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Purpose: We performed simultaneous measurement of herpes simplex virus (HSV) DNA by real-time polymerase chain reaction (real-time PCR) and of HSV-specific secretory IgA antibody (HSV-sIgA) by enzyme-linked immunosorbent assay (ELISA) in tears obtained using Schirmer strips in order to investigate its diagnostic efficacy for herpes simplex keratitis (HSK)., Methods: A total of 59 affected eyes from 59 patients with clinically suspected HSK (HSK group) and 23 eyes from 23 healthy volunteers (control group) were enrolled in this study. The HSK group was divided into five subgroups: dendritic/geographic keratitis, disciform keratitis, necrotizing keratitis, atypical keratitis, and others. The tear samples were taken using Schirmer strips to determine the HSV DNA and HSV-sIgA levels., Results: The overall sensitivity and specificity were 55.8 and 100 % for HSV DNA and 49.2 and 82.6 % for HSV-sIgA. The HSV DNA levels in the disciform keratitis subgroup (median, 3.1 × 10(2) copies/sample) were significantly lower than those in the dendritic/geographic keratitis subgroup (median, 2.3 × 10(4) copies/sample) (P < 0.05, Mann-Whitney test). The HSV-sIgA levels in the disciform keratitis subgroup (median, 50.0 NU/ml) were significantly higher than those in the control group (median, 18.0 NU/ml) (P < 0.05, Steel test). The positive and negative predictive values obtained by simultaneous measurement of HSV DNA and sIgA were 90.9 and 61.3 %, respectively., Conclusion: The combination of laboratory detection of HSV DNA by real-time PCR and of HSV-sIgA by ELISA using tear samples enables higher reliability in diagnosing the subgroups of HSK, although the HSV DNA value is relatively lower in disciform HSK than in dendritic/geographic HSK.

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

Background/aims: To determine the possible roles of impaired corneal sensitivity and reduced tear secretion in various types of corneal epithelial disorders., Methods: A total of 99 patients (179 eyes) with corneal epithelial disorders classified as persistent epithelial defects (PED), corneal erosion, or superficial punctate keratopathy (SPK) and 115 individuals (230 eyes) without apparent ocular surface disorders (controls) were enrolled in a prospective study. Corneal sensitivity was measured with a Cochet-Bonnet esthesiometer, and tear secretion was measured by the Schirmer test in each subject., Results: Corneal sensitivity of eyes in the PED and corneal erosion groups was significantly lower than that in the control group. Schirmer test values for eyes in the SPK group were significantly reduced compared with those in the control group., Conclusion: A loss of corneal sensitivity may contribute to the development of PED and corneal erosion, whereas reduced tear secretion may be a contributing factor for SPK. Both results indicate the importance of corneal sensory innervation to the maintenance of corneal integrity.

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.

Background: The determination of total IgE in tears is useful as a diagnostic tool in allergic conjunctivitis disease (ACD). We evaluated the efficacy of this diagnostic tool for ACD, which is a clinically applicable novel immunochromagraphic method to determine total IgE in tears., Methods: The subjects comprised 4 groups: 15 patients with vernal keratoconjunctivitis (VKC group), 8 patients with atopic keratoconjunctivitis (AKC group), 18 patients with allergic conjunctivitis (AC group), and 7 normal healthy volunteers as a control (control group). Tears were sampled using filter paper, and the total IgE in tears was determined by immunochromatography assay. Semiquantitative determination was carried out by examining the intensity of the colored line using an immunochromatoreader (IgE index). The relationship between IgE indices in tears and total IgE levels in serum or between IgE indices and the clinical scores of ACD was examined., Results: The positive ratio obtained by this novel application of the immunochromatography assay was 38 of the 41 in the patients with ACD and none in the 7 controls. IgE indices for the VKC group, AKC group and AC group were 27.5 +/- 15.6, 19.8 +/- 15.8, and 4.0 +/- 3.1 (mean +/- SD), respectively. IgE indices in tears showed significant correlation with both total IgE levels in serum (P < 0.001, r = 0.76) and clinical scores of ACD (P < 0.001, r = 0.57)., Conclusions: The novel application of the immunochromatography assay to assess the total IgE in tears is a useful clinical tool to investigate ACD.

Purpose: To evaluate the in vivo expression of members of the eotaxin subfamily, eotaxin-1, -2, and -3, at the ocular surface, we analyzed the messenger RNA (mRNA) expression of the eotaxin subfamily in conjunctival epithelium and the protein expression of the eotaxin subfamily in tears of patients with vernal keratoconjunctivitis (VKC) and in those of healthy individuals., Methods: The subjects were 25 patients with VKC (25 eyes) and 11 healthy volunteers (11 eyes) as a control. Tear samples were collected using the Schirmer strip method. Tear samples were eluted, and concentrations of eotaxin-1, -2, and -3 in the tear samples were determined by enzyme-linked immunosorbent assay (ELISA). Concentration of eosinophil cationic protein (ECP) in tears was also determined by chemiluminescent enzyme immunoassay. Conjunctival epithelial cells were obtained from upper tarsal conjunctiva by impression cytology, and eotaxin-1, -2, and -3 mRNA extracted from the impression cytology membrane were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Conjunctival smears, which were obtained by tarsal conjunctival scraping, were stained for eotaxin-2 using immunohistochemical methods., Results: In the ELISA analysis, the expression ratio of eotaxin-1 (P < 0.01) and -2 (P < 0.001) in tears was significantly higher in the VKC group than in the control group. Concentrations of eotaxin-1 and -2 in tears in the VKC group were 0.7 and 1440.5 (median values) pg/ml, respectively. In the VKC group, the concentration of eotaxin-2 in tears was higher than that of eotaxin-1. There was a significant correlation between the concentration of eotaxin-2 and that of ECP in tears in the VKC group (r = 0.53, P < 0.01). Expression of eotaxin-3 protein in tears was not detected in the VKC group or the controls. In the RT-PCR analysis, the positive ratio of eotaxin-1, -2, and -3 mRNA expression in the VKC group was significantly higher than that in the control group (P < 0.01, P < 0.01, P < 0.05, respectively). In the immunohistochemical analysis, positive staining was detected in epithelial-like cells in conjunctival smears obtained from the patients with VKC., Conclusions: We showed that the mRNA expression and the protein production of the eotaxin subfamily at the ocular surface are critical biomarkers when investigating the pathophysiology of eosinophilic inflammation and the effect of antiallergic treatment in patients with VKC.

Purpose: To investigate gelatinase [matrix metalloproteinase (MMP)-2 and MMP-9] expression in the tear fluid of patients with recurrent corneal erosion (RCE)., Methods: Eleven patients with RCE, three patients with traumatic corneal erosion, and 10 control individuals were enrolled in this study. Tear samples from RCE eyes were obtained once, either at the time of recurrence (onset-phase samples, seven samples), or during the remission period (remission-phase samples, four samples). Tear samples from the nonaffected fellow eyes of the RCE patients were also examined once (fellow-eye samples, ten samples from ten patients). In addition, three samples from three patients with traumatic corneal erosion and ten samples from ten control individuals were obtained as well. Tear samples were collected by a modified Schirmer test I method and analyzed by gelatin zymography., Results: Neither the active form of MMP-2 nor that of MMP-9 was detected in the samples from traumatic corneal erosion patients and control individuals. Both active MMP-2 and active MMP-9 were detected in all seven onset-phase samples. Active MMP-2 and active MMP-9 were detected in three of the four remission-phase samples. Although none of the ten fellow eyes had a history of RCE, active MMP-2 and active MMP-9 were detected in three fellow-eye samples., Conclusions: Gelatinase expression was upregulated in the tear fluid of RCE patients. The presence of gelatinase in the affected eye during the remission phase as well as in the nonaffected fellow eye indicates that gelatinase expression in the tear fluid may be related to the recurrence of corneal erosion.

Purpose: To evaluate the significance of soluble interleukin-6 receptor (sIL-6R) in tears as a clinical indicator of disease exacerbation in patients with allergic conjunctivitis disease (ACD)., Methods: The study groups comprised 13 patients (13 eyes) with vernal keratoconjunctivitis (VKC group), 13 patients (13 eyes) with atopic keratoconjunctivitis (AKC group), 11 patients (11 eyes) with giant papillary conjunctivitis (GPC group), and 10 healthy volunteers (10 eyes) as a control. Tear samples were collected by the Schirmer I method using filter paper. Tear samples were eluted and the concentration of sIL-6R in tear samples was determined by enzyme-linked immunosorbent assay. Impression cytology using a nitrocellulose membrane was applied to the upper tarsal conjunctiva, and the mRNA of the signal-transducing molecule glycoprotein 130 (gp130) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was extracted from the membrane and analyzed by semiquantitative reverse transcription-polymerase chain reaction. In the VKC group, the clinical score was graded on the basis of allergic inflammation of the ocular surface, and the correlation between the concentration of sIL-6R and the clinical score was studied., Results: The concentration of sIL-6R in tears in the VKC, AKC, GPC, and control groups was 5.1 +/- 4.7, 1.2 +/- 1.7, 2.6 +/- 2.8, and 0.5 +/- 4.3 (mean +/- SD) ng/ml, respectively. The concentrations of sIL-6R in tears in the VKC and GPC groups were significantly higher than those in the control group (P < 0.001 and 0.05, respectively). The AKC group showed no significant change in the concentration of sIL-6R in tears in comparison with the control group. The gp130/GAPDH mRNA ratio was significantly higher in the patients with ACD than in control individuals (P < 0.05). The concentration of sIL-6R in tears and the clinical score of allergic inflammation of the ocular surface were significantly correlated in the VKC group (r = 0.53, P < 0.01)., Conclusions: We concluded that IL-6/sIL-6R complex trans-signaling via gp130 is an exacerbating factor of ACD, and that the concentration of sIL-6R in tears is a useful clinical biomarker in patients with ACD.

Purpose: To investigate the variations in total secretory IgA (sIgA) and house dust mite (HDM)-specific sIgA antibodies in tears of patients with allergic conjunctival disease (ACD)., Methods: The subjects comprised 40 patients (40 eyes) with ACD and 21 healthy individuals as a control. Patients with ACD comprised three groups: 16 patients (16 eyes) with perennial allergic conjunctivitis (PAC group), 11 patients (11 eyes) with atopic keratoconjunctivitis (AKC group), and 13 patients (13 eyes) with vernal keratoconjunctivitis (VKC group). Tear samples were taken by the Schirmer I method using filter paper. Tear samples were eluted and analyzed for total sIgA and HDM-specific sIgA antibodies in tears by enzyme-linked immunosorbent assay., Results: The mean (+/-SD) value of total sIgA in tears in the control, PAC, AKC, and VKC groups was 1446.7 +/- 1220.1, 706.8 +/- 567.2, 440.4 +/- 151.8, and 460.8 +/- 340.6 microg/ml, respectively. Total sIgA values were significantly lower in the VKC group than in the control group. We defined the HDM index as the ratio of HDM-specific sIgA to total sIgA. The HDM index in the VKC (P < 0.001), AKC (P < 0.001), and PAC (P < 0.001) groups was significantly higher compared with those in the control group., Conclusions: Low levels of total sIgA and high levels of HDM-specific sIgA indicate a local pathogenic factor in ACD.

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 investigate differences in the cytokine and chemokine profiles of patients with vernal keratoconjunctivitis (VKC) or giant papillary conjunctivitis (GPC)., Methods: The study included six patients (six eyes) with VKC, five patients (five eyes) with GPC, and five healthy volunteers (five eyes) as controls. None of the patients had received any anti-allergic treatment prior to this study. One patient with VKC was given a tear examination to evaluate the effect of anti-inflammatory treatment with a steroid on the tear cytokine profile about the treatment. Tear samples were collected with the Schirmer I method, using filter paper. Tear samples were eluted and analyzed by an antibody array system for inflammation-related factors, including cytokines and chemokines., Results: In the patients with VKC, four inflammation-related factors, eotaxin, interleukin (IL)-11, monocyte chemoattractant protein (MCP)-1, and macrophage-colony stimulating factor (M-CSF) increased to four times the values in the control group, and seven inflammation-related factors, eotaxin-2, IL-4, IL-6, interleukin-6 soluble receptor (IL-6sR), IL-7, macrophage inflammatory protein (MIP)-1delta, and tissue inhibitor of metalloproteinases (TIMP)-2, increased to eight times the control values. In the patients with GPC, three inflammation-related factors, IL-6, M-CSF, and monokine-induced gamma interferon (MIG), increased to four times those in the control group, and five inflammation-related factors, eotaxin-2, IL-6sR, IL-11, MIP-1delta, and TIMP-2, increased to eight times the control values. The increase in IL-6sR relative to the controls was statistically significant in both the VKC and GPC groups. The increase in eotaxin-2 was significant only in the VKC group, and that in TIMP-2 was significant only in the GPC group, compared with the controls., Conclusions: The present study demonstrated the presence of crucial cytokines, soluble cytokine receptors, and chemokines in tears of patients with VKC and GPC. In particular, IL-6sR increased significantly in both the VKC and GPC groups, whereas eotaxin-2 increased significantly only in the VKC group. Thus, IL-6sR may play an important pathophysiological role in giant papillary proliferation in VKC and GPC, and eotaxin-2 may play an important role in eosinophilic inflammation in VKC.

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: To determine the pathological role of tumor necrosis factor alpha (TNF-alpha) in ocular surface disorder of patients with Sjögren syndrome (SS), TNF-alpha in the tears of patients with SS were analyzed. In addition, the relationship between TNF-alpha levels in the tear samples and the severity of the corneal epithelial disorder was evaluated., Material and Methods: Tear samples were obtained from the 60 eyes of 30 patients with primary SS and the 60 eyes of 30 normal subjects. Tear samples were analyzed using a sandwich enzyme-linked immunosorbent assay to detect TNF-alpha. Corneal epithelial cell damage was examined with a slit-lamp biomicroscope and scored by the van Bijsterveld scoring system., Results: TNF-alpha was detected in the tears of patients with SS at concentrations ranging from 8 to 1,500 pg/ml in 33 of the 60 eyes of the patients. In contrast, TNF-alpha was not detected in the tears of normal controls. The concentration of TNF-alpha was significantly higher in the tear samples from patients whose clinical scores were two or three points compared with patients whose scores were zero or one point (Mann-Whitney U test: p < 0.03)., Conclusions: TNF-alpha was detected in the tears of patients with SS, and tear TNF-alpha levels showed a significant correlation with the grade of corneal epithelial damage, suggesting that TNF-alpha is a potent mediator in keratoconjunctivitis sicca.

Purpose: To determine the expression of gelatinase A [matrix metalloproteinase (MMP)-2] and gelatinase B (MMP-9) in ocular surface disorders, we carried out examinations of the eyes of patients with such disorders., Methods: The cases consisted of a normal group comprising three eyes as the control and a patient group comprising six eyes of patients with ocular surface disorders. These groups underwent the following examinations. (1) Gelatin zymography: Tear samples taken by using the Schirmer I test were eluted and analyzed by gelatin zymography. (2) Immunohistochemical study: By using the alkaline phosphate-anti-alkaline phosphatase or avidin-biotin complex method, corneal or conjunctival tissue was examined for the presence of MMP-2, MMP-9, and membrane type 1 (MT1)-MMP, which activates MMP-2. (3) Reverse transcription-polymerase chain reaction (RT-PCR): Total RNA was eluted from the specimens, and the expression patterns of MMP-2, MMP-9, and MT1-MMP mRNA were analyzed., Results: (1) Gelatin zymography: Only the proforms of MMP-2 and MMP-9 were detected in the normal group. Both active MMP-2 and active MMP-9 were detected in all studied cases in the patient group. (2) Immunohistochemical study: In the normal group, only MMP-2-positive cells were present in conjunctival epithelium. In the patient group, cells positive for MMP-2, MMP-9 and MT1-MMP were present in the epithelium of the conjunctiva or cornea except in one thermal burn case. (3) RT-PCR: In the normal group, only MMP-2 expression was observed. MMP-2, MMP-9, and MT1-MMP mRNA expression was observed in three cases in the patient group., Conclusions: MMP-2, MMP-9, and MT1-MMP, which activates MMP-2, were expressed in the patients with various ocular surface disorders. In ocular surface disorders, therefore, the expression of gelatinases is upgraded.

Purpose: To investigate the presence of staphylococcal enterotoxin A (SEA) and B (SEB)-specific IgE antibodies in tears from patients with allergic conjunctival disorders., Methods: The study included 8 eyes of 4 patients with perennial allergic conjunctivitis (PAC), 14 eyes of 7 patients with vernal keratoconjunctivitis (VKC), 12 eyes of 6 patients with atopic keratoconjunctivitis (AKC), and 10 eyes of 10 healthy volunteers as controls. Tears were sampled by the method of the Schirmer test I. Sampled tears were eluted and SEA- and SEB-specific IgE antibodies were analyzed by the AlaSTAT-IMMULIZE method., Results: SEA-specific IgE antibodies in tears were positive in 9 of 14 eyes in VKC patients and in 1 of 12 eyes in AKC patients. SEB-specific IgE antibodies in tears were positive in 7 of 14 eyes in VKC patients and in 2 of 12 eyes in AKC patients. Values for antibodies were higher in patients with severe clinical findings. However, all the cases in the normal control and the PAC groups were negative for both antibodies., Conclusion: Our data strongly suggested that staphylococcal enterotoxin may cause type I allergy, and may be an exacerbating factor for vernal keratoconjunctivitis and atopic keratoconjunctivitis.

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 the expression of the active form of gelatinases (gelatinase A: matrix metalloproteinase-2 [MMP-2] and gelatinase B: matrix metalloproteinase-9 [MMP-9]) in ocular surface disorders, we determined the presence of the active form of these gelatinases in the tear fluid of patients with corneal ulcer or ocular burn., Methods: The subjects consisted of the ulcer group comprising 13 eyes, the burn group comprising 6 eyes, and the normal group comprising 10 eyes. Tear samples were taken by the method of the Schirmer test I. The tears were eluted using extraction buffer containing 0.01 M phosphate buffer solution (pH 7.2), 1% Tween 20 and 1 M NaCl, and analyzed by gelatin zymography., Results: Only the proforms of MMP-2, and MMP-9 were detected in the normal group and in uncomplicated herpetic keratitis or herpetic keratitis cases complicated by mixed infection in the ulcer group. Active MMP-9 was detected in mild corneal ulcer cases and mild ocular burn cases. Both active MMP-2 and active MMP-9 were detected in endogenous corneal ulcer cases and severe burn cases, including perforation cases., Conclusions: Both active gelatinases were detected in tears of severe corneal ulcer or severe ocular burn cases. The active form of gelatinase expression may be related to the severity of ulceration.

Purpose: We investigated the efficacy of the tear sampling method using a filter paper to evaluate eosinophil cationic protein (ECP) levels in patients with allergic disorders., Methods: Subjects were an allergic group comprising patients with allergic conjunctivitis, vernal keratoconjunctivitis, or atopic keratoconjunctivitis, a Sjögren group comprising patients with secondary Sjögren syndrome, and a control group comprising healthy volunteers. Tears were sampled using the Schirmer Method I and the sample was eluted from the filter paper in 50 microL of elution solution containing phosphate buffer solution with 0.5 M NaCl + 0.1% Tween 20. Then the ECP concentration in the elution sample was determined by the enzyme-linked immunosorbent assay method., Results: Tear ECP level in the allergic group was significantly higher than the levels in the other two groups (P <.01 for the Sjögren group and P <.001 for the control). Furthermore, the tear ECP level of each allergic disease subgroup in the allergic group was significantly higher than that in the control group., Conclusions: This method of determining tear ECP concentration is useful not only to diagnose allergic conjunctival disorders but also to evaluate their clinical stages.

We performed quantitative analyses of glycosaminoglycans in the tear fluids in a rabbit wound healing model. We ablated rabbit corneal epithelium with trephine and spatula, and sampled tear fluids before the epithelial ablation, and at 3, 24, 48, and 72 hours after it. After an instillation of 200 microliters saline solution in the conjunctival sac, as much tear fluid as possible was collected from the lower cul-del-sac. The glycosaminoglycans in the tears were then treated with chondoroitinase ABC to make fractions of disaccharides. The quantities of disaccharides were determined with high-performance liquid chromatography as weight per unit protein in the tears. The concentrations of delta Di-HA in the tear fluids at 3 and 24 hours were significantly higher than those before the treatment and returned to the initial value at 72 hours after making the epithelial wound. Among the disaccharides of chondroitin sulfate, delta Di-0S and delta Di-6S showed a significant increase at 3 hours after the treatment but delta Di-4S did not show any significant variation. The results suggest that the glycosaminoglycans in the rabbit tear fluids may play an important role in the corneal epithelial wound healing process.