Diego Ponzin

P63 IDENTIFIES KERATINOCYTE STEM CELLS

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3σ has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

Retinitis Pigmentosa: Genes and Disease Mechanisms

Retinitis pigmentosa (RP) is a group of inherited disorders affecting 1 in 3000-7000 people and characterized by abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium of the retina which lead to progressive visual loss. RP can be inherited in an autosomal dominant, autosomal recessive or X-linked manner. While usually limited to the eye, RP may also occur as part of a syndrome as in the Usher syndrome and Bardet-Biedl syndrome. Over 40 genes have been associated with RP so far, with the majority of them expressed in either the photoreceptors or the retinal pigment epithelium. The tremendous heterogeneity of the disease makes the genetics of RP complicated, thus rendering genotype-phenotype correlations not fully applicable yet. In addition to the multiplicity of mutations, in fact, different mutations in the same gene may cause different diseases. We will here review which genes are involved in the genesis of RP and how mutations can lead to retinal degeneration. In the future, a more thorough analysis of genetic and clinical data together with a better understanding of the genotype-phenotype correlation might allow to reveal important information with respect to the likelihood of disease development and choices of therapy.

Analysis of limbal stem cell deficiency by corneal impression cytology

Purpose. The impaired function of corneal epithelial stem cells, located in the limbus, is responsible for corneal surface damage and is clinically characterized by recurrent epithelial defects, conjunctivalization, neovascularization, and corneal opacity. The aim of this study was to investigate corneal limbal stem cell deficiency (LSCD) by means of the impression cytology (IC) technique, using antibodies against cytokeratin 19 (CK19) and cytokeratin 3 (CK3), and to evaluate the diagnostic potential of this approach. Methods. Over a 3-year period (October 1998–June 2001), we collected 113 pairs of IC samples from the eyes of 85 patients with a range of ocular surface diseases and performed an immunocytochemical analysis of CK19 and CK3. Samples with more than 50% cellularity were considered suitable for diagnostic purposes, while samples with less than 50% cellularity were considered with caution. CK19-positive cells in corneal IC were considered an expression of LSCD. We arbitrarily scored LSCD as mild (<25% of CK19-positive cells), moderate (25–50%), and severe (>50%). Results. One hundred thirteen pairs of IC specimens were obtained from 85 patients; 32 patients (37.6%) had alkaline burns, 18 (21.2%) had other chemical or physical corneal injuries, 13 (15.3%) had complications from wearing contact lenses, 8 (9.4%) had severe microbial keratitis, and 14 (16.5%) had suspicious limbal deficit due to other causes. Nine patients underwent bilateral sampling and 12 had to be resampled. Thirteen pairs of IC specimens were obtained during the follow-up of 8 patients who had undergone limbal stem cell transplantation. In 3 of these patients, IC confirmed reversion to corneal immunophenotype (CK3+/CK19−), whereas in 4, residual limbal damage was still evident; 1 patient relapsed. In the remaining 100 pairs of impressions, we found 77 cases of LSCD, whereas in 16 pairs, we did not find LSCD. Seven pairs were defined as “not valuable” because of the poor quality of both CK samples. Diffuse LSCD, moderate or severe in degree, was found in 26 of 32 patients (81.2%) with alkali burns, whereas mild diffuse LSCD or sectoral LSCD was found in 13 of 18 patients (72.2%) with other chemical-physical injuries, in 10 of 13 patients (76.9%) wearing contact lenses, in 7 of 8 patients (87.5%) with severe microbial keratitis, and in 12 of 14 patients (85.7%) with other corneal pathologies. The quality of impressions was assessed in 77 cases and found to be good or discrete for both CKs in 32 cases (41.5%) and poor in 45 (58.5%): in 46.7% of these cases, the IC was poor only for CK19 and in 45.4% only for CK3. Conclusions. Immunocytochemistry for seeking out CK19- and CK3-positive cells on corneal IC is a simple and practical method to investigate LSCD. We believe that this technique could have an important role in evaluating patients undergoing therapeutic penetrating keratoplasty to select those who would benefit from limbal stem cell transplantation. Since sampling has been shown to be a critical point, we believe that any improvement in this area will also help to improve the methodology and will contribute to its wider utilization.

Microkeratome-assisted preparation of ultrathin grafts for descemet stripping automated endothelial keratoplasty.

PURPOSE To compare three microkeratome-assisted techniques for the preparation of ultrathin (UT) grafts for Descemet stripping automated endothelial keratoplasty. METHODS After dissection with a 300-μm microkeratome head in 40 donor tissues, a second cut was performed with a 130-μm head either after manual stromal hydration (group A, n = 10) or osmotic hydration at the eye bank (group B, n = 10) or with a 50- or 90-μm head, depending on residual bed thickness (group C, n = 10); no further dissection was performed in the control group (group D, n = 10). Corneal thickness and endothelial cell (EC) count were determined at all appropriate stages. Statistical analysis was performed using a Fisher exact test. RESULTS Final graft thicknesses in groups A (89.1 ± 34.1μm), B (84.1 ± 18.6 μm), and C (72.1 ± 10.1 μm) were significantly lower than in group D (201.9 ± 25.3 μm) (P < 0.001). EC loss did not differ significantly among the groups. Multiple areas of Descemet detachment were seen in 4 of 10 corneas of group A. CONCLUSIONS All methods proved equally efficient in producing UT grafts, but stromal hydration induced tissue structural changes. EC loss was unaffected by the additional manipulation required to prepare UT grafts.

The CORTES study: Corneal transplant indications and graft survival in an italian cohort of patients

Purpose: To describe the corneal transplantation activity in Italy, to assess the long-term graft survival, and to begin to outline the potential risk factors for graft outcome. Methods: We followed a consecutive series of penetrating (PK) and lamellar (LK) keratoplasties performed with corneas procured and distributed by the Veneto Eye Bank Foundation, which provides about one third of the corneas grafted in Italy each year. Results: Data on 4415 PKs and 489 LKs performed in 174 clinical centers are reported. Keratoconus was the major transplant indication (47% and 66%, respectively, for the 2 groups), followed by regraft (14%) and bullous keratopathy (14%) in the PK group and keratitis (8%) and refractive reasons (4%) for the LKs. In the 2 groups, graft survival, after 1 year, was estimated to be 95% and 93%, respectively, showing a decrease of the survival rate during the second and third years of the study. Graft survival in patients with keratoconus indication was 98% in the PK group and 95% in the LK group for the whole period of observation, whereas the patients with other indications reported a survival rate ranging from 92% after 1 year to 52% after 3 years (PK) and from 89% to 85% (LK). Conclusions: CORTES is the most extensive survey on corneal transplantation in Italy that involves a large cohort of patients and a significant number of surgeons with corneal tissues processed and distributed by a single eye bank. In the first 3 years, a picture of the epidemiology of the corneal transplant has been defined. The graft survival rates were comparable to those reported by other studies for the same follow-up period. However, the follow-up of a sample of this cohort for a further 3 years will allow us to precisely estimate the long-term graft survival and to better evaluate the risk factors related to graft failure.

Limbal stem cell deficiency and ocular phenotype in ectrodactyly-ectodermal dysplasia-clefting syndrome caused by p63 mutations

OBJECTIVE To describe the ocular phenotype in patients with ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome (MIM#604292) and to determine the pathogenic basis of visual morbidity. DESIGN Retrospective case series. PARTICIPANTS Nineteen families (23 patients) affected by EEC syndrome from the United Kingdom, Ireland, and Italy. METHODS General medical examination to fulfill the diagnostic criteria for EEC syndrome and determine the phenotypic severity. Mutational analysis of p63 was performed by polymerase chain reaction-based bidirectional Sanger sequencing. All patients with EEC syndrome underwent a complete ophthalmic examination and ocular surface assessment. Limbal stem cell deficiency (LSCD) was diagnosed clinically on the basis of corneal conjunctivalization and anatomy of the limbal palisades of Vogt. Impression cytology using immunofluorescent antibodies was performed in 1 individual. Histologic and immunohistochemical analyses were performed on a corneal button and corneal pannus from 2 EEC patients. MAIN OUTCOME MEASURES The EEC syndrome phenotypic severity (EEC score), best-corrected Snellen visual acuity (decimal fraction), slit-lamp biomicroscopy, tear function index, tear breakup time, LSCD, p63 DNA sequence variants, impression cytology, and corneal histopathology. RESULTS Eleven heterozygous missense mutations in the DNA binding domain of p63 were identified in all patients with EEC syndrome. All patients had ocular involvement and the commonest was an anomaly of the meibomian glands and lacrimal drainage system defects. The major cause of visual morbidity was progressive LSCD, which was detected in 61% (14/23). Limbal stem cell deficiency was related to advancing age and caused a progressive keratopathy, resulting in a dense vascularized corneal pannus, and eventually leading to visual impairment. Histologic analysis and impression cytology confirmed LSCD. CONCLUSIONS Heterozygous p63 mutations cause the EEC syndrome and result in visual impairment owing to progressive LSCD. There was no relationship of limbal stem cell failure with the severity of EEC syndrome, as classified by the EEC score, or the underlying molecular defect in p63. FINANCIAL DISCLOSURE(S) The authors have no proprietary or commercial interest in any of the materials discussed in this article.

Evaluation of ocular surface disorders: a new diagnostic tool based on impression cytology and confocal laser scanning microscopy

Aim To provide a new tool for the evaluation of altered ocular surfaces by using a combination of impression cytology, laser scanning confocal microscopy and advanced image analysis. Methods The expression of keratin 3 (K3), keratin 12 (K12), keratin 19 (K19) and mucin 1 (MUC1) was analysed by immunofluorescence on both histological sections of nine corneoscleral buttons from normal donors comprising conjunctiva, limbus and cornea and impression cytology specimens from six healthy normal subjects (12 eyes) and 12 patients with chronic ocular surface disorders. Levels of fluorescence expression of the different markers were quantified through quantitative fluorescence immunohistochemistry (Q-FIHC). Results Impression cytology specimens from normal and diseased ocular surfaces showed distinct expression patterns for K12 and MUC1. Healthy corneas expressed only K12 (but not MUC1), while conjunctivalised corneas from patients with limbal stem cell deficiency (LSCD) were characterised by the presence of MUC1 and the disappearance of K12. Similar clear-cut results were not seen with the K3/K19 markers, which showed lack of specificity and overlapping signals in cornea and conjunctiva impression cytology specimens. Conclusions The ability of K12 and of the antibody against MUC1 to discriminate clearly between limbus/cornea and conjunctiva in impression cytology specimens could become a valuable diagnostic tool for ophthalmologists in order to evaluate alterations of the ocular surface and the grading of LSCD.

High-resolution analysis of the human retina miRNome reveals isomiR variations and novel microRNAs

MicroRNAs play a fundamental role in retinal development and function. To characterise the miRNome of the human retina, we carried out deep sequencing analysis on sixteen individuals. We established the catalogue of retina-expressed miRNAs, determined their relative abundance and found that a small number of miRNAs accounts for almost 90% of the retina miRNome. We discovered more than 3000 miRNA variants (isomiRs), encompassing a wide range of sequence variations, which include seed modifications that are predicted to have an impact on miRNA action. We demonstrated that a seed-modifying isomiR of the retina-enriched miR-124-3p was endowed with different targeting properties with respect to the corresponding canonical form. Moreover, we identified 51 putative novel, retina-specific miRNAs and experimentally validated the expression for nine of them. Finally, a parallel analysis of the human Retinal Pigment Epithelium (RPE)/choroid, two tissues that are known to be crucial for retina homeostasis, yielded notably distinct miRNA enrichment patterns compared to the retina. The generated data are accessible through an ad hoc database. This study is the first to reveal the complexity of the human retina miRNome at nucleotide resolution and constitutes a unique resource to assess the contribution of miRNAs to the pathophysiology of the human retina.

Descemet membrane endothelial keratoplasty tissue preparation from donor corneas using a standardized submerged hydro-separation method.

PURPOSE To standardize a novel submerged hydro-separation technique for Descemet membrane endothelial keratoplasty (DMEK) graft preparation from donor corneal tissues. DESIGN Experimental study, laboratory investigation. METHODS SETTING The Veneto Eye Bank Foundation, Venice, Italy. STUDY POPULATION Fifty-four random human donor corneal tissues unsuitable for transplantation. INTERVENTION Donor corneas were laid in a sterile basin partially filled with tissue culture medium. A 25 gauge needle with 1 mL mounted syringe was filled with the tissue culture medium. The needle (with bevel up) was bent to 90 degrees and was inserted in the posterior cornea initiating beneath the trabecular meshwork. It was further advanced toward the midperiphery, ensuring that only the bevel was inserted, considering it as a threshold of insertion. The liquid was injected with a medium to high pressure into the posterior stroma or in the Descemet membrane-stroma interface to create the bubble. The tissues were preserved for 7 days in tissue culture medium at 31°C. Parametrical, physiological and histological analyses were carried out. MAIN OUTCOME MEASURES Larger-diameter tissue, no tissue wastage, reproducibility, and preshipment evaluation. RESULTS Complete detachment was achieved in all the cases without any tissue wastage. Average diameter of the excised graft was 10.80 (±0.28) mm and endothelial cell loss post preservation was 11.48%. Expression of tight junction protein and regular morphology was observed post preservation. No signs of cell apoptosis were seen. Histological analysis showed elimination of residual stroma in most of the cases. CONCLUSIONS The submerged hydro-separation method reduces tissue wastage. It allows preshipment evaluation, thus allowing a validated tissue to be transported from the eye banks to the surgeon. Because of the liquid interface, the peeling of the DMEK graft becomes easy for transplantation.

Anatomy and physiology of the human eye: effects of mucopolysaccharidoses disease on structure and function – a review

The current paper provides an overview of current knowledge on the structure and function of the eye. It describes in depth the different parts of the eye that are involved in the ocular manifestations seen in the mucopolysaccharidoses (MPS). The MPS are a group of rare inheritable lysosomal storage disorders characterized by the accumulation of glycosaminoglycans (GAGs) in cells and tissues all over the body, leading to widespread tissue and organ dysfunction. GAGs also tend to accumulate in several tissues of the eye, leading to various ocular manifestations affecting both the anterior (cornea, conjunctiva) and the posterior parts (retina, sclera, optic nerve) of the eye.