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Mechanisms of MHCII signaling in B lymphocytes
Kotlabová, Klára ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
During the initiation of an antigen-specific immune response, peptide fragments originating from the antigen are presented in complex with MHC class II glycoproteins (MHCgpII) on the surface of the antigen presenting cells (APC). Antigen recognition by T lymphocyte is accompanied by the formation of the molecular structure at the interface with APC called immunological synapse (IS). During this contact, signal transduction is initiated at both, T lymphocyte and APC, sides of the IS. For a long time it was thought that the only function of MHCgpII is presentation of antigen. However, later it was found that stimulation of MHCgpII led to triggering of signals contributing to decision about the further fate of APC. MHCgpII do not have any signaling motifs in their cytoplasmatic domains, and so associated molecules are necessary for the transduction of the signals. This work focuses on B lymphocytes in which the associated molecules are Ig alfa/beta, MPYS, CD19 and CD20. After the stimulation of MHCgpII these proteins mediate signaling events including activation of several families of protein kinases, phospholipase C, mobilization of calcium and activation of transcriptional factors NFAT and AP-1. In B lymphocytes, activities of these pathways may result in proliferation and differentiation but also in the...
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Determination the mechanism of entry F. tularensis into B lymphocytes
Hadámková, Barbora ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Barbora Hadámková Determination the mechanism of entry F. tularensis into B lymphocytes Diploma thesis Charles University, Faculty Of Pharmacy in Hradec Králové Study program: Pharmacy Background: Besides processing the research with basics knowledge of the problem, the main aim of the study was the analysis of mechanism of entrance of intracellular bacteria Francisella tularensis into B cells. Methods: The B cells, which we obtained through peritoneal lavage from mice Balb/c, we blocked using antibodies individual complement receptors, B cell receptor and Fcƴ receptor. The population of the cells was infected by bacteria F. tularensis LVS/GFP opsonized by complement and/or by antibodies. Using flow cytometry we measured the percentage of infection of individual subpopulations of B cell B1a, B1b and B2 and we evaluated the influence of blocking and opsonization on the infection. Results: From the measured data, we can say that the percentage of infected B cells after infection by F. tularensis opsonized by complement is increased. This increase was more distinct in subtype of B cells B1b and B2. On the other hand, the opsonization F. tularensis by antibodies did not affect the infection. We also found out, that blocking of Fcƴ receptor has decrease the infection, if we used for infection of B cells...
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Entry of bacteria Mycobacterium bovis BCG into B lymphocytes
Šamajová, Marianna ; Konečná, Klára (advisor) ; Kubelková, Klára (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...
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Characterization of biological and functional features of a new type of CD27- memory B lymphocytes.
Bajzíková, Martina ; Růžičková, Šárka (advisor) ; Šinkora, Marek (referee)
The increased frequencies of two novel B cell populations defined as IgM+ CD19+ CD27- CD21low CD38low CD24+ and IgM+ CD19+ CD27- CD21low CD38low CD24- in peripheral blood of patients with common variable immunodeficiency (CVID) compared to healthy donors were found. The aim was to search for such B cells in patients with rheumatoid arthritis (RA) and their further characterization. The production of immunoglobulin (Ig) mRNA in single B cells was analyzed using flow cytometry, single cell sorting and RT-PCR, IgVH-specific PCR, cycle sequencing and statistical analysis. The study was focused on analysis of variable regions of the heavy chains of Igs and significant differences in the usage of VH, DH and JH gene segments, mutational frequencies, distribution of silent and replacement mutations, length and composition of CDR3 regions, clonal relation and RAG gene expression in above mentioned B cell populations were found. Because of lack of the surface CD27 molecule being regarded as marker of B cells that have undergone antigen-driven germinal reactions, analyzed populations were considered as naive. However, the pattern and type of mutations suggested that these cells could represent a new type of differentiated memory/antigen- experienced B lymphocytes (in CVID less maturated) with the likely role in...
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The development of swine B cells and the role of gama delta T lymphocytes in immunization of naive immune system.
Štěpánová, Kateřina
Thesis summary The process of B cell lymphogenesis in swine remains uncertain. Some reports indicate that pigs belong to a group of animal that use ileal Peyers's patches (IPP) for the generation of B cells while others point to the possibility that the bone marrow is functional throughout life. The functional subpopulations of B cells in swine are also unknown. Together with other ruminants, and also birds, γδ T cells in swine may account for >70% of all T cells which is in apparent contrast with humans and mice. The purpose of this thesis was to address these discrepancies and unresolved issues. The results disprove the existing paradigm that the IPP is primary lymphoid tissue and that B cells develop in IPP in an antigen-independent manner. On the other hand, it shows that bone marrow is fully capable of B cell lymphogenesis and remains active at least for the same period of time as it had been speculated for the IPP. This thesis also identified functionally different subsets of porcine peripheral B cells, and shows that CD21 molecules can be expressed in differential forms. Finally, this thesis identifies two lineages of γδ T cells that differ in many functional and phenotype features. This finding may explain why γδ T cells constitute of minority of lymphocytes in circulation of humans and mice.
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Markers of transplantation tolerance in kidney transplantation
Krepsová, Eva ; Viklický, Ondřej (advisor) ; Krejčí, Karel (referee) ; Živný, Jan (referee)
Long-term renal graft acceptance still requires long-term immunosuppressive therapy, which is accompanied by many adverse effects. Contrarily insufficient immunosuppression could lead to graft rejection and its failure. Therefore, research continues for biomarkers that reflect a patient's immunological status and thus allowing for individualized immunosuppressive therapy. In our study we showed lower incidence of acute rejection in kidney transplant recipients treated with rabbit anti-thymocyte globulin (rATG) or basiliximab induction within the first three months after transplantation. The rATG induction caused profound decrease of recipient's peripheral blood T and NK cells, as well as transcripts that are exclusively expressed by these cell types together with expansion of regulatory T cells (Tregs) among CD4+ T cells. In rATG group the increase of two transcripts associated with rejection (MAN1A1 and TLR5) was also observed in early post-transplant period. After the basiliximab induction we transiently detected CD4+CD25low/-FoxP3+ cell population along with disappearance of CD4+CD25+FoxP3+ Tregs. Basiliximab induction resulted in a transient increase in CD4+FoxP3+ Tregs, accompanied by the highest peripheral expression levels of markers associated with operational tolerance (FOXP3 and TCAIM)....
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Immune system dysregulation in type 1 diabetes
Paračková, Zuzana ; Šedivá, Anna (advisor) ; Filipp, Dominik (referee) ; Vlková, Marcela (referee)
Type 1 diabetes (T1D) is an autoimmune disease with multifactorial aetiology that involves an attack of self-reactive cytotoxic CD8 lymphocytes on insulin-producing beta cells in the pancreas. In the T1D pathophysiology, both innate and adaptive immunity mechanisms cooperate in the development of inflammation leading to autoimmune destruction. Autoreactive T lymphocytes are the canonical destructors of the beta cells, and B cells produce autoantibodies; the innate immunity cells are considered the initiators of the pathological autoimmune reaction by promoting T and B cell activation. Here, we provide evidence of both innate and adaptive immunity cell types dysregulation in patients with T1D, and that these changes occur before the onset of the disease. The changes in T regulatory lymphocytes (Tregs) and B cell subpopulations occur already in asymptomatic T1D first-degree relatives. During the first year after the onset of the disease, there is a gradual decrease in the neutrophil numbers in the periphery, which probably infiltrate the pancreas. We have focused more closely on the innate immunity dysregulation and its contribution to T1D pathogenesis. Initially, we describe that neutrophil products called neutrophil extracellular traps (NETs) are able to induce IFNγ-producing T cells through...
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Possibilities of prediction and immunointervention in type 1 diabetes
Sklenářová, Jana ; Štechová, Kateřina (advisor) ; Saudek, František (referee) ; Pavlínková, Gabriela (referee)
Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterised by autoimmune destruction of insulin-producing beta cells in the islets of Langerhans. It is a long-term process initiated months or even years prior to the clinical onset. The main role in the pathogenesis is played by T lymphocytes but other cell types are involved as well. The presence of autoantibodies in the circulation is typical even before the disease onset. Nowadays, intensive research is focused on finding individuals at risk and developing an effective prevention. During my postgraduate studies I was involved mainly in the research of T1D prediction and prevention. We investigated the relationship of established autoimmune markers - autoantibodies - and the cellular reactivity to GAD65 and IA2 autoantigens. We discovered that the reaction to autoantigens is very individual and it is influenced by the patient's autoantibody profile. These results could be relevant in planning antigen-specific immunointervention studies and improving their efficacy. We also made an attempt to improve specificity and sensitivity of a beta cell destruction marker (specifically demethylated DNA), which would enable better understanding of the beta cell decline and identification of individuals at risk of T1D development. In...
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Entry of bacteria Mycobacterium bovis BCG into B lymphocytes
Šamajová, Marianna ; Konečná, Klára (advisor) ; Kubelková, Klára (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...
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Determination the mechanism of entry F. tularensis into B lymphocytes
Hadámková, Barbora ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Barbora Hadámková Determination the mechanism of entry F. tularensis into B lymphocytes Diploma thesis Charles University, Faculty Of Pharmacy in Hradec Králové Study program: Pharmacy Background: Besides processing the research with basics knowledge of the problem, the main aim of the study was the analysis of mechanism of entrance of intracellular bacteria Francisella tularensis into B cells. Methods: The B cells, which we obtained through peritoneal lavage from mice Balb/c, we blocked using antibodies individual complement receptors, B cell receptor and Fcƴ receptor. The population of the cells was infected by bacteria F. tularensis LVS/GFP opsonized by complement and/or by antibodies. Using flow cytometry we measured the percentage of infection of individual subpopulations of B cell B1a, B1b and B2 and we evaluated the influence of blocking and opsonization on the infection. Results: From the measured data, we can say that the percentage of infected B cells after infection by F. tularensis opsonized by complement is increased. This increase was more distinct in subtype of B cells B1b and B2. On the other hand, the opsonization F. tularensis by antibodies did not affect the infection. We also found out, that blocking of Fcƴ receptor has decrease the infection, if we used for infection of B cells...
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