PRELP Regulates Cell-Cell Adhesion and EMT and Inhibits Retinoblastoma Progression.

Retinoblastoma (RB) is the most common intraocular pediatric cancer. Nearly all cases of RB are associated with mutations compromising the function of the RB1 tumor suppressor gene. We previously demonstrated that PRELP is widely downregulated in various cancers and our in vivo and in vitro analysis revealed PRELP as a novel tumor suppressor and regulator of EMT. In addition, PRELP is located at chromosome 1q31.1, around a region hypothesized to be associated with the initiation of malignancy in RB. Therefore, in this study, we investigated the role of PRELP in RB through in vitro analysis and next-generation sequencing. Immunostaining revealed that PRELP is expressed in Müller glial cells in the retina. mRNA expression profiling of PRELP-/- mouse retina and PRELP-treated RB cells found that PRELP contributes to RB progression via regulation of the cancer microenvironment, in which loss of PRELP reduces cell-cell adhesion and facilitates EMT. Our observations suggest that PRELP may have potential as a new strategy for RB treatment.

Retinal Pigment Epithelial Detachment Associated with Immunoglobulin A Nephropathy: A Case Report.

Uveitis and scleritis are eye diseases associated with immunoglobulin A (IgA) nephropathy, but reports on retinal pigment epithelial detachment (PED) in relation to IgA nephropathy are scarce. We have experienced a case of PED associated with IgA nephropathy that was improved by pulse steroid treatment. A 68-year-old woman underwent examination for visual loss in the right eye. Her corrected visual acuity was 20/20 on both sides, and serous PED was observed in both eyes. One month later, the PED improved in both eyes but recurred 3 months later. Results of blood examination raised suspicion of IgA nephropathy, and she was referred to a nephrologist. Two weeks later, the PED in both eyes worsened, and a retinal pigment epithelium (RPE) tear appeared in the right eye. A sub-Tenon's injection of triamcinolone acetonide was performed to address the PED, but it was not effective; thus, pulse steroid therapy was performed twice. The PED disappeared from both eyes, and the visual acuity in her left eye was maintained at 20/20, but it decreased to 20/200 in her right eye due to macular atrophy after the RPE tear. The PED had not recurred despite having no improvement in renal function. In conclusion, in IgA nephropathy, deposition of immune complexes on the RPE causes its inflammation, which may lead to PED. In cases of unexplained PED, the possibility of a systemic disease as the cause should be considered.

Poor prognosis of elderly individuals >80 years of age with acute retinal necrosis.

PURPOSE: To report the clinical features and prognosis of acute retinal necrosis (ARN) in elderly (>80 years of age) individuals. METHODS: Six consecutive patients with unilateral ARN who attended the Department of Ophthalmology at Yamaguchi University Hospital between 2014 and 2015 were retrospectively reviewed. Clinical characteristics, causative virus, time from symptom onset to physician visit, visual acuity at presentation and final visit, and treatment were evaluated and compared between the three elderly and three middle-aged (<80 years) patients. RESULTS: Varicella zoster virus (VZV) DNA was detected in aqueous humor by the polymerase chain reaction in all six cases. The mean ± SD time between symptom onset and medical attention was 18.0 ± 8.7 and 8.3 ± 1.5 days in the elderly and middle-aged groups, respectively. All patients were treated with intravenous aciclovir, oral prednisolone, and a nonsteroidal anti-inflammatory drug, and five of the six patients also received oral valaciclovir and underwent vitrectomy. The final best corrected visual acuity of the affected eye was worse for the elderly patients (20/400, hand motion, and light perception negative) than for the middle-aged patients (20/15, 20/50, and 20/25). CONCLUSIONS AND IMPORTANCE: ARN in the elderly individuals of the present study was caused by VZV infection and associated with a poorer visual prognosis compared with that of middle-aged patients. A delay in the onset of antiviral treatment might contribute to the poor prognosis of elderly patients with ARN.

Concentration of acute phase factors in vitreous fluid in diabetic macular edema.

PURPOSE: Diabetic retinal maculopathy is associated with acute and chronic local inflammation. We measured the concentrations of acute phase factors in vitreous fluid of patients with diabetic macular edema (DME) and examined their relations to visual acuity and central retinal thickness (CRT) both before and after vitrectomy. STUDY DESIGN: Retrospective. METHODS: Vitreous fluid was collected during vitreoretinal surgery from 19 patients with DME and 12 control subjects with epiretinal membrane. The concentrations of acute phase factors (α2-macroglobulin, haptoglobin, C-reactive protein, serum amyloid P and A, procalcitonin, ferritin, tissue plasminogen activator, fibrinogen) and vascular endothelial growth factor (VEGF) were measured with multiplex assays. CRT of macular edema was measured by optical coherence tomography (OCT). RESULTS: The levels of serum amyloid P, procalcitonin, ferritin, and fibrinogen in vitreous fluid were increased in DME patients compared with control subjects. The levels of procalcitonin and fibrinogen in DME patients were inversely correlated with visual acuity both before and 3 months after vitrectomy but not 6 months postsurgery. The concentrations of these four factors were not correlated with either CRT or the vitreous levels of VEGF in DME patients. CONCLUSION: Acute phase factors may contribute to local inflammation in DME and may therefore influence disease progression.

Suppression by an RAR-γ Agonist of Collagen Degradation Mediated by Corneal Fibroblasts.

Purpose: To examine the role of retinoic acid receptor (RAR) isoforms in interleukin-1ß (IL-1ß)-induced collagen degradation by corneal fibroblasts. Methods: Primary rabbit corneal fibroblasts embedded in a three-dimensional collagen gel were incubated with or without all-trans retinoic acid (ATRA), the RAR-α agonist Am580, the RAR-ß agonist AC55649, or the RAR-γ agonist R667. Collagen degradation was determined by measurement of hydroxyproline produced in acid hydrolysates of culture supernatants. Matrix metalloproteinase (MMP) expression was evaluated by immunoblot analysis and gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the endogenous nuclear factor (NF)-κB inhibitor IκB-α was examined by immunoblot analysis. Cell proliferation was measured with a bromodeoxyuridine incorporation assay, and cell viability was determined by measurement of the release of lactate dehydrogenase. Results: Interleukin-1ß-induced collagen degradation by corneal fibroblasts was inhibited by ATRA, Am580, and R667 in a concentration-dependent manner but was unaffected by AC55649, with the inhibitory effects of ATRA and R667 being markedly greater than that of Am580. The IL-1ß-induced production of MMP-1, MMP-2, MMP-3, and MMP-9 by corneal fibroblasts was also inhibited by R667 in a concentration-dependent manner. R667 inhibited the IL-1ß-induced phosphorylation of IκB-α but not that of MAPKs. R667 had no effect on the proliferation or viability of corneal fibroblasts. Conclusions: The RAR-γ agonist R667 suppressed MMP production and thereby inhibited collagen degradation by corneal fibroblasts exposed to the proinflammatory cytokine IL-1ß. These effects of R667 may be mediated by the NF-κB signaling pathway.

Combination management by C-arm fluoroscopy and extraocular muscle severance for penetrating ocular trauma with a retrobulbar foreign body.

We report here the successful removal of a retrobulbar metallic foreign body in a patient with penetrating ocular trauma by a transconjunctival approach and combination management with C-arm fluoroscopy and extraocular muscle severance. A 37-year-old man sustained a penetrating injury to the right eye while using an iron hammer. Initial slitlamp examination revealed a corneoscleral laceration, iridocele, anterior chamber collapse, and a traumatic cataract. Visual acuity in the right eye was limited to the perception of hand motion. Computed tomography revealed an orbital foreign body in the retrobulbar area. The patient underwent corneoscleral suturing, severance of extraocular muscles, removal of the foreign body with guidance by C-arm fluoroscopy, pars plana lensectomy, and pars plana vitrectomy. Combination management with C-arm fluoroscopy and extraocular muscle severance may thus be a suitable approach to the removal of a retrobulbar metallic foreign body.

Inhibition by all-trans retinoic acid of collagen degradation mediated by corneal fibroblasts.

BACKGROUND: We examined the effect of all-trans retinoic acid on collagen degradation mediated by corneal fibroblasts. METHODS: Rabbit corneal fibroblasts were cultured with or without all-trans retinoic acid in a three-dimensional collagen gel, and the extent of collagen degradation was determined by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Matrix metalloproteinase expression was examined by immunoblot analysis and gelatin zymography. The abundance and phosphorylation state of the endogenous nuclear factor-kappaB inhibitor IκB-α were examined by immunoblot analysis. Corneal ulceration was induced by injection of lipopolysaccharide into the central corneal stroma of rabbits and was assessed by observation with a slitlamp microscope. RESULTS: All-trans retinoic acid inhibited interleukin-1ß-induced collagen degradation by corneal fibroblasts in a concentration- and time-dependent manner. It also attenuated the release and activation of matrix metalloproteinases as well as the phosphorylation and degradation of IκB-α induced by interleukin-1ß in these cells. Topical application of all-trans retinoic acid suppressed corneal ulceration induced by injection of lipopolysaccharide into the corneal stroma. CONCLUSIONS: All-trans retinoic acid inhibited collagen degradation mediated by corneal fibroblasts exposed to interleukin-1ß, with this effect being accompanied by suppression of nuclear factor-kappaB signalling as well as of matrix metalloproteinase release and activation in these cells. All-trans retinoic acid also attenuated lipopolysaccharide-induced corneal ulceration in vivo. Our results therefore suggest that all-trans retinoic acid might prove effective for the treatment of patients with corneal ulceration.

Necrosis-Induced Sterile Inflammation Mediated by Interleukin-1α in Retinal Pigment Epithelial Cells.

Endogenous danger signals released from necrotic cells contribute to retinal inflammation. We have now investigated the effects of necrotic cell extracts prepared from ARPE-19 human retinal pigment epithelial cells (ANCE) on the release of proinflammatory cytokines and chemokines by healthy ARPE-19 cells. ANCE were prepared by subjection of ARPE-19 cells to freeze-thaw cycles. The release of various cytokines and chemokines from ARPE-19 cells was measured with a multiplex assay system or enzyme-linked immunosorbent assays. The expression of interleukin (IL)-1α and the phosphorylation and degradation of the endogenous nuclear factor-κB (NF-κB) inhibitor IκB-α were examined by immunoblot analysis. Among the various cytokines and chemokines examined, we found that ANCE markedly stimulated the release of the proinflammatory cytokine IL-6 and the chemokines IL-8 and monocyte chemoattractant protein (MCP)-1 by ARPE-19 cells. ANCE-induced IL-6, IL-8, and MCP-1 release was inhibited by IL-1 receptor antagonist and by an IKK2 inhibitor (a blocker of NF-κB signaling) in a concentration-dependent manner, but was not affected by a pan-caspase inhibitor (Z-VAD-FMK). Recombinant IL-1α also induced the secretion of IL-6, IL-8, and MCP-1 from ARPE-19 cells, and IL-1α was detected in ANCE. Furthermore, ANCE induced the phosphorylation and degradation of IκB-α in ARPE-19 cells. Our findings thus suggest that IL-1α is an important danger signal that is released from necrotic retinal pigment epithelial cells and triggers proinflammatory cytokine and chemokine secretion from intact cells in a manner dependent on NF-κB signaling. IL-1α is therefore a potential therapeutic target for amelioration of sterile inflammation in the retina.

Attenuation of EMT in RPE cells and subretinal fibrosis by an RAR-γ agonist.

UNLABELLED: Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells play a key role in the pathogenesis of AMD including the fibrotic reaction. We examined the role of retinoic acid receptor-γ (RAR-γ) in the epithelial-mesenchymal transition (EMT) and other fibrosis-related processes in mouse RPE cells cultured in a type I collagen gel. Transforming growth factor-ß2 (TGF-ß2)-induced collagen gel contraction mediated by the RPE cells was inhibited by the RAR-γ agonist R667 in a concentration- and time-dependent manner. Expression of the mesenchymal markers α-smooth muscle actin and fibronectin, the release of interleukin-6, and the phosphorylation of paxillin, mitogen-activated protein kinases (ERK, p38, and JNK), Smad2, and AKT induced by TGF-ß2 were also suppressed by the RAR-γ agonist. Furthermore, gelatin zymography and immunoblot analysis revealed that the TGF-ß2-induced release of matrix metalloproteinase (MMP)-2, MMP-3, MMP-8, and MMP-9 from RPE cells was inhibited by R667, and the MMP inhibitor GM6001 attenuated TGF-ß2-induced RPE cell contraction. Finally, immunohistofluorescence analysis with antibodies to glial fibrillary acidic protein showed that R667 inhibited the development of subretinal fibrosis in a mouse model in vivo. Our results thus suggest that RAR-γ agonists may prove effective for the treatment of subretinal fibrosis associated with AMD. KEY MESSAGE: RAR-γ agonist R667 suppressed collagen gel contraction mediated by RPE cells. Epithelial-mesenchymal transition (EMT) in RPE cells was inhibited by RAR-γ agonist R667. RAR-γ agonist R667 inhibited fibrosis-related processes in RPE cells. RAR-γ agonists may attenuate AMD-associated fibrosis.

All-trans-retinoic acid inhibition of transforming growth factor-ß-induced collagen gel contraction mediated by human Tenon fibroblasts: role of matrix metalloproteinases.

BACKGROUND/AIMS: Scarring and contraction of the conjunctiva are common complications of many ocular diseases. We investigated the effects of all-trans-retinoic acid (ATRA) on the contractility of human Tenon's capsule fibroblasts (HTFs) cultured in a three-dimensional collagen gel. METHODS: HTFs were cultured in a three-dimensional gel of type I collagen and in the absence or presence of transforming growth factor (TGF)-ß, ATRA, or an inhibitor of matrix metalloproteinases (MMPs). Collagen gel contraction was evaluated by measurement of gel diameter. The release of MMPs and tissue inhibitors of metalloproteinases (TIMPs) into culture supernatants was assessed by immunoblot analysis and gelatin zymography. The release of lactate dehydrogenase activity from HTFs was measured with a colorimetric assay kit. RESULTS: ATRA inhibited TGF-ß-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. TGF-ß induced the release of MMP-1, MMP-2 and MMP-3 by HTFs, and ATRA inhibited these effects of TGF-ß on MMP-1 and MMP-3 release. ATRA also stimulated TIMP-1 release from HTFs in the presence of TGF-ß. Furthermore, TGF-ß-induced collagen gel contraction was blocked by the MMP inhibitor GM6001. ATRA did not exhibit cytotoxicity for HTFs. CONCLUSIONS: ATRA inhibited TGF-ß-induced collagen gel contraction mediated by HTFs, likely in part by attenuating the production of MMP-1 and MMP-3 and by stimulating the production of TIMP-1. ATRA may therefore prove to be of clinical value for inhibition of scar formation in the conjunctiva.

Inhibition by a retinoic acid receptor γ agonist of extracellular matrix remodeling mediated by human Tenon fibroblasts.

PURPOSE: Scar formation is most frequently responsible for the failure of glaucoma filtration surgery. Retinoic acids are vitamin A derivatives that play diverse roles in development, immunity, and tissue repair. The effects of the retinoic acid receptor (RAR) γ agonist R667 on the contractility of human Tenon fibroblasts (HTFs) cultured in a three-dimensional collagen gel as well as on intraocular pressure (IOP) in a rat model of glaucoma filtration surgery were investigated. METHODS: HTFs were cultured in a type I collagen gel, the contraction of which was evaluated by measurement of the gel diameter. The release of matrix metalloproteinases (MMPs) into culture supernatants was assessed with immunoblot analysis and gelatin zymography. Phosphorylation of focal adhesion kinase (FAK) was examined with immunoblot analysis, and production of fibronectin and type I collagen was measured with immunoassays. RESULTS: R667 inhibited transforming growth factor-ß1 (TGF-ß1)-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner, whereas an RARα agonist inhibited this process to a lesser extent and an RARß agonist had no effect. TGF-ß1-induced MMP-1 and MMP-3 release, FAK phosphorylation, and fibronectin and type I collagen production in HTFs were also attenuated by R667. Furthermore, R667 lowered IOP in rats after glaucoma filtration surgery. CONCLUSIONS: R667 inhibited TGF-ß1-induced contraction and extracellular matrix synthesis in HTFs. Such effects might have contributed to the lowering of IOP by R667 in a rat model of glaucoma filtration surgery. RARγ agonists might thus prove effective for inhibition of scar formation after such surgery.

Inhibition by all-trans-retinoic acid of transforming growth factor-ß-induced collagen gel contraction mediated by human tenon fibroblasts.

PURPOSE: Excessive wound contraction can lead to scar formation in the conjunctiva. The effects of all-trans-retinoic acid (ATRA) on the contractility of human Tenon fibroblasts (HTFs) cultured in three-dimensional (3D) collagen gels were investigated. METHODS: Human Tenon fibroblasts were cultured in 3D gels of type I collagen and in the absence or presence of TGF-ß, ATRA, or various inhibitors. Collagen gel contraction was evaluated by measurement of gel diameter. Phosphorylation of various signaling molecules was examined by immunoblot analysis. The formation of actin stress fibers and focal adhesions was detected by laser confocal microscopy. RESULTS: All-trans-retinoic acid inhibited TGF-ß-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. The TGF-ß-induced phosphorylation of focal adhesion kinase (FAK) and formation of stress fibers and focal adhesions in HTFs were attenuated by ATRA. All-trans-retinoic acid also inhibited the TGF-ß-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) as well as that of c-Jun and Smad2/3. Furthermore, TGF-ß-induced collagen gel contraction was blocked by inhibitors of ERK, p38, or JNK signaling. CONCLUSIONS: All-trans-retinoic acid inhibited TGF-ß-induced collagen gel contraction mediated by HTFs, most likely by attenuating the formation of actin stress fibers and focal adhesions as well as signaling by MAPKs, c-Jun, and Smads. All-trans-retinoic acid may therefore prove effective for inhibition of conjunctival scarring through attenuation of the contractility of Tenon fibroblasts.

Inhibition by female sex hormones of collagen gel contraction mediated by retinal pigment epithelial cells.

PURPOSE: Collagen contraction mediated by retinal pigment epithelial (RPE) cells contributes to the pathogenesis of proliferative vitreoretinopathy (PVR). We examined the effects of sex hormones on this process. METHODS: Mouse RPE cells were cultured in a type I collagen gel and exposed to 17ß-estradiol, progesterone, or dehydro-epiandrosterone. Collagen contraction induced by transforming growth factor-ß2 (TGF-ß2) was determined by measurement of gel diameter. Expression of α-smooth muscle actin (α-SMA), as well as phosphorylation of Smad2 and myosin light chain (MLC), was examined by immunoblot analysis. Matrix metalloproteinase (MMP) release was evaluated by gelatin zymography. Fibronectin and interleukin-6 secretion was measured with immunoassays. RESULTS: The female sex hormones 17ß-estradiol and progesterone inhibited TGF-ß2-induced collagen contraction mediated by RPE cells, whereas the male sex hormone dehydro-epiandrosterone had no such effect. The TGF-ß2-induced release of MMP-2 and MMP-9 from RPE cells was also inhibited by 17ß-estradiol and progesterone, and the MMP inhibitor GM6001 attenuated TGF-ß2-induced collagen contraction. Expression of the mesenchymal markers α-SMA and fibronectin, interleukin-6 release, and Smad2 and MLC phosphorylation induced by TGF-ß2 were all inhibited by 17ß-estradiol and progesterone. Immunohistochemical analysis also detected nuclear immunoreactivity for estrogen and progesterone receptors in proliferative fibrocellular membranes of PVR patients. CONCLUSIONS: Female sex hormones inhibited TGF-ß2-induced collagen contraction mediated by RPE cells. This action appeared to be mediated through inhibition both of MMP, α-SMA, and fibronectin expression as well as of Smad2 and MLC phosphorylation. Female sex hormones might thus prove effective for the treatment of PVR.

Role of the JNK signaling pathway in downregulation of connexin43 by TNF-α in human corneal fibroblasts.

PURPOSE/AIM: Proinflammatory cytokines such as tumor necrosis factor (TNF)-α contribute to corneal inflammation. Corneal stromal fibroblasts are connected to each other via gap junctions. We have now examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in TNF-α-induced downregulation of the gap junction protein connexin43 (Cx43) in human corneal fibroblasts. MATERIALS AND METHODS: Cultured human corneal fibroblasts were exposed to TNF-α in the absence or presence of inhibitors of MAPK signaling pathways. Expression of Cx43 was evaluated by immunofluorescence and immunoblot analyses. Gap-junctional intercellular communication (GJIC) was measured with a dye-coupling assay. RESULTS: TNF-α reduced the abundance of Cx43 in human corneal fibroblasts (as revealed by immunoblot analysis) as well as induced the loss of specific staining for this protein (as revealed by immunofluorescence analysis). These effects of TNF-α were attenuated by an inhibitor of c-Jun NH2-terminal kinase (JNK inhibitor II) but not by inhibitors of signaling by extracellular signal-regulated kinase (PD98059) or by p38 MAPK (SB203580). JNK inhibitor II also attenuated the inhibitory effect of TNF-α on GJIC. CONCLUSIONS: The inhibitory effects of TNF-α on Cx43 expression and GJIC in human corneal fibroblasts are mediated, at least in part, by the JNK signaling pathway, which therefore likely plays a role in corneal inflammation.

Protection of human corneal epithelial cells from TNF-α-induced disruption of barrier function by rebamipide.

PURPOSE: TNF-α disrupts the barrier function of cultured human corneal epithelial (HCE) cells. We investigated the effects of the cytoprotective drug rebamipide on this barrier disruption by TNF-α as well as on corneal epithelial damage in a rat model of dry eye. METHODS: The barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance. The distribution of tight-junction (ZO-1, occludin) and adherens-junction (E-cadherin, ß-catenin) proteins, and the p65 subunit of nuclear factor-κB (NF-κB) was determined by immunofluorescence microscopy. Expression of junctional proteins as well as phosphorylation of the NF-κB inhibitor IκB-α and myosin light chain (MLC) were examined by immunoblot analysis. A rat model of dry eye was developed by surgical removal of exorbital lacrimal glands. RESULTS: Rebamipide inhibited the disruption of barrier function as well as the downregulation of ZO-1 expression, and the disappearance of ZO-1 from the interfaces of neighboring HCE cells induced by TNF-α. It also inhibited the phosphorylation and downregulation of IκB-α, the translocation of p65 to the nucleus, the formation of actin stress fibers, and the phosphorylation of MLC induced by TNF-α in HCE cells. Treatment with rebamipide eyedrops promoted the healing of corneal epithelial defects as well as attenuated the loss of ZO-1 from the surface of corneal epithelial cells in rats. CONCLUSIONS: Rebamipide protects corneal epithelial cells from the TNF-α-induced disruption of barrier function by maintaining the distribution and expression of ZO-1 as well as the organization of the actin cytoskeleton. Rebamipide is, thus, a potential drug for preventing or ameliorating the loss of corneal epithelial barrier function associated with ocular inflammation.

Cytokine and chemokine secretion induced by poly(I:C) through NF-κB and phosphoinositide 3-kinase signaling pathways in human corneal fibroblasts.

PURPOSE/AIM: Viral infection of the cornea can result in inflammation and scarring and eventually lead to blindness. Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, induces the secretion of cytokines and chemokines from cultured corneal fibroblasts. We have now investigated the role of nuclear factor (NF)-κB and phosphoinositide 3-kinase (PI3K) signaling pathways in poly(I:C)-induced cytokine and chemokine secretion from corneal fibroblasts. MATERIALS AND METHODS: Human corneal fibroblasts were cultured with poly(I:C) in the absence or presence of IKK-2 inhibitor or LY294002, which are inhibitors of NF-κB and PI3K signaling, respectively. The release of the pro-inflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, IP-10, and RANTES from the cells was measured with an enzyme-linked immunosorbent assay. RESULTS: Poly(I:C) induced the secretion of IL-6, IL-8, IP-10, and RANTES from corneal fibroblasts. Whereas the poly(I:C)-induced secretion of IL-6, IP-10, and RANTES was inhibited by both IKK-2 inhibitor and LY294002, that of IL-8 was blocked only by IKK-2 inhibitor. CONCLUSIONS: The poly(I:C)-induced secretion of IL-6, IP-10, and RANTES from human corneal fibroblasts is mediated by both NF-κB and PI3K signaling pathways, whereas that of IL-8 is mediated by the NF-κB pathway. These signaling pathways thus likely contribute to local inflammation in the corneal stroma induced by viral infection.

Inhibition by medroxyprogesterone acetate of interleukin-1ß-induced collagen degradation by corneal fibroblasts.

PURPOSE: To examine the effect of medroxyprogesterone 17-acetate (MPA) on interleukin-1ß (IL-1ß)-induced collagen degradation by corneal fibroblasts. METHODS: Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels with or without MPA. Collagen degradation was determined by measurement of hydroxyproline after acid hydrolysis. The expression or activity of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) was evaluated by immunoblot analysis or gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts was examined by immunoblot analysis. Cell proliferation and viability were evaluated by measurement of bromodeoxyuridine incorporation and the release of lactate dehydrogenase, respectively. RESULTS: MPA inhibited IL-1ß-induced collagen degradation by corneal fibroblasts in a concentration- and time-dependent manner. MMP expression and activation as well as TIMP expression in corneal fibroblasts exposed to IL-1ß were also inhibited by MPA. MPA had no effect on cell proliferation or viability. MPA inhibited the IL-1ß-induced phosphorylation of p38 MAPK without affecting that of the MAPKs ERK or JNK. IL-1ß-induced MMP expression and activation as well as collagen degradation were also blocked by the p38 MAPK inhibitor SB203580. CONCLUSIONS: MPA inhibited MMP expression and thereby suppressed collagen degradation by corneal fibroblasts induced by IL-1ß. Furthermore, inhibition of p38 MAPK phosphorylation by MPA may contribute to its inhibition of collagen degradation.

Identification of common secreted factors in human corneal fibroblasts exposed to LPS, poly(I:C), or zymosan.

Infection of the cornea with bacteria, viruses, or fungi can result in corneal ulceration. Corneal stromal cells participate in the immune and inflammatory responses to such infection in part by producing various cytokines and chemokines. The effects of lipopolysaccharide (LPS), polyinosinic-polycytidylic acid [poly(I:C)], and zymosan as surrogates for bacteria, viruses, and fungi, respectively, on the release of cytokines and chemokines from cultured human corneal fibroblasts were examined in order to identify common factors in infectious corneal keratitis. The secretion of various cytokines and chemokines by human corneal fibroblasts exposed to LPS, poly(I:C), or zymosan was measured with a multiplex assay system. LPS induced the release of interleukin (IL)-6, IL-8, MCP-1, RANTES, IP-10, eotaxin, and IL-12 from corneal fibroblasts. Poly(I:C) stimulated the secretion of IL-6, IL-8, MCP-1, RANTES, IP-10, eotaxin, MIP-1ß, and interferon-γ, whereas zymosan triggered the production of IL-6, IL-8, and MCP-1. LPS, poly(I:C), and zymosan thus each induced a distinct pattern of cytokine and chemokine release from human corneal fibroblasts, with the release of IL-6, IL-8, and MCP-1 being commonly elicited by all three agents. Our results suggest that IL-6, IL-8, and MCP-1 may therefore play a key role in the inflammatory response to corneal infection.

Klinefelter's syndrome complicated with West syndrome in a 4-month-old boy.

We, for the first time, report a boy with West syndrome associated with Klinefelter's syndrome. He developed episodes of repetitive tonic spasms at the age of 4 months. He had developmental delays and hypsarrhythmia on interictal electroencephalography recording. His karyotype turned out to be 47, XXY, while we failed to observe anomalies in his appearance. Adrenocorticotropic hormone therapy with antiepileptic drugs resulted in cessation of tonic spasms, and his developmental quotient was improved by the age of 1 year. Further studies are needed to determine the causal association between West syndrome and Klinefelter's syndrome.