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Dendritic cells and autoimmune diseases with a view to type 1 diabetes mellitus
Chrástová, Iveta ; Štechová, Kateřina (advisor) ; Krulová, Magdaléna (referee)
Dendritic cells (DC) are professional antigen-presenting cells (APC) that play an essential role in the induction of immune responses. DCs develop from CD34+ hematopoietic stem cells in bone marrow and their role is uptake, processing and presentation of antigens to T cells. DCs can be divided into two distinct subset of cells, myeloid a plasmacytoid DCs. Myeloid DCs (mDC) develop from hematopoietic cells in the presence of GM-CSF and TNF-α or from monocytes in the culture with GM-CSF and IL-4, then with CD40L they mature and produce a large number of IL-12, which is important in driving CD4+ T cell to type Th1. The development of pDC is CD40L and IL-3 dependent and Flt3-L supports this process as well. The essential role of pDC is that they secrete a large amounts of type I IFN in the responses to viruses and so they maintain the antiviral stage. To recognize the viruses pDC express Toll-like receptors 7/9. DCs have on the surface also other groups of receptors, e.g. C-type lectin-like receptors, RIG-I-like receptors and NOD-like receptors. They play the role in the various diseases, mostly autoimmune diseases, in which the immune system recognizes self tissues and activates against them the immune response. Dendritic cells function is that they are competent to activate T cells, in the most cases...
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Th17 lymphocytes and autoimmunity diseases with the intention of diabetes 1. type
Labiková, Jana ; Štechová, Kateřina (advisor) ; Procházková, Jana (referee)
Th17 cells were recently identified as a cell source of IL-17. They turned up to be a T cell lineage independent of previously described Th1 and Th2. The differentiation of naive CD4+ T cells towards Th17 requires the combination of TGFβ (a cytokine essential for the development of anti-inflammatory regulatory T cells) plus IL-6 or IL-21. IL-23 is required for in vivo function and phenotype maintenance of Th17. STAT3 and RORγt were identified as pivotal transcription factors in Th17 differentiation program. Th17 proved to have pro- inflammatory effects and are characterized by the production of IL-17A, IL-17F and IL-22 - cytokines implicated in host defense against certain extracellular pathogens. The cytokine products of Th17 cells act on wide range of cell types. They induce cytokines, chemokines and metalloproteinases and they also mediate neutrophil recruitment and production of antimicrobial peptides. Autoreactive Th17 are highly pathogenic and the production of IL-17 has been detected in several autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, Crohn's disease and type 1 diabetes. These diseases were thought to be mediated by Th1 cells, but it is becoming increasingly clear that the regulation of autoimmunity is influenced at least in some diseases by Th17 cells as well.
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Epigenetic regulation of HLA class II genes and their role in autoimmune diseases
Čepek, Pavel ; Černá, Marie (advisor) ; Štechová, Kateřina (referee) ; Reiniš, Milan (referee)
(EN) Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with HLA (human leukocyte antigen) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of T1D patients and healthy controls. DNA methylation is one of the epigenetic modifications, that regulate gene expression and is known to be shaped by the environment. 61 T1D patients and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulfite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analyzed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr=0.041; DRA normalization, Pcorr=0.052) between healthy controls and T1D patients. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in T1D patients and healthy...
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The incidence of specific islet cell autoantibodies in patients with HNF1A-MODY and HNF4A-MODY
Urbanová, Jana ; Anděl, Michal (advisor) ; Štechová, Kateřina (referee) ; Bém, Robert (referee)
Islet cell autoantibodies are associated with autoimmune insulitis and belong to the diagnostic criteria of Type 1 diabetes mellitus. However, growing evidence suggests that autoantibodies are present in other types of diabetes. Here, we focus on the autoantibody incidence in Czech patients with maturity-onset diabetes of the young (MODY) and analyze their functional relevance in terms of diabetes onset and control. Autoantibodies against glutamic acid decarboxylase 65 (GADA) and protein tyrosine phosphatase islet antigen 2 (IA-2A) were measured in a cohort of 28 Czech patients with MODY (all confirmed by genetic testing). Selected clinical data were correlated to the status and kinetics of autoantibodies. One quarter of patients with MODY examined (7/28; 25%) was positive for GADA or IA-2A. GADA were more prevalent (7/7) than IA-2A (1/7). The incidence of autoantibodies did not correlate with human leukocyte antigen status, nor with particular mutation in MODY genes. The patients who were positive for the autoantibodies developed diabetes later than those who were autoantibody- negative, but had worse glycaemic control. Expression of autoantibodies decreased with any improvement of diabetes compensation. Only one patient did not correspond to the above and displayed signs of combined signs of MODY...
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Immunointerventional therapy of autoimmune diabetes with recent oncet in NOD mice.
Vargová, Lenka ; Saudek, František (advisor) ; Štechová, Kateřina (referee) ; Mráz, Miloš (referee)
Introduction: Type 1 diabetes mellitus is a chronic metabolic disease caused by autoimmune destruction of pancreatic beta cells. The theory of the disease onset is derived from study of a disease course in non-obese diabetic (NOD) mice, in which the diabetes occurs due to a dysregulation of the immune system. Experimental and clinical studies showed that the autoimmunity may be abrogated by immune intervention, which if initiated early enough may at least slow down the ongoing beta cells lost and preserve residual insulin secretion. But immune intervention alone is not sufficient to restore normoglycemia in the majority of cases. Several interventional studies showed that stimulation of proliferation and/or regeneration of beta cells are necessary to restore normoglycemia in animal models. Aim of the study: To find out, if the combination of a potent immunosuppression (murine anti-thymocyte globulin (mATG), gusperimus) together with stimulation of islet regeneration (sitagliptin) will be able to slow down or reverse the course of the disease. Another aim is to identify the mechanism by which the substances act. Material and methods: All experiments were performed in female NODShiLtJ (H2g7 ) mice. The following parameters were examined at day 0, 7, 14 and 28: blood glucose, subpopulations of...
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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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Differentiation of pancreatic stem cells into insulin producing β-cells.
Leontovyč, Ivan ; Saudek, František (advisor) ; Štechová, Kateřina (referee) ; Holáň, Vladimír (referee)
Diabetes mellitus (DM) is a severe and frequent disease with increasing prevalence. It is not possible to achieve long term cure without late complications. Recent advances in cell fate modifications open a pathway to alternative cell therapies for DM cure. My doctoral thesis "Differentiation of pancreatic stem cells into insulin producing β- cells" is focused on the development of a new source of insulin secreting cells for transplantation. Combinatorial testing of numerous potential transcription factors and epigenetic modifiers resulted in a final protocol for the reprogramming pancreatic of exocrine cells into insulin secreting cells. The key transcriptional factors TF (Pdx1, Ngn3 a MafA) were applied in the form of synthetic mRNA. In four independent experiments we applied transcriptional factors in a specific sequence, thus obtaining 14.3 ± 1.9 % insulin positive cells. When challenged in vitro by the glucose levels of 2.5 and 20 mmol/l glucose, respectively, these cells exhibited glucose-sensitivity of insulin secretion (842 ± 72 and 1 157 ± 58 pg insulin/µg DNA/ml, n=5). They also demonstrated a sensitivity of insulin secretion (863 ± 78 and 1 025 ± 66 pg insulin/µg DNA/ml, n=5) to the concentration of depolarization agent KCl applied at 0 and 30 mmol/l, respectively together with 2.5...
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Epigenetic regulation of HLA class II genes and their role in autoimmune diseases
Čepek, Pavel ; Černá, Marie (advisor) ; Štechová, Kateřina (referee) ; Reiniš, Milan (referee)
(EN) Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with HLA (human leukocyte antigen) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of T1D patients and healthy controls. DNA methylation is one of the epigenetic modifications, that regulate gene expression and is known to be shaped by the environment. 61 T1D patients and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulfite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analyzed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr=0.041; DRA normalization, Pcorr=0.052) between healthy controls and T1D patients. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in T1D patients and healthy...
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