|
|
Limbal stem cell transplantation and their utilization for ocular surface reconstruction.
Lenčová, Anna ; Filipec, Martin (advisor) ; Heissigerová, Jarmila (referee) ; Ardan, Taras (referee)
Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...
|
|
|
Expression of endogenic lectins and their glycoligands in the tear fluid, human corneal and conjunctival epithelium under physiological and disease conditions
Hrdličková, Enkela ; Filipec, Martin (advisor) ; Heissigerová, Jarmila (referee) ; Čejková, Jitka (referee)
Purpose: Lectins play an important role in many biological processes. The aim of this work was to analyse mainly the expression of endogenic lectins, such as galectins and plant lectin, e.g. Dolichos biflorus agglutinin (DBA), and their glycoligands in the tear fluid, human corneal and conjunctival epithelium in physiological and disease conditions. Further, we studied the human natural antibody against Galα1,3Gal-R, which is mainly responsible for hyperacute rejection of xenografts transplants. We tried to investigate its localization in human corneal epithelium, lacrimal gland and tears. Material and Methods: Human tissue (lacrimal gland, tear fluid, conjunctiva, cornea, epidermis, keratinocyte and cultured corneal epithelium), as well as porcine tissue (cornea, liver and epidermis) were examined. Endogenous galectins (galectins-1, -3 and -7) were detected using immunohistochemistry methods. Binding sites for galectins, as well as binding sites for plant lectin Dolichos biflorus agglutinin, were localized by lectin histochemistry. Reverse lectin histochemistry was used for the study of binding reactivity of endogenous lectins using labelled (neo)glycoligands. Employing biotinylated natural human IgG anti -galactosides, as well as anti -galactosides, we detected reactive epitopes in human...
|
|
|
Cell and Molecular Characterization of Failed Human Corneal Grafts. The Role of Matrix Metalloproteinases in Recurrent Corneal Melting.
Brejchová, Kristýna ; Jirsová, Kateřina (advisor) ; Smetana, Karel (referee) ; Heissigerová, Jarmila (referee)
The aim of this work was to investigate the contribution of matrix metalloproteinases (MMPs) to recurrent corneal melting. Twenty three melted corneas from seven patients were separated into three groups: a) patients with primary Sjögren's syndrome, b) those with rheumatoid arthritis and c) those with other corneal melting underlying pathologies. Eleven cadaverous corneas served as controls. The presence of MMP-1, -2, -3, -7, -8, -9, and -13 was detected using indirect enzyme immunohistochemistry. The active forms of MMP-2 and -9 and MMP- 3 and -7 were examined by gelatin and casein zymography, respectively. The concentrations of active MMP-1 and -3 were measured using activity assays. Increased immunostaining intensity for MMP-1, -2, -3, -7, -8 and -9 was shown in the corneal epithelium and the stroma of almost all melted corneas from all three groups compared to the negative or slightly positive staining of the controls. In the endothelium, immunostaining for MMP-2 and MMP-9 was increased in most specimens of groups II and III and group I, respectively. A markedly higher level of active MMP-2 was detected in six, and active MMP-9 in all, pathologic specimens compared to the controls. In contrast to the completely negative controls, the proenzymes of MMP-3 and -7 were detected in almost all melted...
|
|
|
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...
|
guest ::
