Tsutomu Inatomi

Cultivated corneal epithelial stem cell transplantation in ocular surface disorders

PURPOSE To investigate the outcome of cultivated corneal epithelial transplantation for severe stem cell deficiencies using denuded amniotic membrane (AM) as a carrier. DESIGN Retrospective, noncomparative case series. PARTICIPANTS Thirteen eyes of 11 patients were studied. These consisted of five eyes with acute Stevens-Johnson syndrome (SJS), two with chronic SJS, one with an acute chemical injury, two with chronic chemical injuries, two with ocular cicatricial pemphigoid, and one with drug-induced pseudopemphigoid. All of these eyes had total stem cell deficiencies. MAIN OUTCOME MEASURES Adaptation of the cultivated corneal epithelium onto the host corneal surface was confirmed 48 hours after surgery. The reconstruction of the ocular surface and visual acuity were measured. METHODS Corneal limbal epithelium from donor corneas was cultivated for 4 weeks on a denuded AM carrier, with 3T3 fibroblast coculture and air lifting. The cultivated corneal epithelium showed four to five layers of stratification and was well differentiated. After conjunctival tissue removal from the cornea up to 3 mm outside the limbus and subconjunctival tissue treatment with 0.04% mitomycin C, cultivated allocorneal epithelium, including the AM carrier, was transplanted onto the corneal surface up to the limbus. Lamellar keratoplasty, using preserved donor graft without epithelium, was performed simultaneously for five chronic-phase patients showing corneal stromal scarring. Systemic immunosuppression was used to prevent allograft rejection. RESULTS In all 13 eyes, the entire corneal surface, on which cultivated allocorneal epithelium had been placed, was free from epithelial defects 48 hours after surgery, indicating complete survival of the transplanted corneal epithelium. Visual acuity improved in all eyes after surgery, and 10 of the 13 eyes were restored to good vision (postoperative visual acuity improved two or more lines) 6 months after the operation. During the follow-up period (mean +/- standard deviation, 11.2 +/- 1.3 months), the corneal surfaces were clear, although three eyes experienced epithelial rejection. CONCLUSIONS Cultivated corneal epithelial transplantation using denuded AM as a carrier can be used for severe stem cell deficiencies.

Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders

Background/aims: To determine outcomes of transplants of cultivated autologous oral epithelial cells in patients with severe ocular surface disorders. Methods: The eyes (n = 6) of four patients with Stevens-Johnson syndrome (three eyes) or chemical burns (three eyes) were studied. Autologous oral epithelial cells, grown for 2–3 weeks on a denuded amniotic membrane carrier in the presence of 3T3 fibroblasts, were air lifted. The resultant sheet was transplanted onto the damaged eye, and acceptance of the sheet by the corneal surface was confirmed 48 hours after surgery. The success of ocular surface reconstruction, graft survival, changes in visual acuity, and postoperative complications were assessed and the quality of the cultivated oral epithelial sheet was evaluated histologically. Results: At 48 hours after transplant, the entire corneal surface of all six eyes was free of epithelial defects indicating complete survival of the transplanted oral epithelium. Visual acuity was improved in all eyes. During follow up (mean 13.8 (SD 2.9) months), the corneal surface remained stable, although all eyes manifested mild peripheral neovascularisation. Conclusions: Autologous oral epithelial cells grown on denuded amniotic membrane can be transplanted to treat severe ocular surface disorders.

Long-term results of autologous cultivated oral mucosal epithelial transplantation in the scar phase of severe ocular surface disorders

Purpose To investigate the long-term outcome of autologous cultivated oral mucosal epithelial transplantation (COMET) for the treatment of the scar phase of severe ocular surface disorders. Participants This study involved 19 eyes of 17 patients who received autologous COMET for total limbal stem-cell deficiency. Methods Autologous cultivated oral mucosal epithelial sheets were created using amniotic membrane as a substrate. Clinical efficacy was evaluated by best-corrected visual acuity and visual acuity at the postoperative 36th month. The clinical results (clinical conjunctivalisation, corneal opacification, corneal neovascularisation and symblepharon formation) were evaluated and graded on a scale from 0 to 3 according to their severity. Clinical safety was evaluated by the presence of persistent epithelial defects, ocular hypertension and infections. Results Autologous cultivated oral mucosal epithelial sheets were successfully generated for all 17 patients. All patients were followed up for more than 36 months; the mean follow-up period was 55 months and the longest follow-up period was 90 months. During the long-term follow-up period, postoperative conjunctivalisation and symblepharon were significantly inhibited. All eyes manifested various degrees of postoperative corneal neovascularisation, but it gradually abated and its activity was stable at 6 months after surgery. Best-corrected visual acuity was improved in 18 eyes (95%) during the follow-up periods, and visual acuity at the postoperative 36th month was improved in 10 eyes (53%). Conclusions These long-term clinical results strongly support the conclusion that tissue-engineered cultivated oral mucosal epithelial sheets are useful in reconstructing the ocular surface of the scar phase of severe ocular surface disorders.

Diagnosis and Treatment of Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis with Ocular Complications

PURPOSE To present a detailed clarification of the symptoms at disease onset of Stevens-Johnson syndrome (SJS) and its more severe variant, toxic epidermal necrolysis (TEN), with ocular complications and to clarify the relationship between topical steroid use and visual prognosis. DESIGN Cross-sectional study. PARTICIPANTS Ninety-four patients with SJS and TEN with ocular complications. METHODS A structured interview, examination of the patient medical records, or both addressing clinical manifestations at disease onset were conducted for 94 patients seen at Kyoto Prefectural University of Medicine. Any topical steroid use during the first week at the acute stage also was investigated. MAIN OUTCOME MEASURES The incidence and the details of prodromal symptoms and the mucosal involvements and the relationship between topical steroid use and visual outcomes. RESULTS Common cold-like symptoms (general malaise, fever, sore throat, etc.) preceded skin eruptions in 75 cases, and extremely high fever accompanied disease onset in 86 cases. Acute conjunctivitis and oral and nail involvements were reported in all patients who remembered the details. Acute conjunctivitis occurred before the skin eruptions in 42 patients and simultaneously in 21 patients, whereas only 1 patient reported posteruption conjunctivitis. Visual outcomes were significantly better in the group receiving topical steroids compared with those of the no-treatment group (P<0.00001). CONCLUSIONS Acute conjunctivitis occurring before or simultaneously with skin eruptions accompanied by extremely high fever and oral and nail involvement indicate the initiation of SJS or TEN. Topical steroid treatment from disease onset seems to be important for the improvement of visual prognosis.

Toll-like receptor 3 gene polymorphisms in Japanese patients with Stevens–Johnson syndrome

Background and aim: Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute-onset mucocutaneous diseases induced by infectious agents and/or inciting drugs. Given the association between the onset of SJS/TEN and infections, the possibility that there is an association between SJS/TEN and a disordered innate immune response was considered. The first line of defence against infection is comprised of evolutionarily conserved sets of molecules, the Toll–like receptors (TLRs). TLR3 recognises double-stranded RNA associated with viral infections. Methods: The Japanese single-nucleotide-polymorphism (JSNP) database reports 7 polymorphisms consisting of 7 SNPs in the human TLR3 gene; 3 of the 7 SNPs are coded in exon regions, (ie, 293248A/G, 293391A/G and 299698T/G), and the other 4 are coded in intron regions, (ie, 294440G/C, 294732C/T, 208036T/C and 298054C/T). These 7 SNPs were analysed in 57 Japanese patients with SJS/TEN with ocular surface complications and in 160 Japanese healthy controls. Results: SNP 299698T/G and the genotype patterns of 293248A/A and 299698T/T were strongly associated with SJS/TEN. Conclusion: The results suggest that polymorphisms in the TLR3 gene could be associated with SJS/TEN in the Japanese population.

Successful Treatment of Stevens-Johnson Syndrome with Steroid Pulse Therapy at Disease Onset

PURPOSE To evaluate the visual prognosis of patients with Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), followed by general and topical high-dose corticosteroids administration from disease onset. DESIGN Prospective, observational case series. METHODS Between May 1, 2003 and June 30, 2005, we enrolled 5 patients with SJS or TEN with ocular complications at the acute stage. Intravenous pulse therapy with methylprednisolone (steroid pulse therapy; 500 or 1000 mg/day for 3 to 4 days) was initiated within 4 days from disease onset. Topically, 0.1% betamethasone was applied over 5 times daily for at least 2 weeks. Visual acuity (VA) and slit-lamp microscopic appearance 1 year from disease onset were evaluated. RESULTS At the first examination, corneal or conjunctival epithelial defects and pseudomembranous conjunctivitis were present in all cases. Skin eruptions dramatically improved after steroid pulse therapy. Although ocular inflammation increased for several days, pseudomembranes disappeared and corneal and conjunctival epithelium regenerated within 6 weeks. At the chronic stage, all eyes had clear corneas with the palisades of Vogt (POV), implying the presence of corneal epithelial stem cells. Best-corrected VA was 20/20 or better in all eyes. Five eyes showed superficial punctate keratopathy. No eye had cicatricial changes except for 1 with slight fornix shortening. No significant adverse effects of steroid occurred during all clinical courses. CONCLUSIONS Steroid pulse therapy at disease onset is of great therapeutic importance in preventing ocular complications. Topical betamethasone also shows great promise for preventing corneal epithelial stem cell loss in the limbal region and cicatricial changes.

Visual Improvement after Cultivated Oral Mucosal Epithelial Transplantation

PURPOSE To report the effectiveness, disease-specific outcomes, and safety of cultivated oral mucosal epithelial sheet transplantation (COMET), with the primary objective of visual improvement. DESIGN Noncomparative, retrospective, interventional case series. PARTICIPANTS This study involved 46 eyes in 40 patients with complete limbal stem cell deficiency (LSCD) who underwent COMET for visual improvement. These LSCD disorders fell into the following 4 categories: Stevens-Johnson syndrome (SJS; 21 eyes), ocular cicatricial pemphigoid (OCP; 10 eyes), thermal or chemical injury (7 eyes), or other diseases (8 eyes). METHODS Best-corrected visual acuity (BCVA) and ocular surface grading score were examined before surgery; at the 4th, 12th, and 24th postoperative week; and at the last follow-up. Data on COMET-related adverse events and postoperative management were collected. The outcomes in each disease category were evaluated separately. MAIN OUTCOME MEASURES The primary outcome was the change in median logarithm of the minimum angle of resolution (logMAR) BCVA at the 24th postoperative week. The secondary outcome was the ocular surface grading score. RESULTS Median logMAR BCVA at baseline was 2.40 (range, 1.10 to 3.00). In SJS, logMAR BCVA improved significantly during the 24 weeks after surgery. In contrast, the BCVA in OCP was improved significantly only at the 4th postoperative week. In 6 of the 7 thermal or chemical injury cases, logMAR BCVA improved after planned penetrating keratoplasty or deep lamellar keratoplasty. Grading scores of ocular surface abnormalities improved in all categories. Of 31 patients with vision loss (logMAR BCVA, >2) at baseline, COMET produced improvement (logMAR BCVA, ≤2) in 15 patients (48%). Visual improvement was maintained with long-term follow-up (median, 28.7 months). Multivariate stepwise logistic regression analysis showed that corneal neovascularization and symblepharon were correlated significantly with logMAR BCVA improvement at the 24th postoperative week (P=0.0023 and P=0.0173, respectively). Although postoperative persistent epithelial defects and slight to moderate corneal infection occurred in the eyes of 16 and 2 patients, respectively, all were treated successfully with no eye perforation. CONCLUSIONS Long-term visual improvement was achievable in cases of complete LSCD. Cultivated oral mucosal epithelial sheet transplantation offered substantial visual improvement even for patients with end-stage severe ocular surface disorders accompanying severe tear deficiency. Patients with corneal blindness such as SJS benefited from critical improvement of visual acuity. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in anymaterials discussed in this article.

Methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis infections in the cornea.

Purpose To describe the incidence and clinical management of corneal infections with methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant Staphylococcus epidermidis (MRSE). Methods The incidence of methicillin-resistant Stuphylococcus (MRS) at the Department of Ophthalmology, Kyoto Prefectural University of Medicine, was reviewed during the 5-year period from January 1996 to December 2000. Clinical aspects of MRS colonization or infection in the eye were investigated. Results Methicillin-resistant S. aureus or MRSE was detected from 30 eyes with ocular diseases; post-keratoplasty (11 eyes), ocular surface disorders without operation (9 eyes), and others (10 eyes). Among the 30 eyes, 12 manifested keratitis. Eight cases (8 eyes) occurred after keratoplasty, including four postoperative cases in patients with Stevens-Johnson syndrome, and two bilateral cases (4 eyes) in patients with acute-phase Stevens-Johnson syndrome. The degree of MRS keratitis was classified into 4 groups: asymptomatic carrier or conjunctivitis, intraepithelial infiltrations, superficial keratitis, and severe keratitis leading to corneal perforation. All cases of keratitis were treated successfully with topical ofloxacin (OFLX), vancomycin (VCM), or arbekacin (ABK). Conclusion Factors associated with ocular MRS colonization were long-term use of antibiotics and/or steroids, and hospitalization. Patients who had undergone keratoplasty or who had Stevens-Johnson syndrome were at increased risk of MRS keratitis. Superficial stromal infiltrations, minimal melting, and minimal stromal scarring are characteristic of MRS keratitis. Therapy for MRS keratitis is summarized. Ofloxacin, VCM, and ABK are effective in the treatment of MRS keratitis. Vancomycin eye ointment is effective as the final choice in serious cases.

The Role of Interleukin-33 in Chronic Allergic Conjunctivitis

PURPOSE The authors discovered a genetic association between the ST2L gene and atopy. The ST2L gene encodes a membrane-bound functional marker for Th2 cells. Recently, a novel Th2 cytokine, interleukin-33 (IL-33), was discovered to be a specific ligand for ST2L. The authors investigated the role of IL-33 in chronic allergic conjunctivitis. METHODS Immunohistochemical analysis was carried out using giant papillae samples obtained from patients with atopic keratoconjunctivitis. The authors used proinflammatory stimuli to clarify IL-33 mRNA/protein-inducing signals with cultured human conjunctival epithelial cells, fibroblasts, human umbilical vascular endothelial cells, and mast cells. These cells were also used to examine the expression of ST2L (IL-33R). Finally, cultured mast cells were stimulated with recombinant IL-33 (rIL-33) to examine the downstream signals. RESULTS The authors found IL-33 protein expression in human vascular endothelial cells in the giant papillae and in the control conjunctivae. IL-33 expression was also observed in conjunctival epithelium of the giant papillae but not in the control conjunctivae. IL-1 beta stimulation upregulated IL-33 mRNA expression in conjunctival fibroblasts. The authors also confirmed mature IL-33 protein expression in ocular resident cells by Western blot analysis. Preferential ST2L expression was observed in human mast cells, and phosphorylation of p38 MAPK and IL-13 mRNA induction was observed in human cultured mast cells after rIL-33 stimulation. Phosphorylation of p38 MAPK was inhibited by soluble ST2 protein. CONCLUSIONS The IL-33-ST2 signaling cascade plays some roles in the pathophysiology of chronic allergic conjunctivitis through the activation of mast cells.

Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency.

PURPOSE To determine the feasibility of cultivated conjunctiva as a viable epithelial sheet for transplantation and corneal resurfacing in eyes with limbal stem cell deficiency (LSCD). METHODS Human corneal epithelial (HCE) and human conjunctival epithelial (HCjE) cells were cultivated on human amniotic membrane (AM) to confluence and then air lifted to allow further stratification and differentiation. Denuded AM and cultivated HCE and cultivated HCjE cells were then transplanted into 18 eyes of rabbits with induced LSCD. The cultivated and engrafted epithelia were examined by transmission electron microscopy (TEM) and immunohistochemistry. Two weeks after transplantation, the eyes were examined by slit lamp biomicroscopy and scored on epithelial integrity, corneal haze, and corneal neovascularization. RESULTS Both cultivated and engrafted HCjE sheets demonstrated confluent epithelial sheets with five to six layers of well-stratified epithelium. TEM examination of engrafted HCjE revealed numerous microvilli, desmosomes, and hemidesmosomes, identical with in vivo corneal epithelium. Immunohistochemical analysis of both HCjE and HCE cells showed the presence of CK3, CK4, and CK12, with absence of Muc5AC. Clinical outcomes for eyes receiving HCjE transplants and HCE transplants were comparable, with most having transparent, smooth corneas, free of epithelial defects. CONCLUSIONS The study showed that microscopically, HCjE cells have features similar to HCE cells, with clinically equivalent outcomes. The ex vivo cultivation of conjunctiva to form transplantable epithelial sheets for corneal replacement is a promising new treatment modality in patients with LSCD.