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

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

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

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&apos;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&apos;s uveitis was associated with a predominant infiltration of neutrophils and increased levels of IFN-␥. CONCLUSIONS. The results demonstrate the presence of serovarspecific LPS in AH, and thus it is likely that endotoxin is a causative factor in leptospiral uveitis. (Invest Ophthalmol Vis Sci. 2008;49:5419 -5424

Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells

The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were immunostained for SOX2, gap junction protein alpha 1 (GJA1), PAX6, α, β and γ-crystallins, followed by a confocal analysis. The functional property of adult stem cells was analysed by their sphere forming ability using cultured lens epithelial cells from different zones. Based on marker expression, the lens epithelium was divided into four zones: the central zone, characterized by a small population of PAX6+, GJA1−, β-crystallin− and γ-crystallin− cells; the germinative zone, characterized by PAX6+, GJA1+, β-crystallin− and γ-crystallin−; the transitional zone, characterized by PAX6+, GJA1+, β-crystallin+ and γ-crystallin−; and the equatorial zone, characterized by PAX6+/−, GJA1+, β-crystallin+, and γ-crystallin+ cells. The putative lens epithelial stem cells identified as SOX2+ and GJA1 membrane expression negative cells were located only in the central zone (1.89 ± 0.84%). Compared to the other zones, a significant percentage of spheres were identified in the central zone (1.68 ± 1.04%), consistent with the location of the putative adult lens epithelial stem cells. In the cataractous lens, an absence of SOX2 expression and a significant reduction in sphere forming ability (0.33 ± 0.11%) were observed in the central zone. The above findings confirmed the presence of putative stem cells in the central zone of the adult human lens epithelium and indicated their probable association with cataract development

In vivo confocal microscopic analysis of normal human anterior limbal stroma.

PURPOSE: To characterize the microarchitecture of anterior limbal stroma in healthy individuals using in vivo confocal microscopy (IVCM) and to correlate it with mesenchymal stem cells (MSCs), a component of the limbal niche. METHODS: The corneal side of the superior limbus was scanned in 30 eyes of 17 normal subjects beyond the basal epithelium, deep into the stroma using an HRT III laser scanning microscope. The IVCM findings were correlated with the immunohistochemical features of MSCs in the anterior limbal stroma. RESULTS: Clusters of hyperreflective structures were observed in the anterior limbal stroma, subjacent to the basal epithelium (depth, 50.2 ± 8.7 μm to 98 ± 12.8 μm), but not in the corneal stroma. The structures showed unique morphology compared with epithelial cells, keratocytes, neurons, and dendritic cells. In parallel, confocal analysis of immunostained sections showed clusters of cells, double positive for MSC-specific markers (CD90 and CD105) in the anterior limbal stroma at a depth of 55.3 ± 12.7 μm to 72 ± 37.6 μm. The organization and distribution of the MSC clusters locates them within the hyperreflective region in the anterior limbal stroma. CONCLUSIONS: The hyperreflective structures, demonstrated for the first time in the human anterior limbal stroma, probably represent an important component of the limbal niche. Our approach of in vivo imaging may pave the way for assessing the limbal stromal health