Annie Joseph

Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche

Background/aims: There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche. Methods: Systematic serial 5–7 μm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the “stem cell” marker ABCG2 transporter protein. Results: Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2. Conclusions: A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.

Stem cell differentiation and the effects of deficiency

AbstractStem cells have several unique attributes, the key features being their potency and plasticity. They have the ability to give rise to multiple cell lineages and to transdifferentiate into totally different cell type(s) when relocated to a novel stem cell niche. Most self-renewing tissues are served by stem cells. At the ocular surface, the corneo-scleral limbus is believed to provide the niche for corneal epithelial stem cells. A large body of circumstantial evidence, both clinical and basic, supports this view. However, specific identification of limbal stem cells has proved elusive. Cytokeratin markers, vimentin, epidermal growth factor receptors, p63, and others have been used to identify epithelial cell populations at the limbus, which could harbour putative stem cells. In contrast, none of the known haematopoietic stem cell markers namely, CD34 and CD133, stain any specific subset of corneal or limbal epithelial cells. Singly or collectively, none of these markers point to any unique cell(s) that could be regarded as stem cells, supporting the notion that the corneal epithelium is served by ‘committed progenitors’ rather than by stem cells. Disease or destruction of the corneo-scleral limbus is associated with consequential events that eventually lead to visual impairment or blindness. Conjunctivalisation and vascularisation of the corneal surface and persistent or recurring epithelial defects are hallmarks of limbal deficiency.

Infliximab in the treatment of refractory posterior uveitis.

PURPOSE To determine the efficacy and safety of infliximab in the treatment of refractory posterior uveitis. DESIGN Noncomparative interventional case series. PARTICIPANTS Five patients with posterior uveitis were treated: 3 had Behçets syndrome, and 2 had idiopathic posterior uveitis. INTERVENTIONS Patients with sight-threatening uveitis refractory to other immunosuppressive agents were treated with infliximab. MAIN OUTCOME MEASURES Intraocular inflammation, by using binocular indirect ophthalmoscopy score, retinal vasculitis, and visual acuity. Adverse effects of infliximab were documented. RESULTS Within 2 weeks of the first infusion of infliximab, 4 of 5 patients showed marked improvement in vitreous haze and visual acuity. By the 6-month follow-up, the same four patients had achieved remission of posterior uveitis and had successfully withdrawn all other immunosuppressive therapy. Further infusions of infliximab were required in 3 patients. One patient developed ocular and systemic tuberculosis, which responded to antituberculous treatment. CONCLUSIONS Infliximab is effective in the treatment of sight-threatening refractory posterior uveitis. However, patients should be thoroughly screened for tuberculosis before treatment and followed up closely during and after therapy with infliximab.

A new classification of ocular surface burns

Ocular burns constitute true ocular emergencies and both thermal and chemical burns represent potentially blinding ocular injuries. Thermal burns result from accidents associated with firework explosions, steam, boiling water, or molten metal (commonly aluminium). Chemical burns may be caused by either alkaline or acidic agents. Common alkaline agents include ammonium hydroxide used in fertiliser production, sodium hydroxide (caustic soda) used for cleaning drains and pipes, and calcium hydroxide found in lime plaster and cement. Alkaline agents are particularly damaging as they have both hydrophilic and lipophilic properties, which allow them to rapidly penetrate cell membranes and enter the anterior chamber. Alkali damage results from interaction of the hydroxyl ions causing saponification of cell membranes and cell death along with disruption of the extracellular matrix. Common acidic agents causing injury include sulphuric acid found in car batteries, sulphurous acid found in some bleaches, and hydrochloric acid used in swimming pools. Acids tend to cause less damage than alkalis as many corneal proteins bind acid and act as a chemical buffer. In addition, coagulated tissue acts as a barrier to further penetration of acid. Acid binds to collagen and causes fibril shrinkage. Historically, it has been recognised that the extent of tissue damage is a prognostic indicator of recovery following ocular surface injury. Recovery of ocular surface burns depends upon the causative agent and the extent of damage to corneal, limbal, and conjunctival tissues at the time of injury. Damage to intraocular structures influences the final visual outcome. Ballen1 first suggested a classification which was later modified by Roper-Hall to provide prognostic guidelines based on the corneal appearance and the extent of limbal ischaemia.2 This classification has become the commonly used benchmark since its introduction in 1965 (Table1). View this table: Table 1 Classification of severity of ocular surface burns by Roper-Hall2 …

Impression cytology of the ocular surface

Impression cytology refers to the application of a cellulose acetate filter to the ocular surface to remove the superficial layers of the ocular surface epithelium. These cells can then be subjected to histological, immunohistological, or molecular analysis. Proper technique is essential as the number of cells sampled can vary considerably. Generally two to three layers of cells are removed in one application but deeper cells can be accessed by repeat application over the same site. Applications for impression cytology include diagnosing a wide range of ocular surface disorders, documenting sequential changes in the conjunctival and corneal surface over time, staging conjunctival squamous metaplasia, and monitoring effects of treatment. It is also a useful investigational tool for analysing ocular surface disease with immunostaining and DNA analysis. It is non-invasive, relatively easy to perform, and yields reliable information about the area sampled with minimal discomfort to the patient. Major ophthalmic centres should develop and introduce this technique into routine clinical practice. This is best achieved with a team approach including the ophthalmologist, pathologist, microbiologist, and the immunologist.

Epithelial cell characteristics of cultured human limbal explants

Aim: To determine the immunohistochemical characteristics of putative corneal epithelial stem cells remaining on limbal explants maintained in culture. Methods: Human limbal explant cultures were generated from 25 residual corneoscleral donor rims following penetrating keratoplasty. Serial sections of these explants were studied using immunohistochemical techniques with a panel of antibodies, on day 0 and 1, 2, and 3 weeks. Results: The number of epithelial cells expressing cytokeratin 19 and vimentin increased with duration in culture, while the number of cells expressing cytokeratin 3 decreased. Connexin 43 expression was lost by 1 week in culture. p63 was expressed by cells that had migrated around the explant and the number of p63 positive cells decreased with longer duration in culture. The explants were initially negative for Ki67, but the epithelial cells were positive at 1 week, and expression of Ki67 was progressively lost with increasing duration in culture. The initial uniform staining of the epithelium for epidermal growth factor receptor and α enolase remained unchanged at 3 weeks. Conclusions: There is an expansion of less differentiated (cytokeratin 3 negative and CK19/vimentin positive) epithelial cells on corneoscleral explants maintained in culture for 3 weeks. The pattern of expression of p63 noted in this study does not support the suggestion that it is a marker of limbal stem cells. The decline in p63 and Ki67 expression among the epithelial cells of the cultured explant button implies that as the epithelial sheet outgrowing from the explant button reaches confluence, the proliferative status of the cells remaining on the explant button declines. These findings are of clinical relevance as explants of limbal tissue are used in limbal stem cell transplantation. There is no information available to date on the fate of epithelial cells on such explants. This study provides some insight into this and suggests that an expansion of the stem cell pool or its progeny may occur in limbal explants.

Failure of amniotic membrane transplantation in the treatment of acute ocular burns

AIM To report the failure of amniotic membrane transplantation (AMT) for ocular surface reconstruction in patients with severe acute chemical and thermal burns. METHODS Four eyes of three patients who suffered severe chemical (n=3) and thermal (n=1) burns were studied. The aim of AMT was to prevent symblepharon formation, promote conjunctival regeneration, inhibit corneal melting by promoting epithelialisation, and to protect the ocular surface while associated lid burns were treated. AMT was used to cover the entire ocular surface of all the severely burnt and ischaemic eyes, 2–3 weeks after the injury. Where indicated, AMT was repeated by itself or in combination with other procedures in all patients. RESULTS Three of the four eyes developed symblepharon and progressive corneal melt requiring urgent tectonic keratoplasty. All four eyes had persistent epithelial defects. Less than 25% of conjunctival regeneration occurred in three eyes. Two eyes autoeviscerated, one patient underwent lid sparing exenteration for a painful blind eye and one eye became phthysical. CONCLUSIONS AMT did not help to restore the ocular surface or preserve the integrity of the eye in all our patients with severe acute burns, when used by itself or in combination with other surgical procedures. This reflects the extreme severity of the ocular burns in these patients and, in turn, draws attention to the fact that the current classification system does not adequately reflect such severity. In the current system such burns would be grouped under grade IV injuries to the eye (more than 50% limbal ischaemia). The prognosis of patients with 100% limbal ischaemia is much worse than patients with just over 50% limbal ischaemia. This inadequacy of the classification system probably also explains the difference between outcomes of management of grade IV burns (with AMT) in this series, compared with others.

The efficacy of sirolimus in the treatment of patients with refractory uveitis.

Aims: To determine the efficacy of sirolimus in the treatment of patients with severe non-infectious uveitis. Methods: Eight patients with severe non-infectious uveitis were recruited to an open study. Inclusion criteria were limited to patients whose disease was not controlled with at least two or more separate steroid sparing immunosuppressants (either because of unacceptable side effects or ineffectiveness of the drug) or who required regular doses of corticosteroids either as high dose systemic or orbital floor injections in order to control their disease. Intraocular inflammation, visual acuity, symptoms, corticosteroid burden, drug toxicity, and side effects were monitored. Results: Sirolimus therapy was effective in five of the eight patients, all of whom had their dose of corticosteroids reduced or discontinued. Treatment in three patients was considered a failure as it caused intolerable side effects and/or failed to control the uveitis. Side effects were common and were typically gastrointestinal or cutaneous in nature. The severity of symptoms was dose dependent in most cases and occurred at trough blood levels above 25 ng/ml. Conclusion: Sirolimus is an effective and potent immunosuppressive treatment in the majority of patients with non-infectious uveitis and can reduce the need for long term supplementary corticosteroid therapy. Further studies are required to establish the long term efficacy and safety of sirolimus alone or in combination with other steroid sparing immunosupressants.

Tacrolimus immunosuppression in high-risk corneal grafts.

Background: Unlike the immune privilege enjoyed by low-risk corneal grafts, high-risk corneal grafts experience rejection rates comparable to liver and kidney transplants. Systemic immunosuppression reduces the risk of rejection in high-risk corneal grafts. Methods: Systemic tacrolimus, a specific T cell inhibitor, was used at a mean daily dose of 2.5 mg to immunosuppress 43 patients undergoing high-risk corneal transplantation. Immunosuppression was continued for a period of 18–24 months after the high-risk corneal graft. Results: During a mean follow-up period of 33.7 months, clarity of the graft was maintained in 65% of patients. Eight patients experienced rejection episodes while on tacrolimus, and this led to graft failure in five patients. Conclusion: Tacrolimus is relatively safe and effective in reducing rejection and prolonging graft survival in patients with high-risk keratoplasty compared with other series where similar immunosuppression was not used.

Ocular surface epithelium induces expression of human mucosal lymphocyte antigen (HML-1) on peripheral blood lymphocytes

Background/aims: Peripheral blood CD8+ lymphocytes that home to mucosal surfaces express the human mucosal lymphocyte antigen (HML-1). At mucosal surfaces, including the ocular surface, only intraepithelial CD8+ lymphocytes express HML-1. These lymphocytes are retained in the intraepithelial compartment by virtue of the interaction between HML-1 and its natural ligand, E-cadherin, which is expressed on epithelial cells. The purpose of this study was to determine whether ocular surface epithelial cells (ocular mucosa) could induce the expression of human mucosal lymphocyte antigen on peripheral blood lymphocytes. Methods: Human corneal and conjunctival epithelial cells were co-cultured with peripheral blood lymphocytes. Both non-activated and activated lymphocytes were used in the experiments. After 7 days of incubation, lymphocytes were recovered and analysed for the antigens CD8/HML-1, CD4/HML-1, CD3/CD8, CD3/CD4, CD3/CD25, CD8/CD25, and CD4/CD25 by flowcytometry. Results: Significant statistical differences were observed in the CD8/HML-1 expression when conjunctival epithelial cells were co-cultured with non-activated and activated lymphocytes (p = 0.04 for each) and when corneal epithelial cells were co-cultured with non-activated lymphocytes (p = 0.03). Significant statistical difference in CD4/HML-1 expression was observed only when conjunctival epithelial cells were co-cultured with activated lymphocytes (p = 0.02). Conclusion: Ocular surface epithelial cells can induce the expression of human mucosal lymphocyte antigen on CD8+ (and to some extent on CD4+) lymphocytes. This may allow the retention of CD8+ and CD4+ lymphocytes within the epithelial compartment of the conjunctiva and play a part in mucosal homing of lymphocytes.