(ENG) This thesis summarizes the results of a project dedicated to adaptive immune system of patients with partial DiGeorge syndrome caused by deletion of 22q11.2. The introduction sets the DiGeorge syndrome into a broader context of international pathophysio- clinical classification of primary immunodeficiencies and goes into detail describing its history, causes, clinical phenotype, therapeutic options and changes of the immune system. The attached manuscripts illustrate the premature aging of the T cell population, but also impaired development of B cells with low class-switched memory and high naïve subpopulations, along with high serum levels of BAFF, a B cell survival factor. The surprising lack of T independent marginal zone- like (MZ-like) B cells is reflected in decreased natural anti-α-Gal antibodies. The faulty B cell maturation and imperfect germinal center response is not caused by a deficit of follicular helper T cells, which are in fact increased in DiGeorge syndrome patients, and in most cases doesn't lead to hypogammaglobulinaemia. Despite the high incidence of autoimmune disease, in particular thyroiditis and thrombocytopenia, and a trend towards hypergammaglobulinaemia in adolescence and adulthood, we saw normal proportion of regulatory T cells (Tregs) and normal expression of...

Primary immunodeficiencies represent a heterogeneous group of hereditary immune system malfunctions with very variable causes and symptoms. Multiparametric flow cytometry has become an important tool in primary immunodeficiency diagnostics and research because it provides detailed information on the phenotype of individual immune cells and their proportions in circulation. We have developed a complex monoclonal antibody panel composed of five eight-color tubes which is designed for immunophenotyping of basic lymphocyte subsets and further analysis of B and T cell subpopulations. We have optimized and standardized the panels so they will identify any changes originating from primary immunodeficiencies and provide comparable data on the level of cooperation between more laboratories. This was achieved by cooperation of six European research facilities which are all parts of the Euroflow consortium. The panels have been validated both on peripheral blood samples from healthy donors and patients with either gentically defined primary immunodeficiency or common variable immunodeficiency. Keywords: T lymphocyte, B lymphocyte, primary immunodeficiency, flow cytometry, immunophenotyping, Euroflow, optimization, standardization

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.

Efficient delivery of genetic material to T-lymphocytes is key in gene therapy using T-lymphocytes with chimeric antigen receptors. Current procedures require the use of potentially dangerous viral vectors or large amount of input material. The diploma thesis therefore focuses on exploring new approaches for gene transfer into T-lymphocytes: use of safe virus-like particles (VLPs) derived from mouse polyomavirus in combination with the amphipathic cationic peptide LAH4. LAH4 has the potential to increase the efficiency of DNA and viral vector transport into cells. The system which combines VLPs and the LAH4 peptide was optimized for the delivery of reporter gene (encoding GFP and luciferase) to the model T-cell line Jurkat. It has been found that Jurkat cells cannot be efficiently transduced by DNA packed into VLPs. When cells were transfected only with DNA and LAH4, consistent results were not obtained, and the transfection efficiency ranged from 0.5 to 19%. The diploma thesis also analysed the effect of phosphorylation of viral structures on gene transfer. The impact of treatment of virus particles by alkaline phosphatase on the infectivity of the virus was studied and it was necessary to analyse the effect of the reaction components. Sublytic concentration of Triton-X100 in the reaction buffer...

T-cell activation is a complex process crucial for a proper function of immune system. It has been extensively studied and its main features are well understood. However, some of the events involved in T-cell signalling are still unclear. After T-cell receptor stimulation, Src-family kinase Lck drives the initiation of signalling by tyrosine phosphorylation. Phosphorylation of several downstream targets is dependent on the redistribution of Lck to the different compartment of the plasma membrane, called lipid rafts. In lipid rafts, active Lck is juxtaposed and activates raft-resident substrates which then trigger downstream signalling. The critical in this process is the mechanism of Lck translocation to lipid rafts which has not been studied so far and represents the topic of great academic and clinical interests. Previously, we identified the adaptor protein RACK1 as a candidate protein mediating the redistribution of Lck to lipid rafts by linking it to the microtubular network. In this thesis, we analysed the structural features and functional role of RACK1 in its interaction with Lck. We show here, using the SYF cell lines expressing the wild type and various mutated forms of Lck, that intact SH3 or SH2 domains of Lck are required for an effective RACK1-Lck complex formation. We also documented...

The influence of interferon gamma and specific antibodies on the infection with E. cuniculi in vivo has been studied. Reconstruction of SCID mice with CD4+ T-lymphocytes from BALB/c mice and from mice with defect gene for interferon gamma was used. Effects of the treatment with mouse recombinant interferon gamma and anti-E. cuniculi sera on survival of E. cuniculi infected SCID mice were monitored. The influence of the immunization with E. cuniculi antigen on the survival of E. cuniculi infected mice with defect gene for interferon gamma was examined.

B-cells of peripheral blood in humans represent a heterogeneous cellular environment displaying many important functions in the immune system. Recently, there is an increasing amount of evidence that B cell subpopulations are involved in the pathogenesis of many different diseases. However, there is little or no knowledge on how the individual differentiation stages of B lymphocytes are involved in pathological processes, and how they are distributed and represented under the physiological state and under pathological conditions. There is a reasonable assumption that, as with dendritic cells, NK / NKT cells and T lymphocytes, also B cell populations will contain minor and/or rare subpopulations reaching relative frequencies in the range of 0.01% to 0, 1 ‰. The primary aim of this thesis was to investigate the extent of phylogenetic and ontogenetic heterogeneity of the peripheral B cell population and lymphopoietic tissues on the basis of a comparative study across different vertebrate species. Another goal of the work was to use polychromatic flow cytometry with 183 individuals, out of them 50 controls and 133 patients with different (immune) pathologies or tumors in order to identify an optimal combination of surface features, and to use it to detect and demonstrate the existence of minor/rare...

The immune system will maintain the integrity of the organism from harmful non-malicious recognizes and protects the body against exo- and endogenous toxic substances and together with the nervous and endocrine system are among regulatory systems of the organism. Recently the evidence has supported the emerging concept of different B cell subpopulations to play a direct or indirect role in a pathogenesis of spectrum of disorders. However, so far the knowledge has been limited mainly in the way of how the specific differentiation stages of B lymphocytes are involved in pathogenesis of diseases and how course of disease, stage, and eventually different treatment can affect B cell homeostasis. This work is focused on the study of peripheral CD27+ B lymphocytes (one of the least explored human B lymphocytes) in healthy controls and patients with various immunopathologies, in this case we present representative results for patients with inflammatory bowel disease. Using polychromatic flow cytometry we examined 31 of peripheral blood samples, including 14 controls, 7 patients with Crohn's disease (CD) and 5 with ulcerative colitis (UC). In 6 patients with CD, we were able to perform immunophenotyping also 2 hours after intravenous administration of infliximab, and in one patient 14 days after drug...

This work deals with creating a hybrid system based on the aggregation of artificial immune system with appropriate heuristics to make the most effective spam detection. This work describes the main principles of biological and artificial immune system and conventional techniques to detect spam including several classifiers. The developed system is tested using well known database corpuses and a comparison of the final experiments is made.

B-lymphocytes are lymphoid cells, which are a part of the adaptive/innate immune system and generate antibodies. Recently, many studies have supported hypothesis that different rather minor B-lymphocyte subpopulations may play a direct and indirect role in immunopathogenesis in human pathologies such as Crohn's disease (CD). The aim of current study was therefore to investigate distribution of frequencies of B lymphocyte subpopulations (from transient to mature effector B cell stages) in peripheral blood of healthy subjects (CO), patients with Crohn's disease (CD) and ulcerative colitis (UC). Thus, using 11-colour flow cytometry we have analysed 30 blood samples of individuals, including 14 healthy controls, 11 patients with Crohn's disease and 5 with UC. In 6 patients with CD we have had an opportunity to analyze blood samples collected 2 hours after an administration of anti-TNF therapy. Higher frequencies of memory B-lymphocytes (CD19+ CD27+ , CD19+ CD20+ CD27+ and CD19+ CD20+ CD27+ IgM+) were found in patients with CD as compared to COs. (20.06±13.58%; 17.61±13.48%; 88.60±20.56% vs. 11.75±26.47%; 11.25±26.50%; and 66.82±22.60%), in case of CD19+CD20-CD27-IgM+ B-lymphocytes the difference was statistically significant (57.15±17.21% in CD vs. 19.59±31.79% in CO; p=0.0341), which is in accordance...