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

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Dendritic Cells (DCs) are professional antigen presenting cells that play a crucial role in the induction of immune tolerance. Although DCs have been a potential target for immunotherapy, the amount of DCs in blood source is limited and ex vivo expansion has been inefficient. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide a great source in cell-based therapy because of their self-renewal ability and pluripotency. My project focuses on generating tolerogenic DCs (tDCs) from human pluripotent stem cells (i.e. hESCs and iPSCs) and their characterization. Specifically, hESCs and hiPSCs were first differentiated to hematopoietic progenitor cells (HPCs) using three different methods (i.e. bone-marrow stromal cell co-culture and two previously reported defined medium methods). The hESC/iPSC-differentiated hematopoietic progenitor cells (HPCs) were characterized by their surface phenotype using flow cytometry. Then the hESC/iPSC-differentiated immature DCs were further expanded and differentiated from the hESC/iPSCdifferentiated CD34+ HPCs with the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin 4 (IL-4). Tolerogenic properties were introduced by treating hESC-differentiated DCs with rapamycin. The treated DCs were characterized for their tolerogenicity by examining their expression of PDL1, PDL2, ICOS and CD40 etc., and their ability to promote regulatory T cells (Treg) differentiation. All these were compared with monocyte-derived tDCs. In summary, this study has examined the potential of using pluripotent stem cells-derived DCs as a cell source for immune tolerance induction therapy. / published_or_final_version / Anatomy / Master / Master of Philosophy

Ng, Sin Man. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 96-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cytomegaloviruses (CMVs) are ubiquitous in nature, having evolved over many millenia with their hosts. While in healthy hosts most infections with CMV are asymptomatic, the virus can cause severe disease in immunocompromised hosts. Thus, the increase in organ transplantation and the HIV/AIDS pandemic have established human CMV (HCMV) as a clinically important pathogen. Indeed, HCMV infections are now the major cause of morbidity and mortality among immunocompromised patients, which has led to more research targeting CMV for effective anti-viral treatment. The discovery that cytomegaloviruses encode several genes which are involved in immune escape has prompted a new area of research, aimed at understanding immune escape mechanisms for exploitation as potential anti-viral therapeutics. By targeting the viral proteins directly, or their receptors in the host, it may be possible to treat CMV disease by agonistic/antagonistic therapy. The first part of this thesis describes the first demonstration of anti-NK1.1 staining in situ to identify NK cells using a modified in vivo perfusion/fixation method. Using this method, we have compared the acute NK1.1+ cellular response to wild-type MCMV infection in the visceral organs of genetically susceptible intra-NK complex recombinant BALB.B6-CT6 (Cmv1s, NK1.1+) mice with resistant C57B⁄J (Cmv1r, NK1.1+) and BALB.B6-Cmv1r mice (Cmv1r, NK1.1+). Expression of viral antigens and the consequences of infection on other cellular subsets, were also analyzed in this study. The data show that in susceptible mice (Cmv1s) MCMV infection is predominent in the marginal zone of splenic white pulp, resulting in local changes in various cellular constituents, including macrophages, NK cells and DC. In the liver, distinct foci of infection were comprised of large numbers of macrophages and NK1.1+ cells surrounding infected cytomegalic cells. In resistant mice (Cmv1r), 6 MCMV infection predominantly affected the red-pulp of the spleen and was associated with increased accumulation of NK1.1+ cells and macrophages at sites of viral infection

A resposta imune adaptativa do tipo Th1 é necessária para proteção contra P. Brasiliensis. Sabendo que células dendrítica são APCs eficientes na ativação da resposta imune mediada por células, investigamos o potencial dessas células em iniciar a resposta imune inata em camundongos suscetíveis (B10.A) e resistentes (A/J) a PCM. Inicialmente, observamos que células dendríticas pulmonares de camundongos B10.A são mais fagocíticas quando comparadas com células de camundongos A/J. Além disso, observamos que a fagocitose na presença de laminarina foi inibida somente em células dendríticas pulmonares de animais B10.A. A produção de citocinas por células dendríticas pulmonares de camundongos A/J mostrou baixa concentração de IL-10, IL-12 e TNF-α. Ao contrário, células dendríticas pulmonares de camundongos B10.A produziram altas concentrações de TNF-α e IL-10, mas, a produção de IL-10 foi significativamente inibida na presença de laminarina. Nós também observamos que células dendríticas pulmonares de camundongos TLR-2KO foram deficientes na produção de IL-10. Além disso, a expressão gênica para TLR-2 aumentou após infecção em camundongos B10.A, mas não nos A/J. Posteriormente, observamos que a capacidade de células dendríticas pulmonares de camundongos suscetíveis em induzir ativação de células T foi diminuída. De acordo com nossos resultados, sugerimos que P. brasiliensis induz células dendríticas regulatórias em camundongos suscetíveis, os quais promovem a produção de IL-10, contribuindo para a suscetibilidade de camundongos B10.A contra a infecção por P. brasiliensis. / An adaptive Th1-type immune response is required for protection against P.brasiliensis. Knowing that DC are the most effective APCs for inducing cellmediated immune responses, it is thus important to investigate lung DC and their potential to initiate an immune response in mice susceptible and resistant to PCM. Initially, we observed that lung DC from susceptible mice were more phagocytic than cells from resistant mice and we observed that phagocytosis in the presence of laminarin was inhibited only in DC from susceptible mice. Cytokines produced by DC from resistant mice showed a low concentration of IL-10, IL-12 and TNF-α. In contrast, DC from susceptible mice produced a high concentration of TNF-α and IL-10, but IL-10 production was significantly inhibited in the presence of laminarin. We also observed that DC from TLR-2KO mice presented a defective production of IL-10. We found that the gene expression for TLR2 is increased after infection in B10.A, but not in A/J mice. Thus, the capacity of lung DC from susceptible mice in inducing T cell activation was decreased. In conclusion, our data suggest that P.brasiliensis induces regulatory DC in susceptible mice, which promotes IL-10 production contributing to the susceptibility of B10.A mice against P.brasiliensis infection.

A resposta imune adaptativa do tipo Th1 é necessária para proteção contra P. Brasiliensis. Sabendo que células dendrítica são APCs eficientes na ativação da resposta imune mediada por células, investigamos o potencial dessas células em iniciar a resposta imune inata em camundongos suscetíveis (B10.A) e resistentes (A/J) a PCM. Inicialmente, observamos que células dendríticas pulmonares de camundongos B10.A são mais fagocíticas quando comparadas com células de camundongos A/J. Além disso, observamos que a fagocitose na presença de laminarina foi inibida somente em células dendríticas pulmonares de animais B10.A. A produção de citocinas por células dendríticas pulmonares de camundongos A/J mostrou baixa concentração de IL-10, IL-12 e TNF-α. Ao contrário, células dendríticas pulmonares de camundongos B10.A produziram altas concentrações de TNF-α e IL-10, mas, a produção de IL-10 foi significativamente inibida na presença de laminarina. Nós também observamos que células dendríticas pulmonares de camundongos TLR-2KO foram deficientes na produção de IL-10. Além disso, a expressão gênica para TLR-2 aumentou após infecção em camundongos B10.A, mas não nos A/J. Posteriormente, observamos que a capacidade de células dendríticas pulmonares de camundongos suscetíveis em induzir ativação de células T foi diminuída. De acordo com nossos resultados, sugerimos que P. brasiliensis induz células dendríticas regulatórias em camundongos suscetíveis, os quais promovem a produção de IL-10, contribuindo para a suscetibilidade de camundongos B10.A contra a infecção por P. brasiliensis. / An adaptive Th1-type immune response is required for protection against P.brasiliensis. Knowing that DC are the most effective APCs for inducing cellmediated immune responses, it is thus important to investigate lung DC and their potential to initiate an immune response in mice susceptible and resistant to PCM. Initially, we observed that lung DC from susceptible mice were more phagocytic than cells from resistant mice and we observed that phagocytosis in the presence of laminarin was inhibited only in DC from susceptible mice. Cytokines produced by DC from resistant mice showed a low concentration of IL-10, IL-12 and TNF-α. In contrast, DC from susceptible mice produced a high concentration of TNF-α and IL-10, but IL-10 production was significantly inhibited in the presence of laminarin. We also observed that DC from TLR-2KO mice presented a defective production of IL-10. We found that the gene expression for TLR2 is increased after infection in B10.A, but not in A/J mice. Thus, the capacity of lung DC from susceptible mice in inducing T cell activation was decreased. In conclusion, our data suggest that P.brasiliensis induces regulatory DC in susceptible mice, which promotes IL-10 production contributing to the susceptibility of B10.A mice against P.brasiliensis infection.