Eckart Bertelmann

Survival of corneal allografts following adenovirus-mediated gene transfer of interleukin-4.

Abstractu2002Background: Genetic manipulation of the donor cornea ex vivo prior to transplantation may allow modulation of the allogeneic immune response following penetrating keratopasty. In this study we investigated the effect of adenovirus-mediated gene transfer of the Th2 cytokine interleukin-4 (IL-4) to rat corneas in an experimental keratoplasty model. Methods: Ex vivo manipulation of Wistar-Furth rat corneas was performed using E1/E3- deleted adenoviral vectors transferring the gene for rat IL-4 (AdrIL-4) under control of the CMV promoter. Following transfection with AdrIL-4 (2×108 pfu) in DMEM/2% FCS for 3 h, donor corneas were transplanted in MHC class I/II-incompatible Lewis rats. Fifty-two Lewis rats were randomly assigned to receive either nontransfected grafts (n=32), AdrIL-4-transfected grafts (n=8), or syngeneic grafts (n=12). Results: The rejection rate of AdrIL-4-transfected grafts (85.7%) could not be reduced as compared to controls (62.9%). In addition, the mean survival time of AdrIL-4-transfected grafts (12.6±4.5 days) did not differ (P>0.05) from that for untreated transplants (14.1±3.8 days). Conclusions: Our results indicate that overexpression of IL-4 is not sufficient to reduce the rejection rate of corneal allografts in an ex-perimental keratoplasty model. Further investigations are necessary to identify the reasons for failure and establish more efficient modulatory approaches.

Efficiency of Cytokine Gene Transfer in Corneal Endothelial Cells and Organ-Cultured Corneas Mediated by Liposomal Vehicles and Recombinant Adenovirus

Purpose: Gene transfer of immunoregulatory cytokines could contribute to reduce rejection of corneal grafts. The aim of our study was to examine the gene transfer efficiency of liposomal vehicles compared to adenoviral vectors for transferring the Epstein-Barr-virus-derived interleukin 10 homologue (viral IL-10, vIL-10) into corneal endothelial cells and organ-cultured human corneas (HC) in vitro. Method: To test liposomal efficiency, 2 lipid formulations (SP-Chol/DOPE 20/80 and DDAB/DOPE 30/70 in various concentrations) were complexed with a plasmid containing the vIL-10 cDNA in an eukaryotic expression vector (pcDSRα-BCRF-I). The complexes were transferred to (1) subconfluent bovine corneal endothelial cells (BCEC) after 1 passage and to (2) HC stored in organ culture. In addition, BCEC and HC were transduced with the recombinant adenoviral vector encoding for vIL-10 (AdvIL-10). Secretion of vIL-10 in the supernatants from both transfected BCEC and HC was measured by specific ELISA. Results: For gene transfer in BCEC, both transfection methods (liposomes and adenovirus) led to high secretion of vIL-10 [>2 ng/ml (liposomes) and <150 ng/ml (adenovirus) per 5,000 initially planted BCEC]. Expression levels in BCEC were dependent on the concentration of applied liposomes. For gene transfer in HC, only the adenoviral transduction technique achieved a high production of vIL-10, whereas liposomal transfection led only to low vIL-10 secretion (4.8 µg/ml vs. 95 pg/ml per quarter of cornea). Conclusion: For transfection of corneal endothelial cells in culture, liposomes can be considered as a safe and useful alternative method of gene transfer avoiding side-effects of viral vectors. However, for transfection of organ-cultured HC, adenoviral vectors are superior to liposomal vehicles.

Intraocular Availability of Topically Applied Mycophenolate Mofetil in Rabbits

The efficacy of mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), in high risk keratoplasties and ocular immune-mediated diseases has been shown in recent years. As peroral administration of MMF can cause various side effects, topical application should be considered. This study investigates the intraocular availability of MMF and MPA in the rabbit eye. An eye drop solution (MMF-CD; 1% MMF/10% hydroxypropyl-beta-cyclodextrin (HP-beta-CD)) or a 1% aqueous suspension (MMF-SP) was instilled into the lower cul-de-sac of the right eye of each rabbit. Rabbits (each group: n = 4) were put down after 30, 60 and 240 min. Aqueous humor, vitreous, cornea, sclera, conjunctiva, iris-ciliary-body, and plasma were isolated. Several extraction procedures were performed in order to quantify the drug by HPLC. The aqueous humor concentration of the active metabolite MPA was 24 microg/mL after 30 min for both preparations. The ratio of the MPA concentrations after 30, 60, and 240 min was 1 : 2 : 0.07 for MMF-CD and 1 : 0.6 : 0.04 for MMF-SP, respectively. MPA levels in the cornea were 90.78 / 56.90 / 4.08 x 10(-6) microg/microg for MMF-CD, whereas MMF-SP resulted in MPA levels of 102.65 / 31.18 / 2.59 x 10(-6) microg/microg at the three time points. As a high concentration of the active drug MPA in cornea and aqueous humor is desired, e.g. following corneal transplantation, the MMF/HP-beta-CD formulation could be an useful topical treatment. Furthermore, the present study shows that MMF-CD is superior to MMF-SP by increasing intraocular availability.

Corneal Permeation Studies of Everolimus Microemulsion

PURPOSEnTo prevent corneal-graft rejection, the topical application of an immunosuppressive drug is an alternative to the systemic application of immunosuppressive drugs or corticosteroids, which may have adverse side effects. The aim of this study was to determine the permeation rate of everolimus through freshly isolated pig cornea (ex vivo).nnnMETHODSnA fluorescence polarization immunoassay with a commercially available assay system was used to quantify everolimus in the acceptor samples of the permeation tests.nnnRESULTSnEverolimus is a poorly soluble drug and was, therefore, incorporated in an eye-administrable microemulsion. The stability of this microemulsion containing 0.1% (1 mg/mL) of the drug was satisfying over a period of 12 months. A concentration of 8.64 ng/mL was already reached 30 min after administration of the microemulsion to the cornea.nnnCONCLUSIONSnThis everolimus-containing microemulsion is a promising ocular formulation for preventing corneal-graft rejection.

Dry Eye Symptoms in Patients after Eyelid Reconstruction with Full-Thickness Eyelid Defects: Using the Tomey TG-1000 Thermographer

Background: Large full-thickness eyelid defects are conventionally reconstructed by either a Hughes flap or Cutler-Beard bridge flap. Since the structure of the eyelid and its components are necessary for the tear film production and stability, we investigated the outcome after eyelid reconstruction focusing on dry eye symptoms using a new thermographic device, the TG-1000. Methods: Seventeen eyes of patients formerly treated with Hughes flaps (n = 16) and a Cutler-Beard bridge flap (n = 1) were compared to untreated healthy eyes (n = 17) regarding the functional and aesthetic outcome. The follow-up ranged from 3 to 63 months (mean 24.88 ± 17.86). Results: There was no significant difference in Schirmer’s test, break-up time and ocular surface temperature (p > 0.05) between patients after full-thickness eyelid reconstruction and a control group. Eleven patients had minor postoperative complications such as notching of the lid margin (11/17), epiphora (1/17), superficial punctate keratitis (6/17), trichiasis (2/17) and a mild tendency to eversion of the lid margin (6/17). More than 75% of the patients rated their postoperative aesthetic outcome as good or even excellent. Conclusion: The new TG-1000 device is a simple and quick tool for screening of dry eye. This study shows that tarsoconjunctival grafts offer good aesthetic and functional outcome with sufficient tear film composition and stability.

Conjunctival Melanoma and BRAF Inhibitor Therapy

Background: BRAF is a proto-oncogene that encodes the protein B-Raf. This is a serine/threonine kinase and part of the mitogen-activated protein kinase (MAPK) pathway. Vemurafenib is a potent inhibitor of the mutant BRAF. It is approved for cutaneous melanoma.Patient/methods: A 80-year-old woman presented with irregular pigmented, hyperaemic upper and lower eye lid changes and alterations in the temporal conjunctiva of the right eye 03/2011. A conjunctival melanoma was detected with expression of BRAF Mutation on exon 15 (by PCR). For causal therapy the only primary surgical option was offered: exenteration of the right orbital. The patient refused this surgical intervention. To stabilize and prevent progression of the lesions, treatment with a BRAF inhibitor (vemurafenib) has been started over 16 month period of time.Results: After successful tumour response and decreasing size complete resection was performed 08/2013. The therapy was terminated due to a controlled tumour situation and progressive deterioration of general condition 09/2013. The progression of conjunctival melanoma could be prevented by this therapy by now.Discussion: To the best of our knowledge it is the first case to show the permanent recovery of a conjunctival melanoma after BRAF inhibitor therapy. Over the course of time there was a significant reduction of the patient’s general condition including weight loss, vomiting, headaches. These side effects should be carefully evaluated in further studies.

Ocular surface temperature gradient is increased in eyes with bacterial corneal ulcers.

Aims: To investigate the ocular surface temperature gradient in eyes with bacterial corneal ulcers. Methods: Prospective examination of 12 eyes with bacterial corneal ulcers (group 1) and 12 control eyes (group 2). Infrared thermal imaging (Tomey TG 1000) was used to study the temperature of the ocular surface. The mean, minimum and maximum temperature of the ocular surface and temperature course over a time period of 10 s of sustained eye opening were evaluated. Furthermore, a correlation between the overall corneal temperature and the temperature at the base of the corneal ulcers was determined. Results: A significant difference between both groups was present. Mean corneal temperature was 35.6°C ± 0.9 in group 1 and 34.8°C ± 0.8 in group 2 (p = 0.033). The temperature course over 10 s of sustained eye opening was –0.6°C ± 0.4 in group 1 and –0.3°C ± 0.2 in group 2 (p = 0.045). There was a close correlation between the mean temperature at the base of the corneal ulcer and the overall corneal temperature (r = 0.92, p < 0.001). Conclusion: Infrared thermal imaging can be used to objectively determine the increased ocular surface temperature in patients with bacterial corneal ulcers. The use of dynamic thermography may offer new options to monitor ocular surface alterations.

Fellow Eye Comparison of Descemet Membrane Endothelial Keratoplasty and Penetrating Keratoplasty.

Purpose: To compare the visual outcomes and postoperative complications in patients undergoing penetrating keratoplasty (PKP) in 1 eye followed by Descemet membrane endothelial keratoplasty (DMEK) in their fellow eye. Methods: A retrospective analysis of 11 patients, who underwent a PKP procedure first in 1 eye and then a DMEK surgery in their fellow eye, was performed. Intraoperative and postoperative complications were recorded. Visual and refractive outcomes were also evaluated, including higher-order aberrations (HOAs) and contrast thresholds. A subjective questionnaire was used to evaluate patient satisfaction. Results: Both uncorrected and best-corrected visual acuities were significantly better in the case of DMEK when compared with that in the case of PKP (0.82 vs. 0.37 logMAR, P = 0.005; 0.61 vs. 0.21 logMAR, P = 0.011, respectively). Postkeratoplasty astigmatism, mean spherical equivalent, and HOAs were also significantly lower in eyes after undergoing DMEK than after undergoing PKP (3.90 vs. 0.89 diopters, P = 0.005; −3.90 vs. −0.68 diopters, P = 0.005; 6.81 vs. 1.71 µm, P = 0.043, respectively). Visual outcome and patient satisfaction were significantly better in those who underwent DMEK (2.91 vs. 4.45, P = 0.011; 3.27 vs. 5.64, P = 0.016, respectively). The estimated time for recovery and rehabilitation was significantly shorter after DMEK (64.0 vs. 9.3 days, P = 0.012). Contrast threshold was better after the DMEK. Ten of 11 patients preferred DMEK procedure. Conclusions: The Patients preferred DMEK to PKP. The reasons for better patient satisfaction after DMEK included better uncorrected visual acuity, better best-corrected visual acuity, avoidance of surgery-induced astigmatism, and lower HOA.

Oral Mucosal Grafts: Old Technique in New Light

Purpose: To present an update on indications and efficacy of oral mucosal grafts in the clinical management of ocular surface diseases focusing on the new developments of the last 5 years. Methods: Pubmed research on the databases of the years 2007 up to 2012 for the key words mucous membrane graft and eye, fornix reconstruction, eyelid reconstruction, and conjunctival replacement. Results: Well-documented clinical indications for oral mucosal grafting to the ocular surface include correction of restricted socket syndrome, ocular adnexal reconstruction after tumor resection, cicatricial ocular surface diseases and therapy-refractory pterygia. New indications are conjunctival insufficiency after filtrating glaucoma surgery and the combination of mucosa and amniotic membrane grafting for fornix reconstruction. Moreover, different strategies for ex vivo mucous epithelial cell expansion are under discussion. Conclusions: Oral mucosal grafting is a viable option for the replacement of the conjunctiva. Advantages include easily accessibility of grafts in sufficient size even for repeated procedures and a high stability of the grafts. On the other hand, nasal mucosal grafts are superior for some indications due to the lack of goblet cells in the oral mucosa.

Rate and Localization of Graft Detachment in Descemet Membrane Endothelial Keratoplasty.

Purpose: To investigate the rate and localization of graft detachment after Descemet membrane endothelial keratoplasty. Methods: Sixty-six consecutive cases operated between June and August 2014 at the Charité–Universitätsmedizin Berlin were examined prospectively 1 week postoperatively. A single masked observer analyzed the rate and localization of graft detachment using optical coherence tomography (OCT), and the rebubbling rate was measured. Localization of graft detachment was correlated to the incision approach. Preoperative data were correlated to the rate of graft detachment and rebubbling. Results: Graft detachment occurred in more than 2 clock hours and with postoperative corneal edema in 33.3% and required rebubbling. In 33.3%, graft detachment occurred in more than 2 clock hours and with postoperative corneal edema and required rebubbling. The mean graft detachment rate was 8.3% per clock hour. A significantly higher graft detachment rate was noted in the inferior clock hours (21.1%, P < 0.0001, 16.7%, P = 0.003). Only higher age of the patient correlated to a higher rate of graft detachment (P = 0.022). No correlation was found between localization of graft detachment and the incision approach (P = 0.615). Conclusions: The graft detachment rate is high after Descemet membrane endothelial keratoplasty, but detachment is usually peripheral, partial and mainly inferior and involves only a few clock hours. Only higher age of the patient is strongly associated with a higher rate of graft detachment. The incision approach is not significantly correlated with the localization of graft detachment. Therefore, the postoperative supine position of the patient seems to be of major importance. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT02020044.