PURPOSE: To assess whether the micronucleus cytome assay (MCyt) reliably detects DNA damage occurring in control and pathological superficial epithelial cells from human conjunctiva. METHODS: Impression cytology samples from the bulbar conjunctiva of 33 healthy controls, eight patients with conjunctival intraepithelial neoplasia (CIN) and eight with mucous membrane pemphigoid (MMP) were examined using the MCyt modified for the ocular surface. RESULTS: The mean number of micronuclei (MNi) in control samples was 0.94 MNi/1000 epithelial cells, with no significant difference between conjunctival quadrants and independent of sex and age. The MCyt assay applied to CIN-affected eyes showed a significantly higher frequency of MNi (18.63/1000 cells), apoptotic cells, nuclear enlargement, multinucleated cells, and keratolysis compared with the corresponding unaffected paired eyes and with the control value. Although the mean MNi frequency in MMP eyes was also higher (1.73 MNi/1000 cells), it did not prove to be statistically different from the control samples. On the other hand, the MMP-affected eyes revealed significantly elevated percentages of cells with snake-like chromatin, multinucleated cells, apoptotic cells, and nuclear buds compared with controls. CONCLUSIONS: Micronucleus cytome assay was adapted as a rapid screening test for genomic instability on the ocular surface. We have determined reference levels for MNi and other nuclear alterations on healthy conjunctiva and demonstrated that particularly frequencies of MNi are significantly elevated in conjunctiva affected by CIN. We demonstrate that MNi are more specific than other nuclear abnormalities and thus can be used for screening of ocular surface neoplasia.

• Klíčová slova
• Conjunctival epithelial cells, Conjunctival impression cytology, Conjunctival intraepithelial neoplasia, Genomic instability, Micronucleus cytome assay, Mucous membrane pemphigoid,
• MeSH
• buněčné jádro MeSH
• epitelové buňky * MeSH
• konjunktiva MeSH
• lidé MeSH
• mikrojaderné testy MeSH
• poškození DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this study was to determine the effect of autologous serum (AS) eye drops on the density of human leucocyte antigen (HLA)-DR-positive epithelial cells and Langerhans cells on the ocular surface of patients with bilateral severe dry eye disease (DED) due to graft-versus-host disease (GvHD) or Sjögren's syndrome (SS). The study was conducted on 24 patients (48 eyes). AS was applied 6-10 times daily for 3 months together with regular artificial tear therapy. HLA-DR-positive cells were detected by direct immunocytochemistry on upper bulbar conjunctiva imprints obtained before and after treatment. The application of AS drops led to a statistically significant increase in the mean density of aberrant HLA-DR-positive conjunctival epithelial cells (p < 0.05) and HLA-DR-positive Langerhans cells (p < 0.05) in the GvHD group. Aberrant HLA-DR-positive epithelial cells in the SS group were decreased non-significantly. All patients reported a significant decrease in the Ocular Surface Disease Index (p < 0.01), which indicates improvement of the patient's subjective feelings after therapy. There was an expected but non-significant decrease of aberrant HLA-DR-positive conjunctival epithelial cells in the SS group only. However, the increased density of HLA-DR-positive cells, indicating slight subclinical inflammation, does not outweigh the positive effect of AS in patients with DED from GvHD.

• MeSH
• dospělí MeSH
• epitel metabolismus   MeSH
• epitelové buňky účinky léků   metabolismus   MeSH
• HLA-DR antigeny metabolismus   MeSH
• imunohistochemie metody   MeSH
• konjunktiva účinky léků   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nemoc štěpu proti hostiteli farmakoterapie   metabolismus   MeSH
• oční roztoky terapeutické užití   MeSH
• počet buněk metody   MeSH
• senioři MeSH
• sérum metabolismus   MeSH
• Sjögrenův syndrom farmakoterapie   metabolismus   MeSH
• syndromy suchého oka farmakoterapie   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HLA-DR antigeny MeSH
• oční roztoky 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

BACKROUND: In vivo corneal confocal microscopy allows the examination of each layer of the cornea in detail and the identification of pathological changes at the cellular level. The purpose of this study was to identify the possible effects of a three-month treatment with autologous serum eye-drops in different corneal layers of patients with severe dry eye disease using corneal confocal microscopy. METHODS: Twenty-six patients with dry eye disease were included in the study. Corneal fluorescein staining was performed. The corneas of the right eyes were examined using in vivo corneal confocal microscopy before and after a three-month treatment with autologous serum drops. The densities of superficial and basal epithelial cells, Langerhans cells, the keratocytes and activated keratocytes, the density of endothelial cells and the status of the sub-basal nerve plexus fibres were evaluated. RESULTS: A significant decrease in corneal fluorescein staining was found after the three-month autologous serum treatment (p = 0.0006). The basal epithelial cell density decreased significantly (p = 0.001), while the density of superficial epithelial cells did not change significantly (p = 0.473) nor did the number of Langerhans cells or activated keratocytes (p = 0.223; p = 0.307, respectively). There were no differences in the other corneal cell layers or in the status of the nerve fibres. CONCLUSIONS: The results demonstrate the ability of corneal confocal microscopy to evaluate an improvement in the basal epithelial cell layer of the cornea after autologous serum treatment in patients with dry eye disease. More studies with longer follow-up periods are needed to elucidate the suitability of corneal confocal microscopy to follow the effect of autologous serum treatment on nerve fibres or other corneal layers in dry eye disease patients.

• Klíčová slova
• Sjögren syndrome, autologous serum, corneal confocal microscopy, dry eye disease, graft-versus-host disease,
• MeSH
• aplikace lokální MeSH
• fluorescein farmakologie   MeSH
• fluorescenční barviva farmakologie   MeSH
• konfokální mikroskopie metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• oční roztoky aplikace a dávkování   MeSH
• počet buněk MeSH
• prospektivní studie MeSH
• rohovka patologie   MeSH
• sérum * MeSH
• syndromy suchého oka diagnostické zobrazování   terapie   MeSH
• výsledek terapie MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH
• Názvy látek
• fluorescein MeSH
• fluorescenční barviva MeSH
• oční roztoky MeSH

AIMS: To assess the impact of autologous serum eye drops on the level of ocular surface apoptosis in patients with bilateral severe dry eye disease. METHODS: This prospective study was conducted on 10 patients with severe dry eye due to graft versus host disease (group 1) and 6 patients with severe dry eye due to primary Sjögren's syndrome (group 2). Impression cytology specimens from the bulbar conjunctiva were obtained before and after a three-month treatment with 20% autologous serum eye drops applied a maximum of 12 times a day together with regular therapy with artificial tears. The percentage of apoptotic epithelial cells was evaluated immunochemically using anti-active caspase 3 antibody. RESULTS: In group 1, the mean percentage of apoptotic cells was 3.6% before the treatment. The three-month treatment led to a significant decrease to a mean percentage of 1.8% (P = 0.028). The mean percentage of apoptotic conjunctival cells decreased from 5.4% before the treatment to 3.8% in group 2; however, these results did not reach the level of significance. CONCLUSION: Three-month autologous serum treatment led to the improvement of ocular surface apoptosis, especially in the group of patients with severe dry eye due to graft versus host disease. This result supports the very positive effect of autologous serum on the ocular surface in patients suffering from severe dry eye.

• Klíčová slova
• apoptosis, autologous serum, caspase 3, conjunctival epithelium, dry eye disease,
• MeSH
• apoptóza fyziologie   MeSH
• epitelové buňky fyziologie   MeSH
• imunohistochemie MeSH
• konjunktiva fyziologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nemoc štěpu proti hostiteli patofyziologie   MeSH
• oční roztoky aplikace a dávkování   MeSH
• prospektivní studie MeSH
• sérum * MeSH
• syndromy suchého oka patofyziologie   terapie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• oční roztoky MeSH

AIM: To assess the impact of autologous serum (AS) eye drops on the ocular surface of patients with bilateral severe dry eye and to draw a comparison between the clinical and laboratory examinations and the degree of subjective symptoms before and after serum treatment. MATERIALS AND METHODS: A three-month prospective study was conducted on 17 patients with severe dry eye. AS eye drops were applied a maximum of 12 times a day together with regular therapy. Dry eye status was evaluated by clinical examination (visual acuity, Schirmer test, tear film breakup time, vital staining, tear film debris and meniscus), conjunctival impression cytology (epithelial and goblet cell density, snake-like chromatin, HLA-DR-positive and apoptotic cells) and subjectively by the patients. RESULTS: The application of AS eye drops led to a significant improvement in the Schirmer test (p < 0.01) and tear film debris (p < 0.05). The densities of goblet (p < 0.0001) and epithelial cells (p < 0.05) were significantly increased, indicating a decrease of squamous metaplasia after AS treatment. A significant decrease (p < 0.05) was found in the number of apoptotic, HLA-DR-positive and snake-like chromatin cells on the ocular surface. A significant improvement was found in all evaluated subjective symptoms. Altogether, the clinical results were improved in 77%, the laboratory results in 75% and the subjective feelings in 63% of the eyes. CONCLUSIONS: We found that three-month AS treatment led especially to the improvement of ocular surface dryness and damage of the epithelium. The improvement of dry eye after AS treatment correlated well with the clinical, laboratory and subjective findings. From the patients' subjective point of view, the positive effect of AS decreased with time, but still persisted up to three months after the end of therapy.

• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• oční roztoky aplikace a dávkování   MeSH
• pohárkové buňky účinky léků   patologie   MeSH
• prospektivní studie MeSH
• rohovkový epitel účinky léků   patologie   MeSH
• senioři MeSH
• spokojenost pacientů * MeSH
• stupeň závažnosti nemoci MeSH
• syndromy suchého oka farmakoterapie   patologie   MeSH
• výsledek terapie MeSH
• zraková ostrost MeSH
• zrakové testy MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oční roztoky 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
• Názvy látek
• biologické markery MeSH
• keratin-19 MeSH
• keratin-7 MeSH
• protilátky MeSH

The goal of our study was to determine whether proteins typical of the human mesothelial cell phenotype, such as mesothelin, HBME-1 (Hector Battifora mesothelial cell-1) protein and calbindin 2, are expressed in the human cornea, especially in endothelial cells. Cryosections and endothelial and epithelial imprints of sixteen human cadaverous corneoscleral discs were used. The presence of proteins was examined using immunohistochemistry and Western blotting, while mRNA levels were determined by qRT-PCR. A strong signal for mesothelin was present in the corneal epithelium, while less intense staining was visible in the endothelium. Similarly, higher and lower mRNA levels were detected using qRT-PCR in the corneal epithelium and endothelium, respectively. HBME-1 antibody strongly stained the corneal endothelium and stromal keratocytes. Marked positivity was present in the corneal stromal extracellular matrix, while no staining was present in the sclera. Calbindin 2 was detected using immunohistochemistry and Western blotting in the corneal epithelium, endothelium and stroma. qRT-PCR confirmed its expression in epithelial and endothelial cells. Three proteins expressed constitutively in mesothelial cells were detected in the human cornea. The possible function of mesothelin in cell-cell contact on the ocular surface is discussed. The presence of HBME-1 protein in the endothelial layer may indicate a still unknown function that could be shared with mesothelial cells of the pleura and peritoneum. The much more pronounced occurrence of calbindin 2 in the corneal epithelium compared to fewer positive endothelial cells explains the higher turnover of epithelial cells compared to the proliferatively inactive endothelium.

• MeSH
• fluorescenční protilátková technika nepřímá MeSH
• GPI-vázané proteiny MeSH
• imunoenzymatické techniky MeSH
• kalbindin 2 MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové glykoproteiny genetika   metabolismus   MeSH
• messenger RNA genetika   MeSH
• mezotelin MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• oční proteiny metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• rohovkový endotel metabolismus   MeSH
• rohovkový epitel metabolismus   MeSH
• S100 kalcium vázající protein G genetika   metabolismus   MeSH
• stroma rohovky metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CALB2 protein, human MeSH Prohlížeč
• GPI-vázané proteiny MeSH
• HBME-1 antigen MeSH Prohlížeč
• kalbindin 2 MeSH
• membránové glykoproteiny MeSH
• messenger RNA MeSH
• mezotelin MeSH
• nádorové biomarkery MeSH
• oční proteiny MeSH
• S100 kalcium vázající protein G MeSH