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Development of an opsonophagocytic assay for the measurement of functional antibody activity against Bordetella pertussis
Brázdilová, Ludmila ; Bumba, Ladislav (advisor) ; Dráber, Peter (referee)
The Gram-negative pathogen bacterium Bordetella pertussis is the infectious agent causing pertussis or whooping cough. The infection is dangerous to infants, often being deadly if untreated. Since whole-cell pertussis vaccines have been replaced by acellular pertussis vaccines, pertussis has become the most prevalent vaccine-preventable disease in developed countries. Therefore, the development of a new generation of pertussis vaccines has become a high priority. Opsonophagocytic assays are one method used to assess the efficacy of new vaccines. The main objective of the thesis is to develop opsonophagocytic killing and uptake assays for the measurement of functional antibody activity against Bordetella pertussis. Neutrophils from mice and humans were isolated by three different methods and used for the assessment of different human and mouse sera in opsonophagocytic killing and uptake assays. Different experimental conditions were tested, including multiplicity of infection and serum dilutions. The opsonophagocytic uptake assay proved to discriminate between naïve and immune sera. Serum from mice vaccinated with the whole-cell pertussis vaccine enhanced opsonophagocytic uptake of B. pertussis cells into neutrophils, while serum from mice immunized with the acellular pertussis vaccine did not....
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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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Photon-upconverting nanoparticles as a novel background-free label in immunoassays
Farka, Z. ; Hlaváček, Antonín ; Mickert, M. J. ; Skládal, P. ; Gorris, H. H.
Photon-upconverting nanoparticles (UCNPs) have become an attractive label in immunoassays because their anti-Stokes luminescence can be excited by the NIR laser and detected in the VIS region without optical background interference. Further advantages of UCNPs include good photostability, large anti-Stokes shifts, and multiple narrow emission bands that can be used for multiplexed detection. We have developed a competitive upconversion-linked immunosorbent assay (ULISA) for detection of the pharmaceutical diclofenac (DCF) in surface waters. Silica-coated UCNPs (50 nm in diameter) with carboxyl groups on the surface were synthesized and conjugated with the secondary anti-IgG antibody. The structure and monodispersity of the nanoconjugates was studied by TEM and agarose gel electrophoresis. Using a highly affine anti-DCF primary antibody, the optimized ULISA provided a detection limit of 50 pg·mL−1.
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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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Genetic and molecular factors influencing the outcome of solid organ transplantation
Pavlova, Yelena ; Slavčev, Antonij (advisor) ; Kalina, Tomáš (referee) ; Mrázek, František (referee)
Since its beginning, graft rejection remains the key problem of solid organ transplantation. This reaction of the recipient's immune system against mismatched antigens of the transplanted organ causes graft damage and consequently loss of its function. Rejection involves cellular (lymphocyte mediated) and humoral (antibody mediated) mechanisms. Among the genetic factors which may have a prognostic value in rejection risk evaluation are the Human Leukocyte Antigens (HLA) genotype, the Killer Immunoglobuline-like Receptor (KIR) gene repertoir, cytokine and other gene polymorphisms. These factors could be screened for before transplantation to find the best possible combination of genetic characteristics of the donor and recipient and to reveal patients with "risky" genotypes, who may need more intensive immunosuppression and more careful post-transplant follow-up. Molecular factors, such as HLA and non-HLA antibodies, soluble CD30 molecule (sCD30), Hepatocyte Growth Factor (HGF) and other cytokines, measured before and/or after transplantation in the recipient's blood may be helpful for rejection risk estimation and may also be used as post-transplant rejection onset markers. In our study, we focused on some of the above mentioned factors. We found that ethnicity plays a significant role in the...
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Study on the induction of intestinal conjugation enzymes by food supplements
Hrdinová, Johana ; Hodek, Petr (advisor) ; Bárta, František (referee)
Cytochrome P450s (CYP) are enzymes, that play an important role in the metabolism of foreign compounds (e.g. drugs, carcinogens, pollutants) and they also participate in the metabolism of hydrophobic endogenic substrates (e.g. sterols, fatty acids, prostaglandins). Cytochromes of subfamily CYP1A are involved in activation of precarcinogens. Whereas the consumption of dietary supplements containing chemopreventive substances (e.g. flavonoids) that can prevent the process of carcinogenesis is significantly increasing in recent years, although these substances may also have a negative effect on the organism. That because these substances can induce the cytochrome P450s and thus increase the risk of activation precarcinogens. In this study had been investigated the inductive effect of some flavonoids compounds - β-naphthoflavone, myricetin, dihydromyricetin and a drug containing an extract of red vine leafs - Antistax®.
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