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The effect of aspartate β-hydroxylase inhibition on immune reactions
Krausová, Kateřina ; Šmahel, Michal (advisor) ; Filipp, Dominik (referee)
Aspartate β-hydroxylase (ASPH) is an enzyme that contributes to tumor progression by enhancing the proliferation, migration and invasiveness of cancer cells. Its expression is mostly negligible in normal cells, whereas it is often overexpressed in cancer cells. An inhibitory effect of ASPH on immune cells, specifically on natural killer cells, has also been demonstrated. Thus, the ASPH enzyme could be a new target for cancer immunotherapy. The aim of this thesis was to show the effect of ASPH inhibition on the immune response. Firstly, it was found that ASPH inhibition contributes to the anti-tumor effect of DNA vaccination. The reduction of tumors induced by ASPH inhibition in combination with DNA vaccination was shown to be mainly caused by CD8+ T lymphocytes. Subsequently, the specific activation of T lymphocytes was confirmed by the ELISPOT assay. In case of non-specific activation of T lymphocytes, ASPH inhibition had no effect on the direct activation of CD8+ T lymphocytes. Finally, it was found that plasmacytoid dendritic cells did not contribute to CD8+ T lymphocyte activation after ASPH inhibition. Thus, the results demonstrate that inhibition of ASPH contributes to the activation of adaptive immunity induced by DNA vaccination, but the mechanism of this activation remains unknown.
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Derivation and characterization of tumor cell lines labeled with fluorescent proteins
Majerová, Miriam ; Šmahel, Michal (advisor) ; Drbal, Karel (referee)
The effective treatment of cancer is hindered by the mechanisms of tumor cells allowing them to escape from immunosurveillance. One such mechanism is the downregulation of MHC I expression by tumor cells. As a result, CD8+ T lymphocytes are not able to eliminate tumor cells. These cells are often characterized by different expression of MHC I, which leads to the heterogeneity of the tumor environment. This thesis describes a production of a model of MHC I heterogeneity in tumors. Expression plasmids carrying genes for FP were created. Tumor cell lines TC-1, TC-1/A9 and TC-1/dB2m with different expression of MHC I molecules were successfully labeled with these plasmids. When monitoring stability of FP expression by these cell lines, a decrease was observed both in vitro and in vivo. The assumption that the cytokine environment of the tumor induces FP expression could not be confirmed because of unstable FP expression. In a tumor from a mixture of TC-1+TC-1/dB2m cell lines, it was possible to distinguish between these two lines based on the expression of β2m. In a mixture of TC- 1/A9+TC-1/dB2m lines, that could not be done due to the heterogeneity of TC-1/A9 MHC I expression. The use of combined immunotherapy showed the greatest impact on immune cell infiltration in tumors from a mixture of TC-1+...
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Pre-clinical model of acute promyelocytic leukemia:/study of the anti-leukemic effect induced by ATRA and DNA vaccination
Pokorná, Kateřina ; Holáň, Vladimír (advisor) ; Stöckbauer, Petr (referee) ; Degos, Laurent (referee)
DOCTORAL THESIS 2012 POKORNA Abstract We have used a well characterized transplantable transgenic mouse model which mimics human acute promyelocytic leukemia (APL), both in its biological characteristics and its response to conventional therapeutic drugs. The aim of our study was to better characterize the efficacy of the combined treatment and to determine molecular markers of clinical outcome. We established a minimal residual disease monitoring based on the high sensitivity of detection of PML-RAR transcripts by polymerase chain reaction (PCR) technology in APL mice. We showed that oncogene-specific PCR-based assays allow, like in patients, the diagnosis, follow-up and prediction of disease evolution. Furthermore, PCR assay was used to assess various tissues and organs for the presence of PML-RAR-positive cells in minimal residual disease free long-term survivors. As expected, majority of mice had no measurable tissue level of PML-RAR demonstrating the efficacy of immunotherapy. However, tracking the oncogene-positive cells reveals for the first time that extramedullary PML-RAR-positive cell reservoirs such as the brain may persist and be involved in the leukemia relapse. We aimed at investigating the immune responses involved in the anti-leukemic effect of the combined immutherapy. To evaluate the...
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DNA vaccination
Plachý, Jiří
We have evalueted the protective activity of v-src-derived DNA vaccines against growth of v-src-induced tumors in congenic chicken lines.
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