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Anti-cytoskeletal antibodies in patients with multiple sclerosis and other neurological diseases
Švarcová, Jana ; Matouš-Malbohan, Ivan (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Průša, Richard (referee)
This thesis focuses on the role of anti-cytoskeletal antibodies in various neurodegenerative diseases. Autoantibodies against different axonal cytoskeletal proteins, such as the light (NF-L) and medium (NF-M) subunits of neurofilament and tubulin (TU), in serum and cerebrospinal fluid may be generated in response to the release of cytoskeleton from damaged neurons. The aim of this study was to assess autoimmune involvement in amyotrophic lateral sclerosis (ALS) as well as to evaluate antibody light and medium neurofilament subunit in serum and cerebrospinal fluid (CSF) of patients with ALS. Furthermore, we were interested in the relationships among these antibodies in the serum and in the CSF as well as between the two anti-NF antibody subtypes. Secondly, the aim was to compare the levels of anti-tubulin antibodies (anti-TU) in cerebrospinal fluid and serum in multiple sclerosis (MS) disease, using bovine tubulin as the antigen in one enzyme-linked immunosorbent assay (ELISA) method (anti-TUb antibodies) and a synthetic neuron-specific octapeptide of tubulin in a second ELISA method (anti-TUs antibodies). In the observed groups of ALS patients, serum levels of anti-NF-L antibodies were higher in ALS patients than in controls and serum anti-NF-L antibodies and intrathecal anti- NF-M antibodies were...
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Laboratory diagnostics of micrometastases in breast cancer patients
Mikulová, Veronika ; Zima, Tomáš (advisor) ; Svoboda, Marek (referee) ; Průša, Richard (referee)
Introduction: The presence of circulating tumor cells (CTC) in the peripheral blood has been associated with worse prognosis and early relapse in breast cancer patients. CTC determination in the peripheral blood has been considered as a liquid biopsy. The aim of this project was to analyze the presence of CTC followed by their molecular characterization with the potential use not only as a new biomarker for real-time monitoring of therapy efficacy but also as a suitable tool for patient's stratification and individualization of treatment for breast cancer. Methods: A total of 54 patients with diagnosed early breast cancer were enrolled into a prospective study. Ten millilitres of peripheral blood were sequentially collected to test for the presence and characterization of CTC during the follow-up of patients. CTC isolation and detection was performed by AdnaTest BreastCancer™ (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC- lysates. cDNA from isolated CTC has been further used for newly optimized qPCR assays for breast tumor and therapy resistance associated genes: TOP1, TOP2A, CSTD, ST6GAL, KRT19 and reference gene actin. qPCR results have been analyzed by Genex software (MultiD Analysis). Results: 195 blood samples have been...
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Research of vitreous and vitreoretinal interface in microvascular retinal disorders focussed on eye complications of diabetes mellitus
Křížová, Libuše ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Sosna, Tomáš (referee)
In this work I present conclusions of clinical-laboratory research focused on the patients with diabetic macular edema (DME). We performed biochemical and immunochemical analyses of vitreous samples that were collected during the pars plana vitrectomy. Moreover, at patients with non-proliferative diabetic retinopathy (NPDR) we assessed morphological characteristics of DME using optical coherence tomography (OCT). According to our findings, the vitreous and serum concentrations of uric acid and glucose were significantly higher in patients with diabetic retinopathy and DME compared to controls. Also total ratio (serum/ vitreous concentration) of uric acid and glucose was in diabetics significantly higher than in controls. The most important determinant of increasing concentration of both uric acid and glucose in the vitreous was the grade of diabetic retinopathy. Moreover, we demonstrated significant correlation between vitreous concentration of uric acid and concentration of the vascular endothelial growth factor (VEGF) in patients with DME and NPDR. We found further, that the volume of the macula (cube volume - CV) computed with the software of Cirrus HD-OCT correlates in diabetics significantly with the vitreous VEGF concentration, but not with uric acid. This OCT parameter could be used to...
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Novel biomarkers in patients with renal disease
Zakiyanov, Oskar ; Kalousová, Marta (advisor) ; Teplan, Vladimír (referee) ; Průša, Richard (referee)
Chronic kidney disease (CKD) and acute kidney injury (AKI) are major public health problems. It is important to be able to identify those at high risk of adverse outcome, CKD progression and associated cardiovascular disease. The aim of the thesis was to study novel promising biomarkers, their relationship to kidney function, chronic inflammation and/or cardiovascular risk - placental growth factor (PlGF), pregnancy associated plasma protein A (PAPP-A), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), soluble receptor for advanced glycation end products (sRAGE), calcium binding protein S100A12 or extracellular newly identified RAGE binding protein (EN-RAGE), and high mobility group box protein-1 (HMGB-1) in patients with renal diseases including CKD, haemodialysis (HD), AKI patients, and healthy controls for comparison. First study revealed that PlGF is elevated in patients with decreased renal function. Second study demonstrated the association of MMP-2 and PAPP-A with proteinuria in patients with CKD. Moreover, serum MMP-2, MMP-9 and PAPP-A levels significantly differed in patients with various nephropathies. EN-RAGE levels are not elevated in patients with CKD, but are related to inflammatory status. PAPP-A, EN-RAGE and HMGB-1 levels are significantly elevated, but sRAGE and PlGF...
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Pathobiochemistry of inhibin A and its application in prenatal screening of chromosomal abnormalities
Loucký, Jaroslav ; Průša, Richard (advisor) ; Procházka, Martin (referee) ; Matouš-Malbohan, Ivan (referee)
Aims: Currently, prenatal testing is based on an ultrasound examination, the testing of certain biochemical markers and most recently, also on the analysis of fragments from the extracellular DNA of the fetus in the mother's blood. The aim of this work was to verify whether inhibin A testing during pregnancy can help improve the results of prenatal screening (risk assessment) for Down syndrome and thus reduce the number of unnecessarily invasive procedures. Methods: The concentrations of inhibin A were measured using chemiluminescent immunoassay with paramagnetic particles on the Access system from Beckman Coulter. Risk assessments of screenings were performed using Alpha software, LMS. The results were compared in two groups of screening tests, a triple test and an integrated test. In the first case, the risks in these two groups were determined without inhibition A and included only high-risk results. Subsequently, the inhibin A results were included in the screening protocols and the existing risks were revised. Results: The first group of screening tests (triple test) included a total of 277 pregnant women. The second group (integrated test) included 91 pregnant women. The resulting risk for pregnant women without the determination of inhibin A was higher or equal to 1:300 (triple test),...
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Biomarkers of early renal injury
Fořtová, Magdaléna ; Průša, Richard (advisor) ; Kalousová, Marta (referee) ; Ryšavá, Romana (referee)
Aims: The thesis deals with the biomarkers of early renal injury, namely albuminuria and neutrophil gelatinase-associated lipocalin (NGAL). The aims in the case of albuminuria were the implementation of HPLC method, comparing HPLC with immunoturbidimetric (IT) method and monitoring the relationship to the diagnosis of diabetes mellitus. The aim of urinary NGAL (and eventually other markers) examination was to verify its reliability in the prediction of acute kidney injury (AKI). Methods: We investigated albuminuria in fresh urine samples in the groups of 636 diabetics and 456 nondiabetics using the HPLC method (Agilent 1200, Agilent Technologies, USA) and immunoturbidimetrically (Cobas Integra 400, Roche Diagnostics); we studied the correlations and relationships between albuminuria and glycated hemoglobin HbA1c. We investigated urinary NGAL by chemiluminescent microparticle immunoassay (Architect i4000, Abbott) in children's groups: 1) after renal transplantation (N = 15), 2) with acute or chronic kidney disease (N = 28); and in adult patient's groups: 1) after cardiac surgery (N = 10) and 2) post angiography (N = 41). Results: Albuminuria determined by HPLC was statistically significantly higher than albuminuria determined by IT. We excluded nonspecificity of the HPLC method. Results indicate...
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Individualization of the treatment of prostate cancer patients based on the immunocytochemical detection of circulating tumor cells
Škereňová, Markéta ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Svoboda, Marek (referee)
Introduction: Together with the introduction of new therapeutic options in castration- resistant prostate cancer (CRPC), an advance in individual disease characterization is required. Since common biopsy methods are not suitable for the majority of CRPC patients, one possible solution is the liquid biopsy that is, the analysis of circulating tumor cells (CTCs) isolated from the cancer patients' blood. Methods: A method based on the immunomagnetic enrichment of CTCs and subsequent PCR detection of tumor-associated genes (AdnaTest, Qiagen) was characterized and used for the detection of CTCs in 41 CRPC patients. Each patient was screened at the time of CRPC diagnosis and after the 3rd cycle of docetaxel therapy. A panel of genes associated with therapeutic decision-making was established and validated. Quantitative PCR (qPCR) method on a BioMark platform (Fluidigm, USA) was used to determine the expression of the gene panel in the CTC-enriched and primary tumor samples and the results were analyzed. Results: CTCs were found in 85% and 45% of CRPC patients before and during the therapy, respectively. The presence of CTCs, as well as EGFR and AR PCR fragments, was associated with a decreased sPSA response and lower survival. The gene expression of the CTC- enriched and primary tumor samples differed...
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Oxidative and carbonyl stress in kidney diseases
Kratochvílová, Markéta ; Tesař, Vladimír (advisor) ; Průša, Richard (referee) ; Zadražil, Josef (referee)
Aims: 1. Determination of AGEs (Advanced Glycation End products) in patients with various types of nephropathy. 2. Association AGEs with nutritional parameters and anemia. 3. Influence of renal parameters on sRAGE (soluble form of Receptor for Advanced Glycation End products) levels. 4. Technics and proceeding methods of the podocytes cultivation. 5. Determination of urine podocytes. Methods: We determined fluorescent AGEs by spectrofluorometry, sRAGE by Enzyme-Linked ImmunoSorbent Assay (ELISA). Podocytes were passaged and identified immunocytochemically. Podocytes in urine were specified by flow cytometry method. Results: 1. We did not find significant differences in AGEs serum levels among various types of nephropathy, even though the pathogenesis differs. 2. The albumin and prealbumin levels positively and haemoglobin levels negatively correlate with AGEs in patients with CKD grade 1-5, without necessity of dialysis. 3. Serum sRAGE levels are increased in patients with decreased renal function independently on the course of renal disease. 4. We implemented the methods and technics of podocyte cultivation. 5. Urine podocytes observation and confirmation that podocyturia relates to disease activity. Conclusion: We confirmed that AGEs serum levels depend more on renal function than the type of...
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Research of vitreous and vitreoretinal interface in microvascular retinal disorders focussed on eye complications of diabetes mellitus
Křížová, Libuše ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Sosna, Tomáš (referee)
In this work I present conclusions of clinical-laboratory research focused on the patients with diabetic macular edema (DME). We performed biochemical and immunochemical analyses of vitreous samples that were collected during the pars plana vitrectomy. Moreover, at patients with non-proliferative diabetic retinopathy (NPDR) we assessed morphological characteristics of DME using optical coherence tomography (OCT). According to our findings, the vitreous and serum concentrations of uric acid and glucose were significantly higher in patients with diabetic retinopathy and DME compared to controls. Also total ratio (serum/ vitreous concentration) of uric acid and glucose was in diabetics significantly higher than in controls. The most important determinant of increasing concentration of both uric acid and glucose in the vitreous was the grade of diabetic retinopathy. Moreover, we demonstrated significant correlation between vitreous concentration of uric acid and concentration of the vascular endothelial growth factor (VEGF) in patients with DME and NPDR. We found further, that the volume of the macula (cube volume - CV) computed with the software of Cirrus HD-OCT correlates in diabetics significantly with the vitreous VEGF concentration, but not with uric acid. This OCT parameter could be used to...
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