In Vivo Confocal Microscopy in Limbal Stem Cell Deficiency After Mesenchymal Stem Cell Transplantation: A Sub-analysis from a Phase I-II Clinical Trial.

INTRODUCTION: The aim of this work is to evaluate the effect of mesenchymal stem cell transplantation (MSCT) and cultivated limbal epithelial transplantation (CLET) therapies on the limbus of patients suffering from limbal stem cell deficiency (LSCD). METHODS: A sub-analysis of a phase I-II randomized, controlled, and double-masked clinical trial was performed to assess the changes in the anatomical structures of the limbus. In vivo confocal microscopy (IVCM) analysis was carried out in LSCD eyes before and 12 months after allogeneic MSCT or CLET. Epithelial phenotype of the central cornea, as well as the presence of transition zones and palisades of Vogt in the limbus, were assessed using Wilcoxon test. RESULTS: Twenty-three LSCD (14 MSCT and nine CLET) eyes were included. The epithelial phenotype of the central cornea improved significantly (p < 0.001) from 15 (eight MSCT, seven CLET) and eight (six MSCT, two CLET) LSCD eyes showing conjunctival and mixed phenotypes, respectively, to eight (five MSCT, three CLET), five (two MSCT, three CLET), and ten (seven MSCT, three CLET) eyes showing conjunctival, mixed, and corneal phenotypes, respectively. Transition areas and palisades of Vogt were observed in at least one quadrant in nine (five MSCT, four CLET) and 16 (nine MSCT, seven CLET), and in four (two MSCT, two CLET) and six (three MSCT, three CLET) LSCD eyes before and after surgery, respectively. Changes in the transition zones and palisades were solely significant (p = 0.046) for the nasal and inferior quadrants, respectively. CONCLUSIONS: MSCT and CLET improved the central corneal epithelial phenotype despite only minor changes in the anatomical structures of the limbus, as detected by IVCM technology. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT01562002.

Corneal Regeneration Using Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived stem cells are a subtype of mesenchymal stem cell that offers the important advantage of being easily obtained (in an autologous manner) from low invasive procedures, rendering a high number of multipotent stem cells with the potential to differentiate into several cellular lineages, to show immunomodulatory properties, and to promote tissue regeneration by a paracrine action through the secretion of extracellular vesicles containing trophic factors. This secretome is currently being investigated as a potential source for a cell-free based regenerative therapy for human tissues, which would significantly reduce the involved costs, risks and law regulations, allowing for a broader application in real clinical practice. In the current article, we will review the existing preclinical and human clinical evidence regarding the use of such adipose-derived mesenchymal stem cells for the regeneration of the three main layers of the human cornea: the epithelium (derived from the surface ectoderm), the stroma (derived from the neural crest mesenchyme), and the endothelium (derived from the neural crest cells).

Phenotypic characterization of patients developing chronic dry eye and pain after refractive surgery: A cross-sectional study.

PURPOSE: To describe the clinical characteristics of patients suffering from chronic dry eye (DE) and pain after refractive surgery (RS). METHODS: Cross-sectional, observational, single-visit study. DE-, pain- and psychological-related symptoms were evaluated with specific questionnaires. DE-related tests evaluated tear osmolarity, conjunctival hyperemia, Meibomian gland dysfunction, tear stability and production, and ocular surface staining. Corneal mechanical sensitivity (Cochet-Bonnet) was measured pre/post topical anesthesia, and symptomatic variation post-anesthesia (anesthetic challenge test) was recorded. When pain was present, it was further categorized as neuropathic or nociceptive based on published criteria. RESULTS: We recruited 104 patients (39.5 ± 9.5 years). Most, 85.6%, had corneal RS as opposed to intraocular RS. Migraines, anxiety, depression (p < 0.0001), and central sensitization syndromes (p = 0.0214) were more frequent post-RS than pre-RS. Persistent DE-symptoms, severe in 86.5% patients, developed in a range of 0-204 months post-RS. Dryness and pain were the two most frequent symptoms. The only DE-related tests showing abnormal values were tear osmolarity (315.2 ± 17.1 mOsm/L; normal ≤308) and tear break-up time (4.1 ± 2.5 s; normal >7). Corneal sensitivity was 55.4 ± 7.0 mm, and decreased (p < 0.0001) after topical anesthesia, 6.0 ± 10.4 mm. However, it remained pathologically elevated, ≥10 mm in 61 (58.7%) patients. The normal symptomatic post-anesthesia improvement was absent in 58 (55.7%) patients. Ocular pain was present in 82 (78.8%) patients, and it was categorized as neuropathic in 66 (80.5%) of them, 63.5% of the entire cohort. CONCLUSIONS: Chronic ocular pain and its neuropathic subtype were diagnosed in 78.8% and 63.5% respectively of patients seeking consultation for persistent symptomatic DE post-RS.

Inflammation-related molecules in tears of patients with chronic ocular pain and dry eye disease.

The purpose of this study was to analyze inflammation- and pain-related molecules in tears of patients suffering from chronic ocular pain associated with dry eye (DE) and/or a previous corneal refractive surgery (RS). Based on history, symptomatology, and clinical signs, the subjects (n = 180, 51.0 ± 14.7 years, 118 females, 62 males) in this cross-sectional study were assigned to one of five groups: DE and chronic ocular pain after RS (P/DE-RS, n = 52); asymptomatic subjects, i.e., without DE and chronic ocular pain, after RS (A-RS, n = 30); DE and chronic ocular pain without previous RS (P/DE-nonRS, n = 31); DE, no pain, and no previous RS (DE-nonRS, n = 35); and asymptomatic subjects with no previous RS (controls, n = 32). The tear concentrations of 20 cytokines and substance P (SP) were analyzed by immunobead-based assay and enzyme-linked immunosorbent assay, respectively. We found that tear levels of interleukin (IL)-10 and SP were increased in the RS groups. There were significant differences in IL-8/CXCL8 among the five groups. Nerve growth factor (NGF) tear levels were significantly higher in P/DE-RS than in DE-nonRS and controls. IL-9 had the highest percentage of detection in the P/DE-RS and P/DE-nonRS groups, while macrophage inflammatory protein (MIP)-1α, IL-2, and interferon (IFN)-γ were higher in the P/DE-RS, A-RS, and P/DE-nonRS groups. IL-17A was detected only in the A-RS group. Moderate correlations were observed in the A-RS, P/DE-nonRS, DE-nonRS and controls groups. A positive correlation was obtained between growth related oncogene concentration and tear break-up time (rho = 0.550; p = 0.012), while negative correlation was found between monocyte chemoattractant protein-3/CCL7 and conjunctival staining (rho = -0.560; p = 0.001), both in the A-RS group. IL-10 correlated positively with ocular pain intensity (rho = 0.513; p = 0.003) in the P/DE-nonRS group. Regulated on Activation Normal T Cell Expressed and Secreted/CCL5 correlated negatively with conjunctival staining (rho = -0.545; p = 0.001) in the DE-nonRS group. SP correlated negatively with corneal staining (rho = -0.559; p = 0.001) in the controls. In conclusion, chronic ocular pain was associated with higher IL-9 tear levels. IL-10, SP, MIP-1α/CCL3, IL-2, and IFN-γ were associated with previous RS. Higher levels of IL-8/CXCL8, MIP-1α/CCL3, IL-2, and IFN-γ were associated with DE-related inflammation, while NGF levels were related to chronic ocular pain and DE in RS patients. These findings suggest that improved knowledge of tear cytokines and neuromodulators will lead to a more nuanced understanding of how these molecules can serve as biomarkers of chronic ocular pain, leading to better therapeutic and disease management decisions.

Epithelial component and intraepithelial lymphocytes of conjunctiva-associated lymphoid tissue in healthy children.

Conjunctiva-associated lymphoid tissue (CALT) plays a key role in protecting the eye surface by initiating and regulating immune responses. The aim of this study was to investigate in healthy children the proportion of intraepithelial lymphocytes (IELs), the degree of viability and/or apoptosis and cell proliferation in three different topographic areas of the conjunctiva. Superior tarsal, superior bulbar, and inferior tarsal-bulbarfornix conjunctival cells were collected by brush cytology (BC) from 24 healthy paediatric subjects (13 boys and 11 girls, mean age 6±2 years) who were to undergo strabismus correction surgery under general anaesthesia. Subsequently, these cells were analysed phenotypically and functionally by flow cytometry (FC). Flow cytometry analysis showed that not all the cells obtained by BC were of the epithelial lineage, but that there was a population of CD45+ cells (IELs) regularly present in the conjunctiva of healthy children. These IELs were mostly T-lymphocytes (CD3+) and B-lymphocytes (CD19+), with higher levels of T-lymphocytes (CD3+) in the upper areas than in the inferior tarsal-bulbar-fornix, where the highest levels of B-lymphocytes (CD19+) were found. In the apoptosis assay, two groups of cell populations were differentiated by cell size and complexity (cytoplasmic granularity), with more complex cells predominating in the upper areas of the conjunctiva and less complex cells being more abundant in the inferior tarsal-bulbar-fornix. Finally, the proliferative capacity of the conjunctival epithelium was significantly higher in the upper tarsal zone than in the rest of the zones analysed. These results suggest that the epithelial component and the IELs of CALT are also regularly present in the conjunctiva of the healthy child, varying in phenotype, viability and cell proliferation according to the different conjunctival regions analysed, which could lead us to believe that each conjunctival zone plays a different, specific role in the regulation of the immune response at the ocular level.

Goals and Challenges of Stem Cell-Based Therapy for Corneal Blindness Due to Limbal Deficiency.

Corneal failure is a highly prevalent cause of blindness. One special cause of corneal failure occurs due to malfunction or destruction of the limbal stem cell niche, upon which the superficial cornea depends for homeostatic maintenance and wound healing. Failure of the limbal niche is referred to as limbal stem cell deficiency. As the corneal epithelial stem cell niche is easily accessible, limbal stem cell-based therapy and regenerative medicine applied to the ocular surface are among the most highly advanced forms of this novel approach to disease therapy. However, the challenges are still great, including the development of cell-based products and understanding how they work in the patient's eye. Advances are being made at the molecular, cellular, and tissue levels to alter disease processes and to reduce or eliminate blindness. Efforts must be coordinated from the most basic research to the most clinically oriented projects so that cell-based therapies can become an integrated part of the therapeutic armamentarium to fight corneal blindness. We undoubtedly are progressing along the right path because cell-based therapy for eye diseases is one of the most successful examples of global regenerative medicine.

Subconjunctival injection of mesenchymal stem cells for corneal failure due to limbal stem cell deficiency: state of the art.

Mesenchymal stem cells (MSCs) have unique and beneficial properties and are currently used to treat a broad variety of diseases. These properties include the potential for differentiation into other cell types, secretion of different trophic factors that promote a regenerative microenvironment, anti-inflammatory actions, selective migration to damaged tissues, and non-immunogenicity. MSCs are effective for the treatment of ocular surface diseases such as dry eye, corneal burns, and limbal stem cell deficiency (LSCD), both in experimental models and in humans. LSCD is a pathological condition in which damage occurs to the limbal epithelial stem cells, or their niche, that are responsible for the continuous regeneration of the corneal epithelium. If LSCD is extensive and/or severe, it usually causes corneal epithelial defects, ulceration, and conjunctival overgrowth of the cornea. These changes can result in neovascularization and corneal opacity, severe inflammation, pain, and visual loss. The effectiveness of MSCs to reduce corneal opacity, neovascularization, and inflammation has been widely studied in different experimental models of LSCD and in some clinical trials; however, the methodological disparity used in the different studies makes it hard to compare outcomes among them. In this regard, the MSC route of administration used to treat LSCD and other ocular surface diseases is an important factor. It should be efficient, minimally invasive, and safe. So far, intravenous and intraperitoneal injections, topical administration, and MSC transplantation using carrier substrata like amniotic membrane (AM), fibrin, or synthetic biopolymers have been the most commonly used administration routes in experimental models. However, systemic administration carries the risk of potential side effects and transplantation requires surgical procedures that could complicate the process. Alternatively, subconjunctival injection is a minimally invasive and straightforward technique frequently used in ophthalmology. It enables performance of local treatments using high cell doses. In this review, we provide an overview of the current status of MSC administration by subconjunctival injection, analyzing the convenience, safety, and efficacy for treatment of corneal failure due to LSCD in different experimental models. We also provide a summary of the clinical trials that have been completed, are in progress, or being planned.

Optimization of Human Limbal Stem Cell Culture by Replating a Single Limbal Explant.

Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.

Clinical and tear cytokine profiles after advanced surface ablation refractive surgery: A six-month follow-up.

Neuropathic dry eye is one of the most frequently seen complications after corneal refractive surgery, however, its incidence decreases in a significant manner along the first six months postoperative, reaching between 10 and 45% incidence. However, little is known on the inflammatory status of the ocular surface during this recovery process. We aim to analyze the clinical and tear molecule concentration changes along six months after advanced surface ablation for myopia correction, in a prospective study including 18 eyes of 18 subjects who bilaterally underwent advanced surface ablation corneal refractive surgery. Clinical variables (uncorrected distance visual acuity, symptoms, conjunctival hyperemia, tear osmolarity, tear stability, corneal fluorescein staining, conjunctival lissamine staining, Schirmer test, and corneal esthesiometry) and a panel of 23 pro and anti-inflammatory cytokines/chemokines concentration in tears preoperatively and at 1, 3 and 6 months postoperatively were evaluated. We found that uncorrected distance visual acuity improved significantly from baseline at 1-month visit, symptoms improved and tear osmolarity decreased significantly from baseline at 3-month visit and there was a decrease in mechanical corneal threshold between 1-month and 3- and 6-month visits. Regarding tear molecules, IL-4, IL-5, IL-6, IL-13, IL-17A, and IFN-γ tear levels were significantly increased at all the three visits, compared to preoperative levels at V0; IL-2 and VEGF were also significantly increased at 1-month and 6-month visits, but not at 3-month visit, whereas IL-9 IL-10 and IL-12 were only significantly increased at 6-month visit. Although we found that there is a recovery in clinical variables at 6 months postoperatively (i.e. neuropathic dry eye was not developed in the sample), ocular surface homeostasis is not completely restored, as it can be seen by the changes in concentration of some pro and anti-inflammatory molecules measured in tears.

Poly-l/dl-lactic acid films functionalized with collagen IV as carrier substrata for corneal epithelial stem cells.

Limbal epithelial stem cells (LESCs) are responsible for the renewal of corneal epithelium. Cultivated limbal epithelial transplantation is the current treatment of choice for restoring the loss or dysfunction of LESCs. To perform this procedure, a substratum is necessary for in vitro culturing of limbal epithelial cells and their subsequent transplantation onto the ocular surface. In this work, we evaluated poly-L/DL-lactic acid 70:30 (PLA) films functionalized with type IV collagen (col IV) as potential in vitro carrier substrata for LESCs. We first demonstrated that PLA-col IV films were biocompatible and suitable for the proliferation of human corneal epithelial cells. Subsequently, limbal epithelial cell suspensions, isolated from human limbal rings, were cultivated using culture medium that did not contain animal components. The cells adhered significantly faster to PLA-col IV films than to tissue culture plastic (TCP). The mRNA expression levels for the LESC specific markers, K15, P63α and ABCG2 were similar or greater (significantly in the case of K15) in limbal epithelial cells cultured on PLA-col IV films than limbal epithelial cells cultured on TCP. The percentage of cells expressing the corneal (K3, K12) and the LESC (P63α, ABCG2) specific markers was similar for both substrata. These results suggest that the PLA-col IV films promoted LESC attachment and helped to maintain their undifferentiated stem cell phenotype. Consequently, these substrata offer an alternative for the transplantation of limbal cells onto the ocular surface.

A proof-of-concept clinical trial using mesenchymal stem cells for the treatment of corneal epithelial stem cell deficiency.

Ocular stem cell transplantation derived from either autologous or allogeneic donor corneoscleral junction is a functional cell therapy to manage extensive and/or severe limbal stem cell deficiencies that lead to corneal epithelial failure. Mesenchymal stem cells have been properly tested in animal models of this ophthalmic pathology, but never in human eyes despite their potential advantages. We conducted a 6- to 12-month proof-of-concept, randomized, and double-masked pilot trial to test whether allogeneic bone marrow-derived mesenchymal stem cell transplantation (MSCT], n = 17) was as safe and as equally efficient as allogeneic cultivated limbal epithelial transplantation (CLET), (n = 11) to improve corneal epithelial damage due to limbal stem cell deficiency. Primary endpoints demanded combination of symptoms, signs, and the objective improvement of the epithelial phenotype in central cornea by in vivo confocal microscopy. This proof-of-concept trial showed that MSCT was as safe and efficacious as CLET. Global success at 6-12 months was 72.7%-77.8% for CLET cases and 76.5%-85.7% for MSCT cases (not significant differences). Central corneal epithelial phenotype improved in 71.4% and 66.7% of MSCT and CLET cases, respectively at 12 months (P = 1.000). There were no adverse events related to cell products. This trial suggests first evidence that MSCT facilitated improvement of a diseased corneal epithelium due to lack of its stem cells as efficiently as CLET. Consequently, not only CLET but also MSCT deserves more preclinical investigational resources before the favorable results of this proof-of-concept trial could be transformed into the larger numbers of the multicenter trials that would provide stronger evidence. (ClinicalTrials.gov number, NCT01562002.).

Prehematopoietic Stem Cell Transplantation Tear Cytokines as Potential Susceptibility Biomarkers for Ocular Chronic Graft-Versus-Host Disease.

Purpose: To determine if cytokine tear levels before hematopoietic stem cell transplantation (HSCT) can help anticipate the occurrence of ocular chronic graft-versus-host disease (cGVHD). Methods: In this pilot study, 25 patients undergoing HSCT were followed prospectively for ≤43 months. After ocular examinations, tears were collected before HSCT. Levels of 19 cytokines (epidermal growth factor [EGF], eotaxin 1/CCL11, fractalkine/CX3CL1, IL-1Ra, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-13, IL-17A, IP-10/CXCL10, IFN-γ, VEGF, TNF-α, and RANTES/CCL5) were measured by multiplex bead assay. A multistate model (MSM) based on four states (HSCT, systemic cGVHD, ocular cGVHD, and death) was developed to identify cytokines associated with each transition probability. Molecules included in the final multivariable model were selected by a supervised principal components analysis. Bootstrap resampling internally validated the final MSM. Model discriminatory ability was determined by time-dependent receiver operating characteristic curves and the corresponding area under the curve (AUC). Results: The final model, based on fractalkine, IL-1Ra, and IL-6 tear levels, accurately influenced the transition between the four different states. The AUC for this model, based on a new variable built upon the combination of these three molecules, was 67% to 80% throughout follow-up and, thus, had good discriminatory ability. Conclusions: In this prospective study, a model based on pre-HSCT tear levels of the inflammatory molecules fractalkine, IL-1Ra, and IL-6 had good prognostic ability for the development of ocular cGVHD after HSCT. These cytokines potentially could act as susceptibility biomarkers for the development of this disease after HSCT.

Therapeutic Effect of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Experimental Corneal Failure Due to Limbal Stem Cell Niche Damage.

Limbal stem cells are responsible for the continuous renewal of the corneal epithelium. The destruction or dysfunction of these stem cells or their niche induces limbal stem cell deficiency (LSCD) leading to visual loss, chronic pain, and inflammation of the ocular surface. To restore the ocular surface in cases of bilateral LSCD, an extraocular source of stem cells is needed to avoid dependence on allogeneic limbal stem cells that are difficult to obtain, isolate, and culture. The aim of this work was to test the tolerance and the efficacy of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) to regenerate the ocular surface in two experimental models of LSCD that closely resemble different severity grades of the human pathology. hAT-MSCs transplanted to the ocular surface of the partial and total LSCD models developed in rabbits were well tolerated, migrated to inflamed tissues, reduced inflammation, and restrained the evolution of corneal neovascularization and corneal opacity. The expression profile of the corneal epithelial cell markers CK3 and E-cadherin, and the limbal epithelial cell markers CK15 and p63 was lost in the LSCD models, but was partially recovered after hAT-MSC transplantation. For the first time, we demonstrated that hAT-MSCs improve corneal and limbal epithelial phenotypes in animal LSCD models. These results support the potential use of hAT-MSCs as a novel treatment of ocular surface failure due to LSCD. hAT-MSCs represent an available, non-immunogenic source of stem cells that may provide therapeutic benefits in addition to reduce health care expenses. Stem Cells 2017;35:2160-2174.

Clinical Features and Prognostic Factors in Presumed Ocular Tuberculosis.

PURPOSE: To characterize the clinical features in patients with presumed ocular tuberculosis (TB) and determine prognostic factors of visual outcomes and complications in this disease. MATERIAL AND METHODS: Retrospective case series of 35 patients (29 females, 6 males) with presumed ocular TB from referral centers in Chile and Spain between 2002 and 2012. Medical records were reviewed, and data regarding clinical features, complications, best-corrected visual acuity (BCVA), duration of disease, extraocular manifestations, and therapy were retrieved. Prognostic factors for low vision (BCVA 20/50 or less), legal blindness (BCVA 20/200 or less), and complications (cataract, glaucoma, and macular lesion) were evaluated. To calculate correlations, we used Spearman's rank correlation test. To determine clinical predictors, we used the binary logistic regression test. RESULTS: Anterior and non-granulomatous uveitis was the most common types of inflammation. Only 2 (5.7%) patients had respiratory symptoms, and 6 (17.1%) patients had an abnormal chest X-ray at diagnosis. All patients received combined antitubercular therapy with a mean duration of 6.9 ± 2.3 months. A longer duration of symptoms at diagnosis was associated with both low vision and legal blindness. Older patients had a higher risk of legal blindness. A longer duration of symptoms as well as anterior inflammation demonstrated an increased risk for cataract formation. The duration of the symptoms and baseline BCVA had a positive correlation with the final BCVA. Prognostic factors of macular lesions were not found. CONCLUSIONS: The diagnosis of ocular TB can be difficult due to the lack of extraocular manifestations and the broad spectrum of ocular features. A longer duration of symptoms at diagnosis was associated with poorer visual outcomes and cataracts. Therefore, efforts should be made to avoid a delay in the diagnosis of ocular TB and to identify prognostic factors for visual outcomes and complications.

Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions.

PURPOSE: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. MATERIALS AND METHODS: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. RESULTS: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. CONCLUSION: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.

Cytokine and chemokine tear levels in patients with uveitis.

PURPOSE: To determine whether the levels of cytokines and chemokines in tears differ in uveitis patients and healthy subjects. METHODS: Ninety-two uveitis patients (mean age 46.4 years) and 157 control healthy subjects (mean age 49.5 years) were recruited. Subjects with ocular surface diseases such as dry eye were excluded from the study. Using multiplex bead-based assays, tears (4 µl) were analysed for the concentration of interleukin (IL)-1ß, IL-1RA, IL-2, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12p70, IL-15, IL-17A, IL-23, epidermal growth factor (EGF), fractalkine/CX3CL1, interferon-γ, IP-10/CXCL10, monocyte chemo-attractant protein (MCP)-1/CCL2, tumour necrosis factor-α, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-ß1, TGF-ß2 and TGF-ß3. Tear molecule levels were compared between the groups and among the different forms of uveitis and disease severity. RESULTS: Epidermal growth factor, IL-1RA, IL-7, IL-8/CXCL8, IP-10/CXCL10, MCP-1/CCL2, TGF-ß2 and VEGF were detected in more than 75% of the samples in both groups. Statistically significant differences in percentage of detection between control and patient groups were found for IL-23, IL-1ß, IL-15, EGF, fractalkine/CX3CL1 and MCP-1/CCL2. The concentrations of IL-1RA, IL-8/CXCL8, fractalkine/CX3CL1, IP-10/CXCL10, VEGF and TGF-ß2 in uveitis tear samples were elevated compared to controls (p < 0.05). Significant differences in tear levels of those molecules and also EGF were also present depending on the anatomic classification of uveitis. CONCLUSION: There were significant differences in the levels of several cytokines and chemokines in tears of patients with uveitis compared with healthy subjects. These results can help understand the underlying pathophysiology of the uveitis and could potentially aid in diagnosis.

Effect of Environmental Conditions on the Concentration of Tear Inflammatory Mediators During Contact Lens Wear.

PURPOSE: To analyze the influence of environmental conditions on the concentrations of tear inflammatory mediators during contact lens (CL) wear. METHODS: Fifty-four CL wearers completed 4 visits combining the bilateral use of omafilcon A or comfilcon A CL and a 90-minute exposure to 2 environmental conditions: standard [50% relative humidity (RH), 23°C, 930 mb] or adverse (5% RH, localized air flow, 23°C, 750 mb). Four microliters of tears was collected by capillarity from each subject. Changes in concentration of epidermal growth factor (EGF); interleukin (IL)-1 receptor antagonist, IL-1ß, IL-2, IL-4, IL-6, and IL-8; tumor necrosis factor (TNF) α; monocyte chemoattractant protein-1; and matrix metalloproteinase (MMP)-9 were analyzed. The effects of the environment, CL type, and symptoms were evaluated using a 3-way mixed analysis of variance with repeated measures. RESULTS: Under the standard condition, EGF significantly increased [0.36; 95% confidence interval (CI), 0.08 to 0.64], and IL-1ß (-0.48; 95% CI, -0.84 to -0.12) and IL-2 (-0.48; 95% CI, -0.87 to -0.09) significantly decreased. Under the adverse condition, IL-6 significantly increased (0.35; 95% CI, 0.09 to 0.62). Comparing conditions, EGF change was significantly lower (P = 0.02) and IL-1ß, IL-2, IL-6, and TNF-α changes were significantly higher (P ≤ 0.04) under the adverse condition. Additionally, IL-1ß significantly decreased with comfilcon A (-0.51; 95% CI, -0.88 to -0.15), being significantly lower (P = 0.01) than the change with omafilcon A (0.06; 95% CI, -0.23 to 0.35). CONCLUSIONS: The secretion of several tear inflammatory mediators during CL wear differs depending on the environmental conditions and the CL type used. These outcomes might help to understand the effect of the environment and CL materials on the ocular surface of CL wearers.

Biomarkers in Ocular Chronic Graft Versus Host Disease: Tear Cytokine- and Chemokine-Based Predictive Model.

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-1ß, 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.

Clinical and Molecular Inflammatory Response in Sjögren Syndrome-Associated Dry Eye Patients Under Desiccating Stress.

PURPOSE: To evaluate the response of the lacrimal function unit in Sjögren syndrome (SS)-associated dry eye patients exposed to 2 simulated daily life environmental conditions. DESIGN: Prospective crossover pilot study. METHODS: Fourteen female SS dry eye patients were exposed for 2 hours to a controlled normal condition (23 C, 45% relative humidity, and air flow 0.10 m/s) and a controlled adverse condition that simulates desiccating stress (23 C, 5% relative humidity, and air flow 0.10 m/s). The following dry eye tests were performed before and after the exposure: tear osmolarity, phenol red thread test, conjunctival hyperemia, fluorescein tear break-up time, corneal fluorescein staining, conjunctival lissamine green staining, and Schirmer test. Levels of 16 molecules were analyzed in tears by multiplex immunobead analysis. RESULTS: Clinical evaluation showed lacrimal functional unit impairment after the desiccating stress: significantly increased tear osmolarity (315.7 ± 3.0 vs 327.7 ± 5.1 mOsm/L, P = .03), conjunctival hyperemia (1.3 ± 0.1 vs 1.6 ± 0.1, P = .05), and corneal staining in temporal (3.5 ± 0.5 vs 4.7 ± 0.4, P = .01) and nasal (3.6 ± 0.5 vs 4.5 ± 0.5, P = .04) areas. Tear concentrations increased for interleukin-1 receptor antagonist (16 557.1 ± 4047.8 vs 31 895.3 ± 5916.5 pg/mL, P = .01), interleukin-6 (63.8 ± 20.2 vs 111.5 ± 29.6 pg/mL, P = .02), interleukin-8 (2196.1 ± 737.9 vs 3753.2 ± 1106.0 pg/mL, P = .03), and matrix metalloproteinase-9 (101 515.6 ± 37 088.4 vs 145 867.1 ± 41 651.5 pg/mL, P = .03). After the simulated normal condition, only a significant increase in nasal corneal staining (2.9 ± 0.5 vs 3.6 ± 0.5, P = .03) was observed. CONCLUSIONS: Even a short exposure to a desiccating environment can produce a significant deterioration of the lacrimal function unit in female SS dry eye patients. The often unnoticed exposure to these conditions during daily life may increase inflammatory activity rapidly, triggering an ocular surface deterioration.

Corneal Sensitivity and Inflammatory Biomarkers in Contact Lens Discomfort.

PURPOSE: The aim of this study was to analyze whether symptoms of discomfort in hydrogel contact lens (HCL) wearers were associated with changes in corneal sensitivity or levels of tear inflammatory mediators. METHODS: Sixty-six subjects were included: 47 HCL wearers, further divided into 24 symptomatic and 23 asymptomatic wearers by the Contact Lens Dry Eye Questionnaire short form and 19 non-contact lens wearers. At least 24 h after HCL removal, we obtained scores from the Ocular Surface Disease Index and mechanical, hot, and cold corneal thresholds using a Belmonte esthesiometer. We collected 4 µl of tears with a capillary micropipette and measured levels of 12 inflammatory markers using a bead-based array: epidermal growth factor, fractalkine, interleukin-10 (IL-10), IL-1ß, IL-1 receptor antagonist, IL-2, IL-4, IL-6, IL-8, monocyte chemoattractant protein 1, tumor necrosis factor-α, and matrix metalloproteinase 9. RESULTS: There were no significant differences between groups in corneal sensitivity thresholds and levels of tear molecules. The following significant correlations were found in the total sample pooled: Ocular Surface Disease Index correlated with mechanical threshold (p < 0.01; rho = -0.324) and epidermal growth factor (p < 0.01, rho = -0.330), and mechanical threshold correlated with heat threshold (p < 0.01, rho = -0.321). CONCLUSIONS: Twenty-four hours after HCL removal, symptoms of discomfort in HCL wearers are not related to changes in corneal sensitivity or tear inflammatory mediator levels. This might indicate either that HCL wear has no effect on these parameters or that ocular surfaces recover from HCL-caused effects during the first hours after HCL removal. However, there were correlations for associations between symptoms, corneal sensitivity, and some molecules in tears.