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Host-microbe interactions and its consequences for intestinal inflammation and carcinogenesis
Kejzlarová, Leona ; Kverka, Miloslav (advisor) ; Smrž, Daniel (referee)
A number of physiological and pathological processes, including the transition from chronic inflammation to cancer, are affected by commensal microbes. However, abundance of microbes and ability to produce active metabolites in the intestine depend on environmental factors, particularly diet. Microbes can influence this process in two ways, by producing genotoxic substances that directly damage the epithelium or by stimulating the inflammatory response. The aim of my thesis was to study the interaction among gut microbiota, diet and the immune system with the subsequent influence on the development of colorectal cancer (CRC) in an experimental mouse model. Animals were fed synthetic diets containing either normal amounts of animal protein (17%; KD) or elevated amounts of animal protein (51%; HPD) throughout the experiments. Two weeks after the diets were introduced, intestinal tumors were induced by administering azoxymethane (AOM) and inducing acute inflammation with 2% sodium dextran sulfate one week after AOM injection. At the end of the experiment I evaluated the number of tumors in the colon and the status of the immune response in the intestine, mesenteric lymph nodes and spleen. To study the effect of macrophages, a similar experiment was performed in animals with depleted macrophages using...
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Mebrane adaptor proteins in hematopoiesis and immune response
Pavliuchenko, Nataliia ; Brdička, Tomáš (advisor) ; Brábek, Jan (referee) ; Smrž, Daniel (referee)
Membrane adaptor proteins are proteins associated with cellular membranes that do not themselves serve as receptors. Instead, they propagate or modify the signals of these receptors by recruiting other signaling and regulatory proteins and arranging them into supramolecular complexes. In this thesis, I sought to describe selected membrane adaptor proteins and their roles in inflammation and regulation of hematopoiesis in mouse models using a reverse genetics approach. The main part of the work focused on the role of the membrane adaptor protein PSTPIP2 in suppressing inflammation. In mice, missense mutations in the Pstpip2 gene causing loss of PSTPIP2 protein lead to the development of autoinflammatory disease chronic multifocal osteomyelitis (CMO) characterized by sterile inflammatory lesions in the bones and adjacent soft tissue. These mice represent a model of the human autoinflammatory disease, chronic recurrent multifocal osteomyelitis. At the molecular level, neutrophils in the absence of PSTPIP2 exhibit pathological hyperactivity of pathways regulating IL-1β and reactive oxygen species (ROS) production, which are both implicated in the etiology of the disease. PSTPIP2 interacts with several signaling regulators, including PEST family protein tyrosine phosphatases (PEST-PTPs) and inositol...
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In vitro modulation of immune cells for adoptive cellular cancer immunotherapy
Kalkušová, Kateřina ; Smrž, Daniel (advisor) ; Kverka, Miloslav (referee)
Cancer is one of the leading causes of death in developed countries. Its traditional treatment is based on surgical removal of the tumor and metastases, radiotherapy, and chemotherapy. Recently, many new therapy options, including immunotherapy, have been investigated. Cancer immunotherapy seems to be a very promising treatment option as it has experienced many successes in the last few decades. However, there is still a number of patients not responding to today's immunotherapy methods. Adoptive cellular immunotherapy is one of those immunotherapeutic methods. This immunotherapeutic modality uses ex vivo prepared immune cells that participate in anti-tumor responses. Nowadays, most research is focused on the use of T cells, although many other cell types are considered, including dendritic cells. This thesis is focused on the modulation of dendritic cells for adoptive cellular cancer immunotherapy. The aim of the practical part is to evaluate the influence of beta2-microglobulin on the maturation of monocyte-derived dendritic cells. Key words: Adoptive cellular immunotherapy, dendritic cells, beta2-microglobulin, cancer
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Surface expression of Tim-3 inhibitory molecule on antigen-specific CD8+ T cells expanded in vitro using dendritic cells for cell-based cancer immunotherapy
Svobodová, Hana ; Smrž, Daniel (advisor) ; Funda, David (referee)
Cancer is the second most common cause of death in the world, and the number of people with the disease increases each year. The therapy of the disease currently stands on four pillars; surgery, chemotherapy, radiotherapy, and immunotherapy. Through the past few years, immunotherapy has become the fastest developing treatment modality. However, despite its unprecedented efficacy in some patients, the majority of patients still does not respond to the therapy. Therefore, there is a need to investigate the mechanisms that make immunotherapy inefficient. Cell-based cancer immunotherapy is the treatment modality which uses live ex vivo-produced tumor-targeting immune cells to treat cancer. One of the mechanisms that may compromise its therapeutic efficacy is the expression of inhibitory molecules on the surface of the produced immune cells. Tim-3 is the inhibitory molecule which attracts attention in recent years. Tim-3 expression in the tumor cells and the tumor-infiltrating immune cells is often associated with worse prognosis and more aggressive forms of the disease. However, its role in the in vitro or ex vivo-produced immune cells is difficult to predict. In this work, an in vitro study model which is based on in vitro-produced antigen-specific CD8+ T cells with high expression of Tim-3 has been...
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Inflammation and cancer in germ-free vs. conventionally reared animals
Čaja, Fabián ; Vannucci, Luca Ernesto (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Smrž, Daniel (referee)
Inflammation is considered as one of the main defence mechanisms of the immune system against threats that occur in the body. When present in its acute form, minimal or no detectable subsequent damage of original affected tissue exists. The more pathological form, chronic inflammation, is associated with permanent damage of the tissue and typically a hallmark of various diseases such as ulcerative colitis or colon carcinogenesis. These two pathologies are evolving in the unique colon microenvironment, where intensive interaction between the host cells and bacteria is present. The aim of our study was to investigate the immunological (ELISA, FACS, RT-PCR) and structural (histology, confocal microscopy) changes in the colon mucosa of Wistar-AVN rats induced by dextran sodium sulphate (DSS) to produce colon colitis and by azoxymethane (AOM) to produce colon carcinogenesis. Conventional (CV) and also germ-free (GF) reared animals were used to investigate the effects of the mucosal inflammation activated by the administered inducers as well as the role of colon microbiota - as promoters of a continuous immune activation - in the modulation of immunity and collagen scaffold remodelling. Our results showed that even in the early period after the induction, both inducers produced a smouldering...
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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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Wnt/beta-catenin and mTOR signaling in regulation of T-cell phenotype and cytotoxic activity for adoptive cellular immunotherapy of cancer
Stakheev, Dmitry ; Smrž, Daniel (advisor) ; Černý, Jan (referee) ; Říhová, Blanka (referee)
1. Abstract (EN) The adoptive cellular immunotherapy (ACI) based on ex vivo produced T cells is a modern treatment modality of cancer. However, the ex vivo production of T cells with high therapeutic efficacy is far to be well established. Wnt/β-catenin and mTOR signaling have been shown to affect both cancer cells and immune cells. Therefore, the modulation of these pathways seems to be perspective for the production of T cells with superior therapeutic efficacy. The aim of our project was to investigate, how interventions into Wnt/β-catenin and mTOR signaling during the ex vivo production of tumor-associated antigen-specific T cells could improve the production of T cells with a desired and controlled phenotype that would best fit for use in ACI of cancer. In the first part of our study, we investigated the role of Wnt/β-catenin inhibition by XAV939 on cancer cell elimination by lymphocytes from patients with localized biochemically recurrent prostate cancer (BRPCa). We found that preconditioning BRPCa lymphocytes with 5 µM XAV939 accelerated the elimination of LNCaP and PC3 cells during the coculturing. However, during subsequent re-coculturing with fresh LNCaP cells, BRPCa lymphocytes were no longer able to eliminate cancer cells unless coculturing and re-coculturing were performed in the presence of...
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Wnt/beta-catenin and mTOR signaling in regulation of T-cell phenotype and cytotoxic activity for adoptive cellular immunotherapy of cancer.
Stakheev, Dmitry ; Smrž, Daniel (advisor) ; Černý, Jan (referee) ; Říhová, Blanka (referee)
1. Abstract (EN) The adoptive cellular immunotherapy (ACI) based on ex vivo produced T cells is a modern treatment modality of cancer. However, the ex vivo production of T cells with high therapeutic efficacy is far to be well established. Wnt/β-catenin and mTOR signaling have been shown to affect both cancer cells and immune cells. Therefore, the modulation of these pathways seems to be perspective for the production of T cells with superior therapeutic efficacy. The aim of our project was to investigate, how interventions into Wnt/β-catenin and mTOR signaling during the ex vivo production of tumor-associated antigen-specific T cells could improve the production of T cells with a desired and controlled phenotype that would best fit for use in ACI of cancer. In the first part of our study, we investigated the role of Wnt/β-catenin inhibition by XAV939 on cancer cell elimination by lymphocytes from patients with localized biochemically recurrent prostate cancer (BRPCa). We found that preconditioning BRPCa lymphocytes with 5 µM XAV939 accelerated the elimination of LNCaP and PC3 cells during the coculturing. However, during subsequent re-coculturing with fresh LNCaP cells, BRPCa lymphocytes were no longer able to eliminate cancer cells unless coculturing and re-coculturing were performed in the presence of...
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Tumor-infiltrating T cells and their role in adoptive cell immunotherapy of cancer
Střížová, Zuzana ; Smrž, Daniel (advisor) ; Vannucci, Luca Ernesto (referee) ; Posová, Helena (referee)
Cancer immunotherapy has become a leading treatment modality in metastatic diseases. Although this novel therapy has changed the therapeutic algorithms and patients' outcomes in multiple malignancies, certain proportions of patients still fail to respond to these approaches. In our studies, we aimed to address the main mechanisms of tumor resistance to cancer immunotherapy. We have systematically defined the main challenges in adoptive cell transfer. We have focused on two key mechanisms of the tumor resistance to immunotherapy: poor trafficking of adoptively transferred immune cells into tumors, and the death receptor-induced apoptosis of the tumor-infiltrating immune cells. In our work, we have gone beyond the tumor tissue and searched for the immune cell populations and novel targets that would help to challenge the two mechanisms of resistance. Our data uncovered the therapeutic potential of the paratumoral tissue compartments and, thus, provided new avenues on how to challenge solid tumors by immunotherapy.
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Tolerogenic dendritic cells as a novel cell-based therapy in type 1 diabetes
Kroulíková, Zuzana ; Funda, David (advisor) ; Smrž, Daniel (referee)
Utilization of tolerogenic dendritic cells (tolDCs) as a cell-based therapy represents a promising strategy in treatment of autoimmune diseases including type 1 diabetes (T1D). Numerous protocols have been established to generate tolDCs ex vivo and their therapeutic effect has been demonstrated in animal models of autoimmune diseases. In this thesis we compared three different variants of such protocols which are based on the combined treatment of bone marrow- derived DCs with vitamin D and dexamethasone applied at different time points of their maturation towards tolDCs. We assessed the efficiency of these protocols in regards of their effect on the expression of co-stimulatory molecules CD40, CD80, CD86, and MHC II and the chemokine receptor CCR7 on the surface of tolDCs. Then, we evaluated the migration pattern of antigen unloaded tolDCs in vivo as well as their effect on the induction of immune responses and cell proliferation of lymph node cells. This was achieved by labelling of tolDCs with membrane dye PKH26 and by following their migration path by flow cytometry after intraperitoneal (i.p) or subcutaneous (s.c.) injection into either left or right side of the body. On day 1, 3, 5, 7, and 9, the presence of PKH26+ tolDCs was examined in spleen, pancreatic, mesenteric, inguinal and axillary...
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