Proton radiotherapy in advanced malignant melanoma of the conjunctiva.

BACKGROUND: The management of conjunctival melanoma is challenging and frequently ends in exenteration. The aim of this retrospective study was to evaluate the long-term results of proton beam radiation with regard to various clinical parameters. METHODS: Eighty-nine patients with extended conjunctival melanoma (≥T2) and multifocal bulbar located tumors (T1c/d) were treated consecutively with proton radiotherapy (dose 45 Gy). The following parameters were assessed: TNM stage, tumor origin, local recurrence, performance of exenteration, occurrence of metastases, overall survival, and potential complications. A time-to-event analysis was preformed to the primary endpoints: relapse, metastasis, exenteration, and death by use of Kaplan-Meier cumulative survival estimates and Cox proportional hazards regression that provides hazard ratios and 95% confidence intervals. RESULTS: The median follow-up time was 4.2 years (max. 21.7 years). Local recurrence and metastatic disease occurred in 33% and 16% of patients, respectively. Exenteration-free survival and overall survival tended to be worse in T3 melanoma. No association between tumor origin and local recurrence, metastatic disease, or overall survival was observed. Main complications after proton radiotherapy were sicca-syndrome (30%), secondary glaucoma (11%), and limbal stem cell deficiency (8%). CONCLUSIONS: In summary, proton radiotherapy in conjunctival melanoma is an effective alternative to exenteration, with a 5-year cumulative probability of eye preservation of 69%.

Gene Therapy for Modulation of T-Cell-Mediated Immune Response Provoked by Corneal Transplantation.

Corneal transplantation (keratoplasty) is the most common type of tissue replacement in the world. The increased rate of graft rejection after keratoplasty is a central problem for repeated transplantations and in inflamed host corneas. It has been shown that apoptosis of grafted epithelium has a role in corneal allograft rejection. This study focused on the T-cell response triggered in BALB/c mice after allogeneic corneal transplantation with and without anti-apoptotic p35-transduced epithelium. To restrict p35 expression to the epithelial cells, modified allogeneic composite grafts were created. As a result, it was found that the proportion of alloreactive CD4+ T cells in postoperatively removed cervical lymph nodes was reduced in the p35-transduced group compared to the allogeneic control group. Diminished priming of the CD4+ T cells was supported by significantly decreased proliferation and lower interferon gamma secretion when compared to allogeneic engraftments. The reduced priming of CD4+ lymphocytes is the first confirmation of the functionality of p35 in the epithelium of corneal grafts to alter the development of the recipient's immune response. Thus, modification of allosensibilization seems to be a promising tool for reducing graft-mediated immune response following corneal transplantation.

Frequent GNAQ, GNA11, and EIF1AX Mutations in Iris Melanoma.

Purpose: The most common malignant intraocular tumors with a high mortality in adults are uveal melanomas. Uveal melanomas arise most frequently in the choroid or ciliary body (97%) and rarely in the iris (3%). Whereas conjunctival and posterior uveal (ciliary body and choroidal) melanomas have been studied in more detail genetically, little data exist regarding iris melanomas. Methods: In our study, we genetically analyzed 19 iris melanomas, 8 ciliary body melanomas, 3 ring melanomas, and 4 iris nevi. A targeted next-generation sequencing approach was applied, covering the mutational hotspot regions of nine genes known to be mutated in conjunctival and uveal melanoma (BRAF, NRAS, KIT, GNAQ, GNA11, CYSLTR2, SF3B1, EIF1AX, and BAP1). Results: Activating GNAQ or GNA11 hotspot mutations were detected in a mutually exclusive fashion in 84% (16/19) of iris melanomas. EIF1AX gene mutations also were frequent, detected in 42% (8/19) of iris melanomas. In 4 iris nevi, one GNAQ mutation was identified. GNAQ, GNA11, EIF1AX, and BAP1 mutations were identified at varying frequencies in ciliary body and ring melanomas. Conclusions: In this most comprehensive genetic analysis of iris melanomas published to date, we find iris melanomas to be related genetically to choroidal and ciliary body melanomas, frequently harboring GNAQ, GNA11, and EIF1AX mutations. Future studies will need to assess if screening mutation profiles in iris melanomas may be of diagnostic or prognostic value.

Clinical outcomes of amniotic membrane transplantation in the management of acute ocular chemical injury.

BACKGROUND: Amniotic membrane transplantation (AMT) has been used in the management of acute ocular chemical burns to promote epithelialisation, reduce inflammation and restore ocular surface integrity. The aim of this study is to analyse the morphological and functional outcomes of patients receiving AMT after ocular chemical burn. METHODS: We performed a retrospective analysis of all patients treated for acute ocular chemical burn between 1998 and 2008 in two participating centres (University of Duisburg-Essen, Germany and Royal Victoria Infirmary, Department of Ophthalmology, Newcastle University, UK). Ocular chemical burns were classified by Roper-Hall and Dua classifications. RESULTS: 72 eyes of 54 consecutive patients aged 37.3 years (±SD 11.6 years) were included in this cohort study. 7 chemical burns were acid burns, 61 were alkaline and 4 were of unknown origin. In 37 eyes (51.4%), AMT was applied within the first 6 days after injury. Mean follow-up time was 36.4 months (median 18.5; 1.3-117.3  months). Overall, 29 eyes (40.3%) achieved a best-corrected visual acuity of LogMAR 0.2 (0.63 decimal) or better at final visit. Complete 360° limbal stem cell deficiency (LSCD) occurred in 33 eyes (45.8%), while partial LSCD occurred in 21 eyes (29.2%). CONCLUSION: AMT is an effective adjunctive treatment in the management of acute ocular chemical burns to support epithelial healing and restore ocular surface integrity with potential to improve vision. However, long-term debilitated vision remained in those with severe burns complicated by LSCD.

Oncogene status as a diagnostic tool in ocular and cutaneous melanoma.

The majority of human tumours can be easily and correctly diagnosed based on clinical information and pathological assessment. In some cases however, correct diagnosis can prove difficult. In such cases, molecular approaches can be of significant diagnostic value. In recent years, the understanding of genetic alterations has greatly increased. In cutaneous melanoma, it is now well recognised, that 70-80% of tumours harbour BRAF and NRAS mutations. These mutations never occur in uveal melanoma. On the other hand activating GNAQ and GNA11 mutations are found in ∼90% of uveal melanomas, and are exceptionally rare in other melanomas (<1%). Here, we demonstrate a number of melanoma cases, where distinguishing if a tumour was of cutaneous or ocular origin was not possible based on clinical and pathological assessment. In these cases there was either atypical clinical presentation or metastasis of unclear primary. Histological distinction between uveal and cutaneous melanomas, especially at the stage of metastasis, is not reliable as they can be morphologically very similar. In all cases we present, a simple genetic assessment of oncogene mutation status was able to clearly define the melanoma type. This type of genetic assessment is of great diagnostic value and due to its simplicity could be performed in routine clinical practice even in smaller institutions.

Frequent TERT Promoter Mutations in Ocular Surface Squamous Neoplasia.

PURPOSE: Ocular surface squamous neoplasia, including intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma (SCC), are one of the most common malignant tumors of the conjunctiva. Little is known of the genetic alterations involved in their pathogenesis. Promoter mutations in telomerase reverse transcriptase (TERT) have been identified in various cancers, including many associated with ultraviolet (UV) exposure. Our study analyzes the mutation rate and clinicopathological associations of TERT promoter mutations in ocular surface squamous neoplasia. METHODS: DNA was isolated and the region of the TERT promoter where hotspot mutations can occur analyzed by Sanger-sequencing in 48 ocular surface squamous neoplasia tumor samples (6 CIN and 42 SCC). An analysis of associations between TERT promoter mutation status and various clinicopathological parameters was performed. RESULTS: We identified TERT promoter mutations in 21 of 48 ocular surface squamous neoplasia samples (43.8%), including 4 in CIN and 17 in SCC. The mutations consisted of 8 Chr.5:1295228C>T, 1 Chr.5:1295228_1295229CC>TT, 5 Chr.5:1295242_1295243CC>TT, and 12 Chr.5:1295250C>T mutations. All mutations were C>T or CC>TT alterations, demonstrating a UV-signature. TERT promoter mutations showed no statistically significant associations with clinicopathological parameters. CONCLUSIONS: Telomerase reverse transcriptase promoter mutations are found in almost half of ocular surface squamous neoplasias and have a mutation profile supporting UV induction as the major source of mutagenesis. We conclude that UV induced TERT promoter mutations leading to aberrant overexpression of telomerase is a major pathogenetic factor in ocular surface squamous neoplasia.

Analysis of SDHD promoter mutations in various types of melanoma.

OBJECTIVES: Recently, recurrent mutations in regulatory DNA regions, such as promoter mutations in the TERT gene were identified in melanoma. Subsequently, Weinhold et al. reported SDHD promoter mutations occurring in 10% of melanomas and being associated with a lower overall survival rate. Our study analyzes the mutation rate and clinico-pathologic associations of SDHD promoter mutations in a large cohort of different melanoma subtypes. METHODS: 451 melanoma samples (incl. 223 non-acral cutaneous, 38 acral, 33 mucosal, 43 occult, 43 conjunctival and 51 uveal melanoma) were analyzed for the presence of SDHD promoter mutations by Sanger-sequencing. Statistical analysis was performed to screen for potential correlations of SDHD promoter mutation status with various clinico-pathologic criteria. RESULTS: The SDHD promoter was successfully sequenced in 451 tumor samples. ETS binding site changing SDHD promoter mutations were identified in 16 (4%) samples, of which 5 mutations had not been described previously. Additionally, 5 point mutations not located in ETS binding elements were identified. Mutations in UV-exposed tumors were frequently C>T. One germline C>A SDHD promoter mutation was identified. No statistically significant associations between SDHD promoter mutation status and various clinico-pathologic variables or overall patient survival were observed. CONCLUSIONS: Melanomas harbor recurrent SDHD promoter mutations, which occur primarily as C>T alterations in UV-exposed melanomas. In contrast to the initial report and promoter mutations in the TERT gene, our analysis suggests that SDHD promoter mutations are a relatively rare event in melanoma (4% of tumors) of unclear clinical and prognostic relevance.

Establishment of a Cell Line From Conjunctival Squamous Cell Carcinoma: PeCa-UkHb-01.

PURPOSE: Until now, no epithelial cell line from conjunctival squamous cell carcinoma (SCC), to our knowledge, has existed; therefore, the establishment of a model cell line would be a useful tool for further studies. In particular, the phenotypic and molecular characterization in comparison to other SCC cells is of high interest because this would enable the development of new treatment options for clinical application in ophthalmic oncology. METHODS: Epithelial cells were isolated from a bulbar conjunctiva SCC obtained from a 74-year-old male, harvested by stepwise trypsinization and named PeCa-UkHb-01. Cell doubling and the number of passages were determined. Short tandem repeats (STR) and karyotype analyses were performed. Semiquantitative real-time PCR and immunocytochemical fluorescence staining were carried out to detect tumor and epithelial cell markers. RESULTS: The cells had an epithelial and conjunctival phenotype. They grew above passage number 50 in a doubling time at approximately 34.5 hours. Short tandem repeat analyses confirmed the cell origin, although loss of heterozygosity occurred. Karyotype analyses revealed a heterogeneous composition of the cell culture and the karyogram itself showed aberrations and changes in the chromosome numbers. Real-time PCR and immunocytochemical fluorescence staining revealed the expression of the stem cell markers such as ABCG2, p63, OCT4, c-MYC, and SOX2 as well as the conjunctival cytokeratin K19. CONCLUSIONS: PeCa-UkHb-01 cells fulfill the criteria of a cell line. They display characteristics of ocular carcinoma cells and therefore the presented cell line might serve for further basic research in ophthalmic oncology.

Trabeculectomy Improves Vessel Response Measured by Dynamic Vessel Analysis (DVA) in Glaucoma Patients.

PURPOSE: To determine the effects of surgical IOP reduction (trabeculectomy) on retinal blood flow parameters in glaucoma patients using Dynamic Vessel Analysis (DVA). METHODS: 26 eyes of 26 patients with progressive primary open-angle glaucoma (POAG) despite maximal topical therapy were examined before and after trabeculectomy. The responses of the retinal vessels to flickering light provocation were measured with DVA the day before surgery and 4 to 6 weeks after trabeculectomy. Between 3 and 4 weeks before surgery all local therapies were stopped and a systemic therapy with acetazolamide and conservative free topic steroidal eye drops was started. RESULTS: In 19 patients (73%), an inadequate response to the flicker stimulation was measured preoperatively. In these patients, the maximum dilation of arteries and veins was reduced significantly as compared to healthy eyes. In this group, the maximum dilation of the arteries following the flicker provocation improved from 1.4% before to 3.8% following trabeculectomy (p<0.01). In retinal veins, this parameter increased from 3.1% to 4.6% (p<0.05). In the 7 patients whose arterial and venous reactions to flickering light provocation preoperatively did not differ from healthy eyes, there was no significant change after surgery. The initial baseline values of arteries and veins (MU) did not deviate significantly in both groups. CONCLUSION: POAG patients with progressive disease and impaired vascular regulation profit from IOP lowering trabeculectomy concerning vascular reactivity and dilative reserve, indicating a possible improvement of retinal perfusion following effective IOP control. Future studies with long-term follow-up must determine the clinical importance of these findings for the treatment of glaucoma patients.

Laser cyclophotocoagulation enhances the regulative capacity of retinal vessels in glaucoma.

PURPOSE: To determine the effects of laser surgical IOP reduction by means of transscleral cyclophotocoagulation (CPC) on retinal blood flow parameters in glaucoma patients using Dynamic Vessel Analysis (DVA). MATERIALS AND METHODOLOGY: 26 patients (average age: 70 years) with a long history of primary open angle glaucoma underwent CPC. The effect on the reactive capacity of retinal vessels was assessed before and 6-8 weeks after CPC by means of the Dynamic Vessel Analyzer (DVA) using flicker light provocation. RESULTS: In our group of POAG patients, IOP was significantly reduced about approximately 20% by CPC while systemic blood pressure and heart rate were not changed. The most obvious differences between the pre- and postoperative DVA measurements could be observed in the maximal dilation of the retinal arteries which increased from 0.75 % (+/- 0.6) to 3.17 % (+/- 0.5) with an average increase of 2.4 % (p<0.01). In addition, the ability of the arteries for constriction improved significantly (p<0.05) while the dynamic responses of the veins and the initial baseline values (MU) of the vessel diameters did not change. CONCLUSIONS: Our results of DVA measurements after an IOP-lowering laser surgical intervention (CPC) reveal a significant recovery of the regulative capacity of retinal arteries in glaucoma patients that has up to now neither been properly documented nor appreciated. Future studies with long-term follow-up must determine the clinical importance of these findings for the treatment of glaucoma patients.

Aphakia correction with retropupillary fixated iris-claw lens (Artisan) - long-term results.

PURPOSE: To evaluate the technique, safety, and efficacy of the retropupillary implantation of iris-claw intraocular lenses in a long-term follow-up study. PATIENTS AND METHODS: This retrospective study included 31 eyes of 31 patients who underwent an Artisan aphakic intraocular lens implantation between January 2006 and February 2011 at the University Hospital Essen, Essen, Germany and at the Zentrum für Augenheilkunde PD Dr Laube, Düsseldorf, Germany. Preoperative data collected included demographics, etiology of aphakia, previous surgeries, preoperative eye pathology, intraocular pressure, clinical signs of endothelial cell loss, and best corrected visual acuity. Operative data and postoperative outcomes included the best corrected visual acuity, lens position, intraocular pressure, pigment dispersion, clinical signs of endothelial cell loss, development of macular edema, and other complications. RESULTS: Thirty-one patients were included. The mean follow-up was 25.2 months (range: 4-48 months). The mean best corrected visual acuity postoperatively was 0.64 logarithm of the minimum angle of resolution (logMAR) and varied from 0 logMAR to 3 logMAR. Some patients had a low visual acuity preoperatively because of preoperative eye pathologies. In 22 patients the visual acuity improved, in two patients the visual acuity remained unchanged, and seven patients showed a decreased visual acuity. Complications were peaked pupils (n=10) and retinal detachment in one case. Four patients showed an iris atrophy and high intraocular pressure was observed only in one patient. Subluxation of the intraocular lens, endothelial cell loss, and macular edema were not observed. CONCLUSION: The presented long-term results demonstrate that retropupillary iris-claw lens implantation is a safe and effective method for the correction of aphakia in patients without capsule support. This surgical procedure has the advantages of a posterior chamber implantation with a low intraoperative and postoperative risk profile.

Amniotic membrane transplantation ineffective as additional therapy in patients with aggressive Mooren's ulcer.

BACKGROUND: Mooren's ulcer is a severe ulcerative inflammation of the cornea. The exact pathogenesis remains unclear. Therefore many therapies of Mooren's ulcer are recommended in literature. To shed more light on the ongoing question of optimal treatment of severe progressive Mooren's ulcer, we here report on a retrospective case series of patients treated with systemic immunosuppressive therapy and additional amniotic membrane transplantation. METHODS: Medical records from seven patients (eleven eyes), 4 male and 3 female, with severe progressive Mooren's ulcer were analysed retrospectively. The mean follow up was 88.4 ± 80.8 months (range 12-232 month). A HLA-typing was performed in all patients. A systemic immunosuppressive therapy was administered in all patients. The amniotic membrane was transplanted after the base of the ulcer was resected. RESULTS: Multiple amniotic membrane transplantations were necessary in six patients. The visual outcome of all patients was poor. No patient achieved a visual acuity better than 20/630 Snellen chart. Five patients were positive for HLA-DQ2 and four patients were positive for HLA-DR17(3). CONCLUSIONS: The aggressive and highly inflammatory form of Mooren's ulcer is difficult to treat and the progression of the disease is hard to influence positively even under systemic immunosuppressive therapy. Therefore, the main intention of therapy is to achieve a stable epithelialized corneal surface without the risk of perforation. Amniotic membrane transplantation is not able to cure severe forms of Mooren's ulcer. However it supports the immunosuppressive therapy in acute situations as in critical corneal thinning.

Conjunctival melanomas harbor BRAF and NRAS mutations and copy number changes similar to cutaneous and mucosal melanomas.

PURPOSE: Conjunctival melanoma is a rare but potentially deadly tumor of the eye. Despite effective local therapies, recurrence and metastasis remain frequent. Once the tumor has metastasized, treatment options are limited and the prognosis is poor. To date, little is known of the genetic alterations in conjunctival melanomas. EXPERIMENTAL DESIGN: We conducted genetic analysis of 78 conjunctival melanomas, to our knowledge the largest cohort reported to date. An oncogene hotspot array was run on 38 samples, screening for a panel of known cancer-relevant mutations. Thirty tumors were analyzed for genome-wide copy number alterations (CNA) using array-based comparative genomic hybridization. Sanger sequencing of selected target mutations was conducted in all samples. RESULTS: BRAF mutations were identified in 23 of 78 (29%) tumors. NRAS mutations, previously not recognized as relevant in conjunctival melanoma, were detected in 14 of 78 (18%) tumors. We found CNAs affecting various chromosomes distributed across the genome in a pattern reminiscent of cutaneous and mucosal melanoma but differing markedly from uveal melanoma. CONCLUSIONS: The presence of NRAS or BRAF mutations in a mutually exclusive pattern in roughly half (47%) of conjunctival melanomas and the pattern of CNAs argue for conjunctival melanoma being closely related to cutaneous and mucosal melanoma but entirely distinct from uveal melanoma. Patients with metastatic conjunctival melanoma should be considered for therapeutic modalities available for metastatic cutaneous and mucosal melanoma including clinical trials of novel agents.

Chemosensitivity of conjunctival melanoma cell lines to target-specific chemotherapeutic agents.

OBJECTIVE: In conjunctival melanoma, local chemotherapy has been based so far on clinical evidence and limited to the therapy of melanoma in situ. Our aim was to define substances that may have the potential to add to therapeutic options in extended local growth and metastatic disease. Two conjunctival cell lines (CRMM-1 and CRMM-2) have been established from recurrent conjunctival melanoma. In this study, we examined the chemosensitivity of these cell lines to different cytotoxic substances. MATERIALS AND METHODS: The cell lines CRMM-1 and CRMM-2 were exposed to chemotherapeutics for 24 h and the IC50 was generated. Sulforhodamin-B assays were used for quantification of in vitro efficacy. Time of exposure and escalating concentrations of the substances were adapted to the experimental setting. RESULTS: Bortezomib, clusianone 502 (nemorosone), ranpirnase, and sorafenib were efficient in inhibiting the growth of conjunctival melanoma cell lines. The IC50 achieved concentrations below or around 10 µM for these substances. CONCLUSIONS: Bortezomib, clusianone 502, ranpirnase, and sorafenib inhibited growth in conjunctival melanoma cell lines efficiently. The new substances may be a suitable alternative for local therapy. New therapeutic options with highly specific targeted agents for metastatic disease have to be evaluated in further experiments.

Comparison of Spectralis-OCT, GDxVCC and GDxECC in assessing retinal nerve fiber layer (RNFL) in glaucomatous patients.

BACKGROUND: Glaucomatous optic neuropathy is characterized by a progressive loss of retinal ganglion cells (RGCs). The defects in the peripapillary retinal nerve fiber layer (RNFL) have been reported to be the earliest sign of glaucoma. We determined the agreement between RNFL thickness assessments from spectral-domain OCT (Spectarlis HRA + OCT; Heidelberg Engeneering, Heidelberg, Germany), scanning laser polarimetry (SLP) with variable cornea compensation (GDxVCC; Carl Zeiss Meditec, Dublin, CA, USA), and SLP with enhanced cornea compensation (GDxECC; Carl Zeiss Meditec, Dublin, CA, USA) in glaucomatous patients. Furthermore, we investigate the influence of typical scan score (TSS) on the results of GDx assessments. METHODS: The enrolled subjects were devided into different groups by modified HODAPP visual field criteria. The peripapillary RNFL thickness was assessed with the three devices . ANOVA test, Pearson and Spearman correlation coefficient, and Bland-Altman plots were used to analyse the RNFL thickness assessments. RESULTS: Ninety-two eyes from 92 glaucomatous subjects were analysed. These were divided into four groups: preperimetric glaucoma (n = 26), mild glaucoma (n = 18), moderate glaucoma (n = 21), and severe glaucoma (n = 27). For Spectralis-OCT, the average RNFL thickness (mean ± SD) was 99.25 ± 26.31 µm, 80.52 ± 16.63 µm, 71.59 ± 21.15 µm, and 63.85 ± 20.86 µm for preperimetric, mild, moderate, and severe glaucoma respectively. For GDxVCC, the corresponding assessments were 52.63 ± 8.18 µm, 52.95 ± 10.20 µm, 46.77 ± 10.62 µm, and 49.70 ± 13.34 µm. For GDxECC, the assessments were 49.35 ± 6.52 µm, 45.92 ± 7.21 µm, 42.19 ± 8.00 µm, and 39.53 ± 8.45 µm. All Spectralis-GDxVCC and Spectralis-GDxECC differences were statistically significant by ANOVA test. The differences between GDxVCC and GDxECC were statistically significant only for severe glaucoma. There was a highly significant correlation between Spectralis-OCT and GDxECC, as well as Spectralis-OCT and GDxVCC, in assessing the RNFL thickness. The best instrument agreement was found between GDxECC and Spectralis-OCT. The RNFL thickness assessed with Spectralis-OCT and GDxECC showed a better correlation to visual field defects than GDxVCC. Evaluating GDx assessments with typical retardation pattern GDxVCC and GDxECC showed very similar RNFL thickness results. CONCLUSIONS: RNFL thickness assessments between GDxVCC, GDxECC, and Spectralis-OCT cannot be directly compared. The assessments are generally higher with Spectralis-OCT than with GDxVCC and GDxECC, because of differences in method of the devices. The atypical retardation pattern has a major impact on the RNFL thickness results of GDx devices. This must be taken into account when evaluating the assessed RNFL thickness results.

Scanning laser topography and scanning laser polarimetry: comparing both imaging methods at same distances from the optic nerve head.

PURPOSE: To compare the performance of scanning laser topography (SLT) and scanning laser polarimetry (SLP) on the rim of the optic nerve head and its surrounding area and thereby to evaluate whether these imaging technologies are influenced by other factors beyond the thickness of the retinal nerve fiber layer (RNFL). MATERIALS AND METHODOLOGY: A total of 154 eyes from 5 different groups were examined: young healthy subjects (YNorm), old healthy subjects (ONorm), patients with normal tension glaucoma (NTG), patients with open-angle glaucoma and early glaucomatous damage (OAGE) and patients with open-angle glaucoma and advanced glaucomatous damage (OAGA). SLT and SLP measurements were taken. Four concentric circles were superimposed on each of the images: the first one measuring at the rim of the optic nerve head (1.0 ONHD), the next measuring at 1.25 optic nerve head diameters (ONHD), at 1.5 ONHD and at 1.75 ONHD. The aligned images were analyzed using GDx/NFA software. RESULTS: Both methods showed peaks of RNFL thickness in the superior and inferior segments of the ONH. The maximum thickness, registered by the SLT device was at the ONH rim where the SLP device tended to measure the lowest values. SLT measurements at the ONH were influenced by other tissues besides the RNFL like blood vessels and glial tissues. SLT and SLP were most strongly correlated at distances of 1.25 and 1.5 ONHD. CONCLUSIONS: While both imaging technologies are valuable tools in detecting glaucoma, measurements at the ONH rim should be interpreted critically since both methods might provide misleading results. For the assessment of the retinal nerve fiber layer we would like to recommend for both imaging technologies, SLT and SLP, measurements in 1.25 and 1.5 ONHD distance of the rim of the optic nerve head.

Chemosensitivity of conjunctival melanoma cell lines to single chemotherapeutic agents and combinations.

OBJECTIVE: Two conjunctival cell lines (CRMM-1 and CRMM-2) have been established from recurrent conjunctival melanoma. The authors examined the chemosensitivity of these cell lines to cytotoxic substances and combinations to identify substances that inhibit cell growth efficiently in vitro. MATERIAL AND METHODS: CRMM-1 and CRMM-2 were exposed to cisplatin, mitomycin C (MMC), all-trans-retinoic-acid (ATRA), fotemustine or imatinib for 24 h. Sulforhodamine-B assays were used to assess the IC(50). Isobolograms were performed to test possible synergism and antagonism with ATRA or imatinib. RESULTS: Cisplatin and MMC were efficient to inhibit the growth of CRMM-1 and CRMM-2. Combination of imatinib with MMC showed additive antitumoral effect on both cell lines. Combined treatment of imatinib with fotemustine or cisplatin resulted in antagonism. Strong antagonisms were also obtained with ATRA and fotemustine or cisplatin in both cell lines. A synergism was found for ATRA and mitomycin or imatinib in CRMM-2, in contrast to CRMM-1, where antagonism was obtained. CONCLUSIONS: Cisplatin and MMC inhibit cell growth in conjunctival melanoma cell lines. The potential of ATRA was evident only in combination with MMC or imatinib in CRMM-2 cells. Imatinib and mitomycin increased their efficiency under combination therapy.

Amniotic membrane induces peroxisome proliferator-activated receptor-γ positive alternatively activated macrophages.

PURPOSE: Amniotic membrane transplantation (AMT) reportedly improves herpetic stromal keratitis (HSK). Here we studied the role of the amniotic membrane (AM) on macrophages. METHODS: BALB/c mice with necrotizing HSK received an AMT or tarsorrhaphy (TAR) as control. Apoptosis of F4/80+ cells was determined using the annexinV/7-AAD system. Macrophage invasion was determined using a cornea invasion assay. Cytokine secretion was quantified by ELISA. Arginase activity was measured by bioassay. Expression of nuclear factor (NF)-κB or peroxisome proliferator-activated receptor (PPAR)-γ related proteins was detected by Western blot analysis, and the expression of costimulatory surface molecules or PPAR-γ by flow cytometry. Lipid accumulation was observed by Oil red O and Sudan B staining. RESULTS: After AMT apoptotic features of corneal macrophages, but also macrophage invasion increased. IL-6, IL-10, IL-12, TNF-α, and NF-κB content in HSK corneas had decreased with AMT. AMT increased expression of PPAR-γ, arginase 1 and 2, and arginase activity in AM-treated HSK corneas. In vitro, NF-κB, cytokine production, costimulatory molecules (CD80, CD86, CD40), phagocytic capacity, proliferation, viability, and accessory function to herpes simplex virus (HSV)-1 specific draining lymph node (DLN) cells were reduced in bone marrow derived macrophages (BM) cocultured with AM, while CD206, CD204, CD163, and CD68, lipid accumulation in the cytoplasm, PPAR-γ expression, and arginase activity was increased. An increase in viability and proliferation was observed in the presence of AM combined with apoptotic cells, compared with AM alone. CONCLUSIONS: Based on these results it can be concluded that the action mechanism of AM is associated with modulation of classically activated macrophages into alternatively activated macrophages or macrophage cell death, probably by engaging lipid metabolism and activating the PPAR-γ pathway, consequently curtailing effector T cell functions. Apoptotic cells induced in the environment with AM support the presence and survival of such macrophages.

A new tool for the transfection of corneal endothelial cells: calcium phosphate nanoparticles.

Calcium phosphate nanoparticles (CaP-NP) are ideal tools for transfection due to their high biocompatibility and easy biodegradability. After transfection these particles dissociate into calcium and phosphate ions, i.e. physiological components found in every cell, and it has been shown that the small increase in intracellular calcium level does not affect cell viability. CaP-NP functionalized with pcDNA3-EGFP (CaP/DNA/CaP/DNA) and stabilized using different amounts of poly(ethylenimine) (PEI) were prepared. Polyfect®-pcDNA3-EGFP polyplexes served as a positive control. The transfection of human and murine corneal endothelial cells (suspensions and donor tissue) was optimized by varying the concentration of CaP-NP and the duration of transfection. The transfection efficiency was determined as EGFP expression detected by flow cytometry and fluorescence microscopy. To evaluate the toxicity of the system the cell viability was detected by TUNEL staining. Coating with PEI significantly increased the transfection efficiency of CaP-NP but decreased cell viability, due to the cytotoxic nature of PEI. The aim of this study was to develop CaP-NP with the highest possible transfection efficiency accompanied by the least apoptosis in corneal endothelial cells. EGFP expression in the tissues remained stable as corneal endothelial cells exhibit minimal proliferative capacity and very low apoptosis after transfection with CaP-NP. In summary, CaP-NP are suitable tools for the transfection of corneal endothelial cells. As CaP-NP induce little apoptosis these nanoparticles offer a safe alternative to viral transfection agents.