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Purpose: To investigate the expression of dectin-1 protein in conjunctival epithelial cells and the expression of dectin-1 and B-cell activating factor belonging to the tumor necrosis factor family (BAFF) mRNA in in vivo conjunctival epithelial cells (CECs) and in vitro cultured CECs, and its difference in topographical change and etiology of disorders., Subjects and Methods: 1. Investigation of dectin-1 and BAFF expression by cytodiagnosis of CECs. The subjects were 12 eyes of 12 healthy volunteers (control group), 6 eyes of 6 patients with Sjögren syndrome (Sjögren group) and 10 eyes of 10 patients with vernal keratoconjuctivitis (VKC group). CECs were sampled by impression cytology using nitrocellulose membrane. The expression of dectin-1 in CECs was detected by immunofluorescence and the quantitative determination of dectin-1 mRNA and BAFF mRNA expression was performed by real-time polymerase chain reaction(real-time PCR). 2. Investigation of dectin-1 and BAFF expression using cultured CECs. Cultured CECs which were divided into an OK-432 addition group (addition concentrations: 0.02, 0.1, 0.5KU/mL), a lipopolysaccharide (LPS) addition group (addition concentrations : 80, 160, 320 microg/mL) and an additive-free group were cultured. Quantitative determination of dectin-1 mRNA and BAFF mRNA expression in cultured CECs was performed by real-time PCR., Results: 1. Investigation of dectin-1 and BAFF expression by cytodiagnosis of CECs. In the control group, there was no significant topographical difference in the expression of dectin-1 and the amount of dectin-1 mRNA among superior, inferior tarsal conjunctiva and temporal bulbar conjunctiva. The levels of dectin-1 mRNA expression were 1.5 (0.1-4.0) [median value (range)] for the control group, 2.6 (1.1-4.8) for the Sjögren group and 3.6 (1.7-16.6) for the VKC group. The VKC group showed a significantly higher level of dectin-1 mRNA than the control group (p < 0.01, Kruskal-Walles H-test). The levels of BAFF mRNA expression were 2.8 (0.2-13.8) [median value (range)] for the control group, 6.3 (2.1-15.1) for the Sjögren group and 11.2 (3.5-70.8) for the VKC group. The VKC group showed a significantly higher level of dectin-1 mRNA than the control group (p < 0.01, Kruskal-Walles H-test). Moreover, regarding the relationship between expression level of dectin-1 mRNA and that of BAFF mRNA in all the subjects, there was a significant correlation between them (r = 0.75, p < 0.001, Spearman's rank coefficient). The levels of dectin-1 mRNA expression in the moderate and severe VKC group 9.2 (2.6-16.6) [median value (range)] were significantly higher than those in mild VKC group 2.8 (1.7-3.8) (p < 0.05, Mann-Whitney U-test). The levels of BAFF mRNA expression in the severe and moderate VKC groups 17.4 (9.1-70.8) [median value (range)] were significantly higher than those in the mild VKC group 4.3 (3.5-11.2) (p < 0.05, Mann-Whitney U-test). 2. Investigation of dectin-1 and BAFF expression by cultured CECs. In the OK-432 addition group, the expression levels of dectin-1 mRNA were increased dose-dependently due to the OK-432 stimulation (p < 0.05, Kruskal-Wallis H-test). Moreover, regarding the relationship between the expression level of dectin-1 mRNA and that of BAFF mRNA in all the cultured conjunctival epithelial cells stimulated by OK-432, there was a significant correlation between them (r = 0.85, p < 0.005, Spearman's rank coefficient)., Conclusions: We concluded that dectin-1 expression in CECs was demonstrated, and expression of both dectin-1 and BAFF in CECs is thought to be involved in pathologic aggravation of allergic inflammatory in patients with VKC.

Purpose: A retrospective study for evaluating the clinical course of patients with vernal keratoconjunctivitis (VKC) treated with topical tacrolimus ophthalmic suspension 0.1% (Tacrolimus)., Subjects and Methods: Subjects were 30 patients (24 men and 6 women) with VKC who were treated with a combined therapy of Tacrolimus and antiallergic ophthalmic solution, and could be followed up for six months. The subjects were divided into two groups: 1. A conversion treatment group in which Tacrolimus was substituted for a steroid ophthalmic solution [21 patients; average age 14.7 +/- 9.44 years (mean +/- SD)] and 2. An additional treatment group receiving Tacrolimus and anti-allergic ophthalmic solution [9 patients; average age 28.2 +/- 7.31 years (mean +/- SD)]. The therapeutic effects of the patients were evaluated chronologically using the ocular clinical score according to the papillae-limbus-cornea grading score and eosinophil cationic protein (ECP) levels in tears., Results: Papillae-limbus-cornea grading scores were significantly decreased from 8 (median) points at instillation initiation to 5 points at the first month after initiation of Tacrolimus treatment (p < 0.01, Steel test). Tear ECP levels were significantly decreased from 3493.6 (median) ng/ml at instillation initiation to 205.6 ng/ml at the first month after initiation of Tacrolimus treatment (p < 0.05, Steel test). During the course, four cases of exacerbation were found among the 30 cases, but no infections of the anterior segment were found., Conclusion: The therapeutic effect of Tacrolimus eye drops for vernal keratoconjunctivitis was remarkable at one month after instillation initiation. For evaluating the effect of treatment and diagnosing exacerbation in VKC treated with Tacrolimus, a follow-up examination using clinical indexes such as the papillae-limbus-cornea grading score and ECP levels in tears is beneficial.

Purpose: To investigate the role of specific secretary IgA antibodies on lipopolysaccharide (LPS-sIgA) and exotoxin A (ETA-sIgA) in tear fluid produced by immunoresponse to P. aeruginosa., Subjects and Methods: Subjects were divided into 3 groups; 41 eyes of 41 normal volunteers without a history of using contact lenses (CL) as controls, 32 eyes of 32 CL users without adverse events as a healthy CL wearer group (CL group), and 12 eyes of 12 patients with CL-related corneal ulcers from which P. aeruginosa was isolated (ulcer group). Tear fluid was obtained using filter paper, and the concentration of LPS-sIgA and ETA-sIgA in samples was determined using enzyme-linked immunosorbent assay (ELISA)., Results: The lower limit of concentration for LPS-sIgA and ETA-sIgA was 1.24 and 1.24 (U/mL), respectively. The positive eyes which exceeded this lower limit in LPS-sIgA were 17 of 22 eyes in the control group (median concentration 2.00U/ml, range 1.28 to 7.20), 15 of 25 eyes in the CL group (median 1.76 U/ml, range 1.24 to 6.92) and 1 of 12 eyes in the ulcer group (1.80 U/ml). The number of eyes which exceeded the lower limit in ETA-sIgA was 36 of 41 eyes in the control group (median concentration 6.56 U/ml, range 1.36 to 182), 28 of 32 eyes in the CL group (median 5.40 U/ml, range 1.56 to 29.20) and 5 of 12 eyes in the ulcer group (median 1.72 U/ml, range 1.40 to 2.16). There was no significant difference in LPS-sIgA and ETA-sIgA between the control group and the CL group, but they were significantly lower in the ulcer group than in the control group (p < 0.01, Steel's test)., Conclusion: Tear fluid in normal, healthy CL wearers contains LPS-sIgA and ETA-sIgA which acts as a barrier to P. aeruginosa infectious keratitis.

Purpose: To investigate the chemokine profile in tears of patients with infectious keratitis., Subjects and Methods: Subjects were 32 eyes of 16 patients with infectious keratitis and 5 eyes of 5 healthy volunteers as a control. The patients with infectious keratitis were classified into two groups of eyes: 10 with bacterial keratitis and 6 with Acanthamoeba keratitis. Tear fluid was obtained from both eyes of the patients with infectious keratitis and from the right eyes of the control subjects using filter paper. Chemokine concentration (unit: Odu/mm2) and its profile in tears was analyzed using an antibody-array., Results: In terms of chemokine profile in the bacterial keratitis group, the expression volume of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in the diseased eyes was significantly higher than in the healthy eyes (p < 0.05). The expression volume of mucosae-associated epithelial chemokines (MECs) in the diseased eyes of the bacterial keratitis group was significantly lower than in the healthy eyes of that group (p < 0.05). In the Acanthamoeba keratitis group, chemokines were not significantly increased in the diseased eyes compared with those in the healthy eyes. However, MCP-1 was increased in tears of the Acanthamoeba keratitis group. Regarding the chemokine ratio, the IL-8/MEC ratio in the diseased eyes of the Pseudomonas keratitis group and the MCP-1/IL-8 in the diseased eyes of the Acanthamoeba keratitis group showed a significantly high level (p < 0.05)., Conclusion: We concluded that the analyses of the chemokine profile and chemokine ratio in the tears of infectious keratitis patients is useful as a clinical tear laboratory test to interpret the pathologic condition of infectious keratitis

Purpose: To investigate, using tear fluid analysis, the effects of topical 0.1% tacrolims therapy on the pathophysiology of vernal keratoconjunctivitis (VKC)., Subjects and Methods: Subjects were 6 eyes of 6 patients with VKC who underwent topical 0.1% tacrolims treatment twice a day and 5 eyes of 5 healthy volunteers as a control. Using the filter paper method, the tear fluid of the patients was sampled both before and 4 +/- 2 weeks after the treatment and once from the control subjects. Eosinophil cationic protein (ECP) in the tears was examined by the chemiimmunoluminescent enzyme immunoassay method and the chemokine profile of the tears was analyzed using an antibody-array., Results: In terms of the chemokine profile, growth related oncogene (GRO) -alpha, eotaxin-2 and thymus and activation-regulated chemokine (TARC) in the VKC were elevated compared with those in the controls, but they decreased significantly after the treatment (p<0.05). ECP concentrations in the tears were 3092 +/- 1658 ng/ml (average +/- S. D.) for the pretreatment base-line and 464 +/- 775 for the posttreatment. ECP values for the pre-treatment time were statistically significantly higher than those for the post-treatment in 5 patients (p<0.05)., Conclusion: Topical tacrolims treatment of VKC can suppress allergic inflammation associated chemokines such as eotaxin-2 and TARC.

Purpose: To evaluate the variation of chemokines in tears of contact lens wearers., Subjects and Methods: The subjects were divided into the three groups: a control group consisting of 26 eyes of 26 healthy volunteers without contact lenses a contact lens group (CL group) consisting of 30 eyes of 30 healthy contact lens wearers without ocular surface disorders, and a giant papillary conjunctivitis group (GPC group) consisting of 9 eyes of 9 patients with giant papillary conjunctivitis caused by contact lens wearing. Tear samples were taken by the modified Schirmer I method using a filter paper. Tear samples were eluted and analyzed for chemokines including interleukin-8 (IL-8), eotaxin-2, and pulmonary and activation-regulated CC chemokine (PARC) by the antibody array method. Concentrations of IL-8, eotaxin-2, and PARC in tears were determined quantitatively by enzyme-linked immunosorbent assay (ELISA)., Results: Using the antibody array method, the expression of IL-8, eotaxin-2, and PARC in the GPC group was 4-fold higher or greater than in the control group. In the measurement by ELISA, IL-8 levels in the GPC group (1154.5 +/- 1739.3 pg/ml) (mean +/- SD) were significantly higher (Kruskal-Wallis test, p < 0.01) than in the control (75.2 +/- 88.7 pg/ml) and CL (153.6 +/- 252.8 pg/ml) groups. The eotaxin-2 levels in tears did not show a statistical difference among the three groups. The PARC level in tears of the GPC group (2859.6 +/- 2299.9 pg/ml) was significantly higher than in the control(589.0 +/- 324.8 pg/ml) and CL (671.7 +/- 536.2 pg/ml) groups (Kruskal-Wallis test, p < 0.05)., Conclusion: Wearing a contact lens per se disorders, does not cause chemokine variation in tears. However, an increase of IL-8 which induces neutrophilic invasion and an increase of PARC which induces lymphocyte invasion play an important roles in increasing the risk factor of GPC when wearing contacts.

Purpose: To evaluate the efficacy of measurement of secretory IgA (sIgA) in tears with tear sampling methods using filter paper, and to review sIgA measurement method for clinical application., Subjects and Methods: Subjects were divided into the following 4 groups: a healthy control group, 29 eyes of 29 subjects; a contact lens group, 15 eyes of 15 subjects; a dry eye group, 13 eyes of 13 subjects; and a herpes group, 6 eyes of 6 subjects. In all subjects the sIgA value in tears was measured. In addition, in eighteen eyes of 18 healthy control individuals, the tear sIgA value was measured three times, morning, noon, and night in one day, and the variation of tear sIgA value was checked. The tears were sampled by the Schirmer 1 method. Schirmer papers were eluted in 200 microl of 0.5 M NaCl and 0.5% Tween 20 in 0.05 M phosphate-buffered solution (pH 7.2). Tear sIgA value was measured by ELISA (enzyme-linked immunosorbent assay)., Results: Tear sIgA value was 1249.0+/-1025.0 microg/ ml in the healthy control group; 1057.4+/-1583.3 microg /ml in the contact len groups; 197.8+/-91.3 microg/ml in the dry eye group; and 759.7+/-467.8 microg/ml in the herpes group. There was no significant difference between the healthy control group and the contact lens group, or the control group and the herpes group. There was a significantly (p<0.0001) low value in the dry eye group in comparison with the healthy control group. In the 18 healthy control individuals, there was a tendency for the tear sIgA value collected at noon to be higher., Conclusion: Measurment of the changes in tear sIgA values caused by inflammation of the lacrimal gland is useful as a clinical test of lacrimal gland function.

Purpose: In the patients with seasonal allergy conjunctivitis (SAC), we investigated the concentration of tear eosinophil cationic protein (ECP) levels, the diagnosis of SAC and the usefulness of studying the disease states., Subjects and Methods: The subjects were patients suffering from conjunctivitis seasonal allergy caused by cedar pollen and a healthy control group, and the study period was three years from 2001 to 2003. We divided the allergy group into 27 eyes of 27 patients that we measured before the pollen season and 44 SAC eyes of 44 patients that we measured during the pollen season. The healthy control group was 23 eyes of 23 healthy adults. Tears were collected by the schirmer I method. The ECP value of specimens was measured by enzyme-linked immunosorbent assay (ELISA) using samples which we eluted in buffer (0.5 M NaCl + 0.5% tris buffer saline with Tween 20) from Schirmer test paper., Results: The measurement range of ELISA was 3.6-180 microg/l. The mean ECP value above the lower limit of ELISA in tears was 5.06 +/- 3.39 microg/l (n = 7) (mean +/- standard deviation) for the healthy control group, 9.32 +/- 6 .68 (n = 11) for the preseason group, and 54.90 +/- 117.74 (n = 29) for the pollen season group. The difference between the healthy group and the seasonal group was highly significant (p < 0.005), but there was no significant difference between the healthy group and the preseason group. We set the cut-off point at the 95th percentile (11.1 microg/l) of the healthy group and considered any value above cut-off as positive. The ECP value was positive in 2.5% (3 eyes out of 27) in the preseason group and 40.9% (18 of 44) in the seasonal group, and there was a significant difference (p < 0.01) between the seasonal group and the healthy group. When the seasonal group was divided into patients who had started treatment before the pollen season and those who began treatment during the season, we found a small ECP difference, but it was not statistically significant., Conclusion: Measurement of the ECP value in seasonal pollen allergy sufferers may be useful for pathological ascertainment, but it dose not provide adequate diagnosis because of the large number of false negative cases.

Purpose: To investigate the role of Staphylococcus aureus in patients with vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) complicated by atopic dermatitis (AD)., Subjects and Methods: Microbiological culture from the conjunctival sac of acute exacerbated patients with VKC and AKC complicated by AD was performed. The subjects were 29 patients (31 eyes) with VKC and AKC who showed acute exacerbated clinical symptoms of conjunctivitis. Antigen specific IgE antibodies to staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) in serum were also examined. In this study, the patients were divided into two groups: An AD group consisting of 22 patients with VKC and AKC complicated by AD, and a control group consisting of 8 patients with allergic conjunctivitis without AD. We also examined SEA/SEB specific IgE antibody in tears from the 5 patients who underwent bacteriological examinations of the conjunctival sac at the same time., Results: Twenty-seven out of 31 eyes were gram-positive in the bacteriological culture from the conjunctival sac (87.1%). Staphylococcus aureus was detected in 21 out of the 27 eyes (77.8%). In the AD group, 2 of the 22 cases were gram-positive for serum SEA specific IgE antibodies, 2 cases of the 22 cases were gram-positive for SEB specific IgE antibodies, and 9 cases were gram-positive for both SEA and SEB specific IgE antibodies. Serum SEA and SEB specific IgE antibodies were all gram-negative in the control group. Either SEA or SEB specific IgE antibody in tears was detected in all of the above 5 patients who underwent bacteriological examinations of the conjunctival sac, and 4 of the 5 were gram-positive. Staphylococcus aureus was isolated in 3 out of the 5 patients, and 1 case was gram-negative., Conclusion: Staphylococcus is one of the exacerbating factors in VKC and AKC. It is important to evaluate both bacteriological examinations of the conjunctival sac and SEA/SEB specific IgE in tears.

Purpose: A histopathological study of lattice corneal dystrophy with L 527 R mutation of exon 12 on transforming growth factor-beta induced gene(TGFBI)., Patient and Methods: The patient was a 66-year-old woman with lattice corneal dystrophy. Gene analysis and histopathological study of corneal tissue obtained at the corneal transplantation were performed. Mutation analysis was done for exon 4, 11, and 12 on the TGFBI gene by direct sequencing. A histological study was performed by light microscopy with hematoxylin-eosin staining, dylon staining, or congo red staining, and by electron microscopy., Results: Gene analysis revealed L 527 R mutation of exon 12 on the TGFBI gene. Light microscopic examination showed dylon-positive and Congo red-positive amyloid deposit from the superficial to deep layer of the corneal stroma. In transmission electron microscopy, the corneal epithelial layer, the basement membrane of the epithelium, Descemet's membrane, and the corneal endothelium were normal, but irregularity of Bowman's membrane and amyloid fibers in the corneal stroma were observed., Conclusion: Lattice corneal dystrophy due to L 527 R mutation showed histopathological findings similar to those of lattice corneal dystrophy type III.

Purpose: To investigate a method for determining antigen specific IgE antibodies in tears of patients with allergic keratoconjunctival disease., Subjects and Methods: Antigen specific IgE antibodies to Japanese cedar pollen or Housedust-Mites in tears of patients with allergic conjunctival diseases were examined. Both eyes in each patient were examined. The right eye was examined in normal healthy volunteers as a control. The number of eyes examined for IgE antibodies to Japanese ceder pollen was 32 eyes with seasonal allergic conjunctivitis(SAC), 12 eyes with perennial allergic conjunctivitis(PAC), 12 eyes with atopic keratoconjunctivitis (AKC), 4 eyes with vernal keratoconjunctivitis (VKC), and 12 eyes of the controls. The number of eyes examined for IgE antibodies to Housedust-Mites was 32 eyes with SAC, 20 eyes with PAC, 30 eyes with AKC, 22 eyes with VKC, and 10 eyes with the control. Tears were sampled by the method of Schirmer Test-1. Sampled tears were eluded with 200 microliters of phosphate buffered solution(pH 7.2, 0.05 M) and analyzed by the AlaSTAT-IMMULYZE method. Recovery rate of absorbance by filter paper for IgE antibodies in tears was determined by ratio to the standard sample of serum solution containing Housedust-Mite specific IgE antibodies with known concentration., Results: Recovery rate of filter paper for IgE antibodies in tears was 83.6%. IgE antibodies in tears to Japanese cedar pollen were detected in 11 of 32 eyes of SAC. In other subjects, IgE antibodies were under the limit of the detection concentration except one eye of a patient with AKC. In the positive cases of Japanese cedar pollen specific IgE antibodies, there was no significant difference in the concentration between the right and the left eyes. IgE antibodies to Housedust-Mites were significantly higher not only in incidence but also in concentration in 18 of 22 eyes with VKC than in that in other in 8 of 20 eyes with PAC and 12 of 30 eyes with AKC., Conclusion: The method for determining antigen specific IgE antibodies in tears is clinically useful to diagnose and to investigate pathophysiology of allergic conjunctival diseases.

Purpose: To investigate conjunctiva-associated lymphoid tissue (CALT) in mouse conjunctiva., Method: We examined normal mice, ranging in age from 4 to 6 weeks, to investigate the presence of follicular tissue histologically. Next, we treated mice by topical instillation of ovalbumin and cholera toxin B, and then examined conjunctival follicles by immunohistological methods., Results: Follicular tissue was present at the nictitating membrane. Both size and amount of follicular tissue was increased by the topical ovalbumin treatment. Immunohistological examination revealed CD4, CD8, and S-100 protein positive cells in follicular tissue. The epithelial layer, corresponding to follicular tissue, had intraepithelial pockets and CD4 positive cells in the pockets., Conclusion: It was concluded that the follicular tissue at the nictitating membrane is CALT in mice.

Purpose: A histopathological study to investigate the efficacy of conjunctival auto-transplantation on alkaline chemical burns of the ocular surface., Material and Methods: An alkaline chemical burn model was developed in one eye of rabbits using 0.1 N NaOH solution. In one group conjunctival transplantation was performed. A control group did not receive conjunctival transplantation. A histological follow-up study was performed by light microscopy with hematoxylin eosin stain and periodic acid Schiff staining by fluoro-microscopy using epithelial Keratin-AE 5 antibody, and by transmission electron microscopy(TEM)., Results: The transplanted group showed scar formation between the transplanted conjunctival tissue and the sclera. At 20 days and 8 weeks after the transplantation, neovascularization and cell infiltration in the subepithelium of the limbus was decreased compared with the transplanted group at 10 days. The control group showed a decrease of cell infiltration in the limbal area compared with the group at 10 days, but conjunctival tissue with thick collagenous tissue and neovascularization instead of scar tissue developed on the injured cornea. AE 5 positive cells were not found in the limbus in either group. In the transplanted group, in TEM, the basement membrane of transplanted conjunctiva showed less irregularity, and degenerated fibroblasts were present at the margin of the scar tissue. In the control group, the basement membrane of the conjunctiva showed an irregular pattern and fibroblasts beneath the conjunctival epithelium had large nuclei., Conclusion: In the transplanted group, scar tissue developed and suppressed cell infiltration, neovascularization, and conjunctival tissue in the injured cornea and secured re-structuring of the limbal tissue.

Purpose: We performed an immunohistochemical study on the development of interstitial keratitis in a rabbit model sensitized by ovalbumin (OA)., Methods: A mixture of OA (5 micrograms/ml) and Freund's complete adjuvant was initially injected subcutaneously (0.4 ml) into the rabbits' back and footpad (0.2 ml). After a week, the rabbits were sensitized by an injection of the same solution into the back (0.5 ml) again. After 4 weeks the rabbits underwent the same treatment as a booster shot. Then a week later OA solution (0.05 ml) was injected into the corneal stroma. We observed the cornea on days 1-3, 7, and 10 after the corneal treatment. Corneas on days 3 and 10 were examined by immunohistological methods using hematoxylin-eosin stain, methylgreen pyronin stain, and by immunohistochemical methods with anti-CD 4, anti-CD 8, and anti-OA antibodies. The localization of specific antibodies was identified by fluorescent-antigen method. Electron-microscopic observation was also done., Results: Rabbits developed corneal opacity and edema on day 1, corneas had on immune ring with a peak intensity on day 3, and vascularization on day 7 after corneal treatment. Microscopic examination revealed infiltration of neutrophils in to the area of the immune ring on day 3 and many plasma cells at the limbus and stroma on day 10. CD 4 positive cells were found at the limbus and stroma on days 3 and 10. CD 8 positive cells were found at the limbus and stroma only on day 10. OA positive findings were observed at the immune ring and its inner area. In the fluorescent-antigen method, specific antibodies were found in plasma cells at the limbus on days 3 and 10., Conclusions: In our experimental model of interstitial keratitis with immune ring and vascularization, the Arthus reaction was dominant. Infiltration of antigen specific plasma cells in the stroma with vascularization caused a modification of pathologic reaction in keratitis.

Purpose: To investigate the immunosuppressive effects of mucosal immune therapy in experimental allergic conjunctivitis., Method: We used 11 white Hartrey guinea pigs divided into two groups. Six animals (treated group) received pretreatment with topical instillation of cholera toxin B (4 micrograms/30 ml) and ovalbumin (10 micrograms/30 ml). The other group of 5 animals served as control. All the animals received intraabdominal injection of ovalbumin (100 g/ml) and aluminium hydroxide (5 mg/ml) repeated twice 2 weeks apart. Allergic conjunctivitis was induced by topical instillation of ovalbumin solution (5 mg/ml) one week after the above procedure., Result: Both groups developed palpebral and bulbar edema with hyperemia 30 minutes after instillation. The allergic reaction was significantly less in score in the treated than in control group (Mann Whitney U-test: p < 0.01). The clinical findings subsiced after 6 hours. The treated group showed less eosinophilic infiltration in the conjunctiva and the limbus, particularly in the conjunctival and the limbus, particularly in the conjunctival epithelium, than in control group at 6 and 24 hours., Conclusion: Pretreatment with topical cholera toxin B and antigen suppresses clinical and histological findings in experimentally induced allergic conjunctivitis.

We histologically examined the existence of follicular dendritic cells (FDCs) in the follicular area of conjunctiva-associated lymphoid tissue (CALT). Animals used in the experiment were Hartley guinea pigs sensitized with a topical application to the eyes of an emulsion of a mixture of ovalbumin and Freund's complete adjuvant in equal amounts. The animals were divided into 4 groups: a group with no eye drops administered (Group A), a group examined 1 week after administration of eye-drops (Group B-1), a group examined 2 weeks after administration (Group B-2), and a group given booster administration of eye-drops (Group C). These animals were examined by methyl green pyronine staining, alpha-naphthylacetate esterase staining, and enzyme-antibody methods against S-100 protein or major histocompatibility complex class II (MHC class II) antigen. Also peroxidase-anti-peroxidase (PAP) was applied to the eyes and the uptake of PAP in CALT was observed histologically. In each group, positive reticular stains to alpha-naphthyl-acetate esterase staining and S-100 protein were found in the CALT follicular area. The positive cells were found to be dendrite cells by immunoelectron microscopy. An uptake of PAP was seen in the CALT follicular area, suggesting the function of trapping and holding the antigen-antibody complex by FDCs. It was concluded that dendritic cells present in the follicular area of CALT were FDCs.

We performed immunohistochemical study on a failed case of keratoepithelioplasty (KEP). A 33-year-old man underwent KEP with lammellar keratoplasty (LKP) for an alkali burn suffered 5 years previously. After several rejections, the cornea developed opacity. We performed surgery a second time with the same method, and investigated the removed keratoconjunctival tissue including the lenticule using enzyme labelled antibodies UCHL-1, MB-1, CD4, CD8, CD54 (ICAM-1), CD68, and HLA-DR. Conjunctival tissue extended to the lamellar corneal graft over the lenticule. Either UCHL-1, CD4, or CD8 positive lymphocytes infiltrated the subconjunctival tissue dominantly, but they were blocked by the lenticule or Bowman's membrane of the graft cornea. In the stroma of the lamellar graft cornea, CD4 or CD8 positive, ICAM-1 or HLA-DR expression cells were detected. It was concluded that the lenticule and Bowman's membrane of the lamellar graft block infiltration of the immune cells.

High endothelial venule (HEV) is an important component of the mucosa-associated lymphoid tissue and plays a role in the homing phenomenon of lymphocytes. We investigated the presence of HEVs in conjunctiva-associated lymphoid tissue (CALT) using guinea pigs sensitized with ovalbumin. Electron microscopy showed characteristic findings for HEVs in the parafollicular area of the CALT. Lymphocytes in the vessels are trapped not only by microprocesses extending from the vascular endothelium but also by the endothelial cells. Some endothelial cells also appear to release lymphocytes. We conclude that HEVs are present also in the CALT.

We compared the distribution of S-100 protein positive cells in normal conjunctiva and in an induced allergic conjunctivitis model using Hartley strain guinea pigs. The animals were sensitized by intraperitoneal injection of ovoalbumin solution with hydroxy aluminum, followed by topical application of the ovoalbumin solution. The primary antibody was antibovine brain S-100 positive antibody, detected by the avidin biotin complex method. S-100 positive cells were present at the limbal corneal epithelium and on the skin of the palpebrae in both the normal group and the allergic conjunctivitis group, indicating the presence of Langerhans cells. In the allergic group, positive cells were also detected in palpebral conjunctiva and the germinal centers of conjunctival follicles. These cells were identified as follicular dendritic cells or interdigitating cells. In conclusion antigen presenting cells were present in the limbal conjunctiva, follicular and parafollicular area of conjunctival follicles, and palpebral skin in the allergic conjunctivitis models.

We performed an immunohistochemical study on the relationship between inflammatory cells and adhesion molecules to investigate inflammatory reaction in allergic conjunctivitis. Guinea pigs were sensitized with an intraperitoneal injection of ovalbumin with hydroxyalminium. Ovalbumin was applied topically to the conjunctiva 3 weeks after the initial treatment. Then the cornea, conjunctiva and lacrymal gland were taken and processed for frozen sections. The specimens were stained with acid Giemsa for inflammatory cells and enzyme-labelled antibody for ICAM-1 and VCAM-1. Neutrophils were dominantly observed 6 hours after the challenge with the antigen, eosinophils at 12 hours, and eosinophils and lymphocytes at 24 hours, respectively. ICAM-1 and VCAM-1 were detected at 6 and 24 hours in the vascular tissue in the subconjunctival tissue and lacrymal gland. There results suggest that adhesion molecules play a role in the infiltration of inflammatory cells in allergic conjunctivitis.

Local immunity on the ocular surface was observed in guinea pigs immunized with horseradish peroxidase (HRP) by determining the activity of a specific antibody in local sites of the conjunctiva by the enzyme-antigen method, which enables a specific antibody to be stained. In addition, immunohistochemical study of the localization of IgA was performed by the enzyme-labelled antibody method. The enzyme-antigen method revealed a number of positive cells in the subconjunctival tissue and the parafollicular area of conjunctival-associated lymphoid tissue. These cells were identified as plasma cells by electron microscopy. The mirror section technique revealed that the cells positive for the enzyme-antigen method were consistent with anti-IgA-positive cells at many sites. Thus the distribution of plasma cells secreting IgA-specific antibody responsible for local immunity on the ocular surface was observed immunohistochemically. The results suggest that immunocytes in the conjunctival-associated lymphoid tissue differentiate and mature into specific antibody-producing plasma cells.

The local immunity of the ocular surface is governed by conjunctival-associated lymphoid tissue (CALT), secretory IgA and immunocytes. The authors performed a histological investigation of the time-course changes in CALT caused by invasion of antigen to the ocular surface through the instillation of horseradish peroxidase (HRP) into the guinea pig eye. We used PAS staining, peroxidase staining, alkaline phosphatase staining. The lymphoepithelial cells of CALT phagocytized HRP 30-60 min after the instillation, and formed intraepithelial pockets 24 hours after instillation. The follicular area of CALT was strongly positive for alkaline phosphatase 2 weeks after instillation. These changes were considered to be the first step in the manifestation of local immunity on the ocular surface. Each staining technique revealed differences between the lymphoepithelium and conjunctival epithelium, suggesting that lymphoepithelium has characteristics different from those of conjunctival epithelium.