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Introduction Some but not all patients with atopic dermatitis (AD) present with allergic conjunctival disease (ACD) including severe types such as atopic keratoconjunctivitis (AKC) with/without giant papillae. We hypothesized that different factors are involved in the severity of ACD and AD. Recently we reported that hsa‐miR‐628‐3p could affect the balance of innate immunity by suppressing pathogen‐associated molecular patterns such as toll‐like receptor 3 (TLR3), RIG‐I, and MDA‐5. We also reported that TLR3 positively regulates ocular surface‐ and skin inflammation such as contact dermatitis and AD. Here we compared the plasma level of miR‐628‐3p in AD patients with severe AKC with giant papillae and/or shield ulcers, with the level in healthy controls and AD patient without AKC or with very mild AKC. Methods We used the plasma from 32 AD patients with severe AKC, from 40 healthy controls, and from 23 AD patient without AKC or with very mild AKC without giant papillae nor shield ulcers. Quantitative microRNA PCR assays were used to measure their plasma level of miR‐628‐3p. Results We found that plasma miR‐628‐3p was upregulated in AD with severe AKC, but not in severe AD without severe AKC, nor in our healthy controls. Conclusion Our new findings suggest that the plasma miR‐628‐3p level may represent a marker to predict the presence of severe AKC in AD patients.

Purpose Our meta-analysis of several ethnic groups (Japanese, Korean, Indian, Brazilian) revealed a significant genome-wide association between cold medicine-related SJS/TEN (CM-SJS/TEN) with severe ocular complications (SOC) and IKZF1 SNPs, suggesting that IKZF1 might be a potential marker for susceptibility to CM-SJS/TEN with SOC. In this study, we examined the association between CM-SJS/TEN with SOC and the IKZF1 SNPs in the Thai population. Methods 57 CM-SJS/TEN with SOC and 171 control samples were collected at Chulalongkorn University and Mahidol University. Genomic DNA samples were genotyped for the IKZF1 SNPs at Kyoto Prefectural University of Medicine in Japan using the TaqMan SNP genotyping assay. Results The four SNPs previously reported to be associated with CM-SJS/TEN with SOC in the Japanese were examined in the Thai samples. Although the number of Thai cases (n = 57) was small, a significant association between CM-SJS/TEN with SOC and IKZF1 SNPs which included rs4917014 (T vs G, OR = 2.9, p = 0.0012, Pc = 0.0049), rs4917129 (T vs C, OR = 2.8, p = 0.0026, Pc = 0.010) and rs10276619 (G vs A, OR = 1.8, p = 0.012, Pc = 0.048) was identified. Conclusion In addition to the Japanese, Korean and Indian populations, Thai cases with CM-SJS/TEN and SOC were significantly associated with IKZF1 SNPs. With our previous report of the critical role of IKZF1 in mucocutaneous inflammation, these results suggest that IKZF1 is important in the pathogenesis of CM-SJS/TEN with SOC.

To develop a pre-emptive genetic test that comprises multiple predisposing alleles for the prevention of phenytoin-related severe cutaneous adverse reactions (SCARs), three sets of patients with phenytoin-SCAR and drug-tolerant controls from Taiwan, Thailand, and Japan, were enrolled for this study. In addition to cytochrome P450 (CYP)2C9*3, we found that HLA-B*13:01, HLA-B*15:02, and HLA-B*51:01 were significantly associated with phenytoin hypersensitivity with distinct phenotypic specificities. Strikingly, we showed an increase in predictive sensitivity of concurrently testing CYP2C9*3/HLA-B*13:01/HLA-B*15:02/HLA-B*51:01 from 30.5-71.9% for selecting the individuals with the risk of developing phenytoin-SCAR in Taiwanese cohorts, accompanied by a specificity of 77.7% (combined sensitivity, 64.7%; specificity, 71.9% for three Asian populations). Meta-analysis of the four combined risk alleles showed significant associations with phenytoin-SCAR in three Asian populations. In conclusion, combining the assessment of risk alleles of HLA and CYP2C9 potentiated the usefulness of predictive genetic tests to prevent phenytoin hypersensitivity in Asians.

Background Our genome-wide 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 up-regulated 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 up-regulated in the epidermis of Ikzf1 Tg compared with wild-type. Conclusion Our findings support the hypothesis that Ikaros might participate in mucocutaneous inflammation. This article is protected by copyright. All rights reserved.

Developing mouse retina has been serving as an ideal model for investigating the molecular mechanism of neural development and angiogenesis, because several significant events associated with these physiological phenomena are drastically occurring in conjunction with retinal development. However, as many genes are influencing on each other to establish mature retina within 21 days from E10 to P12, we must carefully design the experiments, such as in the case of quantitating the amount of altered gene expression toward the establishment of retina by quantitative PCR. As we have seen considerable variations of quantitative results in different developmental stages of retina depending on the reference genes used for compensation, we here attempted to determine a reliable reference gene to accurately quantitate the target genes in each stage. According to the results of in silico prediction and comparison with a database of SAGE, we found that the most stable gene from early to late stages was Sdha, whereas one of the most popular housekeeping genes, Actb, was the one that could mislead the quantitative results even in the adult stage. Consequently, we pointed out the importance of selecting an appropriate reference gene, especially to quantitate the amount of gene expression in the developmental stages of a certain tissue.

One of the major problems encountered in cell transplantation is the low level of survival of transplanted cells due to detachment-induced apoptosis, called anoikis. The present study reports on the chemical synthesis and biological evaluation of water-soluble molecules that protect suspended cells from anoikis. The synthetic molecules bind to and induce clusters of integrins and heparan-sulfate-bound syndecans, two classes of receptors that are important for extracellular matrix-mediated cell survival. Molecular biological analysis indicates that such molecules prolong the survival of suspended NIH3T3 cells, at least in part, by promoting clustering of syndecan-4 and integrin β1 on the cell surface, leading to the activation of small GTPase Rac-1 and Akt. In vivo experiments using animal disease models demonstrated the ability of the molecules to improve cell engraftment. The cluster-inducing molecules may provide a starting point for the design of new synthetic tools for cell-based therapy.

Objective Periodontal ligament (PDL) cells and their substrates play key roles in periodontal regeneration. However, there has been no report on the use of amniotic membrane (AM) as a substrate for culturing PDL cells. In the current study, we conducted an analysis of PDL cells cultivated on AM to determine the distribution of factors responsible for maintaining the characteristics of PDL. Materials and Methods Amniotic membrane was obtained from women undergoing cesarean sections, whereas PDL tissue was obtained from human maxillary third molars. The harvested PDL cells were maintained in explant culture for three or four passages, following which they were cultured on AM. Results After 3 weeks of culture, the PDL cells had grown well on AM. Immunofluorescence showed that these cells were capable of proliferating and potentially maintaining their PDL-like properties. In addition, strong cell–cell adhesion structures, namely desmosomes and tight junctions, were shown to be present between cells. Electron microscopy images showed that the cultured PDL cells had differentiated and proliferated on AM with lateral conjugation and adhesion to AM. Conclusion We conclude that AM may represent a suitable substrate for culturing PDL cells and that PDL cells cultured on AM show sheet formation.

Summary Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but life-threatening severe cutaneous adverse reactions. Recently, strong associations of HLA-B*1502 with carbamazepine-induced SJS/TEN have been found in Han Chinese patients. These associations have been confirmed in several Asian populations, excluding Japanese. SJS patients carrying HLA-B*1508, HLA-B*1511, or HLA-B*1521, which are members of the HLA-B75 type along with HLA-B*1502, were detected in studies in India and Thailand. In the current study, we genotyped the HLA-B locus from 14 Japanese typical and atypical SJS/TEN patients in whom carbamazepine was considered to be involved in the onset of adverse reactions. Although there were no HLA-B*1502 carriers, four patients had HLA-B*1511. Our data suggest that HLA-B*1511, a member of HLA-B75, is a risk factor for carbamazepine-induced SJS/TEN in Japanese.

Enhanced green fluorescence protein (eGFP)-labeled bone marrow (BM) cells were transplanted into syngeneic C57BL/6 (wild-type) mice to investigate the distribution pattern, immunohistochemical characteristics, three-dimensional structure, and ultrastructure of the BM-derived cells in the mouse cornea using a fluorescence microscope, a confocal laser scanning microscope, and a transmission electron microscope. This study provided direct evidence that two morphologically distinct types of BM-derived cells were distributed in the mouse cornea. The majority of the GFP+ cells showed a flattened polygonal form with obtuse angles and these cells were distributed in the corneal stroma. The other type was the GFP+ cells demonstrating slim cell bodies with long and extremely thin dendrites and which were distributed in the corneal epithelium. The immunohistochemical characteristics and ultrastructure of BM-derived cells suggest that most of these cells have a macrophage lineage, whereas some cells in the corneal stroma do not. Interestingly, the direct intimate contact between GFP-labeled BM derived cells and non-GFP-labeled resident cells within the corneal stroma were also clearly visualized at the fine structural level. These data provide new and more detailed insight into the nature of BM-derived cells in the cornea.

Purpose We reported that PTGER3 SNPs were associated with Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) with severe ocular complications (SOC). We also reported that about 80% of our SJS/TEN patients had taken cold medicines within several days before disease onset and designated them cold medicine related-SJS/TEN (CM-SJS/TEN) patients, and that HLA-A*02:06 was significantly associated with CM-SJS/TEN in Japanese and Korean populations. Moreover, we documented that HLA-A*02:06 with TLR3 polymorphisms exerted more than additive effects in SJS/TEN with SOC. In the current study we focused on CM-SJS/TEN with SOC and analyzed an interactive effect between PTGER3 SNPs and HLA-A*02:06 in Japanese and Korean populations. Methods Samples from 132 Japanese patients with CM-SJS/TEN with SOC were collected and 221 healthy Japanese volunteers were also recruited as controls. Samples from 30 Korean patients with CM-SJS/TEN with SOC were collected and 120 healthy Korean volunteers were also recruited as controls. Genotyping of PTGER3 gene SNPs was performed using the TaqMan SNP genotyping assay or the DigiTag2 assay. HLA-A genotyping was performed using using commercial bead-based typing kits, WAK Flow. Results In Japanese population, we found an interaction with additive effects between HLA-A*02:06 and the high-risk genotypes PTGER3 rs1327464 GA or AA (OR = 10.8, p = 2.56 × 10−7). Moreover, we also found an additive effect between HLA-A*02:06 and the high-risk genotypes PTGER3 rs1327464 GA or AA (OR = 14.2, p = 5.58 × 10−6). Conclusions These finding might show that the combination of these two polymorphisms could give improvements for a genetic testing as compared to using only one susceptibility gene.

Purpose: To evaluate the use of sterilized, freeze-dried amniotic membrane (FD-AM) transplantation for pterygium surgery. Methods: This study involved a prospective, non-comparative, interventional case series. Thirteen eyes of 13 patients with primary (eight eyes) or recurrent (five eyes) pterygium were studied. After excision of the pterygium fibrous tissues and application of intraoperative use of mitomycin-C, sterilized FD-AM was sutured over the bare scleral defect. The integrity of the FD-AM graft, epithelialization over the FD-AM, pterygium recurrence and postoperative complications were evaluated. Results: Postoperatively, the FD-AM was well retained in all patients, and complete epithelialization over the transplanted membrane was achieved within 1–2 weeks. All patients demonstrated early resolution of ocular inflammation and there was no recurrence of pterygium in any of the treated patients during the mean follow-up of 13.9 ± 6.0 months. No ocular complications were noted following transplantation. Conclusion: Sterilized FD-AM showed excellent biocompatibility on the human ocular surface. This novel and promising biomaterial may be a useful alternative to conjunctival grafting in the treatment of pterygium.

Purpose: Different from conventional tonometers, the pressure phosphene tonometer (FPT) measures intraocular pressure (IOP) through the upper eyelid without corneal applanation. We evaluated the usefulness of the FPT as a self-tonometer by comparing FPT IOP readings with those obtained with the Goldmann applanation tonometer (GAT). We also evaluated the influence of central corneal thickness (CCT) on IOP measurements obtained with the two different devices. Methods: We confirmed the repeatability of FPT measurements in a preliminary study. The main investigation formed part of a prospective clinical trial, in which IOP was measured in 101 eyes of 101 participants (55 normal and 46 glaucomatous eyes) using GAT and FPT. FPT measurements were self-acquired by each participant. CCT was measured with an ultrasonic pachymeter. The agreement between FPT and GAT measurements was evaluated by the method of Bland and Altman. Using individual IOP and CCT values, we determined the correlation coefficients and performed regression analysis. Results: FPT met the British Standard criteria for reproducibility. Among 101 participants, seven patients with glaucoma were unable to detect the pressure phosphene and to measure IOP using the FPT. By the statistical method of Bland and Altman there was a significant difference between FPT readings self-measured by the remaining 94 participants and GAT readings obtained by an ophthalmologist. There was no correlation between FPT and CCT readings in 65 participants with no prior history of anti-glaucoma medications or glaucoma surgery. Conclusions: Self-tonometer FPT readings differed from GAT readings. However, we suggest that the FPT may be a clinically acceptable device because it enables patients to self-measure their IOP easily and safely. As FPT readings are not affected by CCT, this tonometer may be of clinical and practical value for the at-will measurement of IOP in patients with corneal changes.

Purpose: To examine immune tolerance and corneal ultrastructure following additive corneal xenografts in rodents. Methods: We carried out surgical implantation of excised BALB/c mouse corneal tissue, either freshly isolated (n = 6) or after storage at −20°C for 1 week (n = 7), into the corneas of Wistar rats at approximately mid-stromal depth. Corneal opacity and neovascularization were evaluated postoperatively, and stromal ultrastructure was observed by transmission electron microscopy. Results: Corneal opacification and neovascularization in the weeks after surgery were less prevalent in grafts of frozen-then-thawed tissue than in grafts of fresh tissue. In a well tolerated frozen-then-thawed xenograft, the matrix architecture was normal throughout most of the recipient and donor tissue, but pronounced fibrillar disorganization was evident adjacent to Descemet's membrane. Conclusion: We attribute the improved tolerance of frozen-then-thawed xenografts over fresh xenografts to a reduced cellular immune response.

Purpose: An immunological reaction to a bacterial antigen, such as Mycobacterium tuberculosis or Propionibacterium spp., is suspected to be an initial mechanism in the disorder known as sarcoidosis. We investigated whether or not P. acnes, P. granulosum or M. tuberculosis are present in the vitreous fluid of eyes suffering from uveitis with sarcoidosis. Methods: Using polymerase chain reaction, we analysed the presence of P. acnes, P. granulosum and/or M. tuberculosis DNA in vitreous samples taken from six eyes with sarcoidosis and six control eyes. Results: Among the six uveitis eyes with sarcoidosis, we detected P. acnes DNA in two eyes, P. granulosum DNA in four eyes, and both P. acnes and P. granulosum DNA in one eye, but no Propionibacterium spp. in the control eyes. M. tuberculosis DNA was not present in any of the patient or control eyes. Conclusions: This is the first report indicating the presence of Propionibacterium spp. and/or its DNA in the vitreous fluid of sarcoidic eyes with uveitis. This, therefore, supports the idea that Propionibacterium spp. are involved in the aetiology of uveitis in sarcoidosis.

Background: Patients with severe unilateral ocular surface disease require reconstruction of the damaged ocular surface. We succeeded in culturing primary corneal limbal epithelial cells taken from minimal biopsy and, once grown, transplanting them on denuded amniotic membrane (AM). Methods: Autologous corneal limbal epithelial cells from a 3 mm2 biopsy of the uninjured eye were grown for 3 weeks on a denuded AM carrier. The resultant sheet was then transplanted onto the unilateral severely chemically injured eye. Results: Minimal biopsy showed the autologous cultivated corneal epithelial cells to have 4–5 layers of sufficient stratification and to be well differentiated. At 19 months post-transplantation, the ocular surface epithelium was stable and there were no epithelial defects. Conclusion: We document that it is possible to produce sufficiently stratified, well differentiated, autologous cultivated corneal limbal epithelium on AM from a minimal biopsy of the donor eye and to transplant it onto the injured eye.

Purpose: To evaluate the effects of early systemic administration of betamethasone or cyclosporin A (CsA) on inflammatory cytokine expression and corneal damage after alkali injury. Methods: Sixty-five Wistar rat corneas injured with 1N NaOH were divided into three groups: untreated, betamethasone-treated and CsA-treated. Both agents were administered systemically and daily during the first 7 days after injury. Interleukin (IL)-1α, -6 and -8 concentrations in the injured corneas were measured with ELISA at 2, 4 and 7 days after injury. Corneal damage was evaluated by scoring clinical findings. Results: In untreated injured corneas, IL-1α, -6 and -8 were markedly elevated during the 7-day period following injury. Both betamethasone and CsA significantly suppressed IL-1α and IL-8 at day 4. Only betamethasone significantly suppressed IL-6 at days 4 and 7. Both agents significantly reduced corneal opacity at day 4. Conclusion: Early systemic administration of betamethasone or CsA after alkali injury may be of benefit by suppressing inflammatory cytokine expression in the cornea.

Regenerative medicine via tissue engineering is a newly developed medical field based on the use of somatic stem cells to generate biological substitutes and improve tissue functions. The achievement of these objectives depends on two important factors: stem cells, which are high in proliferability and differentiability, and the substrates that support them. In order to reconstruct the ocular surface in patients with severe ocular surface diseases, we decided to investigate the feasibility of human amniotic membrane as an epithelial carrier, and found that denuded amniotic membrane was the most appropriate substrate for this purpose. To develop the surgical treatment using denuded amniotic membrane as a carrier, we established the tissue engineering system for making transplantable epithelial sheets of either corneal or oral mucosal epithelial cells for ocular surface reconstruction.

Purpose: To introduce a new technique for removing the inner wall of Schlemm's canal using cyanoacrylate. Methods: Two donor eyes were used in this study. A limbal-based scleral flap was dissected. The external wall of Schlemm's canal was opened and the inner wall of Schlemm's canal was exposed. Ethyl-2-cyanoacrylate was applied uniformly to the inner wall. After it had solidified, it was removed with fine forceps. The operated sites were then studied by light microscopy. Results: After cyanoacrylate removal, the aqueous humour was seen percolating through the thin remaining trabecular meshwork. Histopathological findings at the operated sites revealed that the lining of the Schlemm's endothelial cells was irregular. Endothelial cell nuclei were rarely seen along the canal wall. Microperforation to the anterior chamber was not seen. Conclusion: This new technique is potentially a safer method for removing the inner wall of Schlemm's canal than the conventional method.

Background: To report the spontaneous remodelling of retinal veins inadvertently severed during vitreous surgery. Methods: Four diabetic patients who underwent vitrectomy during which retinal veins were damaged were followed for at least 6 months. Haemorrhages from severed veins were controlled by raising intraocular pressure and by endodiathermy of the distal ends of the veins. This was followed by fluid/gas exchange. Results: Remodelling of venous channels in the damaged areas was observed in all eyes. In three of the four eyes we found evidence of collateral veins. In the other eye we noted the connection of two ends of an interrupted vein. No signs of retinal vein occlusion (e.g. superficial retinal haemorrhages and retinal oedema) were observed. Conclusion: These findings suggest that a spontaneous remodelling mechanism to repair damaged venous channels may exist in retina.

Background Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucous membranes of, for example, the ocular surface, oral cavity, and genitals. They are rare but often associated with inciting drugs. Cold medicines including non-steroidal antiinflammatory drugs (NSAIDs) and multi-ingredient cold medications are reported to be important inciting drugs. We investigated the association between HLA genotypes and cold medicine-related SJS/TEN (CM-SJS/TEN) with severe mucosal involvement such as severe ocular surface complications (SOC). We also investigated the association between HLA genotypes and acetaminophen-related SJS/ TEN in patients with CM-SJS/TEN with SOC. Methods Japanese acetaminophen-related SJS/TEN patients consist of 59 patients, they were among the 131 CM-SJS/TEN with SOC. While the specific drugs were not named by all 131 patients, 59 (45.0%) were known to have taken medicines containing acetaminophen. The diagnosis of SJS/TEN with SOC was based on a confirmed history of acute-onset high fever, serious mucocutaneous illness with skin eruptions, and the involvement of at least 2 mucosal sites including the oral cavity and ocular surface. We analyzed HLA-A, -B, and -C of 131 CM-SJS/TEN patients and 419 Japanese controls. Results

Background Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), are severe adverse drug reactions which are very rare, acute, serious, and potentially fatal. They exhibit characteristic symptoms not only in the skin, but also in the mucosal tissues such as ocular surface, oral cavity, and genitals. Although antipyretic analgesics (AAs) are some of the most frequent causative drugs of SJS/TEN, there has been no HLA association studies on these drugs-induced SJS/TEN. The scope of this study is to investigate HLA-type association with AA-related SJS/TEN with severe ocular surface complications (SOCs). Method

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).

Rebamipide eyedrops are approved in Japan for the treatment of dry eye disease. Some patients with allergic conjunctival diseases also manifest dry eye. Earlier we reported that rebamipide suppressed polyI:C-induced inflammatory cytokines in human conjunctival epithelial cells. In the current study we examined the effect of rebamipide eyedrops on the level of interleukin-8 (IL-8), eosinophil cationic protein (ECP), and total IgE on the ocular surface. We prescribed rebamipide eyedrops to patients with atopic keratoconjunctivitis (AKC) who presented with dry eye (6 eyes in 4 AKC patients) and measured the IL-8, ECP, and total IgE levels in their tears before- and 2, and 4–6 weeks after the start of rebamipide treatment. To measure the IL-8 and total IgE levels in their tears we used BD™ CBA Flex sets; ECP measurements were with ELISA. The level of IL-8, ECP, and total IgE in the tears of AKC patients was reduced significantly 4–6 weeks after the start of rebamipide treatment. We also recorded subjective symptoms associated with AKC, e.g. itching, foreign body sensation, and eye mucus discharge, by using a patient questionnaire. Their subjective symptoms associated with AKC were also significantly ameliorated at 2 and 4–6 weeks. Our observations suggest that the anti-inflammatory effects of rebamipide eyedrops help to combat human ocular surface inflammation and that they may be a new effective therapy in patients with AKC. Electronic supplementary material The online version of this article (doi:10.1186/2045-7022-4-40) contains supplementary material, which is available to authorized users.