Geeta K. Vemuganti

Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study

Purpose Ocular burns can damage the corneal epithelial stem cells located at the limbus. This study evaluated the efficacy of xeno-free autologous cell-based treatment of limbal stem cell deficiency. Methods This retrospective study included 200 patients, above 8 years of age, with clinically diagnosed unilateral total limbal stem cell deficiency due to ocular surface burns treated between 2001 and 2010. A small limbal biopsy was obtained from the unaffected eye. The limbal epithelial cells were expanded ex vivo on human amniotic membrane for 10–14 days using a xeno-free explant culture system. The resulting cultured epithelial monolayer and amniotic membrane substrate were transplanted on to the patients affected eye. Postoperative corneal surface stability, visual improvement and complications were objectively analysed. Results A completely epithelised, avascular and clinically stable corneal surface was seen in 142 of 200 (71%) eyes at a mean follow-up of 3±1.6 (range: 1–7.6) years. A two-line improvement in visual acuity, without further surgical intervention, was seen in 60.5% of eyes. All donor eyes remained healthy. Conclusions Autologous cultivated limbal epithelial transplantation using a xeno-free explant culture technique was effective in long-term restoration of corneal epithelial stability and improvement of vision in eyes with ocular surface burns.

Successful Reconstruction of Damaged Ocular Outer Surface in Humans Using Limbal and Conjuctival Stem Cell Culture Methods

When the ocular outer surface is badly damaged, subsequent corneal transplantation fails due to the absence of basal cells that are needed to support the graft. With the realization that the limbus and the conjunctiva have adult stem cells that can be cultured, it has been possible for us to explant culture these on de-epithelized human amniotic membrane, and to graft the resulting viable and transparent epithelium to 125 needy human patients with success. Ultrastructural, histological, biochemical and immunological assays establish the identity of the cells and the tissue formed.

Bilateral infectious keratitis after laser in situ keratomileusis: A case report and review of the literature

OBJECTIVE To report a case of bilateral infectious keratitis after simultaneous bilateral laser in situ keratomileusis (LASIK) and to explore appropriate preventive, diagnostic, and therapeutic measures. DESIGN Interventional case report and literature review. INTERVENTION A 22-year-old woman had bilateral corneal infiltrates after simultaneous bilateral LASIK. The same set of instruments was used for surgery on both eyes. Corneal scrapings from the edge of the infiltrate and underneath the flap were taken for microscopic examination and inoculation on culture media. Treatment consisted of irrigation of stromal bed with amikacin sulphate (2.5%) solution along with half hourly instillation of amikacin (2.5%) and cefazolin (5%) eye drops. MAIN OUTCOME MEASURES Causative organism and response to medical treatment. RESULTS Culture revealed a significant growth of Mycobacterium chelonae from the corneal scrapings of both eyes. There was progressive thinning of corneal stroma in the right eye requiring cyanoacrylate tissue adhesive application. The left eye showed progressive worsening after initial response and required penetrating keratoplasty. CONCLUSIONS The risk of bilateral sight-threatening complications must be kept in mind when contemplating bilateral simultaneous LASIK. Nontuberculous mycobacteria should be considered as an etiologic agent in cases of infectious keratitis occurring after LASIK. Microbiology work-up of a specimen collected directly from the site of lesion can help in early diagnosis and institution of appropriate therapy.

Use of Autologous Cultured Limbal and Conjunctival Epithelium in a Patient with Severe Bilateral Ocular Surface Disease Induced by Acid Injury: A Case Report of Unique Application

Purpose. Reconstruction of the ocular surface in a case of severe bilateral partial limbal stem cell deficiency (LSCD) with extensive symblephara using autologous cultured conjunctival and limbal epithelium. Case report. A 31-year-old woman presented with severe bilateral ocular surface disease with partial limbal stem cell deficiency, symblephara, lid and facial scarring, with a vision of 20/400 and counting fingers at 1 m in both eyes. Limbal and conjunctival tissue was harvested from the healthy-appearing left eye and used to generate two sheets of composite epithelium consisting of central limbal and peripheral conjunctival cells. The limbal tissues were explanted in the central region while the conjunctival tissues were explanted on the periphery of the deepithelialized human amniotic membrane (HAM) and nurtured using human corneal epithelial cell medium. After successful generation of a monolayer from both tissues had been confirmed, the composite of cultivated limbal and conjunctival epithelium with HAM was transplanted in each eye after excision of fibrous tissue and release of symblephara. One year postoperatively, the patient had a best spectacle-corrected visual acuity of 20/40 in the right eye (preoperative acuity 20/400) and counting fingers at 1 m in the left eye (same as preoperative) with a stable ocular surface. Conclusions. Autologous cultured epithelial transplantation is as an excellent option in selected patients with bilateral partial LSCD with small area(s) of healthy limbus in either eye and avoids the attendant risk of rejection and cost and potential toxicity of immunosuppression in allogeneic tissue transplantation. This case also highlights the feasibility of generating a composite culture of limbal and conjunctival epithelium using a single amniotic membrane.

Evaluation of agent and host factors in progression of mycotic keratitis ☆: A histologic and microbiologic study of 167 corneal buttons

PURPOSE To evaluate the host and agent factors in the progression of mycotic keratitis through the microbiologic evaluation and histologic study of human corneal buttons obtained at the time of therapeutic keratoplasty. DESIGN Retrospective noncomparative consecutive case series. MATERIALS One hundred sixty-seven corneal buttons from 148 patients of microbiologically diagnosed and treated cases of mycotic keratitis who underwent therapeutic keratoplasty between January 1995 and May 1998. METHODS Therapeutic penetrating keratoplasty, review of microbiologic results, histopathologic and microbiologic evaluation of the corneal buttons of mycotic keratitis MAIN OUTCOME MEASURES Histologic evaluation of the buttons for morphologic changes, degree and distribution of inflammatory cells, presence or absence of fungal filaments, and their degree and distribution within the corneal buttons. RESULTS The diagnosis of fungal infection was made on corneal scrapings in 36 cases; whereas in 131 (78%), the fungus was grown in cultures and identified as Aspergillus in 55 (42%), Fusarium in 42 (32%), unidentified hyaline fungi in 22 (17%), dematiaceous (unidentified) in 4 (3%), and others in 8 (6%). The mean interval between diagnosis and keratoplasty was 19 (+/-40) days. From the keratoplasty specimen, the fungus was identified at histologic examination in 127 of 167 (76%) buttons and grown by culture techniques in 76 of 115 (66%) buttons. The fungal species identified in the corneal button were Fusarium in 30 (39%); Aspergillus in 25 (33%); unidentified hyaline in 19 (25%), and others in 2 (3%). Fungus-positive corneal buttons had early surgery (mean, 15 days) compared with fungus-negative (39 days) corneal buttons (P = 0.0005), with 93% fungus positivity in the buttons removed within 2 weeks and 42% after 2 months. In the fungus-positive buttons, there was an inverse correlation between the degree, distribution of inflammatory cells, and fungal filaments (r = -0.255, P = 0.024; r = -0.199, P = 0.027), respectively. The factors necessitating an early keratoplasty were heavy fungal load, deeper penetration of fungus, and possibly insufficient inflammation to combat infection. A granulomatous reaction was noted in the posterior stroma and around the fragmented Descemets membrane in 23 buttons (13.8%), independent of fungal species. Inflammation was unaffected by elimination of fungus and increasing interval between diagnosis and treatment. CONCLUSIONS Rapid progression of mycotic keratitis in the early phases is by agent factors such as heavy load and deeper penetration of the fungus, insufficient inflammatory response, and possibly relative ineffectiveness of antifungal agents. Progression in the later phase of mycotic keratitis need not necessarily be agent mediated; it could be either host-modulated, species-related, or drug resistance, thereby suggesting that ideal treatment regimens should include sensitivity-based antifungal therapy aided by in vivo monitoring of fungal filaments.

In vitro culture and expansion of human limbal epithelial cells

Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes ∼2 weeks to establish a confluent monolayer from which ∼3 × 106 cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.

A biomimetic scaffold for culturing limbal stem cells: a promising alternative for clinical transplantation

Limbal tissues can be cultured on various types of scaffolds to create a sheet of limbal‐corneal epithelium for research as well as clinical transplantation. An optically clear, biocompatible, biomimetic scaffold would be an ideal replacement graft for transplanting limbal stem cells. In this study, we evaluated the physical and culture characteristics of the recombinant human cross‐linked collagen scaffold (RHC‐III scaffold) and compared it with denuded human amniotic membrane (HAM). Optical/mechanical properties and microbial susceptibility were measured for the scaffolds. With the approval of the institutional review board, 2 mm fresh human limbal tissues were cultured on 2.5 × 2.5 cm2 scaffolds in a medium containing autologous serum in a feeder cell‐free submerged system. The cultured cell systems were characterized by morphology and immunohistochemistry for putative stem cells and differentiated cell markers. The refractive index (RI) and tensile strength of the RHC‐III scaffold were comparable to human cornea, with delayed in vitro degradation compared to HAM. RHC‐III scaffolds were 10‐fold less susceptible to microbial growth. Cultures were initiated on day 1, expanded to form a monolayer by day 3 and covered the entire growth surface in 10 days. Stratified epithelium on the scaffolds was visualized by transmission electron microscopy. The cultured cells showed p63 and ABCG2 positivity in the basal layer and were immunoreactive for cytokeratin K3 and K12 in the suprabasal layers. RHC‐III scaffold supports and retains the growth and stemness of limbal stem cells, in addition to resembling human cornea; thus, it could be a good replacement scaffold for growing cells for clinical transplantation. Copyright

Autosomal recessive corneal endothelial dystrophy (CHED2) is associated with mutations in SLC4A11

Objective: To map and identify the gene for autosomal recessive congenital hereditary endothelial dystrophy (CHED2, OMIM 217700), a disorder characterised by diffuse bilateral corneal clouding that may lead to visual impairment and requiring corneal transplantation. Methods: Members of 16 families with autosomal recessive CHED were genotyped for 13 microsatellite markers at the CHED2 locus on chromosome 20p13-12. Two-point linkage analysis was carried out using the FASTLINK version of the MLINK program. Mutation screening was carried out by amplification of exons and flanking regions by polymerase chain reaction, followed by direct automated sequencing. Results: Linkage and haplotype analysis placed the disease locus within a 2.2 cM (1.3 Mb) interval flanked by D20S198 and D20S889, including SLC4A11. The maximum limit of detection score of 11.1 was obtained with D20S117 at θ = 0. Sequencing of SLC4A11 showed homozygotic mutations in affected members from 12 of 16 families. Conclusion: These results confirm that mutations in the SLC4A11 gene cause autosomal recessive CHED.

Ophthalmic Applications of Preserved Human Amniotic Membrane: A Review of Current Indications

Preserved human amniotic membrane (AM) is currently being used for a wide spectrum of ocular surface disorders. The AM has a basement membrane, which promotes epithelial cell migration and adhesion. The presence of a unique avascular stromal matrix reduces inflammation, neovascularization and fibrosis. The basic tenets of amniotic membrane transplantation (AMT) are to promote re-epithelialization, to reconstruct the ocular surface and to provide symptomatic relief from surface aberrations. AMT is a useful technique for reconstruction of surface defects resulting from removal of surface tumors and symblephara. AMT has effectively restored a stable corneal epithelium in eyes with, persistent epithelial defects and corneal ulcers. In the setting of acute ocular burns and SJS, AMT has satisfactorily reduced scarring and inflammation. AMT alone may be an effective alternative for partial limbal stem cell deficiency. However remarkable improvements in surface stability have resulted from concurrent AMT and limbal stem cell transplantation, wherein the limbal grafts are obtained from the normal fellow eye, living relative or cadaveric eye. In severe or bilateral cases, well being of the donor eye is a major concern. Currently, the most unique application of preserved human AM in ophthalmology is its use as a substrate for ex-vivo expansion of corneal and conjunctival epithelium. In this novel technique of tissue engineering, epithelial stem cells can be safely harvested and expanded on denuded AM. The resultant composite cultured tissue has been successfully transplanted to restore vision, as well as the structure and function of damaged ocular surfaces.

Cultivated corneal epithelial transplantation for severe ocular surface disease in vernal keratoconjunctivitis

Purpose: To report cultivated epithelial transplantation in 2 patients with vernal keratoconjunctivitis (VKC) with severe ocular surface disease. Methods: Two patients initially diagnosed with burnt-out VKC presented with bilateral photophobia, decreased vision, and corneal neovascularization. The first patient underwent living-related conjunctival-limbal allograft in the left eye and cultivated limbal epithelial cell allotransplant in the right. The second patient underwent unsuccessful amniotic membrane transplantation (AMT) followed by autologous cultivated limbal epithelial cell transplantation in the worse eye. Results: Both patients had onset of VKC in the first decade. Surgical intervention in both led to marked amelioration in symptoms and improvement in vision. In patient 1, vision improved from 20/800 (both eyes) to 20/30 in the right and 20/100 in the left eye at a follow-up of 34 months. In patient 2, it improved from 20/400 to 20/50 after the second procedure, 25 months postoperatively. Histopathology of the excised pannus revealed fibrosis and mononuclear cell infiltrates in all 3 eyes. Conclusions: Severe ocular surface disease may occur in persistent VKC, leading to marked visual loss. AMT alone may be insufficient to restore the ocular surface, and limbal epithelial cell transplantation is warranted.