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Angiogenesis plays a critical role in initiating and promoting several diseases, such as cancer and herpetic stromal keratitis (HSK). Herein, we studied the inhibitory effect of two synthetic stigmasterol derivatives on capillary tube-like structures and on cell migration in human umbilical vein endothelial cells (HUVEC): (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3β-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2). We also studied their effect on VEGF expression in IL-6 stimulated macrophages and in LMM3 breast cancer cells. Furthermore, we investigated the antiangiogenic activity of the compounds on corneal neovascularization in the murine model of HSK and in an experimental model of tumor-induced angiogenesis in mice. Both compounds inhibited capillary tube-like formation, but only compound 1 restrained cell migration. Compound 1, unlike compound 2, was able to reduce VEGF expression. Only compound 1 not only reduced the incidence and severity of corneal neovascularization, when administered at the onset of HSK, but it also restrained the development of neovascular response induced by tumor cells in mice skin. Our results show that compound 1 inhibits angiogenesis in vitro and in vivo. Therefore, compound 1 would be a promising drug in the treatment of those diseases where angiogenesis represents one of the main pathogenic events. Fil: Michelini, Flavia Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Lombardi, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Cátedra de Farmacología; Argentina Fil: Bueno, Carlos Alberto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Berra, Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; Argentina Fil: Sales, María Elena. Universidad de Buenos Aires. Facultad de Medicina. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Alche, Laura Edith. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Angiogenesis plays a critical role in initiating and promoting several diseases, such as cancer and herpetic stromal keratitis (HSK). Herein, we studied the inhibitory effect of two synthetic stigmasterol derivatives on capillary tube-like structures and on cell migration in human umbilical vein endothelial cells (HUVEC): (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S) 3-bromo-5,22,23-trihidroxistigmastan-6-ona (compound 2). We also studied their effect on VEGF expression in IL-6 stimulated macrophages and in LMM3 breast cancer cells. Furthermore, we investigated the antiangiogenic activity of the compounds on corneal neovascularization in the murine model of HSK and in an experimental model of tumor-induced angiogenesis in mice.Both compounds inhibited capillary tube-like formation, but only compound 1 restrained cell migration. Compound 1, unlike compound 2, was able to reduce VEGF expression. Only compound 1 not only reduced the incidence and severity of corneal neovascularization, when administered at the onset of HSK, but it also restrained the development of neovascular response induced by tumor cells in mice skin.Our results show that compound 1 inhibits angiogenesis in vitro and in vivo. Therefore, compound 1 would be a promising drug in the treatment of those diseases where angiogenesis represents one of the main pathogenic events. Fil: Michelini, Flavia Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Lombardi, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Bueno, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Berra, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; Argentina Fil: Sales, María Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Alche, Laura Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Background: Microbial keratitis is an infectious disease of the cornea characterised by inflammation and is considered an ophthalmic emergency requiring immediate attention. While a variety of pathogenic microbes associated with microbial keratitis have been identified, a comprehensive review identifying the diversity of species has not been completed. Methods: A search of peer-reviewed publications including case reports and research articles reporting microorganims implicated in keratitis was conducted. Search engines including PubMed, Scopus and Web of Science with years ranging from 1950-2012 were used. Results: 232 different species from 142 genera, representing 80 families were found to be implicated in microbial keratitis. Fungi exhibited the largest diversity with 144 species from 92 genera. In comparison, 77 species of bacteria from 42 genera, 12 species of protozoa from 4 genera and 4 types of virus were identified as the infectious agents. A comparison of their aetiologies shows reports of similarities between genera. Conclusions: The diversity of microbial species implicated in keratitis has not previously been reported and is considerably greater than suggested by incidence studies. Effective treatment is heavily reliant upon correct identification of the responsible microorganisms. Species identification, the risk factors associated with, and pathogenesis of microbial keratitis will allow the development of improved therapies. This review provides a resource for clinicians and researchers to assist in identification and readily source treatment information.

眼科学教室 堀貞夫教授退任記念特別号

眼科学教室 堀貞夫教授退任記念特別号

眼科学教室 堀貞夫教授退任記念特別号

[抄録]単純へルペスウイルス1型(HSV-1)によるへルペス性角膜炎は，しばしば再発を繰り返し，角膜混濁，視力低下，社会的失明となる場合がある.アシクロビル内服による再活性化抑制効果を検討した報告があるが，臨床的に充分な抑制率ではなかった.今回の研究では，マウスへルペス性角膜炎モデルを用いて，アスコルビン酸(AsA)，亜鉛(Zn)，アデノシンモノフォスフェート(AMP)，ゲルダナマイシン(GM)のHSV-1再活性化抑制効果を検討した.マウス角膜にHSV-1を感染させ， AsA内服群， Zn内服群，これらのコントロールとして生理食塩水(生食)内服群， AMP筋肉内注射(筋注)群，このコントロールとして生食筋注群， GM腹腔内注射(腹注)群，このコントロールとしてジメチルスルホキシド腹注群，計7群に分類し，潜伏感染成立後25日目より各薬剤を投与した.免疫抑制薬と熱ストレスを用いて再活性化し，マウス眼球と三叉神経節を採取し， Plaque AssayとReal-time PCR法を用いて検討した.Plaque Assayによる検討で， GM腹注群は，三叉神経節における陽性サンプル数に有意な減少を認め， Real-time PCRでは， AMP筋注群三叉神経節におけるウイルスDNA検出コピー数に有意な減少を認めた.以上よりGMとAMPは，マウス潜伏感染モデルにおいて， HSV-1再活性化抑制効果を示すと考えられた.

Ocular herpes simplex virus (HSV) infection remains a major cause of corneal blindness. Several topical and oral antiviral medications have been used to treat herpetic keratitis. Advances in topical ophthalmic antivirals have been made over the past several decades. The first antivirals that were discovered were cytotoxic, while the antivirals developed more recently, such as acyclovir and ganciclovir, have exceeded these drugs in both efficacy and tolerability. Commercially available outside of the US since 1996, ganciclovir ophthalmic gel, 0.15% (GCV 0.15%, European tradename: Virgan((R))) is sold in more than 30 countries and has become the standard of care in treating acute herpetic keratitis. GCV 0.15% has been studied in animal models of ocular herpes, in healthy volunteers, and in several clinical studies. It has been found to be safe and effective at treating acute superficial herpetic keratitis. Previous preclinical studies of ganciclovir have shown activity against several common adenovirus strains and one recent clinical study demonstrated clinical effect against adenoviral conjunctivitis. This review is intended to provide a comprehensive overview of the GCV 0.15%, including a brief summary of the etiology and available treatments for ocular HSV, an explanation of GCV 0.15% mechanism of action, a compendium of preclinical and clinical GCV 0.15% studies, and an introduction into new areas of interest involving this drug.

Background This objective of the review and analysis is to demonstrate that acyclovir (ACV) 3% ophthalmic ointment is superior to idoxuridine (IDU) in treating herpetic keratitis (HK) presenting as dendritic and geographic ulcer sub-types. Methods Data sources: Publications in human subjects were identified by searching the Ovid MEDLINE database through April 2011, combining medical subject headings (MESH) “Keratitis, Herpetic/” AND “Acyclovir/” limiting by the key words “topical” OR “ointment” and also restricted to MESH “Administration, Topical/” OR “Ointments/”. The results were cross checked with the references used in the Cochrane Database Syst Rev. 1:1–134, 2009 and GlaxoSmithKline clinical documents related to acyclovir. Study selection: Randomized, double-masked studies in subjects diagnosed with HK with head to head comparator arms of ACV ophthalmic ointment and topical IDU that had actual or calculable healing rates at Day seven. Data extraction: Data independently extracted from identified articles by two authors of this manuscript. Data synthesis: Data from seven randomized, controlled trials (RCT) evaluating 432 subjects that met inclusion criteria (214 were treated with ACV and 218 were treated with IDU) and had Day seven healing rates calculable. All sub-classified lesions were identified as either dendritic ulcers (n = 185) or geographic ulcers (n = 35). The Cochran-Mantel-Haenszel (CMH) method in Biometrics 10:417-51, 1954 and JNCI 22:719-48, 1959, controlling for study, was performed as the primary analysis using SAS v9. Homogeneity was assessed using Breslow-Day-Tarone (BDT) test in IARC 1:1-32, 1980 and Biometrika 72:91-5, 1985. The analysis was performed with outliers removed to assess their impact. Results ACV showed statistically significant greater odds of healing HK at Day seven in all subjects (Odds Ratio 3.95, 95% CI2.60, 6.00, p

The time course for delivery and transport of two major proteins of herpes simplex virus (HSV) has been determined for mature mouse retinal ganglion cell axons in vivo. Twenty-four hours after intravitreal injection of HSV, valacyclovir was introduced into the drinking water of the mice to inhibit subsequent viral replication. Without treatment, viral spread and replication in periaxonal glial cells confound study of axonal transport. At 2 to 5 days after infection, the animals were sacrificed and contiguous segments of the optic pathway were removed. Immunofluorescence microscopy indicated that the number of infected astrocytes was reduced in the proximal optic nerve and eliminated in the optic tract. Western blots of the retina with antibodies for envelope and capsid components, glycoprotein D (gD) and VP5, respectively, revealed that both components were expressed in retinal homogenates by 2 days. Results of reverse transcription-PCR indicated that there was no gD mRNA present in the treated optic tract 5 days after infection. Therefore, we conclude that gD is transcribed from viral mRNA in the retinal ganglion cell bodies. The gD accumulated in the proximal ganglion cell axon by 2 days and reached the most distal segment after 3 days. The VP5 first appeared in the proximal axons at 4 days, about 48 h after the appearance of gD. Thus, gD entered the axon earlier and independent of VP5. These finding confirm the subassembly model of viral transport in neurons and suggest that there is a 4- to 5-day window for initiation of effective antiviral treatment with valacyclovir.

Khalid F Tabbara1,2,3, Noorjehan Al Balushi11The Eye Center and The Eye Foundation for Research in Ophthalmology, Riyadh, 2Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia; 3The Wilmer Ophthalmological Institute of The Johns Hopkins University School of Medicine, Baltimore, Maryland, USAAbstract: Herpetic keratitis is caused by herpes simplex virus (HSV) and is a common cause of corneal blindness. Following a primary ocular herpetic infection, latency of the virus occurs, followed by subsequent recurrences of herpetic keratitis. Such recurrences may lead to structural damage of the cornea. Recurrent herpetic keratitis is a common indication for corneal transplantation. Recurrences of herpetic keratitis in the corneal graft may lead to corneal graft rejection. Several antiviral agents for HSV are available, including the thymidine analogs. Prolonged use of thymidine analogs may lead to toxicity of the ocular surface, including epithelial keratitis, corneal ulcers, follicular conjunctivitis, and punctal occlusions. Availability of topical antiviral agents that are safe and effective in the treatment and prophylaxis of herpetic keratitis is highly desirable. Ganciclovir is a potent inhibitor of members of the herpes virus family. The drug has been used systemically for the treatment of cytomegalovirus (CMV) retinitis. Its hematologic toxicity secondary to systemic administration led to its limited use in herpetic infections. On the other hand, topical ganciclovir has been shown to be as safe and effective as acyclovir in the treatment of herpetic epithelial keratitis. Furthermore, topical ganciclovir can reach therapeutic levels in the cornea and aqueous humor following topical application. Several clinical trials have shown that topical ganciclovir 0.15% ophthalmic gel is safe and effective in the treatment and prophylaxis of herpetic epithelial disease. Long-term use of ganciclovir ophthalmic gel in patients with penetrating keratoplasty following herpetic keratitis has prevented recurrences of the disease. Topical ganciclovir ophthalmic gel is well tolerated, does not cause toxic effects on the ocular surface, and does not cause hematologic abnormalities. Clinical studies have underscored the potential role of ganciclovir ophthalmic gel in the treatment and prophylaxis of herpetic epithelial keratitis. Future randomized, controlled, multicenter, prospective clinical trials are needed to assess the long-term safety and efficacy of topical ganciclovir in the treatment and prevention of herpetic keratitis and uveitis.Keywords: herpetic keratitis, cornea, herpes simplex, ganciclovir, acyclovir

Amniotic membrane transplantation (AMT) promotes rapid epithelialization and reduces stromal inflammation and ulceration in HSV-1 keratitis. 18 patients with non-healing epithelial defect or corneal ulcer caused by herpetic keratitis were included in the study. All patients were treated by AMT. Corneal epithelial cells in patients suffering from herpetic keratitis secreted 5+/-4.8 pg/ml of IL-1alpha and 0.16+/-0.47 pg/ml of IL-1beta (mean+/-SD). IL-1alpha level was significantly higher as compared to controls (p

To describe two cases of herpetic keratitis after corneal collagen cross-linking (CXL) for progressive keratoconus. An 18-year-old male and a 21-year-old male with rapidly progressive keratoconus were treated with CXL. Postoperatively, on the 6(th) and 9(th) days respectively, a dendritic ulcer was observed in the treated eye. The corneal sensation was significantly diminished compared to the fellow eye. Both patients had no prior history of herpetic eye disease or cold sores. The keratitis improved dramatically over the following days after initiation of antiviral therapy. At 4 months, the visual acuity was stable without corneal scarring. Herpetic keratitis could be induced by CXL even in patients with no history of previous herpetic eye disease. Early diagnosis and proper treatment can facilitate the successful management of this rare but important complication.

Herpes simplex virus (HSV) infects both epithelial cells and neuronal cells of the human host. Although HSV assembly has been studied extensively for cultured epithelial and neuronal cells, cultured neurons are biochemically, physiologically, and anatomically significantly different than mature neurons in vivo. Therefore, it is imperative that viral maturation and assembly be studied in vivo. To study viral assembly in vivo, we inoculated wild-type and replication-defective viruses into the posterior chamber of mouse eyes and followed infection in retinal ganglion cell bodies and axons. We used PCR techniques to detect viral DNA and RNA and electron microscopy immunohistochemistry and Western blotting to detect viral proteins in specific portions of the optic tract. This approach has shown that viral DNA replication is necessary for viral DNA movement into axons. Movement of viral DNA along ganglion cell axons occurs within capsid-like structures at the speed of fast axonal transport. These studies show that the combined use of intravitreal injections of replication-defective viruses and molecular probes allows the genetic analysis of essential viral replication and maturation processes in neurons in vivo. The studies also provide novel direct evidence for the axonal transport of viral DNA and support for the subassembly hypothesis of viral maturation in situ.

Introduction. Amniotic membrane transplantation (AMT) promotes rapid epithelialization and reduces stromal inflammation and ulceration in HSV-1 keratitis. In this study we have measured production of inflammatory mediators in corneas of patients with herpetic keratitis, together with anti-inflammatory cytokine secreted by AM. Methods. 18 patients with non-healing epithelial defect caused by herpetic keratitis were included in the study. All patients were treated by AMT. At the time of surgery, corneal cells surrounding epithelial defect were collected and their number was counted under microscope. Cells were cultivated for 24h and production of IL-1 and IL-1 in cells supernatant was measured by immunoassay (R&D Systems, USA). Peace of AM from the same donor as AM used in surgery was also cultivated for 24 hours ; interleukin-1 receptor antagonist (IL-1ra) level in its supernatant was measured by immunoassay. Results. Corneal epithelial cells in patients suffering from herpetic keratitis secreted 5 &plusmn ; ; 4, 8 pg/ml of IL-1 and 0, 16 &plusmn ; ; 0, 47 pg/ml of IL-1 (mean &plusmn ; ; SD). Regression of corneal inflammation and closure of epithelial defects were observed clinically in 88 % of patients after amniotic membrane-transplantation. Vision improved in all but two patients. No serious side effects occurred during the follow up. Amniotic membrane that was used to treat investigated patients contained 339, 87 pg/ml of IL-1ra. Conclusion. In herpetic keratitis pro-inflammatory cytokines IL-1 and IL-1 are secreted from corneal epithelial cells. Beneficial effect of the AMT in such patients could be explained by the fact that AM secretes the natural antagonist of these cytokines - IL-ra.

Nazri Omar,1,2 Charbel T Bou Chacra,1 Khalid F Tabbara1,3,4 1The Eye Center and The Eye Foundation for Research in Ophthalmology, Riyadh, Saudi Arabia; 2Department of Ophthalmology, Universiti Putra Malaysia, Serdang, Malaysia; 3Department of Ophthalmology, King Saud University, Riyadh, Saudi Arabia;4The Wilmer Ophthalmological Institute of Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: The aim of this work was to describe the indications, complications, and outcomes of penetrating keratoplasty (PKP) in Saudi Arabia. Methods: In a retrospective, noncomparative interventional case series, the medical records of patients who underwent PKP from January 2000 to December 2008 and had a minimum follow-up of 6 months were reviewed. All corneas were obtained from eye banks in the US. Indications, complications, and outcomes of surgery were recorded. This study was approved by the institutional review board. Results: Eighty-five consecutive eyes were included in this study. There were 52 (61.2%) males and 33 (38.8%) females. The median age was 35.0 years (range 3–85 years), and the median follow-up period was 24 months (range 6–108 months). The indications for PKP were keratoconus, bullous keratopathy, corneal scars, corneal dystrophy, and corneal regraft. The overall graft survival time was 88.9 months ± 4.9 months (mean ± standard error of mean, 95% confidence interval [CI] 79.4 months -98.4 months) while the 3-year and 5-year cumulative survival rates were 90.7% and 84.3%, respectively. Surgical indication (P = 0.038), immune rejection (P < 0.001), preoperative corneal vascularization (P = 0.022), and perioperative high intraocular pressure (P = 0.032) were associated significantly with corneal graft failure in univariate analysis. Multivariate analysis reduced these significant associations to rejection (P < 0.001) and vascularization (P = 0.009). Relative risk for failure in rejected cornea was 16.22 (95% CI 4.99–52.69) and in vascularized cornea was 3.89 (95% CI 1.36–11.09). At last visit following PKP, 34 (40%) eyes had best spectacle-corrected visual acuity of 20/40 or better, and 51 (60.0%) eyes had 20/80 or better. Best spectacle-corrected visual acuity was worse than 20/400 in 15 (17.6%) eyes. Conclusion: The overall corneal graft survival in a private setting in Saudi Arabia can be excellent. Thorough preoperative evaluation and comprehensive postoperative management are crucial for successful corneal transplantation. A larger multicenter study is recommended to portray the outcome of private corneal transplantation in Saudi Arabia in general. Keywords: cornea, corneal transplantation, corneal dystrophy, corneal scars, bullous keratopathy, keratoconus, herpetic keratitis

A case report of a 68-year-old male who developed acute retinal necrosis (ARN) after Boston type I keratoprosthesis is presented. The procedure was performed for multiple graft failure secondary to herpetic keratitis. Clinical data including visual acuity, color fundus photography, fluorescein angiography, laboratory tests findings, and management are presented. After exclusion of other causes by laboratory workup, the patient was diagnosed with ARN most likely secondary to herpetic infection. Intravenous acyclovir and oral prednisolone were administered to the patient resulting in marked improvement in visual acuity and regression in the size of the retinitis. The patient eventually developed a soft eye and choroidal detachment with light perception vision. In patients with a history of herpetic keratitis or keratouveitis, it is highly advisable to maintain prophylactic systemic antiviral treatment before and after any ocular procedure such as the Boston keratoprosthesis.

Stromal herpetic keratitis has a poor prognosis. To date, corticosteroids appear to be the most efficatious agents against the condition. However, corticosteroids are immuno6uppresive, and may cause recurrences. The recent findings of decreased cellular immunity in the condition offer the possibility of using levamisole as the therapeutic agent. The present investigation was undertaken to evaluate the clinical effects of levamisole on the twenty-one patients with stromal herpetic keratitis. Results were favorable in sixteen. The side effects of levamisole were relatively mild and transient. The results of the present study confirm the efficacy and safety of levamisole for the treatment of stromal herpetic keratitis.