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Preparation and evaluation of nanoparticles for targeted treatment of inflammatory diseases
Wurzel, Natalie ; Holas, Ondřej (advisor) ; Smékalová, Monika (referee)
Charles University, Faculty of Pharmacy in Hradci Králové Department of Pharmaceutical Technology Supervisor: PharmDr. Ondřej Holas, PhD Consultant: Mgr. Barbora Boltnarová Student: Natalie Wurzel Title of Thesis: Preparation and evaluation of nanoparticles for targeted treatment of inflammatory diseases Nanoparticles are used in the health sciences for, among other things, targeted drug delivery. This is used for the therapy of cancer and chronic inflammatory diseases. The aim of this diploma thesis was to prepare nanoparticles suitable for targeted treatment of inflammatory diseases. The task was to determine the ideal concentration of the active substance, which would ensure high encapsulation efficiency and drug loading. The difference between nanoparticles prepared from PLGA 5/5E and PLGA 7/3A was also investigated. Nanoparticles were prepared by the nanoprecipitation method. The matrix of the nanoparticles was composed of PLGA with different ratios of lactic acid and glycolic acid. Acetone was used as the organic solvent. The encapsulated active substance was the anti-inflammatory dexamethasone acetate. The aqueous phase was consisted of a 0.1% aqueous solution of Pluronic F-127. The evaluated parameters of the resulting nanoparticles included size, polydispersity index, zeta potential,...
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The ion channel TRPV1 and its role in dendritic cells and macrophages
Trnková, Nella ; Krulová, Magdaléna (advisor) ; Vlachová, Viktorie (referee)
This bachelor's thesis deals with the TRPV1 channel and its role in cells of the innate immune system, such as dendritic cells and macrophages. TRPV1 is a membrane ion channel that is activated by physical stimuli such as pH and temperature as well as by substances from the vanilloid group. The most significant of these is capsaicin. Activation of the TRPV1 channel has several effects on different types of cells. Based on the literature review, the bachelor's thesis concludes that the TRPV1 channel tends to lead dendritic cells to have an anti- inflammatory effect. However, there is evidence for both an anti-inflammatory and a pro-inflammatory response in macrophages and the role of TRPV1 is unclear in this cell type. Thus, further research is needed to understand the exact mechanisms of modulation of immune responses and to clarify the role of the TRPV1 channel. Key words: ion channels, TRPV1, capsaicin, dendritic cells, macrophages
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Atherosclerosis risk factors and macrophage polarization in adipose tissue
Králová, Anna ; Poledne, Rudolf (advisor) ; Vrablík, Michal (referee) ; Zicha, Josef (referee)
Atherosclerosis is a major cause of cardiovascular disease, and the pro-inflammatory state stimulates the process of atherogenesis. Since the 1990s, it has become clear that adipose tissue has not only an endocrine function, but it is also a very important immune organ due to the presence of various immunocytes with high cytokine secretion. Local resident macrophages represent a major component in the development of this inflammation. Due to our inclusion in the unique transplantation program at IKEM, which ensured availability of peroperatively harvested adipose tissue and its subsequent and immediate analysis, we were able to characterize the different subpopulations of monocytes/macrophages and classify macrophages into pro- and anti-inflammatory populations based on the differential expression of surface markers. From subcutaneous (SCAT), perivascular (PVAT), and visceral (VAT) adipose tissue samples from healthy living kidney donors (LKD), we analyzed the macrophage phenotype after obtaining the stromal fraction by flow cytometry. At the beginning of the experiments, we chose the calprotectin molecule as a proinflammatory marker. However, in subsequent experiments described herein, we chose a different route and selected the CD16 receptor to determine the proinflammatory phenotype. This...
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Fungal Microbiota and Inflammatory Bowel Disease
Vančíková, Sabína ; Kostovčíková, Klára (advisor) ; Dobeš, Jan (referee)
Inflammatory bowel disease (IBD) is a term for the gastrointestinal tract's chronic inflammatory disorders, evincing growing worldwide prevalence. Commonly, IBD is divided into two major subgroups, Crohn's disease (CD) and ulcerative colitis (UC). Despite intensive efforts, IBD etiology and development mechanisms are not fully understood. Gut barrier dysfunction, dysbiosis, and overreactive immune response to gut microbiota appear to have a significant impact. While bacteria are the most studied gut inhabitants, research on the role of fungal microbiota (mycobiota) in IBD lags behind. This diploma thesis aimed to study immune responses to gut mycobiota in IBD. Levels of antifungal antibodies against gut residents and biomarkers relevant to gut barrier damage and microbial translocation were measured in the sera of IBD patients and healthy individuals. Sera of individuals diagnosed with UC, CD, and IBD associated with primary sclerosing cholangitis (PSC-IBD) were used. Since continuous intestinal macrophage activation is a feature of IBD, the effect of selected fungal mycobiota species on the reactivity of peripheral blood-derived macrophages from healthy donors was assessed in vitro. The levels of serum antibodies against mycobiota components were higher in IBD patients than in controls, and the...
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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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Effect of statin treatment on macrophage polarisation in vitro
Muffová, Barbora ; Kauerová, Soňa (advisor) ; Krulová, Magdaléna (referee)
Statins are widely used for their eminent hypolipidemic effect as anti-atherosclerotic drugs from the 90's of the 20th century. Even though there are new approaches, statins are still the first choice in the prevention of cardiovascular diseases. At the beginning of the 21st century, the anti- inflammatory effect independent of lipid-lowering properties was discovered. This diploma thesis deals with the effect of statin treatment on macrophage polarisation in vitro. Macrophages differentiated from blood monocytes were used in this thesis. The effect of statin treatment on the expression of surface markers (CD16, CD15, CD36, CD163, CD206, ABCA-1 and Trem-2) was evaluated by flow cytometry. The qPCR method was used to quantify the effect of statin treatment on the gene expression of inflammatory genes (NFκB, IL-1β, IL-6, TNFα and iNOS), anti- inflammatory genes (Arg-1, TGFβ) and genes which play a role in the adhesion and migration of monocytes and macrophages to vessel intima (VCAM-1 and MCP-1). Griess method was used to evaluate the effect of statin treatment on the inducible NO-synthase activity. Last, but not least, the effect of statin treatment on proteosynthesis of inflammatory cytokines (IL-1β, IL-6 a TNFα) and anti-inflammatory cytokine IL-10 was measured. Flow cytometry results show that...
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The role of macrophages in immunosuppression mediated ny regulatory T cells
Kadlecová, Kristýna ; Holáň, Vladimír (advisor) ; Stříž, Ilja (referee)
Regulatory T cells (Treg) represent one of the most important mechanisms of immunoregulation. Treg suppress immune reactions and prevent overactivation of the immune system. There is a lot of ways of Treg action described, here we have focused on Treg interference with macrophages. The suppressor capacity of a highly purified Treg population was demonstrated in proliferation assays. The level of suppression of effector T cell proliferation differs depending on the presence of macrophages in the culture. Treg suppression has been significantly higher in the presence of macrophages. These observations led to hypotesis that Treg affect directly macrophages. However, using flow cytometry, reduction of expression of costimulatory molecules on macrophages after culture with Treg was not observed. Macrophages precultured with Treg showed a comparable functionality as macrophages cultured alone. Neither flow cytometry nor live cell imaging revealed any cytotoxic activity of Treg towards macrophages. Despite the presence of macrophages, Treg did not suppress effector cell proliferation in a model, where stronger activation of effector cells was induced. Therefore, a new hypothesis was presented - initially observed higher suppression in the presence of macrophages was probably caused by a qualitatively or...
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Macrophages in leishmania - sand fly - host interaction
Kratochvílová, Tereza ; Kolářová, Iva (advisor) ; Fialová, Anna (referee)
Sand flies (order Diptera) are vectors of Leishmania parasites (Trypanosomatida), which are inoculated into the host skin together with the vector saliva. Sand fly saliva plays the important role in the Leishmania transmission; in naive host it supresses the host immune response assisting Leishmania to establish the infection, while in repeatedly bitten host it elicits a protective immune response. The submitted thesis focuses on the effect of sand fly saliva on macrophages, the key cells in the infection control. In the first part of the thesis we established a laboratory model L. major - P. papatasi - Balb/c to describe the protective effect of saliva immunization on Leishmania infection development. Immunized mice were protected against Leishmania infection which was reflected in the ear lesion size, parasite load in the ear dermis and draining lymph nodes but also in cytokine production. On the contrary, produced lower amount of nitric oxide, while arginase activity was comparable with nonimmunized group. The IgG antibodies against saliva served as a marker of exposure to sandflies while IgG antibodies against Leishmania antigens served as a marker of infection severity. The experiments were aimed on the possibility of cross-protectivity in Balb/c mice against L. major between closely related...
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Immunomodulatory properties of vitamin D3
Urbanová, Anna ; Stříž, Ilja (advisor) ; Zajícová, Alena (referee)
1 Abstract Vitamin D3 is important for keeping the right concetration of Ca2+ in plasma. Therefore it is essential for proper bone growth and development. Nevertheless, vitamin D3 has also a number of immunomodulating effects. Our thesis has been targeted on evaluation and comparison of vitamin D3 influence on expression of chosen surface markers (CD14, CD54, HLA-DR, CD16, CD36 and CD163) with THP-1 cells and monocytes gained from human peripheral blood. Other aims have been analysing the vitamin D3 influence on longevity of THP-1 cells and measuring the soluble CD14 and IL-8 production with THP-1 cells under the vitamin D3 influence. The cells have been stimulated with five different concentrations of vitamin D3 for the time 24, 48 and 72 hours. Higher used concetrations of vitamin D3, i.e. 100 nM and 1000 nM have increased the expression of CD14 with THP-1 cells in the time 48 and 72 hours of the stimulation time. With the monocytes from peripheral human blood the increase of the CD14 expression hasn't been remarkable from the physiological point of view. Together with the vitamin D3 concentration increase the sCD14 production with THP1 cells was considerably higher. The sCD14 was the highest in the time 72 hours after the stimulation with the highest used vitamin D3 concetration. The IL-8 quantity with...
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Immunomodulatory effects of macrolide antibiotics
Zemánková, Jana ; Stříž, Ilja (advisor) ; Krulová, Magdaléna (referee)
Macrolide antibiotics are well known not only for their antibacterial properties, but also for their recently discovered anti-inflammatory properties. They are able to significantly suppress destructive and in many cases life-threatening inflammation, an effect which is desired especially in chronic inflammatory diseases. The principle which their act is the modulation of the various components of the immune system. These effects are called "immunomodulatory" and can also include the effect on epithelial cells and their secretory activity, as well as the effect on pathogens which can colonize the airways and contibute to pathogenesis and the emergence of the chronic inflammatory respiratory diseases. This thesis summarizes the most important known mechanisms, by which macrolide antibiotics exert these immunomodulatory effects, and also notes examples of diseases whose treatment is the most clinically significant. Macrolide antibiotics posessing these uniqe anti-inflammatory properties are well tolerated and severe side-effects are rare. However, the most serious risk is the emergence of resistance and that is the main reason why this treatment can not be recommended without reservation. It is up to each doctor to consider the risks and benefits of the treatment in each individual patient.
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