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Immunologic profile of experimental autoimmune encephalomyelitis
Novosádová, Iva ; Fišerová, Anna (advisor) ; Zajícová, Alena (referee)
5 Anglický abstrakt Experimental autoimmune encephalomyelitis (EAE) is widely accepted as a murine model of human multiple sclerosis autoimmune disease. Murine EAE is usually actively induced by immunization with a suitable myelin antigen. Following immunization, CD4+ T helper lymphocytes Th1 and Th17 accumulate in the nervous tissue and via the production of cytokines, they mediate an inflammatory reaction and the subsequent destruction of myelin. The main goal of this study was the induction of EAE with clinically observable symptoms and the observation of changes in the counts and phenotypes of cells, mainly NK and T cells. NK cells express a wide range of inhibitory and activation receptors from the C-lectin-like receptor superfamily. The specific ligand of the activating NKR-P1C isoform is still unknown and thus this receptor's involvement in EAE was also observed. Another goal was the use of medication with regard to the disease progress improvement. For the purposes of this study, two inbred murine strains with distinct NKR-P1 surface expression were used - the SJL/J strain (expressing inhibitory NKR-P1B) and C57BL/6 (expression activating NKR-P1C). SJL mice elicited a relapse-remitting of EAE, while C57BL/6 had chronic EAE. Both mouse strains exerted changes in the counts of NK...
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Carbohydrate dimers in tumor immunotherapy
Krupová, Monika ; Bezouška, Karel (advisor) ; Ryšlavá, Helena (referee)
Carbohydrate dimers in tumor immunotherapy Monika Krupová (Department of Biochemistry, Faculty of Science, Charles University in Prague) One possible approach to tumor immunotherapy is an activation of killer lymphocytes through specific ligands for their surface receptors. CD69 is a molecule greatly widespread among various cells of haematopoietic origin. Since the physiological ligand for this receptor is still unknown, ligand mimetics are used for modulation of its activity. The mimetics tested in this work are based on monomeric or oligomeric carbohydrated attached through two different chemical groups to the central linker of varying length, giving rise to thiourea and triazole series. The ability to precipitate soluble NKR-P1 and CD69 receptors was evaluated in precipitation assays and the optimal length of the linker for NKR-P1 receptor was found to be decyl. On the other hand, cross-linking of CD69 is not so dependent on the length of the linker. The aim of this work was to describe in vitro effect of the tested compounds on cellular signalization, natural killing of leukemic cell lines and activation-induced apoptosis. Compounds of triazole series containing two disaccharides (GalNAc β1→4 GlcNAc) linked by a linker were found to have the strongest effect on the production of...
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The role of LIF gene mutations and the relative distribution of NK cells, NKT and T lymphocytes in follicular fluid and blood of women with different history of infertility
Křížan, Jiří ; Šíma, Petr (advisor) ; Jonáková, Věra (referee) ; Kročová, Zuzana (referee)
Charles University in Prague Faculty of Natural Sciences Summary of Ph.D. thesis The role of LIF gene mutations and the relative distribution of NK cells, NKT and T lymphocytes in follicular fluid and blood of women with different history of infertility Jiří Křížan Prague 2010 1 | P a g e Doctoral degree programs in biomedicine Charles University in Prague and the Academy of Sciences of the Czech Republic Programme: Biomedicine Chairman of the Subject Board: Doc. RNDr. Vladimír Holáň, Dr.Sc. Place of study: Institute of Microbiology, v.v.i., Academy of the Czech Sciences Vídeňská 1083, 142 20 Prague 4 phone: +420 296 442 318 Autor: Mgr. Jiří Křížan Supervisor: RNDr. Petr Šíma, CSc. The dissertation can be found at Dean's Office Faculty of Charles University in Prague 2 | P a g e CONTENTS Contents 2 Summary 3 1. Introduction 5 2. Hypotheses and aims 6 3. Material and methods 7 Material: 7 Methods: 8 4. Results 10 5. Discussion 12 6. Conclusion 15 7. References 16 Bibliography of Autor: 18 1. papers in extenso (thesis background) 18 2. papers in extenso (without regard to thesis) 19 3 | P a g e SUMMARY The aim of the dissertation thesis "The role of LIF gene mutations and the relative distribution of NK cells, NKT, and T lymphocytes in follicular fluid and blood of women with different history of infertility"...
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Study of receptor-ligand pair NKR-P1F and Clrg
Kotýnková, Kristýna ; Man, Petr (advisor) ; Schneider, Bohdan (referee)
Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...
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Studies on interactions between NKR-P1D and Clrb membrane receptors
Hanč, Pavel ; Novák, Petr (advisor) ; Brdička, Tomáš (referee)
Studies on interactions between NKR-P1D and Clrb membrane receptors Interaction between murine NKR-P1D and Clrb receptors was originally described as a novel type of "MHC class-I independent missing-self recognition" and was shown to confer protection from killing by natural killer cells.[1] However, further study brought conflicting results suggesting that NKR-P1D does not binds Clrb strongly if it does at all.[2] In order to address the issues arising from these conflicting results, we have recombinantly expressed the extracellular domains of both receptors in E. coli cells and refolded the proteins in vitro. The quality of refolding was confirmed both by determining the disulphide bonding pattern using FTMS and measuring 1 H/15 N-HSQC spectra. By means of size exclusion chromatography and analytical ultracentrifuge we were unable to provide convincing results for the interaction itself. However, using SPR technique, a weak, specific, pH-dependent interaction was observed. Interaction between the proteins in solution was immobilized using chemical cross-linking technique. Three cross-linking reagents, EDC, DSG and DSS were used. The reaction mixture was separated by means of SDS-PAGE and protein bands corresponding to dimers were digested in gel. Using FT-MS we were able to find peptides from both...
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Recombinant preparation of isotopically labeled receptor rNKR-P1A for NMR studies
Čonka, Martin ; Novák, Petr (referee) ; Bezouška, Karel (advisor)
anglicky NK cells belong to the population of cells which are able to cytotoxic kill certain tumor and cells infected by viruses. This bachelor thesis focuses on the rat's NK cell receptor, especially rNKR-P1A. This protein belongs to the activating receptors and is able to activate the cytotoxic function of NK cells. Physiological ligand and three-dimensional structure of this protein has not been resolved yet. The aim of this work was optimalization of production rNKR-P1A in minimal medium and thereafter producing isotope-labeled proteins. Isotope- labeled proteins could contribute to solving the three-dimensional structure of this rat receptor by using NMR methods.
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In vitro expansion and activation of natural killer cells for cell therapy.
BENEŠOVÁ, Monika
NK cells are part of the non-specific immune response and are one of the main components of antitumor immunity. They do not need antigen stimul for thein activation but recognize damaged ( transformed ) cells by characteristic decreased expression of MHC I molecules. These natural killers become subjekt of many clinical studies based on the use of anti-tumor activity of NK cells for both solid tumors and in hemato - oncological diseases. The aim of this study was to find optimal conditions for in vitro expansion and activation of NK cells. NK cells were isolated from mononuclear cell fraction by magnetic separation and cultured in two types of media SCGM and X - VIVO 10 with the addition of interleukin-2 respectively OKT3 antibody. The influence of the mononuclear cells on proliferation of NK cells was tested. After a 6- day culture , the cells were passaged and growth of NK cells was determined using hematological analyzer and flow cytometry. Expression of the activation markers CD25 and CD336 (NKp44) was observed. All experiments were conducted under conditions of good manufacturing practice. Slightly higher gain of NK cells was observed in SCGM media without a significant differences in the other additives. Much higher number of NK cells was observed in culture with supporting irradiated mononuclear cells. There were no differences in cultures with autologous or allogeneic cells. We found that NK cells with higher proliferative potential express increasingly CD25, unlike the cells with decreased proliferation which had increased expression of CD336 marker. The work led to the definition of the optimal culture conditions for NK cells and became the basis for the further development of the medicinal product from in vitro activated NK cells.
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