Patients with dry eye without hepatitis C virus infection possess the viral RNA in their tears.

PURPOSE: Dry eye is one of the suggested extrahepatic complications associated with hepatitis C virus (HCV) infection. HCV RNA has been detected from the tear fluid of patients with chronic HCV. There has been no literature evidence on the presence of HCV RNA in the tear fluid of patients with dry eye without HCV infection. In this study, tear fluid of patients with dry eye with no HCV infection was screened for the presence of HCV RNA. METHODS: Tear fluid was collected from patients with dry eye (n = 36) and healthy controls (n = 20). Real-time polymerase chain reaction was performed to detect HCV RNA in the tear fluid. Anti-HCV enzyme-linked immunosorbent assay, alkaline phosphatase, and alanine aminotransferase tests were performed in the serum samples collected from 15 patients with dry eye. RESULTS: Viral RNA was detected in 58.3% of the patients. Serum samples collected from 15 patients with dry eye were negative for anti-HCV. Alkaline phosphatase levels were elevated in 12 of 15 patients. Alanine aminotransferase levels were normal in all 15 patients. The odds ratio for the presence of HCV RNA in patients with dry eye was 22.4. CONCLUSIONS: These results indicate a direct correlation between dry eye and HCV in non-HCV patients.

Hepatitis C virus core and NS3 antigens induced conjunctival inflammation via toll-like receptor-mediated signaling.

PURPOSE: Dry eye condition is an extrahepatic manifestation associated with chronic hepatitis C virus (HCV) infection. Since conjunctival inflammation can contribute to the dry eye condition, in the present study we analyzed the conjunctival inflammatory response to HCV core and NS3 proteins. METHODS: We used primary human conjunctival fibroblasts for our study. Cytokines were measured with enzyme-linked immunosorbent assay (ELISA). Toll-like receptor (TLR) and cell adhesion molecule gene expression patterns were analyzed with semiquantitative reverse transcription (RT)-PCR. Immunofluorescence staining was performed for the MyD88, nuclear factor-kappa B (NF-kB), and inducible nitric oxide synthase (iNOS) proteins. Nitric oxide (NO) was measured with the Griess assay; terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed for apoptosis and cell viability, respectively. RESULTS: When exposed to the HCV core and NS3 proteins, the conjunctival fibroblasts secreted interleukin-8 (IL-8), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-10 in a dose-dependent manner. Various TLRs were involved in the innate immune response via MyD88 signaling without NF-kB involvement. The gene expression of cell adhesion molecules such as CD44 and ICAM-1 was upregulated, and the cells secreted NO via iNOS. As the sum of these stress responses, the cells underwent apoptosis, which eventually lead to cell death. CONCLUSIONS: HCV core and NS3 proteins induced conjunctival inflammation that may form the pathogenesis of dry eye condition.

Lacrimal proline rich 4 (LPRR4) protein in the tear fluid is a potential biomarker of dry eye syndrome.

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.

The role of signaling pathways in the expansion of corneal epithelial cells in serum-free B27 supplemented medium.

PURPOSE: To study the influence of serum-free B27 supplemented culture medium on corneal epithelial cells from limbal explants. METHODS: Human limbal tissues obtained from cadaveric donor eyes were used in this study. The morphological characteristics of cultivated epithelial cells were analyzed by phase contrast microscopy. Growth kinetics, bromodeoxyuridine (BrdU) labeling cell proliferation assay, and reverse transcriptase PCR (RT-PCR) for limbus and corneal markers were studied in serum-dependent and serum-free B27 supplemented corneal epithelial culture. The signaling pathway genes were analyzed by RT(2) qPCR profiler array. RESULTS: The corneal epithelial cells morphology and mRNA expression of markers were similar in both the serum-dependent and serum-free B27 supplemented culture. The growth and proliferation of the serum-free B27 supplemented culture was significantly higher than that of the serum-dependent culture. The wnt, hedgehog, survival, NFkB, Jak-Stat, and calcium protein kinase C pathways were highly expressed in the serum-free B27 supplemented corneal epithelial culture. CONCLUSIONS: Most signaling pathway genes are upfolded by B27 supplementation in the corneal epithelial cell culture; it could be an efficient replacement for serum.

Culture & characterisation of limbal epithelial cells & oral mucosal cells.

BACKGROUND & OBJECTIVES: Sources of autologous tissue that can functionally replace the corneal epithelium have been considered as an alternative to allogenous limbal transplants for limbal stem cells deficiency (LSCD). The aim of the present study was to compare the characterization of oral mucosa with limbal epithelial cells by markers using reverse transcriptase polymerase chain reaction (RT-PCR). METHODS: Experiments were performed using oral tissue (n=6) obtained from patients who underwent oral mucosal graft for LSCD. Confluent cultures of limbus and oral mucosa epithelial cells were characterized by the pututative stem cell markers using RT-PCR. The morphological characteristics of cultivated epithelial cells were analyzed by haematoxylin and eosin staining and phase contrast microscopy. RESULTS: Confluent sheets of epithelial cells were seen at the end of 14(th) day resembling the morphological features of limbal epithelia. RT-PCR analysis showed that cultured oral epithelial cells expressed markers such as ABCG2, p63, delta Np63, isoforms of p63, Keratin 3 (K3), membrane protein--Mucin (MUC 1, 4 and 16) and Antimicrobial Peptide--AMP (Human beta Defensin--hBD 1, 2 and 3). INTERPRETATION & CONCLUSIONS: Oral epithelial cultures have morphological features resembling corneal and limbal epithelial cells by expressing similar marker genes. Thus, feasibility of clinical use of oral epithelial cells need be evaluated for allogenous limbal transplants.

Tear ascorbic acid levels and the total antioxidant status in contact lens wearers: a pilot study.

AIMS: The tear ascorbate owing to its high concentration, functions as an effective antioxidant against the oxidative damage of cornea. Contact lens wearers (CLW) are prone to oxidative stress due to the lens-induced hypoxic conditions. A pilot study was done to compare the tear ascorbic acid level and the total antioxidant capacity give as in normal and CLW. MATERIALS AND METHODS: In this study 21 CLW (Mean age 23 +/- 3 years ; M-2, F-19), who were daily wear users, with duration of wear not more than four years, along with age-matched 28 controls (Mean age 28 +/- 3 ; M-15, F-13) were recruited in the study for collection of reflex tears using Schirmer's strip. Ascorbic acid in tears was determined using high-performance liquid chromatography (HPLC), total antioxidant capacity (TAC) and total protein assay by spectrophotometric analysis. RESULTS: CLW showed no significant change in the tear ascorbic acid levels (0.4 +/- 0.26 mM) compared to the control subjects (0.61 +/- 0.59 mM). The amount of ascorbic acid in tears did not correlate with the TAC or the total protein of the tears. The mean TAC in CLW was 0.69 +/- 0.16 mM, with a total protein of 1.35 +/- 0.46 mg/ml while in controls it was 0.7 +/- 0.18 mM and 1.21 +/- 0.47 mg/ml respectively. CONCLUSIONS: Soft contact lens wear did not show any significant change in tear ascorbic acid, TAC and total protein levels compared to controls.

Putative stem cell markers in limbal epithelial cells cultured on intact & denuded human amniotic membrane.

BACKGROUND & OBJECTIVES: The ocular surface is an ideal region to study the epithelial stem cell (SC) biology because of the unique spatial arrangement of stem cells and transient amplifying cells. A major challenge in corneal SC biology is the ability to identify SC in vitro and in situ, and one of the major controversies in the field relates to reliable SC markers. This study was carried out to evaluate and compare the expression of the stem cell associated marker: ABCG2, keratinocyte stem cell marker: p63 and corneal differentiation markers: Cnx43 and K3/K12 on limbal explants cultured on human amniotic membrane (HAM) with intact epithelium and HAM denuded of its epithelium. METHODS: Human limbal biopsies obtained from the cadaveric donor eyes were used in this study. The cells were cultured over the HAM with intact and denuded epithelium. Reverse transcriptase PCR, immunohistochemistry, Western blotting for ABCG2, P63, Cnx43 and K3/K12 were done. RESULTS: The limbal epithelial cells cultured over intact HAM expressed the stem cell associated markers (ABCG2, p63) and showed reduced expression of the differentiation markers (Cnx43 and K3/K12) when compared to limbal epithelial cells cultured over denuded HAM, which expressed more differentiation markers at the end of three weeks. BrdU label retaining cells were observed in the limbal epithelial cells cultured over HAM with epihelium only. INTERPRETATION & CONCLUSIONS: Our results showed that the intact HAM supported the growth of limbal epithelial cells expressing stem cell associated markers, and allowing little differentiation of the limbal cells to cornea phenotype. Further studies are needed to understand the properties of the amniotic epithelium that retains the stemness in the cultured limbal stem cells.