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The human ocular surface epithelium expresses TLR3, which recognizes double-stranded (ds) RNA mimicking polyinosine-polycytidylic acid (polyI:C). Its stimulation induces the secretion of the inflammatory cytokines such as interleukin (IL)-6, IL-8, and type I interferon. The cytoplasmic helicase proteins RIG-I and MDA5 are also expressed on the ocular surface. We investigated the function of TLR3 in the cornea of CD11c− YFP+ and TLR3 KO CD11c− YFP+ mice. We also examined the function of IPS-1, an adaptor molecule common to RIG-I and/or MDA5, in IPS-1 KO CD11c− YFP+ mice. In the central corneal epithelium of CD11c− YFP+ mice, the infiltration of CD11c− YFP+ cells was significantly upregulated 48 h after polyI:C stimulation; it was significantly downregulated in the stromal layer of their central and peripheral cornea. On the other hand, in the corneal epithelium of TLR3 KO CD11c− YFP+- and wild-type mice, the movement of CD11c− YFP+ cells was different from CD11c− YFP+ mice. This suggests that TLR3 knock-out (KO) interferes with their movement from the peripheral- to the central cornea or lymph nodes and that it may be similar in IPS-1 KO CD11c−YFP+ - and wild-type mice. Under normal conditions, the number of CD11c− YFP+ cells in the central and peripheral corneal epithelium, but not in the stromal layer, is significantly greater in TLR3 KO CD11c- YFP+- than CD11c− YFP+ mice. In IPS-1 KO CD11c− YFP+ mice, their number in the stromal layer, but not in the epithelium of the central and peripheral cornea, was significantly greater than in CD11c− YFP+ mice. Our findings suggest that CD11c+ dendritic cell (DC) migration in the corneal epithelium is regulated by TLR3, whereas CD11c+ DC migration in the stromal layer of the cornea is regulated by IPS-1. These observations, together with our earlier findings, imply that TLR3 and IPS-1 contribute distinctly to the regulation of innate immune responses and tissue inflammation elicited by CD11c+ DCs to maintain homeostasis in corneal tissues.

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.

Purpose Cultured human corneal endothelial cells (cHCECs) are anticipated to become an alternative to donor corneas for the treatment of corneal endothelial dysfunction. The aim of this study was to establish proper culture protocols to successfully obtain a reproducibly homogeneous subpopulation (SP) with matured cHCEC functions and devoid of cell-state transition suitable for cell-injection therapy. Methods The presence of SPs in cHCECs was investigated in terms of surface cluster-of-differentiation (CD) marker expression level by flow cytometry, as combined analysis of CD markers can definitively specify the SP (effector cells) conceivably the most suitable for cell therapy among diverse SPs. The culture conditions were evaluated by flow cytometry in terms of the proportion (E-ratio) of effector cells designated by CD markers. Results Flow cytometry analysis identifying CD44-CD166+CD133-CD105-CD24-CD26- effector cells proved convenient and reliable for standardizing the culture procedures. To ascertain the reproducible production of cHCECs with E-ratios of more than 90% and with no karyotype abnormality, the preferred donor age was younger than 29 years. The continuous presence of Rho-associated protein kinase (ROCK)-inhibitor Y-27632 greatly increased the E-ratios, whereas the presence of transforming growth factor-beta/Smad-inhibitor SB431542 greatly reduced the number of recovered cHCECs. The seeding cell density during culture passages proved vital for maintaining a high E-ratio for extended passages. The continuous presence of ROCK-inhibitor Y-27632 throughout the cultures greatly improved the E-ratio. Conclusions Our findings elucidated the culture conditions needed to obtain reproducible cHCECs with high E-ratios, thus ensuring homogeneous cHCECs with matured functions for the treatment of corneal endothelial dysfunction.

PURPOSE To clarify the adherent properties of cultured human corneal endothelial cell (cHCEC) subpopulations (SPs). METHODS Each SP was prepared by controlling the culture conditions or by using magnetic cell separation, and then confirmed by staining with several cell-surface markers. Binding abilities of HCEC SPs were examined by adding the cells to culture plates immobilized with collagens, laminins, or proteoglycans, and then centrifuging the plates. Adhered cells were then evaluated by phase-contrast microscopy. RESULTS The cHCECs were bound to laminin-511, laminin-411, and Type-IV collagen in a concentration-dependent manner, yet weakly bound to Perlecan, Agrin, and TSP-1. Comparison of the influence of cell-suspension vehicles on cHCEC attachment showed that cells suspended in Opti-MEM-I or Opeguard-MA were bound to laminin, yet no binding was observed in cells suspended in BSS-Plus. Next, we compared the adherent properties of HCEC SPs. Both the fully differentiated, mature cHCEC SP and the epithelial-to-mesenchymal-transitioned (EMT)-phenotype SP were found to attach to laminin- or collagen-coated plates. Interestingly, the binding properties to laminins differed among those SPs. Although the level of cells adhered to the laminin-411-coated plate was the same among the cHCEC SPs, the fully differentiated, mature cHCEC SP was significantly more tightly bound to laminin-511 than was the EMT-phenotype SP. CONCLUSIONS The findings of this study suggest that the binding ability of cHCECs to major Descemet's membrane components is distinct among cHCEC SPs, and that Opti-MEM-I and Opeguard-MA are useful cell-suspension vehicles for cell-injection therapy.

Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal transplantation. We previously reported that the adhesion of corneal endothelial cells (CECs) to a substrate was enhanced by the selective ROCK inhibitor Y-27632. It is hypothesized that the inhibition of ROCK signaling may manipulate cell adhesion properties, thus enabling the transplantation of cultivated CECs as a form of regenerative medicine. In the present study, using a rabbit corneal endothelial dysfunction model, the transplantation of CECs in combination with Y-27632 successfully achieved the recovery of corneal transparency. Complications related to cell injection therapy, such as the abnormal deposition of the injected cells as well as the elevation of intraocular pressure, were not observed. Reconstructed corneal endothelium with Y-27632 exhibited a monolayer hexagonal cell shape with a normal expression of function-related markers, such as ZO-1, and Na(+)/K(+)-ATPase, whereas reconstruction without Y-27632 exhibited a stratified fibroblastic phenotype without the expression of markers. Moreover, transplantation of CECs in primates in the presence of the ROCK inhibitor also achieved the recovery of long-term corneal transparency with a monolayer hexagonal cell phenotype at a high cell density. Taken together, these results suggest that the selective ROCK inhibitor Y-27632 enables cultivated CEC-based therapy and that the modulation of Rho-ROCK signaling activity serves to enhance cell engraftment for cell-based regenerative medicine.

The ability to predict anti-tumor immune responses at local tumor growing sites using only peripheral blood specimens would be helpful in determining therapeutic options for patients with solid tumors. Here, we show that the glutathione intracellular content (icGSH) of peripheral monocytes (Mo) correlates positively with T cell infiltration within tumor islets and overall survival in patients with colorectal carcinoma. IcGSH redox status was determined in CD14(+) Mo prior to surgery by staining with monochlorobimane. The tumor-infiltrating T cells (TIL) were quantified as CD45RO(+) T cells in resected tumors using paraffin sections. A positive association was found between the GSH index and TIL in tumor islets (P0.001). The 50% cut-off value for the GSH index, that is the determinant between TIL presence or absence in tumor islets, was calculated to be almost 0.7 through logistic regression analysis. Mo with a GSH index ofor =0.7 were termed reductive (R)-Mo, and those with0.7 were designated as oxidative (O)-Mo. Cox's proportional hazards regression analysis of patients with R-Mo or O-Mo prior to surgery, and the presence or absence of TIL, was found to correlate significantly with the overall survival rate of stage II and III patients. Kaplan-Meier analysis also showed a significant correlation. These results indicate that the Mo icGSH index is a useful biomarker parameter for better understanding the host/tumor relationship prior to surgery, thereby enabling the development of an individual patient-oriented therapeutic strategy.

Aims The essential role of basophils as an initiator of chronic allergic reaction has been elucidated in mouse models. The aim of this present study was to analyse the in situ immunolocalisation of basophils and other relevant inflammatory cells in chronic allergic keratoconjunctivitis. Methods Transmission electron microscopic (TEM) analysis was carried out to examine the existence of basophils in the giant papillae obtained from atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) patients. Cryostat sections of giant papillae were immunostained with basophil-specific antibody BB-1, and with anti-CD4, anti-CD8, anti-CD20, anti-major basic protein (MBP), anti-IgE and anti-FcɛRI-β antibodies. Results TEM analysis confirmed the existence of basophils in the giant papillae. Small clusters of basophils were observed in the substantia propria of giant papillae, especially at the vicinity of vascular endothelium and subepithelial regions. BB-1-positive basophil clusters were surrounded by T cells, B cells, IgE-positive cells and MBP-positive eosinophils. No BB-1-positive basophils were observed in the control conjunctivae. Conclusion Basophils may infiltrate from either vascular endothelium into the giant papillae. The existence of basophils at the centre of inflammatory cells suggests the role of basophils as an initiator of chronic allergic conjunctivitis.

The high-affinity IgE receptor (FcepsilonRI)-beta gene is one of the atopy-associated genes, but its biological significance is largely unknown. In this study, we generated the anti-FcepsilonRI-beta chain antibody to clarify beta-chain protein expression in human mast cells. The FcepsilonRI-beta antibody showed specific binding to a 27 kDa protein with Western blotting and membrane bound immunostaining using cultured mast cells. Monomeric IgE sensitization increased beta-chain expression as well as mature alpha-chain expression in mast cells. Upregulation of beta-chain expression with monomeric IgE treatment suggests possible roles of FcepsilonRI-beta protein as an atopy-related molecule.

Glutathione is the major intracellular redox buffer. We have shown that glutathione redox status, which is the balance between intracellular reduced (GSH) and oxidized (GSSG) glutathione, in antigen-presenting cells (APC) regulates the helper T cell type 1 (Th1)/Th2 balance due to the production of IL-12. Bronchial asthma is a typical Th2 disease. Th2 cells and Th2 cytokines are characteristic of asthma and trigger off an inflammation. Accordingly, we studied the effects of the intracellular glutathione redox status on airway hyperresponsiveness (AHR) and allergen-induced airway inflammation in a mouse model of asthma. We used gamma-Glutamylcysteinylethyl ester (gamma-GCE), which is a membrane-permeating GSH precursor, to elevate the intracellular GSH level and GSH/GSSG ratio of mice. In vitro, gamma-GCE pretreatment of human monocytic THP-1 cells elevated the GSH/GSSG ratio and enhanced IL-12(p70) production induced by LPS. In the mouse asthma model, intraperitoneal injection of gamma-GCE elevated the GSH/GSSG ratio of lung tissue and reduced AHR. gamma-GCE reduced levels of IL-4, IL-5, IL-10, and the chemokines eotaxin and RANTES (regulated on activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid, whereas it enhanced the production of IL-12 and IFN-gamma. Histologically, gamma-GCE suppressed eosinophils infiltration. Interestingly, we also found that gamma-GCE directly inhibited chemokine-induced eosinophil chemotaxis without affecting eotaxin receptor chemokine receptor 3 (CCR3) expressions. Taken together, these findings suggest that changing glutathione redox balance, increase in GSH level, and the GSH/GSSG ratio by gamma-GCE, ameliorate bronchial asthma by altering the Th1/Th2 imbalance through IL-12 production from APC and suppressing chemokine production and eosinophil migration itself.

Background The incidence of allergic symptoms to cedar pollen has reached epidemic proportions in Japan. Intravenous injection of β-1,3-glucan in human subjects is known to induce a T H 1 response, whereas oral uptake does not. Objective It was examined whether orally ingested, superfine dispersed β-1,3-glucan (SDG), easily absorbed by intestinal mucosa, would alleviate allergic symptoms. Methods Allergic patients were orally administrated either SDG (n = 30) or nondispersed β-1,3-glucan (n = 30), and allergic symptoms were assessed clinically in a double-blind, placebo-controlled randomized study. Results SDG alleviated ongoing symptoms of Japanese cedar pollen–induced rhinorrhea, sneezing, nasal congestion, and itchy watery eyes, and its oral uptake before symptom onset exhibited preventive effects. Alleviation of allergic symptoms was evident not only for seasonal allergy to cedar pollen but also for perennial allergy. Oral ingestion of β-1,3-glucan in individuals with allergic tropism could reduce the spontaneous increase in both allergen-specific and total IgE titers. The clinical responses to treatment were well correlated with the capacity of monocytes to bind to β-1,3-glucan. Although SDG reduced allergic symptoms, the oral uptake of nondispersed β-1,3-glucan produced no clinical effects, despite the identical amount of β-1,3-glucan in both preparations. Conclusion We postulate that orally taken β-1,3-glucan prepared in a form easily absorbed by intestinal mucosa is able to alleviate cedar pollen–induced allergic symptoms. Clinical implications Orally effective SDG might greatly contribute to the resolution of epidemic medical problems of seasonal cedar pollen–induced allergy.

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.

Interleukin (IL)-15 is expressed in a variety of inflammatory diseases. However, the contribution of dendritic cell (DC)–derived IL-15 to the development of diseases is uncertain. Using established models of Propionibacterium acnes (P. acnes)– and zymosan-induced liver inflammation, we observed granuloma formation in the livers of wild-type (WT) and RAG-2−/− mice but not in those of IL-15−/− mice. We demonstrate that this is likely caused by an impaired sequential induction of IL-12, IFN-γ, and chemokines necessary for monocyte migration. Likewise, lethal endotoxin shock was not induced in P. acnes– and zymosan-primed IL-15−/− mice or in WT mice treated with a new IL-15–neutralizing antibody. In both systems, proinflammatory cytokine production was impaired. Surprisingly, neither granuloma formation, lethal endotoxin shock, nor IL-15 production was induced in mice deficient for DCs, and adoptive transfer of WT but not IL-15−/− DCs restored the disease development in IL-15−/− mice. Collectively, these data indicate the importance of DC-derived IL-15 as a mediator of inflammatory responses in vivo.

As most solid tumors are hypoxic, dendritic cells (DC) in solid tumors are also exposed to hypoxia. While many adaptation responses of tumor cells to hypoxia are known, it is yet to be determined how hypoxia affects the functions of DC. To explore the effects of hypoxia on the functions of DC, we compared the expression of surface markers, cytokines, chemokine receptors and matrix metalloproteinases (MMP) of human monocyte-derived DC (hmDC) differentiated under hypoxia to those differentiated under normoxia. Both groups of hmDC expressed similar levels of surface markers and cytokines. However, expression of MMP-9 and membrane type-1-MMP, as well as migrating activity, was significantly suppressed in hmDC differentiated under hypoxia compared with their normoxia counterparts. We also demonstrated that trichostatin A restored the production of MMP-9 in hmDC, under hypoxia. Collectively, our findings show that a hypoxic microenvironment suppresses the production of MMP in hmDC, most probably through the deacetylation of promoter regions of MMP, thus suppressing the migrating activity of hmDC. Our results suggest that the hypoxic microenvironment in solid tumor tissues may suppress the function of DC.

Both osteopontin (OPN) and natural killer T (NKT) cells play a role in the development of immunological disorders. We examined a functional link between OPN and NKT cells. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model of T cell-mediated liver diseases. Here, we show that NKT cells secrete OPN, which augments NKT cell activation and triggers neutrophil infiltration and activation. Thus, OPN- and NKT cell-deficient mice were refractory to Con A-induced hepatitis. In addition, a neutralizing antibody specific for a cryptic epitope of OPN, exposed by thrombin cleavage, ameliorated hepatitis. These findings identify NKT cell-derived OPN as a novel target for the treatment of inflammatory liver diseases.

Purpose. To control intraocular pressure in patients treated by trabeculectomy, progressive inflammation, fibroblast proliferation, and the enhanced expression of extracellular matrix (ECM), causes of scar formation at the bleb, must be prevented. Using human Tenon's capsule fibroblasts (TCFs), we examined the effect of APC0576, a suppressor of NF-κB-dependent gene activation of human vascular endothelial cells that does not adversely affect cell viability, on the production of pro-inflammatory chemokine and ECM. Its effect on TCF proliferation was also assessed. Methods. Enzyme-linked immunosorbent assay was used to detect the pro-inflammatory cytokines IL-8 and MCP-1 in the supernatant of TCFs stimulated with IL-1α in the presence or absence of APCO576 and ECM proteins, COOH-terminal peptide of type I procollagen (PIP), fibronectin (FN), and laminin (LN) in supernatants and lysates of TCFs stimulated with IL-1α or TGF-β. The proliferative response of IL-1α-stimulated TCFs was examined using the SF formazan solution reaction. Results. APC0576 significantly suppressed the production by TCFs of IL-8 (p

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.

Murine mature splenic DC with elevated intracellular glutathione, pretreated with IL-18, strikingly augmented the production of IFN-gamma in response to IL-12, whereas intracellular glutathione deprivation ablated this effect of IL-18. Likewise, macrophages with elevated intracellular glutathione augmented IFN-gamma production upon LPS or IL-12+IL-18 stimulation, whereas macrophages with reduced intracellular glutathione showed the reciprocal response. Under hypoxia, macrophages displayed a functional phenotype with decreased intracellular glutathione, i.e. decreased NO and IL-12, and elevated IL-10 production. However, mature DC and macrophages produced an elevated amount of IFN-gamma under hypoxia. Taken together, our results suggest that the intracellular redox status of DC and macrophages may play a pivotal role in local innate immunity, depending on local oxygen tension.

Transgenic (Tg) mice overexpressing human thioredoxin (TRX), a small redox-active protein, were produced to investigate the role of the protein in a variety of stresses. Bone marrow cells from TRX-Tg mice were more resistant to ultraviolet C-induced cytocide compared with those from wild type (WT) C57BL/6 mice. TRX-Tg mice exhibited extended median and maximum life spans compared with WT mice. Telomerase activity in spleen tissues in TRX-Tg mice was higher than that in WT mice. These results suggest that overexpression of TRX results in resistance against oxidative stress and a possible extension of life span without apparent abnormality in mammals.

In vivo lentinan (LNT)-elicited peritoneal macrophages (Mps) showed the reduced release of prostaglandins (PGs), IL-10 and IL-6, while it endowed Mps with the elevated capability to produce IL-12 and nitric oxide (NO) upon in vitro triggering, due to the elevated intracellular glutathione (GSH) content in Mps. Deprivation of intracellular GSH completely ablated the production of IL-12. Conversely, lipopolysaccharide (LPS) induced peritoneal Mps with the reduced intracellular GSH content and the reciprocal profile of mediator production. Mps with the elevated intracelluar GSH is arbitrarily termed as reductive Mp (RMp) and that with reduced amount as oxidative Mp (OMp). OMp was converted to RMp when GSH was replenished with glutathione monoethylester (GSH-OEt). The IL-2 administration in combination with LNT exerted the synergistic induction of RMp, resulting in synergistic augmentation of IL-12, NO and reduction of IL-6 production. It was also confirmed that CD4+T cells derived of LNT-administered mice showed augmented IFN-gamma and reduced IL-4 production upon in vitro anti-CD3 stimulation. Taken together it is concluded that skewing of Th1/Th2 balance to Th1 by a beta-(1-3)-glucan, LNT, is directed through the distinctive production of IL-12 versus IL-6, IL-10 and prostaglandin E2 (PGE2) by Mps, depending on intracellular GSH redox status. To the efficient tumor immunotherapy, it may be one of the critical elements to induce a reductive form of Mps in tumor stromal tissues to maintain Th1 response.

Purpose To elucidate a noninvasive method to qualify and identify cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition and adaptable for cell-based therapy. Methods The variations of cHCECs in their composition of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers and their morphology. The profiles of microRNA (miRNA) in cultured cells or supernatants were detected by 3D-Gene Human microRNA Chips (Toray Industries, Inc.). The profiles were also analyzed for fresh corneal tissues with distinct endothelial cell densities (ECD) with or without gutatta. To validate the 3D-Gene results, quantitative real-time polymerase chain reaction (PCR) was performed. RNAs were extracted from cHCECs transfected with selected miRNA, and target genes were presumed by PCR array (Qiagen). Results Among a variety of morphologically different cHCECs, miRNA expression profiles were distinctively revealed. The one miRNA capable of discriminating CD44- SP from SPs with CD44++∼CD44+++ phenotypes was identified as miR34a. The downregulation of miRNAs in the 378 family paralleled the upregulation of surface CD44 on cHCECs. Interestingly, upregulated miRNAs in the 378 family in corneal endothelium dramatically decreased in the tissues with lower ECD with advanced gutatta, providing new insight on the pathogenesis of Fuchs' endothelial corneal dystrophy. Conclusions The specified cultured SPs sharing the CD44- surface phenotypes with matured HCECs showed the highest expression of miR-378. Conversely, SPs with upregulated CD44+++ showed a reduction of miR-378. Thus, miRNA in cultured cells may serve as an alternative method to qualify cHCECs.

The effects of lentinan, an antitumor polysaccharide, on vascular reactions against vasoactive mediators were investigated in murine systems. Lentinan augmented intradermal reactions against bradykinin. Induction of acute phase proteins (APP) and the vascular dilatation hemorrhage (VDH) reaction on the ears have been reported to reflect the host responses to lentinan. The strain difference in the intensity of skin reactions coincided with those observed in VDH responses and with lentinan-induced antitumor effects against Sarcoma 180. Augmentation of skin reactions was not observed in T-cell-deficient mice. Inhibitors of lipoxygenase, thrombin and plasmin which reduced skin reactions also decreased the incidence of tumor necrosis positive mice among FBL-3-bearing mice treated with lentinan. Furthermore, B10D2 mice treated with fluorouracl (5-FU) and lentinan 10 days after S908.D2 transplantation showed complete tumor regression and augmented skin reactions, whereas augmentation of skin reactions and tumor regression were not observed in mice treated with 5-FU and lentinan 32 days after tumor inoculation. Taken together, these results suggest that these vascular reactions might play crucial roles in antitumor effects of lentinan and that the skin reaction, the convenient method for investigating vascular reactions, is a promising tool to monitor host sensitivity to lentinan in antitumor responses.

PURPOSE. Levels of some cytokines are significantly higher in the vitreous fluid of patients with acute uveitis than in normal vitreous fluid. The authors sought to determine which proinflammatory cytokines were upregulated in the vitreous fluid of patients with ocular sarcoidosis. METHODS. Samples of vitreous fluid were collected from patients with sarcoid uveitis and from nonsarcoid control patients with idiopathic epiretinal membrane. The levels of 27 proinflammatory cytokines were measured with a multiplex beads array system. Postvitrectomy macular thickness was also measured by using spectral domain optical coherence tomography (SD-OCT). To assess the relationship between cytokine levels and disease stage, the authors divided patients into three groups based on macular thickness 1 month after operation. RESULTS. The vitreous levels of 17 cytokines were significantly higher in patients with ocular sarcoidosis than in nonsarcoid controls. Serum levels of interferon c‐induced protein 10 (IP10) were also higher in ocular sarcoidosis patients than in nonsarcoid controls. Conversely, serum levels of interleukin (IL) 15 in ocular sarcoidosis patients were lower than in the control group. Analysis of cytokine levels and macular thickness revealed that IL-1ra, IL-4, IL-8, IFN-c, IP-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1b, and regulated on activation, normal T-cell expressed and secreted (RANTES) were significantly upregulated in patients with thin cystoid macular edema group. CONCLUSIONS. Patients with ocular sarcoidosis had elevated levels of proinflammatory cytokines in vitreous fluids. Different cytokines might contribute to different stages of macular edema. (Invest Ophthalmol Vis Sci. 2012;53:3827‐3833) DOI

The antimetastatic activity of a combination of lentinan and interleukin 2 (IL‐2) was evaluated against spontaneously metastatic 3‐methylcholanthrene‐induced DBA/2.MC.CS.T fibrosarcoma. Although pre‐operative treatment with either IL‐2 or lentinan alone exerted little effect on the reduction of lung metastasis colony numbers (7.1% or 28.4% reduction, respectively), the combination exhibited a synergistic effect (85% reduction). Furthermore, 3 of 13 mice given the pre‐operative combination treatment achieved complete cure, while no mice given saline did. Although the post‐operative combination treatment also reduced the colony number (71% reduction), it caused little prolongation of survival and no mouse achieved complete cure. Synergistic effects were observed between pre‐ and post‐operative treatments with lentinan and IL‐2: 8 of 12 mice were completely cured. The anti‐metastatic activity was abolished in mice treated simultaneously with antibodies to CD4 and CDS antigens, whereas either CD4, CDS, or NK1.1 antibody alone was ineffective. Analysis of the cellular mechanism involved in the antimetastatic activity revealed the involvement of a tumor‐associated antigen‐specific delayed‐type hypersensitivity response. These data suggest that the life‐prolonging effect of the combination of lentinan and IL‐2 is mediated by antigen‐specific T cells and that the combination of pre‐ and post‐operative therapy with lentinan and IL‐2 may be effective to prevent cancer recurrence and metastasis after surgical resection.

Lentinan manifests marked antitumor and antimetastatic activity in numerous tumor/host systems, and prevents chemical and viral carcinogenesis. Modulation of immune or vascular functions by lentinan is involved in its antitumor effects. The impact of lentinan on the functions of macrophages is distinct from that of LPS. One of the effects of lentinan on the vascular system is the vascular dilatation and hemorrhage (VDH) reaction, and the effect can be monitored as augmented skin reactions to vasoactive mediators. Lentinan induces the VDH-like reaction at the tumor site, resulting in the induction of hemorrhagic necrosis and complete regression of the tumor. In contrast to LPS-induced tumor necrosis (Shwartzman's-like reaction), lentinan-induced tumor necrosis is T-cell dependent.

Purpose Compounds regulating intracellular thiol redox status, such as N,N-diacetyl-L-cystine dimethylester (NM(2)), were shown to prolong corneal graft survival in a penetrating keratoplasty (PKP) model. However, the effect of NM(2) on hemangiogenesis and lymphangiogenesis has not been investigated. The effect of manipulating ambient thiol redox status on riskier (higher rejection rate) transplantation models, such as limbal graft survival and hemangiogenesis and lymphangiogenesis in a corneal suture model, were investigated. Methods C57BL/10 mice that received BALB/c corneas were treated by subconjunctival injection of NM(2), and limbal graft survival was assessed. Sutured C57BL/6 received daily intraperitoneal injections of NM(2), glutathione diethylester (GSH-OEt), or PBS. Lymphatic endothelial cell (LEC) and peritoneal mps were treated with NM(2) or GSHOEt, and then VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression were analyzed. Supernatants were collected for analysis of TNF-alpha and VEGF-A levels by ELISA. Results Significantly less cellular infiltration was detected in mice with corneal limbal transplant-treated NM(2)-treated mice. Hemangiogenesis and lymphangiogenesis were suppressed in the NM(2)-treated mice. NM(2) treatment of mps led to reduced levels of VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression compared with PBS- or GSHOEt- treated mps, lower levels of TNF-alpha, and increased secretion of VEGF. Moreover, NM(2)-treated LECs had reduced levels of LYVE-1 and Prox-1. Conclusions Reduction of ambient redox status reduced inflammatory cell infiltrates. Consequently, reduced inflammatory response might have contributed to both the observed prolonged corneal limbal graft survival and the attenuated hemangiogenesis and lymphangiogenesis in cornea.

Although the existence of Fc(epsilon)RI-alphabetagamma(2) and Fc(epsilon)RI-alphagamma(2) receptor subtypes was reported, there has been no direct evidence of these two subtypes of Fc(epsilon)RI in vivo. To investigate the existence of these two subtypes of Fc(epsilon)RI in vivo, the authors evaluated the expression of Fc(epsilon)RI-beta in the giant papillae of chronic allergic conjunctivitis and compared the expression level of Fc(epsilon)RI-beta with control conjunctivae using the anti-human Fc(epsilon)RI-beta antibody.Fc(epsilon)RI-beta expression in giant papillae obtained from patients with atopic keratoconjunctivitis and vernal keratoconjunctivitis in control conjunctivae was evaluated by immunohistochemistry using anti-Fc(epsilon)RI-beta, -alpha, -gamma, and anti-human mast cell tryptase, anti-chymase, anti-basophil, and anti-CD1a antibodies.Statistical analyses revealed that the densities of Fc(epsilon)RI-beta(+) cells, Fc(epsilon)RI-alpha(+) cells, tryptase(+) cells, and Fc(epsilon)RI-beta(+)/tryptase(+) cells were significantly increased in giant papillae compared with controls. There were two types of Fc(epsilon)RI (alphabetagamma(2) and alphagamma(2)) on the mast cells of the giant papillae. The ratio of the Fc(epsilon)RI-beta(+) cell number/Fc(epsilon)RI-alpha(+) cell number in the giant papillae (0.69 +/- 0.08 [mean +/- SD]) was significantly higher than that of the controls (0.07 +/- 0.16). Fc(epsilon)RI-beta/tryptase double immunostaining revealed that 81% +/- 13% of tryptase(+) cells expressed Fc(epsilon)RI-beta. Fc(epsilon)RI-beta(+) cells were preferentially localized within and around epithelial tissue. The authors also found that Fc(epsilon)RI-beta was expressed by basophils but not by Fc(epsilon)RI-alphagamma(2)-positive Langerhans cells in the giant papillae samples.Preferential Fc(epsilon)RI-beta expression observed in the mast cells and basophils of giant papillae suggests important roles of Fc(epsilon)RI-beta in the pathophysiology of atopic keratoconjunctivitis and vernal keratoconjunctivitis.

Interleukin 6 (IL-6) and interleukin 1 (IL-1) were compared as to their ability to induce the proliferation of distinct thymocyte subpopulations. IL-6 functions as a costimulator in IL-1- or IL-2-induced proliferation of adult double-negative (DN) thymocytes, whereas IL-6 alone failed to induce a significant level of proliferation. However, IL-6 alone induced significant proliferation of mature cortisone-resistant thymocytes, whereas IL-1 did not. Instead, IL-1 functioned as a costimulator in IL-6-induced proliferation of mature thymocytes. Finally, both IL-6 and IL-1 were capable of potentiating IL-2-induced proliferation of fetal DN thymocytes. These data suggest that the monokine requirements in thymocyte activation may vary during thymocyte maturation and that IL-6, when compared to IL-1, has a distinct effect on the proliferation of thymocytes.

PURPOSE IkappaBzeta(-/-) mice have been reported to be affected by allergic dermatitis. This study was conducted to analyze the pathophysiological role of IkappaBzeta and to address the functional relevance of Th2-mediated immune responses in the development of ocular surface inflammation and dermatitis by IkappaBzeta(-/-) mice. METHODS BALB/c background IkappaBzeta(-/-) mice were established without individual differences; IkappaBzeta/Stat6 double-knockout (WKO) mice unable to produce Th2 cytokine were created; and microscopic-, histologic-, and immunochemical studies were performed. In IkappaBzeta(-/-) mice the serum IgE levels were examined by ELISA, and quantitative PCR was used to study the gene expression of IFN-gamma, IL4, IL10, TNFalpha, IL6, IL17alpha, and CCL11 in eyelid tissue. RESULTS IkappaBzeta(-/-) mice exhibited a severe inflammatory phenotype on the ocular surface and perioral skin. The inflammatory infiltrates in the perioral skin consisted primarily of CD4(+) and CD8(+) cells; CD4(+) and CD45R/B220(+) cells were mainly detected in the conjunctiva. In eyelid and perioral skin tissue, the expression of IL-17alpha and of Th1 and Th2 cytokines, but not of CCL11, was augmented. IkappaBzeta(-/-) and IkappaBzeta(+/-) mice did not differ significantly in their serum total IgE levels before, 0 to 4 weeks, and 5 to 9 weeks after disease onset. IkappaBzeta/Stat6 WKO mice showed the same or slightly more severe inflammation than did IkappaBzeta(-/-) mice. CONCLUSIONS IgE and Stat6 are not responsible for the immune pathologic response leading to the development of ocular surface and perioral skin inflammation in IkappaBzeta(-/-) mice. IkappaBzeta(-/-) mice may be a suitable model for Stevens-Johnson syndrome, but not for atopic dermatitis.

The existence of adult stem cells or progenitor cells in the human corneal epithelium (i.e., self-renewing squamous cells) has long been suggested, but these cells have not yet been isolated. Here we describe a novel isolation technique using non-tissue culture dishes to enrich progenitor cells, which are able to reconstitute a three-dimensional human corneal epithelial equivalent from single cells in serum-, feeder-, and bovine pituitary extract-free medium. These cells showed original tissue-committed differentiation, a high proliferative capacity, and limited self-renewal. Laminin-5 was measured by mass spectrometric analysis. Pretreatment of cells with anti-laminin-5 antibody demonstrated that laminin-5 was important in allowing corneal epithelial progenitor cells to adhere to non-tissue culture dishes. Hydrophilic tubes (used for cell collection throughout this study) are essential for efficient isolation of adherent corneal epithelial progenitor cells expressing laminin-5. These findings indicate that our new technique using non-tissue culture dishes allows the isolation of progenitor cells from human corneal limbal epithelium and that laminin-5 has a critical role in the adhesion of these cells. Disclosure of potential conflicts of interest is found at the end of this article.

PURPOSE. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute-onset mucocutaneous diseases induced by infectious agents or inciting drugs. The authors previously reported an association between SJS/TEN and IL-4R gene polymorphism that is essential for IL-4 and IL-13 signaling. To examine IL-4 and IL-13 gene polymorphisms and the combination of these polymorphisms with IL-4R polymorphism, the authors performed polymorphism analysis. METHODS. In 76 Japanese SJS/TEN patients with ocular surface complications and 160 healthy controls, the authors analyzed polymorphisms of the promoter - 590C/T in the IL-4 gene and of the promoter -1111C/T and Arg110Gln in the IL-13 gene and assessed Gln551Arg in the IL-4R gene. Because Arg110Gln affects serum IL-13, plasma IL-13 levels were also examined. RESULTS. In the SJS/TEN patients, the Arg110Gln SNP of IL-13 was significantly associated with the disease, and the frequency of Arg110 alleles was significantly higher than that in the controls. Plasma IL-13 tended to be lower in SJS/TEN patients than in the controls. Analysis of the genotype pattern of IL-4R SNP Gln551Arg and IL-13 SNP Arg110Gln showed that the Gln551Gln(A/A>Arg110Arg(G/G) genotype pattern was also associated with SJS/TEN. CONCLUSIONS. IL-13 gene polymorphisms might be associated with SJS/TEN with ocular surface complications. The present findings suggest that SJS/TEN is different from allergic diseases such as atopy and asthma because the ratio of each allele in the IL-13 SNP Arg110Gln was the opposite of the ratio in those diseases. They also reveal that combined polymorphisms in the IL-13/IL-4R signaling pathway were associated with SJS/TEN with ocular surface complications.

Purpose: The expression and function of Toll-like receptor 5 (TLR5) was analysed in human conjunctival epithelial cells (HCjEC). Methods: The expression of TLR5 in HCjEC was studied by reverse transcriptase (RT) PCR and flow cytometry. The amount of interleukin (IL) 6 and IL-8 proteins was determined by ELISA. Messenger RNA expression elicited by stimulation with flagellins derived from Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhimurium, and Bacillus subtilis was assayed by quantitative RT–PCR. The localisation of TLR5 protein in human conjunctival epithelium was detected immunohistochemically. Results: HCjEC expressed TLR5-specific mRNA and TLR5 protein. In HCjEC stimulated with flagellins derived from P aeruginosa and S marcescens, IL-6 and IL-8 production was increased and IL-6 and IL-8 mRNA was upregulated. Flagellins from S typhimurium and B subtilis did not induce the upregulation of these genes and proteins. TLR5 protein was detected on the basolateral but not the apical side of human conjunctival epithelium. Conclusions: Human conjunctival epithelium harbours functional TLR5. Considering the spatially selective basolateral localisation of TLR5 protein, it was postulated that flagellins from ocular pathogenic bacteria induce inflammatory responses when disruption of the epithelial barrier permits their transmigration to the basolateral side but not under healthy physiological conditions on the ocular surface.

PURPOSE Previous reports have shown genetic predisposition for atopic dermatitis (AD). Some of the severe complications of AD manifest in the eye, such as cataract, retinal detachment, and keratoconjunctivitis. This study was conducted to examine the genetic association between the atopy-related genes and patients with ocular complications (ocular AD). METHODS Seventy-eighty patients with ocular AD and 282 healthy control subjects were enrolled in an investigation of the association between the atopy-related genes (FCERB, IL13, and IFNGR1) and ocular AD. Genetic association studies and functional analysis of single nucleotide polymorphisms (SNPs) were performed. RESULTS The -56TT genotype in the IFNGR1 promoter region was significantly associated with an increased risk of ocular AD under recessive models (chi(2) test, raw P = 0.0004, odds ratio 2.57). The -56TT genotype was more common in atopic cataracts. A reporter gene assay showed that, after stimulation with IFN-gamma, the IFNGR1 gene promoter construct that contained the -56T allele, a common allele in ocular AD patients, manifested higher transcriptional activity in lens epithelial cells (LECs) than did the construct with the -56C allele. Real-time PCR analysis demonstrated higher IFNGR1 mRNA expression in the LECs in atopic than in senile cataracts. iNOS expression by IFNGR1-overexpressing LECs was enhanced on stimulation with IFN-gamma and LPS. CONCLUSIONS The -56T allele in the IFNGR1 promoter results in higher IFNGR1 transcriptional activity and represents a genetic risk factor for atopic cataracts.

Background Stevens-Johnson syndrome (SJS) and its severe form, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucous membranes, including the ocular surface, oral cavity, and genitals. These reactions are very rare but are often associated with inciting drugs, infectious agents, or both. Objective We sought to identify susceptibility loci for cold medicine–related SJS/TEN (CM-SJS/TEN) with severe mucosal involvement (SMI). Methods A genome-wide association study was performed in 808 Japanese subjects (117 patients with CM-SJS/TEN with SMI and 691 healthy control subjects), and subsequent replication studies were performed in 204 other Japanese subjects (16 cases and 188 control subjects), 117 Korean subjects (27 cases and 90 control subjects), 76 Indian subjects (20 cases and 56 control subjects), and 174 Brazilian subjects (39 cases and 135 control subjects). Results In addition to the most significant susceptibility region, HLA-A , we identified IKZF1 , which encodes Ikaros, as a novel susceptibility gene (meta-analysis, rs4917014 [G vs T]; odds ratio, 0.5; P = 8.5 × 10 −11 ). Furthermore, quantitative ratios of the IKZF1 alternative splicing isoforms Ik1 and Ik2 were significantly associated with rs4917014 genotypes. Conclusion We identified IKZF1 as a susceptibility gene for CM-SJS/TEN with SMI not only in Japanese subjects but also in Korean and Indian subjects and showed that the Ik2/Ik1 ratio might be influenced by IKZF1 single nucleotide polymorphisms, which were significantly associated with susceptibility to CM-SJS/TEN with SMI.

Background. Healthy C57BL/6 orthotopic corneal allografts in place for more than 8 weeks in BALB/c mice (acceptor8w+) can survive indefinitely due to active suppression of the donor-specific delayed-type hypersensitivity (DTH) response (1). This suggests a state of tolerance in the acceptor mice, however, the mechanism(s) underlying this acceptance remains to be demonstrated. We investigated the relationship between tolerance-induction and the DTH response using murine re-grafting models to explore the possibility of promoting allogeneic corneal regraft acceptance in high-risk graft beds. Methods. Acceptor8w+ BALB/c mice received C57BL/6- or C3H corneal regrafts onto the same eye. Re-grafting models were prepared by inducing corneal neovascularization in the graft beds of naive BALB/c mice 2 weeks before corneal allografting. These mice were intravenously (iv) injected with purified splenic T cells or T-cell-depleted splenocytes from acceptor8w+ mice at the time they received re-grafts of C57BL/6 corneas. We also iv injected acceptor8w+ splenocytes into mice bearing healthy primary corneal allografts for 4 weeks (acceptor4w) and assessed their DTH response to C57BL/6 alloantigen(s). In those experiments, acceptor4w mice received a C57BL/6 corneal regraft onto the same eye. Results. In all acceptor8w+ mice there was indefinite survival of C57BL/6-, but not of C3H regrafts. The iv injection of T cells, but not of T-cell-depleted populations, from acceptor8w+ splenocytes promoted allograft survival. Acceptor4w mice iv injected with acceptor8w+ splenocytes manifested a reduced C57BL/6-specific DTH response and the survival rate of C57BL/6 regrafts was increased from 0% to 87.5%. Conclusion. As donor-specific T cells from acceptor8w+ mice induced prolonged regraft survival, we posit that the active suppression of DTH responses by T cells may have contributed to indefinite allogeneic regraft survival via the induction of corneal allograft tolerance.

The redox status of macrophages (M phi), indexed by intracellular content of glutathione (icGSH), varies sequentially along with disease progression in nonobese diabetic mice. At the stage of early insulitis, M phi skew to oxidative M phi (OM phi) with decreased icGSH, then to reductive M phi (RM phi) with elevated icGSH and to OM phi after the occurrence of diabetes. RM phi or OM phi inducing agents either delayed or accelerated the onset of diabetes in a mutually inverse manner. RM phi or OM phi adoptively transferred exacerbated or ameliorated the disease progression depending on the redox status of M phi of recipient mice. The new paradigm that the sequential conversion of redox status of M phi dictates the pathological progression may provide a new insight on the mechanism underlying autoimmune diabetes.

The distinct thiol redox status in macrophages, either elevated or reduced intracellular content of glutathione (GSH), was confirmed during aging in IL-2 receptor (IL-2R)gamma and Janus family tyrosine kinase (JAK)3 gene-disrupted mice. Oxidative macrophages (OMp) with reduced GSH dominated initially at a younger age in both mice. OMp-dominated JAK3 or IL-2R gamma chain-deficient mice showed shortened life longevity compared with wild-type littermates. These mice elicited spontaneous onsets of inflammatory bowel disease (IBD)-like symptoms accompanied with the conversion of the redox status of macrophages to reductive phenotypes with elevated intracellular GSH. Conversion of OMp to the reductive phenotype by GSH monoethyl ester or by a beta-(1-3)-glucan accelerated the disease onset, concomitant with the skewing from T(h)2 to T(h)1 responses. On the contrary, N,N'-diacetyl cystine dimethylester, which is capable of inducing OMp, delayed the incidence of IBD-like symptoms and improved the survival rate. This implies that the conversion of OMp/T(h)2 to reductive macrophages/T(h)1 may be critical for the disease progression. The study of these mice may provide insight into the mechanisms underlying Crohn's disease and ulcerative colitis.

We have been proposing the functional distinction of two classes of macrophages (Mp), namely the reductive macrophages (RMp) with high intracellular content of glutathione (GSH) and the oxidative macrophages (OMp) with reduced content. At the same time we have been investigating the variation of RMp/OMp balance during aging of mice, especially in relation to the age related onset of autoimmune diseases. In this paper we have investigated the Th1/Th2 balance of thioredoxin (TRX) transgenic (Tg) mice, with prolonged life longevity, during aging in the context of the intracellular redox status of Mp, which has been hypothesized to be crucial in regulating the Th1/Th2 balance. It was confirmed that peritoneal resident Mp of Tg mice showed the higher GSH/GSSG ratios compared with that of age matched wild type (WT) mice. The predominance of RMp was associated with the sustained maintenance of Th1 prevalence during aging until 2 years in Tg mice, whereas WT littermates showed rapid polarization to Th2 around the age of 8 months. The Tg mice showed elevation of IFN-gamma and reduction of IL-10 with moderate change of IL-4 produced by CD4+ T cells. The WT mice showed inverse changes of IFN-gamma/IL-4 and IFN-gamma/IL-10 ratios during aging. In addition, IL-10 production by Mp was dramatically reduced in aged Tg mice. Thus, TRX Tg mice may be useful to investigate the contribution of the anti-oxidant defense mechanism during aging accompanied with increasing oxidative stress.

SUMMARYInterleukin-12 (IL-12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon-γ (IFN-γ) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN-γ and IL-4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL-12 production in human alveolar macrophages (AM). We used both AM from healthy non-smokers obtained by bronchoalveolar lavage and the monocytic THP-1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N-acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)-induced IL-12 secretion by AM. In THP-1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA, whereas L-buthionine-[S,R]-sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN-γ or the helper T cell type 2 (Th2) cytokine IL-4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)-induced secretion of IL-12 in AM was enhanced by IFN-γ but inhibited by IL-4. These results suggest that IFN-γ and IL-4 oppositely affect the GSH/GSSG balance, which may regulate IL-12 secretion from AM in response to LPS.

Peptides of human melanomas recognized by CD8+ CTLs have been identified, but the nature of those of nonmelanoma tumors remains to be elucidated. Previously, we established a gastric signet ring cell carcinoma HST-2 and HLA-A31 (A*31012)-restricted autologous CTL clone, TcHST-2. In the present study, we determined the natural antigenic peptides of HST-2 cells. The purified preparation of acid-extracted Ags was submitted to the peptide sequencer, and one peptide, designated F4.2 (Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile), appeared to be immunogenic. To confirm the antigenicity of F4.2 further, we constructed an expression minigene vector (pF4.2ss) coding adenovirus E3, a 19-kDa protein signal sequence plus F4.2. An introduction of pF4.2ss minigene to HST-2 and HLA-A31(+) allogeneic tumor cells clearly enhanced and induced the TcHST-2 reactivity, respectively. Furthermore, when synthetic peptides of F4.2 C-terminal-deleted peptides were pulsed to HST-2 cells, F4.2-9 (nonamers), but not F4.2-8 or F4.2-7 (octamer or heptamer, respectively), enhanced the reactivity of TcHST-2, suggesting that the N-terminal ninth Trp might be a T cell epitope. This was confirmed by lack of antigenicity when using synthetic substituted peptides as well as minigenes coding F4.2 variant peptides with Ala or Arg at the ninth position of F4.2. Meanwhile, it was indicated that the sixth position Ile was critically important for the binding to HLA-A31 molecules. Thus, our data indicate that F4.2 may work as an HLA-A31-restricted natural antigenic peptide recognized by CTLs.

Purpose Gelatinous drop-like corneal dystrophy (GDLD) is characterized by subepithelial amyloid deposition that engenders severe vision loss. The exact mechanism of this disease has yet to be elucidated. No fundamental treatment exists. This study was conducted to establish an immortalized corneal epithelial cell line to be used as a GDLD disease model. Methods A corneal tissue specimen was obtained from a GDLD patient during surgery. Corneal epithelial cells were enzymatically separated from the cornea and were dissociated further into single cells. The epithelial cells were immortalized by the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcriptase (hTERT) genes. For the immortalized cells, proliferative kinetics, gene expressions, and functional analyses were performed. Results The immortalized corneal epithelial cells continued to proliferate despite cumulative population doubling that exceeded 100. The cells showed almost no sign of senescence and displayed strong colony-forming activity. The cells exhibited a low epithelial barrier function as well as decreased expression of tight-junction-related proteins claudin 1 and 7. Using the immortalized corneal epithelial cells derived from a GDLD patient, we tested the possibility of gene therapy. Conclusions We established an immortalized corneal epithelial cell line from a GDLD patient. The immortalized cells exhibited cellular phenotypes similar to those of in vivo GDLD. The immortalized cells are thought to be useful for the development of new therapies for treating GDLD corneas and for elucidation of the pathophysiology of GDLD.

Lentinan, an antitumor polysaccharide used clinically in Japan, requires the intact T cell compartment to manifest its antitumor effects. The aim of the current study was to clarify the mechanisms playing crucial roles in the T cell requirement in the expression of antitumor effects of lentinan. Lentinan treatment of BDF1 mice transplanted intradermally with FBL-3 induced complete tumor regression and a marked increase in survival time. The antitumor action of lentinan was abolished in mice treated simultaneously with antibodies to CD4 and CD8 antigens, whereas antibody to CD4, CD8 or NK1.1 alone was ineffective. The natural killer, cytotoxic T lymphocyte, and helper T cell activities were already augmented in this FBL-3/BDF1 system and thus further augmentation of these activities by lentinan was not observed. These activities did not correlate with the antitumor activity of lentinan, as was confirmed in lymphocyte subset depletion experiments. On the contrary, the delayed-type hypersensitivity (DTH) response against tumor-associated antigens was triggered by lentinan and was abrogated only in mice treated simultaneously with antibodies to CD4 and CD8 antigens. Furthermore, a non-cytolytic tumor-associated antigen-specific CD4+ T cell clone able to induce the DTH response in concert with lentinan reconstituted the antitumor effects in B6 nude mice when administered with lentinan. These results suggest that, in addition to the augmentation of immune effector cell activity against tumors, infiltration of these cells into the tumor burden initiated by the DTH responses at tumor sites may be involved in eradication of tumors by lentinan.

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a beta 1-3 glucan with beta 1-6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8+ CTL but not by CD4+ T cells or NK1.1+ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.

Among 15 anti-DNA antibody-producing hybridomas derived from a single NZB X NZW F1 mouse, an IgM and an IgG were shown to use the same VH gene of the Q52 family. Using a combination of two primers both consisting of a mixture of oligonucleotides (one complementary to the 5' end of VH segment and one to the 3' end of VH segment of Q52 family) we determined the sequences of several members of germ-line VH genes in the Q52 family derived from NZB and NZW strains. Comparison of the sequences with those of cloned VH cDNA obtained from the hybridomas revealed that the VH sequence of the IgM anti-DNA antibody was identical to that of a cloned NZW germ-line VH gene, except for the priming sites. In contrast, the VH sequence of the IgG counterpart contained somatically mutated nucleotides. Because the IgG anti-DNA antibody showed a higher DNA binding activity than did the IgM antibody, we conclude that these changes in nucleotide sequences were induced and selected through an antigen-driven mechanism as is the case in a normal immune response. It is tempting to speculate that the germ-line encoded, low-affinity IgM autoantibody undergoes somatic mutations and isotype switching, resulting in generation of pathogenic, high-affinity autoantibodies in autoimmune diseases.

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.

Purpose It has been widely accepted that Th1- and IFN-gamma-mediated immune responses are indispensable for corneal allograft rejection in BALB/c hosts. The present study was designed to determine the role of IFN-gamma and IL-17 in the rejection by C57BL/6 hosts, which display high rejection rates. Methods MHC-matched or -mismatched corneal allografts were grafted onto IFN-gamma-knockout (GKO), IFN-gamma-receptor-knockout (GRKO), IL-17-knockout (IL-17KO), or wild-type (WT) C57BL/6 hosts. Graft fates were assessed clinically and histologically. At appropriate time intervals after allografting, RNA was isolated from corneal graft parenchymal and stromal tissues and cervical lymph nodes. The cytokine mRNA levels of Th1, -2, and -17 type were analyzed by real-time PCR. Results No significantly prolonged allograft survival was observed in any combinations. The rejected MHC-mismatched corneas in GKO elicited intensive infiltration of eosinophils, CD11b(+) macrophages, and B cells, but few Gr-1(+)CD11c(-) neutrophils. In contrast, rejected MHC-matched corneas in GKO hosts, as well as GRKO and WT hosts, elicited intensive infiltration of CD11b(+) macrophages and Gr-1(+)CD11c(-) neutrophils, but no B220(+) B cells and eosinophils. At 1 week after MHC-matched allografting, mRNA levels of IL-6 and IL-17A in the lymph node were extensively upregulated in GKO hosts. It is of interest that anti-IFN-gamma treatment did not improve the allograft survival in IL-17KO hosts. Conclusions IFN-gamma and IL-17 play no critical role in the development of minor-specific allograft rejection in C57BL/6 mice. This indicates the presence of sophisticated rejection mechanisms that are still elusive and cannot be ascribed simply to Th1, -2, or -17.