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Study of glycocluster prophylaxis on bacterium adherence model
Áčová, Andrea ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF), as autosomal-recessive inherited disease caused by mutation in CFTR gene, induces an ion channel disorder which triggers big amount of CF patients' health problems. The gravest are complications associated with the respiratory system. Accumulation of thick mucus and altered glycosylation in lung lead to increased adherence of bacterial cells, mainly Pseudomonas aeruginosa (PA) and Burkholderia cepacia complex, to lung epithelial cells of CF patients. The presented thesis, focusing on one of the virulence factors of PA, the PA-IIL lectin, studies the adherence of the PA control strain (PAK; ST 1763) under the influence of anti-PA-IIL chicken IgY antibodies and multivalent glycoconjugates based on fucose, to lung epithelial cells collected from healthy person (NuLi-1) and CF patient (CuFi-1). Before the adherence testing, the presence of PA-IIL in PAK culture was checked by "Western Blotting" followed by immunodetection. For quantitative evaluation of the adherence testing, both lung epithelial and bacterial cells were first fluorescently labeled with PKH dyes. The results of spectrofluorimetric measurement after the adherence testing were also compared to microscopic observation of PAK adherence. Different fractions of chicken anti-PA-IIL antibodies from egg yolks showed...
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Development of model system for study of bacterial adhesion on lung epithelium of CF patients
Nosková, Libuše ; Hodek, Petr (advisor) ; Švédová, Martina (referee)
Cystic fibrosis is an inherited disease bearing a number of health difficulties. The main complication is a chronic colonization and infection of respiratory tract with specific microorganisms - especially Pseudomonas aeruginosa. The lung infection with this microorganism is the most common cause of all of death in these patients. The colonization of respiratory tract is mediated by the series of adhesive structures such as lectin PA-IIL. Currently, the most widely used therapy is an antibiotic treatment. Due to the increasing resistance to antibiotics another methods for treatment are being searched. One possibility is a passive immunization of patients with chicken antibodies. For this purpose, we prepared antibodies against one of the adhesive structures of P. aeruginosa - recombinant lectin PA-IIL. These antibodies be able to recognize a native lectin PA-IIL, expressed by P. aeruginosa. To test the ability of antibodies to prevent adhesion of bacteria on the lung epithelial cells a suitable model system was necessary to develop. The basal components of this system include epithelial cells and P. aeruginosa. Epithelial cells from airways of cystic fibrosis patient were isolated by two methods. One method is based on the direct isolation from the dissected tissue and the second one is a brushing...
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Study the potential of chicken anti-peptide antibodies as means of viral infection prophylaxis
Wojčíková, Lenka ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
In 2020, the WHO declared a global pandemic due to the disease COVID-19, which is caused by the SARS-CoV-2 virus. This virus uses the binding of a surface 'spike' protein to the affected cell's ACE 2 receptor to enter the cell. Vaccination therefore aims at inducing the production of antibodies that inhibit the virus binding. Alternatively, antibodies can be obtained from other organism and used as a means of passive immunisation against COVID-19 disease. The aim of the work was therefore to prepare chicken antibodies, IgY, that would be able to prevent the virus from binding. For the preparation of peptide immunogens, appropriate amino acid sequences have been selected from the RBD domain of SARS-CoV-2 S1 protein, which interacts with the ACE 2 receptor. The synthesized peptides were coupled with the KLH protein carrier, and the resulting conjugate was used to immunize hens. Subsequently, the IgY produced was isolated from the egg yolks and their ability to recognise selected peptides in different types of antigens was demonstrated using the ELISA method. After that, the protective properties of antibodies against the virus were tested ex vivo in cell lines and their efficiency was compared to that of Remdesivir. The antibodies were able to inhibit virus binding to cells in the assays, but the...
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Development of a model system to study adhesion of Burkholderia cenocepacia to epithelial lung cells of patients with cystic fibrosis
Volejníková, Anna ; Bořek Dohalská, Lucie (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF) is a common autosomal recessive disorder that primarily affects epithelial cells in respiratory system. As a consequence of lung damage the patients are chronically colonized by a number of pathogenic microorganisms such as Pseudomonas aeruginosa or Burkholderia cepacia komplex. Due to the seriousness of these bacterial infection and its primary resistance to commonly used antibiotics the suitable therapeutic alternatives are being sought. The passive immunization with yolk antibodies seems to be a suitable alternative. This thesis studied the bacterial adhesion of Burkholderia cepacia, the opportunistic human pathogen, to lung epithelial cells of healthy individuals and patients with CF, respectively. The aim of this study was to develop a model system suitable for this research. Two types of immortalised cell lines have been used for this purpose. CuFi-1 is the cell line derived from patient with CF caused by ∆F508 mutation , NuLi-1 is a healthy epithelial cell line. The strains of B. cenocepacia (ST28 a ST32) were fluorescently labeled with fluorescent cell linker PKH26. Furthermore, the way of fluorescent labeling of cell lines CuFi-1 and NuLi-1 using PKH67 dye was optimized. Using this model system, the adhesion of bacteria to cell line CuFi-1 was up to three times higher...
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Development of an experimental model for yolk antibody prophylaxis of bacterial infections
Hadrabová, Jana ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
Respiratory system of the cystic fibrosis patients is affected by the defect in gene coding for protein transporter for chloride ions - CFTR ("Cystic fibrosis transmembrane conductance regulator"). The main complication of this disease is airways chronic inflammation, in particular caused by bacterium Pseudomonas aeruginosa. Due to asialylation of the lung surfaces the bacterial adhesion is facilitated, for example via lectin PAIIL. The ability of the chicken yolk antibodies to protect lung epithelial cells against Pseudomonas aeruginosa adhesion has been already proven. Therefore this thesis has mainly focused on the influence of the yolk antibodies specific against PAIIL on the development of infection in lungs of experimental animals. The objective was the optimization of the experimental model on which it would be possible to observe the infection development caused by luminescent bacteria strain in vivo using the optical tomography. At first the experiments have been performed on Wistar rats. Since the bacteria colonies in the rat lungs were not detectable in vivo on the available equipment, the rat experimental model showed up as not suitable. Further on only the mouse models were used. Experiments for the inhalation of the antibodies and intratracheal instillation of the bacteria suspension...
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Studium interakcí doplňků stravy s enzymy biotransformace xenobiotik
Bebová, Michaela ; Hodek, Petr (advisor) ; Hansíková, Hana (referee)
Currently, an increasing attention is being paid to phytochemicals as one of the most widely used chemopreventive compounds, generally considered as health-promoting and safe. Flavonoids representing a large group of phytochemicals are present in many dietary supplements formulated from natural sources. The consumption of these concentrated phytochemicals has dramatically increased in the recent decade. It appears, however, that the ingestion of flavonoids might be associated with some adverse effects. Some flavonoids are known modulators of enzymes involved in phase I and phase II metabolism of xenobiotics biotransformation, thus their induction may result in an increase of carcinogen activation. In this study, the effects of selected flavonoid compounds -naphthoflavone, - naphthoflavone, myricetin, and dihydromyricetin, and carcinogens (BaP, PhIP) on phase II metabolism enzymes, sulfotransferases (SULT), have been investigated. To determine the induction of SULT, antibodies for their immunodetection have been developed. Peptide antigens derived from sequences of selected rat sulfotransferases rSULT1A1, 1B1, 1C1, 1C2, 1C1/2, 1E1, and 2A1, were used as KLH conjugates for hen immunization to obtain yolk anti-peptide antibody (IgY). Fractions of IgY were isolated from eggs yolks by simple...
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Development of model system for study of bacterial adhesion on lung epithelium of CF patients
Nosková, Libuše ; Hodek, Petr (advisor) ; Švédová, Martina (referee)
Cystic fibrosis is an inherited disease bearing a number of health difficulties. The main complication is a chronic colonization and infection of respiratory tract with specific microorganisms - especially Pseudomonas aeruginosa. The lung infection with this microorganism is the most common cause of all of death in these patients. The colonization of respiratory tract is mediated by the series of adhesive structures such as lectin PA-IIL. Currently, the most widely used therapy is an antibiotic treatment. Due to the increasing resistance to antibiotics another methods for treatment are being searched. One possibility is a passive immunization of patients with chicken antibodies. For this purpose, we prepared antibodies against one of the adhesive structures of P. aeruginosa - recombinant lectin PA-IIL. These antibodies be able to recognize a native lectin PA-IIL, expressed by P. aeruginosa. To test the ability of antibodies to prevent adhesion of bacteria on the lung epithelial cells a suitable model system was necessary to develop. The basal components of this system include epithelial cells and P. aeruginosa. Epithelial cells from airways of cystic fibrosis patient were isolated by two methods. One method is based on the direct isolation from the dissected tissue and the second one is a brushing...
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