Post-acute phase and sequelae management of epidermal necrolysis: an international, multidisciplinary DELPHI-based consensus.

BACKGROUND: Long-term sequelae are frequent and often disabling after epidermal necrolysis (Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN)). However, consensus on the modalities of management of these sequelae is lacking. OBJECTIVES: We conducted an international multicentric DELPHI exercise to establish a multidisciplinary expert consensus to standardize recommendations regarding management of SJS/TEN sequelae. METHODS: Participants were sent a survey via the online tool "Survey Monkey" consisting of 54 statements organized into 8 topics: general recommendations, professionals involved, skin, oral mucosa and teeth, eyes, genital area, mental health, and allergy workup. Participants evaluated the level of appropriateness of each statement on a scale of 1 (extremely inappropriate) to 9 (extremely appropriate). Results were analyzed according to the RAND/UCLA Appropriateness Method. RESULTS: Fifty-two healthcare professionals participated. After the first round, a consensus was obtained for 100% of 54 initially proposed statements (disagreement index < 1). Among them, 50 statements were agreed upon as 'appropriate'; four statements were considered 'uncertain', and ultimately finally discarded. CONCLUSIONS: Our DELPHI-based expert consensus should help guide physicians in conducting a prolonged multidisciplinary follow-up of sequelae in SJS-TEN.

Mucocutaneous inflammation in the Ikaros Family Zinc Finger 1-keratin 5-specific transgenic mice.

BACKGROUND: Our genomewide association study documented an association between cold medicine-related Stevens-Johnson syndrome/toxic epidermal necrolysis (CM-SJS/TEN) and Ikaros Family Zinc Finger 1 (IKZF1). Few studies examined biological and pathological functions of IKZF1 in mucosal immunity. We hypothesized that IKZF1 contributes to the mucocutaneous inflammation. METHODS: Human skin and conjunctival tissues were obtained for immunohistological studies. Primary human conjunctival epithelial cells (PHCjECs) and adult human epidermal keratinocytes (HEKa) also used for gene expression analysis. We also generated K5-Ikzf1-EGFP transgenic mice (Ikzf1 Tg) by introducing the Ik1 isoform into cells expressing keratin 5, which is expressed in epithelial tissues such as the epidermis and conjunctiva, and then examined them histologically and investigated gene expression of the epidermis. Moreover, Ikzf1 Tg were induced allergic contact dermatitis. RESULTS: We found that human epidermis and conjunctival epithelium expressed IKZF1, and in PHCjECs and HEKa, the expression of IKZF1 mRNA was upregulated by stimulation with polyI:C, a TLR3 ligand. In Ikzf1 Tg, we observed dermatitis and mucosal inflammation including the ocular surface. In contact dermatitis model, inflammatory infiltrates in the skin of Ikzf1 Tg were significantly increased compared with wild type. Microarray analysis showed that Lcn2, Adh7, Epgn, Ifi202b, Cdo1, Gpr37, Duoxa1, Tnfrsf4, and Enpp5 genes were significantly upregulated in the epidermis of Ikzf1 Tg compared with wild type. CONCLUSION: Our findings support the hypothesis that Ikaros might participate in mucocutaneous inflammation.

A whole-genome association study of major determinants for allopurinol-related Stevens-Johnson syndrome and toxic epidermal necrolysis in Japanese patients.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are severe, cutaneous adverse drug reactions that are rare but life threatening. Genetic biomarkers for allopurinol-related SJS/TEN in Japanese were examined in a genome-wide association study in which Japanese patients (n=14) were compared with ethnically matched healthy controls (n=991). Associations between 890 321 single nucleotide polymorphisms and allopurinol-related SJS/TEN were analyzed by the Fisher's exact test (dominant genotype mode). A total of 21 polymorphisms on chromosome 6 were significantly associated with allopurinol-related SJS/TEN. The strongest association was found at rs2734583 in BAT1, rs3094011 in HCP5 and GA005234 in MICC (P=2.44 × 10(-8); odds ratio=66.8; 95% confidence interval, 19.8-225.0). rs9263726 in PSORS1C1, also significantly associated with allopurinol-related SJS/TEN, is in absolute linkage disequilibrium with human leukocyte antigen-B*5801, which is in strong association with allopurinol-induced SJS/TEN. The ease of typing rs9263726 makes it a useful biomarker for allopurinol-related SJS/TEN in Japanese.

Identification of a novel HLA-B allele, HLA-B*5904.

The new human leukocyte antigen (HLA) class I allele, HLA-B*5904 was identified in Japanese individual. HLA-B*5904 differs from HLA-B*5901 by two non-synonymous nucleotide exchanges at codon 163 (ACG to CTG).

Current progress and challenges in ocular surface reconstruction using cultivated epithelial sheet transplantation.

The cultivated epithelial transplantation is a new surgical modality for treating a variety of severe ocular surface disorders. This type of tissue-engineered epithelial sheet provides a rapid epithelial coverage on the corneal surface that reduces inflammation and postoperative complications. Although cultivated corneal epithelial transplantation is an effective surgical strategy, autologous transplantation is limited to unilateral cases. Autologous cultivated oral mucosal epithelial transplantation (COMET) enables surgeons to reconstruct the ocular surface using autologous, non-ocular surface cells, and has opened a new pathway for treating severe, bilateral ocular surface disorders.

Association of Fas Ligand gene polymorphism with Stevens-Johnson syndrome.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute severe blistering diseases of the skin and also two of the most devastating ocular surface diseases leading to corneal damage and loss of vision. The extreme rarity of cutaneous and ocular surface reactions to drug therapies led us to suspect individual susceptibility. SJS/TEN patients in the acute stage were reported to manifest increased serum levels of Fas Ligand (FasL). Thus, we performed SNP association analysis of the FasL gene. METHODS: In 76 Japanese SJS/TEN patients with ocular surface complications and 160 Japanese healthy controls, we examined four SNPs of FasL reported in the Japanese Single Nucleotide Polymorphisms (JSNP) database by sequencing. RESULTS: The SNP rs.3830150 A/G showed a significant strong inverse association with SJS/TEN. Analysis of the genotype pattern of SNPs rs.3830150 and rs.2639614 (rs.3830150 A/A-rs.2639614 G/G) also manifested a strong inverse association with SJS/TEN. CONCLUSION: FasL gene polymorphisms might be associated with SJS/TEN.

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.

Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction.

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo-reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vbeta6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vbeta6-) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vbeta6+ T cells was then determined by FACS analysis, and the division profiles of responder Vbeta6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo-reactive T cells in MLR culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vbeta6+ T cells was significantly reduced in the AM-treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vbeta6+ T cells in cultures with AM than without. In addition, allo-rective T cell synthesis of both Th1 (IL-2 and IFNgamma) and Th2 (IL-6 and IL-10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo-reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT-AMT combination.

Interleukin-6 (IL-6) production by cytokine-stimulated human Müller cells.

PURPOSE: To investigate whether or not human Müller cells synthesize interleukin (IL)-6. METHODS: Using RT-PCR, we first confirmed whether cultured human Müller cells express IL-6 mRNA. Then, to determine Müller cell IL-6 production after stimulation, cultured Müller cells were exposed to various concentrations of IL-1beta (0.2 ng/ml, 2 ng/ml, 20 ng/ml) or lipopolysaccharide (LPS) (0.001 microg/ml, 0.1 microg/ml, 10 microg/ml) in 24 hr assays. In addition, to determine Müller cell time-dependent induction of IL-6 production, cultured Müller cells were exposed to IL-1beta (0.02 ng/ml, 2 ng/ml) or LPS (10 microg/ml) for 6, 12, 24, 36 hr. IL-6 production in supernatants was quantified by ELISA: RESULTS: IL-6 mRNA was expressed in cultured human Müller cells, which produced IL-6 after stimulation with either IL-1beta or LPS for 24 hours. IL-1beta was a significantly more potent stimulator of IL-6 production than was LPS. Exposure of cultured human Müller cells to either IL-1beta or LPS stimulated IL-6 production in a time-dependent fashion. CONCLUSIONS: Our findings indicate that human Müller cells can produce IL-6 when stimulated by IL-1beta or LPS. Müller cell IL-6 production may have an important role in various conditions involving ocular inflammation

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.

Characteristics of the human ocular surface epithelium.

An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, keratin 3, keratin 12, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, ssig-h3, cathepsin L2 (cathepsin V), uroplakin Ib, and Ca(2+)-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmann's dystrophy, gelatinous drop-like corneal dystrophy, and the ssig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, growth factors and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main factors being epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor-ss (TGF-ss), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology-an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.

Expression of sex steroid hormone receptors in human cornea.

PURPOSE: Previously we reported the occurrence of estrogen receptor alpha (ER beta), estrogen receptor beta (ER beta) and androgen receptor (AR) in mouse corneas. The present study was designed to investigate the occurrence of various sex steroid hormone receptors, including ER alpha, progesterone receptor (PR) and AR, in human corneas. METHODS: We used reverse transcription-polymerase chain reaction (RT-PCR) to look for sex hormone receptor mRNAs (ER alpha, PR and AR) in human corneal epithelial cells obtained at autopsy. Next, using an immunocytochemical technique, we localized these receptors in donor human corneas. RESULTS: mRNAs encoding all receptors tested for were found in corneal epithelial cells obtained from male and female donor eyes. Immunocytochemical examination revealed that the receptors were located in the nuclei of corneal epithelial, stromal, and endothelial cells. CONCLUSIONS: Since receptors for both male and female sex hormones are present in human corneas of both genders, we postulate that the receptors may influence the biological functions of corneal cells through direct interaction with specific hormones.

The epidermal growth factor receptor (EGFR): role in corneal wound healing and homeostasis.

To evaluate the role of the epidermal growth factor receptor (EGFR) in corneal epithelial wound healing, the effect of an EGFR inhibitor on epithelial cell proliferation and cell stratification during wound healing was investigated. From 3 days prior to wounding until wound healing was complete, rats were systemically treated with either an EGFR tyrosine kinase inhibitor (ZD1839) at 40 mg kg(-1) day(-1)or 80 mg kg(-1) day(-1), or with vehicle only (control). A single corneal wound was made in the center of 66 rat corneas, using a 6.0 mm glass tube wrapped in tissue paper soaked in n-heptanol. Subsequently, each wound was photographed and measured by a computer-assisted digitizer every 12 hr. To determine the number of cells in S phase, entire corneas were labelled with (3)H-thymidine and subjected to autoradiography at 0, 12, 24 and 48 hr after wounding. Epithelial thickness was also measured at these time points by microscopy. Epithelial wound healing was significantly and dose-dependently delayed following administration of ZD1839. At 24 hr after wounding, the number of S-phase cells in the limbal corneal epithelium was significantly lower in both the treated groups compared with the control group (P < 0.05). In the cornea before wounding (0 hr) and at 48 hr post-wounding, epithelial thickness was also significantly less in treated rats compared with controls (P < 0.05). These results indicate that EGFR inhibition affects epithelial cell proliferation and stratification during corneal epithelial wound healing and may play a role in maintaining normal corneal epithelial thickness.

Vitreous levels of insulin-like growth factor-I in patients with proliferative diabetic retinopathy.

The levels of IGF-I in the vitreous body and the intraocular fluid of patients with proliferative diabetic retinopathy (PDR) were determined using radioimmunoassay (RIA). Eleven vitreous specimens were obtained from the intraocular fluid of eyes of patients with PDR who underwent surgery during the operation. Eleven intraocular fluids from the same patients during reoperations were compared with controls. The expression of IGF-I mRNA in cultured human Muller glial cells was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). The mean IGF-I level in the vitreous samples during initial PDR surgery and reoperation was significantly higher than that found in the vitreous of the control (p < 0.05). The level of IGF-I increased in 6 of the 11 cases. Cultured human Muller cells expressed IGF-I mRNA. The results indicate increased levels of IGF-I both in the initial vitreous and ocular fluid at post-operative re-proliferation. Muller cell is suggested as an origin of local IGF-I production.

Conjunctival inflammation in the chronic phase of Stevens-Johnson syndrome.

AIMS: To understand the immunopathogenesis of the corneal conjunctivalisation in Stevens-Johnson syndrome. METHODS: Conjunctivalised corneas from five patients with Stevens-Johnson syndrome were studied immunohistochemically for several cell surface antigens and two cytokines. Chemical injury specimens were also studied. RESULTS: In all cases, immunohistochemistry revealed LFA-1, CD4, CD8, and CD68 on subepithelial infiltrating cells. Also, HLA-DR and ICAM-1 were found on the surfaces of epithelial cells, subepithelial infiltrating cells, subepithelial fibroblasts, and endothelial cells in blood vessels. IFN-gamma was found in basal epithelial cells; subepithelial cells and subepithelial extracellular matrix CD19 and IL4 were not detected. CONCLUSIONS: The infiltrating cell population in the Stevens-Johnson syndrome samples includes macrophages, CD4 positive T cells, and CD8 positive T cells. The cytokine expression pattern suggests CD4 positive T cells are Th1 cells. The infiltrating cell population is similar in Stevens-Johnson syndrome and chemical injury conjunctivalised corneas.

Growth factor mRNA and protein in preserved human amniotic membrane.

PURPOSE: To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). METHODS: RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80 degrees C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2) in preserved human corneas and in AM both with and without amniotic epithelium. RESULTS: RT-PCR revealed that human AM expresses mRNA for EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. CONCLUSIONS: Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.

Regulatory effects of 1alpha,25-dihydroxyvitamin D(3) on cytokine production by human corneal epithelial cells.

PURPOSE: The topical administration of 1alpha,25-dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3) ] inhibits Langerhans cell (LC) migration and corneal neovascularization in mice. Since the cytokines that induce LC migration [e.g. interleukin-1 (IL-1)] and corneal neovascularization [e.g. interleukin-8 (IL-8)] are produced by human corneal epithelial cells, we investigated the inhibitory effects of 1alpha,25(OH)(2)D( 3) on cytokine production by these cells in vitro. METHODS: In this experiment, human corneal epithelial cells, cultured in DMEM-FBS until confluence, were then switched to serum-free DMEM containing insulin, transferrin, and sodium selenite (DMEM-ITS) for 48 hours. Next, they were cultured with DMEM-ITS containing 1alpha,25(OH)(2)D(3 ) at concentrations of 10(-7) M, 10(-11) M, or 10( -15) M, and vehicle only (0.1% ethanol). After 6 or 12 hours in this culture, the supernatants were collected and concentrations of IL-1alpha, IL-1b, and IL-8 were quantified by ELISA. RESULTS: Significantly lower levels of IL-1alpha and IL-1b were detected in supernatants from cells cultured with 1alpha, 25(OH)(2)D( 3) (10(-7) M, 10(-11) M, and 10(-15) M), compared to cells cultured with vehicle only. This was true at 6 and 12 hours after the addition of 1alpha,25(OH)(2)D(3) (p < 0.05). IL-8 production inhibition by 1alpha,25(OH)(2)D(3), on the other hand, was detected at 6 hours (p < 0.0005) but not at 12 hours (p> 0.1). CONCLUSIONS: 1alpha,25(OH)(2)D(3) inhibits cytokine (IL-1alpha, IL-1b, and IL-8) production by human corneal epithelial cells in vitro. We suspect that 1alpha,25(OH)(2)D(3) can inhibit LC migration and corneal neovascularization, as is seen in ocular surface inflammation.