[Two cases of conjunctival intraepithelial neoplasia in immunosuppressed patients].

UNLABELLED: Background: Conjunctival intraepithelial neoplasia (CIN) is a precursor lesion of conjunctival squamous cell carcinoma (SCC). CIN recurs frequently but progresses less aggressively than SCC. We report 2 cases of recurrent CIN in immunosuppressed patients. CASE 1: A 60-year-old woman had been taking oral immunosuppressive drugs for 30 years for systemic lupus erythematosus. In 2005, both conjunctival tumor and high serum SCC levels were noted. Biopsy revealed right eye CIN and left eye SCC, and extended resection was performed. In 2008, right eye CIN recurred accompanied by high serum SCC levels and another extended resection was performed. The patient was free from recurrence for 1 year until her death. CASE 2: A 43-year-old woman who had been taking oral immunosuppressive drugs for 3 years for nephrotic syndrome. She had twice undergone right corneal transplantation for keratoconus. In 2011, right conjunctival tumor and high serum SCC levels were noted. Biopsy revealed right eye CIN, for which an extended resection was performed. At present she remains free from recurrence. CONCLUSION: CIN can recur in immunosuppressed patients. We suggest that serum SCC levels be monitored to help detect recurrence.

Increase of intracellular Ca2+ by purinergic receptors in cultured rat lacrimal gland myoepithelial cells.

PURPOSE: To isolate and characterize cultured myoepithelial cells (MECs) from rat lacrimal gland and determine which purinergic receptor subtypes are present and functional in MECs. METHODS: Rat lacrimal glands were subjected to collagenase digestion, and MECs were grown. RT-PCR was performed for the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13) on RNA isolated from the MECs. Immunofluorescence experiments were performed with antibodies against MEC markers and P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Proteins from MECs were separated using Western blot analysis techniques. In addition, cells were incubated with Fura 2 tetra acetoxymethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined in response to P2 purinergic agonists. RESULTS: MECs expressed the MEC proteins α-smooth muscle actin, vimentin, α-actinin, and adenylyl cyclase II. RT-PCR, Western blot, and immunofluorescence techniques demonstrated the presence of the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13). The purinergic agonists ATP, benzoylbenzoyl ATP (BzATP), α,ß methylene ATP, UTP, 2-methylthioATP (MeSATP), and ATPγS increased [Ca(2+)](i). As BzATP binds to the P2X(7) receptor, specific characteristics of this receptor were investigated. Neither inhibitors of P2X(7) receptors nor removal of extracellular Mg(2+) or Ca(2+) had an effect on the BzATP-stimulated increase in [Ca(2+)](i). Repeated applications of BzATP desensitized this response. Inhibitors for P2Y(1), P2Y(11), and P2Y(13) each decreased the BzATP-stimulated increase in [Ca(2+)](i) with the P2Y(1) inhibitor most effective. CONCLUSIONS: MECs can be isolated from rat lacrimal glands, and they express P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Surprisingly, BzATP binds the P2Y(1) receptor, which is primarily responsible for the BzATP-stimulated increase in [Ca(2+)](i).

Effect of tacrolimus on chemokine production by corneal myofibroblasts via toll-like receptors, compared with cyclosporine and dexamethasone.

PURPOSE: To investigate the effect of tacrolimus on chemokine production by corneal myofibroblasts compared with that of cyclosporine or dexamethasone. METHODS: We investigated the expression of FK506-binding protein 12, calcineurin, and nuclear factor of activated T cells in corneal myofibroblasts by immunocytochemistry and flow cytometry. Next, we investigated whether toll-like receptor 9 ligand, CpG-DNA, or toll-like receptor 3 ligand, poly (I:C), induced the production of chemokines such as interleukin (IL) 8, Gro-α, and IL-6 by corneal myofibroblasts using enzyme-linked immunosorbent assay. Finally, we assessed the effect of tacrolimus on the production of these chemokines compared with that of cyclosporine or dexamethasone. RESULTS: FK506-binding protein 12, calcineurin, and nuclear factor of activated T cells were constantly expressed by cultured corneal myofibroblasts. CpG DNA and poly (I:C) enhanced the production of IL-8, Gro-α, and IL-6 by corneal myofibroblasts. At a concentration 10 nM/L or higher, dexamethasone strongly inhibited the production of these chemokines. In contrast, cyclosporine concentrations of 10 ng/mL or more enhanced the production of these chemokines by cells stimulated with poly (I:C). On the other hand, tacrolimus (at 10 ng/mL or more) inhibited the production of these chemokines by corneal myofibroblasts stimulated with poly (I:C) but not with CpG-DNA. CONCLUSIONS: These results suggest that tacrolimus and dexamethasone, but not cyclosporine, have a direct immunosuppressive effect on corneal myofibroblasts. Therefore, when inflammatory diseases of the ocular surface are treated by tacrolimus, especially in combination with steroids, caution with regard to the risk of corneal infection may be needed.

The epithelium takes center stage in allergic keratoconjunctivitis.

PURPOSE: To investigate the role of the epithelium in severe allergic conjunctivitis. METHODS: We first investigated the expression of protease-activated receptors (PARs) in cultured human conjunctival epithelial cells and fibroblasts by reverse transcription-polymerase chain reaction. Next, we examined whether mite allergen-stimulated cells release chemokines and whether physiological protease inhibitors such as secretory leukocyte protease inhibitor (SLPI) and α1-antitrypsin can inhibit their production. We also looked at the expression of thymic stromal lymphopoietin (TSLP) in giant papillae of patients with vernal keratoconjunctivitis and examined whether the as Toll-like receptor 3 ligand polyinosinic:polycytidylic acid (poly I:C) can induce expression of TSLP in cultured human conjunctival epithelial cells. RESULTS: PAR 1, PAR2, and PAR3 were expressed in cultured human conjunctival epithelial cells and fibroblasts at mRNA level. These epithelial cells released interleukin (IL) 6 and IL-8, with an upregulation in their gene expression, in response to the serine protease activity of mite allergens. This response was inhibited by SLPI and α1-antitrypsin. Transforming growth factor ß1 decreased the production of SLPI in corneal and conjunctival epithelial cells. TSLP was expressed in giant papillae epithelium in patients with vernal keratoconjunctivitis at mRNA and protein levels. Poly I:C induced expression of TSLP in cultured conjunctival epithelial cells at mRNA level. Costimulation with TSLP and IL-33 had a synergistic effect for IL-13 mRNA expression in cultured human mast cells. CONCLUSIONS: Imbalance between protease of mite allergens and innate protease inhibitors of the epithelium may induce inflammation and disrupt barrier function. Viral infection may induce expression of TSLP via Toll-like receptors and release IL-33 by necrosis. These phenomena promote excessive allergic reactions; hence, the epithelium takes "center stage" in allergic conjunctivitis.

Role of TGF-ß in tissue eosinophilia associated with vernal keratoconjunctivitis.

To determine the role of TGF-ß(1) in the tissue eosinophilia associated with vernal keratoconjunctivitis (VKC), we investigated the immunohistochemical expression of TGF-ß(1) and TGF-ß(1)-related proteins in giant papillae obtained from VKC patients. We also investigated the effect of TGF-ß(1) on production of eotaxin by cultured conjunctival and corneal fibroblasts using ELISA. Finally, the effects of glucocorticoids, cyclosporine, and tacrolimus on eotaxin production by corneal fibroblasts were assessed. Our investigations revealed that eosinophils expressing TGF-ß(1) and TGF-ß(1)-related proteins (such as phosphorylated Smad2, integrin αvß(6), α-smooth muscle actin, type I procollagen, and tenascin-C) were expressed in the giant papillae. TGF-ß(1) and IL-4/IL-13 caused a synergistic increase of eotaxin production in cultured conjunctival and corneal fibroblasts. This effect of TGF-ß(1) and IL-4/IL-13 was inhibited by glucocorticoids, but neither by cyclosporine nor by tacrolimus. In conclusion, TGF-ß(1) has an important role in the tissue eosinophilia associated with VKC.