Abnormal neovascular and proliferative conjunctival phenotype in limbal stem cell deficiency is associated with altered microRNA and gene expression modulated by PAX6 mutational status in congenital aniridia.

PURPOSE: To evaluate conjunctival cell microRNA (miRNAs) and mRNA expression in relation to observed phenotype of progressive limbal stem cell deficiency in a cohort of subjects with congenital aniridia with known genetic status. METHODS: Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age and sex-matched healthy control subjects. RNA was extracted and miRNA and mRNA analyses were performed using microarrays. Results were related to severity of keratopathy and genetic cause of aniridia. RESULTS: Of 2549 miRNAs, 21 were differentially expressed in aniridia relative to controls (fold change ≤ -1.5 or ≥ +1.5). Among these miR-204-5p, an inhibitor of corneal neovascularization, was downregulated 26.8-fold in severely vascularized corneas. At the mRNA level, 539 transcripts were differentially expressed (fold change ≤ -2 or ≥ +2), among these FOSB and FOS were upregulated 17.5 and 9.7-fold respectively, and JUN by 2.9-fold, all being components of the AP-1 transcription factor complex. Pathway analysis revealed enrichment of PI3K-Akt, MAPK, and Ras signaling pathways in aniridia. For several miRNAs and transcripts regulating retinoic acid metabolism, expression levels correlated with keratopathy severity and genetic status. CONCLUSION: Strong dysregulation of key factors at the miRNA and mRNA level suggests that the conjunctiva in aniridia is abnormally maintained in a pro-angiogenic and proliferative state, and these changes are expressed in a PAX6 mutation-dependent manner. Additionally, retinoic acid metabolism is disrupted in severe, but not mild forms of the limbal stem cell deficiency in aniridia.

Development and Assessment of a Simulator for in Vivo Confocal Microscopy in Fungal and Acanthamoeba Keratitis.

BACKGROUND AND PURPOSE: In vivo confocal microscopy (IVCM) is a non-invasive imaging technique that allows morphological analysis as a diagnostic approach of the cornea in real time, thus providing a suspected diagnosis of fungal or amoebic keratitis immediately, whereas culture or PCR require several days or even weeks. Since these infections are rare, it is difficult for ophthalmologists to gain the experience necessary to differentiate infection from normal findings or artefacts. The purpose of this project was to establish a simulator, on which physicians could practice as well as acquiring a database of IVCM images of fungal or amoebic keratitis and respective analyses. PATIENTS AND METHODS: An IVCM simulator was set up with cadaver human corneas, infected with either acanthamoeba, candida or aspergillus. Twenty-one ophthalmologists were trained in IVC microscopy first in a Dry Lab, then practically on the simulator. For evaluation, the participants were asked to fill out a standardized questionnaire, with a pre- and post-course self-assessment. RESULTS: The self-assessed theoretical and practical skills in differentiating infectious from non-infectious keratitis in IVCM significantly increased (p = 0.0001, p = 0.0002, respectively). The barrier to use this technique decreased (p = 0.0474). CONCLUSION: A very simple protocol based on a model of ex vivo corneal mycotic and amoebic infections can be used to train novices in the structured approach and diagnostic use of IVCM for corneal infections.

[Amniotic Membrane Suspension and Autologous Serum - Are they Important for Wound Healing?] / Amnionmembransuspension und autologes Serum ­ spielt der Inhalt eine Rolle für die Wundheilung?

Autologous serum (AS) and amniotic membrane transplantation (AMT) are used in the treatment of several ocular surface diseases. AMT is associated with a surgical intervention. Surgery could be avoided by using eye drops prepared from an amniotic membrane homogenate (AMH) or amniotic membrane suspension (AMS). EGF, bFGF, IL-6 and IL-8 were detected in AMS. However, EGF and bFGF concentrations in AMS were about 1.7-17× lower than in AMH, and IL-6 and IL-8 could not be detected in AMH. 100 % AMS, 15 and 30 % AMH significantly decreased proliferation of human corneal epithelial cells (HCECs) compared to controls (p = <0.002 for all), but 15 and 30 % AMS did not affect proliferation. Migration increased significantly compared to controls with 15 and 30 % AMS (p < 0.001), but did not change significantly with 15 or 30 % AMH (p = 0.153 and p = 0.083). Proliferation of HCECs was significantly greater with 15 % AS than with 30 % AS (p < 0.001). HCEC migration was significantly greater with 30 % AS than with 5 % AS (p < 0.01). In summary, 15 and 30 % AMS and 15 and 30 % AS exhibit the best supportive effect on human corneal epithelial cells. Nevertheless, we always have to keep in mind that individual growth factor concentrations exhibit high inter-individual fluctuations in AS or AMS eye drops.

Crosslinking and corneal cryotherapy in acanthamoeba keratitis -- a histological study.

PURPOSE: Acanthamoeba keratitis is rare, but difficult to treat. Penetrating keratoplasty is performed in therapy-resistant cases. Nevertheless, subsequent recurrences occur in 40 % of the cases. In addition to triple-topical therapy (polyhexamid, propamidinisoethionat, neomycin), treatment alternatives are corneal cryotherapy and/or crosslinking (CXL). The aim of our present histological study was to analyze the persistence of acanthamoebatrophozoites and cysts, the persistence of bacteria, and activation of keratocytes in corneas of acanthamoeba keratitis patients following corneal cryotherapy and/or CXL. PATIENTS AND METHODS: We analyzed histologically corneal buttons (from penetrating keratoplasties) of nine patients with acanthamoeba keratitis, following corneal cryotherapy (two patients) or a combination of crosslinking and corneal cryotherapy (seven patients), using haematoxilin­eosin, periodic acid Schiff (PAS), Gram and alpha-smooth muscle actin (alpha-SMA) stainings. RESULTS: Acanthamoeba trophozoites persisted in three corneas after cryotherapy and CXL. Cysts persisted in one of two corneas following corneal cryotherapy and in six of seven corneas after a combination of CXL and cryotherapy. One cornea showed positive Gram staining, but there were no alpha-SMA positive keratocytes in any of the corneas. CONCLUSIONS: Crosslinking and corneal cryotherapy have only limited impact on killing of acanthamoeba trophozoites, cysts, or bacteria. Corneal cryotherapy and CXL did not stimulate myofibroblastic transformation of keratocytes.

[Effect of Photodynamic Inactivation (PDI) using Riboflavin-Conjugated Antibody against Staphylococcus aureus]. / Effekt photodynamischer Inaktivierung (PDI) mit Riboflavin oder riboflavinkonjugierten Antikörpern gegen Staphylococcus aureus.

PURPOSE: Crosslinking/riboflavin-UVA photodynamic therapy is a potential treatment alternative in antibiotic resistant infectious keratitis. For photodynamic therapy a specific (against bacteria) conjugated antibody may be used in order to increase the effect of the treatment. In our present study we analysed the impact of photodynamic inactivation using riboflavin-conjugated antibody or riboflavin alone on Staphylococcus aureus, in vitro. METHODS: Staphylococcus aureus (S. aureus) was incubated in 1 : 100 diluted riboflavin-conjugated antibody (R-AB) for 30 minutes in darkness. Following UVA-light illumination (375 nm) with an energy dose of 2, 3, 4 and 8 J/cm(2), bacteria were brought to blood agar Plates for 24 hours before colony-forming unit (CFU) counting. In an additional group, we incubated bacteria to 0, 0.05 or 0.1 % riboflavin 5-phosphate as described above followed by illumination using UVA light (375 nm) with an energy dose of 2 J/cm(2), before CFU counting. RESULTS: The number of CFU decreased significantly (inactivation of 36 %, p = 0.022) using 1 : 100 diluted riboflavin-conjugated antibody and 2 J/cm(2) UVA-light illumination, compared to untreated controls. The use of 3, 4 und 8 J/cm(2) energy dose and R-AB in 1 : 100 dilution did not further change the decrease of CFU (inactivation of 39, 39 and 40 %; p = 0.016; p = 0.016; p = 0.015). The use of 0.05 % or 0.1 % riboflavin 5-phosphate alone and UVA-light illumination reduced the CFU count significantly (inactivation of 73 and 55 %; p = 0.002; p = 0.005), compared to untreated controls. CONCLUSIONS: The use of riboflavin-conjugated antibody or 0.05 % or 0.1 % riboflavin 5-phosphate and UVA-light illumination reduces the number of CFU of S. aureus. However, none of these photodynamic therapies reached the necessary 99 % killing rate of these bacteria. Further work is needed to increase the efficacy of riboflavin-conjugated antibodies against antibiotic resistant bacteria.

[Growth Factors and Interleukins in Amniotic Membrane Tissue Homogenate]. / Wachstumsfaktoren und Interleukine in Amniongewebehomogenat.

PURPOSE: Application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy resistant corneal epithelial defects. The purpose of this study was to determine the concentrations of epidermal growth factor (EGF), fibroblast growth factor basic (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-6 (IL-6) and interleukin-8 (IL-8) in amniotic membrane homogenates. METHODS: Amniotic membranes of 8 placentas were prepared and thereafter stored at - 80 °C using the standard methods of the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz. Following defreezing, amniotic membranes were cut in two pieces and homogenized in liquid nitrogen. One part of the homogenate was prepared in cell-lysis buffer, the other part was prepared in PBS. The tissue homogenates were stored at - 20 °C until enzyme-linked immunosorbent assay (ELISA) analysis for EGF, bFGF, HGF, KGF, IL-6 and IL-8 concentrations. RESULTS: Concentrations of KGF, IL-6 and IL-8 were below the detection limit using both preparation techniques. The EGF concentration in tissue homogenates treated with cell-lysis buffer (2412 pg/g tissue) was not significantly different compared to that of tissue homogenates treated with PBS (1586 pg/g tissue, p = 0.72). bFGF release was also not significantly different using cell-lysis buffer (3606 pg/g tissue) or PBS treated tissue homogenates (4649 pg/g tissue, p = 0.35). HGF release was significantly lower using cell-lysis buffer (23,555 pg/g tissue), compared to PBS treated tissue (47,766 pg/g tissue, p = 0.007). CONCLUSION: Containing EGF, bFGF and HGF, and lacking IL-6 and IL-8, the application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy-resistant corneal epithelial defects.

[CD34 and alpha-smooth muscle actin expression of keratocytes following photodynamic inactivation (PDI)]. / CD34 und Alpha-smooth-Muscle-Actin-Keratozyten-Expression nach photodynamischer Inaktivierung (PDI).

PURPOSE: Photodynamic inactivation (PDI) may be a potential treatment alternative in therapy-resistant infectious keratitis. PDI may eliminate the microorganisms from the infected cornea by damage caused through free oxygen radicals, or even by supporting different stages of activation of keratocytes and inflammatory cell response. The purpose of this study was to determine the impact of PDI on activation of human keratocytes in culture. METHODS: Primary human keratocytes were isolated by digestion in collagenase A (1 mg/mL) from human corneal buttons, and cultured in DMEM/Ham's culture medium supplemented with 10% foetal calf serum. Keratocytes underwent illumination (670 nm) for 13 minutes following exposure to 0, 50, 150 and 250 nMol/ml concentrations of the photosensitizer chlorin e6 (Ce6) in the culture medium. Twenty-four hours after treatment CD34 and α-smooth-muscle actin expression of the cells was analysed using flow-cytometry (FACS). RESULTS: Using Ce6 or illumination only, α-smooth-muscle actin expression of the cells did not change significantly. Twenty-four hours after PDI the percentage of CD34-positive keratocytes did not change significantly using 50-250 nM Ce6, however, the percentage of α-smooth-muscle actin-positive keratocytes decreased significantly at 250 nM Ce6 (p = 0.01). CONCLUSIONS: As a short-term effect, PDI seems to inhibit myofibroblastic transformation of keratocytes, but does not have an impact on activation of CD34-positive keratocytes.With this impact PDI possibly may reduce the antimicrobial defence of keratocytes.

Regulation of soluble VEGFR-2 secreted by microvascular endothelial cells derived from human BPH.

BACKGROUND: Recently, it was reported that the soluble vascular endothelial growth factor receptor-2 (sVEGFR-2) is secreted by microvascular endothelial cells from human BPH (HPECs). The purpose of this study was to investigate the modulation of sVEGFR-2 by common endothelial cell stimulators. In addition, the physiological role of sVEGFR-2 with regard to the VEGF-stimulated proliferation of HPEC was investigated. METHODS: HPECs were isolated and cultured from fresh BPH tissue. After the incubation of HPECs either with adenosine triphosphate (ATP), interleukin (IL)-6, IL-8 or IL-12, the secretion of sVEGFR-2 was measured by enzyme-linked immunosorbent assay. For measurement of HPEC proliferation influenced by sVEGFR-2, VEGF-stimulated HPEC was cultured with/without sVEGFR-2. Cell proliferation was assessed with the Alamar Blue method. RESULTS: The sVEGFR-2 secretion was increased by ATP and decreased by IL-12 and IL-8, respectively. IL-6 did not show any significant effect on sVEGFR-2 secretion of HPECs. HPEC proliferation was significantly inhibited by sVEGFR-2. CONCLUSIONS: In this study, our data suggest that the secretion of sVEGFR-2 by microvascular endothelial cells from prostate origin is influenced by multiple endothelial cell stimulators. Furthermore, our data suggest that sVEGFR-2 acts as an antiangiogenic factor.