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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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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, characterization and use of anti-orphan cytochrome P450 antibodies
Hrdinová, Johana ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
The cytochromes P450 (P450s) are important enzymes involved in metabolic pathways, which use exogenous and endogenous substances as their substrate for various enzymatic reactions. These enzymes can also use precarcinogens as their substrate and activate them into carcinogens, which leads to a cancer development. If the P450s are induced, the cancer risk increases. Some chemopreventive compounds may induce the P450s and thus be harmful to the human body. Therefore it is necessary to pay enough attention to a study of the mechanism of action of P450s and the influence of the chemopreventive compounds on the activity of cytochromes P450. mRNA expression of most of the P450s isoforms is detected in a number of healthy (nontransformed) tissues, viz. liver, brain, heart, colon, kidney or placenta. Nevertheless there are a few P450s isoforms which mRNAs are expressed at relatively low levels in the nontransformed tissues, whereas the expression in the transformed tissues is significantly higher. One of these P450s is CYP2W1, which can be used as a prognostic marker for colorectal cancer - therefore it is useful to be able to detect a presence of this enzyme in various tissues. A detection of P450s can be accomplished by using a method Western blot. In this method, the immunodetection is achieved by using...
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Effect of inhaled IgY and Fab fragments on the lung inflammation
Majerová, Barbora ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
Human respiratory system is in constant contact with heterogeneous environmental agents, which can possibly become initiators of inflammatory responses. Mechanical barriers and balance between the innate and adaptive immunity provide protection from external agents without initiation of any inflammatory response. Under certain circumstances, overreaction of the immune system might occur and leads to the production of various inflammatory cytokines and mediators such as TNF-α and GM-CSF factors or interleukins IL-1ß and IL-6. Respiratory system of the human suffering from cystic fibrosis is unable to defend against bacterial infections effectively. Chicken egg immunoglobulins (IgY) could eventually become a suitable tool for the protection from opportunistic pathogens, because they, when bound to antigen, don't activate the complement cascade. One method of determining the immunogenicity of the studied agent is the exposure of experimental animal and further measurement of inflammatory cytokine concentration. In this thesis, the potential inflammatory effect of IgY, IgY-derived Fab fragments and ovalbumin was determined after an exposure of Wistar rats by long-term inhalation. Correct implementation of inhalation experiment was confirmed via production of specific antibodies against the inhaled...
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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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