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Cancer Immunotherapy exploiting engineered antibody fragments against prostate-specific membrane antigen
Das, Gargi ; Bařinka, Cyril (advisor) ; Vaněk, Ondřej (referee) ; Ormsby, Tereza (referee)
Prostate cancer (PCa) remains a leading cause of male cancer-related mortality, necessitating thus the development of novel therapeutic approaches as conventional treatments have limited efficacy. Prostate-specific membrane antigen (PSMA) is an established biomarker for both imaging and therapy of PCa, as it is highly upregulated in neoplastic PCa tissues and metastatic castration- resistant prostate cancer. Consequently, immunological targeting of PSMA has gained significant attention as a therapeutic platform for the management of the disease. The thesis is focused on engineering of antibody fragments and fusion proteins derived from the high affinity anti-PSMA 5D3 monoclonal antibody that can be used as immune cell engagers to target and eliminate PSMA-positive cells. To this end, we engineered 5D3 single chain variable fragments (scFv) that were subsequently fused to anti-CD3 scFv and CP33 sequences, creating thus immune cell engagers targeting T-cells (BiTE) and monocytes (5D3-CP33), respectively. The engagers were expressed in insect cells, purified to homogeneity and their biophysical and functional characteristics evaluated using size exclusion chromatography, differential scanning fluorimetry, ELISA and flow cytometry. Ensuing cell-based assays revealed that both BiTE and 5D3-CP33 can...
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Mannan-BAM, TLR ligands, and anti-CD40 immunotherapy in established murine pancreatic adenocarcinoma: understanding therapeutic potentials and limitations
VENHAUEROVÁ, Anna
The aim of this RNDr. Thesis is focused on understanding therapeutic potentials and limitations of the antitumor MBTA immunotherapy which is based on synergy of TLR agonists, anti-CD40, and phagocytosis stimulating ligands anchored into the tumor cell membranes. In this study, immunotherapy was tested in murine pancreatic adenocarcinoma Panc02 model. Firstly, the short-term and long-term efficacy of MBTA therapy was tested using established subcutaneous Panc02 tumors two times larger than in previous study. Secondly, the work is devoted to better understanding of the adaptive immunity involvement focusing on CD4+ and CD8+ T lymphocytes during the therapy and their effect on tumor volume reduction, long-term survival and resistance against tumor rechallenge. Subsequently, the ability of immunological memory to cross over the blood-brain barrier confirming its potential applicability in metastatic brain tumors was examined. Moreover, the antigen specificity of the immunological memory was evaluated. Finally, the potential of MBTA therapy to cure metastatic disease, represented by bilateral Panc02 mouse model, was studied. In this case, the MBTA therapy manifested a lower therapeutic response. Therefore, it was combined with diverse therapeutic approaches, such as intratumoral application of anti-CTLA-4 antibody, heat-killed Listeria monocytogenes, chemoablation using EtOH, targeting the tumor microenvironment by hyaluronidase, simultaneous injections of MBTA therapy in primary and secondary distant tumors, and its combination with RT. Despite all these combinations, our results showed that only simultaneous application of MBTA therapy into both tumors has potential for the treatment of the bilateral Panc02.
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Interaction of Borrelia sp. with HL-60 cells and monocytes and cultivation of Anaplasma phagocytophilum in HL-60 cell culture
Marková, Lucie ; Hulínská, Dagmar (advisor) ; Beranová, Jana (referee)
Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are causative agents of Lyme disease and human granulocytic anaplasmosis. Their common vector in Europe are the ticks from the genus Ixodes. In our work, we focused on interaction of innate immune cells with the causative agent of Lyme diseases, that are insubstitutable in their function in the early phase of the disease. Anaplasma phagocytophilum is hard to cultivate, the only possibility is to cultivate it in cell cultures. Successful cultivation of Anaplasma phagocytophilum acquired from patients in our geographic area is crucial for following experiments and for diagnostics too. In our experiments, we used validated cell cultures of HL-60 cells, canine monocytes DH82 and murine monocytes P388D1. During our studies of interaction of the causative agent of Lyme diseases with cells, we used two strains of different species Borrelia. Borrelia garinii M192 and Borrelia burgdorferi sensu stricto B31. These strains vary in virulence. The strain M192 is virulent, but the strain B31 lost its virulence by passages. We specialised in study of morphological changes using light microscopy (observation of dyed and fixed preparates and observation in dark field), eventually by transmision electron microscopy. During our experiments, we concluded that HL-60...
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy
Rajtmajerová, Marie ; Drbal, Karel (advisor) ; Brdička, Tomáš (referee)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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Analysis of lysosomes of Trichomonas vaginalis
Zimmann, Nadine ; Tachezy, Jan (advisor) ; Walochnik, Julia (referee) ; Field, Mark (referee)
Lysosomes represent the central degradative compartment of eukaryote cells. Harboring a variety of acid hydrolases at acidic pH, this organelle is designed for the degradation and recycling of material for cellular homeostasis and sustenance. Studies on mammalian lysosomes have been extensive and revealed a long list of lysosomal proteins. While the function of most of these remains elusive, it is not surprising that a large subset have been found to be hydrolases. However, little is known about the biogenesis and function of this organelle in parasitic protists, and even less about its role in secretion. This work aimed to shed light on the (phago-)lysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting, and involvement in hydrolase secretion. Our studies revealed a lysosomal proteome of 462 proteins in 21 functional classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. The identification of a large set of proteins involved in vesicular trafficking and cytoskeleton rearrangement indicates a dynamic phagolysosomal compartment. Our research, as well as the research of others, have identified several hydrolases also in the secretome, including the cysteine protease TvCP2. However, previously the mode...
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Proportional and functional characteristics of particular neutrophil subpopulations in umbilical cord and peripheral blood
Miková, Eliška ; Hrdý, Jiří (advisor) ; Krulová, Magdaléna (referee)
Early postnatal period is characterised by generally immature phenotype of the newborn's immune system. The maturation of the immune system including setting appropriate regulatory responses is occurring during this period and encountering pioneering bacteria colonizing neonate plays an important role. In the early days after birth, the immune system of a newborn is very limited, and the adaptive part is mostly represented by antibodies transferred from the mother by cord blood (CB) in the womb and then by colostrum and mother's milk after labour. Therefore, innate immunity plays a key role in defence (against pathogens) in neonates and is largely represented by neutrophils. This study aims to better understand neutrophil biology and phenotype in umbilical CB, compared to neutrophils from peripheral blood (PB) of mothers and healthy non pregnant women (referred to as HC). The assessment of neutrophil phenotype based on surface markers was performed using flow cytometry. Expression of genes linked to antimicrobial function was measured using quantitative PCR. Functional properties of neutrophils, metabolic activity during activation and phagocytosis, and suppressive properties were assessed using the SeaHorse machine and flow cytometry, respectively. Here we confirm the presence of immature CD16low...
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Virulence factors of Entamoeba histolytica
Novotná, Monika ; Tachezy, Jan (advisor) ; Tůmová, Pavla (referee)
The parasitic protist Entamoeba histolytica causes intestinal disease called amoebiasis (amoebic dysentery), which is one of the most significant diseases worldwide, mainly in developing countries. The goal of this bachelor thesis is to summarize current knowledge about virulence factors of E. histolytica. It is primarly focused on adhesive lectin Gal/GalNAc, cysteine proteases, phagocytosis of apoptotic cells, amoebapore forming pores in the membranes of the target cells and trogocytosis. Keywords: virulence factors, Entamoeba histolytica, parasite, protist, amoebiasis, lectin Gal/GalNAc, cysteine proteinase, phagocytosis, trogocytosis, amoebapore
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Molecular interactions of Staphylococcus aureus with the host
Cabrnochová, Marie ; Melter, Oto (advisor) ; Vopálenský, Václav (referee)
The aim of this thesis is to summarize S. aureus interactions with selected mechanisms of innate host immunity especially interactions with neutrophils and processes on the cell level which lead to host colonization. S. aureus surface proteins MSCRAMM interact with host cell surface proteins such as fibrinogen, keratin and thereby mediate adhesion to the host cell, which is an essential point for colonization of the host cell. The central mechanism of innate immunity against any S. aureus infection is the interaction of the pathogen with neutrophils, which produce neutrophil extracellular traps and phagocytes S. aureus cells. A crucial role in the elimination of bacterial cells in the phagosome of neutrophils is lysis by the antimicrobial peptides and degradation of bacterial biomolecules by the oxygen radicals. S. aureus defence mechanisms against action of immune system are considered to be virulence factors, due to its contribution to the establishment of the infection. These mechanisms are based on cell wall modification, inhibition of neutrophil chemotaxis, and production of enzymes that inhibit the effect of antimicrobial peptides, lysozyme, oxygen and nitrogen radicals. Expression of virulence factors of a particular S. aureus strain and host-specific risk factors can lead through successful...
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy
Rajtmajerová, Marie ; Drbal, Karel (advisor) ; Brdička, Tomáš (referee)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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Lymnaeid snails: hemocyte activities and their changes caused by Trichobilharzia infections
Jindrová, Zuzana ; Horák, Petr (advisor) ; Bilej, Martin (referee)
Molluscs as well as all other invertebrates rely on innate immune response only. Their internal defense system is capable of destroying most pathogens. However, there are some exceptions, e.g. some snails serve as intermediate hosts for some trematodes. Trematodes are able to develop inside these snails due to intervention in the snail internal defense system. The submitted thesis describes hemocyte activities of two lymnaeid snails, Lymnaea stagnalis a Radix lagotis, and the influence of Trichobilharzia regenti infection on R. lagotis hemocytes. Hemocytes of both species exposed to various chemicals produced different amounts of H2O2 and NO. The response varied between both lymnaeid species. The amount of circulating hemocytes was elevated in R. lagotis snails due to T. regenti infection. However, the infenction attenuated hemocyte activities monitored by us. Hemocyte basal NO production was decreased as well as phagocytosis of bacteria, cell adherence and pseudopodia formation. Toxicity of L. stagnalis plasma against T. regenti miracidia was also described. Mechanisms used by trematodes to interact with the snail internal defense system will help us to understand why one species is suitable for the develepment of the trematode whereas another closely related species kills it. Powered by TCPDF (www.tcpdf.org)
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