55 research outputs found
Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2
Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.−339_361dup for CHED1 and c.−370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.−274T>G and c.−307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies
Presence of snake-like chromatin in epithelial cells of keratoconjunctivitis sicca followed by a large number of micronuclei
Objective: To evaluate the number of micronuclei in snake-like chromatin (SLC) cells in the conjunctival epithelium of keratoconjunctivitis sicca (KCS) patients. To elucidate possible correlations between SLC cell numbers and KCS intensity.
Study Design: Impression cytology specimens from the bulbar conjunctiva of healthy controls and KCS patients were harvested and divided into 3 groups: group 1, controls; group 2, KCS SLC\u96 negative; and group 3, KCS SLC\u96positive. The number of micronuclei (MNi) in SLC-negative and SLC-positive epithelial cells of each group was counted.
Results: The number of MNi in SLC-negative cells of groups 1 and 2 did not exceed 1 MNi/1,000 cells. A significant increase in the frequency of micronuclei in the upper bulbar conjunctiva was noted in SLC-positive (14.75\ub18.09 MNi/1,000 cells) as well as SLC-negative cells (4.0\ub13.83 MNi/1,000 cells) of group 3.
Conclusion: We demonstrate here that the presence of MNi in the conjunctival epithelium of KCS patients could be a characteristic feature accompanying SLC cells. The fact that increased numbers of SLC cells correlate with impaired values in clinical tests as well as decreased goblet and epithelial cell densities confirms that the presence of SLC cells correlates with KCS intensity
Descemet membrane endothelial keratoplasty with a stromal rim in the treatment of posterior polymorphous corneal dystrophy
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/mm2 to 1012 cells/mm2. 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
The spectrum of cytokeratins expressed in the adult human cornea, limbus and perilimbal conjunctiva
The aim of this study was to detect a
spectrum of cytokeratins (CK) present in the adult
human cornea, limbus and perilimbal conjunctiva.
Cryosections from seven corneo-scleral discs were
fixed, and indirect immunofluorescent staining was
performed using antibodies directed against CK1-CK10
and CK13-CK20. The percentage of positive cells was
calculated in the epithelium of the cornea, limbus and
perilimbal conjunctiva. Quantitative real time RT-PCR
(qRT-PCR) was used to detect CK6 and CK18
expression in the corneal and conjunctival epithelium.
The most intense staining present throughout the
cornea was observed for CK3, CK5 and CK14; CK19
was found at the corneal periphery only. CK4 and
CK10/13 revealed mild to moderate positivity mostly in
the superficial layers of the cornea. The suprabasal cell
layers of all examined areas showed a strong positivity
for CK16. A heterogeneous staining pattern with a
centrifugal decrease in the signal was observed for CK8
and CK18. CK5/6, CK14 and CK19 were present in the
limbus, where a positive signal for CK3 was observed in
the suprabasal and superficial cells only. In contrast to
the cornea, CK15 appeared in the basal and suprabasal
layers of the limbus. The perilimbal conjunctiva showed
strong immunostaining for CK10/13, CK14 and CK19.
A moderate signal for CK7 was detected in the
superficial layers of the conjunctiva. qRT-PCR
confirmed CK6 and CK18 expression in the corneal and
conjunctival epithelium.
The detailed characterization of the corneal, limbal
and perilimbal conjunctival epithelium under normal
circumstances may be useful for characterizing the
changes occurring under pathological conditions
Distribution of an analgesic palmitoylethanolamide and other N-acylethanolamines in human placental membranes.
BackgroundHuman amniotic and amniochorionic membranes (AM, ACM) represent the most often used grafts accelerating wound healing. Palmitoylethanolamide, oleoylethanolamide and anandamide are endogenous bioactive lipid molecules, generally referred as N-acylethanolamines. They express analgesic, nociceptive, neuroprotective and anti-inflammatory properties. We assessed the distribution of these lipid mediators in placental tissues, as they could participate on analgesic and wound healing effect of AM/ACM grafts.MethodsSeven placentas were collected after caesarean delivery and fresh samples of AM, ACM, placental disc, umbilical cord, umbilical serum and vernix caseosa, and decontaminated samples (antibiotic solution BASE 128) of AM and ACM have been prepared. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used for N-acylethanolamines analysis.ResultsN-acylethanolamines were present in all studied tissues, palmitoylethanolamide being the most abundant and the anandamide the least. For palmitoylethanolamide the maximum average concentration was detected in AM (350.33 ± 239.26 ng/g), while oleoylethanolamide and anandamide were most abundant in placenta (219.08 ± 79.42 ng/g and 30.06 ± 7.77 ng/g, respectively). Low levels of N-acylethanolamines were found in serum and vernix. A significant increase in the levels of N-acylethanolamines (3.1-3.6-fold, P ConclusionsThe presence of N-acylethanolamines, particularly palmitoylethanolamide in AM and ACM allows us to propose these lipid mediators as the likely factors responsible for the anti-hyperalgesic, but also anti-inflammatory and neuroprotective, effects of AM/ACM grafts in wound healing treatment. The increase of N-acylethanolamines levels in AM and ACM after tissue decontamination indicates that tissue processing is an important factor in maintaining the analgesic effect
The presence of lysyl oxidase-like enzymes in human control and keratoconic corneas
Purpose: Lysyl oxidases, a family comprising
lysyl oxidase (LOX) and four LOX-like enzymes
(LOXL1-4), catalyse the cross-linking of elastin and
collagen fibrils. Keratoconus (KC) is characterized by
progressive thinning leading to irregular astigmatism,
resulting in significant visual impairment. Although the
pathogenesis of KC remains unclear, one of the current
hypotheses is based on alterations in the organization
and structure of collagen fibrils. To extend existing
general knowledge about cross-linking enzymes in the
human cornea, in the present study we have focused on
the detection of LOXL enzymes.
Method: The localization and distribution of
LOXL1-4 were assessed in cryosections of 7 control
donors (three males and three females; 25-68 years;
mean age 46±17.6 years) and 8 KC corneas (5 males and
3 females; 25-46 years; mean age 31.3±7.5 years) using
indirect fluorescent immunohistochemistry (IHC). The
specimens were examined using an Olympus BX51
microscope (Olympus Co., Tokyo, Japan) at a
magnification of 200-1000x. Western blot analysis of 4
control and 4 KC corneas was performed for all tested
enzymes.
Results: All four LOX-like enzymes were present in
all layers of control corneas as well as in the limbus and
conjunctiva. Almost no differences between control and
pathological specimens were found for LOXL1. A lower
staining intensity of LOXL2 was found using IHC and
Western blot analysis in KC specimens. Decreases of the
signal and small irregularities in the staining were found
in the epithelium, keratocytes and extracellular matrix,
where a gradual anterior-posterior weakening of the
signal was observed. LOXL3 IHC staining was lower in
the corneal stromal extracellular matrix and keratocytes
of KC samples. No prominent differences were detected
using IHC for LOXL4, but a slight decrease was
observed in KC corneas using Western blot analysis.
Conclusion: We presume that the decrease of
LOXL2 in KC corneas is more likely a consequence of
the associated pathological processes (activation of
stromal cells due to tissue weakening and consequent
structural changes) than a direct cause leading to KC
development. At this time, we are unable to provide a
coherent explanation for the observed decrease of
LOXL3 and LOXL4 in KC corneas
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
The OV-TL 12/30 Clone of Anti-cytokeratin 7 Antibody as a New Marker of Corneal Conjunctivalization in Patients with Limbal Stem Cell Deficiency
Compared with cytokeratin (CK)19, CK7 is the more reliable marker for distinguishing between the corneal and conjunctival epithelium, particularly in patients with limbal stem cell deficiency
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