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- Azem, Josef, 1961
(author)
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Approaches to analyses of cytotoxic cells. And studies of their role in H. pylori infection
- 2005
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Doctoral thesis (other academic/artistic)abstract
- Helicobacter pylori infection causes chronic gastritis that may progress to peptic ulcers or gastric adenocarcinoma and thereby cause major world-wide health problems. Previous studies have shown that CD4+ T cells and the production of the cytokine IFN- × are important components of the immune response to H. pylori in humans. However, the roles of NK cells and CD8+ T cells ¡V which are major IFN- × producers and have cytotoxic function - are less clear.The aim of this thesis was to develop methods to study NK cells and CD8+ T cells in the context of human H. pylori infection, and to study their role in the immune response toH. pylori in infected subjects.To this end, we first evaluated ways to activate NK cells by H. pylori antigens. This was done by purifying NK cells from peripheral blood, and stimulating the cells by combinations of stimulatory cytokines such as IL-12 and lysate from H. pylori. Furthermore, parallel experiments were performed when the NK cells were separated from the bacterial antigens by an epithelial cell layer. To develop methods to activate and study H. pylori-reactive CD8+ T cells, we initially evaluated whether dendritic cells (DC), B cells or monocytes pulsed with H. pylori antigens could efficiently activate CD8+ T cells from H. pylori infected or non-infected individuals. In order to study proliferation of CD8+ T cells in cell cultures, the cells were stained using the fluorescent dye CFSE, which allows analysis of proliferation of single cells in a mixed cell population. Furthermore, the presence of cytotoxic activity among the H. pylori-reactive CD8+ T cells was analysed mainly by the cytotoxicity-related molecules granzyme A and B, and IFN- ×. This was done using intracellular analysis of granzyme B and IFN- × expression, and by analysing the secretion of granzyme A and B into the supernatants by H. pylori-activated CD8+ T cells.Our results show that highly purified NK cells can be activated by H. pylori antigens, and that there is a synergistic effect of H. pylori and IL-12 in the activation of NK cells. Furthermore, we show that in H. pylori-infected individuals, there are H. pylori-reactive memory CD8+ T cells that proliferate, produce IFN- × and secrete granzyme A after activation. We show that these cells can be activated both by B cells and DC pulsed with H. pylori antigens, but for practical purposes, B cells are preferable to use as APC.In conclusion, in this thesis we show that cytotoxic cells may contribute to the immunity against H. pylori in infected individuals by production of IFN- ×; but there are also indications that cytotoxic activity is involved. These findings may be of importance for the further study of NK-cell and cytotoxic T lymphocytes (CTLs) activity in subgroups of H. pylori-infected individuals, especially in relation to protection against H. pylori-induced gastric cancer development.
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| 2. |
- Azem, Josef, 1961, et al.
(author)
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B cells pulsed with Helicobacter pylori antigen efficiently activate memory CD8+ T cells from H. pylori-infected individuals
- 2005
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In: Clin Immunol. - : Elsevier BV. ; 118:2-3, s. 284-91
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Journal article (peer-reviewed)abstract
- Helicobacter pylori infection causes chronic gastritis that may progress to peptic ulcers or gastric adenocarcinoma and thereby cause major world-wide health problems. Previous studies have shown that CD4+ T cells are important in the immune response to H. pylori in humans, but the role of CD8+ T cells is less clear. In order to study the CD8+ T cell response to H. pylori in greater detail, we have evaluated efficient conditions for activation of CD8+ T cells in vitro. We show that H. pylori-reactive CD8+ T cells can be activated most efficiently by B cells or dendritic cells pulsed with H. pylori antigens. We further show that the majority of CD8+ T cells in H. pylori-infected gastric mucosa are memory cells, and that memory CD8+ T cells sorted from peripheral blood of H. pylori-infected individuals respond 15-fold more to H. pylori urease compared to memory cells from uninfected subjects. We conclude that CD8+ T cells do participate in the immune response to H. pylori, and this may have implications for the development of more severe disease outcomes in H. pylori-infected subjects.
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| 3. |
- Sun, Jia-Bin, 1948, et al.
(author)
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Vaccination with dendritic cells pulsed in vitro with tumor antigen conjugated to cholera toxin efficiently induces specific tumoricidal CD8+ cytotoxic lymphocytes dependent on cyclic AMP activation of dendritic cells.
- 2004
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In: Clinical immunology (Orlando, Fla.). - : Elsevier BV. - 1521-6616. ; 112:1, s. 35-44
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Journal article (peer-reviewed)abstract
- We investigated the development of CD8+ tumor-specific cytotoxic lymphocytes (CTL) and protection against tumor growth after vaccination with bone marrow-derived dendritic cells (DC) pulsed with a model protein ovalbumin conjugated to cholera toxin (OVA-CT) in B6 mice using E.G7 tumor cells expressing OVA(257-264) peptide (SIINFEKL) as target cells in vitro and in vivo. Vaccination with OVA-CT-pulsed DC concurrently induced strong CTL in vitro activity and anti-E.G7 tumor protection in vivo in WT, NK-depleted and CD4-deficient mice as well as in IL-12-/- and IFN-gamma-/- mice but not in CD8-deficient mice. Importantly, activation of CTL by OVA-CT-pulsed DC was dependent on CT-induced activation of adenylate cyclase and increased cAMP production by DC associated with increased expression of MHC class I and co-stimulatory molecules (CD80, CD86 and CD40). These results show that vaccination with DC pulsed with antigens (Ag) conjugated to CT induces a strong CTL response and suggest that conjugation of tumor Ag to CT for DC vaccination represents a promising approach for tumor vaccination and immunotherapy.
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| 4. |
- Wising, Catharina, 1973, et al.
(author)
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Induction of apoptosis/necrosis in various human cell lineages by Haemophilus ducreyi cytolethal distending toxin.
- 2005
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In: Toxicon : official journal of the International Society on Toxinology. - : Elsevier BV. - 0041-0101. ; 45:6, s. 767-76
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Journal article (peer-reviewed)abstract
- We investigated the impact of highly purified Haemophilus ducreyi cytolethal distending toxin (HdCDT) on the apoptosis and necrosis of various human cells; including myeloid cells, epithelial cells, keratinocytes, and primary fibroblasts. The levels of apoptosis and necrosis induced in these cells were compared to those induced by HdCDT in human T cells and in the Jurkat T cell line. Levels of caspase-3 activity were measured, and membrane changes like phosphatidylserine (PS) translocation was evaluated after double-staining with the fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) using flow cytometry. HdCDT induced various degrees of apoptosis and necrosis in dose- and time-dependent manners in cells of various lineages. Early and late apoptosis (annexin V-stained cells) were induced in more than 90% of T cells and monocytes after treatment with 100 ng/ml HdCDT for 24 and 48 h, respectively. The corresponding numbers for epithelial cells, keratinocytes, and fibroblasts were 26-32% after treatment with 100 ng/ml HdCDT for 48 h. HdCDT appears to eliminate effectively by inducing apoptosis those cells that are involved in immune responses. Epithelial cells, keratinocytes and fibroblasts, which are important for the healing of chancroid ulcers, are eliminated by apoptosis or necrosis after contact with HdCDT, albeit slower and to a lesser extent than T cells.
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| 5. |
- Yun, C. H., et al.
(author)
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Natural killer cells and Helicobacter pylori infection: bacterial antigens and interleukin-12 act synergistically to induce gamma interferon production
- 2005
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In: Infect Immun. - 0019-9567. ; 73:3, s. 1482-90
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Journal article (peer-reviewed)abstract
- Helicobacter pylori is known to induce a local immune response, which is characterized by activation of lymphocytes and the production of IFN-gamma in the stomach mucosa. Since not only T cells, but also natural killer (NK) cells, are potent producers of gamma interferon (IFN-gamma), we investigated whether NK cells play a role in the immune response to H. pylori infection. Our results showed that NK cells were present in both the gastric and duodenal mucosae but that H. pylori infection did not affect the infiltration of NK cells into the gastrointestinal area. Furthermore, we could show that NK cells could be activated directly by H. pylori antigens, as H. pylori bacteria, as well as lysate from H. pylori, induced the secretion of IFN-gamma by NK cells. NK cells were also activated without direct contact when separated from the bacteria by an epithelial cell layer, indicating that the activation of NK cells by H. pylori can also occur in vivo, in the infected stomach mucosa. Moreover, the production of IFN-gamma by NK cells was greatly enhanced when a small amount of interleukin-12 (IL-12) was added, and this synergistic effect was associated with increased expression of the IL-12 receptor beta2. It was further evident that bacterial lysate alone was sufficient to induce the activation of cytotoxicity-related molecules. In conclusion, we demonstrated that NK cells are present in the gastroduodenal mucosa of humans and that NK cells produce high levels of IFN-gamma when stimulated with a combination of H. pylori antigen and IL-12. We propose that NK cells play an active role in the local immune response to H. pylori infection.
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