii (ScCWP) in mice infected with S. schenckii strictu sensu. The BMDCs were stimulated with PSCs and analyzed for surface expression of costimulatory molecules and the secretion of proinflammatory cytokines. Subsequently, healthy mice were vaccinated with one or two doses of BMDCs to assess their immunogenicity, and finally the effect of BMDCs on S. schenckii infected mice was evaluated. Our results show that the ScCWPs were able to activate BMDCs. Immunization of healthy mice with ScCWPs-stimulated BMDCs induced a Th17 profile immune response, with increased T... (Complete abstract click electronic access below) / Doutor

D. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Cholera is a waterborne disease caused by toxigenic strains of Vibrio cholerae. The spread of cholera in developing countries has largely been imputed to the unavailability of proper water treatment and sanitary infrastructure as well as poor hygiene. In order to prevent the contraction and spread of cholera the use of solar disinfection (SODIS) to treat water in waterborne endemic communities has been recommended by the World Health Organization (WHO). SODIS is a water sterilizing method that relies on natural sunlight to improve the microbiological quality of water. During SODIS the culturability of the water contaminating microorganisms is inactivated by the ultraviolet component of solar radiation. The success of SODIS treatment of water in alleviating the risks associated with the contraction of waterborne diseases such as cholera has been attributed to the effectiveness, with which the water is treated, simple application as well as low cost of materials required. Currently SODIS research has been dominated by studies geared towards understanding how the microbial inactivation occurs, enhancement of the disinfection process and health impact assessments. However, little to no research has been directed towards exploring the role played by the immune system following the consumption of the solar irradiated water pathogens such as V. cholerae. SODIS of microorganisms in water results in immunologically important microbial states and components that could induce an immune reaction or response. In view of the role of dendritic cells in shaping an immune response, the effect that solar irradiated V. cholerae in water has on the immunochemistry of the dendritic cells in vitro was investigated. Prior to the stimulation of the dendritic cells with the solar irradiated cultures of V. cholerae, the first objective required an evaluation on the impact that solar irradiation has on the production and secretion of the cholera toxin by V. cholerae in water. The results from this evaluation showed that solar ultraviolet radiation was incapable of inducing the secretion of the cholera toxin. Furthermore, there was extensive DNA degradation in the solar irradiated cultures of V. cholerae. The second objective was to investigate the ability for solar irradiated cultures of V. cholerae in water to induce the phenotypic maturation of immature dendritic cells in vitro. In order to achieve this objective, solar and non-solar irradiated, chemically/ heat inactivated and phosphate buffered saline (PBS) prepared cultures of V. cholerae as well as lipopolysaccharide (LPS) and cholera toxin-β (CTB) subunit were each used to stimulate immature dendritic cells. After 48 hours of stimulation the dendritic cells were assessed for the expression of CD54, CD80, CD83, CD86, MHC-I and MHC-II on their cell membrane. The results showed an increase in the expression of all the maturation phenotypic markers with CD54, CD86 and MHC-I being the most prominent ones on the surface of the dendritic cells stimulated with solar irradiated cultures of V. cholerae. The third objective was to assess the profile of the cytokines and chemokines secreted by the dendritic cells following their stimulation with solar and non-solar irradiated, chemically/heat inactivated and PBS prepared cultures of V. cholerae as well as LPS and CTB subunit. After 48 hours of dendritic cell stimulation the tissue culture media from each treatment was quantitatively and qualitatively analysed for the presence of interleukin (IL)-1α, IL-1β, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, IL-23, IL-27, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) and tumor necrosis factor (TNF)-α. The analysis revealed that solar irradiated cultures of V. cholerae induced dendritic cells to secrete significant levels of pro-inflammatory cytokines in comparison to the unstimulated dendritic cells. Furthermore the profile of the cytokines and chemokines secreted by the dendritic cells in response to the solar irradiated cultures of V. cholerae in water was similar to that required to induce a T- helper (Th) Th2 immune response. The fourth objective was to assess the expression of the toll like receptor (tlr) genes by the dendritic cells following their stimulation with solar and non-solar irradiated, chemically/heat inactivated and PBS prepared cultures of V. cholerae as well as LPS and CTB subunit. After 48 hours of stimulation total RNA was extracted from the dendritic cells and subjected to real time quantitative polymerase chain reaction (RT qPCR) assay for tlr 1, 2, 3, 4, 5, 6, 9, 11, 12 and 13. The results showed no significant increase or decrease in the expression of most tlr genes in comparison to the unstimulated dendritic cells. This observation is synonyms with dendritic cell maturation. Taken together these findings show that solar irradiated cultures of V. cholerae were able to induce the maturation of immature dendritic cells in vitro. Furthermore dendritic cells stimulated with solar irradiated cultures of V. cholerae produced pro-inflammatory cytokines and chemokines. The results from this study suggests that the consumption of SODIS treated could provide immunological benefits.

Food allergies are an increasing health problem throughout the developed world. Among these, peanut allergy is particularly significant, due to its exceptional severity and frequent lifelong duration. Much of its aetiology remains unclear. In particular, it remains unknown why, unlike other food allergies, peanut allergy incidence correlates poorly with average dietary peanut consumption. A popular explanation for this discrepancy is that peanut allergy is more common in regions where predominantly dry-roasted (DR) peanuts are consumed, leading to speculation that DR-induced chemical modifications may contribute to pathological T_h2 responses in humans. Yet to date, no research group has demonstrated an enhanced immunogenicity of DR peanuts relative to raw in a murine model of sensitisation. This thesis begins with the hypothesis that dry-roasting does indeed alter the chemical composition of peanut proteins in such a way as to increase immunogenicity and allergenicity. To test this hypothesis robustly, I have first addressed flaws in previous studies by developing a methodology to thoroughly characterise samples of raw and DR peanut protein, as well as purifying samples of individual peanut allergens. Using these samples, I have demonstrated an enhanced immunogenicity of DR peanut protein relative to raw, in intragastric, subcutaneous and epicutaneous models of mouse sensitisation, and furthermore, that such enhanced responses feature a pronounced T_h2 bias and functional IgE production. I will present evidence that this difference is not caused by either protein aggregation or the presence of other non-protein substances, but is due to an intrinsic property of the DR peanut proteins. I will go on to clarify candidate molecular mechanisms of this effect, examining several putative receptors and probing the effects of roasting on dendritic cell binding and interactions of peanut proteins. I conclude in light of these investigations that the dry-roasting hypothesis remains the most plausible explanation for the epidemiological distribution of peanut allergy, although many additional questions remain regarding the nature of the chemical modifications produced by roasting and the molecular basis of their recognition by the immune system.

Dendritic cells (DC) are sentinels of the immune system that display an extraordinary capacity to present antigen to naïve T cells and initiate immune responses. DCs are distributed throughout the lungs in the conducting airways of the tracheobronchial tree and in the parenchymal lung, and play a pivotal role in controlling the immune response to inhaled antigens. The respiratory surface is continually exposed to potentially injurious particulates and pathogenic organisms, to which tightly regulated innate and adaptive immunological responses are made. The airways are usually sterile in healthy individuals. However, patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) have increased susceptibility to microbial infections and increased neutrophil elastase (NE) in lung secretions. This thesis was designed to test the hypotheses that; (i) excess NE may result in a dysregulation of lung DCs function in pulmonary chronic diseases, and (ii) the natural NE inhibitors in the respiratory system are able to rescue the NE-mediated dysregulation of DCs and potentially enhance their antigen presenting activity. The data in this thesis demonstrate that purified human NE down-regulated murine bone marrow (BM)-derived DC co-stimulatory molecules (CSM; CD40, CD80 and CD86), which was due to its proteolytic activity. NE-treated LPS-matured DCs were less efficient at presenting ovalbumin (OVA) peptide to naïve OVAspecific transgenic (D011.10) T cells. In addition, immature DCs (iDC) simultaneously treated with LPS and NE failed to mature fully and produced significantly less IL-12 and TNF-α than DCs matured in the presence of LPS alone. Similarly, treatment of mature DC (mDC) with pooled and individual COPD and CF sputum samples caused a reduction in CD80 and CD86 levels (but not CD40) which positively correlated with the NE concentration present in the samples. The demonstration that NE could adversely affect DC phenotype and function suggested that augmentation of NE inhibitors could reverse this process and preserve DC function in inflammatory microenvironments. Over-expression of an NE specific inhibitor (elafin) in the lungs of mice (using either replication-deficient adenovirus [Ad] or elafin transgenic [eTg] mice) increased the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung DCs in in vivo models. Replication-deficient Ad vectors encoding NE inhibitors, namely elafin, secretory leukocyte protease inhibitor (SLPI) and α1-protease inhibitor (α1-PI), were also used to infect DCs in vitro, to further study the effect of these NE-inhibitors on DCs in isolation. These findings suggest that purified NE and NE-containing lung inflammatory secretions are powerful down-regulators of DC maturation, resulting in reduced capacity of these potent APCs to efficiently present antigens; whereas, NE inhibitors could boost immunity by increasing the activation state and/or number of DCs.

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: The aim of this study was to investigate the effect of MBV and Coley’s Toxin on dendritic cells in vitro. The dendritic cell system of antigen presenting cells is the initiator and modulator of the immune response. The principle function of the dendritic cells is to present antigens to resting naïve T lymphocytes: these cells are the only APCs that prime naïve T cells and only mature DCs can carry out this function.Previous studies done on dendritic cells showed that bacterial peptides can induce the maturation of dendritic cells. With the results of these studies in mind we hypothesized that these two vaccines will also induce the maturation of dendritic cells. Chapter 1 is a literature review on the immune system explaining the organs and cells of the immune system. Chapter 2 includes a full description of DCs, the MBV and Coley’s toxin. Also included in this chapter is a short explanation of the principle of the technique being used for the identification and maturation of both mDCs and pDCs, namely the technique of flow cytometry. Chapter 3 describes the method for the phenotypic identification of DCs: the subsets are distinguished by their absence of expression of several lineage markers for lymphocytes, monocytes and NK cells and the expression of CD11c (in the case of myeloid DCs) and CD123 (in the case of plasmacytoid DCs). The inclusion of HLA-DR in addition to the previous described markers allows the discrimination of CD123+ DCs from basophils. The assay requires three tubes per sample which enables quick analysis of these rare subsets with a small sample volume. This assay was applied to peripheral blood samples obtained from healthy individuals and individuals with cancer, HIV and HIV and TB co-infected patients. Our results showed that the maturation status of DCs in HIV and lymphoma were low but those measured in the case of HIV + TB patients were even higher than in the control group. Chapter 4 and 5 describe the in vitro activation and maturation status of DCs following their incubation with bacterial-derived products. Interactions between DCs and microbial pathogens are fundamental to the generation of innate and adaptive immune responses and upon contact with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of human DCs to MBV and Coley’s Toxin. Previous studies showed DCs can be activated with killed Streptococcus pyogenes. With this study in mind it was hypothesized that the MBV and Coley’s Toxin used in this study might modulate DC maturation. The results of this study showed that the MBV and Coley’s toxin did induce the maturation of both pDCs and mDCs as measured by increased surface expression of costimulatory molecules such as CD80 and CD83. Chapter 6 presents the measurement of cytokines released after the PMBCs had been were incubated with Coley’s Toxin and Mixed Killed bacteria. The BD™ Cytometric Bead Array (CBA) flex set was used for the simultaneous detection of multiple soluble analytes. The results indicated that both Coley’s Toxin and the MBV activated the DCs and subsequently induced TH1 as well as a TH2 responses in the T cells present in the cell cultures. Finally, a general conclusion discussing the significance and implications of our results as well as possible future research required is discussed in Chapter 7. DCs are potent antigen presenting cells (APCs) which play a critical role in the regulation of the immune response. There is great interest in exploiting DCs to develop immunotherapies for cancer, chronic infections, immunodeficiency diseases and autoimmune diseases. / AFRIKAANSE OPSOMMING: Die doel van die studie was om die effek van ‘n gemengde bakteriële vaksiene en Coley se toksiene op dendritiese selle te toets in vitro. Die dendritiese sel sisteem speel ‘n belangrike rol in die modulering en reaksie van die immuun sisteem.Die hoof funksie van dendritiese selle is om antigene bloot te stel aan naïewe ongeaktiveerde T selle. Slegs volwasse dendritiese selle kan die T selle aktiveer. Vorige studies het bewys dat bakteriële peptiedes die veroudering van die dendritiese selle kan induseer. Met die resultate in gedagte het ons gehipotiseer dat die twee vaksienes ook die maturasie van dendritiese selle kan induseer. Hoofstuk 1 is ‘n literatuur studie wat handel oor die organe en selle van die immuun sisteem. Hoofstuk 2 gee n volle beskrywing van dendritiese selle, die gemengde bakteriële vaksiene en Coley se toksiene. Ingesluit in die hoofstuk is die beskrywing van die prinsiep van die tegniek, vloei sitometrie, wat gebruik word vir die identifikasie en veroudering status van die dendritiese selle. Hoofstuk 3 beskryf ‘n vloei sitometrie metode vir die fenotipiese identifikasie van dendritiese selle. Dendritiese sel tipes kan onderskei word deur die afwesigheid van sekere merkers vir limfosiete, monosiete en NK selle. Plasmasitoïede dendritiese selle druk CD123 uit en miloïede dendritiese selle druk CD11c uit. HLA DR is ook ingesluit saam met die bogenoemde merkers om die dendritiese selle te onderskei van basofiele. Vir elke toets word slegs drie buise geprosesseer en dus kan die subklasse vinning geanaliseer word. ʼn Klein volume bloed word benodig vir die toests. Perifêre bloed is gebruik vir die toets op bloed monsters van 10 gesonde individue en individue met kanker, HIV en HIV en TB. Die resultate van die studie het getoon dat die maturasie status van die dendritiese selle in HIV en limfoom was, maar in die geval van HIV en TB pasïente was die maturasie status selfs hoër as die van die kontrole groep. Hoofstuk 4+5 beskryf die aktivering en maturasie status van die dendritiese selle na inkubasie met die bakteriële produkte. Interaksie tussen dendritiese selle en patogene speel ‘n belangrike rol in die aktivering van die immuunstelsel. Wanneer dendritiese selle in aanraking kom met bakterieë of bakteriële komponente, matureer die dendritiese sel wat lei tot the uitdrukking van stimulerings molekules, HLA molekules end die uitskeiding van sitokiene. Die uitdrukking van die molekules lei tot limfosiet ontwikkeling en differensiasie. In die studie het ons gekyk na die reaksie van menslike dendritiese selle in die teenwoordigheid van die gemende bakteriële vaksiene en Coley se toksiene. Vorige studies het bewys dendritiese selle word geaktiveer deur Streptococcus pyogenes. Met die resultate in gedagte het ons gehipotetiseer dat die gemengde bakteriële vaksiene en Coley se toksiene ook die maturasie van dendritiese selle kan induseer. Die resultate van die studie het bewys dat die gemengde bakteriële vaksiene en Coley se toksiene die veroudering van beide pDCs en mDCs induseer. Die uitdrukking van verouderings merkers CD80 en CD83 is gemeet. Hoofstuk 6 beskryf ‘n vloei sitometrie metode om die sitokiene te meet wat afgeskei word nadat selle geinkubeer het in die teenwoordigheid van Coley se toksiene en die gemengde bakteriële vaksiene.Die BDTM CBA Flex set metode het dit moontlik gemaak om meer as een sitokiene te meet in net een buis Die resultate het getoon dat albei die vaksienes ‘n TH1 en TH2 reaksie veroorsaak. Laastens volg‘n algemene afleiding waar ons kyk na die toepassing en implikasies van die resultate asook toekomstige navorsings moontlikhede,word bespreek in Hoofstuk 7 Dendritiese selle speel ‘n kritiese rol in die regulering van die immuun reaksie. Verdere studies kan nou gedoen word om dendritiese selle terapeuties toe te pas vir die behandeling van kanker, autoimmuniteit, immuun onderdrukkende siektes en kroniese siektes.

Thesis (MSCMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2005. / There is an urgent need for a safe, effective, affordable human immunodeficiency virus type 1 (HIV-1) vaccine that induces both cellular and humoral immunity. A popular strategy for vaccine design is the use of plasmid DNA encoding HIV-1 genes for priming vaccinations followed by either viral vector or recombinant protein boosting. DNA-based vaccines are attractive because they are safe, easily administered and can induce both cellular and humoral immune responses. In order for DNA vaccination to induce a potent immune response it is necessary for plasmid-encoded genes to be targeted to dendritic cells (DCs) as these are the key antigen presenting cells in natural HIV infection. The immunogenicity of all potential vaccine candidates needs to be assessed in animal models prior to entry into human trials. Nonhuman primates are the best alternative to humans for assessment of vaccine immunogenicity and protective efficacy. In order to clearly understand how DNA vaccines interact with DCs, suitable in vitro DC culture systems for nonhuman primates need to be developed. This study investigated the culture and characterisation of chacma baboon DCs in vitro, and was the first to assess the effect of various transfection methods on baboon DC maturation and function. The study also evaluated the efficacy of a candidate HIV-1 subtype C DNA vaccine at the level of baboon DC transfection, gene transcription and antigen presentation. Generation of immature DCs (iDCs) in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) was accompanied by a loss in the monocyte marker CD14. Expression of the markers CD80 and CD83 was observed on a minority of iDCs, whereas CD86 was expressed on almost all iDCs. Following maturation, all these markers were expressed on an increased number of cells, a pattern of marker expression and upregulation that is similar to that observed in both human and macaque DCs. Transfection of baboon DCs by passive pulsing, lipofection and electroporation was evaluated and compared in several ways. Transfection efficiency, cytotoxicity, the effect of the transfection on DC maturation and subsequent presentation of plasmidencoded antigen to memory T lymphocytes was examined. Baboon DCs lipofected with pDNA efficiently took up HIV-1 subtype C plasmid DNA, transcribed plasmid-encoded genes into mRNA, translated the mRNA into protein, processed the protein and presented peptide antigens to antigen-specific memory T cells. The other methods of transfection were less effective than lipofection due to either decreased transfection efficiency or increased cell cytotoxicity. However, neither lipofection nor passive pulsing in any way negatively impacted on DC marker, CD83, or costimulatory molecule, CD80 and CD86, upregulation. Both methods were found to be as effective as a standard cytokine maturation cocktail in inducing DC maturation. Transfected DCs were also found to be more potent inducers of allogeneic T cell stimulation than their untransfected counterparts, which would appear to indicate enhanced major histocompatibility complex (MHC) expression concurrent with DC maturation marker expression. Lipofection with candidate HIV-1 subtype C vaccine plasmid DNA constructs led to antigen-specific expansion of autologous memory T cells, a finding which indicates the effective expression of plasmid-encoded HIV genes in baboon DCs. This study highlights the functional activity of in vitro generated baboon DCs and provides the groundwork for future studies addressing targeting of plasmid DNA to DCs and enhancement of expression of plasmid-encoded antigens in DCs. A more detailed evaluation of baboon DC interaction with simian immunodeficiency viruses/chimeric simian human immunodeficiency viruses (SIVs/SHIVs) may also reveal how the course of infection in this primate differs from that seen in the macaque or chimpanzee and also how it relates to HIV-1 infection in humans.

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy