Francisco C. Figueiredo

Amniotic membrane transplantation for acute chemical or thermal burns

PURPOSE To determine whether preserved human amniotic membrane (AM) can be used to treat ocular burns in the acute stage. DESIGN Prospective, noncomparative, interventional case series. PARTICIPANTS Thirteen eyes from 11 patients with acute burns, 10 eyes with chemical burns and 3 with thermal burns of grades II-III (7 eyes) and grade IV (6 eyes), treated at 7 different facilities. METHODS Patients received amniotic membrane transplantation (AMT) within 2 weeks after the injury. MAIN OUTCOME MEASURES Integrity of ocular surface epithelium and visual acuity during 9 months of follow-up. RESULTS Ten patients were male and one patient was female; most were young (38.2 +/- 10.6 years). For a follow-up of 8.8 + 4.7 months, 11 of 13 eyes (84.63%) showed epithelialization within 2 to 5 weeks (23.7 +/- 9.8 days), and final visual acuity improved > or = 6 lines (6 eyes), 4 to 5 lines (2 eyes), and 1 to 3 lines (2 eyes); only one eye experienced a symblepharon. Eyes with burns of grade II to III showed more visual improvement (7.3 +/- 3 lines) than those with burns of grade IV (2.3 +/- 3.0 lines; P < 0.05, unpaired t test). In the group with grade II or III burns, none had limbal stem cell deficiency. All eyes in the group with grade IV burns did experience limbal stem cell deficiency. CONCLUSIONS Amniotic membrane transplantation is effective in promoting re-epithelialization and reducing inflammation, thus preventing scarring sequelae in the late stage. In mild to moderate burns, AMT alone rapidly restores both corneal and conjunctival surfaces. In severe burns, however, it restores the conjunctival ocular surface without debilitating symblepharon and reduces limbal stromal inflammation, but does not prevent limbal stem cell deficiency, which requires further limbal stem cell transplantation. These results underscore the importance of immediate intervention in the acute stage of eyes with severely damaged ocular surface. Further prospective randomized studies including a control group are required to determine the effectiveness of AMT in acute chemical and thermal burns of the eye.

13 years of cultured limbal epithelial cell therapy: A review of the outcomes

The cornea is the clear tissue at the front of the eye which enables the transmission of light to the retina for normal vision. The surface of the cornea is composed of an epithelium which is renewed by stem cells located at the periphery of the cornea, a region known as the limbus. These limbal stem cells can become deficient as a result of various diseases of the eyes surface, resulting in the blinding disease of limbal stem cell deficiency. The treatment of this disease is often difficult and complex. In 1997, it was proposed that a small amount of limbal tissue containing limbal stem cells could be culture expanded and then transplanted. Since then various case reports and case series have been reported showing promising results. Here, we review the outcomes of this procedure over the past 13 years with the aim of highlighting the best culture and surgical techniques to date. J. Cell. Biochem. 112: 993–1002, 2011.

Correlations between commonly used objective signs and symptoms for the diagnosis of dry eye disease: clinical implications

Purpose:  To evaluate the relationship between signs and symptoms of dry eye disease (DED) in a clinic‐based population.

Differentiation of Human Embryonic Stem Cells into Corneal Epithelial‐Like Cells by In Vitro Replication of the Corneal Epithelial Stem Cell Niche

Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiating into any cell type of the body. It has long been known that the adult stem cell niche is vital for the maintenance of adult stem cells. The cornea at the front of the eye is covered by a stratified epithelium that is renewed by stem cells located at its periphery in a region known as the limbus. These so‐called limbal stem cells are maintained by factors within the limbal microenvironment, including collagen IV in basement membrane and limbal fibroblasts in the stroma. Because this niche is very specific to the stem cells (rather than to the more differentiated cells) of the corneal epithelium, it was hypothesized that replication of these factors in vitro would result in hESC differentiation into corneal epithelial‐like cells. Indeed, here we show that culturing of hESC on collagen IV using medium conditioned by the limbal fibroblasts results in the loss of pluripotency and differentiation into epithelial‐like cells. Further differentiation results in the formation of terminally differentiated epithelial‐like cells not only of the cornea but also of skin. Scanning electron microscopy shows that some differences exist between hESC‐derived and adult limbal epithelial‐like cells, necessitating further investigation using in vivo animal models of limbal stem cell deficiency. Such a model of hESC differentiation is useful for understanding the early events of epithelial lineage specification and to the eventual potential application of epithelium differentiated from hESC for clinical conditions of epithelial stem cell loss.

Successful Clinical Implementation of Corneal Epithelial Stem Cell Therapy for Treatment of Unilateral Limbal Stem Cell Deficiency

The corneal epithelium is maintained by a population of stem cells known as limbal stem cells (LSCs) due to their location in the basal layer of the outer border of the cornea known as the limbus. Treatment of limbal stem cell deficiency (LSCD) has been achieved with transplantation of ex vivo expanded LSCs taken from a small biopsy of limbus. This is a relatively new technique, and as such, specific national or international guidance has yet to be established. Because of the lack of such specific guidance, our group has sought to minimize any risk to the patient by adopting certain modifications to the research methodologies in use at present. These include the replacement of all non‐human animal products from the culture system and the production of all reagents and cultures under Good Manufacturing Practice conditions. In addition, for the first time, a strictly defined uniform group of patients with total unilateral LSCD and no other significant ocular conditions has been used to allow the success or failure of treating LSCD to be attributable directly to the proposed stem cell therapy. A prospectively designed study with strict inclusion and exclusion criteria was used to enroll patients from our database of patients with unilateral LSCD. Eight eyes of eight consecutive patients with unilateral total LSCD treated with ex vivo expanded autologous LSC transplant on human amniotic membrane (HAM) with a mean follow‐up of 19 (RANGE) months were included in the study. Postoperatively, satisfactory ocular surface reconstruction with a stable corneal epithelium was obtained in all eyes (100%). At last examination, best corrected visual acuity improved in five eyes and remained unchanged in three eyes. Vision impairment and pain scores improved in all patients (p < .05). This study demonstrates that transplantation of autologous limbal epithelial stem cells cultured on HAM without the use of non‐human animal cells or products is a safe and effective method of reconstructing the corneal surface and restoring useful vision in patients with unilateral total LSCD. STEM CELLS 2010;28:597–610

LOSS OF CORNEAL EPITHELIAL STEM CELL PROPERTIES IN OUTGROWTHS FROM HUMAN LIMBAL EXPLANTS CULTURED ON INTACT AMNIOTIC MEMBRANE

BACKGROUND The corneal epithelium is renewed by stem cells located at the limbus, the so-called limbal stem cells (LSCs). Absence, damage or loss of the LSC population leads to the painful and blinding condition of LSC deficiency (LSCD). Ex vivo expansion of LSCs is an increasingly well recognized treatment modality for LSCD. One method of ex vivo expansion of LSCs involves the culture of limbal explants on amniotic membrane (AM). The purpose of this study was to analyze the outgrowths from human cadaveric limbal explants cultured on AM for properties associated with LSCs. In particular, the expression of putative stem cell markers and the colony-forming efficiency of the different zones of the outgrowth were studied. METHODS The limbal explants were expanded in the standard way used for clinical transplantation and the outgrowths were divided into three zones depending on proximity to the explant (inner, middle and outer zones). The colony-forming efficiencies (CFEs) of cells from each zone were calculated. In addition, the expression of DeltaNp63, ABCG2 (both putative positive LSC markers) and cytokeratin K3 (marker of corneal differentiation) were assessed using quantitative reverse transcription PCR (RT-PCR). Immunohistochemistry on paraffin-embedded sections was also performed to demonstrate protein localization and allow further confirmation of the quantitative RT-PCR results. RESULTS Successful cultures for both the explant outgrowths and the CFE calculations were obtained in every case. CFE showed a successive decline in zones further away from the explant (p < 0.00005). Quantitative RT-PCR revealed that the expression of the positive putative LSC markers DeltaNp63 and ABCG2 also showed a steady decrease in the zones furthest from the explant (p < 0.05 and p < 0.005, respectively). The expression of cytokeratin K3 was increased in zones furthest from the explant (p < 0.005). Immunohistochemistry on paraffin-embedded sections of intact ex vivo-expanded limbal epithelium for the putative positive marker p63 and cytokeratin K3 confirmed the findings of the quantitative RT-PCR and CFE results. CONCLUSIONS We demonstrate for the first time that outgrowths from human limbal explants, a widely used technique in ex vivo expansion of LSCs for clinical transplantation, show a steady decline in a wide range of stem cell properties with distance from the central explant. These findings support the importance of proximity of stem cells to their niche environment in maintaining their undifferentiated state. These findings suggest the need for modifications of existing techniques to ensure maximum numbers of LSCs following ex vivo expansion protocols, which will then ensure the success of subsequent engraftment.

Opinions on risk factors and management of corneal graft rejection in the United kingdom.

Purpose: To determine the opinions regarding risk factors and practice preferences for corneal graft rejection by members of the Bowman Club (UK) and to compare them with those of members of the Castroviejo Society (USA). Methods: A questionnaire was sent in 1999 to members of the Bowman Club (UK), who were responsible for two thirds of all corneal grafts undertaken annually. The survey included 8 questions identical to those given to members of the Castroviejo Society (USA) in a survey carried out in 1989. Results: Thirty-six out of 40 surgeons replied. Factors considered by respondents to be high risk for corneal graft rejection were previous corneal graft rejection in the operated eye (97%), significant corneal vessels (97%), and previous herpetic eye disease (94%). The preferred routine preoperative treatment in “high-risk” patients included no treatment (47%), topical corticosteroids (33%), and oral prednisolone (22%). In postoperative “high-risk” patients, 100% of surgeons used topical and 42% used oral corticosteroids. Immune suppression agents were used by 44% of respondents, the majority (92%) using cyclosporine A. In previous herpes simplex patients, 47% of surgeons used oral and 14% used topical antivirals preoperatively, whereas 75% used oral and 47% used topical postoperatively. Conclusion: This study documents the perceived risk factors and management of corneal graft rejection by corneal surgeons in the UK. It showed wide variation in practice preferences, allowing individual surgeons a comparison with peer practice. It highlights the need for greater use of postoperative antiviral prophylaxis in the presence of previous herpetic corneal pathology.

Successful Application of Ex Vivo Expanded Human Autologous Oral Mucosal Epithelium for the Treatment of Total Bilateral Limbal Stem Cell Deficiency

Ocular surface reconstruction with ex vivo expanded limbal stem cells (LSCs) is a widely used clinical treatment for patients with limbal stem cell deficiency (LSCD). This is not applicable to patients with bilateral LSCD where there are no remaining LSCs. Cultivated oral mucosa epithelium (OME) has been used as an alternative source of autologous epithelial stem cells for ocular reconstruction in few clinical trials. However, successful generation of stratified OME epithelium has only been achieved in the presence of animal feeder cells and/or animal‐derived products in the culture media, likely to contribute to increased risk of pathogen transmission and graft rejection. In this study, we report generation of multilayered OME epithelium that shares many of the characteristics of corneal epithelium using a fully compliant good manufacturing practice, feeder‐ and animal product‐free method. Proof of concept was achieved by transplantation of autologous ex vivo expanded OME in two patients with histologically confirmed bilateral total LSCD that resulted in successful reversal of LSCD in the treated eye up to 24 months. Stem Cells 2014;32:2135–2146

Bacterial susceptibility to topical antimicrobials and clinical outcome in bacterial keratitis.

PURPOSE To investigate the relationship between the susceptibility of bacteria to topical antimicrobials and clinical outcome in microbial keratitis. METHODS Clinical outcome data were collected from patients with microbial keratitis from whom a bacterium had been isolated during the period 2003 to 2006. The minimum inhibitory concentration (MIC) was determined for the isolates against 10 antimicrobials. The determinants of the primary clinical outcome, the ratio of healing time (closure of epithelial defect) to ulcer size (HT/UA), was analyzed in a general linear model. RESULTS Complete clinical outcome and MIC data were available for 421 patients. Sixteen (4%) patients required enucleation and 23 (5%) surgical treatment; in 382 (91%) the ulcer healed with intensive topical antimicrobial therapy. There were significant correlations between HT/UA and organism type (P = 0.001), nearest distance of the ulcer to the limbus (0.02), and MIC of the first antimicrobial used or lowest MIC of combined therapy (P = 0.006). In a model including patients who received monotherapy with a fluoroquinolone who had no subsequent change in their treatment and whose ulcers healed without surgical intervention, there were significant linear associations between clinical outcome and MIC for Pseudomonas spp. (P = 0.047), Staphylococcus aureus (P = 0.04), and Enterobacteriaceae (P = 0.045), but not for Streptococcus spp. (P = 0.85) and coagulase-negative staphylococci (CNS) (P = 0.88). CONCLUSION With fluoroquinolone monotherapy, there was significant association between the MIC of the antimicrobial prescribed and the clinical outcome with all bacteria except CNS and Streptococcus spp. The approach used in this study, if used prospectively, could allow topical breakpoint susceptibility concentrations to be determined for individual antimicrobial and bacterial combinations.

Stem cell therapies for ocular surface disease.

Transparency of the cornea on the front surface of the eye is essential for vision. A variety of blinding ocular surface diseases involve the cornea. This review focusses on vision loss caused by disruption of the integrity and function of the outermost corneal layer (the epithelium) and the stem-cell-based therapeutic strategies in use and under development to restore sight in affected patients.