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Regulation of the B7-H1 (CD274) inhibitory molecule expression on tumour and immune cells.
Hrušková, Veronika ; Reiniš, Milan (advisor) ; Krulová, Magdaléna (referee)
The co-stimulatory/inhibitory molecules of family B7 that are expressed on the surface of antigen-presenting and other cells, including tumour cells, play an important role in immune responses regulations. Owing to binding on the T-lymphocyte receptors, they regulate their positive and also negative responses. An important co-stimulatory member of B7 family is B7-H1 molecule. This molecule is presented in many different tissues, not only on haematopoietic cells. However, it is also expressed on tumour cells. Its two binding receptors are in the cytoplasmic membrane of T-cells. The first receptor is unknown and the second receptor is PD-1 (CD279). The linkage between B7-H1 - PD-1 regulates negatively proliferation, differentiation and cytokine secretion of T-cells. This negative regulation is crucial for the constitution of peripheral immune tolerance and also for escape of tumour cells from T-lymphocyte based anti-tumour responses. Although the mechanisms underlying the B7-H1 cell-surface expression have been intensively studied, and they are not fully understood, especially in tumour cells. IFNγ and TNFα regulate B7-H1 expression on transcription level as well as on post- transcription level. Binding sites for transcription factors IRF-1 and NF-κB are located in B7- H1 promoter region. The IRF-1...
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Impact of pattern and functional properties of tumor-infiltrating immune cells for clinical outcome of head and neck cancer
Hladíková, Kamila ; Špíšek, Radek (advisor) ; Plzák, Jan (referee) ; Reiniš, Milan (referee)
Head and neck squamous cell carcinoma encompasses a complex and heterogeneous group of malignant diseases. Originally, this tumor type was associated with tobacco and alcohol consumption. However, a significantly expanding subset of tumors associated with oncogenic human papillomavirus infection arising in deep tonsillar crypts was identified within the last decades. Due to the essential role of the immune system in antiviral and anticancer immune response, the prognosis of patients is significantly influenced by the volume, composition and functional capacity of the immune infiltrate. The immunosuppressive landscape of head and neck cancer leads to unfavorable outcome of patients and decreased efficacy of immunotherapy. The response rate to standard treatment is high, however, standard therapy is accompanied by considerable toxicity influencing the quality of life. In 2016, the first immunotherapeutics for the treatment of patients with recurrent squamous cell carcinoma of the head and neck were approved - the anti-PD-1 immune checkpoint inhibitors nivolumab and pembrolizumab. This type of therapy, based on mitigation of immunosuppression, shows strong efficacy and less toxicity in combination with other therapies. Therefore, anti-PD-1 immunotherapy was recently approved in the first-line...
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Construction of various types of vaccines based on the structural proteins of mouse polyomavirus and analysis of immune response after their administration to mice
Hrušková, Veronika ; Forstová, Jitka (advisor) ; Roubalová, Kateřina (referee) ; Reiniš, Milan (referee)
Conclusion Humoral and cellular immune responses developed in mice after intranasal delivery of model mouse polyomavirus derived VLPs carrying epitope of enhanced green fluorescence protein (EGFP) Model chimeric EGFP-VLPs were purified and used for immunization of mice. Immune response of immunized animals was examined. No specific antibodies against EGFP protein, but high titers of specific antibodies against major structural protein VP1 were developed in the sera of immunized animals. Splenocytes derived from immunized animals secreted IL-2 and IFN-γ after their antigen (EGFP or VP1) restimulation. Proliferation of CD4+, but not CD8+ T cells from immunized mice after the stimulation with both EGFP and VP1 was observed. No EGFP specific cytotoxic activity of splenocytes from immunized mice was detected. The presentation of EGFP-VLPs in the context ofMHC class II was blocked by inhibitors of endo-lysosomal as well as proteasomal compartments. Changes in the numbers of CD25+Foxp3+ subpopulation of CD4+ T cells were observed in the spleens if immunized mice. Chimeric VLPs derived from mouse polyomavirus carrying epitopes of human Bcr-Abl fusion protein (Bcr-Abl VLPs) Chimeric Bcr-Abl VLPs carrying 171 amino acids sequence of Bcr-Abl protein (containing Bcr-Abl breakpoint region) were prepared. Chimeric VLPs...
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Epigenetic regulation of expression of immunoactive genes on tumor cells
Hejhal, Tomáš ; Reiniš, Milan (advisor) ; Šmahel, Michal (referee)
The aim of this master thesis was to identify epigenetically silenced genes and to determine molecular effects of DNA methyltransferases inhibitor (5-aza-2'-deoxycytidine) and histon deacetylases inhibitor trichostatin A (TSA) on gene expression in cancer cell lines TC-1/A9 and RVP3. Epigenetic events play an important role in tumorigenesis and also in escape of cancer cells from immune surveillance. I analyzed global changes in gene expression profiles of two cell lines by microarray analysis with a special attention paid to immunoactive genes. The experimental model used for this purpose were murine tumor cell lines, MHC class I deficient. I identified epigenetic regulation of several genes that are involved in cancer and immune system interactions. These data demonstrate that epigenetic agents are capable to activate expression of genes that are important for antigen presentation, cell adhesion and apoptosis. Powered by TCPDF (www.tcpdf.org)
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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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Depletion of Treg cells for potentiation of cancer treatment with HPMA copolymer-bound cytostatic drug conjugates"
Dvořáková, Barbora ; Kovář, Marek (advisor) ; Reiniš, Milan (referee)
Tumor diseases are severe problem worldwide with increasing number of patients suffering from various types of malignancies. Many of approved therapeutics cause serious side toxicities. Therefore, there are intensive efforts to improve cancer treatment protocols. The aim of this study was to deplete regulatory T (Treg) cells without affecting other immunocompetent cells playing a positive role in tumor eradication. Treg cells were reported to hamper anti-tumor immunity and promote tumor growth and survival. Thus, their selective elimination could lead to induction of anti-tumor responses and tumor rejection if combined with chemotherapy with selected N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer-bound drug conjugates. Original approach was to deplete of Treg cells without the use of anti-CD25 mAb that has been widely exploited for Treg cell elimination; however, its long-term persistence in circulation together with inhibitory effect on activated effector cells (CD25+ ) are its main disadvantages. Thus, Treg cells were sensitized to cell cycle-specific cytostatic drugs via application of IL-2/anti-IL-2 JES6.1 mAb immunocomplexes that induce vigorous selective proliferation of this cell population. Subsequent application of cell cycle-specific cytostatics showed steep decrease of Treg cell...
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T-cell immunotherapy of cancer
Táborská, Pavla ; Smrž, Daniel (advisor) ; Funda, David (referee) ; Reiniš, Milan (referee)
T cell immunotherapy of cancer Abstract Cancer is the second leading cause of death worldwide. Patients diagnosed at the late stages of the disease have limited treatment options. Traditional treatment modalities such as surgery, chemotherapy and radiotherapy also have limited efficacy at the late stages of the disease. Passive cancer cellular immunotherapy, namely the adoptive cell transfer, is a promising treatment modality in patients with late and refractory forms of the disease. The objective of the presented work is the development of the T cell-based immunotherapy of prostate cancer. The work addresses 3 parts of the T cell preparation for immunotherapy: enrichment, expansion, and modulation. The first part of the study investigates new ways how to enrich the patients' lymphocytes with T cells reactive to tumor-associated antigens. The second part of the study establishes a protocol for the extensive expansion of the enriched cell cultures. The last part of the study examines new approaches for modulating the phenotype of the enriched and expanded antigen reactive T cells. The work was summarized in 3 primary-authored publications, each of which addressed the individual parts of the cell preparation for T cell-based immunotherapy. Keywords CD8+ T cells, cytokine starvation, ex vivo expansion,...
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Molecular mechanisms of apoptosis induction by taxanes in breast cancer cells
Jelínek, Michael ; Kovář, Jan (advisor) ; Brábek, Jan (referee) ; Reiniš, Milan (referee)
Taxanes are cytostatic routinely used for the treatment of solid breast, ovarian, prostate, head and neck tumors and other types of tumors. Resistance of tumor cells to the effect of taxanes represents serious obstacle for the employment of taxanes in the treatment of tumors. This resistance can be associated, among other things, with lower rate of apoptosis induction in cancer cells or also with increased level of transporters transporting taxanes out of the cell. In this PhD thesis we tried: (1) to contribute to elucidation of the role of molecular mechanisms of apoptosis induction by taxanes in cells of human breast cancer. Specifically, it meant to contribute to elucidation of the role of initiator caspase -8 a - 9 and mainly of initiator caspase-2. Next, to contribute to elucidation of the role of executioner caspase -3 - 6, and -7 and selected proteins of the Bcl-2 family. (2) To contribute to elucidation of molecular mechanisms of resistance of human breast cancer cells to taxanes. Specifically, it meant to describe the role of selected functional groups in taxane structure in bringing about and overcoming resistance to taxane and next to contribute to elucidation of the role of P-glycoprotein (ABCB1 transporter) in the resistance to individual taxanes. 1) We found that caspase-2 represents...
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Epigenetic regulation of HLA class II genes and their role in autoimmune diseases
Čepek, Pavel ; Černá, Marie (advisor) ; Štechová, Kateřina (referee) ; Reiniš, Milan (referee)
(EN) Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with HLA (human leukocyte antigen) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of T1D patients and healthy controls. DNA methylation is one of the epigenetic modifications, that regulate gene expression and is known to be shaped by the environment. 61 T1D patients and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulfite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analyzed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr=0.041; DRA normalization, Pcorr=0.052) between healthy controls and T1D patients. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in T1D patients and healthy...
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