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Cellular and molecular mechanisms of immunoregulatory action of stem cells and their effect on adaptive immune cells
Boháčová, Pavla ; Holáň, Vladimír (advisor) ; Hrdý, Jiří (referee) ; Kalina, Tomáš (referee)
Regulation of immune reactions represents an entire system of maintenance of homeostasis, self-tolerance, and host defense. Regardless of intensive research, the cellular and molecular insights into immunomodulation remain incomplete. Therefore, we aimed to study different approaches to modulate the immune system, primarily focused on the induction, expansion, and activation of immunoregulatory cells. We analyzed the therapeutic effect of the combined action of mesenchymal stem/stromal cells (MSCs) and immunosuppressive drugs on the balance among T cell populations. We found that MSCs ameliorated unfavorable effects of immunosuppressants on T cell activation. As a result of this approach, T cell development was altered from the T helper (Th) 1, Th2, and Th17 cell polarization to anti-inflammatory regulatory T cell-mediated response. Additionally, we studied the effect of the immunoregulatory action of MSCs on B cells. We evaluated the impact of cytokine-primed MSCs on the induction of interleukin (IL)-10-producing B cells. Results revealed that interferon (IFN)-γ- and IL-4-primed MSCs suppressed the production of IL-10 by activated B cells. This suppression was dependent on cell-to-cell contact. In the case of IFN-γ-primed MSCs, the inhibition of IL-10 secretion involved the cyclooxygenase-2...
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The Mechanism of Pathogenesis of Experimental Autoimmune Uveitis and Possilbilities of Their Regulation
Klímová, Aneta ; Heissigerová, Jarmila (advisor) ; Pitrová, Šárka (referee) ; Holáň, Vladimír (referee)
Introduction:Uveitis in an ocular inflammation affecting mostly people of working age. Uveitis is responsible for severe visual impairment despite of expanding new therapeutics. The animal models of uveitis were established, because the wide clinical variability of uveitis limits the studies in human medicine. The goal our project was to establish a reproducible model of experimental autoimmune uveitis in Czech Republic, and further on this model to observe the frequency of CD3+ and F4/80+ cells in retina, to assess the influence of microbial environment on intensity of intraocular inflammation and to test the therapeutical possibilities. Material and methods: The C57BL/6J mice were immunized by retinal antigen (IRBP 1-20, interphotoreceptor retinoid binding protein), enhanced by complete Freund's adjuvant and pertussis toxin and mild posterior autoimmune uveitis was induced. The mice were bred in conventional and germ-free (gnotobiotic) conditions. The uveitis intensity was evaluated in vivo biomicroscopically and post mortem histologically on hematoxylin eosin stained sections according to the standard protocol. The histological eye specimen were analyzed also by imunohistochemisty and by flow cytometry. Each experiment was performed for 35 days. The conventional mice with uveitis were treated...
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Heat shock proteins - their role in diagnosis and prognosis of pregnancy-related complications
Dvořáková, Lenka ; Hromadníková, Ilona (advisor) ; Holáň, Vladimír (referee) ; Lipoldová, Marie (referee)
Heat shock proteins provide a universal stress response to cells, for example, exposed to high temperature, heavy metals, extreme pH, infection, inflammation, oxygen radicals, or in case of lack of nutrients and oxygen. We assumed that stress conditions associated with pregnancy-related complications will result in a change in gene expression profile of heat shock proteins we selected for the study. Heat shock protein mRNA levels (Hsp27, Hsp60, Hsp70, Hsp90α and HspBP1) were tested in placental tissue, whole peripheral venous blood and maternal plasma in women with the following pregnancy-related complications - preeclampsia (PE), fetal growth restriction (FGR), gestational hypertension (GH), spontaneous preterm birth (PTB), preterm premature rupture of membranes (PPROM) and normal pregnancies. We also investigated whether the severity of the disease had any impact on hsp gene expression in particular biological samples. In placental tissue, overexpression of Hsp27, Hsp90α and HspBP1 was found in patients with mild preeclampsia (that does not require immediate termination of pregnancy if properly treated), and in women with late onset of preeclampsia with clinical manifestation after 34th week of gestation. Concerning preterm birth, overexpression of Hsp27 and Hsp60 was observed in both groups (PTB...
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Donor specific T cell alloreactivity in kidney transplant recipients with borderline changes
Šilhová, Markéta ; Hrubá, Petra (advisor) ; Holáň, Vladimír (referee)
After kidney transplantation the recipient's immune system responds to the donor's antigens and the graft rejection occurs. Borderline changes are a frequent diagnosis after kidney transplantation, representing only mild rejection signs. Some patients with borderline changes undergo progression to rejection. The identification of these at- risk patients by biomarkers will allow enhanced treatment and help to prevent the development of rejection. The aim of my work was to verify biomarkers of rejection in patients with borderline changes. Chemokines CXCL9, CXCL10 and CCL17 in urine/serum of 40 patients with subclinical borderline changes at 3 months and in 25 patients with early borderline changes were determined by ELISA. At 3 months, the higher CXCL10 level predicted rejection with AUC=0.749, p=0.024. High levels of CXL10 had also been found in patients with BKV infection. We did not confirm the relationship between rejection and the CXCL9 and CCL17. In the early posttransplant period the levels of CXCL10 and CXCL9 were elevated in all patients and therefore couldn't be used to predict rejection. The alloreactivity was examined using IFN-γ ELISPOT (n=38). No association between the frequency of IFN-γ producing cells after stimulation with donor cells or CMV peptides and the development 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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Differentiation of pancreatic stem cells into insulin producing β-cells.
Leontovyč, Ivan ; Saudek, František (advisor) ; Štechová, Kateřina (referee) ; Holáň, Vladimír (referee)
Diabetes mellitus (DM) is a severe and frequent disease with increasing prevalence. It is not possible to achieve long term cure without late complications. Recent advances in cell fate modifications open a pathway to alternative cell therapies for DM cure. My doctoral thesis "Differentiation of pancreatic stem cells into insulin producing β- cells" is focused on the development of a new source of insulin secreting cells for transplantation. Combinatorial testing of numerous potential transcription factors and epigenetic modifiers resulted in a final protocol for the reprogramming pancreatic of exocrine cells into insulin secreting cells. The key transcriptional factors TF (Pdx1, Ngn3 a MafA) were applied in the form of synthetic mRNA. In four independent experiments we applied transcriptional factors in a specific sequence, thus obtaining 14.3 ± 1.9 % insulin positive cells. When challenged in vitro by the glucose levels of 2.5 and 20 mmol/l glucose, respectively, these cells exhibited glucose-sensitivity of insulin secretion (842 ± 72 and 1 157 ± 58 pg insulin/µg DNA/ml, n=5). They also demonstrated a sensitivity of insulin secretion (863 ± 78 and 1 025 ± 66 pg insulin/µg DNA/ml, n=5) to the concentration of depolarization agent KCl applied at 0 and 30 mmol/l, respectively together with 2.5...
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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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