For the treatment of bilateral limbal stem cell deficiency (LSCD), cell therapy with transplantation of cultivated oral mucosa epithelial cells (COMET) is a promising alternative. Although not yet established, current protocols on the cultivation of oral mucosal epithelial cell (OMECs) sheets are based mainly on substrates and xenobiotic additives that may lead to variable outcomes and undesirable immune responses by the patient. The aim of this study was to characterize OMECs cultivated in xenobiotic-free media (XF) seeded on fibrin gel, in comparison to conventional complex (COM) medium. Oral mucosal biopsies were retrieved from 31 donors. After cultivation in COM or XF medium, OMECs were compared based on growth kinetics, morphology, cell size and viability. Using immunofluorescence and gene expression analyses, the degree of stemness, proliferation and differentiation was evaluated in OMEC cultures. Our findings showed that although OMECs showed a similar morphology and viability, and comparable growth kinetics, immunofluorescence revealed the preservation of stemness (p63 + p40 positivity in cells ≤11 μm) and proliferation in both COM and XF. Gene expression analyses showed that keratin (K)13 and K15 expression levels were significantly higher in XF (adj. p < 0.001), but otherwise COM and XF-treated OMECs had comparable transcriptional profiles in a panel of stemness, proliferation and differentiation genes. These results demonstrate the feasibility of culturing OMECs on fibrin gel without xenogeneic additives, while maintaining their undifferentiated state and preserving stemness. In conclusion, both in terms of results and methodology, the procedures presented here are suitable for implementation in clinical practice.

• MeSH
• buněčná diferenciace MeSH
• buněčné kultury * MeSH
• deficit limbálních kmenových buněk MeSH
• dospělí MeSH
• epitelové buňky * metabolismus   účinky léků   MeSH
• fibrin * MeSH
• gely MeSH
• kmenové buňky * metabolismus   cytologie   MeSH
• kultivační média MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• limbus corneae * cytologie   patologie   metabolismus   MeSH
• nemoci rohovky patologie   farmakoterapie   metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• rohovkový epitel metabolismus   cytologie   účinky léků   patologie   MeSH
• senioři MeSH
• transplantace kmenových buněk metody   MeSH
• ústní sliznice * cytologie   MeSH
• viabilita buněk MeSH
• xenobiotika farmakologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• kmenové buňky metabolismus   patologie   MeSH
• lidé MeSH
• limbus corneae metabolismus   patologie   MeSH
• nemoci rohovky genetika   metabolismus   patologie   MeSH
• rohovkový epitel metabolismus   patologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

Artificial corneas (keratoprostheses) and biosynthetic collagen-based corneal equivalents are surgical implants designed to ease the global burden of corneal blindness. However, keratoprostheses in many cases fail due to development of fibrous retro-corneal membranes (RCM). Fibrous membranes which develop in the anterior chamber after prosthesis implantation do so on a matrix of fibrin. This study investigated fibrin deposition and RCM formation after full-thickness collagen-based hydrogel implants and compared them with syngeneic and allogeneic corneal grafts in mice. Fibrin cleared from the anterior chamber within 14 days in both allo- and syn-grafts but, persisted in hydrogel implants and developed into dense retro-corneal membrane (RCM) which were heavily infiltrated by activated myofibroblasts. In contrast, the number of CD11b+macrophages infiltrating the initial deposition of fibrin in the anterior chamber (AC) after hydrogel implantation was markedly reduced compared to syn- and allo-grafts. Inoculation of mesenchymal stem cells prior to collagen gel implant promoted clearance of gel-associated fibrin from the anterior chamber. We propose that a failure of macrophage-mediated clearance of fibrin may be the cause of RCM formation after collagen-based hydrogel implants and that mesenchymal stem cell therapy promotes clearance of fibrin and prevents RCM formation. STATEMENT OF SIGNIFICANCE: The manuscript addresses the potential value of bone marrow-derived mesenchymal stem cell therapy for retro-corneal membrane (RCM) formation in full-thickness transplantation of biosynthetic corneal equivalents. This work reports the pathophysiological changes in the anterior chamber of the mouse eye following full-thickness recombinant human cross-linked collagen-based hydrogel implants in which persistent fibrin promotes the development of dense RCM. Furthermore, pre-treatment with mesenchymal stem cells reduces RCM formation and enhances corneal transparency.

• MeSH
• alografty MeSH
• bioprotézy * MeSH
• hydrogely * chemie   farmakologie   MeSH
• membrány umělé * MeSH
• mezenchymální kmenové buňky metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši transgenní MeSH
• myši MeSH
• nemoci rohovky metabolismus   terapie   MeSH
• rohovka * MeSH
• transplantace izogenní MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

In various corneal injuries (such as chemical burns or irradiation of corneas with UVB radiation) and ocular diseases (e.g. dry eye disease, keratokonus, bullous keratopathy, Fuchs' endothelial dystrophy), the expressions of malondialdehyde (a marker of lipid peroxidation) and nitrotyrosine (a marker of oxidative stress) appeared in cells of individual corneal layers and conjunctival cells (dry eye disease). This is in contrast to healthy corneas in which negligible levels of malondialdehyde and no expressions of nitrotyrosine are present. The injured or diseased corneas reveal decreased capacity of antioxidants (enzymatic as well as non-enzymatic), whereas the levels of pro-oxidants (e.g. oxidases that generate reactive oxygen species) remain at physiological levels or even increase, leading to the antioxidant/prooxidant imbalance and oxidative stress. Oxidative stress in the cornea stimulates generation of pro-inflammatory cytokines, proteolytic enzymes and enzymes that generate nitric oxide (nitric oxide synthases). An abundant amout of reactive oxygen species and nitric oxide lead to the formation of toxic reactive products contributing to tissue damage. This review aims to summarize immunohistochemical changes in severe corneal injuries and diseases in which oxidative stress has been proved.

• MeSH
• lidé MeSH
• malondialdehyd metabolismus   MeSH
• nemoci rohovky metabolismus   patologie   MeSH
• oxidační stres * MeSH
• poranění rohovky metabolismus   patologie   MeSH
• radiační poranění patologie   MeSH
• rohovka metabolismus   patologie   MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The UVB-irradiated cornea is damaged by oxidative stress. Toxic oxygen products induced by UVB radiation in the cornea are insufficiently removed by antioxidants, whose numbers decrease with increasing UVB irradiation. In addition, the UVB-irradiated cornea suffers from hypoxic conditions because damaged corneal cells cannot utilize oxygen normally, although the supply of oxygen to the cornea is unchanged (normal). This contributes to attenuated re-epithelialization, corneal neovascularization and apoptotic cell death. Our previous publications reported that trehalose applied on the corneal surface during irradiation significantly suppressed UVB-induced corneal oxidative damage. The results of this study provide for the first time important evidence that trehalose applied on the surface of corneas for two weeks following repeated UVB irradiation (312 nm, daily dose 0.5 J/cm2) accelerated corneal healing, restored corneal transparency and suppressed corneal neovascularization. Compared to buffered saline treatment, following which caspase-3, nitrotyrosine, malondialdehyde and urokinase-type plasminogen activator were still strongly expressed in the corneal epithelium two weeks after irradiation and corneal neovascularization was evident, apoptotic cell death was already significantly reduced after one week of trehalose application. The expression of other markers of injury returned to normal levels during two weeks of trehalose treatment. In conclusion, our results show that trehalose accelerated healing of the UVB irradiated cornea, very probably via suppression of hypoxia-response injury. In addition, immunohistochemical results on corneal cryostat sections corresponded with those obtained using corneal impression cytologies, thus confirming that corneal impression cytologies are useful for diagnostic purposes.

• MeSH
• aktivátor plazminogenu urokinázového typu metabolismus   MeSH
• apoptóza účinky léků   MeSH
• biologické markery metabolismus   MeSH
• cytodiagnostika metody   MeSH
• experimentální radiační poranění farmakoterapie   metabolismus   patologie   MeSH
• hojení ran účinky léků   MeSH
• imunohistochemie metody   MeSH
• injekce nitrooční MeSH
• kaspasa 3 metabolismus   MeSH
• králíci MeSH
• malondialdehyd metabolismus   MeSH
• nemoci rohovky farmakoterapie   metabolismus   patologie   MeSH
• oxidační stres MeSH
• patologická angiogeneze farmakoterapie   MeSH
• rohovkový epitel účinky záření   MeSH
• trehalosa aplikace a dávkování   terapeutické užití   MeSH
• tyrosin analogy a deriváty   metabolismus   MeSH
• ultrafialové záření MeSH
• zmrazené řezy metody   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE: To present cytokeratin (CK)7 (OV-TL 12/30 clone) as a newly identified, reliable marker for distinguishing between the conjunctival and corneal surface epithelia, which will contribute to the precise diagnosis of limbal stem cell deficiency (LSCD). METHODS: Corneal and conjunctival epithelial imprints from 12 cadaveric bulbi and from 9 patients with clinically diagnosed LSCD were used for CK7 and CK19 immunocytochemistry. Specimens on nitroacetate cellulose filter papers obtained from the patients were stained with a combination of periodic acid-Schiff (PAS) and Gill's modified Papanicolaou stains, to assess the presence of goblet cells (GCs). RESULTS: CK7 was present in almost all superficial conjunctival epithelial cells from the cadaveric specimens. No immunostaining was observed on the corneal surface. A prominent sharp border of stain was found between the positive conjunctiva and the completely negative epithelium of the central cornea. A more gradual centrifugal decrease in the number of positive cells between the conjunctiva and cornea was observed for CK19. Several CK19-positive cells were detected in the central corneal epithelium. All corneal specimens from affected eyes (unilateral as well as bilateral LSCD patients) revealed strong positivity for CK7, and GCs were present in only 78% of patients. CONCLUSIONS: In cases in which GCs are severely decreased or are absent from the conjunctival surface, the detection of CK7 (OV-TL 12/30 clone) clearly confirms the overgrowth of the conjunctival epithelium over the cornea. Moreover, CK7 is a more reliable marker for distinguishing between the corneal and conjunctival epithelia compared with CK19.

• MeSH
• biologické markery MeSH
• dospělé kmenové buňky patologie   MeSH
• dospělí MeSH
• keratin-19 imunologie   metabolismus   MeSH
• keratin-7 genetika   imunologie   metabolismus   MeSH
• konjunktiva metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• limbus corneae metabolismus   patologie   MeSH
• mrtvola MeSH
• nemoci rohovky metabolismus   patologie   MeSH
• protilátky imunologie   MeSH
• rohovkový epitel metabolismus   patologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• specificita protilátek MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• dospělé kmenové buňky patologie   MeSH
• financování organizované MeSH
• keratin-7 genetika   imunologie   metabolismus   MeSH
• lidé MeSH
• nemoci rohovky metabolismus   patologie   MeSH
• protilátky imunologie   MeSH
• rohovkový epitel metabolismus   patologie   MeSH
• Check Tag
• lidé MeSH

• MeSH
• gama-krystaliny chemie   MeSH
• krevní nemoci klasifikace   komplikace   MeSH
• lidé MeSH
• nemoci rohovky diagnóza   metabolismus   patologie   MeSH
• oční symptomy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• kazuistiky MeSH

Metodou tkáňových řezů bylo dokázáno, že hyaluronidása nemění metachromatické vlastnosti fibril, skládajících rohovku a natravuje patologicky nahromaděné interfibrilární mucopolysacharidy. Tyto hrají důležitou roli při r|ohovkových zánětech a degenerativních změnách, snižujících transparenci. Jejich zmnožení je způsobeno buď isolovaným vzestupem interfibrilárních mucopolysacharidů nebo změnou chemické struktury

• MeSH
• glykosaminoglykany MeSH
• hyaluronoglukosaminidasa MeSH
• nemoci rohovky metabolismus   patofyziologie   patologie   MeSH

Popis jednostranné tukové dystrofie rohovky u 37letého muže. Diagnóza dle klin. obrazu a mikroskopického průzkumu rohovky (perfor. keratoplastika). Otázka patogenesy. Mechanismus původu tuků v rohovce při primární tuk. d|ystrofii, přítomnost cholesterolu. Hypotesa o ztučnění rohovky následkem úbytku cholinu pro zvýšenou potřebu syntezy acetylcholinu. Účast nervové složky. Konečné mínění, že onemocnění u pacienta vyvoláno nervovou porucho|u ve smyslu hyperfunkce při současné poruše tukové přeměny

• MeSH
• dospělí MeSH
• lidé MeSH
• metabolické nemoci MeSH
• nemoci rohovky epidemiologie   metabolismus   MeSH
• tuky metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH