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Genotoxic stress and senescence in tumour cells: impact on the tumour growth and anti-tumour immunity.
Sapega, Olena ; Reiniš, Milan (advisor) ; Brábek, Jan (referee) ; Šmahel, Michal (referee)
Premature cellular senescence is the process of permanent cell cycle arrest in response to various inducers, such as DNA damage, oxidative stress, chemotherapy agents, and irradiation. Senescent cells produce and secrete numbers of cytokines, chemokines, growth factors, which compose specific senescence-associated secretory phenotype (SASP). Senescence is considered to be an important barrier against tumor progression. On the other hand, senescent cells can also exert protumorigenic effects in their microenvironment. Based on this concept, the major aim of this thesis was to determine tumor cells senescence in terms of different inducers, namely chemotherapeutic agent docetaxel (DTX) and cytokines IFNγ and TNFα, and to demonstrate the role of immunotherapy in senescent cells elimination. Our results show that DTX-induced senescent cells can exert a tumor-promoting effect when co-injected with proliferating cells in mice. Importantly, we demonstrate that IL-12-based immunotherapy suppresses senescence-accelerated tumor growth. These results suggest that IL-12-based immunotherapy can be effectively used in anti-tumor therapy mainly in a case when the microenvironment is altered by the presence of tumor senescent cells. On the other hand, the data we obtained in vitro show that bystander or...
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Adoptive transfer of tumor-specific lymphocytes for cancer immunotherapy
Vávrová, Kateřina ; Bartůňková, Jiřina (advisor) ; Němečková, Šárka (referee) ; Reiniš, Milan (referee)
Prostate cancer is the second leading cause of cancer death in men in Europe and the US. In the context of previous preclinical experiments and clinical studies there are certain assumptions predicating successful application of immunotherapy in the treatment of patients with prostate cancer. Promising results have been achieved by a combination of different treatment modalities which provide a synergistic antitumor effect. One of these combinatorial options is the use of antitumor vaccines and adoptive T cell transfer. The topic of this thesis is to provide a fresh insight into the past and current trends following the long-term candidate's department program in the field of anti-tumor immunotherapy. The experimental part of this thesis revolves around our own results published in this field. The introductory chapter delivers a basic overview of cellular mechanisms of anti-tumor immunity and the role of individual immune components in these processes. Following chapters are dedicated to current immunotherapeutic approaches with emphasis on the adoptive T cell transfer and implication of this technology in the treatment of prostate cancer. The results section describes the establishment of our protocol for adoptive T cell transfer as well as the protocol for ex vivo enrichment of human T cell...
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Identification of prognostic biomarkers for immunotherapy of ovarian carcinoma
Quaiserová, Lenka ; Palich Fučíková, Jitka (advisor) ; Říhová, Blanka (referee) ; Reiniš, Milan (referee)
Ovarian cancer belongs to the gynecological malignancies with the worst prognosis, mainly due to the late diagnosis of this disease and limited therapeutic options for patients. Despite the undeniable progress in surgical and chemotherapy treatment, the mortality of this disease is still rather high. For this reason, several preclinical and clinical studies have been involved in identification of new treatment strategies (including immunotherapy) and characterization of new prognostic and predictive biomarkers to help determine the development of the clinical condition of patients or their response to treatment. The aim of this thesis was to better understand the role of the immune system in the tumor microenvironment (TME) of high-grade serous ovarian cancer (HGSC) and its possible prognostic role in the treatment of patients. Our results show that the presence of activated DC-LAMP+ dendritic cells in the TME is associated with the induction of anti-tumor T helper type 1 response (Th1) and cytotoxic response. Surprisingly, the resulting effector activity of the cytotoxic T lymphocytes (CTLs) is not inhibited by the presence of programmed death-ligand 1 (PD-L1), programmed cell death (PD- 1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and lymphocyte-activation gene 3 (LAG-3), as...
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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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TET1 overexpression, DNA hypomethylation and aberrant expression of human endogenous retrovirus ERVWE1 in germ cell tumors
Benešová, Martina ; Trejbalová, Kateřina (advisor) ; Černá, Marie (referee) ; Reiniš, Milan (referee)
TGCTs are tumors of male germ cells. They comprise of seminomas and non-seminomas (embryonal carcinoma, yolk sac tumor, choriocarcinoma, and teratoma). GCT types differ in the stage of differentiation, from undifferentiated seminoma to more differentiated non-seminomas. In our studies, we aimed to characterize specific epigenetic features of GCT types that enable transcription derepression of the human endogenous retrovirus ERVWE1 in these tumors. We detected upregulated mRNA expression of TET1-3 dioxygenases in GCTs, especially of TET1 in seminomas. Moreover, seminomas showed low global levels of 5mC and 5hmC. TET1 knock-down in a seminoma-derived cell line resulted in a decreased amount of 5hmC and unchanged 5mC level. These results stress the dynamics of cytosine modifications, which has not been precisely described yet. Further, we observed high level of ERVWE1 transcript together with efficient RNA splicing in seminomas. Detected ERVWE1 transcription is independent of the expression of other examined endogenous retroviruses. ERVWE1 transcription derepression corresponds with the low global level of 5mC detected in seminomas, which involves extensive DNA hypomethylation of the ERVWE1 promoter. We propose the high TET1 dioxygenase expression as s marker of undifferentiated GCTs. Furthermore, we...
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Senescent cells and their elimination by the immune system
Novotný, Ondřej ; Reiniš, Milan (advisor) ; Mrázková, Blanka (referee)
Cell senescence is a type of cell cycle arrest in which the spectrum of the expressed genes changes specifically, also a change in the shape, size and other properties of a cell occurs. Senescent cells secrete a specific set of substances that affect the surrounding tissue, immune system and themselves. All this due to the induction of signalling pathways, inherent to individual types of senescence. The senescent cells accumulate in the body both during pathological conditions and during the natural process of aging and tissue renewal, with varying intensity depending on the type of tissue and organism. The consequence of their presence in the body is often ambivalent - for example, they are an effective mechanism of defence against tumour growth, but at the same time they can be its cause. The positive elimination of senescent cells usually has a positive effect - the immune system is responsible for this in vivo. Studies mapping the natural rate of accumulation and elimination of senescent cells in individual organs, together with new immunotherapeutic elimination procedures, are an important tool for developing new approaches to treating a wide range of human diseases and potentially to prolong human life.
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Gradual Molecular Changes in Primary Porcine Cells Expressing Mutated Huntingtin
Šmatlíková, Petra ; Motlík, Jan (advisor) ; Trejbalová, Kateřina (referee) ; Reiniš, Milan (referee)
Huntington's disease (HD) is inherited fatal disorder caused by CAG triplet expansions in the huntingtin gene resulting in the expression of mutated huntingtin protein (mtHtt). The main symptoms of HD are neurodegeneration, osteoporosis, testicular degeneration, loss of muscle tissue and heart muscle malfunction, weight loss, metabolic changes, and sleeping disturbances. Since huntingtin protein (Htt) has a role in several biological processes, many molecular mechanisms, like oxidative stress, mitochondrial dysfunction, DNA-damage, and others, are affected by mtHtt. However, its exact pathogenic mechanisms in HD are still not well understood. Transgenic minipig model of HD (TgHD) serves an opportunity to isolate unlimited number of primary cells and unlike primary cells obtained from HD patients, often in the late stages of the disease, the TgHD minipig model allows to monitor molecular changes occurring gradually with age and progression of the disease. Thus, TgHD minipig model and primary cells isolated from it play an important role in investigating and understanding the underlying mechanistic cause of HD. We focused on molecular and cellular changes in primary cells isolated from TgHD minipigs and their wild type (WT) controls at different ages (24, 36, and 48 months). In mesenchymal stem cells...
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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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Development of therapeutic vaccine against HPV-16 induced tumor- influence of E7 antigen modification on cell madiated immune response
Macková, Jana ; Němečková, Šárka (advisor) ; Eckschlager, Tomáš (referee) ; Reiniš, Milan (referee)
Conclusions We constructed the vaccines carrying IIPV-16 E7 antigen based on .B. pertussisCyaA toxin We introduced ELISPOT and MHC-I totaÍner assays for testing of cell-mediarcdimmrmeresponsein themousemodel We tested cell-mediated immune response following immunization with differentkinds of thevaccinescarryingthemodifiedHPV-16 E7 antigen: o Recombinantadenylďe cyclase toxoid CyaA336IE7 is able to induce E7 specific CDE- cellular immuneresponsein mice and proteď them againstTC-l firmorgrowth o Some DNA vaccines carying the E7 genefusď to anothergeneare able to induoe betteranti-tumorimmuneresponsethan non-modified E7 genein mice @NA vaccinesevďuated accordingto themagpitude of CTL responseinduced: ETCTGG.GUS > ETGGGHSP, ETHSP >> cP-E7 >E7) o Fusion of E7 with W hemagglutininleadsto cell-surfaceexpressionof E7 and following vaccinďion with W.E7.HA it induces Th-2 polarized immuner€spons€ type ďong with the absenceof anti.tumor Th-l response o Co-expression of IL-12 from double recombinant vaccinia virus (W.IL-l2-sig/E7|LAI\D) reducesCD8- cellular immuniý inducedby Sig/E7lLAMP o Immunization with dendritic cells transducedby rW improves the efficacy of rW vaccination o Combined immunization increases vaccination effectiveness (CyaA336/E7+MVA- Sig/E7lLAMP, DNA-SigETGGG/LAMP+cellular vaccine) I7
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