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Polymeric-drug Conjugates in Treatment of Head and Neck Squamous Cell Carcinoma
Betka, Jaroslav ; Říhová, Blanka (advisor) ; Prausová, Jana (referee) ; Reiniš, Milan (referee)
Malignant diseases are after cardiovascular diseases the second most common cause of death in the developed countries. The number of patients newly diagnosed with cancer is constantly rising, on average by 2.5% yearly. This trend also applies to squamous cell carcinoma of the head and neck, which is the sixth most common oncological disease. Its occurrence is most influenced by two external risk factors: smoking and abuse of alcohol; latest research indicates that for the emergence of neoplasia is crucial not only the amount of and duration of the use of a harmful substance but also the time of day when a harmful substance is used as well as psychological dependence on the substance. But recently, we are witnessing growth of the frequency of occurrence of the squamous cell carcinoma of the pharynx with non-smokers and younger age groups in connection with presence of the HPV infection in tumor. The forms of so far most commonly used anticancer therapy (surgery, radiotherapy, chemotherapy) have, despite progressions over the last decades, lead to significant improvement only with some types of cancer but in the case of squamous cell carcinoma of the head and neck is the total probability of five-year survival still low, approximately 40%. Therefore we are turning our attention to the new forms of...
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Epigenetic mechanisms in the regulation of the B7-H1 and IRF-1 expression in tumour cells.
Hrušková, Veronika ; Reiniš, Milan (advisor) ; Krulová, Magdaléna (referee)
Interferon γ is an important T-cell helper type 1 (Th1) cytokine involves in antimicrobial immunity. It is a part of the inflammatory immune response in the site of infection. However, for its proper function, the regulation of immunity is necessary to avoid injury of the tissue caused by long-term inflammation. While interferon γ triggers expression of proinflammatory genes, it also regulates genes which inactivate immune response. The B7-H1 molecule belongs among these inhibitory regulators. Furthermore, antitumour effect of interferon γ is well-known as well. After extensive experiments, interferon γ was tested as an immunotherapeutic drug against melanomas in clinical trials. However, the trials had to be terminated prematurely because of unsuccessful results. It started to be clear that interferon γ could have also a protumour effect. Interferon γ upregulates the expression of B7-H1 molecule which aids tumour in escape from immunity. The B7-H1 molecule possesses a binding site for interferon regulatory factor 1 (IRF-1) in its promoter region. This IRF-1 is induced by interferon γ - JAK/STAT signalling pathway. In our previous research, we observed interferon γ induced DNA demethylation of promoters in genes that are involved in antigen presenting machinery. Additionally, DNA methylation of...
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Epigenetic mechanisms in the regulation of antigen presentation and anti-tumour immunity.
Vlková, Veronika ; Reiniš, Milan (advisor) ; Plachý, Jiří (referee) ; Němečková, Šárka (referee)
Veronika Vlková: Epigenetic mechanisms in the regulation of antigen presentation and anti-tumour immunity. Charles University in Prague, Faculty of Science, Molecular and Cellular Biology, Genetics and Virology Dissertation, 2014 Reversible downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, is frequently associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFN-γ. Here we describe association of DNA demethylation of selected antigen-presenting machinery gene regulatory regions upon IFN-γ treatment with MHC class I upregulation on tumour cells thus demonstrating that IFN-γ acts as an epigenetic modifier. Our results cast more light on the role of DNA methylation in tumour cell escape from specific immunity. Treatment of MHC class I deficient tumour by epigenetic modifiers sensitized neoplasia to the immunotherapy. Our data also provide knowledge about differentiation cancer chemotherapies, especially for use in combination with other drugs to achieve lower immunosuppressive function of tumour microenvironment. In addition, our data provide evidence that besides the known targets of epigenetic agents or immunoregulatory antibodies other...
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DNA methylation changes in patients with acute myeloid leukemia
Hájková, Hana ; Haškovec, Cedrick (advisor) ; Hejnar, Jiří (referee) ; Reiniš, Milan (referee)
DNA methylation is a well-established epigenetic mechanism regulating gene expression. It has essential functions in cells under physiological as well as pathological conditions. In acute myeloid leukemia (AML), aberrant DNA methylation has been confirmed in the pathogenesis and progression of the disease. Changes in DNA methylation of promoters, or other regions, are studied primarily with respect to pathways that are involved in tumor transformation and DNA methylation impact on prognosis. Clinical importance of DNA methylation has been confirmed by a number of recent publications. According to the Cancer Genome Atlas (TCGA) Research Network, mutations of genes involved in DNA methylation are found in 44% of AML patients at diagnosis. However, the impact of these mutations on specific DNA methylation and gene expression remains controversial. We examined 79 AML patients at diagnosis for DNA methylation of 12 selected genes (CDKN2B, CALCA, CDH1, ESR1, SOCS1, MYOD1, DAPK1, TIMP3, ICAM1, TERT, CTNNA1, EGR1) - some of them proved as tumor suppressor genes and 24 HOX genes, and in parallel for mutations in DNMT3A. We observed lower levels of DNA methylation (P<0.0001) as well as lower numbers of concurrently hypermethylated genes (P<0.0001) in patients with DNMT3A mutations. Our study of the impact of...
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Role of microbiota and gut inflammation in the pathogenesis of experimental colorectal cancer
Klimešová, Klára ; Tlaskalová - Hogenová, Helena (advisor) ; Vítek, Libor (referee) ; Reiniš, Milan (referee)
Mucosal surface of the gut is in continuous contact with foreign compounds derived from diet as well as from commensal or pathogenic microorganisms. Thousands of years of symbiosis resulted in tight cooperation between the host and its microbiota. Microbiota composition and metabolism actively influence host's physiological as well as pathological processes. Chronic inflammation is characterized by prolonged active inflammatory response associated with tissue damage. This status results from accumulation of defects in various factors including gut barrier functions as well as mechanisms of innate and adaptive immunity. It's commonly accepted that chronic inflammatory diseases of the gastrointestinal tract like IBD, are associated with an increased risk of CAC development. Two publications related to this thesis deal with modulatory effects of peroral administration of components of commensal bacteria or probiotics on intestinal inflammation. Using acute or chronic model of DSS-induced colitis, we demonstrated that oral treatment of BALB/c mice with membranous fraction of the commensal, Parabacteroides distasonis, as well as with lysate of probiotic bacterium Lactobacillus casei DN-114 001 significantly reduces the severity of intestinal inflammation. Moreover, the treatment was associated with reduction of...
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Molecular mechanisms of fibroblastoid cell phenotype transitions:dedifferentiation of myofibroblasts and influencing of invasiveness and metastasis of sarcoma
Kosla, Jan ; Dvořák, Michal (advisor) ; Peková, Soňa (referee) ; Reiniš, Milan (referee)
Fibroblasts are the principal cellular component of the connective tissue. They are a heterogeneous group of cells which contribute to the structure of connective tissue and wound healing by their ability to produce extracellular matrix (ECM). Fibroblasts and cells derived from them are involved in many pathological processes such as formation of malignant tumors and fibrosis. Tumor progression which finally leads to metastasis is a serious biomedical problem. There is a growing body of the recent evidence showing an important role of the tumor stroma and its interaction with cancer cells in cancer progression. Tumor stroma comprises mainly of myofibroblasts and their products, namely ECM, soluble factors, and enzymes. Myofibroblasts contribute more or less to all steps of cancer progression. Furthermore myofibroblasts play a key role in fibrosis, another serious human disease which is not efficiently treatable and which is associated with cancer progression. These facts made us to search for molecular means capable of eliminating the myofibroblastic phenotype. We succeeded to entirely dedifferentiate primary myofibroblasts by concomitant inhibition of TGFβ signaling and perturbation of MAPK signaling in a chick model that we have introduced. Malignant fibroblasts form sarcomas. ECM is the first...
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Studies of the Mouse polyomavirus: properties of the minor structural proteins, requirements for virion productive trafficking to the nucleus and observed side effects of DNA transfection.
Huérfano- Meneses, Sandra ; Forstová, Jitka (advisor) ; Mělková, Zora (referee) ; Reiniš, Milan (referee)
In this thesis, we aimed to investigate the role of Mouse polyomavirus (MPyV) minor structural proteins, VP2 and VP3 in the viral life cycle as well as to study specific aspects of the productive trafficking of the virus. The thesis is divided into 3 chapters as follows: chapter 1 addresses questions about the interaction of the minor structural proteins, VP2 and VP3, of MPyV with host cells and their role during late stages of virus infection. In the chapter 2, aspects concerning the role of late endosomes, recycling endosomes and actin cytoskeleton in MPyV productive infection are addressed and in the chapter 3, results of studies of interferon (IFN) responses induced after DNA nucleofection are presented. The study in chapter 3 was preceded by a microarray analysis primary performed to reveal cell responses to the presence of the minor proteins within host cells. We showed that both MPyV minor structural proteins, VP2 and VP3, when produced individually (without VP1) are highly cytotoxic, inducing fast, caspase dependent apoptosis. Immuno-electron and confocal microscopy revealed affinity of both proteins to endocellular membranes. Both VP2 and VP3 were found to be bound to damaged ER and mitochondrial membranes. Interaction of the proteins with membranes causing physical damage of organelles...
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DNA methyltransferase inhibitors and their effect on the activation of genes being of importance to a presentation of the antigen in tumour cells
Cebová, Magdaléna ; Reiniš, Milan (advisor) ; Poljaková, Jitka (referee)
DNA methylation is one of the epigenetic modulations that can be associated with tumour diseases. DNA methyltransferases act as catalysers of the process of DNA methylation and their inhibition could be used as a possible approach to the therapy of tumours. DNA methyltransferase inhibitors are classified into two groups, nucleoside inhibitors that are incorporated into DNA and subsequently form covalent bonds with DNA methyltransferase, and non-nucleoside inhibitors whose action is limited to inhibiting the catalytic site of the DNA methyltransferase. The first objective of this bachelor thesis was to compare the toxicity of DNA methyltransferase inhibitors in mouse tumour cell lines. The results show that the toxicity of nucleoside inhibitors is much higher (due to their incorporation into the DNA) than that of non-nucleoside inhibitors. The second objective was to compare the effect of DNA methyltransferase inhibitors on the expression of MHC (major histoncompatibility complex) class I glycoproteins on the surface of mouse tumour cells. Reduced expression of MHC class I glycoproteins is known to be one of the mechanisms used by the tumour cells to escape the immune system. Our laboratory has shown that some inhibitors (5-azacytidine) increase the expression of MHC I class molecules in MHC...
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Gene Modified Cellular Vaccines against bcr-abl-transformed Cells
Petráčková, Martina ; Vonka, Vladimír (advisor) ; Forstová, Jitka (referee) ; Reiniš, Milan (referee)
of PhD. Thesis Gene Modified Cellular Vaccines against bcr-abl-Transformed Cells In our laboratory we are focused on the development of therapeutic vaccines against chronic myeloid leukaemia (CML), using as a model system mouse bcr-abl-transformed B210 and 12B1 cells. Both these cells are of early B-cell lineage, express BCR-ABL protein and induce leukaemia after i.v. inoculation; the 12B1 cells also induce solid lymphoma-like tumours after s.c. inoculation. Several types of experimental vaccines directed against these bcr-abl-transformed cells were developed in our laboratory. Since it has been recognized that BCR-ABL protein does not carry the immunodominant epitope, our attention switched to the development of cell-based vaccines that are capable of inducing immune responses against a whole complex of tumour-associated antigens. The present work describes studies on experimental cellular vaccines based on the B210 or 12B1 cells, gene-modified to express either IL-2 or GM-CSF or IL-12. For the transfection of these cells, an optimised electroporation method was used. The vaccines were tested in our mouse BALB/c model. All the cell lines derived from B210 cells secreting IL-2, GM-CSF or IL-12 were non-oncogenic. The oncogenicity of the IL-2 producing 12B1 sublines was reduced and most of the...
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Immunotherapy of HPV16 - associated cancers and regulation of antitumour immune response
Štěpánek, Ivan ; Reiniš, Milan (advisor) ; Lipoldová, Marie (referee) ; Němečková, Šárka (referee)
The MHC class I status of tumour cells during immunotherapy is often underestimated. It represents one of important tumour escape mechanisms and thus can contribute to the failure of most of the cancer clinical trials that are usually based on the induction of cytotoxic T cell responses. Epigenetic changes in the promoters of genes involved in the MHC class I Ag presentation can result in decreased expression of the cell surface MHC molecules on tumour cells. Thus, epigenetic modifiers can restore an expression of the MHC class I molecules and make tumours visible to the CD8+ effector cells. Besides the epigenetic changes on the tumour cells, epigenetic modulators affect cells of the immune system such as dendritic cells (DC). Tumour cells can escape from the immune response not only by changes in the cancer cells, but also by influencing, expanding and/or activating immunoregulatory cell populations, such as regulatory T cells (Treg). This thesis focuses on the potential of the DC-based vaccines against HPV-16-associated tumours with a different MHC class I expression, on the combination of cancer immunotherapy with the treatment using epigenetic modifiers, with special attention paid to their effects on DC, and, finally, on the impacts of the anti-CD25 antibody (used for Treg elimination) on Treg and NKT...
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