Stanislav Matuska

Limbal Stem-Cell Therapy and Long-Term Corneal Regeneration

BACKGROUND Corneal renewal and repair are mediated by stem cells of the limbus, the narrow zone between the cornea and the bulbar conjunctiva. Ocular burns may destroy the limbus, causing limbal stem-cell deficiency. We investigated the long-term clinical results of cell therapy in patients with burn-related corneal destruction associated with limbal stem-cell deficiency, a highly disabling ocular disease. METHODS We used autologous limbal stem cells cultivated on fibrin to treat 112 patients with corneal damage, most of whom had burn-dependent limbal stem-cell deficiency. Clinical results were assessed by means of Kaplan-Meier, Kruskal-Wallis, and univariate and multivariate logistic-regression analyses. We also assessed the clinical outcome according to the percentage of holoclone-forming stem cells, detected as cells that stain intensely (p63-bright cells) in the cultures. RESULTS Permanent restoration of a transparent, renewing corneal epithelium was attained in 76.6% of eyes. The failures occurred within the first year. Restored eyes remained stable over time, with up to 10 years of follow-up (mean, 2.91+/-1.99; median, 1.93). In post hoc analyses, success--that is, the generation of normal epithelium on donor stroma--was associated with the percentage of p63-bright holoclone-forming stem cells in culture. Cultures in which p63-bright cells constituted more than 3% of the total number of clonogenic cells were associated with successful transplantation in 78% of patients. In contrast, cultures in which such cells made up 3% or less of the total number of cells were associated with successful transplantation in only 11% of patients. Graft failure was also associated with the type of initial ocular damage and postoperative complications. CONCLUSIONS Cultures of limbal stem cells represent a source of cells for transplantation in the treatment of destruction of the human cornea due to burns.

Acanthamoeba keratitis with perforation after corneal crosslinking and bandage contact lens use

A 32-year-old man with keratoconus developed corneal melting 5 days after riboflavin/ultraviolet-A corneal collagen crosslinking (CXL). Corneal scraping was positive for Acanthamoeba. The patient was unaware that he was wearing a bandage contact lens and repeatedly rinsed his face and eyelids with tap water. Because of corneal perforation, a large therapeutic keratoplasty à chaud was performed. Although CXL is considered a safe procedure, this case emphasizes the potential risks. We discuss the potential effects of deepithelialization, contact lens placement, instillation of topical nonsteroidal antiinflammatory drugs and anesthetic agents, and the possible role of apoptosis when performing CXL treatment for keratoconus.

Biological parameters determining the clinical outcome of autologous cultures of limbal stem cells

AIM Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the biological parameters instrumental for their clinical success. METHODS We report a long-term multicenter prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically evaluated both by parametric and nonparametric methods. RESULTS Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have been observed and the regenerated epithelium was completely devoid of any 3T3-J2 contamination. CONCLUSION Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.

Isolation and genotyping of Acanthamoeba strains from corneal infections in Italy

Acanthamoeba keratitis (AK) is a corneal disease caused by members of a genus of free-living amoebae and is associated predominantly with contact lens (CL) use. This study reports 16 cases of culture-proven AK diagnosed in northern Italy. Genotype identification was carried out with a PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques. A 405 bp region of the 18S rRNA gene (ASA.S1) including diagnostic fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the nuclear small-subunit rRNA gene sequence excluding the highly variable DF3 region. Phylogenetic analysis was also performed on the sequences obtained. All patients complained of monolateral infection; 11 (68.75%) admitted improper CL disinfection. In 14/16 (87.5 %) subjects, corneal scrapings were stained with calcofluor white and haematoxylin and eosin and, in ten cases (62.5 %), microscopy was positive for Acanthamoeba cysts. In vitro culture on 3 % non-nutrient agar plates was obtained in all cases (100 %), whereas cloning and axenic growth were positive for 14 amoebic stocks (87.5 %). PCR analysis had 100 % sensitivity and specificity compared with in vitro axenic culture, showing positive amplification from 15 isolates. All Acanthamoeba strains belonged to the T4 genotype, the main AK-related genotype worldwide. These results confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK on biological samples. Genotyping allowed inclusion of all isolates in the T4 group, thus demonstrating the prevalence of this genotype in northern Italy.

Deep anterior lamellar keratoplasty using an original manual technique

Aims To evaluate the clinical findings and visual outcomes of deep anterior lamellar keratoplasty (DALK) using an original manual dissection technique. Methods 288 eyes (268 patients) with corneal pathologies without endothelial involvement were treated by DALK using an original manual dissection technique guided by a calibrated knife incision based on ultrasonic pachimetry values. Clinical records were examined retrospectively at 2 months, 6 months, 1 year and 2 years. The following outcomes were measured: visual acuity, topographic parameters, endothelial cell density and recipient stromal residue thickness. Results At the 2-year postoperative follow-up, the mean logarithm of the minimum angle of resolution best spectacle corrected visual acuity (BSCVA) was 0.131±0.087 and topographic astigmatism was 2.87±1.57 diopters. In 12 cases (4.2%) a perforation of Descemets membrane required conversion of the procedure to penetrating keratoplasty. Mean optical coherence tomography (OCT) residue thickness (measured in 82 eyes with OCT Visante) was 31.63±24.57 μm; lower values of recipient residue thickness were significantly associated with higher BSCVA (Spearman coefficient 0.635, p< 0.001). Conclusion DALK using a dry manual dissection technique provides visual, refractive and clinical results comparable to other deep lamellar techniques. Eyes with lower values of recipient residue thickness are associated with better visual acuity.

Severe keratitis following corneal cross‐linking for keratoconus

performed and the patient underwent proton beam radiotherapy. Two years after proton beam radiotherapy, the tumour thickness was 3.5 mm but the patient developed a recurrent vitreous haemorrhage and neovascular glaucoma requiring enucleation. Pathological examination showed a partially necrotic choroidal melanoma of epithelioid cell type, with vascular hyalinization from the radiotherapy. The mitotic count was 1 ⁄ 40 high power fields. Numerous macrophages were seen. The sclera adjacent to the tumour showed collagen degeneration and calcification (Fig. 2). Apical tumour recurrence after ruthenium plaque radiotherapy is rare, even with eccentric plaque placement (Damato et al. 2005; Lommatzsch et al. 2000; Summanen et al. 1993). This case therefore raises the possibility that treatment failure occurred because of intrascleral calcification beneath the tumour. We suggest that this finding should prompt a higher dose of brachytherapy or an alternative form of radiotherapy, such as proton beam radiotherapy.

Bilateral Acanthamoeba keratitis with late recurrence of the infection in a corneal graft: A case report

Purpose To report a case of bilateral Acanthamoeba keratitis with late, atypical recurrence after penetrating keratoplasty à chaud. Methods A 23-year-old contact lens wearer was treated for bilateral Acanthamoeba keratitis and underwent penetrating keratoplasty in the right eye for descemetocele with impending risk of perforation. The postoperative course was uneventful and topical steroids were combined with neomycin and propamidine. Two months after the operation in the right eye the patient presented with active infection in the left eye. One month later recurrence appeared in the right eye, as a central corneal infiltrate in the graft. Results Recurrences in both eyes were successfully treated with a combination of hexamidine and neomycin, and with polyhexamethylene biguanide respectively. The right eye was regrafted three months after the recurrence and penetrating keratoplasty was done two years later in the left eye. Both grafts were successful and remained clear. There has been no further recurrence in the long-term follow-up. Conclusions Recurrence of Acanthamoeba keratitis after penetrating keratoplasty à chaud may occur even several months after the operation and the manifestation may be atypical. Current antiamoebal therapy was effective and regrafting in the quiet eye was successful.

Double-Biguanide Therapy for Resistant Acanthamoeba Keratitis

Aims: To report the clinical and diagnostic findings of a patient with Acanthamoeba keratitis resistant to both polyhexamethylene biguanide (PHMB)-hexamidine and chlorhexidine-hexamidine treatment. Methods: Slit-lamp biomicroscopy, corneal cell scraping and histopathology were performed on a 39-year-old woman presenting with corneal ulcer in her left eye. Results: The patient was successfully treated with PHMB-chlorhexidine association therapy. Subsequent perforating keratoplasty remained clear at the last follow-up visit after 7 months and increased visual acuity to 20/20 with correction. Conclusions: This case emphasizes the proteiform aspects of Acanthamoeba drug resistance, and suggests that PHMB-chlorhexidine association might represent an additional option for cases resistant to standard therapy.

Corneal collagen cross-linking in paediatric patients affected by keratoconus

Background/aims To evaluate the effectiveness of corneal collagen cross-linking (CXL) in paediatric patients. Methods Fifty-two eyes of 43 paediatric patients with progressive keratoconus were enrolled in a prospective cohort study. Corneal CXL was performed using a conventional technique with instillation of 0.1% riboflavin solution containing dextran 20% for 30 min during the soaking phase and during the 30 min ultraviolet A irradiation (3 mW/cm2). Visual outcomes, topographic keratometry, maximum keratometry (K-max), refractive astigmatism, demarcation line and endothelial cell density were measured postoperatively. Results A significant decrease of K-max from 59.30±7.08 to 57.07±6.46 (p<0.001) was observed 2 years after treatment. Uncorrected visual acuity improved from 0.59±0.41 LogMAR (logarithm of the minimum angle resolution) to 0.46±0.33 LogMAR (p=0.06) 2 years after the procedure, while best spectacle corrected visual acuity improved from 0.17±0.11 LogMAR to 0.15±0.12 LogMAR (p=0.17). Twenty-five eyes had K-max values of 60 dioptres (D) or greater. In this subgroup, K-max significantly decreased from 64.94±4.99 D to 62.25±4.42 D at 2 years (p<0.001). The demarcation line of the CXL treatment had a mean value of 249±74 µm and did not show a significant correlation with K-max flattening (Spearman r=0.019, p=0.899). Endothelial cell density remained stable 2 years after the procedure, changing from 2800±363 to 2736±659 cells/mm2 (p=0.90). Conclusion CXL is an effective treatment for avoiding keratoconus progression in paediatric patients. The procedure is safe and successful in stabilising keratoconus in eyes with more advanced forms of the disease, characterised by topographic K-max values greater than 60 D.

Autologous cultivated limbal stem cell transplantation after failed previous limbal graft

Purpose To describe a case of unilateral limbal stem cell deficiency (LSCD) with previously failed autologous graft, resolved by ocular surface reconstruction using cultured autologous limbal stem cells from the contralateral eye. Case Report A 35-year-old patient presented to our clinic with LSCD due to a unilateral alkali burn. The patient had received a previous limbal graft from the contralateral eye that had failed to impede corneal conjunctivalization. We decided to repeat limbal stem cell transplantation using an ex vivo cultivation procedure to reduce the risk of tissue harvesting on the healthy fellow eye. A small limbal biopsy (1.5 × 1.5 mm) near the previously excised limbus was performed. Stem cells were then isolated and cultured on fibrin and a 3T3 feeder cell layer using a standard protocol. Four months later, the cultivated cells on fibrin were grafted after pannus removal. In the subsequent months, the ocular surface stabilized and inflammation decreased. Two years later, the patient underwent large tectonic lamellar keratoplasty for severe corneal thinning involving the entire cornea, and 6 months later central penetrating keratoplasty and extracapsular cataract extraction with intraocular lens implantation and pupilloplasty was performed. Following reconstruction, the patient showed improved best-corrected vision from count fingers to 20/200 due to amblyopia, and the ocular surface was stable with a transparent corneal graft. Conclusions Ex vivo limbal stem cell transplantation is a valid technique for treating LSCD and can be utilized for treating patients who have had previous failed limbal grafts.