Katsuhiko Shinomiya

Pharmacokinetics of Bevacizumab after Topical, Subconjunctival, and Intravitreal Administration in Rabbits

PURPOSE To investigate the pharmacokinetics of bevacizumab in rabbits for three different routes of administrations: intravitreal injection, subconjunctival injection, and eye drops. METHODS Pigmented rabbits received bevacizumab in one eye by topical eye drops (1.25 mg/0.05 mL six times daily for the first 7 days), single subconjunctival injection (1.25 mg/0.05 mL), or single intravitreal injection (1.25 mg/0.05 mL). Bevacizumab concentrations in plasma and ocular tissues in the treated and fellow eyes were determined by sandwich enzyme-linked immunosorbent assay at 1, 2, 4, and 12 weeks after administration. RESULTS After intravitreal injection in the treated eye, the mean maximum concentrations (C(max)) of bevacizumab in the iris/ciliary body and retina/choroid were 109,192.6, and 93,990.0 ng/g, respectively, whereas after subconjunctival injection, the C(max) was 1418.7 and 295.8 ng/g, respectively. In the fellow eyes, when the drug was administered by intravitreal injection, the C(max) was 753.6 ng/g in the iris/ciliary body and 224.2 ng/g in the retina/choroid and by subconjunctival injection was 1192.9 and 187.0 ng/g, respectively. With eye drops, only a small level of bevacizumab was detected in the iris/ciliary body and retina/choroid. Systemic exposure to bevacizumab was at the same level when administered by intravitreal or subconjunctival injection. CONCLUSIONS Intravitreal injection of bevacizumab was the most effective route of administration for intraocular tissue. Also, bevacizumab injected subconjunctivally was transported into the intraocular tissues of the treated eyes at an effective level. Both intravitreal and subconjunctival injections of bevacizumab resulted in high plasma concentrations. Bevacizumab was distributed into the intraocular tissues in fellow eyes via the systemic circulation. This treatment may be effective for blocking vascular endothelial growth factor activity.

Simultaneous Analysis of Multiple Cytokines in the Vitreous of Patients with Sarcoid Uveitis

PURPOSE Levels of some cytokines are significantly higher in the vitreous fluid of patients with acute uveitis than in normal vitreous fluid. The authors sought to determine which proinflammatory cytokines were upregulated in the vitreous fluid of patients with ocular sarcoidosis. METHODS Samples of vitreous fluid were collected from patients with sarcoid uveitis and from nonsarcoid control patients with idiopathic epiretinal membrane. The levels of 27 proinflammatory cytokines were measured with a multiplex beads array system. Postvitrectomy macular thickness was also measured by using spectral domain optical coherence tomography (SD-OCT). To assess the relationship between cytokine levels and disease stage, the authors divided patients into three groups based on macular thickness 1 month after operation. RESULTS The vitreous levels of 17 cytokines were significantly higher in patients with ocular sarcoidosis than in nonsarcoid controls. Serum levels of interferon γ-induced protein 10 (IP-10) were also higher in ocular sarcoidosis patients than in nonsarcoid controls. Conversely, serum levels of interleukin (IL) 15 in ocular sarcoidosis patients were lower than in the control group. Analysis of cytokine levels and macular thickness revealed that IL-1ra, IL-4, IL-8, IFN-γ, IP-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1β, and regulated on activation, normal T-cell expressed and secreted (RANTES) were significantly upregulated in patients with thin cystoid macular edema group. CONCLUSIONS Patients with ocular sarcoidosis had elevated levels of proinflammatory cytokines in vitreous fluids. Different cytokines might contribute to different stages of macular edema.

Immunohistochemical analysis of inflammatory limbal conjunctiva adjacent to Mooren's ulcer

Background/aims To examine the characteristics of infiltrating cells in conjunctival tissues adjacent to the peripheral corneal ulcers of Moorens ulcer. Methods This study involved four eyes of four patients with Moorens ulcer and who were considered to be in need of surgical treatment. The patients’ resected conjunctival tissues were embedded and frozen. The tissue sections were then subjected to H&E and immunohistochemical staining. The stained sections were observed and the characteristics of the infiltrating cells in the conjunctival tissues were pathologically examined. Results In all patients, infiltration of inflammatory cells was observed in the submucosal connective tissue of the conjunctiva. Immunohistochemical analysis revealed inflammatory cell infiltration into the submucosal layer of the conjunctiva that was mainly composed of CD3-positive and CD45RO-positive cells. Some of these cells also showed positive reactivity with CD4, yet very few cells showed positive reactivity with CD8. In addition, infiltration of the cells indicating CD68 positivity was frequent in a few cases. Conclusions In the four Moorens ulcer cases, infiltrating cells in the submucosa of the conjunctival tissues adjacent to the ulcerative cornea were found to be mainly composed of helper T lymphocytes and macrophages. Our results show that helper T cells and macrophages contribute to the pathogenesis of Moorens ulcer.

Cyclosporin A eye drops inhibit fibrosis and inflammatory cell infiltration in murine type I allergic conjunctivitis without affecting the early-phase reaction.

Purpose: To understand the mechanisms of action of cyclosporin A eye drops in severe allergic diseases such as vernal keratoconjunctivitis, the inhibitory effects of cyclosporin A eye drops on fibrosis and inflammatory cell infiltration in murine allergic conjunctivitis were evaluated. Methods: BALB/c mice that had been actively sensitized with ovalbumin were challenged with ovalbumin on days 10–14 after initial sensitization. Cyclosporin A (0.1%) or betamethasone (0.1%) eye drops were instilled 1, 4, and 7 hours after each challenge. Ocular tissue was harvested for histological evaluation 24 hours after the last challenge, and conjunctival tissue was collected for the measurement of collagen content and quantitative PCR 8 hours after the last challenge. Results: Scores for fibrosis and inflammatory cell infiltration and collagen content in the conjunctiva were higher after 5 days of antigen challenge than in normal non-challenged conjunctiva. Instillation of cyclosporin A or betamethasone reduced the antigen-induced increases in scores for fibrosis and inflammatory cell infiltration in the conjunctiva, and cyclosporin A significantly reduced the antigen-induced increase in conjunctival collagen content. Betamethasone also showed a tendency to reduce the increase in collagen content. Cyclosporin A and betamethasone decreased the numbers of CD3+ and CD4+ T-cells and eosinophils in the conjunctiva, but did not affect the number of mast cells. Neither type of eye drop suppressed the increase in vascular permeability that occurred for 30 minutes after the last antigen challenge. In quantitative PCR, cyclosporin A suppressed the expression of IL-4 and IL-5 mRNA but did not suppress the expression of transforming growth factor (TGF)-β 1, whereas betamethasone suppressed the expression of IL-4, IL-5, and TGF-β 1. Conclusion: The results suggest that cyclosporin A eye drops inhibited fibrosis and inflammatory cell infiltration by the suppression of Th2 cytokine production in repeatedly antigen-challenged conjunctiva without affecting the early-phase reaction.

Cyclosporine A Eye Drops Inhibit the Early-Phase Reaction in a Type-I Allergic Conjunctivitis Model in Mice

PURPOSE The effects of cyclosporine A eye drops on the early-phase reaction were investigated in a type-I allergic conjunctivitis model. METHODS Mice were actively sensitized with ragweed (RW) absorbed on aluminium hydroxide gel and challenged with RW for 10 days (single challenge model) or 10-14 days (repetitive challenge model) after the first sensitization. For the evaluation of itching, ovalbumin was used as an antigen instead of RW. The effects of cyclosporine A eye drops on increased vascular permeability, mast cell degranulation, and itching were evaluated and compared with those of other anti-allergic eye drops. RESULTS In the single challenge model, cyclosporine A eye drops significantly inhibited the increase in vascular permeability and histological evaluations showed suppressed degranulation of mast cells. Disodium cromoglycate (DSCG) eye drops showed only a slight tendency to inhibit the increase in both pathophysiological parameters. Ketotifen or betamethasone eye drops significantly inhibited the increase in vascular permeability. The order of potency in the single challenge model was ketotifen > cyclosporine A > betamethasone. In the repetitive challenge model, cyclosporine A eye drops significantly inhibited the increase in vascular permeability and DSCG eye drops showed only slight inhibition. Ketotifen or betamethasone significantly inhibited the increase in vascular permeability. The order of potency in the repetitive challenge model was cyclosporine A > betamethasone > ketotifen. The effect of cyclosporine A eye drops on the itch-scratch response was studied. Cyclosporine A and DSCG significantly reduced the itch-scratch response in the single and repetitive challenge models; the effect of cyclosporine A in the repetitive challenge model was more potent than in the single challenge model. CONCLUSIONS Those results suggest that administration of cyclosporine A eye drops inhibit the early-phase reaction in type-I allergic conjunctivitis, which may be mediated by the suppression of mast cell degranulation. This action of cyclosporine A eye drops may be involved in the therapeutic effect of cyclosporine A on allergic conjunctivitis.

Establishment of a Human Corneal Epithelial Cell Line Lacking the Functional TACSTD2 Gene as an In Vitro Model for Gelatinous Drop-Like Dystrophy

PURPOSE Gelatinous drop-like corneal dystrophy (GDLD) is characterized by subepithelial amyloid deposition that engenders severe vision loss. The exact mechanism of this disease has yet to be elucidated. No fundamental treatment exists. This study was conducted to establish an immortalized corneal epithelial cell line to be used as a GDLD disease model. METHODS A corneal tissue specimen was obtained from a GDLD patient during surgery. Corneal epithelial cells were enzymatically separated from the cornea and were dissociated further into single cells. The epithelial cells were immortalized by the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcriptase (hTERT) genes. For the immortalized cells, proliferative kinetics, gene expressions, and functional analyses were performed. RESULTS The immortalized corneal epithelial cells continued to proliferate despite cumulative population doubling that exceeded 100. The cells showed almost no sign of senescence and displayed strong colony-forming activity. The cells exhibited a low epithelial barrier function as well as decreased expression of tight-junction-related proteins claudin 1 and 7. Using the immortalized corneal epithelial cells derived from a GDLD patient, we tested the possibility of gene therapy. CONCLUSIONS We established an immortalized corneal epithelial cell line from a GDLD patient. The immortalized cells exhibited cellular phenotypes similar to those of in vivo GDLD. The immortalized cells are thought to be useful for the development of new therapies for treating GDLD corneas and for elucidation of the pathophysiology of GDLD.

Interaction Between Conjunctival Epithelial Cells and Mast Cells Induces CCL2 Expression and Piecemeal Degranulation in Mast Cells

PURPOSE Intraepithelial mast cells are observed in giant papillae tissue samples obtained from patients with atopic keratoconjunctivitis (AKC)/vernal keratoconjunctivitis (VKC). We examined the roles of interaction between the conjunctival epithelial cells and mast cells. METHODS The interaction between human mast cells and conjunctival epithelial cells (HCjE) was investigated using a coculture model. Protein array analysis, ELISA, and real-time PCR were performed to test the interaction. Tissue samples (n = 6) from giant papillae were resected for therapeutic purposes, and subjected to immunohistological analysis of CCL2 expression. Recombinant CCL2 (10 ng/mL) was reacted with the cultured human mast cells and ultrastructural analysis was performed. A ragweed (RW)-induced mouse experimental allergic conjunctivitis model was used to examine ccl2 mRNA expression and mast cell morphology. RESULTS Protein array and real-time PCR analyses showed that CCL2 protein/mRNA expression was induced by mast cell-HCjE coculture. Upregulation of CCL2 mRNA was observed in mast cells, whereas in situ CCL2 expression was observed at the conjunctival epithelium of the giant papillae by immunohistochemistry. Ultrastructural analysis showed that recombinant CCL2 treatment induced piecemeal degranulation (PMD) in the mast cells. Ultrastructural analysis of tissues from the giant papillae showed PMD of mast cells within the conjunctival epithelial cells. The RW-induced experimental allergic conjunctivitis model showed increased ccl2 mRNA expression and PMD morphology in the conjunctivae. CONCLUSIONS Mast cell-conjunctival epithelial cell interaction induces CCL2 expression and subsequent PMD.

Intravital imaging of the cellular dynamics of LysM-positive cells in a murine corneal suture model

Background Corneal suturing is a surgical procedure used in patients with corneal trauma or transplants. It was reported that endogenous neutrophils are brightly labelled in gene-targeted mice expressing enhanced green fluorescent protein (eGFP) under the control of the endogenous lysozyme M promoter (LysM-eGFP mice). Methods We applied intravital imaging methods to analyse in vivo the dynamics of LysM-positive granulocytes (neutrophils) in LysM-eGFP mice with corneal sutures and examined their role in the elicitation of neutrophil infiltration. Results We found that in the presuturing state, neutrophils strongly positive for LysM were located in the periphery of the corneal stromal layer; none were present in the centre of the cornea. After introducing a corneal suture, neutrophils accumulated in limbal vessels and then migrated to the corneal side and the conjunctival side, suggesting that they derived from limbal vessels. Thereafter they accumulated towards the central corneal area, arriving at the suture about 7 h after its placement. Although corneal sutures may elicit the continuous infiltration of neutrophils, their number was markedly decreased by day 1 after suture removal and continued to decrease thereafter. Conclusions Our results showed that corneal sutures may elicit the continuous infiltration of neutrophils.

Characterization of monocarboxylate uptake and immunohistochemical demonstration of monocarboxylate transporters in cultured rabbit corneal epithelial cells.

This study aimed to characterize the mechanisms of monocarboxylate uptake by cultured rabbit corneal epithelial cells (RCECs) using l‐ and d‐lactic acids as model substrates.

Allogeneic Sensitization and Tolerance Induction After Corneal Endothelial Cell Transplantation in Mice.

PURPOSE We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance. METHOD The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice. RESULTS The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%). CONCLUSIONS The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.