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.

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). 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. Fourteen out of the 15 molecules were reliably detected in 1 μl 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-1β, interferon gamma (IFN-γ), 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-α) 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. 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 develop a tear molecule level-based predictive model based on a panel of tear cytokines and their correlation with clinical features in ocular chronic graft versus host disease (cGVHD). Methods: Twenty-two ocular cGVHD patients and 21 healthy subjects were evaluated in a controlled environmental research laboratory (CERLab). Clinical parameters were recorded, and tears were collected. Levels of 15 molecules (epidermal growth factor [EGF], IL receptor antagonist [IL-1Ra], IL-1β, IL-2, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-17A, interferon inducible protein [IP]-10/CXCL10, IFN-γ, VEGF, TNF-α, eotaxin 1, and regulated on activation normal T cell expressed and secreted [RANTES]) were measured by multiplex-bead assay and correlated with clinical parameters. Logistic regression was used to develop a predictive model. Leave-one-out cross-validation was applied. Classification capacity was evaluated in a cohort of individuals with dry eye (DE) of other etiologies different from GVHD. Results: Epidermal growth factor and IP-10/CXCL10 levels were significantly decreased in ocular cGVHD, positively correlating with tear production and stability and negatively correlating with symptoms, hyperemia, and vital staining. Interleukin-1Ra, IL-8/CXCL8, and IL-10 were significantly increased in ocular cGVHD, and the first two correlated positively with symptoms, hyperemia, and ocular surface integrity while negatively correlating with tear production and stability. Predictive models were generated, and the best panel was based on IL-8/CXCL8 and IP-10/CXCL10 tear levels along with age and sex, with an area under the receiving operating curve of 0.9004, sensitivity of 86.36%, and specificity of 95.24%. Conclusions: A predictive model based on tear levels of IL-8/CXCL8 and IP-10/CXCL10 resulted in optimal sensitivity and specificity. These results add further knowledge to the search for potential biomarkers in this devastating ocular inflammatory disease.

Acta Ophthalmol. 2010: 88: e250–e258: Purpose:  There is growing evidence for the existence of an ‘immune tone’ in normal tears. The aim of this study was to determine the levels of a large panel of cytokines and chemokines in tears obtained from healthy subjects. These levels can then serve as baseline values for comparison with patients suffering from ocular surface diseases.Subjects and Methods:  Nine healthy subjects participated in this study, and normal ocular surface health was documented by the results of a dry eye questionnaire, Schirmer strip wetting, and vital staining of the cornea. Four microliters of tears were collected from each eye and analysed separately with multiplex bead‐based assays for the concentration of 30 cytokines and chemokines.Results:  Twenty‐five cytokines/chemokines were detected. CCL11/Eotaxin1, GM‐CSF, G‐CSF, IFN‐γ, IL‐2, IL‐3, IL‐4, IL‐5, IL‐10, IL‐13, IL‐12p70, IL‐15, CX3CL1/Fractalkine, TNF‐α, epidermal growth factor, and CCL4/MIP‐1β were present at 5–100 pg/ml. IL‐1β, IL‐6, IL‐7A, CXCL8/IL‐8, and CCL2/MCP‐1 were present at 100–400 pg/ml. IL‐1Ra, CXCL10/IP‐10 and vascular endothelial growth factor were present at more than 1000 pg/ml.Conclusion:  Multiplex bead‐based assays are convenient for cytokine/chemokine detection in tears. Fracktalkine has been detected in human healthy tears for the first time. The knowledge of cytokine/chemokine concentrations in tears from normal subjects is an important reference for further comparison with patients suffering from ocular surface diseases. Variability in their levels can reflect a phenomenon of potential importance for the understanding of the ocular surface cytokine pattern.