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Tolerogenic dendritic cells as immune interventions in prevention or therapy of type 1 diabetes
Petrovčíková, Diana ; Funda, David (advisor) ; Hrdý, Jiří (referee)
The main aim of this work is to refer a recent summary of the opportunities and pitfalls of the application of tolerogenic dendritic cells in the prevention or therapy of type 1 diabetes (T1D). Tolerogenic dendritic cells (TolDCs) represent a potential tool for the treatment of allergies, transplant rejections and autoimmune diseases, including T1D, due to their capability to specifically inhibit autoimmune reactions without causing general immunosuppression. TolDCs represent a specific group of dendritic cells and are essential in establishing central and peripheral tolerance. This work presents a helpful guide to better understanding the physiology of tolerogenic DCs and an overview of in vitro generation attempts. In addition, the route of application and migration to target organs has been described. Type 1 diabetes (T1D) is a chronic disease resulting from immune-mediated destruction of the insulin-producing beta cells in the pancreas. Animal models have been invaluable in testing innovative medical treatments since the early testing of insulin in dogs almost a century ago. Animal models of type 1 diabetes (T1D) enable the study of the mechanisms underlying its pathogenesis and the potential development of therapeutic interventions. However, there are still significant gaps in our general...
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Effects of gluten-free diet on immune parameters in Parkinson's disease
Jandová, Mônica ; Funda, David (advisor) ; Dobeš, Jan (referee)
Tato studie zkoumá možné, i malé, vlivy bezlepkové diety v porovnání se standardní dietou na rozvoj Parkinsonovi nemoci s využitím 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinem (MPTP) indukovaném myším modelu Parkinsonovi nemoci, a samců kmene C57Bl6. Tento výzkum zahrnuje zavedení jak akutního tak chronického myšího MPTP modelu, a řadu navazujících flow cytometrických analýz zaměřených na regulační T buňky, cytokiny - interleukin 10 (IL-10) a interferon gama (IFN-), gamma/delta T buňky, a NK buňky ve slizničních a sytémových lyfatických orgánech. Pro posouzení vlivu na chování jse použili test otevřeného pole, který nám umožnil posoudit lokomotorickou aktivitu a explorativní chování myší v závislosti na podávané dietě. Dále jsem použili také imunofluorescenční barveni pro validaci efektu MPTP, které nám poskytlo vizuální potvrzeni neuroanatomických změn indukovaných neurotoxinem. První výsledky naznačují malé ale slibné známky pozitivních efektů bezlepkové diety. Nicméně tyto změny lze pouze opatrně interpretovat, protože je potřeba opakovaných a podrobnějších experimentů k posouzení zazanmenaných efektů. Spojení behaviorálních testů, imunohistologie a imunologických analýz představuje vícečetný metodicky přístup k této problematice, který nám může poodhalit komplexní interakce diety, střeva,...
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Effect of early postnatal supplementation by probiotic bacteria Escherichia coli O83:K24:H31 on proportional and fucntional characteristics of selected cellular population
Věcek, Jan ; Hrdý, Jiří (advisor) ; Funda, David (referee)
The hygiene hypothesis proposes that exposure to microorganisms during the postnatal period is crucial for proper immune system development and may help to prevent development of autoimmune diseases and allergies. Probiotics, live microorganisms with beneficial health effects, could be a safe way to promote the appropriate maturation of the immune system. Early postnatal administration of a specific probiotic strain, Escherichia coli O83:K24:H31 (EcO83), reduces the incidence of allergies later in life. To understand the immunomodulatory features of EcO83, we conducted a bioinformatic analysis of its genome and compared it to two other strains, E. coli Nissle and E. coli K12. Our analysis identified unique genes in EcO83 related to propionate and galactose metabolism, as well as genes that may enhance its ability to thrive in the gastrointestinal tract. Moreover, we transformed EcO83 with luciferase enzymes and observed that it effectively colonizes the gastrointestinal tract of newborn mice but not adult mice. Further analysis of mice treated with EcO83 revealed that the probiotic promotes the expression of genes involved in tight junction formation and increases costimulatory molecules on dendritic cells in the mesenteric lymph nodes (MLN). Induced RORγt+ Tregs in MLN displayed increased...
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Identification of new regulators of proinflammatory signaling pathways
Dráberová, Helena ; Štěpánek, Ondřej (advisor) ; Krulová, Magdaléna (referee) ; Funda, David (referee)
Identification of new regulators of proinflammatory signaling pathways Helena Dráberová Protein 4.1R has been described in immune system as regulator of migration and cell adhesion, but was also shown to play a role in activation of T lymphocytes. Polymorphism in gene ORMDL-3 is associated with asthma risk in children and correlates with increased ORMDL-3 expression. This disertation thesis describes the function of proteins 4.1R and ORMDL-3 in activation of mast cells after stimulation of FcεRI receptor. IL-17 is a proinflammatory cytokine that plays a role in immune response against fungal and yeast infections. IL-17 however also plays a role in the pathology of autoimmune diseases such as reumatoid arthritis, psoriasis and multiple sclerosis. IL-17 signaling is tightly regulated, however the exact mechanism has not been described. This disertation thesis describes the IL-17R complex by mass spectrometry and analyze the function of its known and newly discovered components in cells deficient in individual proteins by method CRISPR-Cas9. Last part focuses on the discovery of new subunit of IL-17RC protein CMTM4, which role in IL-17 signaling has not been described so far. CMTM4 stabilizes IL-17RC and is required for its surface expression. In vitro data are supported by data from autoimmune model of...
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Effect of bacterial monoassociations (Akkermansia muciniphila) on development of type 1 diabetes and immune parameters in ex-germ-free NOD mice
Němec, Dušan ; Funda, David (advisor) ; Zadražil, Zdeněk (referee)
Type 1 diabetes (T1DM) is an autoimmune condition affecting around 0,4 % of general population and its prevalence is still increasing. T1DM is a multifactorial disease, and it develops under the forces of various environmental and hereditary factors. Gut microbiota is recently one of the most relevant environmental features of autoimmunity, including T1DM. Healthy gut microbiota is characterized especially by its variability. However, there has been an effort to determine critical bacteria that can either drive or suppress T1DM development. Akkermansia muciniphila is among those potentially protective bacteria. This diploma thesis examined changes of immune parameters, such regulatory T cells, NK cells, γδ T cells and expression of IFNγ, IL-10 and IL-17, and their correlation with T1DM onset in A. muciniphila- monoassociated ex-germ-free NOD mice compared to germ-free (GF) and specific-pathogen- free (SPF) controls. Furthermore, the second part of the thesis, NOD-SCID adoptive transfer provided an insight into whether diabetogenic ability of NOD mice-derived splenocytes differ in A. muciniphila vs GF and SPF controls. Minor differences were found in immune parameters among various cell populations, with the most prominent increased IL-10 expression in A. muciniphila-monoassociated mice compared to...
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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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The role of gut microbiome in the development of type 2 diabetes
Movsesian, Lilit ; Cahová, Monika (advisor) ; Funda, David (referee)
In recent decades, ample evidence of the impact of intestinal microbiota and metabolome on the development of type 2 diabetes and other metabolic disorders such as obesity has been obtained. This thesis provides an overview of known information about this correlation and aims to highlight the importance of further research to identify prospective treatments. Bacterial taxa and metabolites most associated with the incidence of diabetes, as well as those associated with improved glucose and insulin tolerance, are considered. The thesis also compares similarities, differences and contradictions of the discussed studies. Key words: type 2 diabetes, obesity, microbiome, metabolome
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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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Roles of environmental factors and microbiome in type 1 diabetes
Novotná, Kateřina ; Funda, David (advisor) ; Cahová, Monika (referee)
Type 1 diabetes mellitus (T1DM) is an insulin-dependent autoimmune disease. Its onset is characterized by an autoreactive self-destruction of β-cells within pancreatic islets. T1DM is influenced by multiple genetic predispositions, but since the incidence of the disease has increased dramatically in the past decades, especially in developed, western-type countries, the importance of the environmental factors has become obvious. There are various significant environmental influences that need to be addressed in the equation of variables. This bachelor thesis deals with the environmental variables and their mechanisms in T1DM and focuses on several areas of interest. It introduces frequently used spontaneous animal model of T1DM, pathogenetic mechanisms and T-cells in T1DM as well as regulatory immune cells and their mechanisms, in the light of hygiene and another hypothesis. Next it addresses the role of intestinal microbiota, dietary factors, mucosal immunity, their mechanisms and interactions in T1DM and extends to other, less researched, but important environmental variables such as circadian rhythm in connection with circadian gene expression depending on the rhythmicity of light/dark rotation and timing of food intake throughout the day, psychological/oxidative stress, and the effects of...
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