Evidence for endotoxin as a causative factor for leptospiral uveitis in humans

Purpose: To understand the pathogenic mechanism of leptospiral uveitis by determining the profile of infiltrating cells, the levels of cytokines, and the causative factor in aqueous humor (AH). Methods: AH and blood samples were collected from 22 patients with leptospiral uveitis that was confirmed by microscopic agglutination test (MAT). Nine patients with Behçet's uveitis, 10 with phacolytic uveitis, and 13 with age-related cataract were included as control subjects. A cytometric bead array was used to estimate human inflammatory and Th1/Th2 cytokines. The level of endotoxin in AH was estimated by limulus amebocyte lysate (LAL) test and by dot blot analysis using a leptospiral serovar lipopolysaccharide (LPS)-specific monoclonal antibody. Results: Except for one patient with leptospiral uveitis, AH from all other patients and control subjects was negative for Gram-negative bacterial endotoxin by LAL test. However, a significant level of serovar Copenhageni LPS was observed in AH of patients with leptospiral uveitis seropositive for the same serovar by MAT, in contrast to its absence in all control subjects. A selective infiltration of neutrophils as well as a significant increase in the levels of protein and cytokines IL-12p70, TNF, IL-6, IL-8, and IL-10 was observed in AH of patients with leptospiral uveitis. Phacolytic uveitis was associated with a high proportion of activated macrophages and increased levels of IL-6 and IL-8, whereas Behçet's uveitis was associated with a predominant infiltration of neutrophils and increased levels of IFN-γ. Conclusions: The results demonstrate the presence of serovar-specific LPS in AH, and thus it is likely that endotoxin is a causative factor in leptospiral uveitis

A subset of human limbal epithelial cells with greater nucleus-to-cytoplasm ratio expressing high levels of p63 possesses slow-cycling property

Purpose: The purpose of this study was to evaluate the subset of limbal epithelial cells with greater nucleus-to-cytoplasm (N/C) ratio expressing high levels of p63 for their slow-cycling property, a characteristic feature of stem cells (SCs). Methods: Limbal and peripheral corneal explant cultures were pulse labeled with 5-5-bromo-2'-deoxyuridine (BrdU) for 5 days, followed by a period of 3-week chase. Cultured explants were cryosectioned and stained for BrdU. The epithelial cells in the outgrowth and those remaining on the explant were isolated and subjected to cytospin and double immunostaining for BrdU and p63, followed by identification of label-retaining cells (LRCs) and quantification of p63 expression using confocal microscopy. Results: A distinct population of small cells with large N/C ratio expressing high levels of p63 retained the BrdU label after 21-day chase. Further, this population of LRCs, negative for the differentiation marker K3, was observed in the epithelial outgrowth of limbal but not in that of peripheral cornea. LRCs were seen to migrate along the cut edge of limbal explants in culture and were also observed as clusters of small cells in the outgrowth, which contained cells with the ability to form holoclone colonies. Conclusions: These results demonstrate that the small cells with large N/C ratio and high levels of p63 have BrdU label retaining slo-cycling property, thus confirming that these 2 parameters in combination may serve as a precise marker for identification and quantification of ex vivo-expanded limbal SCs. This method would be useful to standardize the optimal culture conditions that can maintain and expand SCs for therapeutic applications

High expression of p63 combined with a large N/C ratio defines a subset of human limbal epithelial cells: implications on epithelial stem cells

Purpose: To characterize human limbal epithelial cells based on the expression levels of nuclear protein p63 and the nucleus-to-cytoplasm (N/C) ratio. Methods: Limbal, peripheral, and central corneal epithelia were separated from the stroma by Dispase II and subsequently were treated with trypsin to obtain single-cell suspensions. Cytospin smears of the cell suspensions were double immunostained for p63 and then stained for any one of the markers (acidic cytokeratins [AE1], K5, K3, or connexin 43 [C×43]). They were counterstained with propidium iodide. More than 100 cells from each zone were analyzed for p63 expression levels and nuclear/cellular area using quantitative confocal microscopy. Results: A gradient of p63-positive cells was observed in corneal and limbal epithelial cells. The percentage of p63-positive cells and the level of p63 expression were significantly higher in the limbal than in the peripheral or central corneal epithelium. Two-parameter (p63 levels and N/C ratio) analysis revealed the presence of a distinct population of small cells with higher levels of p63 and a large N/C ratio in the limbal epithelium. Such limbal epithelial cells were positive for AE1 and K5 but negative for K3 and C×43. Conclusions: These results suggest that this distinct group of small cells in the limbal epithelium with greater N/C ratio, expressing high levels of nuclear protein p63, probably represent corneal epithelial stem cells

Hsa-miR-150-5p inhibits Wnt-beta-catenin signaling in human corneal epithelial stem cells

Purpose: In our earlier study, we identified hsa-miR-150-5p as a highly expressed miRNA in enriched corneal epithelial stem cells (CESCs). In this study, we aimed to understand the molecular regulatory function of hsa-miR-150-5p in association with the maintenance of stemness in CESCs. Methods: The target mRNAs of hsa-miR-150-5p were predicted and subjected to pathway analysis to identify targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-150-5p mimic, inhibitor, or scrambled sequence using Lipofectamine 3000. The transfected cells were analyzed to determine (i) their colony-forming potential; (ii) the expression levels of stem cell (SC) markers/transcription factors (ABCG2, NANOG, OCT4, KLF4, and ΔNp63), the differentiation marker (Cx43), and the hsa-miR-150-5p predicted targets (JARID2, INHBA, AKT3, and CTNNB1) by qPCR; and (iii) the expression levels of ABCG2, p63α, Cx43, JARID2, AKT3, p-AKT3, β-catenin, and active β-catenin by immunofluorescence staining and/or western blotting. Results: The ectopic expression level of hsa-miR-150-5p increased the colony-forming potential (8.29% ± 0.47%, p < 0.001) with the ability to form holoclone-like colonies compared with the control (1.8% ± 0.47%). The mimic-treated cells had higher expression levels of the SC markers but reduced expression levels of Cx43 and the targets of hsa-miR-150-5p that are involved in the Wnt-β-catenin signaling pathway. The expression levels of β-catenin and active β-catenin in the inhibitor-transfected cells were higher than those in the control cells, and the localized nuclear expression indicated the activation of Wnt signaling. Conclusions: Our results indicate a regulatory role for hsa-miR-150-5p in the maintenance of CESCs by inhibiting the Wnt signaling pathway

Immune status of health care personnel &amp; post vaccination analysis of immunity against rubella in an eye hospital

Background &amp; objectives: Congenital rubella syndrome (CRS) accounts for a significant amount of mortality and morbidity in India. Rubella vaccination is not included in our national immunization programme. Occupational exposure of the health care personnel to rubella infection is well known. This study aims to assess the serological status of health care workers against rubella virus in Aravind Eye Care System, Madurai and to follow the immune response in the seronegative individuals after vaccination

A Comparative Genome-Wide Transcriptome Analysis of Glucocorticoid Responder and Non-Responder Primary Human Trabecular Meshwork Cells

Aim: To investigate genes and pathways involved in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. Methods: Using paired human donor eyes, human organ-cultured anterior segment (HOCAS) was established in one eye to characterize GC responsiveness based on intra ocular pressure (IOP) change and, in the other eye, primary HTM cell culture was established. For RNA sequencing, total RNA extracted from GC-responder (GC-R) and non-responder (GC-NR) cells after dexamethasone (DEX) or ethanol (ETH) treatment for 7d was used. Differentially expressed genes (DEGs) were compared among five groups and validated. Results: In total, 616 and 216 genes were identified as significantly dysregulated in Group #1 and #2 (#1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR), respectively. Around 80 genes were commonly dysregulated in Group #3 (overlapping DEGs between #1 and #2), whereas 536 and 136 genes were uniquely expressed in GC-R (#4) and GC-NR HTM (#5) cells, respectively. Pathway analysis revealed that WNT signaling, drug metabolism cytochrome p450, cell adhesion, TGF-β signaling, and MAPK signaling were associated with GC responsiveness. Conclusion: This is the first study reporting distinct gene signatures and their associated pathways for GC-R and GC-NR HTM cells. WNT and MAPK signaling are potential therapeutic targets for the management of GC-induced glaucoma

Hsa-miR-143-3p inhibits Wnt-β-catenin and MAPK signaling in human corneal epithelial stem cells

Our previous study demonstrated hsa-miR-143-3p as one of the highly expressed miRNAs in enriched corneal epithelial stem cells (CESCs). Hence this study aims to elucidate the regulatory role of hsa-miR-143-3p in the maintenance of stemness in CESCs. The target genes of hsa-miR-143-3p were predicted and subjected to pathway analysis to select the targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-143-3p mimic, inhibitor or scrambled sequence using Lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential, (ii) expression of stem cell (SC) markers/ transcription factors (ABCG2, NANOG, OCT4, KLF4, ΔNp63), (iii) differentiation marker (Cx43), (iv) predicted five targets of hsa-miR-143-3p (DVL3, MAPK1, MAPK14, KRAS and KAT6A), (v) MAPK signaling regulators and (vi) Wnt-β-catenin signaling regulators by qPCR, immunofluorescence staining and/or Western blotting. High expression of hsa-miR-143-3p increased the colony forming potential (10.04 ± 1.35%, p < 0.001) with the ability to form holoclone-like colonies in comparison to control (3.33 ± 0.71%). The mimic treated cells had increased expression of SC markers but reduced expression of Cx43 and hsa-miR-143-3p targets involved in Wnt-β-catenin and MAPK signaling pathways. The expression of β-catenin, active β-catenin and ERK2 in hsa-miR-143-3p inhibitor transfected cells were higher than the control cells and the localized nuclear expression indicated the activation of Wnt and MAPK signaling. Thus, the probable association of hsa-miR-143-3p in the maintenance of CESCs through inhibition of Wnt and MAPK signaling pathways was thus indicated

Short and long-term effect of dexamethasone on the transcriptome profile of primary human trabecular meshwork cells in vitro

In the quest of identifying newer molecular targets for the management of glucocorticoid-induced ocular hypertension (GC-OHT) and glaucoma (GCG), several microarray studies have attempted to investigate the genome-wide transcriptome profiling of primary human trabecular meshwork (TM) cells in response to dexamethasone (DEX). However, no studies are reported so far to demonstrate the temporal changes in the expression of genes in the cultured human TM cells in response to DEX treatment. Therefore, in the present study, the time-dependent changes in the genome-wide expression of genes in primary human TM cells after short (16 hours: 16 h) and long exposure (7 days: 7 d) of DEX was investigated using RNA sequencing. There were 199 (118 up-regulated; 81 down-regulated) and 525 (119 up-regulated; 406 down-regulated) DEGs in 16 h and 7 d treatment groups respectively. The unique genes identified in 16 h and 7 d treatment groups were 152 and 478 respectively. This study found a distinct gene signature and pathways between two treatment regimes. Longer exposure of DEX treatment showed a dys-regulation of Wnt and Rap1 signaling and so highlighted potential therapeutic targets for pharmacological management of GC-OHT/glaucoma