Search

Inhibition of fibrosis is indispensable for maintaining filtering blebs after glaucoma filtration surgery (GFS). The purpose of this study was to investigate the ability of a pluripotent epigenetic regulator OBP-801 (OBP) to ameliorate extracellular matrix formation in a rabbit model of GFS. Rabbits that underwent GFS were treated with OBP. The gene expression profiles and intraocular pressure (IOP) were monitored until 30 postoperative days. The bleb tissues were evaluated for tissue fibrosis at 30 postoperative days. In in vitro models, OBP interfered the functions of diverse genes during the wound-healing process. In in vivo GFS models, the expressions of TGF-β3, MMP-2, TIMP-2 and 3, LOX, COL1A and SERPINH1 were significantly inhibited at 30 postoperative days in the OBP group compared with those in the vehicle control group. OBP treatment involving subconjunctival injection or eye drops showed no adverse effects, and reduced levels of α-SMA and collagen deposition at the surgical wound site. OBP maintained the long-lived bleb without scar formation, and IOP was lower at 30 postoperative days compared with the vehicle control group. These findings suggest that OBP is an effective and useful candidate low-molecular-weight agent for improving wound healing and surgical outcomes in a rabbit model of GFS.

This study investigated the pathophysiological features of pachychoroid neovasculopathy (PNV) and neovascular age-related macular degeneration (nAMD) by analysing and comparing cytokine profiles in aqueous humour (AH) collected from 18 PNV, 18 nAMD and 11 control patients. Responses to intravitreal injection of aflibercept were also analysed in the PNV and nAMD groups. In the PNV group, vascular endothelial growth factor (VEGF)-A was significantly lower than in the nAMD group (p = 0.03) but was almost identical to that in the control group (p = 0.86). The nAMD group showed positive correlations between interleukin (IL)-6 and IL-8 (r = 0.78, p r = 0.68, p = 0.002) and IL-8 and MCP-1 (r = 0.68, p = 0.002). In the nAMD group, eyes with dry maculae one month after the first aflibercept injection showed significantly lower VEGF-A and placental growth factor (PlGF) at baseline than those with wet maculae (p = 0.02 for both). However, there was no significant difference between dry and wet maculae in the PNV group. The results suggest that angiogenic factors and proinflammatory cytokines may play the distinct roles in the pathogenesis of PNV and nAMD.

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 The injection of cultured human corneal endothelial cells (cHCECs) into the anterior chamber (AC) is a newly developed modality for the successful treatment of corneal endothelium dysfunction. Here, we investigated whether or not cHCECs could be labeled using quantum dots (QDs) composed of semiconductor nanoparticle octa-arginine (R8) to trace injected cHCECs and examined the utility of in vivo fluorescence imaging to analyze the dynamics and accumulation of QD-labeled injected cHCECs in a corneal endothelial dysfunction mouse model. Methods The cHCECs, either of high quality or with cell-state transition, were labeled by adding a mixture of QDs655 and R8. The labeling efficiency and the unchanging of the cell phenotypes by the labeling was confirmed by flow cytometry. The labeled cHCECs were injected into the AC of either healthy mice or mice with corneal endothelium damaged by cryogenic treatment. The kinetics of the injected cHCECs was traced quantitatively via multiphoton confocal laser microscopy. Results QD labeling induced no morphologic change in the cHCECs or in the expression of the functional markers of cHCECs (i.e., Na+/K+-ATPase and zonula occludens-1). The injected cHCECs-QDs were quantitatively detected, and the retention of cHCECs-QDs was evident, from 3 to 48 hours post cell injection on the posterior surface in the cryogenically injured corneal endothelium mouse model eyes, yet not in the noninjured healthy control eyes. Conclusions The findings of this study show that in the field of regenerative medicine, QD labeling of cells presents a convenient and sensitive method of finely monitoring the fate of injected cells in vivo.

Dysfunction of the corneal endothelium reduces the transparency of the cornea and can cause blindness. Because corneal endothelial cells have an extremely limited proliferative ability in vivo, treatment for corneal endothelial dysfunction involves the transplantation of donor corneal tissue. Corneal endothelium can also be restored via intraocular injection of endothelial cells in suspension after their expansion in vitro. Yet, because quality assessment during the expansion of the cells is a destructive process, a substantial number of the cultured cells are lost. Here, we show that the 'spring constant' of the effective interaction potential between endothelial cells in a confluent monolayer serves as a biomarker of the quality of corneal endothelial cells in vitro and of the long-term prognosis of corneal restoration in patients treated with culture-expanded endothelial cells or with transplanted corneas. The biomarker can be measured from phase contrast imaging in vitro and from specular microscopy in vivo, and may enable a shift from passive monitoring to pre-emptive intervention in patients with severe corneal disorders.

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.

The corneal endothelium maintains corneal transparency; consequently, its dysfunction causes severe vision loss. Tissue engineering-based therapy, as an alternative to conventional donor corneal transplantation, is anticipated to provide a less invasive and more effective therapeutic modality. We conducted a preclinical study for cell-based therapy in a primate model and demonstrated regeneration of the corneal endothelium following injection of cultured monkey corneal endothelial cells (MCECs) or human CECs (HCECs), in combination with a Rho kinase (ROCK) inhibitor, Y-27632, into the anterior chamber. We also evaluated the safety and efficacy of Good Manufacturing Practice (GMP)-grade HCECs, similar to those planned for use as transplant material for human patients in a clinical trial and we showed that the corneal endothelium was regenerated without adverse effect. We also showed that CEC engraftment is impaired by limited substrate adhesion, which is due to actomyosin contraction induced by dissociation-induced activation of ROCK/MLC signaling. Inclusion of a ROCK inhibitor improves efficiency of engraftment of CECs and enables cell-based therapy for treating corneal endothelial dysfunction as a clinically relevant 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.

Corneal endothelial cells (CECs) show poor regenerative ability in humans, and noncompensatory damage of CECs causes irreversible corneal haziness in cases of bullous keratopathy. Although corneal transplantations provide considerable clinical benefits, allograft rejection, primary graft failure, and the shortage of donor corneas are problems that still need to be overcome. The establishment of new treatment therapies is the key to solving these problems, and we have attempted to establish a new clinical intervention for corneal endothelial dysfunction. We previously demonstrated that the inhibition of Rho/Rho kinase (ROCK) signaling by Y-27632, a specific ROCK inhibitor, promoted cell adhesion, inhibited apoptosis, and enhanced cell proliferation in cultured primate CECs. These results raise the possibility that the ROCK inhibitor might serve as a new tool for establishing an effective culture method for newly emerging cultivated CEC transplantation therapies. Moreover, because Y-27632 enhances cell proliferation in vitro, we hypothesized that the use of a ROCK inhibitor could be a new pharmacological intervention for the treatment of corneal endothelial dysfunction. We demonstrated that the topical instillation of a ROCK inhibitor promotes corneal endothelium wound healing in an animal model. This indicates that use of a ROCK inhibitor is a less invasive and novel therapy that should prove promising for the treatment of corneal endothelial dysfunction.

Gelatinous drop-like dystrophy (GDLD) is a rare autosomal recessive form of corneal dystrophy characterized by subepithelial amyloid depositions on the cornea. Previous clinical and laboratory observations have strongly suggested that epithelial barrier function is significantly decreased in GDLD. Despite the decade-old identification of the tumor-associated calcium signal transducer 2 (TACSTD2) gene as a causative gene for GDLD, the mechanism by which the loss of function of this causative gene leads to the pathological consequence of this disease remains unknown. In this study, we investigated the functional relationship between the TACSTD2 gene and epithelial barrier function. Through the use of immunoprecipitation and a proximity ligation assay, we obtained evidence that the TACSTD2 protein directly binds to claudin 1 and 7 proteins. In addition, the loss of function of the TACSTD2 gene leads to decreased expression and change in the subcellular localization of tight junction-related proteins, including claudin 1, 4, 7, and ZO1 and occludin, both in diseased cornea and cultured corneal epithelial cells. These results indicate that loss of function of the TACSTD2 gene impairs epithelial barrier function through decreased expression and altered subcellular localization of tight junction-related proteins in GDLD corneas.

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.

Objective This study aimed to evaluate in situ tissue-engineered esophagus in a canine model after experimental resection and replacement of a full circumferential defect of the intrathoracic esophagus. Methods Two types of scaffolding were fabricated. In the KF(+) group (n = 6), oral keratinocytes and fibroblasts cultured on human amniotic membrane were sheeted on polyglycolic acid felt with smooth muscle tissue and were then rolled around tubes. In the KF(−) group (n = 6), the same procedure was followed, but the keratinocytes and fibroblasts were omitted. Both scaffolds were wrapped in omentum and implanted in the abdomen. In the KF(+) group, at 3 weeks after implantation, the scaffold developed into a tube with a well-differentiated lumen of stratified squamous cells surrounded by a thick smooth muscle-like tissue (in situ tissue-engineered esophagus). A part of the esophagus was resected and replaced by the graft in the same dogs. Results In the KF(−) group, strictures developed after esophageal replacement, with almost complete obstruction within 2 to 3 weeks. In contrast, in the KF(+) group, the in situ tissue-engineered esophagus showed good distensibility and the dogs remained without feeding problems through 420 days. Esophageal peristalsis transferred food to the stomach, despite the absence of peristaltic activity in the in situ tissue-engineered esophagus itself. The thickness of the squamous epithelial layer and the smooth muscle layer of the in situ tissue-engineered esophagus were similar to that of the adjacent native esophagus. Conclusion The in situ tissue-engineered esophagus can successfully replace the intrathoracic esophagus, and this procedure may offer a promising surgical approach to esophageal diseases.

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.

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.

Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs) has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs) is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β) receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na(+)/K(+)-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.

Purpose Cultured human corneal endothelial cells (cHCECs) are anticipated to become an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, cHCECs reportedly tend to exhibit chromosomal abnormality during in vitro cell division, thereby hampering their use in the clinical setting. The purpose of this study was to clarify whether a specified subpopulation (SP) of heterogeneous cHCECs would exhibit aneuploidy, whereas other SPs would not. Methods The presence of SPs in cHCECs was analyzed on the basis of surface cluster of differentiation (CD) antigen CD166, CD105, CD44, CD26, and CD24 expression levels by flow cytometry. Cytogenetic examination was performed for 23 lots of cHCECs, either as whole-cell preparations (bulk) consisting of mixed SPs or as a semipurified SP by magnetic activated cell sorting (MACS). The HCEC donors ranged from 9 to 69 years of age and the culture passages from primary to fifth passage. Results Flow cytometry analysis demonstrated the presence of at least three cHCEC SPs. One SP, purified by MACS, with surface expression of CD166+, CD105-, CD44-, CD24-, and CD26- did not show any aneuploidy in 50 cells. However, CD166+, CD44+++, CD24-, and CD26+ cHCEC SPs showed sex chromosome loss in all cells (60 cells), whereas CD166+, CD44+++, CD24+, and CD26- SPs exhibited, albeit partly, trisomy on chromosomes 6, 7, 12, and 20. Conclusions We found that cHCEC aneuploidy is linked to specified SPs present in cHCECs and that the SP sharing the surface phenotype with mature HCECs in corneal tissues was devoid of the karyotype abnormality.

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.

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.

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.

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.

Although hypovasculature is an outstanding characteristic of pancreatic cancers, the tumor cells survive and proliferate under severe hypoxic, glucose-deprived conditions caused by low blood supply. It is well known that the hypoxia-inducible factor-1 pathway is essential for the survival of pancreatic cancer cells under hypoxic conditions. To discover how pancreatic cancer cells adapt to glucose deprivation as well as hypoxia, we sought glucose deprivation–inducible genes by means of a DNA microarray system. We identified 63 genes whose expression was enhanced under glucose-deprived conditions at >2-fold higher levels than under normal glucose conditions. Among these genes, asparagine synthetase (ASNS) was studied in detail. Although it is known to be associated with drug resistance in leukemia and oncogenesis triggered by mutated p53, its function is yet to be determined. In this study, we found that glucose deprivation induced the overexpression of ASNS through an AMP-activated protein kinase–independent and activating transcription factor-4–dependent manner and that ASNS protects pancreatic cancer cells from apoptosis induced by glucose deprivation itself. ASNS overexpression also induced resistance to apoptosis triggered by cisplatin [cis-diammine-dichloroplatinum (CDDP)] and carboplatin, but not by 5-fluorouracil, paclitaxel, etoposide, or gemcitabine. We show that glucose deprivation induces the activation of c-jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK) in a mock transfectant but not in an ASNS transfectant. Consequently, an inhibitor of JNK/SAPK decreased the sensitivity of pancreatic cancer cells to apoptosis by glucose deprivation and CDDP. These results strongly suggest that ASNS is induced by glucose deprivation and may play a pivotal role in the survival of pancreatic cancer cells under glucose-deprived conditions. [Cancer Res 2007;67(7):3345–55]

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.

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.

Purpose To evaluate the effect of Rho kinase (ROCK)-inhibitor eye drops on a corneal endothelial dysfunction primate model and human clinical case series of corneal endothelial dysfunction. Methods As a corneal-endothelial partially injured model, the corneal endothelium of seven cynomolgus monkeys was damaged by transcorneal freezing; 10 mm of rock inhibitor Y-27632 was then applied topically 6 times daily. The phenotype of the reconstructed corneal endothelium was evaluated by immunohistochemical analysis and noncontact specular microscopy. For clinical study, the effect of Y-27632 eye drops after transcorneal freezing was evaluated in eight corneal endothelial dysfunction patients: four central corneal edema patients and four diffuse corneal edema patients. Results Slit-lamp microscopy revealed that both Y-27632-treated and -nontreated corneas became hazy after transcorneal freezing, and then recovered their transparency within 4 weeks. ROCK inhibitor Y-27632 promoted recovery of corneal endothelial cell density and wound healing in terms of both morphology and function. The percentage of ZO-1 and Na(+)/K(+)-ATPase positive cells in the regenerated area in the Y-27632 group was significantly higher than in the controls. Noncontact specular microscopy revealed that corneal endothelial cell density was significantly higher in the Y-27632 group compared with the controls at 4 weeks; cell density reached approximately 3000 cells/mm(2), as opposed to 1500 cells/mm(2) in the control group. In addition to the animal study findings, the clinical study findings showed that Y-27632 eye drops effectively improved corneal edema of corneal endothelial dysfunction patients with central edema. Conclusions These findings show that rock inhibitor Y-27632 eye drops promote corneal endothelial wound healing in a primate animal model and suggest the possibility of Y-27632 as a novel therapeutic modality for certain forms of corneal endothelial dysfunction. (http://www.umin.ac.jp/ctr/ number, UMIN000003625.).

Purpose Proliferative eye diseases, such as proliferative vitreoretinopathy and proliferative diabetic retinopathy, are caused partly by fibrotic change of retinal pigment epithelial cells (RPECs). The purpose of our study was to examine the effect of the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist on the fibrotic change of primate RPECs. Methods Monkey RPECs (MRPECs) isolated from a cynomolgus monkey eye were subcultured. To induce fibrotic change, MRPECs were cultured with TGF-β2 (3 ng/mL), and also cultured in the coexistence of TGF-β2 and the PPAR-γ agonist pioglitazone (30 μM). The phenotype of the cultured MRPECs was evaluated by phase contrast microscopy and immunocytochemical analysis. The phosphorylation of Smad2/Smad3 proteins was examined by Western blot analysis. Results Primary MRPECs were cultured as a monolayer with a hexagonal cell shape, and positive expression of ZO-1, Na(+)/K(+)-ATPase, and RPE65 was confirmed. Cell morphology and the expression of these markers were maintained in the presence of pioglitazone, whereas the cells were elongated and the expression of these markers was reduced in its absence. Conversely, the expression of phalloidin, α-smooth muscle actin, and fibronectin was reduced in the presence of pioglitazone, whereas it was increased in the absence. Western blot assay demonstrated that phosphorylation of Smad2/Smad3 proteins was suppressed by pioglitazone. Conclusions The PPAR-γ agonist pioglitazone inhibited the fibrotic change of primary MRPECs through the suppression of TGF-β signaling. Pioglitazone might prove to be a clinically applicable and effective pharmaceutic treatment for proliferative eye diseases.

Purpose Subretinal fibrosis causes damage to visual acuity, especially if the lesion is in the macula, as is frequently observed in advanced age-related macular degeneration. Exudate leukocytes form abnormal vessels that initiate regional inflammation accompanied with local glial proliferation and matrix production. The purpose of this study was to establish an animal model of focal subretinal fibrosis. Methods Macrophage-rich peritoneal exudate cells (PECs) were injected into the subretinal space of C57BL/6 or MCP-1 knockout (KO) mice. Seven days later, the size of the subretinal fibrotic tissue was evaluated by the adherent area of glial fibrillary acidic protein (GFAP)-positive retinal glial cells on choroidal flat mounts. Myofibroblastic changes and collagen synthesis were detected by α-smooth muscle actin (α-SMA) and Masson trichrome staining of the histologic section, respectively. α-SMA expression was also examined on retinal pigment epithelium (RPE) cells during co-culture with activated macrophages. Results Subretinal fibrous tissue was observed by funduscopy in PEC-injected mice after 7 days. The tissue consisted of a monotonous, low-cell-density area that expressed α-SMA with collagen synthesis. Both steroid and antioxidant treatment can reduce residual glia. Because PEC-injected MCP-1 KO mice showed less residual glia, not only exogenous macrophages, but also intrinsic macrophages were activated. The macrophages directly induced myofibrotic changes in RPE cells in vitro. Conclusions Activated macrophages form subretinal fibrosis when they are placed in the subretinal space and induce myofibrotic changes in RPE cells.

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. The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs. METHODS. MCECs of cynomolgus monkeys were cultured in a medium containing 10 M Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry. RESULTS. The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin Vpositive apoptotic cells was decreased at 24 hours. CONCLUSIONS. The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation. (Invest Ophthalmol Vis Sci. 2009;50:3680‐3687) DOI:10.1167/iovs.082634

PURPOSE The ocular surface epithelium is part of the mucosal defense system. Because transcription factor NF-kappa B in mucosal epithelial cells plays a central role in regulating the genes that govern the onset of mucosal inflammatory responses, we examined the role of a regulator of NF-kappa B, I kappa B zeta, in murine ocular surface inflammation. METHODS The eyes of I kappa B zeta(-/-) mice were analyzed biomicroscopically and histologically. I kappa B zeta expression in normal mouse cornea and conjunctiva was examined by RT-PCR. The results were compared with those obtained in other tissues by real-time PCR. I kappa B zeta mRNA on the ocular surface and in other mucosal tissues was localized by in situ hybridization. RESULTS I kappa B zeta(-/-) mice manifested chronic inflammation, specifically in the ocular surface, but not in other tissues. In normal mice, I kappa B zeta was expressed in a variety of mucosal tissues. The I kappa B zeta transcript was predominantly distributed in the epithelia of these tissues. As inflammatory symptoms progressed on the ocular surface of I kappa B zeta(-/-) mice, inflammatory cells, mainly CD45R/B220(+) and CD4(+) cells, intensely infiltrated the submucosa of the conjunctival epithelia. This infiltration was accompanied by an almost complete loss of goblet cells in the conjunctival epithelia. CONCLUSIONS The authors postulate that I kappa B zeta in the ocular surface epithelia negatively regulates the pathologic progression of ocular surface inflammation.

The cytotoxic ability of interleukin 2 (IL2)-induced T cells and the effect on their cytotoxicity of serum suppressive factors obtained from cancer patients were examined. Peripheral blood mononuclear cells (PBL) of healthy adults were IL2-cultured for 72 h either together with a gastric carcinoma cell line MKN-28 or with phytohemagglutinin, then IL2-cultured for another 11 days. Cytotoxicity of the MKN-28 precultured lymphocytes against MKN-28 was particularly increased. Presence during preculture or during cytotoxicity assay of sera from gastric cancer patients having the same histological type as MKN-28 suppressed the cytotoxicity. However, the serum suppressive effect on cytotoxicity of the IL2-cultured lymphocytes was far milder than that on PBL cytotoxicity.

Despite the enormous efforts of researchers around the world, promising cancer chemotherapeutics do not as yet exist, excluding some agents acting against certain cancer types with special characteristics and against certain leukemias. Chemotherapeutic agents possessing direct cytocidal activities on target cancer cells intend to kill the total mass of cancer, but they clearly also have accompanying detrimental toxic side effects on various lymphoid cells and bone marrow cells which have recently been considered to have relevant roles in host defense-surveillance mechanisms against cancer and they, therefore, limit the effectiveness of immunotherapy.

MANY polysaccharides obtained from different origins have recently been found to inhibit the growth of transplantable solid tumours in mice1–7.

POLYSACCHARIDE preparations from bamboo leaves1, bagasse2,3 and wheat straw4 inhibit the growth of sarcoma 180 implanted subcutaneously in mice. It was proposed that this activity was host mediated, not cytocidal.