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Roles of antigen presenting cells in regulation of Th17 response against Candida albicans
Böhmová, Helena ; Dobeš, Jan (advisor) ; Kostovčíková, Klára (referee)
Candida albicans is a common human pathobiont that inhabits mucosal surfaces throughout the body. In healthy individuals, it behaves as a benign member of the microfora. However, in immunocompromised individuals Candida becomes pathogenic and causes extensive mucosal infections. In the most severe cases, Candida translocates into the bloodstream and causes life-threatening deep tissue infections. Although the innate immune components involved in early anti-Candida immune response are relatively well defned, our knowledge regarding adaptive T cell responses to Candida is limited. Several populations of antigen-presenting cells (APCs) have been implicated in the induction of protective Th17 response against Candida - including innate lymphoid cells type 3 (ILC3s), conventional dendritic cells (cDCs) and CX3CR1+ mononuclear phagocytes (MNPs). The cellular and molecular mechanisms by which Candida-specifc T cells are induced have not yet completely been identifed. Presented thesis focuses on the involvement of direct antigen presentation by these APC populations in mounting the anti-Candida adaptive immune response. Furthermore, this is investigated in the context of both gastrointestinal colonization and bloodstream infection by C. albicans. In the frst part, published data concerning the immune...
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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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Immunomodulation of dendritic cells by adenylate cyclase toxin from B. pertussis
Jáňová, Hana ; Adkins, Irena (advisor) ; Brdička, Tomáš (referee)
Adenylate cyclase toxin (CyaA) produced by the causative agent of whooping cough Bordetella pertussis, is a key virulence factor important for colonization of the host. CyaA targets preferentially myeloid phagocytes expressing CD11b/CD18 integrin. By elevating cytosolic cAMP in the host cells, CyaA interferes with their phagocytic, chemotactic and oxidative burst capacities. Furthermore, CyaA modulates the secretion of cytokines and the maturation state in LPS-stimulated dendritic cells (DC) by affecting the expression of costimulatory molecules. In this study, we investigated the effects of CyaA on the capacity of murine bone-marrow DC to prime CD4+ and CD8+ T cells in response to ovalbumin epitopes delivered by the CyaA-AC- toxoid, as a model antigen. Further, we examined the possible impact of CyaA on the antigen uptake and processing for MHC class I and II-restricted presentation by DC, as we previously observed a decreased T cell stimulatory capacity of CyaA-treated DC in response to soluble ovalbumin. We found out that the high levels of cAMP generated by CyaA in LPS-stimulated DC account for the decreased presentation of ovalbumin epitopes carried by CyaA-AC- toxoid on MHC class I and II molecules, thereby impairing the CD8+ and CD4+ T cell responses. Whereas CyaA did not influence the...
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Analysis of the resistence of B cell antigen receptor signaling to the inhibition of Src-family kinases
Borna, Šimon ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
Signalling through antigen specific receptors BCR and TCR is crucial for the development and the function of T cells and B cells. Although much is known about their signalling pathways a number of observations still remain to be clarified. In my thesis, I focused on the roles of Src-family kinases (SFKs) in the initiation of BCR- and TCR-mediated signalling. Several studies have suggested that in contrast to TCR signalling, BCR signal transduction could be initiated independently of SFKs or with only a minimal activity of these kinases. We used genetic approach to study the differences between TCR and BCR signalling apparatuses combined with inhibition of SFKs by pharmacological approach. Using this experimental set up, we show that the differences in the roles of SFKs and in the activities of SFKs needed for the initiation of BCR and TCR signalling are likely based on different composition or architecture of BCR and TCR. We further show that the SFK activity required for the initiation of TCR signalling is lower if ZAP-70 kinase is substituted with Syk kinase, which most likely reflects the different molecular mechanisms of Syk and ZAP-70 kinase activation. Key words: Src-family kinases, BCR receptor, TCR receptor, PP2, B cells, T cells, BCR signalling, TCR signalling.
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Translational control in immune response.
Hlaváček, Adam ; Valášek, Leoš (advisor) ; Čáp, Michal (referee)
Immune reaction often requires a prompt modification of gene expression that in turn alters cellular physiology. There are an increasing number of articles supporting a critical role of translational control in this aspect of cellular biology. The aim of this work is to present some of cellular and molecular mechanisms that connect translational control and immune reaction in immune and somatic cells and can be possibly misused by some viruses. Perhaps not surprisingly, many immunologically relevant translational control mechanisms are similar to those acting during the stress response. Over the years it has been documented that the T cells, dendrocytes, Natural killer cells and macrophages utilize translational control for their immunological activation following stimulation. Combination of general and gene-specific translational control mechanisms enables fast changes in proteome and physiology that are characteristic for immune cell activation. The overall impact of translational control on immune response is further illustrated by the fact that it acts upon each stage of life of immune cells - from their activation, through survival, to a programmed cell death. Even in some non-immune cells the translational control plays an important role with respect to immunity, as these cells are known to have an...
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The role of structural motifs in the localisation of T-cell plasma membrane proteins
Glatzová, Daniela ; Cebecauer, Marek (advisor) ; Brábek, Jan (referee) ; Rozbeský, Daniel (referee)
Plasma membrane of T cells is abundant in diverse receptors and other molecules orchestrating immune responses. Numerous studies demonstrate that the localisation of proteins in the cell is non-random and that mislocalisation either in the context of plasma membrane at nanoscale or with respect to the cell interior can lead to the protein malfunction and subsequent aberrant T- cell response. In my first Ph.D. project we focused mainly on the role of the transmembrane domain length and amino acid composition, proximal sequences and the presence or absence of palmitoylation on the localisation of transmembrane adaptor proteins LAT, PAG and NTAL in T cells. We showed that plasma membrane localisation of PAG and NTAL is controlled by the amino acid composition of their TMD and is palmitoylation independent. We propose that NTAL localisation to the plasma membrane is, despite its suboptimal length, facilitated by the electrochemical asymmetry of its TMD. Among transmembrane adaptor proteins, LAT was the most interesting one. Dependency of LAT plasma membrane localisation on palmitoylation in combination with unusual amino acid composition of its TMD led us to investigate it in a separate project. My first author Ph.D. project was thus to elucidate the role of highly conserved helix-breaking amino acids,...
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Immune reactions induced by SARS-CoV-2 infection
Krausová, Kateřina ; Šmahel, Michal (advisor) ; Šroller, Vojtěch (referee)
Coronavirus disease 2019 (COVID-19) pandemic caused by newly discovered Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe health and economic problems all over the world. The disease severity depends mainly on the host's immune response to SARS-CoV-2. This virus uses many mechanisms for escape from the host's immune system. The major evasion mechanisms include suppression of interferon production at the early phase of infection, exhaustion of natural killer cells and induction of a cytokine storm. After the innate immune response, mechanisms of adaptive immunity join the defense against the virus. Patients with severe cases have a significant reduction in the amount of both helper CD4+ T cells and cytotoxic CD8+ T cells. On the contrary, these patients have an increased level of antibodies. Even though there have been many findings about immune reactions to SARS-CoV-2 in the year after its discovery, there are still many unknowns. Vaccines, which are successful at preventing COVID-19, have been developed in a short time. However, an important remaining question for further research is the longevity of immune memory after vaccination or after suffering from COVID-19.
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Effect of gluten-free diet on potentially regulatory immune mechanisms in human type 1 diabetes
Císařová, Radka ; Funda, David (advisor) ; Zadražil, Zdeněk (referee)
Type 1 diabetes (T1D) is an autoimmune disease, whose incidence is rising every year, and its prevention or a cure does not exist. T1D is influenced by multiple genetic factors but environmental factors represent the major contributor to the recent almost epidemic increase of T1D incidence worldwide, primarily in developed countries. Amongst these factors belong for example enteroviral infections, microbiota dysbiosis or gluten-free diet (GFD). GFD has been proven to have a protective effect in NOD mice, which is a spontaneous model of T1D, and a beneficial effect on glycemic control in humans, when administered after T1D onset. This diploma thesis examined changes of regulatory and potentially regulatory T-cells and their cytokines in peripheral blood mononuclear cells (PBMC) of T1D children, who underwent 12-month intervention trial of GFD. Secondly, the thesis assessed if the influence of GFD on immune regulatory functions can be transferred by colonization of germ-free NOD mice with gut microbiota of these children. We have found that intervention with GFD increases percentage of Tr1 cells and IL-10 producing CD4+ T-cells in PBMC of T1D children. Furthermore, the beneficial effect on immune regulation can be at least partially transferred to NOD mice by the colonization with human microbiota...
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Lyme borreliosis diagnostics using in vitro cellular immune response testing
Prokopová, Tereza ; Drbal, Karel (advisor) ; Melter, Oto (referee)
Lyme borreliosis is a multisystemic disease affecting skin, joints, heart and central nervous system. The disease is caused by spirochetes of Borrelia burgdorferi sensu lato complex. These bacteria are spread by ticks of Ixodes genus. In 2016 there were almost 4,000 newly infected individuals reported in the Czech Republic. Contemporary serological diagnostics of Lyme borreliosis is not sensitive nor specific enough and does not even correlate with the pathology of the disease in the early or late phases. For the correct diagnosis of the disease it is necessary to detect the pathogen and its genotype. For this reason we had aimed at two goals. Through the digital droplet PCR (ddPCR) method we detected Borrelia-specific DNA and its genotype. The detection limit of borrelial DNA was set on gDNA samples isolated from the tick. Detection threshold for the initial amount of 1 ng of tick gDNA is at the range of 10-17 g of specific borrelial DNA. Borrelia spp. coinfection was detected in 5 out of 12 tested samples. The most frequent type was B. garinii which was detected in 5 samples. On the basis of published sequences for virulent factors we have designed specific primers in conserved regions of the genes flanking their variable segments to be PCR amplified. Gene variability will be monitored through...
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Analysis of the resistence of B cell antigen receptor signaling to the inhibition of Src-family kinases
Borna, Šimon ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
Signalling through antigen specific receptors BCR and TCR is crucial for the development and the function of T cells and B cells. Although much is known about their signalling pathways a number of observations still remain to be clarified. In my thesis, I focused on the roles of Src-family kinases (SFKs) in the initiation of BCR- and TCR-mediated signalling. Several studies have suggested that in contrast to TCR signalling, BCR signal transduction could be initiated independently of SFKs or with only a minimal activity of these kinases. We used genetic approach to study the differences between TCR and BCR signalling apparatuses combined with inhibition of SFKs by pharmacological approach. Using this experimental set up, we show that the differences in the roles of SFKs and in the activities of SFKs needed for the initiation of BCR and TCR signalling are likely based on different composition or architecture of BCR and TCR. We further show that the SFK activity required for the initiation of TCR signalling is lower if ZAP-70 kinase is substituted with Syk kinase, which most likely reflects the different molecular mechanisms of Syk and ZAP-70 kinase activation. Key words: Src-family kinases, BCR receptor, TCR receptor, PP2, B cells, T cells, BCR signalling, TCR signalling.
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