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Interaction of human immune cells with ultrasmall nanoparticles
Javorová, Pavlína ; Hubálek Kalbáčová, Marie (advisor) ; Krulová, Magdaléna (referee)
The application of nanoparticles in the field of theranostics requires knowledge of the specific interaction of nanoparticles with the immune system. One of the first cells with which nanoparticles interact when given to the body are cells of the mononuclear phagocytic system. The aim of this diploma thesis is to prepare an in vitro study that describes the effect of two types of gold and three types of silicon ultra-small nanoparticles on immune cells. Immune cells are presented in the form of primary PBMCs isolated from whole blood , and cells of monocytic cell line THP-1 in the form of monocytes and differentiated macrophages. During the experiments with primary cells, emphasis is placed on maintaining the concept of personalized protein corona. After characterization of the immune cells used, cells are subsequently stimulated with ultra-small nanoparticles and the influence of these nanoparticles on cell metabolism, viability, degree of differentiation and secretion of pro- and antiinflammatory cytokines is monitored. The outcome takes into account further use of the tested nanoparticles in the field of biomedicine. Key words: primary monocytes, cell lines, differentiation, macrophages, cytokines, cytotoxicity
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Monocyte adhesion to endothelium and atherogenesis
Kauerová, Soňa
Despite the availability of effective therapy of hypercholesterolemia and hypertension, cardiovascular mortality continues to be very high in the Western world. Inflammatory changes occurring in the arterial wall as well as in the adipose tissue play a major role in the development of atherosclerosis. Macrophages are involved in the process of atherogenesis as early as atherosclerosis begins to develop, when, still as monocytes, they migrate and adhere to the arterial wall as a result of endothelial activation and stimulation by pro-inflammatory substances. Adipose tissue has long been recognized as an important endocrine organ, with part of adipose tissue made up by a large amount of macrophages capable of producing a large number of pro-inflammatory cytokines, which contribute to the development of low-grade chronic inflammation important in the development of atherosclerosis. In samples of subcutaneous, visceral and perivascular adipose tissue (SAT, VAT, and PVAT, respectively) obtained from healthy subjects (living kidney donors, LKD), we analyzed macrophages and their polarization, gene expression of pro-inflammatory cytokines and the effect of substances released by VAT on the level of monocyte adhesion to the endothelium. In some analyses, we included samples of SAT, VAT and PVAT obtained...
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Detection and characterization of macrophages in the tumors of viral and non-viral etiology
Dalewská, Natálie ; Tachezy, Ruth (advisor) ; Krulová, Magdaléna (referee)
Head and neck cancers are etiologically associated with smoking and alcohol consumption. Part of these tumors is induced by HPV and their incidence is increasing in the last decade. Patients with virally induced tumors have better prognosis even though they are usually diagnosed with tumors in advanced stage. One of the possible explanations may be better stimulation of the immune system by viral antigens. Macrophages are cells of the innate immune system which belong to professional phagocytes. They are called TAM upon infiltration to the tumor where they represent heterogeneous group of cells. Two main phenotypes are antitumor M1 and protumor M2 macrophages. TAMs are a major component of tumor microenvironment of many types of tumors, one of them are also head and neck cancers. In my thesis I focused on the immunohistochemical detection of M1 and M2 macrophages in the head and neck tumors of viral and non-viral etiology and at the same time RT-qPCR analyses of gene expression of macrophage-associated and/or immunosuppressive genes IDO1, ARG1, CD163, NOS2 a PTGS2 was performed. My data showed that HPV- negative tumors had higher number of M2 macrophages with typical markers CD163, ARG1 and PTGS2. It is known that patients with these tumors have worse prognosis of the disease. Due to high...
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Aplikace mesalazinu do peritoneální dutiny potkana
Hönigová, Kateřina
The objective of this diploma thesis was to determine the influence of the application of 5-aminosalicylic acid (5-ASA) in the peritoneal cavity on the initiation and course of the inflammatory reaction. For these purposes, the rat was chosen as the model animal on which the model of intraperitoneal lavage was applied. The rat’s peritoneal cavity is easily accessible and reflects the functions and reactions of the immune system. For the purposes of the experiment, the rats were divided into 3 groups; in the first group, the peritoneal cavity was assessed in its physiological condition, without any prior application of the substance. PBS was applied to the second group of rats, and the evaluation of the absolute numbers of cells followed after 4 and 24 hours using the Bürker counting chamber and optical microscopy. In the last group, mesalazine was applied, the exposure of which was 4 hours for one half of the group and 24 hours for the other. These time intervals were followed by the evaluation of the absolute number of cells. In order to determine the differential numbers of cells for all the samples, the coated glasses were coloured and evaluated. The greatest statistically relevant difference was identified in the case of the neutrophil population, where the neutrophil share increased from 0-5 % in the intact cavity to up to 35 % in the cavity after the PBS application. Out of all the experimental groups, the population of lymphocytes was relatively stable; the share of macrophages was, statistically, considerably lower for the groups after the mesalazine application. These results indicate that the application of PBS as an inert substance did not cause such a major reaction regarding the influx of neutrophils as the application of 5-ASA.
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Monocyte adhesion to endothelium and atherogenesis
Kauerová, Soňa ; Králová Lesná, Ivana (advisor) ; Kraml, Pavel (referee) ; Kuneš, Jaroslav (referee)
Despite the availability of effective therapy of hypercholesterolemia and hypertension, cardiovascular mortality continues to be very high in the Western world. Inflammatory changes occurring in the arterial wall as well as in the adipose tissue play a major role in the development of atherosclerosis. Macrophages are involved in the process of atherogenesis as early as atherosclerosis begins to develop, when, still as monocytes, they migrate and adhere to the arterial wall as a result of endothelial activation and stimulation by pro-inflammatory substances. Adipose tissue has long been recognized as an important endocrine organ, with part of adipose tissue made up by a large amount of macrophages capable of producing a large number of pro-inflammatory cytokines, which contribute to the development of low-grade chronic inflammation important in the development of atherosclerosis. In samples of subcutaneous, visceral and perivascular adipose tissue (SAT, VAT, and PVAT, respectively) obtained from healthy subjects (living kidney donors, LKD), we analyzed macrophages and their polarization, gene expression of pro-inflammatory cytokines and the effect of substances released by VAT on the level of monocyte adhesion to the endothelium. In some analyses, we included samples of SAT, VAT and PVAT obtained...
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Differentiation of human M2 monocytes/macrophages and their role in kidney transplantation
Čápová, Barbora ; Stříž, Ilja (advisor) ; Krulová, Magdaléna (referee)
The system of mononuclear phagocytes includes macrophages that modulate their phenotype based on microenvironmental signals. Their properties vary considerably in a differentiated stage. M1 macrophages, which are classically activated (typically by IFN-γ), are involved in phagocytosis and produce some pro-inflammatory cytokines, that can stimulate other immune cells. A phenotypically different cell population are M2 macrophages, which are alternatively activated by exposure by Th2 cytokines. M2 macrophages produce preferentially anti-inflammatory cytokines IL-10, TGF-β and participate in repair and tissue healing. The main aim of this study was to standardize a model of differentiation of THP-1 cells and human monocytes towards the M2 phenotyp using an in vitro model. This is represented in particular by increased expression of CD163 and CD206 molecules. The second aim was to assess the dynamics of expression (and co-expression) of CD163 and CD206 molecules in monocytes of patients after kidney transplantation. Expression of surface markers was determined by flow cytometry. Both THP-1 cells and human monocytes, isolated from buffy coat fraction, were stimulated by IL-4, TNF-α, TGF-β and IL-10. Changes in CD163 and CD206 expression were measured after day 1, day 3 and day 6 of stimulation. The most...
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Perivascular adipose tissue in health and disease
Pasáková, Kateřina ; Králová Lesná, Ivana (advisor) ; Janovská, Petra (referee)
Adipose tissue is the biggest endocrine organ in a human body. Its function and composition have been intensively studied due to the rising number of overweight people. The cells of adipose tissue are called adipocytes. The substances that are produced by them are termed adipokines - cytokines secreted by adipocytes. It is possible to distinguish several types of adipose tissue based on its composition - white, brown and transient beige, or based on its location - visceral, subcutaneous and perivascular. Perivascular adipose tissue surrounds most of the blood vessels. Its effect can be inflammatory or anti-inflammatory and it is involved in the process of regulation of vascular tone. Pathology of perivascular adipose tissue can lead to a development of diseases which can directly or indirectly affect the cardiovascular system, and thus it may represent one of the main causes of death in the Western world.
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Regulatory mechanisms affecting etiopathogenesis of obesity, insulin resistance and type 2 diabetes mellitus.
Cinkajzlová, Anna ; Haluzík, Martin (advisor) ; Krčma, Michal (referee) ; Kříž, Jan (referee)
Subclinical inflammation plays a pivotal role in the development of obesity- related complications including type 2 diabetes mellitus, hypertension and cardiovascular diseases. Macrophages are considered important players participating in the initiation and progression of subclinical inflammation within as well as outside of adipose tissue. The first part of this dissertation thesis was focused on macrophage characterization and their quantitative and qualitative changes accompanying metabolic improvements after bariatric surgery. We have demonstrated that the number of macrophages in subcutaneous adipose tissue is reduced regardless of their M1 or M2 polarization manifested as positivity of both the M1- associated CD40 antigen and the M2-associated CD163 and CD206 antigens 6 months after bariatric surgery. Thus, as suggested by previously published data, subcutaneous adipose tissue macrophages seem to have a mixed phenotype. We further confirmed a higher number of non- classical monocytes, which play a role in the control of vascular integrity, in obese subjects as well as a classical monocyte-derived origin of CD163 positive monocytes. Our data also support the previous suggestion of the soluble form of CD163 antigen being a suitable marker of metabolic complications of obesity. The second part...
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