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B- and T- lymphocyte subpopulations in lymphocyte-associated immunodeficiencies
Šinkorová, Vendula ; Kalina, Tomáš (advisor) ; Javorková, Eliška (referee)
The antigen-specific immunity consists of cells called T and B lymphocytes. These cells together with cells of non-specific (innate) immunity begin their development in fetal liver and later in bone marrow from the common progenitor, the hematopoietic stem cell. Both B and T lymphocyte lineages then undergo differentiation which is regulated by many cytokines and transcriptional factors and leads to very heterogeneous cohort of subsets. Because the immune system is not only protecting the organism from infections and malignant growth but also from itself, lymphocyte differentiation must pass many checkpoints where B and T clones are strictly selected. Cells of both lineages closely communicate with each other and also with cells of innate immunity. If, due to mutation of protein encoding genes, disturbance of differentiation or malfunction of effector activities providing some of these functions occurs, an immune system malfunction called immunodeficiency arises. Multiparametric immunophenotyping followed by flow cytometry examination has been proven one of the most suitable techniques for studying lymphocyte subsets and lymphocyte- associated immunodeficiencies. Here we describe examples of primary lymphocyte- associated immunodeficiencies, how they affect individual lymphocyte subsets, what it...
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Development of selected CD markers and their role in the phylogenesis of human immune system
Podolská, Tereza ; Růžičková, Šárka (advisor) ; Vinkler, Michal (referee)
In the first part of the thesis we investigated the origin of selected surface CD markers of human, namely CD19, CD20, CD21, CD24, CD27 and CD38 molecules. In addition, nucleotide and amino acid sequences of these molecules were compared using in silico approach. Bioinformatic databases of sequences of selected molecules at DNA, mRNA and protein level, such as GeneBank, NCBI BLAST, Homologene and OrthoDB, have been used. The intent was to identify at the domain level the first organism in which it is possible to find the searched molecule. At the N-terminal domain of the CD38 of birds, a sequence showing significant similarity to the archaebacterial flagellin domain was found. This flagellin sequence in the CD38 avian molecule is located in the region of transmembrane domain, indicating that the occurrence of this sequence might be related to the formation of the transmembrane domain. The approach used here could be implemented in comparative hybridization studies as a tool in the preparatory non-laboratory phase of the research of the presence of paralogs and orthologs in phylogenetically old species. Keywords: CD marker, immunocyte, B lymphocyte, innate and adaptive immunity, sequence database, amino acid, nucleotide
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Development of selected CD markers and their role in the phylogenesis of human immune system
Podolská, Tereza ; Růžičková, Šárka (advisor) ; Vinkler, Michal (referee)
In the first part of the thesis we investigated the origin of selected surface CD markers of human, namely CD19, CD20, CD21, CD24, CD27 and CD38 molecules. In addition, nucleotide and amino acid sequences of these molecules were compared using in silico approach. Bioinformatic databases of sequences of selected molecules at DNA, mRNA and protein level, such as GeneBank, NCBI BLAST, Homologene and OrthoDB, have been used. The intent was to identify at the domain level the first organism in which it is possible to find the searched molecule. At the N-terminal domain of the CD38 of birds, a sequence showing significant similarity to the archaebacterial flagellin domain was found. This flagellin sequence in the CD38 avian molecule is located in the region of transmembrane domain, indicating that the occurrence of this sequence might be related to the formation of the transmembrane domain. The approach used here could be implemented in comparative hybridization studies as a tool in the preparatory non-laboratory phase of the research of the presence of paralogs and orthologs in phylogenetically old species. Keywords: CD marker, immunocyte, B lymphocyte, innate and adaptive immunity, sequence database, amino acid, nucleotide
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Differences in immunophenotype of CD27+ B lymphocyte subpopulations in healthy controls and patients with various immunopathologies
Valatová, Pavla ; Růžičková, Šárka (advisor) ; Petanová, Jitka (referee)
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...
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Rare B lymphocyte populations in the context of peripheral B lymphopoiesis in humans
Grígelová, Andrea ; Růžičková, Šárka (advisor) ; Hrušák, Ondřej (referee)
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...
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Immunophenotype differences in non-memory B lymphocyte populations in healthy controls and patients with immunopathologies
Polák, Milan ; Růžičková, Šárka (advisor) ; Šplíchal, Igor (referee)
B-lymphocytes are a subset of immune cells that can be distinguished mainly by carrying clonally diversified membrane-bound immunoglobulin specialized to specific antigen recognition. Together with other immunocytes B-lymphocytes play a central role in adaptive immune system which takes part in defense of the host against wide variety of pathogens. 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. That is the reason for the thesis to be focused on an analysis of B-cell population profile changes in disease, identification of any association present among specific B-cell subpopulations, as well as association between these subpopulations and clinical parameters. Using polychromatic flow cytometry we analyzed frequencies of 11 B-cell subpopulations including stages of transient B-lymphocytes through effector antibody-producing plasma cells. We examined 81 individuals including 22 healthy controls and 59 patients with...
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Eight color flow cytometry test development for primary imunodeficiency patients
Šinkorová, Vendula ; Kalina, Tomáš (advisor) ; Brdička, Tomáš (referee)
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
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B- and T- lymphocyte subpopulations in lymphocyte-associated immunodeficiencies
Šinkorová, Vendula ; Kalina, Tomáš (advisor) ; Javorková, Eliška (referee)
The antigen-specific immunity consists of cells called T and B lymphocytes. These cells together with cells of non-specific (innate) immunity begin their development in fetal liver and later in bone marrow from the common progenitor, the hematopoietic stem cell. Both B and T lymphocyte lineages then undergo differentiation which is regulated by many cytokines and transcriptional factors and leads to very heterogeneous cohort of subsets. Because the immune system is not only protecting the organism from infections and malignant growth but also from itself, lymphocyte differentiation must pass many checkpoints where B and T clones are strictly selected. Cells of both lineages closely communicate with each other and also with cells of innate immunity. If, due to mutation of protein encoding genes, disturbance of differentiation or malfunction of effector activities providing some of these functions occurs, an immune system malfunction called immunodeficiency arises. Multiparametric immunophenotyping followed by flow cytometry examination has been proven one of the most suitable techniques for studying lymphocyte subsets and lymphocyte- associated immunodeficiencies. Here we describe examples of primary lymphocyte- associated immunodeficiencies, how they affect individual lymphocyte subsets, what it...
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