|
| |
|
|
Regulatory roles of PAG and CSK in FcɛRI signaling of mast cells
Potůčková, Lucie ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Holáň, Vladimír (referee)
8 1 ABSTRACT (EN) This thesis is focused mainly on understanding mechanisms of regulatory roles of C-terminal Src kinase (CSK) and phosphoprotein associated with glycosphingolipid- enriched microdomains (PAG) in the high-affinity IgE receptor (FcɛRI)-mediated signaling of murine mast cells. FcɛRI activation is initiated by aggregation of the receptor by complexes of multivalent antigen with IgE, followed by activation and enhanced activities of protein tyrosine kinases, phosphatases, adaptor proteins and number of other signal transduction molecules. The signaling events result in mast cell degranulation and release of variety of proinflammatory mediators, responsible for initiation of allergy and other inflammatory diseases. Understanding the function of key regulatory molecules controlling FcεRI-mediated mast cell activation, degranulation, and cytokines production could have therapeutic impact. CSK is a major negative regulator of Src family tyrosine kinases (SFKs) that play a critical role in various immunoreceptor signaling events. However, its function in mast cell activation has not been completely understood. Because of its cytoplasmic localization, CSK was assumed to be brought to the vicinity of the plasma membrane- bound SFKs via binding to membrane-bound adaptors and PAG was a major candidate....
|
|
| |
|
|
Targeted differentiation and transdifferentiation of stem cells and their therapeutic application
Chudíčková, Milada ; Holáň, Vladimír (advisor) ; Bernášková, Klára (referee) ; Hrdý, Jiří (referee)
Mesenchymal stem cells (MSCs) naturally differentiate into cells of tissues of mesodermal lineage: cartilage, bone or adipose tissue. As a result of specific environmental stimuli, MSC are able to transdifferentiate into cells of endodermal or ectodermal lineage. Also, MSCs are able to regulate the inflammatory processes and to support healing and regeneration. These properties make MSCs suitable in cell-based therapy and tissue engineering. Characteristics of MSCs (for example differentiation and proliferative potential and cytokine secretion profile) can vary slightly depending on their origin. These differences can be further amplified by the effects of specific environments. Thus, to obtain maximal benefit, it is important to select MSCs optimal for a particular environment. The main goal of this thesis was to design in vitro protocol for transdifferentiation of MSCs into neuron-like cells. For this application, the adipose tissue-derived MSCs seemed to be optimal, due to their higher production of basic fibroblast growth factor, one of the important factors in neural development. The resulting cells acquired typical neuron-like morphology, expressed genes for neuron-specific markers and produced neuron-specific proteins. Further, the resulting cells showed immunomodulatory properties similar...
|
|
|
Study of opioid receptors
Cechová, Kristína ; Hudeček, Jiří (advisor) ; Holáň, Vladimír (referee)
1 ABSTRACT In this Thesis, we studied properties of μ-, δ-, and κ-opioid receptors in lymphocytes isolated from rat spleen. This splenocytes were exposed to mitogen concanavalin A or opiate morphine and cultivated for 48 hours. Under physiological conditions, level of opioid receptors in immune cells is very low. Due to various factors such as presence of opioids, mitogens, long-term exposition to stress, expression of these receptors can be amplified. In this study we demonstrated, that concanavalin A causes up-regulation of μ-, δ- and κ-opioid receptors in lymphocytes isolated from rat spleen. In control cells no significant signal of μ- or δ-receptors was observed. In contrast, κ-opioid receptors were detected already in control cells. Concanavalin A stimulation caused a 2.4 - fold increase of these receptors. In lymphocytes treated with morphine only μ-opioid receptors were up-regulated, whereas in control cells, there was no signal for these receptor type. δ-opioid receptors were not detected in control or morphine treated cells. κ-opioid receptors were determined in control and also in morphine affected lymphocytes but the amount of these receptors wasn't changed by morphine. Detection of μ-, δ- and κ-opioid receptors using Western blot technique in lymphocytes isolated from rat spleen, that were...
|
|
|
Local production of cytokines after treatment with stem cells of damaged ocular surface
Kössl, Jan ; Holáň, Vladimír (advisor) ; Kovář, Marek (referee)
The damage of ocular surface represents one of the most common causes of decreased quality of vision or even blindness. If the injury is extensive and includes the region of limbus, niche of limbal stem cells (LSC), LSC deficiency occurs and the natural corneal regeneration is stopped. Conjunctival epithelium migrates into the injured area. Neovascularization, local inflammation and corneal opacity occur. Corneal transplantation is an insufficient treatment in such case. If the injury is bilateral, the allogenic limbal graft or LSC transplantation is required. In such cases systemic immunosuppressive drugs with many negative side-effects must be administered. The search for an adequate autologous substitution is important for avoid immunosuppressive medication. Mesenchymal stem cells (MSC) represent a perspective substitution for the reason of their immunomodulatory properties and the capability to differentiate in many cell types. There is possibility to isolate sufficient number of these cells from adipose tissue or bone marrow which are relatively easily accessible. Our goal was to observe local production of cytokines and other molecules which are present in inflammatory reaction after the chemical burn of the murine cornea and after the treatment with stem cells growing on nanofiber scaffold....
|
|
|
Mechanisms of immunoregulatory action of IL-10-producing B lymphocytes in dependence on the cytokine environment
Boháčová, Pavla ; Holáň, Vladimír (advisor) ; Stříž, Ilja (referee)
Regulatory B lymphocytes (Bregs) represent a small heterogeneous subpopulation of B cells which participate in a regulation of immune responses by the antibody-independent mechanisms. The main mechanisms of Breg action is a production of anti-inflammatory cytokines or a direct cell contact through their surface molecules. This study deals with an induction of suppressive Bregs from mouse spleen B cells in vitro. We were aiming for a description of an influence of the selected cytokines to the induction of Bregs from lipopolysaccharide (LPS)-stimulated B cells and a determination of the mechanism of Breg action. We also analyzed the ability of inducted Bregs to produce interleukin-10 (IL) and to express genes for Fas ligand (FasL) and programmed death ligand 1 (PD-L1) molecules. We found that only two cytokines, IL-12 and IFN-γ, supported development of Bregs in a population of LPS-stimulated B cells. IFN-γ enhanced production of IL-10 and gene expression of FasL and PD-L1. Furthermore, we analyzed effects of Bregs on macrophages and their following action on T cells. Expression of costimulatory molecules CD80 and CD86, gene expression of IL-1α and the production of IL-6 were tested to determine the effects of macrophages on T cells. Macrophages influenced by Bregs had decreased ability to stimulate...
|
|
|
The study of food allergy in patients and experimental model
Goliáš, Jaroslav ; Tučková, Ludmila (advisor) ; Holáň, Vladimír (referee) ; Prokešová, Ludmila (referee)
Food allergy belongs among the most frequent disorders and its incidence is continuously rising over the last two decades in the developed world. Although the methods used in the diagnostics of food allergies are high sensitive, they have low specificity, which is affected by a purity of used extracts. Therefore, it is important to develop new proteomic procedures for isolation of food allergens in the pure and the biologically active forms, thereby improving the diagnostics of food allergies. Another approach for studying allergies is using an experimental model, which can help us to clarify the mechanisms of allergic response and the acquired findings employ in prophylaxis or allergy treatment. In the first part, we have developed a new proteomic procedure for isolation of wheat allergens in the purified form. By this procedure, using Rotofor, HPLC and electrophoretic methods, we identified 27 potential wheat allergens, from which 7 were newly identified: endogenous α-amylase/subtilisin inhibitor, trypsin/α-amylase inhibitor CMX1/CMX3, TLP, XIP-1, β-glucosidase, class II chitinase, and 26 kDa endochitinase. Further, we showed that isolated allergens (α-amylase 0.19, LTP, TLP, and wheatwin) retained their biological activity and were capable to activate basophils (BAT). In the second part, we...
|
|
|
Immunogenicity of stem cells and their derivatives
Doležalová, Nikola ; Holáň, Vladimír (advisor) ; Kubinová, Šárka (referee)
Immunogenicity of stem cells and their derivatives Nikola Doležalová Abstract Stem cells (SCs) have the potential to be used in regenerative medicine on the basis of their differentiation capacity and promising immunological properties, including low expression of histocompatibility antigens and costimulatory molecules, or secretion of suppressive cytokines. Their immunogenicity has often been ignored in the past but it is becoming clear that rejection of genetically incompatible SCs represents a very common issue. At present, SCs are extensively studied from the immunological point of view, since it represents a critical aspect of the safety of SC therapy. This thesis presents an overview of current knowledge about immunogenicity of SCs and their derivatives, including both pluripotent SCs (embryonic and induced pluripotent SCs) and adult SCs (mesenchymal, limbal, neural, haematopoietic and umbilical cord blood SCs). The expression of immunologically relevant molecules on their surface and interaction with the immune cells in vitro and in vivo will be discussed, together with suggestions for overcoming the immunological barriers for transplantation. Detailed analysis of these aspects necessarily has to precede the safe clinical translation of SC therapies.
|
|
|
Possibilities of using stem cells for treatment of ocular surface injuries
Kössl, Jan ; Holáň, Vladimír (advisor) ; Drbal, Karel (referee)
Ocular surface defects are one of the most common causes of impaired vision or even blindness. Corneal transplantation is the first choice of the treatment of these defects. If the damage is extensive and includes the limbus, niche of limbal stem cells (LSCs), LSC deficiency (LSCD) occurs and reparation with regeneration of cornea is impaired. The only way to treat LSCD is limbal transplantation or transplantation of autologous LSCs from the healthy eye. In cases of bilateral LSCD there are no autologous LSCs available. The use of allogeneic LSCs is associated with the requirement of systemic or local administration of immunosuppressive drugs which have often negative side-effects and outcomes remain uncertain. The alternative for treatment of ocular surface defects and LSCD is finding a new source of an appropriate autologous stem cell substitute which are e.g. mesenchymal stem cells (MSCs). These cells can be obtained from bone marrow or adipose tissue of the particular patient. MSCs can be easily cultivated ex vivo and can be transferred onto the damaged ocular surface using appropriate scaffold. Here they can differentiate to corneal epithelial cells, have immunomodulatory properties and produce numerous trophic and growth factors. The experiments with MSCs on animal models of mechanically or...
|
guest ::
RSS feed.