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Methods of molecular diagnostics of parasitic helminths
Dejmková, Tereza ; Leontovyč, Roman (advisor) ; Škorpíková, Lucie (referee)
Helminthiases represent a significant health risk, therefore the use of effective and rapid diagnostic methods is essential, including molecular methods, which are summarized in presented thesis. The biology of important human helminth pathogens is summarized in the introductory part. The DNA isolation, visualisation and molecular methods are described in the following parts. The section about molecular methods is divided into two parts. The first part is focused on PCR and its modifications, the second part describes methods of isothermal DNA amplification, which represent more practical and effective alternative to PCR. The advantages, disadvantages of all methods and examples of helminthiases to which they have been applied are also discussed. The final part is about the impact of the COVID-19 pandemic on the development of molecular diagnostic methods, during which were previously established protocols modified or new molecular methods were applied to increase the efficiency. The presented thesis brings an overview of molecular methods that were used to this day for diagnosis of helminthiases. At the same time the thesis discusses the impact of the COVID-19 pandemic on the development of molecular diagnostics and shows the possible future development of molecular diagnostic methods in the field of...
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Immunomodulatory properties of helminth-produced molecules and their effect during autoimmune and chronic inflammatory diseases
Moravcová, Johana ; Šmídová, Barbora (advisor) ; Kolářová, Iva (referee)
Parasitic helminths produce excretion/secretion products (ESP) that affect the host's immune system to prevent damage or exclusion of the parasite. In recent decades, individual ESP molecules have been the focus of research for the treatment of autoimmune and chronic inflammatory diseases due to their immunomodulatory potential. Diseases that have been investigated in this context include ulcerative colitis, multiple sclerosis, type 1 diabetes, rheumatoid arthritis and allergic asthma. Among the pleiad of helminths and their immunomodulatory molecules, it is worth mentioning Acanthocheilonema viteae (Av17), Ancylostoma caninum (NIF), Ascaris lumbricoides (Al-CPI), Brugia malayi (BMCys), Clonorchis sinensis (CsStefin-1), Fasciola hepatica (FhHDM-1), Heligmosomoides polygyrus (HpTGM), Schistosoma japonicum (SjCystatin) and Schistosoma mansoni (Omega-1). The main effects of these molecules on the host immune system include affecting the function of dendritic cells and macrophages, influencing cytokine production and reducing the Th1 immune response, which usually leads to alleviation of disease symptoms. Based on the current state of knowledge, it is not yet certain how the molecules will work in treating patients and whether they will have the same effect when administered in the long term. Despite...
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Immune response of the paratenic host to Toxocara canis infection, possible influence on the course of experimental autoimmune encephalomyelitis
Novák, Jan ; Horák, Petr (advisor) ; Fajfrlík, Karel (referee) ; Ditrich, Oleg (referee)
i Univerzita Karlova 1. lékařská fakulta Studijní program: Doktorské studijní programy v biomedicíně Studijní obor: Biochemie a patobiochemie RNDr. Jan Novák Imunitní odpověď paratenického hostitele na infekci Toxocara canis, možné ovlivnění průběhu experimentální autoimunitní encefalomyelitidy Immune response of the paratenic host to Toxocara canis infection, possible influence on the course of experimental autoimmune encephalomyelitis Abstrakt dizertační práce Školitel: prof. RNDr. Petr Horák, Ph.D. Konzultant: prof. RNDr. Libuše Kolářová, CSc. Praha, 2022 ii ABSTRACT The most complex interactions between host and infectious agent are generated during helminth infections, which represent a significant source of serious health problems worldwide. Because many helminths migrate after entering the host, these infections are characterized by the gradual development of a range of clinical symptoms. These are not only due to the damage to various organs but also to the modified host's immune response. The published studies show that, on the one hand, the immune response is stimulated in order to eliminate the parasite, but on the other hand, helminths possess a number of mechanisms that may lead to immune modulation and thus ensure their long-term survival in the host. An indirect consequence of such a...
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Molecular and serologic diagnosis of infections caused by Trichobilharzia
Vaščiková, Michaela ; Horák, Petr (advisor) ; Ditrich, Oleg (referee)
Cercariae of the genus Trichobilharzia can penetrate not only the skin of definitive hosts (ducks), but they are also able to penetrate the skin of accidental hosts (mammals). As a result of the penetration, the inflammatory response known as cercarial dermatitis appears. The goal of our thesis is to detect parasite DNA in the serum and cerebrospinal fluid of infected ducks, and also in the serum of infected mice. By using PCR with primers designed for a tandem repeated sequence, we were able to detect 1 femtograms of parasite DNA isolated from sera of infected ducks. We were able to amplify parasite DNA only from 16 samples of sera and cerebrospinal fluid of the infected ducks, but we were not able to do so with the serum of mice. Sera of infected mice were also tested by ELISA and Western blot. The homogenates of T. regenti (TRhc), T. szidati (TShc) and S. mansoni (SMhc) cercariae were selected as an antigen. The results showed progressive increase in the level of IgM antibody from 10 days after 1st infection and also increase of the level of IgG from the 2nd infection. 10 days after the 4th infection, the level of IgM and IgG gradually declined, but the level of antibodies 100 days after the 4th infection was still higher if compared to uninfected mice. Results from Western blot analysis...
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Immune response against skin-penetrating helminths with a focus on schistosomes
Revalová, Alena ; Macháček, Tomáš (advisor) ; Leštinová, Tereza (referee)
Breaching the vertebrate skin and overcoming the local immunity represents a critical step in the life cycles of many helminths. This bachelor thesis summarized the current knowledge of the skin immune response against schistosomes. Both innate and adaptive immune mechanisms are activated soon after the infection. Despite certain differences between mice and humans, complement, granulocytes and especially CD4+ T-lymphocytes are considered as key players in anti-schistosomal immunity of both species. However, several aspects of the host immune response, such as the initial source of cytokine IL-4, IL-10 or expression pattern of certain co- receptors remain unclear and warrant further research. A comprehensive understanding of the host immune response in the skin as well as the respective parasite immune evasion strategies is needed to boost vaccine development. Keywords: immune response, skin, lymph nodes, helminths, schistosomes
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Differentiation of totipotent germinal cells in larvae of bird schistosomes
Peštová, Jitka ; Horák, Petr (advisor) ; Chanová, Marta (referee)
This thesis aims to explore the larval development of a bird fluke Trichobilharzia regenti in its intermediate hosts, as well as the processes of differentiation of its embryonal cells and the differentiation between sporocystogenesis and cercariogenesis in sporocysts, with the ultimate goal to find out whether it is possible to find multiple generations of daughter sporocysts throughout the development of avian schistosomes in the intermediate hosts, just like in the case of human schistosomes of genus Schistosoma. Five developmental stages of daughter sporocysts, and ten developmental stages of cercariae have been defined. The first developmental stage in both larvae is the germinal cell. It divides and gives rise to a cell agregate. Afterwards an envelope (primitive epithelium) is formed around the embryo and subsequently, the embryo elongates. At this stage, the development of the two larvae undergoes different pathways. We can distinguish daughter sporocyst from cercaria in the phase, when the tegument is completed. The daughter sporocyst acquires characteristic vermiform appearance, and its body cavity contains plenty of germinal cells. For cercariae with an developed tegument, presence of the penetration glands is characteristic. Key words: Trichobilharzia regenti, germinal cells, mother...
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Application of mass spectrometry methods for identification and localization of important molecules from parasitic helminths
Siegel, Tomáš ; Kašný, Martin (advisor) ; Harant, Karel (referee)
The thesis summarises the fundamental technological solutions, processes and principles of mass spectrometry with special focus on their use in parasitology. In the first part, the particular phases of processing of the biological sample by using mass spectrometry are defined - from ionisation to the evaluation of results. In the text, the various methodological approaches are presented mainly on the published examples concerning the identification of molecules from parasitic helminths, especially proteins of fasciolid flukes (e.g. Fasciola hepatica). The second part of the thesis comprises the methods of identification and localization of molecules by using mass spectrometry imaging and the use of this combined technique in experimental biology, parasitology, is discussed. There is also proposed the procedure including the combination of laser microdissection and mass spectrometry techniques which could be applied for identification and localisation of important proteins of the fluke Fascioloides magna.
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Influence of tissue helminths on the development of cancers in model organisms
Schreiber, Manfred ; Horák, Petr (advisor) ; Kolářová, Libuše (referee)
Mesocestoides corti and Taenia crassiceps are tapeworms, larvae of which are characterized by their ability to reproduce asexually. In this work, the effect of infection by M. corti and T. crassiceps in BALB/c, C57BL/6J and ICR mice on the growth and metastasis of B16F10 melanoma tumors was investigated. Although an increase in metastatic activities was observed after intravenous administration of melanoma cells to M. corti-infected mice, both tapeworms showed a strong suppressive effect on the size and number of tumors and metastases formed when the cells were administered intraperitoneally. This, in some cases, led to a complete elimination of tumor cells. In vitro cultivation of B16F10 cells in the presence of larval excretory-secretory products led to a decrease in their viability but an increase in their migration ability. Flow cytometry proved that M. corti infection has an effect on the increased number and proportion of macrophage populations in the peritoneum of ICR mice. Our work confirmed the anti-tumor effect of T. crassiceps infection in mice and introduced M. corti as a new helminth species capable of influencing cancer. Key words: helminths, cestodes, cancers, Mesocestoides corti, Taenia crassiceps
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Immune response against skin-penetrating helminths with a focus on schistosomes
Revalová, Alena ; Macháček, Tomáš (advisor) ; Leštinová, Tereza (referee)
Breaching the vertebrate skin and overcoming the local immunity represents a critical step in the life cycles of many helminths. This bachelor thesis summarized the current knowledge of the skin immune response against schistosomes. Both innate and adaptive immune mechanisms are activated soon after the infection. Despite certain differences between mice and humans, complement, granulocytes and especially CD4+ T-lymphocytes are considered as key players in anti-schistosomal immunity of both species. However, several aspects of the host immune response, such as the initial source of cytokine IL-4, IL-10 or expression pattern of certain co- receptors remain unclear and warrant further research. A comprehensive understanding of the host immune response in the skin as well as the respective parasite immune evasion strategies is needed to boost vaccine development. Keywords: immune response, skin, lymph nodes, helminths, schistosomes
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