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Antimicrobial peptides in infections with grammnegative bacteria.
Tocháčková, Petra ; Jílek, Petr (advisor) ; Andrýs, Ctirad (referee)
Bc. Petra Tocháčková Antimicrobial peptides in infections with grammnegative bacteria Diploma thesis - abstract Charles University in Prague, Faculty of Pharmacy in Hradec Králové Pharmacy Background: Aim of this thesis was a development of methods for detection of gene activation of antimicrobial peptides (beta defensins 1 and 2 - pBD1 and pBD2) in ileum during infection of gnotobiotic piglets with gramnegative bacteria. Salmonella enterica serovar Typhimurium strain LT2 and its R mutants (rfaG- and rfaL-) were selected as representants of gramnegative bacteria of different virulence. Methods: 1) Suggestion of systems for detection of pBD1 and pBD2 (software), 2) Purification of total RNA (commercial columns), quantification (spectrophotometry), and estimation of its purity (ratio A280/A260), 3) Reverse transcription (mixture of random n-mers and oligo d(T)m), 4) Quantification of cDNA (real time polymerase chain reaction - LNA probes), and 5) Normalization of gene transcription for pBD1 and pBD2 against beta actin, cyclophilin A or to both together. Results: We used a method reducing risk of contamination by genomic DNA and obtaining of false positive results for purification of total RNA. We suggested detection systems for estimation of pBD1 and pBD1 with efficiency approx. 100% (comparable with...
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Levels of umbilical cord blood pathogen pattern receptors and proinflammatory molecules - the relation to histological chorioamnionitis
Mitášová, Eva ; Andrýs, Ctirad (advisor) ; Procházka, Martin (referee) ; Ulčová-Gallová, Zdeňka (referee)
The umbilical cord blood (UCB) represents the earliest haematological sample of the fetus/neonate without invasive intervention or any risk for mother and her child during sampling. From the UCB we are able to determine a lot of information about the clinical status of newborn. The fetal response to preterm birth with IAI is characterised by increased levels of proinflammatory cytokines in the UCB. Our aim was therefore to investigate the association of IAI and immunological parameters interleukin IL-6, IL-8, matrix metalloproteinase 8 (MMP-8), pentraxin 3 (PTX3), soluble form of Toll-like receptor 2 (sTLR2) and soluble form of scavenger receptor CD163 (sCD163). The strengths of our study include quantification of sTLR2, sCD163, PTX3 and MMP-8 in UCB and their relationship to IAI that has never been examined. There was a significant increase in inflammatory markers IL-6 and sCD163 in UCB in infants exposed to preterm premature rupture of membranes (PPROM) and to histological chorioamnionitis (HCA) compared to those without intraamniotic inflammation (IAI). There was significant differences in IL-6, IL-8, MMP-8 and sCD163 levels in those with and without funisitis in women with PPROM. However, no significant differences were observed between levels of sTLR2 and PTX3 in UCB in subjects with and...
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The effect of Goeckerman therapy on selected immune markers in patients with psoriasis
Kondělková, Kateřina ; Andrýs, Ctirad (advisor) ; Weigl, Evžen (referee) ; Posová, Helena (referee)
1 Summary The effect of Goeckerman therapy on selected immune markers in patients with psoriasis Psoriasis is a systemic inflammatory disorder with skin manifestation. Classical approach to treat psoriasis is Goeckerman therapy (GT) which is based on daily application of pharmaceutical coal tar on affected skin with subsequent exposition to UV light. The aim of this study was to observe selected immunological markers and cell subpopulations in patients with psoriasis and to elucidate the effect of GT on these parameters. We evaluated T regulatory cells (Treg), membrane CD163 expression on monocytes, serum levels of CD163 (sCD163), membrane TLR2 and TLR4 expression on monocytes and serum levels of TLR2 (sTLR2). The immunofluorescent analysis was performed on erythrocyte lysed peripheral blood using a flow cytometer FC500 Cytomics (Beckman Coulter) and FACSCalibur (Becton Dickinson). The level of soluble markers was detected by sandwich enzyme-liked immunosorbent assay (ELISA). There was no significant difference in the relative number of Treg cells in the peripheral blood of healthy blood donors and patients with psoriasis before initiation of GT. The relative number of Treg is significantly increased in psoriatic patients after GT compared to the pre-treatment level. In comparison with levels in controls...
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Selected factors of the complement system in the diagnosis of intraamnial inflammation in a patient with preterm prelabor rupture of fetal membranes
Sejkorová, Ilona ; Andrýs, Ctirad (advisor) ; Kopřivová, Kateřina (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Ilona Sejkorová Supervisor: prof. RNDr. Ctirad Andrýs, Ph.D. Title of Thesis: Selected factors of the complement system in the diagnosis of intraamnial inflammation in a patient with preterm prelabor rupture of fetal membranes (PPROM) Objective: The aim of the study was to evaluate concentrations of selected markers in amniotic fluid in pregnancies complicated by PPROM with respect to the presence of the microbial invasion of the amniotic cavity (MIAC), intraamnial inflammation (IAI) and intraamnial infection. Among the evaluated biomarkers were complement component C8, terminal complement complex (TCC), the regulatory protein of the complement system protectin (CD59). Methods: One hundred sixty-four women with singleton pregnancies complicated by PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Concentrations of selected molecules were determined by enzyme immunoassay test (ELISA). MIAC was detected by a polymerase chain reaction. Interleukin 6 levels were determined in amniotic fluid of patients by immunochromatographic assay. IAI was defined as an amniotic fluid ilnterleukin-6 concetration ≥745 pg/ml. Intraamnial infection...
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Changes of Immune Parameters Induced by Intraamnial Inflammation
Souček, Ondřej ; Andrýs, Ctirad (advisor) ; Lokaj, Jindřich (referee) ; Šimetka, Ondřej (referee)
Changes of immune parameters induced by intraamnial inflammation Abstract Intraamniotic infection plays an important role in the etiology of preterm birth and can lead to a serious threat to fetal health. The diagnostic approach is based on direct microbiological detection of an infectious agent or indirect detection by determining various biomarkers, which concentration increases during intraamniotic infection. Due to the nature of the infection, these parameters are determined from amniotic fluid, which makes this diagnosis difficult for both the doctor and the mother and routinely unavailable. The dissertation comments the published results of a scientific team whose aim was to identify suitable markers of infection, determine their concentration in amniotic fluid and test their diagnostic potential in cervical fluid, ie biological material that can be collected non- invasively. Amniotic and cervical fluid samples were taken from women with singleton pregnancies with preterm labor and that were complicated by intraamniotic inflammation and infection in part of the cohort. It was found that among the tested molecules there is a statistically higher concentration of calreticulin, cathepsin G, CD11b, FcgammaBP and MIP1α in amniotic fluid during intraamniotic infection. Significantly higher levels of...
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SARS-CoV-2 in Care Home Břevnice
Myslivcová, Lenka ; Andrýs, Ctirad (advisor) ; Jankovičová, Karolína (referee)
The aim of this diploma thesis was to describe information about SARS-CoV-2 virus and coronavirus disease (COVID-19), to evaluate the course of the disease in the Home with a special regime in Břevnice, in which the epidemic took place at the beginning of the first wave of COVID-19 pandemic in spring 2020. Another goal was to perform an antibody analysis and evaluate the obtained data. I divided the diploma thesis into three main parts: theoretical, experimental and discussion. In the theoretical part, I worked with the literature and described information related not only to COVID-19, but also to other serious infections caused by human coronaviruses. In the experimental part, which I performed in the immunological laboratory of the Department of Joint Laboratories at Havlíčkův Brod Hospital, I dealt with the issue of the clinical course of the disease, the severity of the disease and possible consequences after the infection. I also dealt with methods for the determination of antibodies, the principles of which are described in Chapter 4.5. The diagnostic methods and procedures used are described in Chapter 5.3 Laboratory Assays. From the obtained data, I prepared graphs and tables and processed data on the clinical course of the infection in the Břevnice Home, both for the clients and the...
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Allergic phenotypes in patients with respiratory allergy evaluated by microchip ISAC technique
Porubová, Petra ; Andrýs, Ctirad (advisor) ; Drahošová, Marcela (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Bc. Petra Porubová Supervisor: prof. RNDr. Ctirad Andrýs, Ph.D. Title of diploma thesis: Allergic phenotypes in patients with respiratory allergy evaluated by microchip ISAC technique Key words: allergic inflammation, allergen specific IgE, component resolved diagnostics, microchip, ISAC Background: The aim of this diploma thesis was to evaluate the sensitization profile in the group of pacients with allergic rhinitis and/or bronhial asthma and to identify possible differences between patients with different disease severity. Methods: ImmunoCAP ISAC was used to determine specific IgE antibodies against 112 allergen components. The evaluated group included 152 patients (57.9 % women) from the allergology outpatient office of the Department of Clinical Immunology and Allergology, University Hospital Hradec Králové. Results: The most common allergens in this group of patients were the components Phl p 1 (68,4 %) and Bet v 1 (65,1 %). Differences in the number of positive and negative reactions were demonstrated between patients with different severity of allergic rhinitis. Correlations were found between the number of positive/negative reactions and the severity of bronchial asthma. And the...
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Bazotests and lymphocyte transformation tests linked to drug intolerance
Honsárková, Tereza ; Krčmová, Irena (advisor) ; Andrýs, Ctirad (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Science Candidate: Bc. Tereza Honsárková Supervisor: prim. MUDr. Inrena Krčmová, CSc. Title of diploma thesis: Bazotests and lymphocyte transformation tests linked to drug intolerance Basophil activation tests and lymphocyte transformation tests are methods that allow drug allergy diagnosis. The right diagnosis is crucial for treatment. Drug allergy is often associated with β-lactam antibiotics hypersensitivity and especially with penicillins. The aim of this diploma thesis is to provide actual statistics based on selected patient medical reports. Those patients were completely clinically and laboratory examined. The main condition for data selection was the execution of skin tests for suspicion of drug allergy caused by penicillin-like antibiotics at the Institute of Clinical Immunology and Allergology of the Faculty Hospital Hradec Králové in 2017. Patients' anamnesis, skin tests and laboratory examination focused on basophil activation tests and lymphocyte transformation was composed. Statistics of 25 patients shows beneficial informations. Delayed manifestations are the most prevalent (63 %) in patients which is interesting. However the patients with positive skin test on major determinant had acute...
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