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Antibody response of anseriform birds against avian schistosomes
HAVELKOVÁ, Markéta
Flukes of the genus Trichobilharzia are typical parasites using aquatic gastropods as intermediate hosts and birds as definitive hosts. However, infectious stages in open water are able to penetrate the skin of a mammal, including humans, and thereby induce a hypersensitivity reaction known as swimmer's itch. Infection with neurotropic species T. regenti in ducks induces an increase in level of IgY antibody which recognize certain parasitic antigens. In this work, these parameters in two other relative but visceral species, T. szidati and T. franki, and cross-reactivity of antibodies in serum of infected duck against heterologous parasitic antigen by ELISA and Western blot were characterized. Antibodies of ducks infected with T. szidati showed very low reactivity with all three parasitic antigens in compare to ducks infected with T. franki or T. regenti. The previously identified strongly immunogenic T. regenti antigens have been evaluated as species non-specific for all trichobilharzia species studied, but useful for avian fluke infection in wild individuals diagnosis.
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Preparation of fusion ligands and evaluation of their binding to NK cell receptors
Nepokojová, Tereza ; Vaněk, Ondřej (advisor) ; Grantz Šašková, Klára (referee)
Natural killer cells (NK cells) are an important part of innate immunity. On their surface they express a complex group of receptors that use different signalling motifs to activate or inhibit NK cell cytotoxic activity. NK cells are capable to kill aberrant cells (namely, viral, infected, and tumour cells) by using special cytotoxic mechanisms to trigger apoptosis. The activating receptors recognize tumour or stress-induced ligands, e.g., NKG2D receptor recognizes the MICA ligand and NKp30 recognizes the B7-H6 ligand. Therefore for human immune system it is only natural that cancer cells are destroyed by NK cells. The current therapeutic goals in the treatment of cancer are primarily focused on strengthening the body's own natural ability to fight with cancer and one possible way is stimulation of NK cells to win this deadly fight. In addition to NK cells, antibodies are also widely used for the treatment of cancer, as well as other immune-related disorders. Most of them are monoclonal antibodies, but antibody fragments are getting attention and are being tested more and more in recent years. This work describes the preparation of three bifunctional fusion proteins: B7-H6-L-aHER2, MICA-L-aHER2, and aHER2-L-MICA, which contain immunoligands for the activating receptors of NK cell and VHH fragment...
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The Frequency of HLA Antigens in the Group of Plasma Donors of the Transfusion Department, University Hospital, Hradec Králové
Nováková, Petra ; Řeháček, Vít (advisor) ; Jílek, Petr (referee)
Author: Petra Novakova Title: The frequency of HLA antigens in the group of plasma donor of the Transfusion Departmen, University Hospital, Hradec Králové Bachelor thesis Charles University, Faculty of Pharmacy in Hradec Kralove Field of study: Medical Laboratory Technician Background: The major objective of this study is the statistical data processing in order to determine the frequency of HLA antigens in the group of plasma donors. Methods: The serological typing of HLA antigens using the lymphocytotoxic test was performed among plasma donors of the Transfusion Department of the University Hospital Hradec Kralove. On a sample group of 200 plasma donors was determined the frequency of HLA antigens by blood group and gender on the basis of a serological examination. The collected data of the observed sample of aforementioned group were processed using Microsoft Excel in relation to the gender and blood group of the donor. Results: It was discovered that within the observed sample there is the highest frequency of HLA-A antigens 2, 1 and 3; HLA-B antigens 7, 44, 35 and HLA-C antigens 7, 6, 4. The most frequented combination of HLA antigens within the observed sample of 200 plasma donors regardless blood group and gender are combinations of HLA-A 1,2 (15x) HLA-A 2,3 (15x), HLA-B 8,44 (8x), HLA-B 7,...
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Protein domains utilizable for development of binding molecules
Dobešová, Petra ; Malý, Petr (advisor) ; Veverka, Václav (referee)
Small protein domains represent basic building blocks of naturally occurring proteins. Many of them exhibit excellent stability, lack disulfide bonds and their structures, therefore, represent attractive tools for generation of artificial binding molecules. First step in the production of novel binding proteins is the definition of a basic domain structure, called "scaffold", which is identified using in silico approaches, resulting in discovery of mutable amino acid residues. Then, randomization of such residues leads to design of a highly complex combinatorial library as a key tool for targeted selection of protein variants. Based on chosen selection approach, the particular protein variants can be tested for their ability to recognize the target molecule with high specificity and binding affinity. Small binding proteins lack post-translation modifications, exhibit thermal stability, are resistant to many organic solvents and can be produced on a mass scale in bacterial host cells. In addition, they can be easily modified and used in vivo with excellent tissue penetration. Due to these beneficial properties, small artificial binding proteins are extraordinary useful biotechnological tools and represent a promising alternative to monoclonal antibodies. The aim of this work is to summarize our knowledge on...
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Occurrence of HLA-B27 antigen in patients examined for suspected rheumatological diseases
Lipenská, Zuzana ; Řeháček, Vít (advisor) ; Jílek, Petr (referee)
Author: Zuzana Lipenská Title: Occurrence of HLA-B27 antigen in patients examined for suspected rheumatological diseases Bachelor's Thesis Charles University in Prague, Faculty of Pharmacy in Hradec Kralove Field of study: Medical Laboratory Technician Background: The aim of the study of HLA B27 antigen in patients investigated for suspected rheumatological disease in 2015. Methods: Patient samples examined for the presence of antigen HLA-B27 in the HLA laboratory of the Transfusion Department, University Hospital Hradec Kralove are processed using the serological method. First is used the isolation of peripheral blood lymphocytes using a density gradient, thereafter lymphotoxical test with peripheral lymphocytes. Results: In 2015 were examined 1,329 patients. 207 persons had the HLA-B27, 1122 persons did not. Of the total number of examined patients were examined 754 women and 575 men. Positive was found 108 women and 99 men. The highest incidence of HLA-B27 was observed in the group of 20-29 years old, another slight increase was in the group of 10-19 years old and 40-49 years old. The examined persons had mainly a group diagnosis of disease of the musculoskeletal system with the most frequently diagnosed dorsalgia (M54). Conclusions: The association between HLA-B27 antigen and the deseases has...
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Immunocomplexes of IL-2 and anti-IL-2 mAbs as a novel class of selective and extremely potent immunostimulators
Tomala, Jakub ; Kovář, Marek (advisor) ; Smetana, Karel (referee) ; Špíšek, Radek (referee)
vi ABSTRACT IL-2 has been used in cancer therapy and also for other applications like treatment of chronic viral infections or as an adjuvant for vaccines. However, treatment with IL-2 is rather difficult due to its severe side effects. These toxicities, associated with high-dose treatment necessary for IL-2 to function, have been found the most limiting factor for IL- 2 applications. Further, particular anti-IL-2 monoclonal antibodies (mAb) can actually increase biological activity of IL-2 rather than block it. Binding of IL-2 to anti-IL-2 mAb creates a superagonistic immunocomplexes which have dramatically higher and selective biological activity in comparison to free IL-2 in vivo. Such approach may finally over- come the difficulties associated with administration of IL-2, thus opening brand new scopes for IL-2 and its application not only in the field of tumor therapy. We have shown that IL-2 immunocomplexes composed of IL-2 and anti-IL-2 mAb S4B6 (IL-2/S4B6) stimulate predominantly cells expressing CD122 and CD132 (dimeric IL-2 receptor), i.e. NK and MP CD8+ T cells, with Treg, T and NKT cells being expanded as well. IL-2/S4B6 are able to drive the expansion of activated naive CD8+ T cells into functional memory-like CD8+ T cells. Moreover, these immunocomplexes exert therapeu- tical potential alone...
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Preparation of Monoclonal Antibodies Against VP2 Protein of Human Polyomaviruses
Vochyánová, Klára ; Drda Morávková, Alena (advisor) ; Růžičková, Šárka (referee)
Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...
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Humoral response of specific hosts to bird schistosome antigens
Turjanicová, Libuše ; Mikeš, Libor (advisor) ; Kolářová, Libuše (referee)
This thesis focuses on humoral immune response of specific hosts to antigens of various developmental stages of bird schistosomes T. regenti and T. szidati, and follows up on previous research of antibody response in non-specific hosts (mouse, human). Sera of experimentally infected and hunted-down wild ducks were examined using the ELISA and western blot methods. The sera samples were taken in predefined intervals. Results of the ELISA analysis show the process of humoral immune response after infection by bird schistosomes. The level of specific antibodies IgY against homogenate of T. regenti cercariae increased significantly 20 d.p.i. in ducks infected by T. regenti. Such reaction wasn't observed in ducks infected by T. szidati. Slight changes in level of specific antibodies IgM against T. szidati cercariae homogenate were observed 10 d.p.i. only in fully immunocompetent ducks and in reinfected ducks. Examination of hunted-down wild ducks didn't prove infection by bird schisosomes; this conclusion was confirmed by results of the ELISA analysis. IgY antibodies from ducks infected by T. regenti demonstrated strong reactions with 2 antigens in ranges 49-47 kDa and 47-45 kDa. Other reactions, which were recognized, have not been observed in all specimen. An Western blott with homogenate from 7 days...
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The testing of celiac disease in connection with osteoporosis
SLADKÁ, Romana
My Bachelor´s thesis deals with the examination of celiac disease in connection to osteoporosis. The aim of the thesis was to specify the level of antidotes against tissue transglutaminase in the class IgA and IgG in the serum with osteoporosis diagnosed patients, to interpret, based on the results of the study, possible link between osteoporosis and celiac disease as well as to compare the outcome with scientific data. I focused on the present knowledge of osteoporosis and celiac disease in the first part. As for celiac disease, I described its history and characteristics. I also paid attention to diagnostics of celiac disease with the assistance of laboratory serological tests ELISA. I mentioned the free-gluten diet treatment after that. As regards osteoporosis, I described its history, symptoms, forms, perils and complications which the disease causes. I conducted the methodical part of my work in the biochemical haematological laboratory Stafila where I work, with its seat in České Budějovice. As for measurements, I took them in the department of microbiology, where immunologic tests are also taken. I carried out the screening of celiac disease from serum, namely the determination of antidotes against tissue transglutaminase in the class IgA and IgG, with the aid of ELISA - a sandwich method. I took measurements with 58 osteoporosis diagnosed patients. I obtained the sera thanks to the supervisor of my thesis Mrs. Marie Ládová, who is at the head of an osteological outpatient department in České Budějovice. I stated the serological markers of celiac disease with the assistance of enzymatic immunoanalysis conducted on automatic analyzer Nexgen Four, which was provided by the firm Test-Line. I describe the analyzer, its specification, checking and calibration of the gadget together with determination checks in this part of the thesis. Additionally, there is described the principle of the method ELISA- sandwich, which is non-competitive enzymatic immunoanalysis. At the end of the section there is depicted the methodical procedure of my work, the preparation of working solutions and checks, the preparation and dilution of samples for analysis and working procedure for semiquantitative interpretation with positivity index and for quantitative interpretation U/ml. I continued with my own measurements. while respecting standard operations procedures of the laboratory Stafila, a limited liability company. The results of my measurements are processed in the third part of the thesis. The acquired results are given in a table: quantitative interpretation of the antidote against tTg IgA and IgG (U/ml) level in the sera of the osteoporosis diagnosed patients. Then I presented a graph interpreting the proportional relation of the antidote against tTg IgA and IgG levels in the sera of the osteoporosis diagnosed patients. There are included calibration graphs for tissue transglutaminase IgA and IgG and tables of the obtained data of checks for the methods in the results. 3 out of the 58 examined samples of osteoporotic patients were positive, they had the level of antibodies higher than 22 U/ml. One of the samples had marginal figures, which means it had the antidotes against tTg IgA an IgG between 18 and 22 U/ml. Positive results were found with patients born in 1976 and 1978. A patient born in 1962 appeared to have a marginal result. The acquired antidote figures against tissue transglutaminase in the class IgA and IgG are presented proportionally in the graph 1. 3 out of the 58 measured samples were positive, which represents 5%, and 1 sample was marginal, which is 2%. My analysis is based on the availability of the examined material and the potential of our laboratory. Celiac disease is an illness which was not paid much attention to in the past. Hypothesis about the link between osteoporosis and celiac disease has been proved.
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