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The role of memory T cells in transplant immunity
Straňavová, Lucia ; Viklický, Ondřej (advisor) ; Zajícová, Alena (referee) ; Vlas, Tomáš (referee)
Memory T cells represent a specific subpopulation of cells formed during the first encounter with antigen. The main role of these cells is to elicit faster and more effective secondary response during reinfections. In transplant immunity, they may affect graft survival directly with donor-specific memory T cells or with cross-reactive virus-specific memory T cells. In this study, we focused on donor-specific and CMV-specific memory/effector T cells. We were interested in the effect of immunosuppressive therapy on the frequency of these cells in periphery. We found that the immunosuppression, prophylaxis and length of dialysis did not significantly affect the number of CMV-reactive cells 6 months after transplantation. We were also interested in the cross-reactivity between CMV and donor antigens, so-called heterologous immunity, which we verified by analyzing the TCR-β repertoire using next- generation sequencing (NGS) in CMV and donor-reactive T cells. Functional cross-reactive T cell clones (shared the same TCR-β sequence) were then found both in the peripheral blood of pre-transplant patients and in the post-transplant graft biopsy. We were also interested if long-term dialysis treatment affects immune memory. Dialysis therapy is often associated with the presence of poorly defined immune system...
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The effect of opioids on immunoregulatory and migratory properties of mesenchymal stem cells.
Echalar, Barbora ; Zajícová, Alena (advisor) ; Funda, David (referee)
Opioids are one of the oldest analgesics used to relieve pain. Besides their therapeutic properties, they also have negative side effects, which include impaired tissue regeneration. Therefore, it can be assumed that opioids also have a negative effect on stem cells which are responsible for tissue healing. One of stem cell populations involved in wound regeneration are mesenchymal stem cells (MSCs). MSCs are undifferentiated, multipotent cells that could be find in almost all tissues. They have immunoregulatory properties and they can migrate to the site of inflammation or injury where they contribute to healing of tissues. Therefore, the purpose of this thesis was to evaluate the effect of morphine and methadone on properties and migration of MSCs. Their effect on the metabolic activity of MSCs and also on the production of cytokines and growth factors was measured. The effect of these opioids on the immunoregulatory properties of MSCs acting on both innate and adaptive immune cells in vitro was studied. The effect of morphine on expression of adhesive molecules on MSCs was also examined. Furthermore, the effect of morphine on migration properties of systemically administered exogenous MSCs in vivo was investigated in mouse models. Distribution of MSCs to individual organs and to the site of...
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Use of the nanofiber scaffold for transfer of stem cells onto the injured ocular surface in mouse experimental model
Kössl, Jan ; Zajícová, Alena ; Heřmánková, Barbora ; Javorková, Eliška ; Boháčová, Pavla ; Holáň, Vladimír
Corneal damage is one of the most common causes of impaired vision or even blindness. When the injury is more extensive and the limbal region is involved, the natural regeneration of the cornea is not sufficient. Such damage can lead to the limbal stem cell deficiency (LSCD). The only option for LSCD treatment is transplantation of the limbal tissue or a transfer of limbal stem cells (LSCs) cultured from the healthy eye. The allogenic transplantation of the limbus or cultivated LSCs with a systemic administration of immunosuppressive drugs is needed in the case of bilateral LSCD. Nevertheless, the cell therapy is very promising approach for LSCD treatment. Transplantation of mesenchymal stem cells (MSCs) seeded on an appropriate scaffold turned out to be a suitable therapy of the LSCD. In our experimental model of LSCD we use nanofiber scaffold for MSC and LSC cultivation and for transplantation of these cells onto the chemically injured mouse eye. MSCs have immunosuppressive and immunomodulatory properties. We showed that MSCs have the ability to inhibit production of molecules associated with the inflammation and support epithelial regeneration in the damaged cornea. These inhibitory properties were confirmed in both in vitro and in vivo mouse model. Results thus showed beneficial effects of stem cell transplantation for murine corneal healing and for suppression of a local immune reaction which can impede the healing process. Such similarity of in vivo and in vitro results allows us further experiments to clarify mechanisms of MSC regenerative and healing properties after the transplantation onto the injured cornea.
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Nanofiber scaffolds for local delivery of stem cells and immunosuppressive drugs for therapeutic purposes
Zajícová, Alena ; Kössl, Jan ; Heřmánková, Barbora ; Boháčová, Pavla ; Holáň, Vladimír
Cell-based therapy of local tissue injuries or damages requires application of stem cells and inhibition of harmful inflammatory reaction which could impede the healing process. To increase the effectiveness of this therapy, a local administration of drugs can avoid their side effects associated with a systemic treatment. A local therapy requires suitable carriers, which can transfer the cells and drugs to the site of injury. As a promising carriers turned out nanofiber scaffolds prepared by electrospinning technology from various types of polymers. The main advantage of this technology is a possibility to define properties of nanofiber scaffolds, optimal for the growth and transfer of stem cells, and which could incorporate various types of immunosuppressive drugs. Here we describe the formation and use of nanofiber scaffolds prepared by needleless electrospinning technology from poly (L-lactic acid) (PLA) which are loaded with immunosuppressive drug Cyclosporine A (CsA). We show that CsA-loaded nanofibers effectively and selectively inhibit proliferation of activated T cells and suppress the production of T cell cytokines in vitro. Simultaneously, these nanofiber scaffolds enable growth of mesenchymal stem cells (MSCs) and thus can serve as stem cell carriers. Moreover, using an experimental mouse model of skin transplantation, we showed that covering skin allografts with MSC-seeded and CsA-loaded nanofibers significantly inhibited the local production of pro-inflammatory cytokines IL-2, IL-17 and IFN-gamma, and supported healing. Thus, nanofiber scaffolds seeded with stem cells and loaded with CsA can serve as carriers of cells and drugs for a local cell therapy and for simultaneous effective immunosuppression.
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GENE EXPRESSION AND IMMUNOLOGICAL RESPONSE IN MICE EXPOSED TO ZnO NANOPARTICLES
Rössner ml., Pavel ; Vrbová, Kristýna ; Strapáčová, S. ; Rössnerová, Andrea ; Ambrož, Antonín ; Brzicová, Táňa ; Líbalová, Helena ; Javorková, Eliška ; Zajícová, Alena ; Holáň, Vladimír ; Kulich, P. ; Večeřa, Zbyněk ; Mikuška, Pavel ; Coufalík, Pavel ; Křůmal, Kamil ; Čapka, Lukáš ; Dočekal, Bohumil ; Šerý, Omar ; Machala, M. ; Topinka, Jan
We analyzed gene expression changes in the lungs and the immunological response in splenocytes of mice exposed by inhalation of ZnO nanoparticles - NP. Adult female ICR mice were treated for three days and three months, respectively. Analysis of differential expression in genes involved in oxidative stress was conducted using quantitative RT-PCR. The potential immunotoxic and immunomodulatory effects of ZnO NP were analyzed by phenotyping and cytokine production by splenocytes after three months exposure. Three days exposure resulted in down-regulation of GCLC, GSR, HMOX-1, NQO-1, NF-kB2, PTGS2 and TXNRD1 mRNA expression, three months exposure increased the expression of these genes. Three months exposure caused a significant decrease in the percentage of granulocytes in the spleen cells, and affected the production of IL-10 and IL-6 by lipopolysaccharide-stimulated leukocytes. In summary, our study revealed changes in the expression of genes involved in the oxidative stress response following acute ZnO NP exposure. Subchronic ZnO NP exposure induced immunomodulatory effects in the spleen.
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Intensive care of patient with metformin intoxication
Zajícová, Alena ; Povolná, Pavla (advisor) ; Hocková, Jana (referee)
Metformin is the first-line oral antidiabetic drug for patients with type 2 diabetes mellitus. As the rate of new diagnosed cases of diabetes increases year-on-year, the number of patients who are prescribed metformin rises as well. In the Czech Republic only, 858,010 patients were treated for diabetes in 2015. 786,586 of them were treated for the type 2 diabetes mellitus. Metformin intoxication, called also MALA (metformin- associated lactic acidosis) after its main symptom, is a rare complication (≤ 10 cases per 100,000 patients per year) in the metformin treatment, but the mortality is high (up to 50%). Metformin-induced lactic acidosis develops when the contraindications of metformin therapy are not respected or in the event of an acute disease linked with dehydration and hypoxia. Patients with MALA are hospitalized at the int ernal medicine intensive care unit or the department of anesthesiology and critical care with multiple organ failure. This thesis aims at determining whether procedural nursing care standards, implemented in the form of a nursing process, are actively used in practice on intensive care beds. Methodology: The empirical part is compiled in the form of a qualitative research, as a case study of a female patient suffering from metformin intoxication, caused by a suicide...
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Effect of growth factors and bioactive substances on mesenchymal stem cell differentiation.
Blahnová, Veronika ; Daňková, Jana (advisor) ; Zajícová, Alena (referee)
Mesenchymal stem cells are a population of multipotent cells, which have the ability to restore damaged tissues derived from mesoderm. Under the influence of wide range of growth factors, hormones and other bioactive molecules they can differentiate for example into chondrocytes, osteocytes, adipocytes, myocytes or insulin producing cells. The differentiation is induced even by contacts with neighboring cells or with extracellular matrix. Tissue engineering often uses especially growth factors. Growth factors act through specific surface receptors, which mediate cell-to-cell and cell-to-extracelular matrix communication and influence many cellular processes such as growth, proliferation, differentiation and others. Due to their specific impact growth factors are used individually or in combinations in tissue engineering applications. Substances stimulating cells to proliferate and differentiate can be added right to the culture medium, when cultivated in vitro, or can be loaded into a scaffold onto which cells are later seeded. The determination of exact growth factors combinations and concentrations influence on MSCs differentiation would enable more efficient use of MSCs in regenerative medicine. The aim of this thesis is to summarise present knowledge about the effect of growth factors and other...
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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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Impacts of chemotherapy on imunoregulatory gene expression in the tumor microenvironment
Paračková, Zuzana ; Reiniš, Milan (advisor) ; Zajícová, Alena (referee)
Tumor microenvironment is an area, where the local immunosuppressive effects dominate and prevents the immune system to perform its physiological functions. The cells infiltrating the microenvironment have an important function among many cell types since they produce a large quantity of factors suppressing the immune response. In our work, we monitored the immune changes in the microenvironment during tumor growth and chemotherapy. For these purposes, we utilized the methods for analysis of the proportion and phenotype of the distinct populations of immunocytes and for analysis of the total level of expressions of selected genes associated with immunosuppression or with distinct populations of immunocytes. The aim of our work was to discover, using two types of mouse tumors (TRAMP-C2 and TC-1/A9), how 5-azacytidine (5AC), a cytostatic drug with epigenetic activity, affects the proportion of leukocytes infiltrating the tumor microenvironment and, further, whether these changes are accompanied by decreased expression of immunosuppressing genes. In addition, we have also focused on the changes of relative expression of genes encoding markers of lymphoid lines and, on other immunoregulating genes, encoding IL-6, IL-10, IL-12, IL-4 and IFNγ cytokines, in the microenvironment of these tumors....
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