Cintia S. De Paiva

Characterization of putative stem cell phenotype in human limbal epithelia.

This study evaluated proposed molecular markers related to stem cell (SC) properties with the intention of characterizing a putative SC phenotype in human limbal epithelia. Human corneal and limbal tissues were cut in the vertical and horizontal meridians for histology, transmission electron microscopy (TEM), and immunostaining. Semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) and in situ hybridization were used to evaluate gene expression. TEM showed that the limbal basal cells were small primitive cells. Immunostaining disclosed that p63, ABCG2 and integrin α9 were primarily expressed by the basal epithelial cells of limbus. Antibodies against integrin β1, epidermal growth factor receptor (EGFR), K19, enolase‐α, and CD71 stained the basal cells of the limbus more brightly than the suprabasal epithelia. Integrin α6, nestin, E‐cadherin and connexin 43 did not stain the limbal basal cells, but the suprabasal epithelia of the cornea and limbus showed strong immunoreactivity. K3 and involucrin stained only corneal and limbal superficial cells. RT‐PCR showed higher levels of p63, ABCG2 and integrin α9 mRNA, but lower levels of K3, K12 and connexin 43 expressed in the limbal epithelia than the corneal epithelia. In situ hybridization showed that p63 transcripts were located in basal layer of the limbal epithelium. This work suggests that the basal epithelial cells of the limbus are p63, ABCG2 and integrin α9 positive, and nestin, E‐cadherin, connexin 43, involucrin, K3, and K12 negative, with relatively higher expression of integrin β1, EGFR, K19, and enolase‐α. This putative SC phenotype may facilitate the identification and isolation of limbal epithelial SCs.

Tear Cytokine Profiles in Dysfunctional Tear Syndrome

PURPOSE To compare tear cytokine and chemokine concentrations in asymptomatic control and Dysfunctional Tear syndrome (DTS) patients and determine the correlations between tear inflammatory mediators and clinical severity. DESIGN Prospective observational cohort study. METHODS Concentrations of epidermal growth factor (EGF), interleukin (IL)-1 alpha (1alpha), 1 beta (1beta), 6, 10, 12, and 13, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and chemokines: IL-8 (CXC); macrophage inflammatory protein-1 alpha (MIP-1alpha) (CCL3); and regulated upon activation, normal T-cell expressed and secreted (RANTES CCL5) were measured by a multiplex immunobead assay in an asymptomatic control group and DTS patients with and without meibomian gland disease (MGD). Spearman correlations between tear cytokines and severity of irritation symptoms and ocular surface signs were calculated. RESULTS Tear concentrations of IL-6, IL-8 and TNF-alpha were significantly higher in DTS with and without MGD and EGF was significantly reduced in the DTS without MGD group compared with the control group. MIP-1alpha was greater in entire DTS and DTS without MGD groups than the control group and RANTES was greater in DTS with MGD than the control and DTS without MGD groups. IL-12 was significantly higher in the DTS with MGD than the DTS without MGD subgroup. Significant correlations were observed between IL-6 and irritation symptoms and between a number of cytokines and chemokines and clinical parameters. CONCLUSIONS As predicted, patients with DTS have higher levels of inflammatory mediators in their tears that show correlation with clinical disease parameters. Furthermore, different tear cytokine/chemokine profiles were observed in DTS patients with and without MGD groups.

ABCG2 Transporter Identifies a Population of Clonogenic Human Limbal Epithelial Cells

ABCG2, a member of the ATP binding cassette (ABC) transporters, has been identified as a molecular determinant for bone marrow stem cells and proposed as a universal marker for stem cells. This study investigates ABCG2 expression and its potential as a marker that identifies human limbal epithelial stem cells. ABCG2 expression was evaluated by immunofluorescent and immunohistochemical staining, laser scanning confocal microscopy, flow cytometry, and semiquantitative reverse transcription–polymerase chain reaction. Cells selected from primary limbal epithelial cultures by flow cytometry with ABCG2 monoclonal antibody (mAb) or Hoechst 33342 dye staining were evaluated for their gene expression and colony‐forming efficiency (CFE). ABCG2 protein was mainly located in the basal cells of limbal epithelia but not in the limbal suprabasal and corneal epithelia. ABCG2 staining was also observed in primary limbal epithelial cultures. Limbal epithelia express higher levels of ABCG2 and ΔNp63 mRNAs than corneal epithelia. Labeling with ABCG2 mAb yielded 2.5%–3.0% positive cells by flow cytometry. The ABCG2‐positive cells exhibited greater CFE on a 3T3 fibroblast feeder layer than ABCG2‐negative cells. A side population (SP) was detected by the Hoechst 33342 exclusion assay. SP cells displayed stronger expression of ABCG2 and ΔNp63 mRNA and greater CFE than the non‐SP cells. In conclusion, these findings demonstrate that ABCG2 transporter was exclusively expressed by limbal basal cells and that the ABCG2‐positive and SP cells possess enriched stem cell properties, suggesting for the first time that ABCG2 could serve as a marker to identify the putative limbal epithelial stem cells.

Desiccating Stress Induces T Cell-Mediated Sjögren’s Syndrome-Like Lacrimal Keratoconjunctivitis

Chronic dry eye syndrome affects over 10 million people in the United States; it is associated with inflammation of the lacrimal gland (LG) and in some cases involves T cell infiltration of the conjunctiva. We demonstrate that environmental desiccating stress (DS) elicits T cell-mediated inflammation of the cornea, conjunctiva, and LG, but not other organs in mice. The lacrimal keratoconjunctivitis (LKC) was mediated by CD4+ T cells, which, when adoptively transferred to T cell-deficient nude mice, produced inflammation in the LG, cornea, and conjunctiva, but not in any other organ. Adoptively transferred CD4+ T cells produced LKC even though recipients were not exposed to DS. LKC was exacerbated in euthymic mice depleted of CD4+CD25+forkhead/winged helix transcription factor+ regulatory T cells. The results suggest that DS exposes shared epitopes in the cornea, conjunctiva, and LG that induce pathogenic CD4+ T cells that produce LKC, which under normal circumstances is restrained by CD4+CD25+forkhead/winged helix transcription factor+ regulatory T cells.

Matrix Metalloproteinase-9 Knockout Confers Resistance to Corneal Epithelial Barrier Disruption in Experimental Dry Eye

Altered corneal epithelial barrier function is the cause for ocular irritation and visual morbidity in dry eye disease. Increased matrix metalloproteinase (MMP)-9 activity has been observed in the tear fluid of dry eye patients. To determine the pathogenic role of MMP-9 in the corneal epithelial disease of dry eye, the effects of experimentally induced dry eye on corneal epithelial morphology and barrier function were compared in MMP-9 knockout mice and their wild-type littermates. Dry eye was created through cholinergic blockade and exposure to a desiccating environment. The tear fluid MMP-9 concentration increased in response to dryness in wild-type mice. Corneal epithelial permeability to three different-sized molecules increased in dry eye wild-type mice, but not in MMP-9 knockout mice. Topical administration of active MMP-9 to dry eye MMP-9 knockout mice significantly increased corneal epithelial permeability. Compared to MMP-9 knockout mice, wild-type mice showed greater desquamation of differentiated apical corneal epithelial cells that expressed the tight junction protein occludin in response to dryness. This was accompanied by an increase in lower sized (50 kd) occludin in the corneal epithelia of wild-type mice. These findings could be replicated in cultured human corneal epithelial cells that were treated with active MMP-9. These studies indicate that increased MMP-9 activity on the ocular surface in response to dryness disrupts corneal epithelial barrier function. This appears to be because of accelerated loss of tight junction bearing superficial corneal epithelial cells, perhaps by proteolytic cleavage of occludin.

Production and Activity of Matrix Metalloproteinase-9 on the Ocular Surface Increase in Dysfunctional Tear Syndrome

PURPOSE To evaluate production and activity of metalloproteinase (MMP)-9 on the ocular surface of patients with dysfunctional tear syndrome (DTS) and determine any correlation between MMP-9 activity and clinical parameters. METHODS Forty-six patients with newly diagnosed DTS and 18 control subjects were recruited. Complete ocular surface examinations were performed. Tear MMP-9 activity was assessed with an MMP-9 activity assay in 1 microL of unstimulated tear fluid. Using conjunctival epithelial cells from 19 patients with DTS and 16 controls, levels of MMP-9 and its regulating cytokine mRNA transcripts were evaluated by semiquantitative real-time PCR. RESULTS Each of four DTS severity-based groups had significantly higher mean MMP-9 activities than did the control group, which was 8.39 +/- 4.70 ng/mL. The DTS4 group had the highest MMP-9 activity (381.24 +/- 142.83 ng/mL), for which the mean was significantly higher than that of other DTS groups. In addition, patients with DTS had significantly higher levels of IL-1beta, IL-6, TNF-alpha, and TGF-beta1 mRNA transcripts in their conjunctival epithelia than did the control subjects. Tear MMP-9 activities showed significant correlation with symptom severity scores, decreased low-contrast visual acuity, fluorescein tear break-up time, corneal and conjunctival fluorescein staining, topographic surface regularity index (SRI), and percentage area of abnormal superficial corneal epithelia by confocal microscopy. CONCLUSIONS Tear MMP-9 activity was significantly higher in patients with DTS. This activity was associated with increased mRNA expression of MMP-9 and its regulating genes and correlated strongly with clinical parameters. MMP-9 appears to be a potentially useful biomarker for diagnosing, classifying, and monitoring DTS.

Corneal epitheliopathy of dry eye induces hyperesthesia to mechanical air jet stimulation.

PURPOSE To evaluate corneal sensation in different groups: normal subjects, dry eye patients, and patients with and without dry eye after laser in situ keratomileusis (LASIK) using the modified Belmonte gas esthesiometer. DESIGN A retrospective, clinic-based, case-control study. METHODS We evaluated 20 normal subjects, 20 dry eye patients, 20 post-LASIK patients without dry eye, and six post-LASIK patients with dry eye. The corneal sensation was measured with the modified Belmonte gas esthesiometer that uses two different stimuli to assess mechanical and polymodal receptors on the corneal surface. Mechanoreceptors were assessed by 2-second pulsed air jets of variable intensity. Polymodal receptors were measured by stimulating the corneal with 2-second pulsed air jets of varying concentrations of CO(2), a gas that is converted to carbonic acid on contact with the corneal surface. The main outcome measure was determining corneal sensation. RESULTS The mean +/- standard deviation (+/- SD) age was similar in all groups. The mean mechanical threshold was 61.50 +/- 20.07 ml/min in the normal group (n = 20), 34.60 +/- 21.09 ml/min in the dry group (n = 20, P <.05 vs normal), 99.50 +/- 47.40 ml/min in the post-LASIK group (n = 20, P <.01 vs normal), and 50.00 +/- 15.49 ml/min in the post-LASIK patients with keratitis sicca (n = 06, P <.05 vs post-LASIK). The percentage of CO(2) to elicit discomfort was similar in all groups (P >.05). No sex-related differences were noted (P >.05). There was a significant inverse correlation between the threshold of mechanical stimulation and the severity of corneal fluorescein staining. CONCLUSIONS The Belmonte modified noncontact esthesiometer is a sophisticated instrument that can assess different types of corneal sensory receptors. Patients with dry eye were hypersensitive to the air jet stimulus of this instrument, and this appears to be due to altered corneal epithelial barrier function. Profound hypoesthesia was observed after LASIK and similar to dry eye, post-LASIK patients with dry eye were sensitized. These findings provide new insight into the hypersensitivity to environmental stresses, particularly air drafts experienced by dry eye patients.

Cell Size Correlates with Phenotype and Proliferative Capacity in Human Corneal Epithelial Cells

This study investigated whether cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Primary cultured human corneal epithelial cells were sorted by flow cytometry based on forward scatter profile in comparison with the profile of beads of known size. Four fractions (A, B, C, and D) of cells ranging in size from 10 to 16, 17 to 23, 24 to 30, and ≥31 μm in diameter, respectively, were collected to evaluate their 5‐bromo‐2‐deoxyuridine (BrdU) label retention properties, cell phenotype, and clonal growth capacity on a 3T3 fibroblast feeder layer. Among these four populations, cell size was shown to positively correlate with the expression of the differentiation markers keratin (K) 3, K12, and involucrin and inversely with the levels of stem cell–associated markers ΔNp63 and ABCG2 and with colony‐forming efficiency (CFE) and growth capacity. Population A with the smallest size, accounting for 11.0% ± 4.5% of the entire population, contained the greatest number of BrdU label‐retaining slow‐cycling cells, displayed the highest percentage of cells immunopositive to p63 and ABCG2 and negative to K3 and involucrin, expressed the highest levels of ΔNp63 and ABCG2 mRNA and the lowest levels of K3, K12, and involucrin, and possessed the highest CFE and growth capacity. These findings suggest that cell size correlates with cell differentiation phenotypes and proliferative capacity in human corneal epithelial cells. The smallest cells in population A seem to be enriched for putative stem cells, and small cell size may represent one of the important properties of adult corneal epithelial stem cells.

Assessing the severity of keratitis sicca with videokeratoscopic indices

PURPOSE To determine the correlation between the regularity indices of the Tomey TMS-2N computerized videokeratoscopy (CVK) instrument (Tomey, Waltham, MA) with conventional measures of dry eye symptoms and disease. DESIGN A retrospective, clinic-based, case-control study. PARTICIPANTS A total of 16 eyes of 16 asymptomatic normal subjects and 74 eyes of 74 patients with reports of ocular irritation. METHODS Corneal surface regularity and potential visual acuity indices of the Tomey TMS-2N CVK instrument were evaluated in patients with ocular irritation symptoms and in normal subjects. MAIN OUTCOME MEASURES The surface regularity index (SRI), surface asymmetry index (SAI), potential visual acuity index (PVA), and irregular astigmatism index (IAI) of the Tomey TMS-2N were compared between normal and dry-eye patients. Severity of dry-eye symptoms was assessed with a validated questionnaire. Schirmer 1 test (without anesthesia), biomicroscopic meibomian gland evaluation with a composite severity score (MGD score), fluorescein tear break-up time (TBUT), and corneal fluorescein staining were performed. The correlations between CVK indices of the Tomey TMS-2N and the symptom severity score, Schirmer 1 test, MGD score, TBUT, and corneal fluorescein staining score were studied. RESULTS Dry-eye patients had greater mean symptom severity scores, lower Schirmer 1 test scores, greater MGD scores, more rapid TBUT, and greater total corneal fluorescein staining scores (P < 0.001 for all parameters). The SRI, SAI, and IAI were all significantly greater in dry-eye patients than normal subjects. These were 0.46 +/- 0.36 (normal) versus 1.09 +/- 0.76 (dry) for the SRI (P = 0.0017), 0.30 +/- 0.15 (normal) versus 0.90 +/- 1.09 (dry) for the SAI (P = 0.0321), and 0.42 +/- 0.28 (normal) versus 0.56 +/- 0.24 (dry) for the IAI (P = 0.0321). The PVA index was significantly lower in the dry-eye patients (0.89 +/- 0.13) than normal eyes (0.68 +/- 0.23; P = 0.0008). The SRI, SAI, and IAI were positively correlated with total and central corneal fluorescein staining scores (P < 0.00001 for all indices). An SRI (> or =0.80), SAI (> or =0.50), and IAI (> or =0.50) had sensitivities in predicting total corneal fluorescein staining (score > or = 3) of 89%, 69%, and 82%, respectively. The specificity of these indices was 80%, 78%, and 82%, respectively. In all 90 eyes, the mean SRI was greater in subjects older than 50 years (P = 0.012) compared with younger patients, whereas no age effect was noted in the dry-eye patients. The SRI and PVA index showed better correlation with symptoms of blurred vision than the best-corrected visual acuity. CONCLUSIONS Patients with ocular irritation have an irregular corneal surface that may contribute to their irritation and visual symptoms. Because of their high sensitivity and specificity, the regularity indices of the Tomey TMS-2N have the potential to be used as objective diagnostic indices for dry eye, as well as a means to evaluate the severity of this disease.

Epithelial-immune cell interaction in dry eye.

Dry eye is a potent stimulus of both innate and adaptive immune systems. At the nexus of the dry eye inflammatory/immune response is the dynamic interplay between the ocular surface epithelia and the bone marrow-derived immune cells. On the one hand, ocular surface epithelial cells play a key initiating role in this inflammatory reaction. On the other hand, they are targets of cytokines produced by activated T cells that are recruited to the ocular surface in response to dry eye. This interaction between epithelial and immune cells in dry eye will be thoroughly reviewed.