Interleukin-13 increases the stemness of limbal epithelial stem cells cultures.

This study aimed to determine the effect of interleukin-13 (IL13) on the stemness, differentiation, proliferation, clonogenicity, and morphology of cultured limbal epithelial cells (LECs). Human limbal explants were used to culture LECs up to the second passage (P0-P2) with or without IL13 (IL13+ and IL13-, respectively). Cells were analyzed by qPCR (for the expression of ΔNp63α, BMI-1, keratin (K) 3, K7, K12, K14, K17, mucin 4, and MKI67) and immunofluorescence staining for p63α. The clonogenic ability was determined by colony-forming assay (CFA), and their metabolic activity was measured by WST-1 assay. The results of the CFA showed a significantly increased clonogenic ability in P1 and P2 cultures when LECs were cultured with IL13. In addition, the expression of putative stem cell markers (ΔNp63α, K14, and K17) was significantly higher in all IL13+ cultures compared to IL13-. Similarly, immunofluorescence analysis showed a significantly higher percentage of p63α positive cells in P2 cultures with IL13 than without it. LECs cultures without IL13 lost their cuboidal morphology with a high nucleocytoplasmic ratio after P1. The use of IL13 also led to significantly higher proliferation in P2, which can be reflected by a higher ability to reach confluence in P2 cultures. On the other hand, IL13 had no effect on corneal epithelial cell differentiation (K3 and K12 expression), and the expression of the conjunctival marker K7 significantly increased in all IL13+ cultures compared to the respective cell culture without IL13. This study showed that IL13 enhanced the stemness of LECs by increasing the clonogenicity and the expression of putative stem cell markers of LECs while maintaining their stem cell morphology. We established IL13 as a culture supplement for LESCs, which increases their stemness potential in culture, even after the second passage, and may lead to the greater success of LESCs transplantation in patients with LSCD.

Ex vivo expansion and characterization of human corneal endothelium for transplantation: a review.

The corneal endothelium plays a key role in maintaining corneal transparency. Its dysfunction is currently treated with penetrating or lamellar keratoplasty. Advanced cell therapy methods seek to address the persistent global deficiency of donor corneas by enabling the renewal of the endothelial monolayer with tissue-engineered grafts. This review provides an overview of recently published literature on the preparation of endothelial grafts for transplantation derived from cadaveric corneas that have developed over the last decade (2010-2021). Factors such as the most suitable donor parameters, culture substrates and media, endothelial graft storage conditions, and transplantation methods are discussed. Despite efforts to utilize alternative cellular sources, such as induced pluripotent cells, cadaveric corneas appear to be the best source of cells for graft preparation to date. However, native endothelial cells have a limited natural proliferative capacity, and they often undergo rapid phenotype changes in ex vivo culture. This is the main reason why no culture protocol for a clinical-grade endothelial graft prepared from cadaveric corneas has been standardized so far. Currently, the most established ex vivo culture protocol involves the peel-and-digest method of cell isolation and cell culture by the dual media method, including the repeated alternation of high and low mitogenic conditions. Culture media are enriched by additional substances, such as signaling pathway (Rho-associated protein kinase, TGF-ß, etc.) inhibitors, to stimulate proliferation and inhibit unwanted morphological changes, particularly the endothelial-to-mesenchymal transition. To date, this promising approach has led to the development of endothelial grafts for the first in-human clinical trial in Japan. In addition to the lack of a standard culture protocol, endothelial-specific markers are still missing to confirm the endothelial phenotype in a graft ready for clinical use. Because the corneal endothelium appears to comprise phenotypically heterogeneous populations of cells, the genomic and proteomic expression of recently proposed endothelial-specific markers, such as Cadherin-2, CD166, or SLC4A11, must be confirmed by additional studies. The preparation of endothelial grafts is still challenging today, but advances in tissue engineering and surgery over the past decade hold promise for the successful treatment of endothelial dysfunctions in more patients worldwide.

The micronucleus cytome assay - A fast tool for DNA damage screening in human conjunctival epithelial cells.

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.

A comprehensive analysis of the expression, epigenetic and genetic changes of HNF1B and ECI2 in 122 cases of high-grade serous ovarian carcinoma.

High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer, with a poor prognosis; however, most studies concerning ovarian carcinoma have focused mainly on clear cell carcinoma. The involvement of hepatocyte nuclear factor 1ß (HNF1B) in the carcinogenesis of HGSC has not yet been fully elucidated. To the best of our knowledge, the present study is the first to analyse the expression of the possible downstream target of HNF1B, enoyl-CoA (Δ) isomerase 2 (ECI2), in HGSC. The present study performed a comprehensive analysis of HNF1B mRNA and protein expression, and epigenetic and genetic changes, as well as an analysis of ECI2 mRNA and protein expression in 122 cases of HGSC. HNF1B protein expression was detected in 28/122 cases, and was positively associated with lymphovascular invasion (P=0.025). Protein expression of ECI2 was detected in 115/122 cases, but no associations with clinicopathological variables were revealed. Therefore, ECI2 does not seem to function as a suitable prognostic marker for HGSC. In the sample set, a positive correlation between HNF1B and ECI2 protein expression was detected (P=0.005). HNF1B mRNA was also positively correlated with HNF1B protein expression (P=0.001). HNF1B promoter methylation was detected in 26/67 (38.8%) of cases. A novel pathogenic somatic HNF1B mutation was detected in 1/61 (1.6%) of the analysed HGSC cases. No other correlations between the examined SNPs (rs4430796, rs757210 and rs7405776), HNF1B promoter methylation, HNF1B/ECI2 expression or clinicopathological characteristics were found.

HNF1B, EZH2 and ECI2 in prostate carcinoma. Molecular, immunohistochemical and clinico-pathological study.

Hepatocyte nuclear factor 1 beta (HNF1B) is a tissue specific transcription factor, which seems to play an important role in the carcinogenesis of several tumors. In our study we focused on analyzing HNF1B in prostate carcinoma (PC) and adenomyomatous hyperplasia (AH), as well as its possible relation to the upstream gene EZH2 and downstream gene ECI2. The results of our study showed that on an immunohistochemical level, the expression of HNF1B was low in PC, did not differ between PC and AH, and did not correlate with any clinical outcomes. In PC, mutations of HNF1B gene were rare, but the methylation of its promotor was a common finding and was positively correlated with Gleason score and stage. The relationship between HNF1B and EZH2/ECI2 was equivocal, but EZH2 and ECI2 were positively correlated on both mRNA and protein level. The expression of EZH2 was associated with poor prognosis. ECI2 did not correlate with any clinical outcomes. Our results support the oncosuppressive role of HNF1B in PC, which may be silenced by promotor methylation and other mechanisms, but not by gene mutation. The high expression of EZH2 (especially) and ECI2 in PC seems to be a potential therapeutic target.

Ex vivo cultivated oral mucosal epithelial cell transplantation for limbal stem cell deficiency: a review.

Destruction or dysfunction of limbal epithelial stem cells (LESCs) leads to unilateral or bilateral limbal stem cell deficiency (LSCD). Fifteen years have passed since the first transplantation of ex vivo cultivated oral mucosal epithelial cells (COMET) in humans in 2004, which represents the first use of a cultured non-limbal autologous cell type to treat bilateral LSCD. This review summarizes clinical outcomes from COMET studies published from 2004 to 2019 and reviews results with emphasis on the culture methods by which grafted cell sheets were prepared.

Aberrant HLA-DR expression in the conjunctival epithelium after autologous serum treatment in patients with graft-versus-host disease or Sjögren's syndrome.

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.

Discontinuous transcription of ribosomal DNA in human cells.

Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.

Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants.

To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]-P2). Cells were characterized by alcian blue/periodic acid-Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0-P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.

Germline mutation in the TP53 gene in uveal melanoma.

We performed comprehensive molecular analysis of five cases of metastasizing uveal malignant melanoma (UM) (fresh-frozen samples) with an NGS panel of 73 genes. A likely pathogenic germline TP53 mutation c.760A > G (p.I254V) was found in two tumor samples and matched nontumor tissue. In three cases, pathogenic BAP1 mutation was detected together with germline missense variants of uncertain significance in ATM. All cases carried recurrent activating GNAQ or GNA11 mutation. Moreover, we analyzed samples from another 16 patients with primary UM by direct Sanger sequencing focusing only on TP53 coding region. No other germline TP53 mutation was detected in these samples. Germline TP53 mutation, usually associated with Li-Fraumeni syndrome, is a rare event in UM. To the best of our knowledge, only one family with germline TP53 mutation has previously been described. In our study, we detected TP53 mutation in two patients without known family relationship. The identification of germline aberrations in TP53 or BAP1 is important to identify patients with Li-Fraumeni syndrome or BAP1 cancer syndrome, which is also crucial for proper genetic counseling.

Frequency of Complications During Preparation of Corneal Lamellae Used in Posterior Lamellar Keratoplasty Using the Pneumodissection Technique (Big Bubble).

PURPOSE: To determine the frequency of formation of various types of bubbles and the potential impact of donor and lamella parameters on this frequency, and to identify possible risk factors of unsuccessful "big-bubble" creation in preparation of pre-Descemet endothelial keratoplasty and Descemet membrane endothelial keratoplasty with peripheral stromal support. METHODS: Donor age and sex, death to preservation time (DPT), storage time, presence of corneal scars (mainly a condition after cataract surgery), and endothelial cell density of 256 donor corneas were assessed before Descemet membrane endothelial keratoplasty with peripheral stromal support or pre-Descemet endothelial keratoplasty lamella preparation using the big-bubble technique. RESULTS: Mean donor age was 62.3 ± 8.5 years (28.3% women and 71.7% men). Mean endothelial cell density of the donor graft was 2866 ± 255 cells/mm. Mean DPT was 10.12 ± 4.88 hours, and mean storage time of the transplant before surgery was 6.5 ± 4.8 days. Corneal scars were present in 17 donor grafts (6.6%) after cataract surgery. Eleven corneas were devalued because of Descemet membrane rupture during preparation (4.3%). In 182 corneas, standard bubble type I was created (71.7%); in 27 corneas, bubble type II was created; eventually, both types of bubbles formed simultaneously (10.5%); in 47 corneas, no bubble was created (18.4%). CONCLUSIONS: We identified higher endothelial cell density, shorter DPT, and the presence of corneal scars after cataract surgery as risk factors threatening successful bubble formation. The only risk factor for creating type II bubbles was higher donor age in our study.

The enzymatic de-epithelialization technique determines denuded amniotic membrane integrity and viability of harvested epithelial cells.

The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture.

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion.

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.

Active transforming growth factor-ß2 in the aqueous humor of posterior polymorphous corneal dystrophy patients.

PURPOSE: Posterior polymorphous corneal dystrophy (PPCD) is characterized by abnormal proliferation of corneal endothelial cells. It was shown that TGF-ß2 present in aqueous humor (AH) could help maintaining the corneal endothelium in a G1-phase-arrest state. We wanted to determine whether the levels of this protein are changed in AH of PPCD patients. METHODS: We determined the concentrations of active TGF-ß2 in the AH of 29 PPCD patients (42 samples) and 40 cadaver controls (44 samples) by ELISA. For data analysis the PPCD patients were divided based on either the molecular genetic cause of their disease as PPCD1 (37 samples), PPCD3 (1 sample) and PPCDx (not linked to a known PPCD loci, 4 samples) or on the presence (17 samples) or absence (25 samples) of secondary glaucoma or on whether they had undergone penetrating keratoplasty (PK, 32 samples) or repeated PK (rePK, 7 samples). RESULTS: The level of active TGF-ß2 in the AH of all PPCD patients (mean ± SD; 386.98 ± 114.88 pg/ml) in comparison to the control group (260.95 ± 112.43 pg/ml) was significantly higher (P = 0.0001). Compared to the control group, a significantly higher level of active TGF-ß2 was found in the PPCD1 (P = 0.0005) and PPCDx (P = 0.0022) groups. Among patients the levels of active TGF-ß2 were not significantly affected by gender, age, secondary glaucoma or by the progression of dystrophy when one or repeated PK were performed. CONCLUSION: The levels of active TGF-ß2 in the AH of PPCD patients are significantly higher than control values, and thus the increased levels of TGF-ß2 could be a consequence of the PPCD phenotype and can be considered as another feature characterizing this disease.

Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review.

The use of amniotic membrane in ophthalmic surgery and other surgical procedures in the fields of dermatology, plastic surgery, genitourinary medicine and otolaryngology is on the increase. Furthermore, amniotic membrane and its epithelial and mesenchymal cells have broad use in regenerative medicine and hold great promise in anticancer treatment. Amniotic membrane is a rich source of biologically active factors and as such, promotes healing and acts as an effective material for wound dressing. Amniotic membrane supports epithelialization and exhibits anti-fibrotic, anti-inflammatory, anti-angiogenic and anti-microbial features. Placentas utilised in the preparation of amniotic membrane are retrieved from donors undergoing elective caesarean section. Maternal blood must undergo serological screening at the time of donation and, in the absence of advanced diagnostic testing techniques, 6 months postpartum in order to cover the time window for the potential transmission of communicable diseases. Amniotic membrane is prepared by blunt dissection under strict aseptic conditions, then is typically transferred onto a nitrocellulose paper carrier, usually with the epithelial side up, and cut into multiple pieces of different dimensions. Amniotic membrane can be stored under various conditions, most often cryopreserved in glycerol or dimethyl sulfoxide or their mixture with culture medium or buffers. Other preservation methods include lyophilisation and air-drying. In ophthalmology, amniotic membrane is increasingly used for ocular surface reconstruction, including the treatment of persistent epithelial defects and non-healing corneal ulcers, corneal perforations and descemetoceles, bullous keratopathy, as well as corneal disorders with associated limbal stem cell deficiency, pterygium, conjunctival reconstruction, corneoscleral melts and perforations, and glaucoma surgeries.

The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

Apoptosis of conjunctival epithelial cells before and after the application of autologous serum eye drops in severe dry eye disease.

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.

Expression, Epigenetic and Genetic Changes of HNF1B in Endometrial Lesions.

Hepatocyte nuclear factor 1-beta (HNF-1-beta) is a transcription factor involved in cancerogenesis of various tumors, including endometrioid carcinoma. We performed comprehensive analysis of HNF-1-beta in lesions of the endometrium, including protein expression and genetic and epigenetic changes. Expression of HNF-1-beta was analyzed immunohistochemically in 320 cases including both tumor and non-tumor endometrial lesions. Promoter methylation and genetic variants were evaluated, using bisulphite and direct sequencing, in 30 (18 fresh frozen, 12 FFPE tumors) endometrioid carcinomas (ECs) and 15 ovarian clear cell carcinomas (OCCCs) as a control group. We detected expression of HNF-1-beta in 28 % of ECs (51/180 cases), 26 % of serous carcinoma (7/27 cases), 83 % of endometrial clear cell carcinoma (15/18 cases), 93 % of hyperplastic polyps with atypias (13/14 cases), 100 % of hyperplastic polyps without atypias (16/16 cases), 88 % of hyperplasias with atypias (14/16 cases), 91 % of hyperplasias without atypias (10/11 cases), and in ≥80 % of different normal endometrium samples. The control group of OCCCs showed HNF-1-beta expression in 95 % (18/19 cases). Methylation in promoter region was detected in 13.3 % (4/30) of ECs, but not in corresponding normal tissue where available, nor in OCCCs (0/15 cases). Mutation analysis revealed truncating variant c.454C > T (p.Gln152X) in one EC and missense variant c.848C > T (p.Ala283Val) was detected in one OCCC. In conclusion, expression of HNF-1-beta was detected in various extents in all types of lesions analyzed, nevertheless its strong expression was mostly limited to clear cell carcinomas. Biological significance of genetic and epigenetic changes needs further investigation.

Identification of six novel mutations in ZEB1 and description of the associated phenotypes in patients with posterior polymorphous corneal dystrophy 3.

Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584*) and c.1717_1718del; p.(Val573Phefs*12) in two Czech families, c.1176dup; p.(Ala393Serfs*19), c.1100C>A; p.(Ser367*), c.627del; p.(Phe209Leufs*11) in three British families and a splice site mutation, c.685-2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs*3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5-52.7 diopters, steep keratometry 48.1-54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation.

The application of autologous serum eye drops in severe dry eye patients; subjective and objective parameters before and after treatment.

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.