Evaluation of collagen derived antiangiogenic factors and matrix metalloproteinases in anterior lens epithelial cells of pediatric eyes with persistent fetal vasculature.

Purpose: To measure levels of collagen-derived antiangiogenic factors (arresten, canstatin, tumstatin, endostatin) and matrix metalloproteinases (MMP-2 and MMP-9) in anterior lens epithelial cells (LECs) and anterior capsules of children with cataract and persistent fetal vasculature (PFV) as cases and cataract without PFV as controls. Methods: Anterior capsules harboring LECs were collected from pediatric cataract patients with (n = 13) and without PFV (n = 13) during surgery. Samples were immediately subjected to RNA extraction and cDNA preparation. Quantitative real time PCR was performed to determine the mRNA levels of antiangiogenic factors and matrix metalloproteinases. GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase) and ß Actin were used as the housekeeping control. The mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between controls and cases. The non-parametric Mann-Whitney U test was applied for statistical evaluation. P values < 0.05 were statistically significant. Results: The relative mRNA levels of arresten, canstatin, tumstatin, endostatin, MMP-2 and MMP-9 in cases were 6.20E-03 ± 0.003, 1.49E-01 ± 0.02, 1.70E-01 ± 0.007, 3.20E-03 ± 0.003, 1.11E-03 ± 0.0009 and 3.72E-04 ± 0.0001. The mRNA levels of arresten was 1.6 times lower (P = 0.01) while mRNA levels of MMP-2, tumstatin and canstatin were 4, 2.5, and 2.3 times higher in cases than in controls. No change was observed in mRNA levels of MMP-9 and endostatin (P = 0.82). Conclusion: A significant difference in the levels of arresten, canstatin, tumstatin, and MMP-2 was found in LECs with PFV.

Trichostatin A Restores Expression of Adherens and Tight Junction Proteins during Transforming Growth Factor ß-Mediated Epithelial-to-Mesenchymal Transition.

PURPOSE: Adherens junctions and polarity markers play an important role in maintaining epithelial phenotype but get altered during the epithelial-mesenchymal transition (EMT). Alterations of these markers during EMT of lens epithelial cell (LEC) can lead to vision compromising conditions. The aim of this study was to examine if Trichostatin-A (TSA), a histone deacetylase inhibitor, can prevent EMT by restoring the adherens junction complex in LEC. METHODS: Fetal human lens epithelial cell line (FHL124) was used. Cells were treated with 10 ng/ml TGF-ß2 in the presence or absence of TSA. Real time-PCR and western blotting were carried out for HDAC1, HDAC2, CDH1 (E-cad), TJP1 (ZO-1) and CTNNB1 (ß-cat). Level of histone acetylation was analyzed by western blotting. Chromatin Immunoprecipitation was carried out to study the level of acetylated histone H4 and HDAC2 at the promoter regions of CDH1, TJP1, and CTNNB1. E-cad, ZO-1, and ß-cat were localized using immunofluorescence. Kruskal-Wallis test was used for statistical analysis. RESULTS: TSA down-regulated HDAC1 and HDAC2 and led to an increase in global acetylation. The mRNA and protein levels of E-cad, ZO-1, and ß-cat decreased during EMT but were up-regulated by TSA treatment. TSA also helped in stabilizing these proteins at cell-cell junctions during EMT. TSA decreases association of HDAC2 at the promoter regions of adherens junction genes while increasing histone H4 acetylation status. CONCLUSION: TSA increases histone acetylation and restores the adherens junction complex in LECs. TSA helps in preventing EMT and thus shows potential against lens fibrosis and vision compromising conditions.

DOPA and DHN pathway orchestrate melanin synthesis in Aspergillus species.

Melanins are high molecular weight hydrophobic pigments that have been studied for their role in the virulence of fungal pathogens. We investigated the amount and type of melanin in 20 isolates of Aspergillus spp.; A. niger (n = 3), A. flavus (n = 5), A. tamarii (n = 3), A. terreus (n = 3), A. tubingensis (n = 3), A. sydowii (n = 3). Aspergillus spp. were identified by sequencing the internal transcribed spacer (ITS) region. Extraction of melanin from culture filtrate and fungal biomass was done and followed by qualitative and quantitative analysis of melanin pigment. Ultraviolet (UV), Fourier transformed infrared (FT-IR), and electron paramagnetic resonance (EPR) spectra analyses confirmed the presence of melanin. The melanin pathway was studied by analyzing the effects of inhibitors; kojic acid, tropolone, phthalide, and tricyclazole. The results indicate that in A. niger and A. tubingensis melanin was found in both culture filtrate and fungal biomass. For A. tamarii and A. flavus melanin was extracted from biomass only, whereas melanin was found only in culture filtrate for A. terreus. A negligible amount of melanin was found in A. sydowii. The maximum amount of melanin from culture filtrate and fungal biomass was found in A. niger and A. tamarrii, respectively. The DOPA (3,4-dihydroxyphenylalanine) pathway produces melanin in A. niger, A. tamarii and A. flavus, whereas the DHN (1,8-dihydroxynaphthalene) pathway produces melanin in A. tubingensis and A. terreus. It can be concluded that the amount and type of melanin in aspergilli largely differ from species to species.

Microscopic evaluation, molecular identification, antifungal susceptibility, and clinical outcomes in fusarium, Aspergillus and, dematiaceous keratitis.

PURPOSE: Fusarium, Aspergillus, and Dematiaceous are the most common fungal species causing keratitis in tropical countries. Herein we report a prospective study on fungal keratitis caused by these three fungal species. METHODOLOGY: A prospective investigation was undertaken to evaluate eyes with presumed fungal keratitis. All the fungal isolates (n = 73) obtained from keratitis infections were identified using morphological and microscopic characters. Molecular identification using sequencing of the ITS region and antifungal susceptibility tests using microdilution method were done. The final clinical outcome was evaluated in terms of the time taken for resolution of keratitis and the final visual outcome. The results were analyzed after segregating the cases into three groups, namely, Fusarium, Aspergillus, and Dematiaceous keratitis. RESULTS: Diagnosis of fungal keratitis was established in 73 (35.9%) cases out of 208 cases. The spectra of fungi isolated were Fusarium spp. (26.6%), Aspergillus spp. (21.6%), and Dematiaceous fungi (11.6%). The sequence of the ITS region could identify the Fusarium and Aspergillus species at the species complex level, and the Dematiaceous isolates were accurately identified. Using antifungal agents such as fluconazole, natamycin, amphotericin B, and itraconazole, the minimum inhibitory concentrations (MICs) for Fusarium spp. were >32 µ g/mL, 4-8 µ g/mL, 0.5-1 µ g/mL, and >32 µ g/mL, respectively. Antifungal susceptibility data showed that Curvularia spp. was highly resistant to all the antifungal agents. Overall, natamycin and amphotericin B were found to be the most effective antifungal agents. The comparative clinical outcomes in all cases showed that the healing response in terms of visual acuity of the Dematiaceous group was significantly good when compared with the Fusarium and Aspergillus groups (P < 0.05). The time required for healing in the Fusarium group was statistically significantly less when compared with the Aspergillus and Dematiaceous groups. CONCLUSION: This study demonstrates important differences in microscopic features of scraping material and antifungal susceptibility between the three groups. Early and accurate identification coupled with the MIC data, and thereby appropriate treatment is crucial for complete recovery.

Estrogen mediated protection of cytoskeleton against oxidative stress.

BACKGROUND & OBJECTIVES: Cytoskeletal proteins are deregulated during oxidative stress and cataract formation. However, estrogen which protects against cataract formation and harmful effects of oxidative stress has not been tested on the cytoskeleton of lens epithelial cells (LECs). The current study was undertaken to assess if the protection rendered to LECs by estrogen was mediated by preserving the cytoskeletal proteins. METHODS: Oxidative stress was induced by 50 µM of H 2 O 2 in cultured goat LECs (gLECs) and effect of 1 µM 17ß-estradiol (E 2 ) was tested. After treatment, morphological analysis of cells was carried out using haematoxylin-eosin staining and cell density was also quantified. Cell viability was determined using Hoechst (Ho), YO-Pro (YP) and propidium iodide (PI). F-actin and vimentin were localized using phalloidin and anti-vimentin antibody, respectively, and viewed under fluorescence microscopy. Vimentin was further analysed at protein level by Western blotting. RESULTS: H 2 O 2 led to increased condensation of nucleus, cell death and apoptosis but these were prevented with pre- and co-treatment of E 2 with increase in cell viability (P<0.001). E 2 also prevented H 2 O 2 mediated depolymerization of cytoskeleton but was not able to reverse the changes when given after induction of oxidative stress. INTERPRETATION & CONCLUSIONS: Our findings showed that E 2 helped in preventing deteriorating effect of H 2 O 2 , inhibited cell death, apoptosis and depolymerisation of cytoskeletal proteins in LECs. However, the exact mechanism by which estrogen renders this protection to cytoskeleton of lens epithelial cells remains to be determined.

Incision integrity and postoperative outcomes after microcoaxial phacoemulsification performed using 2 incision-dependent systems.

PURPOSE: To compare incision integrity and clinical outcomes of 2 microcoaxial phacoemulsification systems. SETTING: Iladevi Cataract & IOL Research Centre, Ahmedabad, India. DESIGN: Prospective randomized clinical trial. METHODS: Eyes were randomized to have phacoemulsification using a 1.8 mm clear corneal incision (CCI) system (Group 1, Stellaris system) or a 2.2 mm CCI system (Group 2, Intrepid Infiniti system). Incision enlargement at end of surgery was measured. At the conclusion of surgery, trypan blue was applied over the conjunctival surface, anterior chamber aspirate withdrawn, and ingress into anterior chamber measured. Postoperative observations included evaluation of the CCI using anterior segment optical coherence tomography (AS-OCT), change in central corneal thickness (CCT), and anterior segment inflammation at 1 day, 1 week, and 1 month and endothelial cell loss and surgically induced astigmatism (SIA) at 3 months. RESULTS: Incision enlargement (P<.001) and trypan blue ingress in the anterior chamber (mean 1.7 log units ± 0.6 [SD] versus 3.8 ± 0.6 log units, P<.001) was significantly greater in Group 1 (n = 50) than in Group 2 (n = 50). On AS-OCT, endothelial misalignment and gaping were more frequent in Group 1 at 1 day (P=.001) and 1 week (P=.018). There were no significant differences in SIA, change in CCT, endothelial cell loss, or anterior segment inflammation (P>.05). CONCLUSION: At the end of surgery, it is not the initial incision size alone but also the distortion of the incision during subsequent stages of surgery that determine the integrity of the CCI.

Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells.

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n=52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (n=11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student's t-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.

Matrix metalloproteinase-2 and -9 activities in the human lens epithelial cells and serum of steroid induced posterior subcapsular cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase (MMP)-2 and MMP-9 activities in patients with steroid induced posterior subcapsular cataract (PSC). METHODS: This prospective, observational study comprised of 156 patients having either steroid induced PSC (n=50) or non-steroidal PSC (n=106) were performed to evaluate the level of MMP-2 and MMP-9 activities in the lens epithelial cells (LECs) and the serum. Anterior lens capsules harboring LECs were obtained during phacoemulsification and peripheral blood was collected from patients before administration of anesthesia. Serum was separated by centrifugation at 10,000× g for 15 min at 4 °C. The LECs and serum samples were processed to analyze MMP-2 and MMP-9 activities using succinylated gelatin assay. Quantitative real time-PCR (qRT-PCR) was performed to determine the mRNA levels of MMP-2 and MMP-9 in LECs. The mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between cases with steroid induced PSC and cases with non-steroidal PSC. MMP-2 and MMP-9 levels were also compared in the two groups using immunolocalization. RESULTS: The level of MMP-2 and MMP-9 activity was found to be high in LECs and serum of cases with steroid induced PSC. Further in all steroid induced cases, a 1.4 fold increase was observed in MMP-2 activity in LECs and a 1.4 fold increase in MMP-9 activity in the serum. Both qRT-PCR and immunolocalization showed increased expression of MMP-2 and MMP-9 activity. CONCLUSIONS: MMP-2 and MMP-9 activity in both LECs and serum was significantly higher in cases with steroid induced PSC. The possible use of MMP-9 as a non-invasive biomarker in ascertaining the presence of steroid induced PSC should be evaluated using a larger sample size.

Fungal scleral keratitis caused by Phomopsis phoenicicola.

We report a case of scleral keratitis caused by Phomopsis phoenicicola. Pterygium surgery was a predisposing factor, and the patient was treated with natamycin and fluconazole eye drops and oral fluconazole. The fungus was identified by sequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal DNA (rDNA) locus and confirmed on the basis of its typical pycnidia and conidia.

Effect of vitamin C and E activity on surgically removed cataractous human lens epithelium cells.

Oxidative stress has been proposed as a common underlying mechanism of cataractogenesis. Experimental and observational data suggest that micronutrients like vitamin C and vitamin E with antioxidant capabilities may retard the development of age-related cataract. Effect of these factors on lens epithelium cells, center of lens metabolic activities, is not completely elucidated. The aim of present study was to examine the effect of vitamin C and E on surgically removed lens epithelium cells of patients with cataract. Capsulorhexis samples were collected from 170 patients, admitted for cataract surgery. Catalase specific activity was estimated in lens epithelium cells with and without vitamin (C or E) treatment at different concentration for different time duration. Student's t-test was employed for data analysis. We observed that in ex-vivo condition, a) both vitamin C and E bring about a decrease in catalase activity in lens epithelial cells. b) vitamin C showed toxic effect at high concentration. c) 100µM was the optimum concentration at which both vitamins showed maximum antioxidant activity. It was concluded that both vitamin C and E has direct effect on lens epithelium cells. At optimum concentration, they can reduce oxidative stress in these cells thus can support to prevent or delay cataract development.

Effect of stromal hydration of clear corneal incisions: quantifying ingress of trypan blue into the anterior chamber after phacoemulsification.

PURPOSE: To use trypan blue as a quantifiable ingress tracer to determine whether stromal hydration reduces ocular surface fluid ingress at the end of phacoemulsification. SETTING: Iladevi Cataract and IOL Research Centre, Memnagar, Ahmedabad, India. METHODS: A prospective randomized study included 80 eyes having phacoemulsification through 2.2 mm incisions. These eyes were divided into 2 equal groups: 1 had stromal hydration (surgery completed by injecting fortified balanced salt solution [BSS Plus] to hydrate the lateral walls and internal entry of incision) and the other had no stromal hydration. One half milliliter of 0.0125% sterile trypan blue was instilled on the ocular surface and allowed to remain for 2 minutes. One-tenth milliliter of aqueous fluid was aspirated from the anterior chamber, and its optical density was measured using ultraviolet spectrophotometry. Logs of dilution of trypan blue were used for statistical analysis using the nonparametric Mann-Whitney U test. RESULTS: There was a statistically significant decrease and difference between groups in mean dilution of trypan blue in the aqueous aspirate (P<.001). The mean was 1:11,337 in the stromal hydration group and 1:220 in the no stromal hydration group. Logs of mean dilution of trypan blue had statistically significant lower values in the stromal hydration group than in the no stromal hydration group (3.21 and 2.14, respectively) (P<.001). CONCLUSIONS: Stromal hydration of clear corneal incisions reduced ingress into the anterior chamber of the trypan blue instilled on the ocular surface. Clinically, these findings may have a beneficial effect in reducing the risk for postoperative endophthalmitis.