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Activating antitumor immune response using bispecific fusion proteins
Chytrá, Gabriela ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
Natural killer (NK) cells are lymphocytes of the innate immune system that recognize and eliminate transformed and potentially harmful cells in a mechanism termed immunosurveillance. Malignant cells strive to escape immunosurveillance, and if successful, oncological disease develops. To restore immune recognition, immunotherapy utilizing NK cell-directed therapeutic fusion proteins can be employed. Therapeutic fusion proteins target tumour markers expressed on the surface of malignant cells and, at the same time, stimulate immune response through binding to NK cell activating receptors, for example receptor NKG2D or NKp30. A relevant example of a tumour marker is the HER2 receptor, which is often overexpressed in several types of cancer, most notably breast carcinoma. This thesis describes the preparation of several bispecific fusion proteins with potential use in immunotherapy. Bispecific fusion proteins consist of an NK cell activating ligand (ligand MICA or B7-H6) and nanobody targeting selected tumour marker (receptor HER2), which are connected by flexible glycine-serine linker. The constructs of fusion proteins were prepared in two configurations - with nanobody located on the N-terminus and the ligand on the C-terminus and vice versa. In addition, bispecific fusion proteins introducing...
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Immunological profile of patients with multiple sclerosis
Šubjak, David ; Fišerová, Anna (advisor) ; Růžičková, Šárka (referee)
Immunological profile of multiple sclerosis patients Abstrakt Multiple sclerosis is an autoimmune neurodegenerative disease affecting predominantly the white matter of the CNS and the spinal cord. The mechanism of disease progression is not yet fully understood. In this study we focused on a comparison of selected immunological markers between patients with multiple sclerosis who were naïve newly diagnosed, subsequently treated with Avonex (IFNβ1a) and healthy donors. The T cells (particulary cytotoxic CD8+ T cells) are the major population involved in pathogenesis of MS causing the demyelization of axons. Subpopulation of CD161+ Th cells has a potential to be very important in this process. We focused on the role of NK cells phenotype and function in autoimmune response of patients and their changes during the therapeutic intervention. Using flow cytometry we analyzed the distribution of NK, NKT, T cells and monocytes with special regard to the expression of CD161 and NKG2D molecules on their surface. We observed increase counts of CD161+ cells in subpopulations NK CD56bright , NK CD56dim , Th, Tc CD8bright , Tc CD8dim and decrease counts of NKG2D+ cells in subpopulations NK CD56bright , NK CD56dim , NKT, Th, Tc CD8bright , Tc CD8dim and monocytes. The decreased cytotoxic activity of NK cells in naïve MS...
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Preparation of fusion domains of human immunoreceptors for their utilization in immunotherapy
Cmunt, Denis ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
The functions of the immune system include immunosurveillance of transformed cells, i.e., the ability to eliminate these cells before they become harmful to the organism. If the transformed cells succeed to escape the immune system surveillance, an oncological disease develops. The tumour immunotherapy aims to stimulate the immune system mechanisms to fight against the tumour. Lately, there's an interest in using NK cells in the immunotherapy of tumours. These cells appertain to the innate immune system and participate in immunosurveillance. When an NK cell encounters a target cell, its activation depends on the integration of signals from the surface activating and inhibiting receptors which bind ligands on the surface of the target cell. Upon activation, NK cell exhibits a cytotoxic response against the target cell. The use of NK cells in immunotherapy includes, among others, the testing of bispecific fusion proteins which can bind a tumour surface antigen by one part and NK cell activating receptor by the other part. Thus, these fusion proteins mediate a contact between both cells and trigger the cytotoxic response. This work presents a preparation of bispecific fusion proteins which consist of an activating ligand MICA (for the receptor NKG2D) or B7H6 (for the receptor NKp30), and a nanobody...
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Preparation of fusion ligands and evaluation of their binding to NK cell receptors
Nepokojová, Tereza ; Vaněk, Ondřej (advisor) ; Grantz Šašková, Klára (referee)
Natural killer cells (NK cells) are an important part of innate immunity. On their surface they express a complex group of receptors that use different signalling motifs to activate or inhibit NK cell cytotoxic activity. NK cells are capable to kill aberrant cells (namely, viral, infected, and tumour cells) by using special cytotoxic mechanisms to trigger apoptosis. The activating receptors recognize tumour or stress-induced ligands, e.g., NKG2D receptor recognizes the MICA ligand and NKp30 recognizes the B7-H6 ligand. Therefore for human immune system it is only natural that cancer cells are destroyed by NK cells. The current therapeutic goals in the treatment of cancer are primarily focused on strengthening the body's own natural ability to fight with cancer and one possible way is stimulation of NK cells to win this deadly fight. In addition to NK cells, antibodies are also widely used for the treatment of cancer, as well as other immune-related disorders. Most of them are monoclonal antibodies, but antibody fragments are getting attention and are being tested more and more in recent years. This work describes the preparation of three bifunctional fusion proteins: B7-H6-L-aHER2, MICA-L-aHER2, and aHER2-L-MICA, which contain immunoligands for the activating receptors of NK cell and VHH fragment...
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Mechanisms of specific immune response interactions with tumor cells.
Kaššák, Filip ; Hořejší, Václav (advisor) ; Černý, Jan (referee)
Interactions between the immune system and tumors have been among the highlights of present immunological research. An extensive body of new knowledge recently substantiated the long-presumed concept of cancer immunosurveillance. Immune system searches the organism for cells expressing tumor antigens or cellular stress signals and destroys them. T-cells, NK-cells and dendritic cells, as well as cytokine signaling and direct cell cytotoxicity play dominant role in this process. However, a fraction of nascent tumors can evade these mechanisms and create a dynamic equilibrium, gradually sculpting its phenotype by clonal selection. Eventually, tumor cells escape immune control by concealing themselves from recognition or by actively subjugating local immune response. This immunosubversion results in formation of immunosuppressive tumor microenvironment by recruiting protumorigenic cell populations, such as Treg cells, macrophages and myeloid derived suppressor cells. Soluble signaling molecules, as well as surface- expressed immune checkpoint molecules are exploited by tumor cells for inhibition of anti-tumor immunity. Highly effective therapeutic antibodies blocking these checkpoints have been developed for clinical use, with many more in current trials. Several other promising immunotherapeutic...
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NK cell involvement in the pathogenesis of autoimmune arthritis
Richter, Jan ; Fišerová, Anna (advisor) ; Hromadníková, Ilona (referee) ; Turánek, Jaroslav (referee)
Rheumatoid arthritis (RA) is a worldwide problem representing one of the most prevalent autoimmune diseases in the world. Despite the commonness of the disea- se, its pathogenesis has not been fully described. Immune cells ranging from antigen- presenting cells to T, B and NK cells playing various roles participate in the rheumatic process. In this work we concentrated on NK cells expressing a repertoire of activating and inhibitory receptors which influence their function in health and disease. We focused on the analysis of NK cell function and described its possible modulation by rheumatic autoantigens and multivalent glycodendrimers bearing 4 (GN4C) or 8 (GN8P) N-acetyl glucosamine moieties. The effect on NK cells and the glycosylation pathways was further studied in vitro. Finally, an in vivo study was performed on an animal model of RA - col- lagen-induced arthritis (CIA) to assess the effect of the compounds on clinical develop- ment of the disease and selected immune parameters. Comparison of NK cell cytotoxicity in patients suffering from RA, other inflam- matory diseases and healthy donors showed its impairment particularly in RA patients. Peripheral blood NK cells reacted to GN8P glycoconjugate by inhibition of their effector function in CD161 high-expressing samples. The MGAT5...
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Recombinant production of bovine NK cell receptors
Böerová, Nikola ; Kavan, Daniel (advisor) ; Šulc, Miroslav (referee)
NK cells, which are part of the innate immune system, are increasingly gaining attention, especially due to their cytotoxic ability to kill tumor cells of certain lines and certain viral, bacterial or parasitic infestation of the body. They lay a role in organ transplantation, the fight against HIV and other autoimmune diseases. NK cells have been studied since the 70th of the 20th century, but the structures and physiological ligands of their receptors remain only partially understood, as does the exact role of these cells in the organism. They communicate with others through their receptors, that recognize the lack of expression of MHC class I glycoproteins on the surface of target cells, thereby preventing cell recognition by cytotoxic lymphocytes. This diploma thesis deals with the research of receptors from cattle (Bos taurus), which is not a traditional laboratory animal and my task was to contribute to research of the structure of this group of proteins. I dealt with the recombinant production of some of the most important representatives of NK receptors CD69, NKRP1 and NKG2D in bacterial cells. The findings published in this thesis are a continuation of my bachelor thesis and together can be beneficial for further research into structural proteins and thus may help as in veterinary medicine...
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Immunological profile of patients with multiple sclerosis
Šubjak, David ; Fišerová, Anna (advisor) ; Růžičková, Šárka (referee)
Immunological profile of multiple sclerosis patients Abstrakt Multiple sclerosis is an autoimmune neurodegenerative disease affecting predominantly the white matter of the CNS and the spinal cord. The mechanism of disease progression is not yet fully understood. In this study we focused on a comparison of selected immunological markers between patients with multiple sclerosis who were naïve newly diagnosed, subsequently treated with Avonex (IFNβ1a) and healthy donors. The T cells (particulary cytotoxic CD8+ T cells) are the major population involved in pathogenesis of MS causing the demyelization of axons. Subpopulation of CD161+ Th cells has a potential to be very important in this process. We focused on the role of NK cells phenotype and function in autoimmune response of patients and their changes during the therapeutic intervention. Using flow cytometry we analyzed the distribution of NK, NKT, T cells and monocytes with special regard to the expression of CD161 and NKG2D molecules on their surface. We observed increase counts of CD161+ cells in subpopulations NK CD56bright , NK CD56dim , Th, Tc CD8bright , Tc CD8dim and decrease counts of NKG2D+ cells in subpopulations NK CD56bright , NK CD56dim , NKT, Th, Tc CD8bright , Tc CD8dim and monocytes. The decreased cytotoxic activity of NK cells in naïve MS...
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