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 1β (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).
promoter methylation was detected in 26/67 (38.8%) of cases. A novel pathogenic somatic
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.
Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant genetically heterogeneous disorder. Nineteen Czech PPCD pedigrees with 113 affected family members were identified, and 17 ...of these kindreds were genotyped for markers on chromosome 20p12.1- 20q12. Comparison of haplotypes in 81 affected members, 20 unaffected first degree relatives and 13 spouses, as well as 55 unrelated controls, supported the hypothesis of a shared ancestor in 12 families originating from one geographic location. In 38 affected individuals from nine of these pedigrees, a common haplotype was observed between D20S48 and D20S107 spanning approximately 23 Mb, demonstrating segregation of disease with the PPCD1 locus. This haplotype was not detected in 110 ethnically matched control chromosomes. Within the common founder haplotype, a core mini-haplotype was detected for D20S605, D20S182 and M189K2 in all 67 affected members from families 1-12, however alleles representing the core mini-haplotype were also detected in population matched controls. The most likely location of the responsible gene within the disease interval, and estimated mutational age, were inferred by linkage disequilibrium mapping (DMLE+2.3). The appearance of a disease-causing mutation was dated between 64-133 generations. The inferred ancestral locus carrying a PPCD1 disease-causing variant within the disease interval spans 60 Kb on 20p11.23, which contains a single known protein coding gene, ZNF133. However, direct sequence analysis of coding and untranslated exons did not reveal a potential pathogenic mutation. Microdeletion or duplication was also excluded by comparative genomic hybridization using a dense chromosome 20 specific array. Geographical origin, haplotype and statistical analysis suggest that in 14 unrelated families an as yet undiscovered mutation on 20p11.23 was inherited from a common ancestor. Prevalence of PPCD in the Czech Republic appears to be the highest worldwide and our data suggests that at least one other novel locus for PPCD also exists.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Keratoconus (KC) is an eye disease characterized by the progressive thinning and protrusion of the cornea, which results in the loss of visual acuity. This disorder remains poorly understood, ...although recent studies indicate the involvement of genetic and environmental factors. Recently, we have found that the distribution of the cross-linking enzyme lysyl oxidase (LOX) is markedly decreased in about 63 % of keratoconic specimens. Similarly, LOX activity is significantly reduced by 38 % compared to control tissue. Nearly 70 systemic disorders have been reported in association with KC, most of them affecting the extracellular matrix. In this review we attempted to ascertain whether any KC-associated diseases exhibit signs that may reflect LOX impairment. We hypothesized that very similar changes in the extracellular matrix, particularly at the level of collagen metabolism, including LOX impairment in mitral leaflets, may reflect an association between KC and mitral valve prolapse. Moreover, this putative association is supported by the high frequency of Down syndrome in both diseases. Among other disorders that have been found to coincide with KC, we did not find any in which the LOX enzyme may be directly or indirectly impaired. On the other hand, in cases where KC is present along with other connective tissue disorders (Marfan syndrome, Ehlers–Danlos syndrome and others), KC may not arise as a localized manifestation, but rather may be induced as the result of a more complex connective tissue disorder.
A 20-year-old patient, diagnosed with posterior polymorphous corneal dystrophy, developed corneal edema for which he underwent Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) ...in the right eye. No intra- or postoperative complications were noted. At the last follow-up 2 years and 9 months after the procedure, the best corrected visual acuity was 1.0 and endothelial cell density declined from 3533 cells/mm 2 to 1012 cells/mm 2 . Despite the endothelial cell loss, DMEK-S appears to be a good alternative to other surgical techniques for the treatment of corneal endotheliopathies, and it may be of benefit to young patients.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Purpose: To assess the impact of Acanthamoeba keratitis (AK) and amniotic membrane transplantation (AMT) in corneal explants on presence of two multifunctional endogenous lectins, i.e. galectins-1 ...and -7.
Methods: Ten corneal explants from AK patients (five with previous AMT and five controls without this treatment) and seven specimens of disease-free control cornea were processed by indirect fluorescent immunohistochemistry.
Results: Immunostaining for both galectins was obtained in the epithelium, stroma and the endothelial layer of all controls, with the strongest positivity in the epithelium. Significantly decreased intensity for galectin-1 was recorded in the epithelium of corneal explants from patients with AK and AMT. The signal for galectin-7 was significantly decreased in the epithelium of AK patients and normalized after AMT.
Conclusions: AMT has a marked impact on presence of the two galectins in opposite directions, encouraging complete profiling for this family of endogenous effectors.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Posterior polymorphous corneal dystrophy (PPCD) is a hereditary bilateral disorder affecting Descemet's membrane and the endothelium. The aim of the present study was to determine the spectrum of ...cytokeratin (CK) expression in cells on the posterior surface of the cornea in PPCD patients. Ten corneal buttons and one specimen of the trabecular meshwork (TM) from PPCD patients who underwent graft or glaucoma surgery were used, as well as six corneal buttons and two TM specimens obtained from healthy donors as controls. Cryosections were fixed and indirect immunofluorescent staining was performed using antibodies directed against a wide spectrum of cytokeratins (CKs). The number of positive cells and the intensity of the staining were assessed using fluorescent microscopy. All 10 PPCD corneal specimens had areas of endothelium displaying typical endothelial morphology as well as areas consisting of layers two to six cells thick with both flat endothelial-like cells and polygonal cells with round nuclei and a large cytoplasm. Both of these morphologically distinct cell types showed strong immunostaining for CK7, CK19, CK8 and CK18, while weaker positive signals were observed for CK1, CK3/12, CK4, CK5/6, CK10, CK10/13, CK14, CK16 and CK17. PPCD endothelium was completely negative for CK2e, CK9, CK15, and CK20. Focal positivity was detected in PPCD TM for CK4, CK7 and CK19. CK8 and CK18 were the only CKs expressed in control endothelium. PPCD and control epithelium displayed similar staining patterns. The distinct positivity for CK3/12, CK4, CK5/6, CK10/13, CK14, CK16 and CK17 was observed in aberrant PPCD endothelium for the first time. We demonstrate that the abnormal endothelium of PPCD patients expresses a mixture of CKs, with CK7 and CK19 predominating. In terms of CK composition, the aberrant PPCD endothelium shares features of both simple and squamous stratified epithelium with a proliferative capacity. The wide spectrum of CK expression is most probably not indicative of the transformation of endothelial cells to a distinct epithelial phenotype, but more likely reflects the modified differentiation of metaplastic epithelium.
To investigate the sensitivity of human Schwann cells to cisplatin (cis-DDP), different approaches to estimate DNA damage were used: the comet assay, morphological evaluation of the granular ...condensation of nuclear chromatin and the terminal transferase-mediated dUTP nick-end-labelling (TUNEL) method. The number of micronuclei (MNi), as a sign of cisplatin-induced genotoxicity, was counted. DNA damage assessed by the comet assay was already evident after 1.5
μM cisplatin treatment at all exposure times (24, 48, and 72
h). Initial morphological changes characterised by the granular condensation of nuclear chromatin were detectable after 24
h exposure to 25
μM cis-DDP, while an increased number of apoptotic cells, determined by the TUNEL method, was noted after 48
h exposure to the same concentration. The first significant increase in the number of MNi was observed in cells treated with 75
μM cis-DDP for 24
h. We demonstrate that the comet assay is a highly sensitive method for measuring cisplatin induced DNA damage. Morphological observation revealed advanced as well as less prominent alterations in the nuclear chromatin. In contrast, the TUNEL method detected only those cells with advanced DNA fragmentation.
Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the ...biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H2O2 in nM per μg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic samples (2.60 ± 2.23 nM H2O2/μg of total protein) was more than 2.5-fold lower than in control tissue (6.83 ± 2.53 nM H2O2/μg of total protein), and the decrease was statistically significant (p = 0.0178). The location of lysyl oxidase in the healthy cornea, limbus and perilimbal conjunctiva was described. We hypothesize that the restricted lysyl oxidase distribution in keratoconic corneas, and particularly the decrease of total lysyl oxidase activity in cultured keratoconic fibroblasts, is one potential reason for the inadequate collagen cross-linking that is a hallmark of this disease.
► Presence and distribution of lysyl oxidase in the cornea, limbus and conjunctiva. ► Lower presence and changes in lysyl oxidase distribution in keratoconic corneas. ► Decreased activity of lysyl oxidase in culture medium of keratoconic fibroblasts.
Although stem cell activity represents a crucial feature in corneal and ocular surface homeostasis, other cells populating this region and the neighboring zones might participate and influence local ...microenvironment. Mast cells, the long-lived and tissue-sited immune cells, have been previously reported in corneoscleral specimens. Herein, mast cells were investigated in corneoscleral tissues and related to microenvironment protein expression.
Twenty-six (14 male/12 female; older than 60 years) human corneoscleral specimens were sectioned for light and fluorescent immunostaining (CD45, p63, Ck-3/7/12/19, tryptase/AA1, and chymase/CC1). Corneal, limbal, and conjunctival squares were produced for molecular and biochemical analysis. Statistical comparisons were carried out by ANOVA.
Toluidine blue staining identified metachromatic intact or degranulated mast cells in the area below the palisades' Vogt (Ck-3/12-positive epithelium and underneath p63 immunoreactivity). Tryptase immunoreactivity was observed close to palisades' Vogt, whereas no specific signal was detected for chymase. Tryptase/AA1 transcripts were quantified in limbal and conjunctival RNA extracts, whereas no specific amplification was detected in corneal ones. Few mediators were overexpressed in limbal extracts with respect to corneal (Neural cell adhesion molecule (NCAM), Intercellular adhesion molecule 3 (ICAM3), Brain-derived Neurotrophic factor (BDNF), and neurotrophin 3 (NT3); P < 0.00083) and conjunctival (NCAM, ICAM3, and NT3; P < 0.05) protein extracts. A trend to an increase was observed for Nerve Growth Factor (NGF) in limbal extracts (P > 0.05).
The specific observation of tryptase phenotype and the interesting protein signature of microenvironment (adhesion molecules, growth factors, and neurotrophins), known to partake mast cell behavior, at least in other areas, would provide additional information to better understand this crucial zone in the framework of ocular surface healthiness.
Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine ...serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.
•Both non-xenogeneic media maintain a high number of stem cells and low level of apoptosis in LEC cultures on both substrates.•hSM combined with fibrin scaffold yields higher growth rates and proliferation of LEC cultures compared to hPLM.•Fibroblast-like cell contamination of LEC cultures is less pronounced in hSM combined with fibrin scaffold than in hPLM.•The growth onset and harvest of LEC cultured in hSM on fibrin scaffold are comparable with that of COM.
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