Amalia Enríquez‐de‐Salamanca

In the field of predictive, preventive and personalised medicine, researchers are keen to identify novel and reliable ways to predict and diagnose disease, as well as to monitor patient response to therapeutic agents. In the last decade alone, the sensitivity of profiling technologies has undergone huge improvements in detection sensitivity, thus allowing quantification of minute samples, for example body fluids that were previously difficult to assay. As a consequence, there has been a huge increase in tear fluid investigation, predominantly in the field of ocular surface disease. As tears are a more accessible and less complex body fluid (than serum or plasma) and sampling is much less invasive, research is starting to focus on how disease processes affect the proteomic, lipidomic and metabolomic composition of the tear film. By determining compositional changes to tear profiles, crucial pathways in disease progression may be identified, allowing for more predictive and personalised therapy of the individual. This article will provide an overview of the various putative tear fluid biomarkers that have been identified to date, ranging from ocular surface disease and retinopathies to cancer and multiple sclerosis. Putative tear fluid biomarkers of ocular disorders, as well as the more recent field of systemic disease biomarkers, will be shown.

PURPOSE: Inflammatory molecules have been demonstrated in the tear film of patients with severe dry eye disease (DED). However, little attention has been paid to the most frequent moderate forms of DED. This study analyzes tear cytokine levels and their clinical correlations in patients with moderate evaporative-type DED due to meibomian gland disease (MGD). METHODS: Twenty three evaporative-type DED patients (46 eyes) of mild-to-moderate intensity and nine healthy subjects (18 eyes) were recruited. Two symptom questionnaires were self-answered and multiple DED-related clinical tests were performed. Unstimulated tears from each eye were isolated and were not pooled. Levels of 15 cytokines and chemokines were measured by multiplex bead analysis, compared with control levels, and correlated with clinical tests. RESULTS: Fourteen out of the 15 molecules were reliably detected in 1 microl of unstimulated tears from DED patients. Epidermal growth factor (EGF), fractalkine/CX3CL1, interleukin (IL) 1-receptor antagonist (Ra), IL-8/CXCL8, interferon inducible protein (IP)-10/CXCL10, and vascular endothelial growth factor (VEGF) were found in 94%-100% of samples; IL-6 in 65% (significantly more detected in older patients); IL-1beta, interferon gamma (IFN-gamma), and IL-10 in 30%-48%; IL-17 in 13%; granulocyte macrophage colony stimulating factor (GM-CSF), IL-13, and tumor necrosis factor alpha (TNF-alpha) in 2%-9%; and IL-5 was never detected. EGF, fractalkine/CX3CL1, IL-1Ra, IP-10/CXCL10, and VEGF levels were significantly increased compared to normal controls. Pain was correlated with IL-6 and IL-8/CXCL8. Tear break-up time correlated inversely with IL1-Ra. Schirmer test and tear lysozyme levels negatively correlated with IL-1Ra, IL-8/CXCL8, fracktalkine/CX3CL1, IL-6, IP-10/CXCL10, and VEGF had the same tendency. Conjunctival staining correlated negatively with EGF and positively with IL-6. CONCLUSIONS: In this sample of moderate evaporative-type DED patients, five inflammatory molecules were elevated. Fracktalkine was demonstrated to be present and elevated in tears in human DED. IL-1Ra, IL-6, IL-8/CXCL8, and EGF levels correlated with pain and with clinical parameters measuring tear stability, tear production or ocular surface integrity. These results suggest that inflammation plays a role not only in severe DED but also in moderate evaporative DED.

PURPOSE: To study the in vitro and in vivo interaction of chitosan nanoparticles (CSNPs), a new particulate drug carrier, with epithelial cells on the ocular surface. METHODS: CSNPs labeled with fluorescein isothiocyanate-bovine serum albumin were produced by ionotropic gelation. Human conjunctival epithelial cells (IOBA-NHC) were exposed for 15, 30, 60, and 120 minutes to three different CSNP concentrations. Immediately after treatment and after a 24-hour recovery period in culture medium, cell survival, and viability were measured. The association of CSNPs with IOBA-NHC cells was investigated by confocal microscopy. The influence of temperature and the effect of metabolic inhibition were studied by fluorometry. The in vivo uptake and acute tolerance of the ocular surface to CSNPs were evaluated in rabbits. RESULTS: Cell survival and viability of CSNP-exposed cells were equivalent to that of the control. Uptake of CSNPs was continuous for the 2-hour duration of these experiments and was temperature dependent. Metabolic inhibition by sodium azide had no effect on CSNP uptake. The rabbit ocular surface showed no signs of inflammation or alteration after CSNP exposure compared with the control. Fluorescence microscopy of rabbit eyeball and lid sections confirmed in vivo uptake by conjunctival and corneal epithelia. CONCLUSIONS: CSNPs were internalized by IOBA-NHC cells by an active transport mechanism that did not compromise cell viability. Moreover, these nanoparticles were well tolerated by the ocular surface tissues. These facts add further support for the potential use of these colloidal systems to delivery drugs to the ocular surface.

PURPOSE: To characterize a new nontransfected, spontaneously immortalized epithelial cell line from normal human conjunctiva (IOBA-NHC), both morphologically and functionally, to determine whether the differentiated phenotype of conjunctival epithelial cells is preserved. METHODS: Outgrowing cells from explanted conjunctival tissue were successively passaged and preliminarily characterized at passage 3 to assess epithelial origin. The cells were further characterized at passages 15 to 20, 40, 60, and 100 by analyzing (1) proliferation and in vitro behavior (viability, plating efficiency, colony forming efficiency and colony size, and Ki-67 protein expression), (2) karyotype and G-banding, (3) epithelial marker expression (cytokeratins, desmoplakins, EGF receptor), (4) absence of contaminating cell types, (5) expression of conjunctival differentiation markers (mucin gene expression), and (6) functional capability in response to proinflammatory stimuli. IOBA-NHC cells were analyzed by light and electron (transmission and scanning) microscopy, immunohistochemistry, electrophoresis and Western blot analysis, flow cytometry, and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: IOBA-NHC cells showed high proliferative ability in vitro and typical epithelial morphology. Cytokeratins and GalNAc, GluNAc, mannose, and sialic acid residues were immunodetected in these cells. No contaminating cell types were found. MUC1, -2, and -4, but not -5AC or -7 mucin genes were expressed in every cell passage tested. Exposure of cells to inflammatory mediators (IFNgamma and/or TNFalpha) resulted in increased expression of intercellular adhesion molecule (ICAM)-1 and HLA-DR. CONCLUSIONS: Morphologic and functional characterization of the nontransfected, spontaneously immortalized IOBA-NHC cell line shows that this new cell line may be a useful experimental tool in the field of ocular surface cell biology.