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Phenotypic and functional characterization of leukocytes in patients with immune system dysregulation
Fejtková, Martina ; Kanderová, Veronika (advisor) ; Vlková, Marcela (referee) ; Balounová, Jana (referee)
Inborn errors of immunity (IEI) are a heterogenic group of diseases of the immune system causing dysregulations of both innate and adaptive immunity. New altered immune-related genes are discovered every year, nowadays reaching over 400. (Tangye et al., 2020) Here three new autoinflammatory disorders of IEI patients are described. Toll-like receptors (TLR) 7 and 8 are endosomal receptors in the innate immune response against external pathogens and endogenous autoantigens. A dysregulation in TLR7 and TLR8 in mice causes autoimmunity and inflammation, however, in humans, the immunopathology of TLR8 and TLR7 remains unclear. We identified a novel X-linked c.1715G>T mutation in TLR8 that leads to autoimmune haemolytic anaemia and autoinflammation in male twins caused by dysregulation in TLR8 and TLR7 response especially in myeloid cells (low TLR8 protein expression, cross-reactivity of TLR8 for TLR7 ligands and enhanced TLR7 response). Hematopoietic cell kinase (HCK) belongs to the Src family of kinases and is involved in myeloid cell migration, adhesion and degranulation. Kinase activity in the immune response must be strictly regulated. In HCK, this regulation is based on C-terminal inhibitory tyrosine, which when phosphorylated, HCK kinase activity is switched off. We identified a heterozygous...
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Leukemia cell signaling and signaling of non-malignant B-cells
Kanderová, Veronika ; Kalina, Tomáš (advisor) ; Klener, Pavel (referee) ; Otáhal, Pavel (referee)
Acute leukemia (AL) is the most common pediatric cancer. Approximately 90 - 100 children is diagnosed every year in the Czech Republic. Acute leukemia is a complex disease that is pathologically manifested at the DNA, mRNA, protein and cellular level. Leukemic cells aberrantly express molecules that are found in other cell types under physiological conditions and their functional involvement in leukemic cells is unknow. We found that aberrantly expressed CEACAM6 increases the expression and affinity of integrins, increases the phosphorylation of intracellular kinases Akt, p38MAPK and p44/42 MAPK and triggers apoptosis in B-cell precursor acute lymphoblastic leukemia cells. Adaptor molecule NTAL, aberrantly expressed in T-cell acute lymphoblastic leukemia, signals through intracellular kinase p44/42 MAPK and potentiates corticosteroid induced apoptosis. Current leukemia research is focused mainly on monitoring of mutations at the DNA level, however, the functional consequences of these changes on cellular machineries are not straightforward. Since proteome analysis can provide link between gene sequence and cellular physiology, proteomics will contribute to elucidate mechanism of disease, prognosis and response to treatment. Protein microarrays technology is of major interest for basic proteomic research as...
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Cell signaling aberrations in primary immunodeficiencies
Fejtková, Martina ; Kanderová, Veronika (advisor) ; Vlková, Marcela (referee)
Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...
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Leukemia cell signaling and signaling of non-malignant B-cells
Kanderová, Veronika
Leukemia cell signaling Acute leukemia (AL) is the most common pediatric cancer. Approximately 90 - 100 children is diagnosed every year in the Czech Republic. Acute leukemia is a complex disease that is pathologically manifested at the DNA, mRNA, protein and cellular level. Leukemic cells aberrantly express molecules that are found in other cell types under physiological conditions and their functional involvement in leukemic cells is unknow. We found that aberrantly expressed CEACAM6 increases the expression and affinity of integrins, increases the phosphorylation of intracellular kinases Akt, p38MAPK and p44/42 MAPK and triggers apoptosis in B- cell precursor acute lymphoblastic leukemia cells. Adaptor molecule NTAL, aberrantly expressed in T-cell acute lymphoblastic leukemia, signals through intracellular kinase p44/42 MAPK and potentiates corticosteroid induced apoptosis. Current leukemia research is focused mainly on monitoring of mutations at the DNA level, however, the functional consequences of these changes on cellular machineries are not straightforward. Since proteome analysis can provide link between gene sequence and cellular physiology, proteomics will contribute to elucidate mechanism of disease, prognosis and response to treatment. Protein microarrays technology is of major...
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Cell signaling aberrations in primary immunodeficiencies
Fejtková, Martina ; Kanderová, Veronika (advisor) ; Vlková, Marcela (referee)
Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...
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Immunomodulatory properties of mesenchymal stem cells from patients with amyotrophic lateral sclerosis and healthy donors
Matějčková, Nicole ; Javorková, Eliška (advisor) ; Kanderová, Veronika (referee)
Mesenchymal stem cells (MSC) possess a multilineage differentiation potential and have the ability to regulate reactivity of the immune system. They are usually isolated and expanded from the bone marrow, adipose tissue or umbilical cord. MSC represent promising cell population for the treatment of some severe diseases, such as amyotrofic lateral sclerosis (ALS), due to the combination of regenerative and immunomodulatory properties. The aim of this study is to compare MSC from ALS patients and healthy donors in their phenotype, proliferative activity and mainly their immunomodulatory properties. The assessment of impact of the disease on the properties of MSC is important for their autologous use in clinical trials. In this study we used MSC isolated from bone marrow of 14 ALS patients and 15 patients undergoing mostly orthopedic surgery as control group. We also used MSC stimulated for 24 hours by poinflammatory cytokines. Cells were compared in terms of immunophenotype, differentiation in adipocytes and osteoblasts, metabolic activity, expression of selected genes for immunomodulatory molecules and for inhibition of lymphocyte proliferation. Further experiments were focused on evaluation of immunomodulatory properties of MSC. The effect of MSC on peripheral blood mononuclear cells stimulated...
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Leukemia cell signaling and signaling of non-malignant B-cells
Kanderová, Veronika ; Kalina, Tomáš (advisor) ; Klener, Pavel (referee) ; Otáhal, Pavel (referee)
Acute leukemia (AL) is the most common pediatric cancer. Approximately 90 - 100 children is diagnosed every year in the Czech Republic. Acute leukemia is a complex disease that is pathologically manifested at the DNA, mRNA, protein and cellular level. Leukemic cells aberrantly express molecules that are found in other cell types under physiological conditions and their functional involvement in leukemic cells is unknow. We found that aberrantly expressed CEACAM6 increases the expression and affinity of integrins, increases the phosphorylation of intracellular kinases Akt, p38MAPK and p44/42 MAPK and triggers apoptosis in B-cell precursor acute lymphoblastic leukemia cells. Adaptor molecule NTAL, aberrantly expressed in T-cell acute lymphoblastic leukemia, signals through intracellular kinase p44/42 MAPK and potentiates corticosteroid induced apoptosis. Current leukemia research is focused mainly on monitoring of mutations at the DNA level, however, the functional consequences of these changes on cellular machineries are not straightforward. Since proteome analysis can provide link between gene sequence and cellular physiology, proteomics will contribute to elucidate mechanism of disease, prognosis and response to treatment. Protein microarrays technology is of major interest for basic proteomic research as...
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Leukemia cell signaling and signaling of non-malignant B-cells
Kanderová, Veronika
Leukemia cell signaling Acute leukemia (AL) is the most common pediatric cancer. Approximately 90 - 100 children is diagnosed every year in the Czech Republic. Acute leukemia is a complex disease that is pathologically manifested at the DNA, mRNA, protein and cellular level. Leukemic cells aberrantly express molecules that are found in other cell types under physiological conditions and their functional involvement in leukemic cells is unknow. We found that aberrantly expressed CEACAM6 increases the expression and affinity of integrins, increases the phosphorylation of intracellular kinases Akt, p38MAPK and p44/42 MAPK and triggers apoptosis in B- cell precursor acute lymphoblastic leukemia cells. Adaptor molecule NTAL, aberrantly expressed in T-cell acute lymphoblastic leukemia, signals through intracellular kinase p44/42 MAPK and potentiates corticosteroid induced apoptosis. Current leukemia research is focused mainly on monitoring of mutations at the DNA level, however, the functional consequences of these changes on cellular machineries are not straightforward. Since proteome analysis can provide link between gene sequence and cellular physiology, proteomics will contribute to elucidate mechanism of disease, prognosis and response to treatment. Protein microarrays technology is of major...
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