Development and validation of an in vitro model of dendritic cell identification and activation

dc.contributor.advisorBouic, P. J. D.
dc.contributor.authorClark, Anel
dc.contributor.otherStellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.en
dc.descriptionThesis (MScMed)--Stellenbosch University, 2008.en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANSE 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
dc.format.extentix, 214 leaves : ill.
dc.publisherStellenbosch : Stellenbosch University
dc.subjectDendritic cellsen_ZA
dc.subjectMolecular immunologyen_ZA
dc.subjectAntigen presenting cellsen_ZA
dc.subjectTheses -- Medicineen_ZA
dc.subjectDissertations -- Medicineen_ZA
dc.titleDevelopment and validation of an in vitro model of dendritic cell identification and activationen_ZA
dc.rights.holderStellenbosch Universityen_ZA

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