Mitsuru Sawa

Antibody Array-Generated Cytokine Profiles of Tears of Patients with Vernal Keratoconjunctivitis or Giant Papillary Conjunctivitis

PurposeTo investigate differences in the cytokine and chemokine profiles of patients with vernal keratoconjunctivitis (VKC) or giant papillary conjunctivitis (GPC).MethodsThe 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.ResultsIn 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)-1δ, 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-1δ, 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.ConclusionsThe 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. Jpn J Ophthalmol 2006;50:195–204

A 10-Year Review of Penetrating Keratoplasty

PURPOSE To survey the changes in indications for penetrating keratoplasty (PKP) and re-evaluate the risk factors for allograft rejection and graft failure. METHODS We evaluated the records of 396 eyes of 335 patients who had undergone PKP at the Tokyo University Hospital between 1987 and 1997. Clinical results were analyzed by the Kaplan-Meier life table method and the log-rank test. RESULTS The overall rates of graft survival and rejection-free graft survival at 10 years were 72.2% and 76.8%, respectively. The rates of graft survival and rejection-free graft survival were 98.8% and 86.6% in keratoconus, 87.0% and 56.5% in herpetic keratitis, 76.9% and 73.1% in corneal dystrophy and degeneration, 69.4% and 80.6% in nonherpetic keratitis, 62.5% and 75.0% in chemical burns, 61.8% and 72.1% in regrafting, and 51.1% and 79.8% in bullous keratopathy, respectively. The graft survival rates were statistically higher in the PKP alone group than in the combined operation group. The graft survival and rejection-free graft survival rates were statistically higher in the first operation group than in the regrafted group, and in the avascular cornea group than in the vascular cornea group. CONCLUSIONS We recognized changes in indications for PKP. Combined operation, reoperation, and vascularization of recipient cornea were risk factors for graft failure.

Effect of retinol palmitate as a treatment for dry eye: A cytological evaluation

Vitamin A is known to regulate the proliferation and differentiation of corneal epithelial cells and preserved conjunctival goblet cells and has been used in the treatment of disease of the eye such as dry eye and superior limbic keratoconjunctivitis for some time. This study was undertaken in order to evaluate the efficacy of retinol palmitate aqueous ophthalmic solution under development for the treatment of dry eye failing to respond to the conventional therapy with artificial tears or cornea-protective drugs. Retinol palmitate ophthalmic solution was applied repeatedly for 4 consecutive weeks. Before and after instillation therapy, brush cytology (Cytobrush-S) was performed and cytodiagnosis was made for keratinized cells, nonkeratinized cells, goblet cells and inflammatory cells on samples prepared using an automated smear apparatus (ThinPrep). In dry eye, an increase in goblet cells (1.3+/-2.6-->2.1+/-1.8 cells/slides), a decrease in keratinized cells (11.2+/-16.5-->5.2+/-10.9 cells/300 cells) and, hence, an increase in nonkeratinized cells (287.3+/-16.6-->293.4+/-11.4/300 cells) were found after treatment with retinol palmitate. As to inflammatory cells, there was no change from the pretreatment baseline (1.4+/-1.4-->1.4+/-1.3 cells/300 cells). These results demonstrate that brush cytology suggests the efficacy of retinol palmitate ophthalmic solution in dry eye treatment.

Pretranscriptional Regulation of Tgf-β1 by PI Polyamide Prevents Scarring and Accelerates Wound Healing of the Cornea After Exposure to Alkali

Corneal alkali burns are a serious clinical problem that often leads to permanent visual impairment. In this process, transforming growth factor (Tgf)-β1 is upregulated and involved in the response to corneal injury and the process of corneal stromal scarring. To develop an efficient compound to inhibit Tgf-β1 in the cornea, we designed GB1201, a pyrrole-imidazole (PI) polyamide targeting rat Tgf-β1 gene promoter to the activator protein-1 (AP-1) binding site. GB1201 showed a high binding affinity to the target DNA sequence in the gel mobility shift and Biacore assays. GB1201 significantly inhibited the rat Tgf-β1 gene promoter activity in HEK (human embryonic kidney) 293 cells in a concentration-dependent manner. Topically administrated GB1201 was distributed immediately to the nuclei of all cell layers of the cornea and remained for 24 hours. A corneal alkali burn model in rats was used to evaluate the therapeutic efficacy of GB1201. GB1201 suppressed the upregulation of Tgf-β1 in the burned cornea, both in the mRNA and protein levels. Moreover, daily treatment with GB1201 for a week significantly improved the corneal tissue wound healing, reduced corneal stromal scarring, and prevented corneal haze formation. Our data suggest that PI polyamide may open new opportunities for therapeutic intervention in the treatment of chemically burned corneas.Corneal alkali burns are a serious clinical problem that often leads to permanent visual impairment. In this process, transforming growth factor (Tgf)-beta1 is upregulated and involved in the response to corneal injury and the process of corneal stromal scarring. To develop an efficient compound to inhibit Tgf-beta1 in the cornea, we designed GB1201, a pyrrole-imidazole (PI) polyamide targeting rat Tgf-beta1 gene promoter to the activator protein-1 (AP-1) binding site. GB1201 showed a high binding affinity to the target DNA sequence in the gel mobility shift and Biacore assays. GB1201 significantly inhibited the rat Tgf-beta1 gene promoter activity in HEK (human embryonic kidney) 293 cells in a concentration-dependent manner. Topically administrated GB1201 was distributed immediately to the nuclei of all cell layers of the cornea and remained for 24 hours. A corneal alkali burn model in rats was used to evaluate the therapeutic efficacy of GB1201. GB1201 suppressed the upregulation of Tgf-beta1 in the burned cornea, both in the mRNA and protein levels. Moreover, daily treatment with GB1201 for a week significantly improved the corneal tissue wound healing, reduced corneal stromal scarring, and prevented corneal haze formation. Our data suggest that PI polyamide may open new opportunities for therapeutic intervention in the treatment of chemically burned corneas.

Active form of gelatinases in tear fluidin patients with corneal ulcer or ocular burn

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.

Metalloproteinases in corneal diseases: degradation and processing.

Abstract: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with the potential to degrade all types of extracellular matrix. The ADAM (a disintegrin and metalloproteinase) family of peptidases was recently identified as cleaving the extracellular domain of transmembrane proteins. This was termed ectodomain shedding. We investigated the MMP expression in patients with corneal diseases and the potential role of ADAMs in corneal pathophysiology. We detected upregulation of the active form of MMP-2 and MMP-9 in the tear fluid from patients with corneal melting or recurrent corneal erosion. Using human corneal epithelial cells, we observed ADAM17-dependent ectodomain shedding of soluble tumor necrosis factor receptor 1 and soluble interleukin-6 (IL-6) receptor (sIL-6R). The production of sIL-6R was also induced by messenger RNA splicing in the human corneal epithelial cells. IL-6/sIL-6R-induced signal transducer and activator of transcription 3 phosphorylation was observed in cultured human corneal fibroblasts, suggesting that IL-6 trans-signaling induced inflammatory cellular signaling in the human corneal fibroblasts. We demonstrated that MMPs are significantly upregulated in collagen-destructive disorders of the cornea. Additionally, we observed that ectodomain shedding by ADAMs in corneal epithelial cells mediated the production of soluble cytokine receptors. Trans-signaling of IL-6 can induce an inflammatory response in corneal stroma, indicating the significance of IL-6 trans-signaling in ocular surface inflammation. Thus, MMPs and ADAMs play an important role in the pathophysiology of corneal diseases.

Comparison of topical dexamethasone and topical FK506 treatment for the experimental allergic conjunctivitis model in BALB/c mice.

PurposeTo evaluate the efficacy of topical dexamethasone and topical FK506 treatment on allergic inflammation in conjunctiva, we performed a comparative study using a mouse experimental allergic conjunctivitis model.MethodsThe experimental allergic conjunctivitis model was developed by ovalbumin (OVA) instillation after OVA sensitization by intraperitoneal application of a mixed solution of OVA and aluminum hydroxide. The Balb/c mice of the experimental allergic conjunctivitis model were divided into three groups, depending on whether or not they received topical treatment and which type of treatment they received. The allergy group comprised six mice without topical treatment; the dexamethasone (Dx) group comprised six mice receiving topical 0.1% dexamethasone ointment treatment; and the FK506 (FK) group comprised six mice receiving topical 0.1% FK506 ointment treatment. The negative control group comprised six mice with neither sensitization nor topical treatment. For the histological examination, frozen sections of the eyelids, eyeballs, and lacrimal glands were stained with acid Giemsa stain or, in the immunohistochemical method, with alkaline phosphatase–antialkaline phosphatase (APAAP), for CD4. Densities of eosinophils and CD4-positive lymphocytes in the conjunctival tissue were counted. Productive C ε gene expression in the conjunctival tissue, including the lacrimal gland, was also evaluated by reverse transcriptase polymerase chain reaction (RT-PCR).ResultsIn the histological study, the cell density of eosinophils infiltrating into subconjunctival tissues was 66.3 ± 34.7 cells/mm2 (mean ± SD) for the allergy group, 2.5 ± 4.2 cells/mm2 for the Dx group, 4.2 ± 8.6 cells/mm2 for the FK group, and 3.7 ± 7.6 cells/mm2 for the negative control group. The cell density of CD4-positive cells infiltrating into subconjuctival tissues was 145.9 ± 66.4 cells/mm2 for the allergy group, 10.0 ± 12.9 cells/mm2 for the Dx group, 10.1 ± 15.5 cells/mm2 for the FK group, and 21.7 ± 17.8 cells/mm2 for the negative control group. The allergy group showed significant differences in the density of eosinophils and CD4-positive cells compared with those of the Dx (P < 0.0001) and FK (P < 0.0001) groups. In the RT-PCR study, the expression of productive C ε gene was positive in the Dx group, but negative in the allergy group, the FK group, and the negative control group.ConclusionsTopical Dx and FK506 treatments were both efficacious in suppressing eosinophil and lymphocyte infiltration into subconjunctival tissue in the mouse experimental allergic conjunctivitis model. However, the influence of their IgE productive mechanisms seemed to differ, causing different efficacy of productive C ε gene expression in the conjunctival tissue. Jpn J Ophthalmol 2005;49:205–210© Japanese Ophthalmological Society 2005

Histological Study of Conjunctiva-associated Lymphoid Tissue in Mouse

PURPOSE To investigate conjunctiva-associated lymphoid tissue (CALT) in the mouse conjunctiva by histological methods. METHODS The presumed follicular tissue in the conjunctiva of normal mice, age ranging from 4 to 6 weeks, was histologically investigated by the hematoxylin-eosin staining method. Next, we treated the mice with topical instillation of a combined solution of ovalbumin and cholera toxin B to investigate the morphological changes of conjunctival follicles to antigen challenge. The treated mice underwent sequential clinical examinations, and the conjunctival follicular tissue was examined by an immunohistochemical method using anti-CD4 antibody, anti-CD8 antibody, and anti-S-100 antibody. RESULTS Follicular tissue was present on the mouse nictitating membrane. Both size and number of follicular tissue areas increased with topical ovalbumin treatment. Immunohistochemical study revealed CD4, CD8, and S-100 positive cells in the follicular tissue. The epithelial layer, corresponding to follicular tissue, demonstrated intra-epithelial pocket and the presence of CD4-positive cells in the intra-epithelial pocket. CONCLUSION Follicular tissue at the nictitating membrane is CALT in the mouse.

Clinical Evaluation of Total IgE in Tears of Patients with Allergic Conjunctivitis Disease Using a Novel Application of the Immunochromatography Method

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.

Concentration of Soluble Interleukin-6 Receptors in Tears of Allergic Conjunctival Disease Patients

PurposeTo 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).MethodsThe 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.ResultsThe 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).ConclusionsWe 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. Jpn J Ophthalmol 2007;51:332–337