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Monitoring of sublingual allergen immunotherapy using serum cytokine levels
Brzáková, Adéla ; Řezáčová, Vladimíra (advisor) ; Andrýs, Ctirad (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Bc. Adéla Brzáková Supervisor: Mgr. Vladimíra Řezáčová, Ph.D. Title of thesis: Monitoring of sublingual allergen immunotherapy using serum cytokine levels Keywords: allergen immunotherapy, cytokine, ELISA, grass pollen allergy Background: The aim of this thesis was to find out whether there are detectable changes in the levels of IFNγ, IL-4 and IL-5 during allergen immunotherapy and whether these cytokines could thus serve as biomarkers of the efficiency of this treatment. Methods: Selected cytokines were determined using Quantikine® HS ELISA Human IFN-γ Immunoassay, Quantikine® HS ELISA Human IL-4 Immunoassay and Quantikine® ELISA Human IL-5 Immunoassay. The monitored population consisted of 32 polinotics, 19 of which were administered allergen immunotherapy and 13 of which constituted the control group. As part of the follow-up, both groups also continuously recorded the severity of their symptoms using a visual analog scale. Results: In the group of treated patients, a decrease in IL-4, IL-5 and IFNγ concentrations was observed during treatment, but no significant differences in the values of these cytokines were found between patients treated with Oralair and control individuals. Using...
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Monitoring of sublingual allergen immunotherapy using serum cytokine levels
Brzáková, Adéla ; Řezáčová, Vladimíra (advisor) ; Andrýs, Ctirad (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Bc. Adéla Brzáková Supervisor: Mgr. Vladimíra Řezáčová, Ph.D. Title of thesis: Monitoring of sublingual allergen immunotherapy using serum cytokine levels Keywords: allergen immunotherapy, cytokine, ELISA, grass pollen allergy Background: The aim of this thesis was to find out whether there are detectable changes in the levels of IFNγ, IL-4 and IL-5 during allergen immunotherapy and whether these cytokines could thus serve as biomarkers of the efficiency of this treatment. Methods: Selected cytokines were determined using Quantikine® HS ELISA Human IFN-γ Immunoassay, Quantikine® HS ELISA Human IL-4 Immunoassay and Quantikine® ELISA Human IL-5 Immunoassay. The monitored population consisted of 32 polinotics, 19 of which were administered allergen immunotherapy and 13 of which constituted the control group. As part of the follow-up, both groups also continuously recorded the severity of their symptoms using a visual analog scale. Results: In the group of treated patients, a decrease in IL-4, IL-5 and IFNγ concentrations was observed during treatment, but no significant differences in the values of these cytokines were found between patients treated with Oralair and control individuals. Using...
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Role of IL-34 in the pathogenesis of autoimmune rheumatic diseases
Reichlová, Petra ; Andrés Cerezo, Lucie (advisor) ; Brdička, Tomáš (referee)
Interleukin-34 (IL-34) is a cytokine described in 2008 using proteomic analysis. As IL-34 does not share any sequence similarity with other interleukins, it fell into the group so-called "orphan" cytokines. IL-34 binds to three receptors: CSF-1R, syndecan-1 and PTP-ζ. These interactions influence signal pathways which lead to proliferation, differenciation of monocytes or to an increased expression of other cytokines. Up-regulated expression of IL-34 has been observed in many autoimmune rheumatic diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögrenʼs syndrome or systemic scleroderma. These findings indicate that IL- 34 may be involved in the pathogenesis of these diseases and may become one of the diagnostic markers or even the target of medical therapy in the future. Key words: interleukin-34, cytokine, autoimmune rheumatic diseases, rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, Sjögrenʼs syndrome
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The expression of interleukin 20 and its role in rheumatoid arthritis
Yadollahi, Benjamin ; Šenolt, Ladislav (advisor) ; Krulová, Magdaléna (referee)
Rheumatoid arthritis (RA) is a chronic autoimmune disease that is associated with formation of autoantibodies, activation of inflammatory cascade and up-regulation of several cytokines. These processes lead to persistent synovial inflammation, joint damage and systemic manifestations. The aim of this diploma thesis is to characterize the role of a novel cytokine interleukin-20 (IL-20) in the pathogenesis of RA and to investigate its involvement in different stages of the disease as a potential surrogate biomarker. In this work, several methods including Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry and Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) have been employed. We demonstrated increased expression of IL-20 in the synovial tissue of RA compared with control osteoarthritis (OA) patients. Along with the up-regulation at sites of inflammation, concentrations of IL-20 were higher in the synovial fluid compared with circulating levels of IL-20. Furthermore, serum and synovial fluid IL-20 levels significantly correlated with RA disease activity. Synthesis of IL-20 was significantly increased in peripheral blood mononuclear cells (PBMCs) and synovial fibroblasts upon stimulation with some TLR ligands and pro-inflammatory cytokines. Although not regulating PBMCs functions in...
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Significance of prolactin as peripheral cytokine in dysbalance of immune system
Janatová, Kateřina ; Černá, Marie (advisor) ; Holáň, Vladimír (referee)
Background: Interactions between the neuroendocrine and immune system play an importatnt role in maintaining homeostasis. This communication is mediated by cytokines, neurotransmiters and hormones through endocrine, paracrine and autocrine signaling. Prolactin (PRL), hormone of anterior pituitary, is produced by a number of other tissues and cells of immune system. On periphery, PRL is cytokine. Sepsis is an inflamatory response of the organism to severe infection, Th1 immune response is activated and PRL could participate in it. Toll-like receptors (TLR) play a key role in a recognition of bacteial components and mediate a systemic response (with PRL secretion) during infection. It is supposed that activated immune system leads to increasing of PRL, TLR2 and TLR4 gene expression. We detected PRL, TLR2 a TLR4 mRNA levels in monocytes from patiens with system inflammation. We studied influence of single nucleotide polymorphism (SNP -1149 G/T) in PRL gene promotor, it supposed that G allele increases PRL expression. Materials and Methods: For the pilot study 30 patients diagnose with severe infectious event. Collectoin of patiens blood samples was performed consequently three times. Control group comprised 40 healthy individuals. One blood sample was taken from each healthy subject. For testing of...
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The role of Kit ligands in hematopoiesis of Danio rerio
Oltová, Jana
Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...
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Increasing affinity of Interferon gamma receptor 1 to Interferon gamma by combining molecular modeling and experimental methods
Mikulecký, Pavel
Protein-protein interactions play an important role in nearly all processes of the living cells and the function of many proteins is dependent on their specific interactions with other biomolecules. A reliable tool to modulate these interactions would be invaluable for the development of molecules suitable for diagnostics, medicine, and biotechnology. In this work, we aimed to study the specificity of interactions in the model system of Interferon gamma receptor 1 (IFNgR1) and its natural ligand Interferon gamma (IFNg), important in innate immunity. We searched for mutations within the interferon receptor molecule IFNgR1 to modulate (increase as well as decrease) its affinity to IFNg by in silico analysis of the existing crystal structures of the complex between IFNgR1 and IFNg. We modeled amino acid substitutions and gauged how they influenced the interaction using empirical force field implemented in software FoldX. All selected promising IFNgR1 variants were expressed in Escherichia coli, purified to homogeneity, characterized, and kinetics of their interactions with IFNg was measured by Surface Plasmon Resonance (SPR). The first set of IFNgR1 variants included mutations on the interface of the IFNg/IFNgR1 complex. According to our SPR measurements, the affinity of most of these receptor...
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Modulation of interactions between interleukins and their receptors
Nepokojová, Tereza ; Schneider, Bohdan (advisor) ; Obšilová, Veronika (referee)
Scaffolds are proteins with high conformational stability, allowing us to implement multiple mutations into specific parts of the protein. Even with these mutations, the structural integrity of the protein is maintained as well as its physical-chemical properties. These mutations give the specific scaffold new properties. In most cases it is the binding specificity towards previously chosen target. The biggest advantages of scaffolds are their small size, stability, low-cost manufacturing, and easiness of preparation. Scaffold utilized in this thesis is unique for having two binging surfaces designed on which it can be mutated. Each of those two surfaces can be separately mutated to develop a binging site for two different proteins. In our case these mutations led to binding two nonidentical receptors of a human cytokine. Mutations are made with a use of yeast display, one of the methods of directed evolution. The main focus of this thesis is changing an expression system of the binding proteins from the yeast system to a bacterial one, their production and purification followed by characterization of those binding proteins using biophysical methods. These methods were used to evaluate structural and thermal stability, and binding affinity to both receptors of the beforementioned binding proteins....
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Cytokine expression in regressive melanoma on porcine MeLiM model
Miltrová, Veronika ; Skalníková, Helena (advisor) ; Krulová, Magdaléna (referee)
Cutaneous melanoma is a very aggressive cancer with increasing incidence. It originates from transformed pigmented skin cells (melanocytes). The main risk factor for melanoma development is exposure to UV light and repeated sunburns. In approximately 10 % of cases, melanoma occurs on hereditary basis. Patients with cutaneous melanoma diagnosed in early stages have very good prognosis, with surgical resection of the primary tumour being mostly sufficient for treatment. In contrast, the advanced melanoma stages with metastases are often progressive and refractory to conventional therapies. Cutaneous melanoma is referred to as an immunogenic tumour that is frequently infiltrated by cells of the immune system. Tumours with immune cell infiltration show better prognosis. Spontaneous regression may occur. Over the last few years, progress has been made in the treatment of melanoma using checkpoints molecules (anti-CTLA-4 and anti-PD-1) to activate patients own immune system to recognize tumour lesions. In the tumour microenvironment, cytokines play an important role, enabling communication between cells and regulation of cell proliferation and migration and thus the tumour development. Cytokines (IL-2, IFNα) can be used in adjuvant therapy of melanoma. This work analysed levels of expressed cytokines in...
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The role of Kit ligands in hematopoiesis of Danio rerio
Oltová, Jana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...
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