Sonja Klebe

Endothelial cell loss after phacoemulsification: relation to preoperative and intraoperative parameters

PURPOSE To evaluate the effect of the location of the corneoscleral tunnel incision as well as preoperative and intraoperative parameters on total and localized endothelial cell loss. SETTING Department of Ophthalmology, Humboldt-University of Berlin, Berlin, Germany. METHODS Fifty consecutive patients scheduled for routine cataract surgery were selected prospectively for this clinical trial. Preoperatively, the axial length, anterior chamber depth, lens thickness, and astigmatism were measured. Phacoemulsification time and relative energy as well as total surgical time were recorded. With a specular microscope, endothelial cell counts were determined centrally, superiorly, and temporally preoperatively and 6 weeks and 6 and 12 months postoperatively. RESULTS After 12 months, the mean overall central endothelial cell loss in all eyes was 8.5%. The mean endothelial cell loss was 11.9% in the lateral quadrant and 11.4% in the superior quadrant. There were no significant differences between superior and temporal surgical approaches in intraoperative parameters of phacoemulsification time, relative intensity of phacoemulsification, and surgical time. There were no significant differences in central endothelial cell loss or in the area localized in the quadrant of the positions of the corneal surgical site. The only risk factors found significant for higher endothelial cell loss were shorter axial length and longer phacoemulsification time. CONCLUSIONS The location of corneoscleral incisions for phacoemulsification can be chosen according to the preoperative astigmatism without inducing additional adverse effects on the corneal endothelium. Shorter eyes have a significantly higher risk for greater endothelial cell loss.

Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10.

Background. Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. Methods. The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. Results. Sheep corneal endothelium was found to be essentially amitotic. Transfection of >70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 ⩾300 days) compared with control corneas (median 20.5 days, range 18–32 days, P =0.011). Conclusion. Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma

BACKGROUND Malignant pleural mesothelioma (MPM) is recalcitrant to treatment and new approaches to therapy are needed. Reduced expression of miR-15/16 in a range of cancer types has suggested a tumour suppressor function for these microRNAs, and re-expression has been shown to inhibit tumour cell proliferation. The miR-15/16 status in MPM is largely unknown. MATERIALS AND METHODS MicroRNA expression was analysed by TaqMan-based RT-qPCR in MPM tumour specimens and cell lines. MicroRNA expression was restored in vitro using microRNA mimics, and effects on proliferation, drug sensitivity and target gene expression were assessed. Xenograft-bearing mice were treated with miR-16 mimic packaged in minicells targeted with epidermal growth factor receptor (EGFR)-specific antibodies. RESULTS Expression of the miR-15 family was consistently downregulated in MPM tumour specimens and cell lines. A decrease of 4- to 22-fold was found when tumour specimens were compared with normal pleura. When MPM cell lines were compared with the normal mesothelial cell line MeT-5A, the downregulation of miR-15/16 was 2- to 10-fold. Using synthetic mimics to restore miR-15/16 expression led to growth inhibition in MPM cell lines but not in MeT-5A cells. Growth inhibition caused by miR-16 correlated with downregulation of target genes including Bcl-2 and CCND1, and miR-16 re-expression sensitised MPM cells to pemetrexed and gemcitabine. In xenograft-bearing nude mice, intravenous administration of miR-16 mimics packaged in minicells led to consistent and dose-dependent inhibition of MPM tumour growth. CONCLUSIONS The miR-15/16 family is downregulated and has tumour suppressor function in MPM. Restoring miR-16 expression represents a novel therapeutic approach for MPM.

Common variants near ABCA1 , AFAP1 and GMDS confer risk of primary open-angle glaucoma

Primary open-angle glaucoma (POAG) is a major cause of irreversible blindness worldwide. We performed a genome-wide association study in an Australian discovery cohort comprising 1,155 cases with advanced POAG and 1,992 controls. We investigated the association of the top SNPs from the discovery stage in two Australian replication cohorts (932 cases and 6,862 controls total) and two US replication cohorts (2,616 cases and 2,634 controls total). Meta-analysis of all cohorts identified three loci newly associated with development of POAG. These loci are located upstream of ABCA1 (rs2472493[G], odds ratio (OR) = 1.31, P = 2.1 × 10−19), within AFAP1 (rs4619890[G], OR = 1.20, P = 7.0 × 10−10) and within GMDS (rs11969985[G], OR = 1.31, P = 7.7 × 10−10). Using RT-PCR and immunolabeling, we show that these genes are expressed within human retina, optic nerve and trabecular meshwork and that ABCA1 and AFAP1 are also expressed in retinal ganglion cells.

Sarcomatoid mesothelioma: a clinical-pathologic correlation of 326 cases.

Sarcomatoid mesothelioma is the least common, but most aggressive of the three major histological types of mesotheliomas. This study comprises 326 cases of sarcomatoid mesotheliomas among 2000 consecutive malignant mesothelioma cases received in consultation (16%). Patients included 312 men (96%) and 14 women (4%), with a median age of 70 years (range 41–94 years). Most tumors were pleural (319; 98%), and 7 were peritoneal (2%). Some desmoplastic features were identified in 110 cases (34%), and 70 (21%) were classified as desmoplastic. Rare subtypes included two cases with a lymphohistiocytoid pattern (<1%) and eight heterologous mesotheliomas (2%). Labeling for cytokeratins (CKs) was observed in 261/280 cases (93%), and for calretinin and vimentin in 31 and 91%, respectively. Pleural plaques were present in 79% of cases for which information was available, and asbestosis was diagnosed in 34/127 cases (27%). Median survival was 3.5 months. Fiber analysis was performed in 61 cases. The median asbestos body count was 1640/g wet lung tissue (by light microscopy). Amosite fibers were the most commonly identified fibers using energy-dispersive X-ray analysis and were significantly higher in the sarcomatoid cases, as were uncoated fibers using scanning electron microscopy. This study represents the largest series of sarcomatoid and desmoplastic malignant mesotheliomas to date and confirms the diagnostic usefulness of CK immunohistochemistry. The relationship with asbestos exposure—particularly amosite—and an association with pleural plaques and less often asbestosis is confirmed.

Low Calretinin Expression and High Neutrophil-To- Lymphocyte Ratio Are Poor Prognostic Factors in Patients with Malignant Mesothelioma Undergoing Extrapleural Pneumonectomy

Introduction: Survival after extrapleural pneumonectomy (EPP) is variable in patients with malignant pleural mesothelioma (MPM), and there are no validated prognostic factors that could be used preoperatively. We investigated the calretinin and D2-40 expression and the neutrophil-to-lymphocyte ratio (NLR), an index of systemic inflammation as potential preoperative prognostic factors. Methods: Consecutive patients who underwent EPP were included in this retrospective study. Potential prognostic factors such as age, gender, histological subtype, baseline laboratory parameters including NLR, and immunohistochemical staining for calretinin and D2-40 were evaluated. Overall survival (OS) from the date of surgery was determined by the Kaplan-Meier method. The prognostic value of the variables was examined using Cox regression, and significant factors (p < 0.05) were entered into a multivariate model to determine their independent effect. Results: A total of 85 patients were included: median age 58 years; 80% men; 77% epithelial and 23% biphasic MPM. The median OS was 19.7 months. The following variables were predictive of longer OS: female gender (p = 0.02), epithelial subtype (p = 0.04), low NLR (p < 0.01), and high calretinin score (p < 0.001). In a multivariate analysis, only NLR ≥3 (hazard ratio 1.79; 95% confidence interval: 1.04–3.07; p = 0.04) and calretinin score ≤33 versus more than 67% (hazard ratio 4.72; 95% confidence interval: 1.97–11.32; p < 0.01) remained independent predictors. The addition of calretinin score increased the explained variation by 10.1%. Conclusions: Both low calretinin expression and high NLR were independently associated with poor prognosis in patients with MPM undergoing EPP, and the calretinin score seemed to improve the accuracy of the prognostic model.

Increased Circulating miR-625-3p: A Potential Biomarker for Patients With Malignant Pleural Mesothelioma

Introduction: We investigated the ability of cell-free microRNAs (miRNAs) in plasma and serum to serve as a biomarker for malignant mesothelioma (MM). Methods: Using miRNA microarrays, we profiled plasma samples from MM patients and healthy controls. miRNAs with significantly different abundance between cases and controls were validated in a larger series of MM patients and in an independent series of MM patients using quantitative real-time polymerase chain reaction. Levels of candidate miRNAs were also quantified in MM tumor samples. Results: We compared cell-free miRNA profiles in plasma from MM patients with healthy controls. Reviewing 90 miRNAs previously reported to be associated with MM, we found that the levels of two miRNAs, miR-29c* and miR-92a, were elevated in plasma samples from MM patients. In addition, we identified 15 novel miRNAs present at significantly higher levels in the plasma of MM patients. Further analysis of candidate miRNAs by real time-quantitative polymerase chain reaction confirmed that one of them, miR-625-3p, was present in significantly higher concentration in plasma/serum from MM patients and was able to discriminate between cases and controls, in both the original and the independent series of patients. MiR-625-3p was also found to be up-regulated in tumor specimens from a group of 18 MM patients, who underwent extrapleural pneumonectomy. Conclusion: Our data confirm the potential of miR-29c* and miR-92a as candidate tumor markers and reveal that miR-625-3p is a promising novel diagnostic marker for MM.

Challenges and controversies in the diagnosis of mesothelioma: Part 1. Cytology-only diagnosis, biopsies, immunohistochemistry, discrimination between mesothelioma and reactive mesothelial hyperplasia, and biomarkers

The detection of neoplastic invasion remains the linchpin for a clear diagnosis of malignant mesothelioma. Cytology-only diagnosis of epithelioid mesothelioma on aspirated effusion fluid remains controversial. A major problem is poor sensitivity, although cytodiagnosis is achievable in many cases at a high order of specificity, especially when a large volume of effusion fluid is submitted for cytological evaluation, enabling the preparation of cell-block sections for immunohistochemical investigation and when the cytological findings can be correlated with imaging studies to assess the anatomical distribution of the lesion and evidence of nodularity of the pleural disorder and, in some cases, to demonstrate evidence of invasion. Although ‘positive’ and ‘negative’ immunohistochemical markers have proved remarkably effective in distinguishing between epithelioid mesothelioma and secondary carcinoma and other malignant tumours metastatic to serosal membranes, no mesothelial marker has 100% sensitivity and specificity for mesothelioma diagnosis, so that panels of ‘positive’ antibodies and markers with negative predictive value are required. At present, no tissue or serum marker (including the molecular detection of p16/CDKN2A) has been proved to have sufficient specificity, consistency and reproducibility that it can replace evidence of invasion as the decisive marker for diagnosis when there is any uncertainty concerning a diagnosis of epithelioid mesothelioma and in the case of atypical fibrous lesions of the pleura (especially collagen-rich lesions, namely fibrous pleuritis vs desmoplastic mesothelioma), in which even the assessment of invasion can be problematical as illustrated in part 2 of this review.

Evaluation of mesoporous silicon/polycaprolactone composites as ophthalmic implants.

The suitability of porous silicon (pSi) encapsulated in microfibers of the biodegradable polymer polycaprolactone (PCL) for ophthalmic applications was evaluated, using both a cell attachment assay with epithelial cells and an in vivo assessment of biocompatibility in rats. Microfibers of PCL containing encapsulated pSi particles at two different concentrations (6 and 20 wt.%) were fabricated as non-woven fabrics. Given the dependence of Si particle dissolution kinetics on pSi surface chemistry, two different types of pSi particles (hydride-terminated and surface-oxidized) were evaluated for each of the two particle concentrations. Significant attachment of a human lens epithelial cell line (SRA 01/04) to all four types of scaffolds within a 24h period was observed. Implantation of Si fabric samples beneath the conjunctiva of rat eyes for 8 weeks demonstrated that the composite materials did not cause visible infection or inflammation, and did not erode the ocular surface. We suggest that these novel composite materials hold considerable promise as scaffolds in tissue engineering with controlled release applications.

Prolongation of Sheep Corneal Allograft Survival by Transfer of the Gene Encoding Ovine IL-12-p40 but Not IL-4 to Donor Corneal Endothelium

Immunological rejection is the major cause of human corneal allograft failure. We hypothesized that local production of IL-4 or the p40 subunit of IL-12 (p40 IL-12) by the grafted cornea might prolong allograft survival. Replication-deficient adenoviral vectors encoding ovine IL-4 or p40 IL-12 and GFP were generated and used to infect ovine corneas ex vivo. mRNA for each cytokine was detected in infected corneas, and the presence of secreted protein in corneal supernatants was confirmed by bioassay (for IL-4) or immunoprecipitation (for p40 IL-12). Sheep received uninfected or gene-modified orthotopic corneal allografts. Postoperatively, untreated corneas (n = 13) and corneas expressing GFP (n = 6) were rejected at a median of 21 and 20 days, respectively. Corneas expressing IL-4 (n = 6) underwent rejection at 18.5 days (p > 0.05 compared with controls) and histology demonstrated the presence of eosinophils. In contrast, corneas expressing p40 IL-12 (n = 9) showed prolonged allograft survival (median day to rejection = 45 days, p = 0.003). Local intraocular production of p40 IL-12 thus prolonged corneal graft survival significantly, but local production of the prototypic immunomodulatory cytokine IL-4 induced eosinophilia, inflammation, and rejection. These findings have important implications for the development of novel strategies to improve human corneal graft survival.