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HLA neshody u pacientů po opakované transplantaci ledviny a incidence akutní buněčné a protilátkami zprostředkované rejekce.
Karasová, Alexandra ; Slavčev, Antonij (advisor) ; Ambrůzová, Zuzana (referee)
Kidney transplantation is the most appropriate treatment for end-stage kidney failure. The risk of graft failure in retransplanted patients is generally higher than in first-transplant patients due to immunological and non-immunological reasons. An important risk factor to consider for retransplant patients is their sensitization, i.e. the presence of antibodies directed to HLA antigens of previous donor(s). For that reason, a project called Forbidden (Non-acceptable) Antigens was launched by IKEM with the aim of reducing the incidence of acute cellular and antibody-mediated rejection in retransplant patients. Work on the project was carried out between the years 2011-2013. Forbidden antigens were defined as mismatched HLA antigens of previous kidney donor(s) against which patients waiting for retransplantation produced antibodies. The aim of this diploma thesis is to evaluate whether the incidence of rejection is lower in patients with forbidden HLA antigens in comparison with a control cohort, where no forbidden antigens are defined. 234 patients (162 males and 72 females) were included in the study. Almost all tested patients were producing HLA antibodies (90.2%) and forbidden antigens were determined in 71.4% of patients. In a control group of 267 patients waiting for their first transplantation, the...
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Epigenetic regulation of HLA class II genes in relation to senescence of organism
Říhová, Adéla ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Slavčev, Antonij (referee)
Introduction: Glycoproteins of the major histocompatibility complex (MHC) are an irreplaceable part of immune response regulation and immune homeostasis maintenance. The regulation of the expression plays an important role in adaptive immune response. Recently, DNA methylation in regulatory areas, crucial for DNA availability to transcription factors, is one of the most researched mechanisms of this type of regulation. The DNA methylation is, among others, related to the aging processes. Increased predisposition age-related immunosenescence in higher age could result from the changes in methylation status of regulatory areas of MHC class II genes. Aims: The aim of this thesis is to analyze the methylation status of regulatory areas of DQB1 gene and to compare the differences between generations and specific alleles. The differences in the levels of DQB1 gene mRNA transcription between generations and specific alleles is also compared. Methods: Both DNA and RNA were isolated from blood samples obtained from donors of three different age groups. DNA was genotypized and modified by bisulfite conversion. The regulatory areas of DQB1 genes were then amplified and subcloned into bacteria. The positive clones were selected and subjected to DNA methylation analysis. RNA was reverse transcribed into cDNA...
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Laboratory Diagnostics and HLA Typization of Patients with Rheumatoid Artritis.
Škoda, Marek ; Půtová, Ivana (advisor) ; Slavčev, Antonij (referee)
This work studies the relationship between DRB1 alleles and production of clinically most important autoantibodies in patients with rheumatoid arthritis (RA). A group of 81 patients with RA were diagnosed in immunological laboratory and genotyped. The prevalence of most often tested analytes were determined: antinuclear antibodies (ANA), rheumatoid factor (RF), anti-CCP antibodies and anti-MCV antibodies. HLA-DRB1 alleles were detected using PCR-SSP low resolution. The aim of this study was to determine the frequency of DRB1 alleles in Czech patients and to investigate the relationship between DRB1 alleles and production of particular antibodies. The frequency of HLA-DRB1*01 and HLA-DRB1*04 alleles was significantly increased in RA patients compared to healthy subjects. In contrast, the frequency of DRB1*15 allele was significantly reduced. Studying the relationship between DRB1 alleles and presence of antibodies showed a significantly increased frequency of DRB1*04 allele in patients with positive ACPA antibodies (anti-CCP, anti-MCV). Regarding other antibodies (ANA, RF), no relationship between their production and presence of DRB1 alleles was found. Comparison of anti-CCP and anti-MCV antibodies levels between groups of RA patients with the presence and absence of DRB1*04 alleles showed no difference....
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Immunopathological and immunogenetic aspects of haematopietic stem cell and solid organ transplantations
Jindra, Pavel ; Boudová, Ludmila (advisor) ; Fakan, František (referee) ; Slavčev, Antonij (referee)
The genetic diversity of KIR genes and genotypes resembles of the HLA. Although the genes encoding KIR and HLA are located in different chromosomes and segregate independently, there is some evidence of some kind of co-evolution. Therefore, one could expect reduced KIR diversity within the HLA restricted population. A total of 41 unrelated individuals homozygous for ancestral HLA haplotype AH8.1 (HLA-A*0101-Cw*0701-B*0801- DRB1*0301-DQB1*0201), were typed for KIR genes. Over all, fourteen different genotypes were identified. The observed frequencies of KIR genes and genotypes composition generally mirror the published frequencies in Caucasians. Non-framework genes with frequency of more than 90 % included KIR2DL1, KIR2DL3, KIR3DL1, KIR2DS4 and KIR2DP1. Except for the KIR2DS4, all activating genes presented frequencies bellow 50 %. KIR2DS5 was the least frequent among activating genes (17 %), whereas KIR2DL5 (37 %) among inhibitory ones. The most frequent (39 %) was AA genotype. 22 individuals (54 %) had a copy of KIR haplotype A and B (AB genotype), whereas 3 (7%) were homozygous for B (BB genotype). Nine of 14 reported genotypes occurred only in one individual. Comparing with published and recorded genotypes (www.allelefrequencies.net), 5 genotypes were reported in less than 20 individuals worldwide and...
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ELISpot methodology and prediction of acute rejection after renal transplantation.
Rybáková, Kateřina ; Slavčev, Antonij (advisor) ; Mrázek, František (referee)
Transplantation is the best therapeutic solution for patients with chronic renal failure. Due to the great advances in immunosuppressive therapy in the last decades, graft and patient survival have improved significantly. On the other hand, immunosuppressive therapy has serious side effects - too strong immunosuppression may lead to infection or malignancies, conversely insufficient immunosuppression may lead to graft rejection. Due to the grave consequences of acute rejection, the main goal of cooperation of clinicians and transplant immunologists is to stratify patients into groups with low, moderate and high risk of rejection based on the evaluation of various immunologic risk factors. There are reports in the literature that the numbers (frequencies) of interferon gamma (IFNγ) producing cells before transplantation may be helpful to identify patients with high risk of acute cellular rejection and to predict long-term survival of the graft. In this retrospective study we determined the pre-transplant frequencies of activated donor specific T lymphocytes producing IFNγ after short stimulation (24 hrs) by ELISpot (Enzyme-linked immunosorbent spot assay). The results were correlated with the incidence of acute cellular (ACR) and antibody-mediated (AMR) rejection and with other risk factors. In our...
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Epitopes of HLA antigens and their relevance for organ transplantation program
Šutta, Adrián ; Slavčev, Antonij (advisor) ; Hájková, Michaela (referee)
and Key words This diploma thesis is focused on assessing the potential benefit of HLA epitopes for the prediction of de novo antibody production at kidney transplant recipients. The topic and patient selection criteria were selected in accordance with the 18th International HLA and Immunogenetics workshop (IHIWS), which is taking place in May 2022 in the Netherlands, where our data will also be contributed. Our aims were to compare HLA antigens mismatches (counted as total number of mismatched alleles) defined on the high-resolution level by NGS sequencing, HLA eplets mismatches defined by HLA matchmaker, and amino acid mismatches defined by HLA EMMA in their capacity to predict de novo antibody production and compare these results to other works by different authors from this field. We have identified N= 28 patients who developed de novo antibodies and N= 19 who didn't develop de novo antibodies in 5 years follow up their transplant. These two cohorts were compared based on all three approaches and correlation between number of mismatches and number of patients with de novo antibodies were made using ROC curves. Superiority of eplet mismatches over HLA antigen mismatches (total number of mismatched alleles) defined on high resolution was not detected. The HLA epitopes identified by the HLA...
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Regulation of HLA class II genes expression
Zajacová, Marta ; Černá, Marie (advisor) ; Černý, Jan (referee) ; Slavčev, Antonij (referee)
HLA class II genes are known to be highly polymorphic, even in the regulatory non-coding gene regions. Polymorphism in the promoter region potentially forms a strong basis for an uneven allele-specific expression. Even though it is known that the amount of HLA class II molecules on the cell surface has a significant role in shaping immune response, HLA class II expression polymorphism has not yet been thoroughly measured. The thesis aims to shed light into allele-specific mRNA expression and promoter DNA methylation of HLA class II genes. Two studies, each addressing different aspects of the HLA class II allele expression regulation, were conducted. Study A examines the DNA methylation of 10 DQA1 promoter and its effect on the DQA1 mRNA expression. DNA methylation in whole blood cells was determined with bisulfite sequencing and mRNA expression was measured using RT-qPCR. Even though inter-allelic differences in overall methylation were observed (the most methylated alleles were DQA1*02:01 and *04:01), the expected negative correlation between the DQA1 promoter DNA methylation density and the allele expression was not observed. We suggest that the genetic polymorphism in the region (especially region upstream of position -400, which is almost completely methylated in all alleles) may lead to different...
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Complement-binding antibodies in patients after organ transplantation and their clinical relevance
Kovandová, Barbora ; Slavčev, Antonij (advisor) ; Krulová, Magdaléna (referee)
The diagnosis of antibody-mediated rejection after liver transplantation is complicated, due to the fact that the clinical and pathological signs of this life- threatening complication are often overlapping with non-immunological symptoms, like biliary obstruction, ischemia, thrombosis and others. Furthermore, the transplanted liver is to a great extent resistant to this type of rejection. Like in the transplanted kidney and heart, the main pathological factors of graft injury are antibodies directed to the mismatched HLA antigens of the organ donor, i.e. donor- specific antibodies. Besides analysis of HLA specificity of antibodies, research lately has been directed to define whether these antibodies are complement-binding or not. Literature data on this are however up till now limited. Therefore, the aim of this diploma thesis was to study the clinical relevance of complement-binding antibodies against HLA antigens in patients after liver transplantation. Our preliminary results suggest that there might be a correlation between the presence of complement-binding antibodies and the development of antibody-mediated rejection. This finding may play a role for improvement of the prognosis of patients after liver transplantation. Key words HLA, antibodies, C4d, complement, liver transplantation
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Regulation of HLA class II genes expression
Zajacová, Marta ; Černá, Marie (advisor) ; Černý, Jan (referee) ; Slavčev, Antonij (referee)
HLA class II genes are known to be highly polymorphic, even in the regulatory non-coding gene regions. Polymorphism in the promoter region potentially forms a strong basis for an uneven allele-specific expression. Even though it is known that the amount of HLA class II molecules on the cell surface has a significant role in shaping immune response, HLA class II expression polymorphism has not yet been thoroughly measured. The thesis aims to shed light into allele-specific mRNA expression and promoter DNA methylation of HLA class II genes. Two studies, each addressing different aspects of the HLA class II allele expression regulation, were conducted. Study A examines the DNA methylation of 10 DQA1 promoter and its effect on the DQA1 mRNA expression. DNA methylation in whole blood cells was determined with bisulfite sequencing and mRNA expression was measured using RT-qPCR. Even though inter-allelic differences in overall methylation were observed (the most methylated alleles were DQA1*02:01 and *04:01), the expected negative correlation between the DQA1 promoter DNA methylation density and the allele expression was not observed. We suggest that the genetic polymorphism in the region (especially region upstream of position -400, which is almost completely methylated in all alleles) may lead to different...
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