Search

Pollinosis, or allergic conjunctivitis and rhinitis induced by pollen, is one of the most common diseases worldwide. In Japan, Japanese cedar (Cryptomeria japonica) pollinosis is a predominant allergic condition that affects more than one-third of all Japanese individuals. Pharmacological treatments of allergic conjunctivitis include administration of antiallergic eye drops containing an antihistamine or mast cell stabilizer. However, these topical treatments provide transient relief from symptoms. The only available curative treatment for allergic diseases is allergen-specific immunotherapy. Sublingual immunotherapy for pollinosis has been found to be effective for suppression of ocular and nasal symptoms, but patient compliance is low. Oral administration of staple foods engineered to express allergens is a possible means of delivering antigens for immunotherapy, and its convenience would be expected to improve compliance. With the aim of developing more convenient, effective, and safe immunotherapies for allergic diseases, we have generated rice-based edible vaccines expressing antigens derived from dust mites or pollen from Japanese cedar, birch, or ragweed. In this study, we summarize the results of our immunotherapy studies using transgenic rice. Oral immunotherapy with transgenic rice seeds containing hypoallergenic modified forms of Japanese cedar pollen antigens was effective for both preventing allergic conjunctivitis and suppressing established disease in mice. Oral administration of transgenic rice seeds is thus a promising approach to immunotherapy for conjunctivitis and rhinitis induced by Japanese cedar pollen.

Purpose: The synthesis of cytokines and adhesion molecules by corneal fibroblasts contributes to the innate immune response to corneal infection. The effects of the antimicrobial peptide LL37 on cytokine and adhesion molecule expression induced by bacterial lipopolysaccharide (LPS) in human corneal fibroblasts were examined., Methods: The release of the cytokines IL-6 and IL-8 into culture supernatants and the expression of intercellular adhesion molecule (ICAM)-1 at the cell surface were measured with ELISAs and by flow cytometry. The abundance of mRNAs was quantitated by RT and real-time PCR analysis, and the phosphorylation of signaling proteins was examined by immunoblot analysis. The subcellular localization of ICAM-1 and the transcription factor nuclear factor (NF)-κB was determined by immunofluorescence analysis. Neutrophil infiltration in a mouse model of LPS-induced keratitis was evaluated by immunohistofluorescence analysis., Results: The antimicrobial peptide LL37 inhibited the up-regulation of IL-6, IL-8, and ICAM-1 both at protein and mRNA levels in corneal fibroblasts induced by LPS without affecting those elicited by TNF-α. Furthermore, LL37 attenuated the LPS-induced phosphorylation of the NF-κB inhibitor IκBα and the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun NH2-terminal kinase, as well as the translocation of NF-κB to the nucleus in corneal fibroblasts. Lipopolysaccharide-induced keratitis in mice was also suppressed by topical application of LL37., Conclusions: The inhibition of LPS-induced cytokine and adhesion molecule expression in human corneal fibroblasts by LL37 suggests that this peptide might promote the resolution of corneal inflammation associated with bacterial infection.

Background/aims: To determine whether oral immunotherapy with transgenic rice seeds expressing hypoallergenic modified antigens suppresses cedar pollen-induced allergic conjunctivitis by eliciting immune tolerance in mice., Methods: BALB/c mice were fed once a day for 20 days with 220 mg of transgenic rice expressing modified Japanese cedar pollen allergens Cry j 1 and Cry j 2 or with non-transgenic rice seeds as a control. They were then sensitised with two intraperitoneal injections of Japanese cedar pollen in alum before challenge twice with pollen in eye drops. Twenty-four hours after the second challenge, the conjunctiva, spleen, and blood were isolated for histological analysis, cytokine production assays, and measurement of serum immunoglobulin E concentrations, respectively., Results: The numbers of eosinophils and total inflammatory cells in the conjunctiva were significantly lower in mice fed the transgenic rice than in those fed non-transgenic rice. The clinical score evaluated at 15 min after antigen challenge was also significantly lower in mice fed the transgenic rice than in those fed non-transgenic rice. The serum concentrations of both total and allergen-specific immunoglobulin E were also significantly lower in mice fed the transgenic rice. Oral vaccination with transgenic rice resulted in significant down-regulation of the allergen-induced production of interleukin (IL)-2, IL-4, IL-5, IL-12p70, interferon-γ, and IL-17A by splenocytes., Conclusions: Oral immunotherapy with transgenic rice expressing modified Japanese cedar pollen allergens suppressed pollen-induced experimental allergic conjunctivitis in mice by eliciting immune tolerance. This novel prophylactic approach is potentially safe and effective for allergen-specific oral immunotherapy in allergic conjunctivitis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Introduction: The objective of this study was to compare the efficacy and safety of olopatadine versus epinastine in healthy Japanese adults with a history of allergic conjunctivitis to Japanese cedar pollen., Methods: This Phase IV double-blind randomized controlled clinical trial comprised three clinical visits over 30 days. Screening tests were performed to identify subjects with a history of allergic conjunctivitis to Japanese cedar pollen in terms of skin sensitivity and positive bilateral reactions to a conjunctival allergen challenge (CAC) with Japanese cedar pollen at Visit 1, and confirmation by a positive bilateral CAC reaction at Visit 2. At Visit 3, the subjects were randomized to receive one drop of olopatadine HCl ophthalmic solution 0.1% (olopatadine) in the left or right eye (1:1 ratio). All subjects received one drop of epinastine HCl ophthalmic solution 0.05% (epinastine) in the contralateral eye as an active control. Five min later, the subjects underwent bilateral CAC tests with one drop of the allergen solution at the concentration that elicited positive reactions at Visits 1 and 2. Efficacy outcomes included the severity of ocular itching at 5, 7, and 15 min and the severity of conjunctival hyperemia at 7, 15, and 20 min after the CAC test, as graded by the investigator by biomicroscopy., Results: Fifty people participated in this study (25 per group). Olopatadine significantly reduced ocular itching at 7 and 15 min (both p<0.05) and conjunctival hyperemia at 7 and 20 min (p=0.0010 and p<0.05, respectively) after allergen exposure compared with epinastine. There were no adverse events for either treatment., Conclusion: The results of this single-dose study suggest that olopatadine is superior to epinastine in terms of suppressing ocular itching and hyperemia induced by Japanese cedar pollen during CAC tests. Further studies are needed to confirm these findings in real-life settings.

Background: The objective of this study was to investigate the efficacy of topical 0.1% tacrolimus in treating refractory allergic conjunctivitis with proliferative lesions and/or corneal involvement., Methods: This prospective observational study included 1436 patients with refractory allergic conjunctivitis whose condition had responded poorly to conventional antiallergic drugs and/or topical steroids and/or topical cyclosporine. All patients received tacrolimus eye drops twice daily during the study period. Ten clinical signs and six clinical symptoms were rated on a four-grade scale. The primary endpoint was the change from baseline in total clinical signs and symptoms score at the last observation or following 6 months of treatment., Results: Total signs and symptoms score significantly decreased after 1 month of treatment (p<0.001). Giant papillae and corneal lesions were also reduced by tacrolimus eye drop use (p<0.001). The drug proved effective in patients whose condition did not respond well to topical cyclosporine therapy. About 50% of all patients using topical steroids were weaned. The most common adverse reaction was a transient burning sensation (3.20%)., Conclusions: Tacrolimus eye drops are highly effective in treating refractory allergic conjunctivitis with proliferative lesions and/or corneal involvement, and may reduce or replace topical steroid use., Trial Registration Number: UMIN 000008640., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Disruption of corneal epithelial barrier function by inflammation may contribute to the development of dry eye. The effects of rebamipide, a drug used for the treatment of dry eye, on barrier function and cytokine expression in a human corneal epithelial (HCE) cell line were examined., Methods: Barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance (TER). The subcellular localisation of the tight junction protein zonula occludens (ZO)-1 was examined by immunofluorescence analysis. The release of cytokines was determined with ELISAs, and the intracellular abundance of cytokine mRNAs was quantitated by reverse transcription and real-time PCR analysis. Degradation of the nuclear factor-κB inhibitor IκBα was detected by immunoblot analysis., Results: Rebamipide increased TER of HCE cells in a concentration-dependent manner as well as attenuating the loss of TER and the disappearance of ZO-1 from the cell surface induced by tumour necrosis factor α (TNFα). Rebamipide also suppressed TNFα-induced expression of interleukin-6 and interleukin-8 at the mRNA and protein levels and inhibited the TNFα-induced degradation of IκBα., Conclusions: The upregulation of barrier function and the anti-inflammatory effects of rebamipide, together with its mucin secretagogue activity, may contribute to the effectiveness of this drug for the treatment of dry eye.

Background/aims: To investigate the effect of Ag doses and administration time points on oral tolerance induction in experimental allergic conjunctivitis (EAC)., Methods: BALB/c mice were actively sensitised twice with ovalbumin (OVA) in alum, and then challenged twice with OVA in eye drops. Twenty minutes after the last challenge, the clinical appearance was evaluated. Twenty-four hours later, the conjunctivas, spleens and blood were collected for histological analyses, cytokine production assays, and measurement of serum Ig levels, respectively. The mice were fed with a high or low dose of OVA before sensitisation (prophylactic treatment). To assess the effect of therapeutic treatment, the high-dose Ag was administered after sensitisation. Control groups received phosphate-buffered saline without OVA. To determine the role of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in prophylactic low-dose oral tolerance, mice were injected intraperitoneally with neutralising antibodies during the entire experimental period. To assess the role of regulatory T cells, neonatally thymectomised mice were injected with depleting anti-CD25 monoclonal antibodies before the low-dose prophylactic treatment., Results: OVA-feeding was suppressive even when the mice were treated soon after Ag sensitisation. Both the low and high doses of oral OVA suppressed EAC. High-dose treatment significantly suppressed EAC-related cytokine production and serum Ig levels. IL-10 and TGF-β were less likely to be involved in prophylactic low-dose oral tolerance induction but regulatory T cells played a role., Conclusions: Prophylactic and early therapeutic treatment of OVA feeding suppressed EAC. Both high and low doses of oral OVA induced oral tolerance but with different mechanisms.

Purpose: To evaluate the efficacy of subjective symptoms, objective findings, and of a total tear IgE test kit in the diagnosis of allergic conjunctival diseases (ACD)., Subjects and Methods: The subjects were 223 patients with ACD, diagnosed according to the clinical practice guidelines for allergic conjunctival diseases in 28 medical facilities in Japan. The ACD patients were divided into the following five groups: 84 patients with seasonal allergic conjunctivitis (SAC), 52 patients with perennial allergic conjunctivitis (PAC), 41 patients with atopic keratoconjunctivitis (AKC), 38 patients with vernal keratoconjunctivitis (VKC) and 8 patients with giant papillary conjunctivitis (GPC). All cases were examined for clinical ACD, subjective symptoms, objective findings by the clinical score and by a total tear IgE test. An eosinophil examination by conjunctival smear was also performed in 87 ACD patients., Results: The most prevalent subjective symptoms were itching (81.6%) and hyperemia (77.6%). The clinical scores of the objective findings in the patients with SAC, PAC, AKC, VKC and GPC were 16.3 +/- 3.8 (mean +/- SD) points, 16.2 +/- 2.8, 19.8 +/- 6.5, 23.1 +/- 5.3, and 21.4 +/- 3.9, respectively. In the total tear IgE test 72.2% of the ACD patients were positive; i.e., the ratios were 61.9% in SAC, 65.4% in PAC, 80.5% in AKC, 94.7% in VKC, and 75.0% in GPC. In the examination of eosinophils in the 87 ACD patients, 42.5% (37 eyes) were positive, i.e., the ratio was 20.0% in SAC, 36.8% in PAC, 53.3% in AKC, 75.0% in VKC and 33.3% in GPC. The rate of agreement between the total tear IgE test and the examination of eosinophils was kappa = 0.28 (Cohen kappa coefficient)., Conclusion: Because of the high positive ratio in patients quasi-definitively diagnosed with ACD, we conclude that the total tear IgE test is useful as an auxiliary method for diagnosing ACD.

Purpose: To establish a specific quality of life (QOL) questionnaire for Japanese allergic conjunctival disease (ACD) (Japanese allergic conjunctival disease QOL questionnaire: JACQLQ)., Subjects and Methods: A multicenter study was conducted in 521 patients with ACD and 127 healthy volunteers (total 648 cases). The JACQLQ ver. 0 was developed by modifying the Japanese rhino-conjunctivitis QOL questionnaire (JRQLQ). The participants were asked to complete the questionnaire, and objective scores were determined by an ophthalmologist using a slit lamp. To confirm validity, item and factor analyses were conducted and correlation coefficients were calculated., Results: The items were grouped into four subscales (Daily activity, Psychological well-being, Eye symptoms, Nasal symptoms) after factor analysis. The JACQLQ had good item-internal consistency (Cronbach's alpha: 0.846-0.934). QOL scores were correlated with eye itching, eye irritation and tearing. Objective scores were correlated with eye redness, eye itching and eye irritation. Face scores were correlated with eye itching, eye irritation and eye redness., Conclusion: The JACQLQ is a useful tool for assessing disease specific QOL in ACD.

Purpose: To evaluate the effectiveness and safety of topical cyclosporine 0.1% in patients with vernal keratoconjunctivitis (VKC)., Methods: All patients who were prescribed topical cyclosporine 0.1% during the 2 year period after market launch were registered and followed up for 6 months. We assessed the severity of 6 subjective ocular symptoms, such as itching and 10 objective signs, such as giant papillae in the tarsal conjunctiva., Results: All scores for symptoms and signs in 2597 patients with VKC significantly decreased throughout a 6-month follow-up. For total symptoms scores, mean changes from baseline at 1, 3, and 6 months were -4.0, -4.8, and -5.1, respectively. Total sign scores were -4.6, -6.1, and -6.5, respectively. Thirty percent of topical steroid users were able to discontinue the steroids within 3 months. Adverse drug reactions (ADRs) were found in 7.44% of patients, the most common ADR being eye irritation. Ocular infections were observed in 1.44% of patients. During long-term use of topical cyclosporine 0.1%, there was no increase in the incidence of ADR and no late-onset ADRs., Conclusions: Topical cyclosporine 0.1% is an effective and safe treatment for VKC over the long term, provided care is exercised to avoid the occurrence of infection.

Purpose: Interleukin (IL)-17-producing Th17 cells play a crucial role in the development of experimental autoimmune uveoretinitis (EAU). Recent studies revealed that the production of cytokines such as IL-17 is controlled by microRNA (miRNA). Here the authors investigated the expression of miRNA in the eye during the development of EAU., Methods: To induce EAU, B10.RIII mice were injected with interphotoreceptor retinoid binding protein peptide 161-180 with adjuvants. Control mice received adjuvants alone. Seven, 14, 21, or 28 days after immunization, eyes were harvested for histologic analysis, miRNA array, quantitative real-time polymerase chain reaction (qRT-PCR) for cytokines and miRNAs, and in situ hybridization for miRNAs. Expression levels of cytokines and miRNAs were compared with control mice., Results: No histologic changes were observed in eyes collected at day 7. At day 14, EAU was most severe, and thereafter retinal structure was gradually destroyed. Retinal inflammation was not observed in control mice. IL-17A and IL-17F were significantly higher in the eyes of EAU-induced mice at day 7. Array analysis followed by qRT-PCR revealed that in EAU-induced mice, miRNA-142-5p and miRNA-21 were significantly higher, whereas miRNA-182 was significantly lower. These changes could be detected 7 days after EAU induction. In situ hybridization analysis for these miRNAs confirmed qRT-PCR data., Conclusions: Expression changes in three miRNAs could be detected in the eye before histologic EAU. Kinetic changes of these miRNAs in the eye paralleled those of IL-17. The possibility that miRNAs can affect IL-17 suggests that miRNAs in the retina regulate the development of EAU.

Purpose: Antigen (Ag)-presenting cells (APCs) participate in the development of allergic conjunctivitis (AC). However, the conjunctival APCs that take up Ag in AC have not been identified. We sought to clarify the phenotypes of conjunctival APCs that take up, process, and present Ag to T cells during the development of experimental AC., Methods: Splenocytes from naïve ovalbumin (OVA)-specific T cell receptor transgenic (DO11.10) mice were stimulated with either OVA, gold, or OVA-gold to evaluate stimulation-induced proliferation. Naïve DO11.10 mice were subconjunctivally injected with PBS, gold, or OVA-gold. Twenty-four hours later, conjunctivas were harvested for immunohistochemistry and electron microscopy to identify cells that engulfed OVA-gold., Results: Stimulation of DO11.10 splenocytes with OVA-gold but not gold alone induced similar levels of proliferation as OVA alone. Subconjunctival injection of OVA-gold, but not gold alone, induced infiltration of eosinophils and CD4-positive T cells into the conjunctiva. The cells that took up OVA-gold into their cytoplasma expressed Cluster of Differentiation (CD) 11b, CD68, major histocompatibility complex (MHC) class II., Conclusions: It appears that conjunctival macrophages are APCs in the development of experimental AC.

Objective: To investigate the histological and clinical relation between eyes with true exfoliation (TEX) and a double-ring sign (DRS)., Study Design: Retrospective clinical case study., Participants: Twenty-four eyes of 22 patients who underwent cataract surgery at Kochi Medical School Hospital and its affiliated hospitals during the period from April 1994 to September 2007., Methods: Twenty-four anterior lens capsules excised in cataract surgery underwent a histological examination and their clinical backgrounds were investigated. The TEX group consisted of 13 eyes with capsular delamination on the anterior lens capsule confirmed by slit-lamp microscopy before the surgery, and the DRS group consisted of 11 eyes that showed a double-ring sign during capsulorrhexis., Results: Twenty-two eyes underwent phacoemulsification and aspiration after uneventful capsulorrhexis. Two eyes that showed phacodonesis underwent extracapsular cataract extraction. In all specimens the capsular delamination was confirmed. In some specimens from both groups, vesicle spaces were observed in the capsule and the underlying epithelium. In the TEX group, 8 eyes had capsular complications, such as pseudoexfoliation and phacodonesis, and in the DRS group, 2 eyes had phacodonesis (p = 0.016, student's t test)., Conclusions: In spite of different clinical processes leading to capsular delamination, there were not any specific histological differences distinguishing the TEX group from the DRS group. The incidence of capsular complications was significantly higher in the TEX group than in the DRS group.

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.

The immune response is regulated, in part, by effector cells whose activation requires multiple signals. For example, T cells require signals emanating from the T cell antigen receptor and co-stimulatory molecules for full activation. Here, we present evidence indicating that IgE-mediated hypersensitivity reactions in vivo also require cognate signals to activate mast cells. Immediate hypersensitivity reactions in the conjunctiva are ablated in mice deficient in eotaxin-1, despite normal numbers of tissue mast cells and levels of IgE. To further define the co-stimulatory signals mediated by chemokine receptor 3 (CCR3), an eotaxin-1 receptor, effects of CCR3 blockade were tested with an allergic conjunctivitis model and in ex vivo isolated connective tissue-type mast cells. Our results show that CCR3 blockade significantly suppresses allergen-mediated hypersensitivity reactions as well as IgE-mediated mast cell degranulation. We propose that a co-stimulatory axis by CCR3, mainly stimulated by eotaxin-1, is pivotal in mast cell-mediated hypersensitivity reactions.

Background: We showed previously that alpha-galactosylceramide (alpha-GalCer) treatment elevated splenic CD4+CD25+Foxp3+ T-cell numbers and suppressed the development of experimental allergic conjunctivitis (EC). Here, we investigated whether CD4+CD25+Foxp3+ T cells mediate the suppressive effects of alpha-GalCer treatment on EC., Methods: To deplete CD4+CD25+Foxp3+ T cells, neonatal mice were thymectomized and intraperitoneally injected with anti-CD25 Ab. At 6 weeks of age, these mice were immunized with ragweed (RW) in aluminum hydroxide. Ten days later, the mice were challenged with RW in eye drops and 24 hours later, the conjunctivas and spleens were harvested for histological and flow cytometric analyses, respectively. alpha-GalCer or vehicle was injected 2 hours prior to RW challenge. In addition, alpha-GalCer was injected into thymus-intact EC-developing mice that had not been treated with anti-CD25 Ab., Results: alpha-GalCer treatment significantly suppressed EC in the thymus-intact mice that had not been treated with anti-CD25 Ab. In contrast, alpha-GalCer treatment of thymectomized and anti-CD25 Ab-treated mice did not affect the severity of EC or splenic CD4+CD25+Foxp3+ T-cell numbers. However, alpha-GalCer treatment did significantly increase splenic CD4+CD25+Foxp3+ T-cell numbers in thymectomized mice that had not received anti-CD25 Ab., Conclusions: alpha-GalCer treatment during the effector phase of EC increased CD4+CD25+Foxp3+ T-cell numbers, which in turn suppressed the development of EC.

Purpose: To evaluate retrospectively the management and prognosis of Merkel cell carcinoma of the eyelid in Japanese patients., Subjects and Methods: Cases diagnosed as Merkel cell carcinoma of the eyelid from January 1993 to February 2005 in 111 institutions in Japan were included in this retrospective study. Management and prognosis were evaluated., Results: The total number of cases enrolled was 21(5 male and 16 female patients). Excision of the tumor was carried out in 18 cases. Two cases were treated with either irradiation or local injection of interferon after biopsy of the tumor. After initial treatment, there were recurrences in 3 cases; local recurrence in one case and nodal metastasis in two cases. No patient died because of Merkel cell carcinoma of the eyelid., Conclusions: Excision with wide surgical margins with irradiation is recommended as the first choice of treatment for Merkel cell carcinoma of the eyelid. Because the number of patients was only 21 and the duration of observation was short, further investigation is necessary to determine the optimal management and more accurate prognosis for Merkel cell carcinoma.

Background: CD25+ regulatory T (T reg) cells play a suppressive role in experimental autoimmune uveoretinitis as well as experimental airway inflammation but their involvement in the development of allergic conjunctivitis (AC) remains unclear. We therefore investigated whether T reg cells play a role in the development of experimental AC (EC)., Methods: BALB/c and C57BL/6 mice were actively immunized with ragweed (RW). The mice were treated with an anti-CD25 Ab (PC61) or control normal rat IgG (nrIgG) either 2 days prior to active immunization or during the induction phase (days 0, 2, 4, 6 and 8). Ten days after active immunization, the mice were challenged with RW-containing drops. Twenty-four hours after the challenge, the conjunctivas were harvested for histological analysis of eosinophil infiltration, and the spleens were harvested for cell culture for splenocyte transfer. Cultured splenocytes were transferred into syngeneic mice, and 4 days after the transfer, the recipient mice were challenged with RW. Twenty-four hours after the challenge, conjunctivas were collected for histological analysis., Results: Pretreatment with PC61 did not affect EC in either strain of mice; however, treatment with PC61 during the induction phase significantly suppressed EC in C57BL/6 mice. In contrast, transfer of RW-primed splenocytes from mice treated with PC61 induced EC that was significantly more severe regardless of strain and treatment protocol., Conclusions: The finding that T reg cells play a suppressive role in the development of EC in splenocyte transfer experiments suggests that modulation of T reg cells may be a possible therapy for AC.

Background: We present the clinical case reports of elderly Vogt-Koyanagi-Harada (VKH) disease patients, including the oldest patient at onset to date., Cases: Four patients with VKH disease, whose ages at onset were 68 to 89 years, were treated at Kochi Medical School Hospital between December 2002 and September 2004., Observations: Four elderly patients were diagnosed with VKH disease according to the International Revised Diagnostic Criteria for VKH disease. Two were treated with corticosteroid pulse therapy, but the other two could not tolerate high-dose steroid therapy and were treated with only topical corticosteroids. All had recurrences, which were treated effectively. None of these patients experienced recurrence after sunset glow fundus was observed in the follow-up period., Conclusions: VKH disease is thought to be more common in younger people, but it is not as uncommon in elderly people as is generally believed. Among elderly VKH disease patients, some cannot tolerate high-dose corticosteroid therapy, so we need to treat these patients with only topical corticosteroids., ((c) Japanese Ophthalmological Society 2007.)

Purpose: The severity of allergic conjunctivitis (AC) correlates with the degree of eosinophil infiltration into the conjunctiva, which is believed to be mediated by chemokines and adhesion molecules. The adhesion molecule very late antigen (VLA)-4 and its ligand, vascular cell adhesion molecule (VCAM)-1, are known to play important roles in eosinophil infiltration. However, the expression and function of VLA-4 in AC have not been investigated in detail. We sought to characterize VLA-4-expressing cells in the conjunctivas of mice that are developing experimental AC (EC) and to determine whether the interaction between VLA-4 and VCAM-1 is needed for the infiltration of eosinophils into the conjunctiva in AC., Methods: EC was induced in Balb/c mice by active immunization with ragweed (RW) or adoptive transfer of RW-primed splenocytes, followed by RW challenge. Twenty-four hours after RW challenge, the conjunctivas were harvested. The conjunctivas from naive mice or mice developing EC were evaluated for VLA-4 and VCAM-1 expression by immunohistochemistry and immunofluorescent analyses. To investigate whether the interaction between VLA-4 and VCAM-1 is needed for the genesis of AC, mice developing EC were treated with anti-VLA-4 or anti-VCAM-1 antibodies two h before and after RW challenge. As a control, EC-developing mice were treated with normal rat IgG. Twenty-four hours after RW challenge, the conjunctivas were harvested for histological analysis., Results: Upon induction of EC, VLA-4-expressing cells infiltrated the conjunctiva but the constitutive VCAM-1 expression around conjunctival vessels was not augmented. Immunofluorescent analyses demonstrated that most of the T cells infiltrating the conjunctiva expressed VLA-4 but only half of the infiltrating eosinophils expressed it. Treatment with both anti-VLA-4 and anti-VCAM-1 antibodies significantly suppressed the infiltration of eosinophils into the conjunctiva that was induced by either active immunization or splenocyte transfer., Conclusions: These results confirm that VLA-4-expressing cells infiltrate the conjunctiva and that the interaction between VLA-4 and VCAM-1 is needed for the development of EC.

Purpose: The goal of the present study was to investigate the role of platelet activating factor (PAF) and PAF receptor (PAF-R) in the recruitment of eosinophils into the conjunctiva in the course of PAF induced conjunctivitis. Eosinophils are important players in the immediate hypersensitivity reactions and in allergic conjunctivitis. PAF-R is expressed in many ocular tissues including conjunctival cells. Although it is known that PAF is one of the most potent chemotactic agents for the recruitment of eosinophils, factors responsible for it in conjunctivitis are not clear. Colocalization analysis has been employed to quantify the degree of colocalization of major basic protein (MBP) and PAF-R antigens in the course of PAF induced conjunctivitis., Methods: A 1% solution of PAF was applied in eye drops to male Brown Norway rats. Eyes were harvested with intact conjunctivas at different time points and examined using histology, immunohistochemistry, confocal immunofluorescence microscopy, and reverse transcription-polymerase chain reaction. PAF-R and MBP (a marker of eosinophils) antibodies have been used for immunohistochemical studies. Quantitative analysis of the colocalization of PAF-R and major basic protein (MBP) antigens was performed., Results: Instillation of PAF caused a time dependent recruitment of eosinophils. Eosinophils revealed PAF-R in the intact state. An influx of eosinophils into the conjunctiva was caused by the interaction of PAF with PAF-R and, possibly, with MBP antigen. PAF appeared to enhance the expression of PAF-R by eosinophils and to act toward the PAF-R directly, without chemokine mediation., Conclusions: Quantitative colocalization analysis helped to determine that the recruitment of eosinophils in PAF induced conjunctivitis is accomplished via direct action of PAF toward the PAF-R. It also ensured an objective evaluation of the changes of the degree of colocalization of MBP and PAF-R antigens and the degree of PAF-R expression in dynamics, the findings not otherwise obtainable using qualitative approaches alone.

The NF-kB family transcription factor c-Rel is a critical molecule for inducing expression of cytokine genes by T cells. Here, we report that a deletion of the C-terminal end, similar to the deletion in the highly oncogenic chicken v-Rel gene, renders c-Rel hyperactive toward cytokine gene promoters. At the same time, this mutation dramatically reduced c-Rel activity in induction of IkB-alpha mRNA expression. Moreover, ectopic expression of IkB-alpha, along with the C-terminal truncated c-Rel, abrogates hyperactivity of this mutant. IkB-alpha co-expression did not affect the function of wild-type c-Rel. The data demonstrate that the C-terminal end of c-Rel has specific activity for IkB-alpha mRNA expression and is dispensable for IL-2 gene expression.

Conjunctival papillary formation and corneal damage, which are seen in severe types of allergic conjunctival diseases, are mediated by eosinophils. Eosinophils themselves are not able to recognize a specific antigen (Ag) and thus, Ag-specific T cells are thought to be important for eosinophilic infiltration into the conjunctiva. Cytokines, which are produced by Ag-specific T cells followed by Ag recognition, are considered to be involved in eosinophilic infiltration. Therefore, we investigated the involvement of cytokines, which are produced by immunocompetent cells and conjunctival cells, in the infiltration of inflammatory cells into the conjunctiva, using an animal model for allergic conjunctival diseases (experimental immune-mediated blepharoconjunctivitis, EC). The peak of expression of Th 2 cytokines such as interleukin (IL)-4 in the rat conjunctiva was 6 and 12 hours after Ag challenge. In contrast, expression of Th1 cytokines such as interferon (IFN)-gamma persisted up to 48 hours after Ag challenge. The kinetic change of IL-4 was concordant with eosinophilic infiltration into the conjunctiva and that of IFN-gamma was in accord with infiltration of macrophages. To investigate the roles of these cytokines, we induced conjunctivitis in cytokine knockout mice. The infiltration of inflammatory cells was attenuated in IL-4 knockout mice, whereas it was augmented in IFN-gamma knockout mice. To further elucidate the roles of these cytokines, we induced and analyzed EC in Brown Norway rats. Eosinophilic infiltration was increased in EC induced by the transfer of T cells, which were stimulated by IL-4. In addition, systemic treatment with IFN-gamma inhibited eosinophilic infiltration in EC induced by active immunization, but did not affect infiltration of inflammatory cells in EC induced by passive immunization. These results demonstrate that IL-4 and IFN-gamma are involved in the infiltration of eosinophils and macrophages, respectively. In addition, IFN-gamma exerts its suppressive effects on the development of EC only during the induction phase of EC. Further detailed studies investigating the roles of cytokines in the conjunctiva will elucidate the developing mechanism of allergic conjunctival diseases. These studies will provide important clues for a therapeutic approach in targeting cytokines.

Background: How the early phase allergic reaction affects the late phase reaction remains unclear. We examined this issue with an experimental model of allergic conjunctivitis that permits the two reactions to be disconnected from each other., Methods: Experimental immune-mediated blepharoconjunctivitis (EC) was initiated in Brown Norway rats by transferring ovalbumin (OVA)-specific T cells and then challenging with OVA-containing eye drops. To induce early phase reaction, a mast-cell activator, C48/80, was challenged together with or without OVA. Rats were evaluated clinically and eyes were harvested for histologic examination and for evaluation of chemokine expression by reverse-transcriptase PCR., Results: The rats challenged with OVA alone developed the T-cell-mediated late phase reaction histologically, but not clinically, in the absence of early phase reaction. While rats challenged with C48/80 with or without OVA exhibited clinical signs of the early phase reaction, the clinical late phase reaction was observed only in the OVA+C48/80 group. Eosinophilic infiltration into the conjunctiva during the late phase reaction of the OVA+C48/80 group markedly exceeded that of rats challenged with either OVA or C48/80 alone. RANTES (regulated on activation, normal T-cell expressed and secreted), an eosinophil attractant, was expressed both in the OVA+C48/80 and OVA groups, while eotaxin was expressed at equivalent levels in all three groups., Conclusion: The mast-cell-mediated early phase reaction potentiates the T-cell-mediated late phase reaction, and RANTES is involved in eosinophilic infiltration induced by antigen-specific T cells. Other molecules induced by allergen-specific T cells activated in an as yet unknown manner by the mast cells may be responsible for the infiltration of eosinophils.

Genetic background determines the histological features of experimental immune-mediated blepharoconjunctivitis (EC) in rats, which is a model for human allergic conjunctivitis (AC). A great number of lymphocytes predominate in EC of Lewis rats, while less lymphocytes and more eosinophils are present in that of Brown Norway (BN) rats. Although this difference could be attributed to their systemic Th1/Th2 dominancy, it remains unclear whether some regulatory mechanisms may exist in the inflammatory site in the conjunctiva. Here, we aim to investigate this hypothesis by comparing the expression levels of inflammatory mediators in the conjunctiva between the two strains. EC was induced in Lewis and BN rats by transfer of ovalbumin (OVA)-specific CD4(+) T-cell lines followed by eye drops of OVA as antigen challenge, and then was clinically and histologically evaluated. Reverse-transcription (RT)-PCR was performed to compare the expressions of cytokines and cytokine receptors (Rs) in conjunctivas of both strains of rats either with or without EC. To confirm the biological significance of interferon (IFN)-gamma R expression, phosphorylation of signal transducers and activators of transcription (STAT)-1 was examined in the conjunctivas, followed by subconjunctival injection of IFN-gamma. BN T cells contained interleukin (IL)-4 and IFN-gamma, while Lewis T cells expressed no IL-4. Transfer of those cells induced more severe EC in Lewis rats. RTPCR using naive conjunctivas detected more IL-4, IFN-gamma, and IFN-gamma R beta-chain RNA expression in BN rats. After the EC induction, BN rats expressed significantly higher amounts of IFN-gamma R beta-chain, and upregulation of interferon regulatory factor (IRF)-1 was observed. Phosphorylation of STAT-1 was more remarkable in BN rats. The findings demonstrate differential expression of IFN-gamma R and signaling through IFN-gamma in the conjunctiva between the two strains. This may be due to differences in histopathological character between the two strains.

Purpose: Aluminum hydroxide (Al) is an adjuvant to induce Th2 immune responses. The aim of this study is to investigate the effects of pretreatment with Al on experimental autoimmune uveoretinitis (EAU), a Th1 disease model., Methods: Lewis rats were pretreated with an uveitogenic peptide (#29)/Al, phosphate-buffered saline (PBS)/Al or PBS. EAU was then induced by immunization of #29 in complete Freund's adjuvant (CFA) with intravenous injection of Bordetella pertussis. Three weeks later, EAU was evaluated histologically and antigen-specific cellular immune responses were assessed., Results: EAU was exacerbated in the PBS/Al group and attenuated in the #29/Al group, compared to the control PBS group. Antigen-specific cellular proliferation and interferon-gamma production were augmented in the PBS/Al group and suppressed in the #29/Al group., Conclusions: Suppression or exacerbation of EAU by the pretreatment in this study is related to inhibition or augmentation of antigen-specific Th1 immunity.

Background: Sarcoidosis is a granulomatous disease causing uveitis. Although steroid therapy is usually effective for many patients, some are resistant to this therapy. In such cases, vitrectomy may be chosen as the therapeutic method to treat intraocular inflammation., Case: A 26-year-old Japanese man was diagnosed as having sarcoidosis by clinical findings and histological examination of bronchoalveolar lavage., Observation: One year after diagnosis, vitreous opacity worsened and pars plana vitrectomy (PPV) was performed. Six months later, recurrence of vitreous opacity appeared and severe retinal exudative changes with proliferative vitreoretinopathy developed. One year after the first operation, PPV was repeated and the epiretinal proliferative tissue was removed and examined., Conclusions: Histologic examination of the specimen showed scattered noncaseating granulomatous inflammation mainly consisting of epithelioid histiocytes and lymphocytes. Plasma cells as well as T cells were identified and the predominance of CD8-positive T cells was demonstrated. Taking everything into consideration, a new finding of CD8 predominance in the epiretinal proliferative tissue was demonstrated.

Background: Experimental immune-mediated blepharoconjunctivitis (EC) in Brown Norway (BN) rats, which is inducible by transfer of antigen-specific T cells, is a model for human allergic conjunctivitis. We investigated the possible inhibition of EC in BN rats by topical application of FK506, which is an immunosuppressive agent that mainly targets T cells., Methods: To induce EC by active immunization, ovalbumin (OVA) adsorbed to alum was injected into the hind footpads of BN rats. Three weeks after the initial immunization, rats were challenged with OVA by eye drops. Twenty-four hours later, lids including conjunctivas, lymph nodes (LNs), and sera were harvested for histology or reverse transcriptase PCR, proliferation assays, and measurement of IgE titer, respectively. For passive immunization, rats were intravenously injected with 10 million of in vitro-stimulated OVA-primed LN cells. Four days after the transfer, rats were challenged with OVA and evaluated as above. The rats were divided into two groups. One group received topical FK506 treatment three times per day from 15 to 21 days after active immunization or from 1 to 4 days after transfer. The other group was treated with vehicle as above., Results: FK506 treatment suppressed infiltration of both lymphocytes and eosinophils in the conjunctiva either by active or passive immunization (P<0.002). No differences were noted in antigen-specific cellular and humoral immune responses. Concerning cytokine expression in the conjunctiva, a prominent difference was noted only with IL-4, which was more abundantly detected in the vehicle-treated group., Conclusion: Topical FK506 treatment suppressed EC in BN rats, possibly by inhibition of IL-4 in the conjunctiva.

Abstract: Nitric oxide (NO) inhibits T cell proliferation. We demonstrate that the action of NO on T cell proliferation is different for Lewis and Brown Norway (BN) rats. Splenocytes from Lewis rats consistently showed higher proliferation against concanavalin A than splenocytes from BN rats did. In contrast, NO production was higher in BN rats than in Lewis rats. A depletion of adherent cells increased proliferation in BN rats to a level similar to that in Lewis rats. Thus NO produced by adherent splenocytes could be considered to inhibit proliferation. The addition of NG-monomethyl-L-arginine, a potent inhibitor of NO production, increased proliferation in Lewis rats, but much less so in BN rats. Similar results were obtained by the addition of anti-interferon (IFN)-gamma. It is surprising that, low doses of sodium nitroprusside, an NO donor, increased proliferation in BN rats but not in Lewis rats. To investigate the mechanism of differential NO production between the two strains, splenocytes were stimulated with IFN-gamma. The early signaling event evaluated by the phosphorylation of Stat-1 was similar in both strains, whereas inducible NO synthase (iNOS) mRNA expression seemed more sustained in BN rats. Thus the differential production of NO might be related to the differential transcriptional regulation of iNOS. Altogether, genetic background might be involved in sensitivity to the inhibitory function of NO for T cell proliferation and NO production.