Expression analysis of human pterygium shows a predominance of conjunctival and limbal markers and genes associated with cell migration.

PURPOSE: Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. To increase our understanding of the cells types involved in pterygium, we have used expressed sequence tag analysis to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration. METHODS: An unnormalized unamplified cDNA library was prepared from 15 pooled specimens of surgically removed pterygia as part of the NEIBank project. Gene expression patterns were compared with existing data for human cornea, limbus, and conjunctiva, and expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium. RESULTS: Sequence analysis of 2,976 randomly selected clones produced over 1,800 unique clusters, potentially representing single genes. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Immunofluorescence of Krt12 and 13 in the normal ocular surface showed specificity of Krt12 in cornea and Krt13 in conjunctival and limbal epithelia, with a fairly sharp boundary at the limbal-corneal border. In the pterygium there was a patchy distribution of both Krt12 and 13 up to a normal corneal epithelial region specific for Krt12. Immunoglobulins were also among the prominently expressed transcripts. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. A number of genes highly expressed in cornea were not found in pterygium (several small leucine-rich proteoglycan family members) or were expressed at considerably lower levels (ALDH3A1 and decorin). CONCLUSIONS: The expression pattern of keratins and other markers in pterygium most closely resemble those of conjunctival and limbal cells; some corneal markers are present, notably Krt12, but at lower levels than equivalent conjunctival markers. Our data are consistent with the model of pterygium developing from the migration of conjunctival- and limbal-like cells into corneal epithelium. Identification of genes with roles in cell migration suggests potential therapeutic targets. In particular, the ability of polyamine analogues to reduce migration in primary cultures of pterygium presents a possible approach to slowing pterygium growth.

Accumulation of p53 protein in pterygia is not accompanied by TP53 gene mutation.

Previously, we reported that pterygial epithelial cells show positive p53 staining by immunohistochemistry, and that they do not demonstrate apoptosis. We wished to determine whether the accumulation of p53 protein was caused by missense mutations in exons 5-8 of the TP53 gene, as is frequently the case in malignant tumours that contain high levels of abnormal p53. From 11 pterygia, epithelial cells were isolated by laser capture microdissection, or manually, in order to reduce the contribution of TP53 from normal cells. DNA from pterygial epithelial cells was amplified across exons 5-8 in 10 pterygia and across exons 5,7 and 8 in another pterygium. In 2 pterygia, all translated exons (2-11) were sequenced. No mutations were found, although normal polymorphisms in codon 72 were readily detected in 2 pterygia. RT-PCR was used to compare amounts of TP53 mRNA isolated from normal conjunctiva and pterygia from eight additional patients. We detected an approximate two-fold increase of TP53 RNA in pterygia compared to that in normal conjunctiva. Western blotting was used to compare amounts of p53 protein in pterygia and normal conjunctiva. Consistent with our previous immunohistochemical studies, amounts of p53 protein in pterygia, detected by the western blotting, were elevated compared to those detected in normal conjunctiva and corneal limbal epithelium. However, the TP53 gene in pterygia is not mutated, and therefore, the elevated levels of p53 protein must result from a different mechanism than that seen in malignant tumours containing TP53 missense mutations. The increased amount of p53 protein in pterygial cells does not cause apoptosis or block cell proliferation, suggesting that these normal p53 functions are inactivated in pterygia.

A new method for measuring progression in patients with ocular cicatricial pemphigoid.

OBJECTIVES: To describe a method to measure the progression of ocular cicatricial pemphigoid and to compare its facility with traditional methods used to measure the progression of the disease. METHODS: The proposed method consists of measuring (in millimeters) the total relative inferior conjunctival surface available in 3 gaze positions. This method was used to monitor 7 eyes of 4 patients with ocular cicatricial pemphigoid over 2 years. The changes in the conjunctival measurements from baseline were compared with the changes documented by traditional methods. RESULTS: During the study, 2 eyes remained stable (changes, <3 mm), 2 had a decrease of 10 mm or more, and 3 had a change in measurements between 4 and 9 mm. With the proposed method, we demonstrated the detection of more subtle changes in the conjunctiva of all patients. Patients who had changes between 4 and 9 mm easily underwent staging by the traditional systems when the new technique was used as a reference. CONCLUSION: The proposed method offers an objective variable that can be used in consecutive visits to detect subtle progression or disease control in patients with ocular cicatricial pemphigoid.

Measurement of mRNAs for TGFss and extracellular matrix proteins in corneas of rats after PRK.

PURPOSE: To assess the role of the transforming growth factor (TGF)ss system in formation of corneal haze after excimer laser photorefractive keratectomy (PRK), levels of mRNAs for three TGFss isoforms (TGFss1, TGFss2, and TGFss3), the TGFss type II receptor (TssRII), and extracellular matrix (ECM) genes including fibronectin (FN), collagen I, collagen III, and collagen IV were measured in rat corneas. METHODS: Corneas were graded for corneal haze at 0, 1.5, 7, 21, 42, and 91 days after PRK. Total RNA was isolated from pooled corneas, and the levels of mRNAs were measured using competition-based quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Severe corneal haze developed by day 42 and persisted to day 91. Levels of TGFss1 mRNA were high in rat corneas before PRK and remained relatively constant. In contrast, levels of TGFss2 and TGFss3 mRNAs were very low in normal corneas, increased 300-fold and 25-fold, respectively, on day 21, and remained elevated on day 91. Levels of mRNA for TssRII increased, with a peak elevation of 50-fold on day 42 after PRK. Levels of mRNAs for ECM proteins also increased. Fibronectin mRNA was nondetectable in normal corneas but rapidly increased to 675 copies/cell on day 7 and remained elevated to day 91. Collagen III mRNA levels peaked on day 21 with a 700-fold increase compared with a very low level of expression in normal cornea, and then decreased on day 91. Expression of collagen I mRNA lagged expression of collagen III mRNA and peaked at day 42 after PRK with a 1200-fold increase over normal cornea. In contrast, mRNA for collagen alpha(1)IV, a major component in basement membranes, remained relatively stable through day 21 and then increased slightly on days 42 and 91. CONCLUSIONS: The synchronized increase in mRNA synthesis for both the TGFss system and key ECM genes supports the hypothesis that TGFss is a key growth factor promoting stromal haze formation in corneas after PRK and suggests that limiting TGFss system may reduce corneal scarring after excimer laser ablation.

The BioMask for treatment of corneal surface irregularities with excimer laser phototherapeutic keratectomy.

PURPOSE: To assess the characteristics of BioMask as a potential masking agent for use with the excimer laser. METHOD: We addressed ablation rate, smoothness, ease of use, dioptric shift, treatment of standardized irregular topography, and ability of BioMask to induce dioptric change in vivo. RESULTS: BioMask ablates at a rate of 0.28 microm per pulse. The BioMask conforms to the base curve of a contact lens in the excimer blank, eye bank eye, and rabbit eye with a r2 of 0.9982, 0.9844, 0.9858, respectively. We are readily able to create 20 diopters of flattening or steepening (r2 = 0.9944). Standardized irregular topography generation in the rabbit eye and then removal with BioMask was successful. The central corneal topography of the rabbit cornea showed predictable changes with various contact lens base curves with the BioMask (r2 = 0.875). CONCLUSIONS: BioMask has excellent potential as an ablatable mask material in the treatment of superficial corneal scars.

Surgical correction of moderate myopia: which method should you choose? I. Radial keratotomy will always have a place.

This set of "Viewpoints" articles examines the relative merits of radial keratotomy (RK), photorefractive keratectomy (PRK), and laser assisted in-situ keratomileusis (LASIK). Drs. Rowsey and Morley review advances in RK techniques, long-term results, and complications, and explain why RK will remain a viable method for correction of moderate myopia, notably its minimal cost. Drs. Steinert and Bafna review both PRK and LASIK, discussing techniques and results and comparing their advantages and disadvantages with each other and with RK. Dr. Dutton, as "Viewpoints" section editor, summarizes clinical, technologic, and economic aspects of all three techniques, concluding that all will find a place among refractive surgeons for some time to come.

Comparison of three computerized videokeratoscopy systems with keratometry.

PURPOSE: To compare the reproducibility of computerized videokeratoscopy systems by using normal eyes and calibrated objects. METHODS: We evaluated the reproducibility of three commercially available videokeratoscopes [EyeSys, TechnoMed C-Scan, and PAR Corneal Topography System (CTS)] with the manual keratometer (Bausch & Lomb) by using calibration spheres and 10 normal subjects (20 eyes). All videokeratoscopy and keratometer results were obtained by one investigator (R.M.). Each eye and calibration sphere were submitted to 10 serial examinations by using each system. The average K of all points within the central 3.0 mm of the topography systems (central 3.0 mm) was compared with the average K of the manual keratometer. RESULTS: All videokeratoscopy systems correlated well with each other and manual keratometry when accessing aspheric and spherocylinder calibration balls. EyeSys central keratometry clinical results had the strongest correlation with the average keratometry results at 35%, followed by PAR-CTS at 25% and C-Scan at 5%. Among the videokeratoscopy units, EyeSys and PAR-CTS had the strongest correlation at 65%. The correlation between the TechnoMed C-Scan and both the EyeSys and PAR-CTS systems was 25%. There was a statistically significant difference (p < 0.05) between the systems when analyzing the results obtained from clinical subjects. The average keratometry (K) difference of human eyes between videokeratoscopy systems is <0.35 diopters (D) (p < 0.05), which may be clinically significant. The average manual K reading (42.97 D) is statistically significantly flatter (p < 0.05) than each of the videokeratoscopy units (EyeSys = 43.49 D; PAR = 43.48 D; C-Scan = 43.83 D). Comparing the 10 measurements of each eye or calibration object in the same videokeratoscopy system verified that the devices give reproducible results. The average standard deviation (ASD) of the keratometer was 0.10 D. The ASD of the videokeratoscopy units was 0.05 D for the EyeSys, 0.29 D for the PAR-CTS, and 0.31 D for the C-Scan systems. CONCLUSION: Based on this study, we should not assume that the results of different topography systems can be interchanged in clinical studies.

The immunology of corneal graft rejection.

Corneal transplantation is the most successful of organ transplants due to the fact that the eye is an immunologically privileged site, and the cornea is an immunologically privileged tissue. The factors responsible for this include presence of the blood-aqueous barrier, the avascularity of the cornea, the absence of classic antigen-presenting cells (APCs) in the central cornea, inhibitory factors in the aqueous humor, the phenomenon known as anterior chamber-associated immune deviation (ACAID), and the intraocular expression of Fas ligand. Loss of ocular immune privilege can occur with breaching of the blood-ocular barrier, corneal neovascularization, migration of classic APCs to the center of the cornea, loss of inhibitory factors in aqueous humor, abrogation of ACAID, and loss of Fas ligand expression within the anterior chamber. The purpose of this review is to analyze these events and how they relate to corneal graft rejection. A discussion on future research and therapeutic modalities is provided.

Cystic epithelial downgrowth following clear-corneal cataract extraction.

Epithelial downgrowth is a serious complication of intraocular surgery. It is characterized by the diffuse or cystic proliferation of surface epithelium inside the eye. Advances in microsurgical technique that result in smaller surgical wounds and permit greater precision in wound closure should reduce the incidence of this complication. We report a case of epithelial downgrowth presenting 3 years after an uncomplicated clear-corneal cataract extraction with insertion of a posterior chamber silicone lens. The epithelial down-growth was amenable to surgical correction because the well-defined cyst could be excised en bloc.

The Tampa Trephine technique with cat corneal endothelium.

The Tampa Trephine (Martin Marietta Speciality Components, Largo, FL, U.S.A.) penetrating keratoplasty technique uses a 7.0-mm corneal donor button with six rectangular 1 x 2-mm tabs of Bowman's layer, 75 microns in thickness, which are inserted into the recipient stroma beneath Bowman's layer. We evaluated the safety of the Tampa Trephine tissue-trephination method on the cat corneal endothelium combining vital staining and scanning electron microscopy, comparing it with the standard Weck trephination technique. The Tampa Trephine tissue trephination produces a donor button with a 6.7-mm diameter central area of normal endothelium. Localized peripheral areas of cellular loss, endothelial and Descemet's tears, endothelial detachment, and folding along the border of the trephination were observed with the Tampa Trephine method, all located in an area of < or = 150 microns, adjacent to the edge of the button. Standard trephination induced a localized peripheral area of endothelial damage < 50 microns in extension from the donor edge. A theoretic maximal 8.4% peripheral endothelial cell loss is induced with the Tampa Trephine trephination method, compared with a 2.8% loss with the standard procedure. The peripheral location of the alterations after the Tampa Trephine does not hinder the viability of the corneal endothelium, as it has been clinically observed.

Use of a mirror needle holder with transsclerally sutured posterior chamber intraocular lenses.

A mirror needle holder has been designed to facilitate the passage of a needle through the ciliary sulcus during transscleral fixation of posterior chamber (PC) intraocular lens (IOLs). Two human postmortem eyes were used to demonstrate the efficacy of this mirror used as the needle holder. This method was compared with the current method of passing sutures without view of the ciliary sulcus. The mirror needle holder demonstrated precise passage of the needle into the ciliary sulcus by direct visualization of the ciliary processes and sulcus. This method may avoid the complications associated with transsclerally sutured PC IOLs during penetrating keratoplasty and secondary IOL placement.

Refractive corneal surgery with the Draeger rotary microkeratome in human cadaver eyes.

BACKGROUND: Instrumentation for performing a uniform lamellar keratoplasty has been undergoing various stages of refinement. Reliable reproduction and uniform thickness and diameter of lamellar resections is required before lamellar refractive keratoplasty can be considered safe and effective. METHODS: The authors used the Draeger rotary microkeratome with mechanical blade advance for lamellar dissections in 61 human cadaver eyes prepared by injecting Swinger-Kornmehl (SK) solution into the anterior chamber to a pressure of 35 to 40 mm Hg and by soaking for 30 minutes in SK solution. Spacer sizes of 0.25 to 0.40 units were utilized using an anterior lamellar disc diameter estimate between 8.0 and 8.5 mm and a stromal lamellar disc diameter estimate between 5.5 and 6.5 mm. Preoperative pachometry, anterior and stromal lamellar disc thicknesses, and anterior and stromal lamellar disc diameters were measured. RESULTS: The Draeger unit created anterior lamellar thickness between 100 and 268 microns. Stromal lamellar disc thicknesses were consistently between 90 and 161 microns. The continuous, unidirectional, rotary blade and the uniform mechanical advance of the instrument produced a generally uniform bed as evaluated by scanning electron microscopy, although undulations were still present. CONCLUSION: The Draeger microkeratome produced regular lamellar dissections; however, predictability of the thickness of the lenticules varied 10% to 20%, and of the diameter, 1.5% to 15%. Predictability improved with experience. This variability may reduce predictability of refractive outcome.