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Anti-tumour necrosis factor activity in saliva of various tick species and its appearance during the feeding period
REZKOVÁ, Markéta
Tumour necrosis factor (TNF) is one of the most important pro-inflammatory cytokines of the innate immune system. We compared the presence of anti-TNF activity in saliva or salivary gland extract from 11 tick species from the family Ixodidae and demonstrated it in genera Ixodes and Haemaphysalis. We also tested dynamics of this specific activity in Ixodes ricinus saliva. The highest effect of saliva appears at the end of feeding period. Significance of anti-TNF activity for tick feeding and transmission of tick-borne pathogens is discussed.
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Increasing affinity of Interferon gamma receptor 1 to Interferon gamma by combining molecular modeling and experimental methods
Mikulecký, Pavel ; Schneider, Bohdan (advisor) ; Šulc, Miroslav (referee) ; Vaněk, Ondřej (referee)
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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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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Impact of Toll-like receptor 4 polymorphism on pro-inflammatory responsiveness in great tit (Parus major)
Vinklerová, Jitka ; Bryja, Josef (advisor) ; Černý, Jan (referee)
Toll-like receptor 4 (TLR4) belongs among chief bacteria-sensing Pattern recognition receptors. Endotoxin (lipopolysaccharide, LPS) recognition by TLR4 triggers signalling leading to release of cytokines that direct leukocyte infiltration into the inflammatory site and cause swelling. Effector mechanisms that ensure pathogen elimination include phagocytosis and oxidative burst. It has been repeatedly reported that the polymorphism in TLR4 may affect host resistance to various diseases. TLR4 may be, therefore, an important molecule in host-parasite co-evolution. Herein, I focused on TLR4 amino acid substitution Q549R which is associated with ornamentation in great tits. In tits I describe immune responsiveness to LPS stimulation on morphological and molecular level and examine effects of the Q549R substitution on inflammation and general body condition. In LPS- treated individuals I found decrease in heterophil-lymphocyte ratio (H/L) that might be caused by attraction of the blood-borne cells into the inflamed tissue. This is in striking contrast with increase in H/L in PBS-treated animals resulting from the stress response. There was no effect of Q549R on general condition and haematological parameters but I revealed a significant effect of the interaction between host Q549R genotype and the type...
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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 effect of tick saliva on the interactions between \kur{Borrelia afzelii} spirochetes and murine dendritic cells.
SLÁMOVÁ, Martina
Interaction between mouse dendritic cells (DCs) and Borrelia afzelii spichochetes was studied on three different levels: phagocytosis of borrelia by DCs, production of cytokines by borrelia-activated DCs and the ablilty of DCs to activate CD4+ T cells. The effect of Ixodes ricinus saliva on each of these levels was examined. Tick saliva was shown to decrease the number of phagocosing DCs. The ability of borrelia-activated DCs to induce both proliferation and IL-2 production in specific CD4+ T cells was significantly reduced by tick saliva. And surprisingly, we have shown an inhibitory effect of I. ricinus saliva on the production of both Th1 (IL-6 and TNF-{$\alpha$}) and Th2 (IL-10) cytokines. Our data reveal a complex inhibitory effect of tick saliva on DC function.
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